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Full text of "Practical organic and biochemistry"

PRACTICAL ORGANIC AND 
BIO-CHEMISTRY 



BY THE SAME AUTHOR. 

THE CHEMICAL CHANGES AND PRODUCTS 
RESULTING FROM FERMENTATION. 

8vo. 6s. 6d. net. 

MONOGRAPHS ON BIOCHEMISTRY 

EDITED BY 

R. H. A. PLIMMER, D.Sc., AND F. G. HOPKINS, F.R.S., D.Sc. 
Royal 8vo. 

THE NATURE OF ENZYME ACTION. By W. M. BAYLISS, 
M.A., D.Sc., F.R.S. 55. 6d. net. 

THE CHEMICAL CONSTITUTION OF THE PROTEINS. 
By R. H. A. PLIMMER, D.Sc. 

Part I., ANALYSIS. 6s. net. Third Edition. 

Part II., SYNTHESIS. 45. net. Second Edition. 

THE GENERAL CHARACTERS OF THE PROTEINS. 
By S. B. SCHRYVER, Ph.D., D.Sc. 

THE VEGETABLE PROTEINS. By THOMAS B. OSBORNE, 
Ph.D. 45. net. 

THE SIMPLE CARBOHYDRATES AND THE GLUCO- 
SIDES. By E. FRANKLAND ARMSTRONG, D.Sc., Ph.D. 
53. 6d. net. 

THE FATS. By J. B. LEATHES, F.R.S., M.A., M.B.. F.R.C.S. 

ALCOHOLIC FERMENTATION. By ARTHUR HARDEN. 
Ph.D., D.Sc., F.R.S. 4 s. 6d. net. 

THE PHYSIOLOGY OF PROTEIN METABOLISM. By 
E. P. CATHCART, M.D., Ch.B., D.Sc. 

SOIL CONDITIONS AND PLANT GROWTH. By EDWARD 
J. RUSSELL, D.Sc. (Lond.). With Diagrams. 6s. 6d. net. 

OXIDATIONS AND REDUCTIONS IN THE ANIMAL 
BODY. By H. D. DAKIN, D.Sc., F.I.C. 

THE SIMPLER NATURAL BASES. By GEORGE BARGER, 
M.A., D.Sc. 6s. 6d. net. 

NUCLEIC ACIDS. Their Chemical Properties and Physio- 
logical Conduct. By WALTER JONES, Ph.D. 45. net. 

LECITHIN AND ALLIED SUBSTANCES. THE LIPINS. 
By HUGH MACLEAN, D.Sc., M.D. 

THE RESPIRATORY EXCHANGE OF ANIMALS AND 
MAN. By AUGUST KROGH, Ph.D. 6s. 6d. net. 
Other Volumes are in preparation. 

LONGMANS, GREEN AND CO., 

LONDON, NEW YORK, BOMBAY, CALCUTTA. AND MADRAS. 




Hemoglobin 1:100. 



Oxyhwrnoglobin 1:100. 



Carboxyhcemoglobin 1:100. 



Methmmoglobin : neutral. 



Hti'matin in acid alcohol. 



H (emochromogcn in alkaline 
solution, 1:100. 

Htematoporphyrin in l/o 



Hcematoporphyrin in 5/o 



Urobilin. 



Uroelythrin. 



Chlorophyll in living nettle 
leaf 



a Chlorophyll in ether. 



b Chlorophyll in ether. 



Carotin in alcohol. 



Xanthophyll in alcohol. 



ABSORPTION SPECTRA. 



M?hy 

p ' 

PRACTICAL ORGANIC 



'H. ' 1 



AND BIO-CHEMISTRY 



f BY 

R^ H^A^PLIMMER 



READER IN PHYSIOLOGICAL CHEMISTRY, UNIVERSITY OF LONDON, UNIVERSITY COLLEGE 



WITH COLOURED PLATE AND OTHER ILLUSTRATIONS IN THE TEXT 



NEW AND REVISED EDITION 




LONGMANS, GREEN AND CO. 

39 PATERNOSTER ROW, LONDON 

FOURTH AVENUE & 30xH STREET, NEW YORK 
BOMBAY, CALCUTTA, AND MADE AS 

1918 



PREFACE TO REVISED EDITION. 

FOR this edition the text has been thoroughly revised. 
Several sections have been rewritten and some new methods 
of preparation and analysis have been incorporated. 

R. H. A. P. 

November, 1917. 



PREFACE. 

IN this edition the same method is adopted as was followed 
in the former edition, entitled " Practical Physiological 
Chemistry," but further experience in teaching has led me 
to believe that the book would be improved and made more 
useful if its scope were extended. New sections upon organic 
chemistry and organic substances found in plants, together 
with methods used in more advanced work, are therefore in- 
cluded. 

As the basis of the book is Organic Chemistry this term 
is used in its title, and since the subject is also treated from 
the botanical side, the term Bio-Chemistry is substituted for 
Physiological Chemistry, a term too often restricted to the aspect 
of the animal side of the subject. 

The book is still intended mainly for medical students, but 
it contains the essentials for all students of Biology. The 
medical student will gain rather than lose by having additional 
matter, though it may not be of immediate use to him. A 
survey of the sections upon plant compounds should give him 
a wider outlook and a deeper insight into the wonderful sub- 
stances connected with the phenomena of Life, and withal, 
they may be useful for reference. 

In order to help the student in using the book the essentials 
of the subject are printed in large type, while the botanical and 
more advanced portions are printed in small type. The more 
important practical experiments suitable for a preliminary course 
are indicated by asterisks and correspond to the small type of 
the former edition. 

I am much indebted to Dr. C. Lovatt Evans for his 
assistance in the sections on " The Function of Haemoglobin " 
and "The Analysis of Blood Gases". Mr. W. W. Reeve, 
B.Sc., has kindly assisted me with the proofs. 

R. H. A. P. 



PREFACE TO FORMER EDITION. 

THIS book was originally compiled as a handbook for prac- 
tical work in Physiological Chemistry at University College, 
London, since no single text or class book covered the 
complete course, or treated Physiological Chemistry as part 
of the subject of organic Chemistry, or even as an in- 
dependent subject. 

The present book must still be regarded mainly as a 
compilation. It represents an attempt to give to the worker 
a nearly complete statement of the whole subject. Each 
section has a short explanatory summary of the essential 
points, so as to connect the various sections together. The 
essential points are illustrated by the practical experiments, 
which are printed in different type. 

The illustrations are also compiled from various sources. 
These are mentioned underneath each figure. The illustra- 
tions of apparatus not so mentioned have been drawn from 
my own sketches. For those of the osazone crystals, haemin, 
and tyrosine, I am indebted to Miss V. G. Sheffield, who 
has also kindly helped in reading the proof sheets. 

In most physiological chemistry laboratories the strengths 
of the reagents employed are very various, e.g. dilute acetic 
acid may be i per cent., or 2 per cent, or 5 per cent., or even 
10 per cent. In order that all workers may employ a reagent 
of standard concentration, a list of reagents has been carefully 
drawn up and is appended. 



CONTENTS. 

I'AOE 

DEFINITION i 

RECOGNITION OF AN ORGANIC COMPOUND .2 

ISOLATION AND PREPARATION OF PURE ORGANIC COMPOUNDS. CRITERIA 

OF THEIR PURITY 3 

I. Purification of a Liquid by Distillation. Determination of the Boiling- 
point, 4 ; II. Separation of Liquids, 8 ; Evaporation of Liquids, 
13;. III. Separation of Solid and Liquid. Filtration, 15; IV. 
Purification of Solids by Crystallisation, 1 7 ; Determination of the 
Melting-point, 23 ; V. Separation of Solids, 25 ; Isolation of Solids 
from Solution, 26. 

COMPOSITION OF ORGANIC COMPOUNDS . . . . . . .28 

A. Elementary Composition. Detection of the Elements, 28 ; B. Quantita- 
tive Composition. Estimation of the Elements, 31 ; C. Calculation 
of Results, 41 ; D. Determination of Molecular Weight, 42. 

IDENTIFICATION OF AN ORGANIC COMPOUND 46 

HYDROCARBONS 49 

A. Saturated, 49 ; B. Unsaturated (a) Olefines, 54, (b) Acetylenes, 56. 
HALOGEN DERIVATIVES OF THE HYDROCARBONS 57 

Monohalogen Derivatives. Alkyl Halides, 57 ; Dihalogen Derivatives, 
58 ; Trihalogen Derivatives. Chloroform, 59. lodoform, 62. 

ALCOHOLS 63 

Methyl Alcohol, 63 ; Ethyl Alcohol, 64 ; Propyl Alcohols, 68 ; Butyl 
Alcohols, 68 ; Amyl Alcohols, 69 ; Higher Alcohols, 70. 

ESTERS . . . . . . . . . . . . -71 

Esters of Inorganic Acids, 71 ; Esters of Organic Acids, 72 ; Hydrolysis 
of Esters, 74. 

ETHERS. ETHYL ETHER . . . . . .76 

MERCAPTANS AND SULPHIDES . 78 

ALDEHYDES So 

Formaldehyde, 80; Acetaldehyde, 80; Estimation, 85; Chloral, 86; 
Chloral Hydrate, 86. 

KETONES. ACETONE 88 

THE FATTY ACIDS . . 93 

Formic Acid, 94 ; Acetic Acid, 96 ; the Higher Fatty Acids, 99. 

HALOGEN SUBSTITUTION DERIVATIVES OF THE FATTY ACIDS . . ' . too 

ACID OR ACYL CHLORIDES ' . 101 

ACID ANHYDRIDES . 102 



CONTENTS 



I'AOE 



UNSATURATED ALCOHOLS, ALDEHYDES AND FATTY ACIDS . . .103 

HYDROXY-, KETO- AND DIBASIC ACIDS .106 

AMINES 124 

AMIDES 129 

THE AMINO ACIDS .... 137 

BETAINES 150 

CYANOGEN COMPOUNDS 152 

GUANIDINE AND ITS DERIVATIVES .164 

Dl-, TRI- AND POLYHYDRIC ALCOHOLS . . I?3 

FATS AND OILS. WAXES. LECITHINS . 175 

THE CARBOHYDRATES ... .183 

THE MONOSACCHARIDES .184 

PROPERTIES AND REACTIONS OF THE MONOSACCHARIDES . .190 
A. Glucose, 190 ; B. Fructose, 194 ; C. Galactose, 195 ; D. Mannose, 
195 ; E. Pentoses, 195 ; F. Glycuronic Acid, 196. 

THE DISACCHARIDES : CANE SUGAR; LACTOSE; MALTOSE . .197 
TRISACCHARIDES AND TETRASACCHARIDES . 202 
CHITIN AND CHONDROITIN . . . 203 
THE POLYSACQHARIDES : STARCH ; DEXTRINS ; GLYCOGEN ; CELLULOSE 205 
GLUCOSIDES *. 214 

ESTIMATION OF CARBOHYDRATES .... .216 

A. Estimation by Means of the Polarimeter, 216; B. Estimation by 
Reduction of Copper Salts, 22 1 ; C. Estimation by Fermentation, 
233 Estimation of Pentoses, 234 ; Estimation of Disaccharides, 
235 ; Estimation of Polysaccharides, 236. 

CARBOCYCLIC COMPOUNDS. ..<. . . . . . 237 

AROMATIC COMPOUNDS 238 

Benzene and its Monosubstitution Derivatives, 240 ; Disubstitution Deriva- 
tives of Benzene, 260 ; Trisubstitution Derivatives of Benzene, 
270; Tetrasubstitution Derivatives of Benzene, 273. 

TANNINS . . .' . 274 

HETEROCYCLIC COMPOUNDS . 276 

UREIDES . ... . x . . . . . . 277 

A. Ureides of Monobasic Acids, 277 ; B. Ureides of Hydroxy and Alde- 
hyde Acids, 277 ; C. Ureides of Dibasic Acids, 279. 

PYRIMIDINES 282 

GLYOXALINE OR IMINAZOLE DERIVATIVES . .... 284 

PURINES 286 

NUCLEIC ACIDS 299 

FURFURANE, OR FURANE, AND ITS DERIVATIVES 303 

THIOPHENE AND ITS DERIVATIVES 304 

PYRROLE AND ITS DERIVATIVES . 304 

PYRIDINE AND ITS DERIVATIVES 307 



CONTENTS xi 



PAGE 



HYDROAROMATIC COMPOUNDS . . ... . 310 

The Inositols . . . . . . . . . . .310 

The Terpenes . . . . . . . . . . .312 

The Cholesterols . . . . . . . . . . -319 

Bile Acids . ... 322 

COMPLEX AROMATIC COMPOUNDS . . 330 

THE ANTHOXANTHINS . 338 

THE ANTHOCYANS . . 340 

INDOLE AND ITS DERIVATIVES 342 

QUINOLINE AND ISOQUINOLINE ......... 350 

THE ALKALOIDS -351 

THE PROTEINS 361 

The General Reactions of the Proteins, 365 ; A. Colour Reactions, 365 ; 
B. Coagulation Reactions, 368 ; C. Precipitation Reactions, 368. 
Derivatives of Proteins, 370 ; Metaproteins, 370 ; Proteoses and Peptones, 
371 ; Separation of Proteoses and Peptones, 372 ; Peptone, 373. 

APPENDIX TO PROTEINS. COLLOIDS AND COLLOIDAL SOLUTIONS . .374 

^ENZYMES. FERMENTATION 391 

Localisation of the Enzymes and the Chemical Changes in the Organism, 
396 ; Demonstration of the Action of Enzymes, 399 ; the Cataly- 
tic Action of Enzymes, 413 ; the Synthetical Action of Enzymes, 
415; the Measurement of the Activity of Enzymes,. 416. 
Appendix to Digestion 

I. The Acids in the Gastric Contents, 427 ; II. The Constituents of Bile, 
429; III. Gall Stones, 431. 

THE INDIVIDUAL GROUPS OF PROTEINS . 432 

Protamines ............ 432 

Histones ............ 433 

Coagulable Proteins. Albumins. Globulins ...... 435 

The Coagulable Proteins of Egg- White, 436 ; Blood, 438 ; Milk, 446 ; 

Muscle, 447 ; Other Animal Tissues, 449 ; Plants, 450. 
Gliadins and Glutelins . . . . . . . . . -452 

The Scleroproteins ..... .... 454 

Phosphoproteins . . . . . . . . . . -457 

Milk, 457 ; Egg- Yolk, 463. 
Nucleoproteins . . . . . . . . . . . 465 

Glucoproteins . . . . . . . ., . . . 468 

Chromoproteins ........... 472 

Blood, 472. The Reactions of Haemoglobin in Defibrinated Blood. Spec- 
troscopic Examination of Haemoglobin and its Derivatives, 478. 
Estimation of Haemoglobin, 486. Crystals of Oxyhaemoglobin and 
Derivatives, 491. The Function of Haemoglobin as Carrier of 
Oxygen, 497. The Blood as Carrier of Carbon Dioxide, 510. 
THE CHEMICAL CONSTITUTION OF HJEMIN AND H^MATOPORPHYRIN . 516 
THE PIGMENTS OF LEAVES. THE CHLOROPHYLLS AND CAROTINOIDS . 519 
METABOLISM. INTEGRATION OF THE CHEMICAL PROCESSES . . . 533 

COMPOSITION OF THE COMMONER TISSUES USED AS FOOD- STUFFS FOR 

ANIMALS 537 



xii CONTENTS 

I' A OK 

ANALYSIS OF NORMAL URINE . . 539 

Micro-Methods for the Analysis of the Nitrogenous Constituents of Urine 556 

Appendix to Urine .......... 564 

i. The Pigments, 564; 2. Urinary Sediments, 565 ; 3. Urinary Calculi, 
568 ; 4. Inborn Errors of Metabolism, 569 ; 5. Pathological 
Urines: (i) Diabetic, 570; (2) Protein, 571; (3) Blood, 572; 
(4) Bile, 572. 

ANALYSIS OF TISSUES ... 573 

A. The Inorganic Constituents . . . . . . . 573 

B. Proteins ............ 575 

C. Nitrogenous Extractives . . . . . . . . . 577 

D. Carbohydrates . . . . . . . . . -585 

E. Lactic Acid ........... 590 

F. Aceto-acetic Acid .......... 593 

G. /3-Hydroxybutyric Acid ......... 597 

H. Fat . . t ..'.... . 601 

I. Cholesterol .'."'. . . . . . . . . . 603 

TABLES . . 605 

LIST OF REAGENTS . . . . ' . 610 

INDEX 618 



DEFINITION. 

THE substances composing the organic material connected with the 
phenomena of life, and the great majority of the products of vital 
activity, are mixtures of compounds of the element Carbon. 

From these substances the chemist has isolated numerous pure 
carbon compounds and prepared others ; he has also synthesised carbon 
compounds from their elements. 

About 1 50,000 carbon compounds are now known. The possibility 
of their existence is due to the unique property which the element 
carbon possesses of being able to combine with itself; compounds are 
known which contain in their molecules from one up to sixty atoms 
of carbon directly joined together. 

Of these 150,000 carbon compounds only a small number are 
directly concerned in vital processes. 

The chemistry of all the carbon compounds is termed organic 
chemistry. 

The chemistry of those carbon compounds which are the con- 
stituents of living matter and are concerned in vital processes is 
physiological or biological chemistry ; the term physiological chemistry 
more frequently refers to the compounds and their functions in 
animals ; the term biological chemistry comprises the compounds and 
their functions in both plants and animals. The changes which they 
undergo and the functions which they fulfil in the living plant or 
animal form the subject of chemical physiology. 

Though a distinction can be made between biological chemistry 
and chemical physiology, the two subjects are so closely interrelated 
that they are essentially only different aspects of the same subject. 
No biological change can be followed until a knowledge of the chemical 
properties of the substances involved has first been acquired. Chemi- 
cal physiology is thus dependent on biological chemistry, or Bio- 
chemistry. Bio-chemistry is the branch of organic chemistry which 
deals with the natural organic compounds and with the functions of 
these compounds in nature, 

I 
I 



RECOGNITION OF AN ORGANIC COMPOUND. 

Organic compounds are distinguished from inorganic compounds 
by being combustible : on heating they generally char, sometimes 
take fire, and on prolonged heating completely sburn away leaving 
no ash. Inorganic compounds when heated do not char and they 
leave a residue. A mixture of an organic compound and an inorganic 
compound will also char and leave a residue. There are a fe^w excep- 
tions to this general rule : oxalic acid and its salts amongst the organic 
compounds do not char on heating ; amongst the inorganic com- 
pounds the ammonium salts volatilise leaving no ash. An oxalate 
leaves a residue of the oxide of the metal with which it is combined. 

The following experiments exemplify these statements : 

1 . A small piece of paraffin wax heated upon platinum foil will melt, take 
fire, and will completely burn away leaving no residue. 

2. A crystal of cane sugar heated in the same way will melt, char, and 
on further heating will disappear completely. 

3. A few crystals of common salt heated on platinum foil will melt, and 
unless heated very strongly, e.g. with a blowpipe flame, will remain as a solid 
white mass when allowed to cool. 

4. A small piece of soap heated as above will char, the vapours evolved 
may take fire, and when the charred particles have all vanished a white or 
nearly white residue will remain. 

Note. It is in this way that substances composed of organic and inorganic 
matter are recognised. The composition of the inorganic residzie is found out by 
the usual methods of inorganic analysis after the organic matter has been destroyed 
by heating. 

5. No appreciable change will be seen on heating a little oxalic acid or an 
oxalate, e.g. calcium oxalate. 

6. Ammonium chloride volatilises on heating and leaves no residue. 

7. To prove the presence of carbon in oxalic acid or in an oxalate the sub- 
stance is heated in a small glass tube and the gases evolved are passed into 
lime or baryta water. A precipitate of calcium or barium carbonate indicates 
the presence of carbon. 

8. On heating ammonium chloride as in 7, there is no formation of 
carbonate. 



ISOLATION AND PREPARATION OF PURE 
ORGANIC COMPOUNDS. 

CRITERIA OF THEIR PURITY. 

The organic material which composes all animal and vegetable 
cells consists of a mixture of numerous compounds. In order to in- 
vestigate their evolution and their degradation in nature it is necessary 
to separate these compounds from one another and to prepare each of 
them in a state of purity. The pure substance can then be analysed 
and its chemical and physical properties ascertained. Knowledge of 
the pure compounds shows their chemical relationship to one another 
and an idea of their role in nature is obtained. This idea is proved or 
disproved by an investigation of the changes which the organism, as a 
whole or individual portions of it, can effect in these substances. 

In the study of the chemical properties of the compounds, other 
compounds are formed by their interaction. These compounds also 
require isolation and purification. The principal operations in organic 
and biological chemistry will thus consist in the isolation and prepara- 
tion of pure compounds. 

The methods of separating organic compounds are based upon 
differences in the properties of the substances under investigation. 
These differences are taken advantage of as much as possible ; some- 
times they are so gross that the separation is simple, sometimes they are 
so small that the separation is of extreme difficulty, and in these cases 
a separation can only be effected when sufficient material is available. 

Solid organic compounds are more numerous than liquid ; gases are 
comparatively rare. 

Solids are most usually separated and purified by solution in water 
or organic solvents. The methods for the manipulation of liquids are 
therefore most frequently used and are described first. 



4 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

I. PURIFICATION OF A LIQUID BY DISTILLATION- 
DETERMINATION OF THE BOILING-POINT. 

A liquid is purified by distillation. The criterion of the purity of 
a liquid is its boiling-point. A pure liquid has a constant boiling- 
point. 

A. Drying and Cleaning of Apparatus. 

Since organic liquids are very frequently not miscible with water 
all the apparatus which is used with them must be dry. 

The apparatus may be dried in an oven for some time, but on 
removal to prevent deposition of aqueous vapour a current of air is 
blown through them whilst they are hot and during cooling. The 
current of air is most conveniently got from bellows, or by suction with 
a pump. The tubing, or if the glass vessel be narrow, a glass tube 
inserted in the tubing, is placed inside the vessel so that the farthest 
extremity is dried and cooled first. 

More usually since the vessels are wet with water, they are rinsed 
with alcohol after draining as much water away as possible, then with 
ether after again draining. A current of air from the bellows or water- 
pump is drawn through to evaporate the ether. The vessel may be 
warmed in a luminous flame, when no more ether is present and 
the vessel is not quite dry, and air driven through as above. It is im- 
portant that all the ether be evaporated, since it may become ignited 
or form an explosive mixture inside the vessel. 

Apparatus which contains charred matter may be cleaned by 
oxidising it away with potassium bichromate and sulphuric acid, or 
by heating in it a mixture of concentrated sulphuric and nitric acids, 
washing with water and proceeding as above to dry it. 

B. Distillation of a Liquid and Determination of its Boiling- 
point. 

The liquid is placed in a clean, dry fractionating or distilling flask 
a round-bottom flask with a side tube in its neck * of suitable size 
so that only about half or at most two-thirds of the space is filled. 
Some small pieces of unglazed porcelain, or porous earthenware, or 
pieces of platinum, are added to ensure steady boiling without bump- 
ing. The neck of the flask is closed with a well-fitting cork - which 

1 With liquids of high boiling-point the flask should have the side tube low down so 
as to prevent decomposition by the high temperature. 

2 Rubber corks are dissolved by many organic liquids and consequently are not used, 
except in special gases. 



PREPARATION OF PURE COMPOUNDS 



5 



is bored to carry a thermometer. The position of the thermometer 
is so adjusted that the bulb is just below or opposite the side tube and 
not touching the walls. The side tube is connected by a cork to a 
clean, dry condenser, which is supported by a clamp, and a slow stream 
of cold water is allowed to flow through the condenser. A receiver 
(flask) is placed at the other end of the condenser (Fig. i). 

A water condenser is not used for liquids boiling above 120 ; the 
vapours are condensed by being passed through a simple tube (the 
inner tube of the above condenser). The vapours of liquids boiling at 
very high temperatures are condensed in the side tube of the distilling 
flask. If the vapours condense to a solid on cooling, the solid is melted 
by a flame so that the liquid runs into the receiver and does not block 
up the side tube. 




FIG. i. 

Liquids boiling below 100 are heated on a water-bath, liquids 
boiling above 100 are heated directly with a flame, which is moved 
round and round under the bottom of the flask until boil ing. begins. 
When boiling commences it must be kept on continuously and 
vigorously and not interrupted by the removal of the flame or by 
draughts. 

When the vapour from the boiling liquid reaches the thermometer, 
the temperature is seen to rise rapidly, and then becomes stationary 
at a definite temperature. This is the boiling-point of the liquid. 
Drops of condensed liquid are usually seen to fall from the end of the 
thermometer into the flask. The heating is continued until all the 
liquid boiling at this temperature has distilled over into the receiver. 
The portion remaining in the distilling' flask contains the impurities. 



6 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Towards the end of the distillation the thermometer may be seen 
to rise slowly and the last portion to distil i higher than the first por- 
tion. Most pure liquids boil over a range of -5 or i or sometimes 
more. 

Chloroform and aniline may be used as examples. 



Correction for Boiling-point. 

A correction should be applied when the mercury in the thermometer 
passes outside the neck of the flask as this portion is cooled below the tem- 
perature of the vapour of the liquid. The following amount is added to the 
observed temperature T 

(T - /) x -000154 

where n is the number of scale divisions projecting, / the temperature of the 
air recorded by another thermometer held at a point about the centre of the 
projecting thread and screened from heat radiation from below by a sheet of 
cardboard. 

A short thermometer registering only 50 and which can be completely 
inserted in the vapour is used for more accurate determinations. Sets of 
thermometers registering intervals of 50 between o and 360 can be pur- 
chased. 

Determination of the Boiling-point of Small Quantities. 

When insufficient liquid is available for distillation its boiling-point can be 
determined by placing it in a test tube and heating it through an opening in 
a sheet of asbestos. The thermometer is held in the vapour. 

If only a few drops of liquid are available the boiling-point can be ascer- 
tained by introducing it into a small test tube, attaching the test tube to a 
thermometer by a rubber band and heating the two together in a beaker con- 
taining sulphuric acid. In the liquid is placed a piece of capillary tubing, 
a melting-point tube (p. 24) which is sealed near the end immersed in the 
liquid. As the temperature of the bath rises bubbles escape from the capillary 
and ascend through the liquid. At the boiling-point they form a continuous 
stream and the temperature of the bath is noted. Several determinations are 
made with fresh pieces of capillary and the mean will give the boiling-point 
of the liquid. 



PREPARATION OF PURE COMPOUNDS 



C. Distillation in Vacuo. 

Liquids which boil at high temperatures and decompose on distillation 
under atmospheric pressure can frequently be distilled under reduced pressure. 

The liquid is distilled from a fractionating flask, the side tube of which is 
inserted in another fractionating flask, or other stout vessel with a side tube, 
which acts as receiver. This is kept cold by allowing a stream of cold water 
to run over it ; the water is collected in a funnel, which serves as a support 
to the receiver and it runs thence to the waste. The vacuum is produced by 
connecting the side tube of the receiver with a water pump or a mechanical 
pump. A gauge is in connection between the apparatus and the pump by a 
T piece so that the pressure can be ascertained. 

To ensure continuous ebullition without bumping a slow stream of air, 
or carbon dioxide from a Kipp apparatus if the liquid tends to oxidise or de- 
compose in the air, is passed through the liquid by inserting in the cork a tube 
with a long capillary which reaches almost to the bottom. The supply of air 
is regulated by means of a screw pinch on a piece of pressure-tubing placed 
on the end of the glass tube. The apparatus is shown in Fig. 2. 




TO PUMP 



FIG. 2. 

A distilling flask with a double neck as in Fig. 7 (p. 1 1) is more convenient 
than a simple distilling flask ; the neck with the side tube carries the thermo- 
meter, the other neck the capillary. 

The heating of the flask during" distillation in vacuo is best effected by 
means of a bath ; overheating is prevented, and the flask and its contents can 
be completely immersed which ensures a uniform temperature. 

Rubber corks are used in vacuum distillation as they more easily prevent 
leakage in of air. If they are attacked by the vapours of the liquid they 
may be protected by placing a piece of cork between their ends and the 
vapours. 



PRACTICAL ORGANIC AND BIO-CHEMISTRY 



another. 



II. SEPARATION OF LIQUIDS. 
A. Mechanically. 

(a) Two liquids, if they are not miscible, are easily separated from one 
They are placed in a tap funnel or separating funnel (Fig. 3), 
either cylindrical or pear-shaped according to the 
volume, the stopper is removed and the heavier 
liquid run out and collected. The Blighter liquid is 
poured out through the top so as to avoid contamina- 
tion by coming in contact with drops of the other 
liquid remainingjin the stem. 

() Two liquids, if they are miscible, may not both 
be soluble in the same solvent ; such a solvent must 
not be miscible with one of the constituents. On 
shaking the mixture with the solvent in a separating 
funnel, the mixture will separate into two layers. In 
shaking up two liquids in a separating funnel, the 
stopper of the funnel is held in the palm of one hand, 
and as usually an increase of pressure occurs, especi- 
ally if ether and water be the liquids, the tap of the 
funnel is occasionally opened after allowing the liquids to settle at the 
other end. After thoroughly shaking, the liquids areiallowed to separate, 
the stopper is removed and the heavier liquid is run out. The insoluble 
liquid is shaken up once or twice more with more solvent, and is puri- 
fied by drying, if necessary, and distillation. The solvent, if water be 
one of the liquids, is dried by shaking it, or allowing it to stand for 
12-24 hours with solid calcium chloride, or sodium sulphate, or potas- 
sium carbonate. It is separated from the soluble constituent by frac- 
tional distillation, or treated as the case necessitates. 

As an example, a mixture of equal parts of alcohol (50 c.c.) and 
chloroform may be made. On shaking up with two volumes of water 
in a separating funnel, the chloroform separates and sinks. It is re- 
moved and the other constituents are poured out. The chloroform is 
returned and shaken once more with water. It is run out and the 
moisture is removed by shaking or standing with some calcium chloride 
from which it is filtered and then distilled. 




PREPARATION OF PURE COMPOUNDS 



B. Fractional Distillation. 

A mixture of two or more miscible liquids may be separated from 
one another by fractional distillation if their boiling-points differ by 
20-30. 

The mixture is distilled as described previously (p. 4), preferably 
from a flask with its side tube high in the neck and the thermometer is 
carefully watched. The first portion which distils will consist mainly 
or entirely of the more volatile constituent which has a higher vapour 
pressure ; the last portion will consist of the less volatile constituent 
having a lower vapour pressure or higher boiling-point. Between 
these portions there may be a small intermediate fraction consisting 
of a mixture of the liquids. The three portions or fractions are 
collected in separate receivers. 

E.g.- 

A mixture of equal volumes of chloroform and aniline will give on 
fractional distillation a fraction boiling at 61 (almost pure chloroform), 
an intermediate fraction, and a final fraction boiling at 183 (almost 
pure aniline). During the distillation of such a mixture when the 
boiling-point of a constituent exceeds 120 the water 
should be run out of the condenser. 

Redistillation of the first and last fractions will 
give each of them in a state of purity. 

If a mixture of liquids contains constituents which 
have boiling-points fairly close to one another, the 
fractionation must be repeated several times until each 
fraction is found to have a constant boiling-point. 

The separation of such a mixture is greatly facili- 
tated by the use of a fractionating column or still-head. 
This is simply a device to lengthen the neck of the 
distilling flask so that the higher boiling fractions are 
exposed to the air and condensed before they reach 
the condenser and run back into the flask. Numerous 
forms have been invented ; two efficient forms are 
those of Hempel and of Young (pear still-head, Fig. 4). 

The former consists of a glass tube filled with 
glass beads and a side tube. The latter consists of a 
piece of glass tubing upon which are blown 2, 3, 4 or 
more bulbs of a pear shape, and a side tube. The 
liquid is placed in a round-bottom flask, the fractional 



FIG. 4. 



column inserted and this in turn connected by its side tube to the 
condenser. The mixture in the flask, to which several small pieces of 



10 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

porous earthenware have been added, is heated over a gauze at such a 
rate that the condensed liquid comes over drop by drop. Fractions 
are collected at 5 or 10 ranges of temperature. 

The redistillation of each fraction is carried out as follows : In 
the apparatus which has been washed out and dried fraction I is put 
which boiled, say, at 90-95, and distilled till the thermometer shows 
95, when the distillation is stopped and fraction II, 95-100, is added 
to the remainder in the flask. On distilling, fractions below 95 are 
collected in the first receiver and the second fraction is distilled till 
the temperature reaches 100. Distillation is stopped and the next 
fraction added. The process is continued until all the fractions have 
been redistilled. Finally the main fractions boiling at the extremes 
will be obtained. 

A mixture of benzene, toluene and xylene, such as occurs in coal 
tar, may be taken in illustration. 

Constant Boiling Mixtures. 

It frequently happens that two liquids form a mixture which has 
a constant boiling-point and behaves like a single liquid. Such a 
mixture cannot be separated by fractional distillation, although more 
or less separation may be possible by distilling at a different pressure. 

Such a mixture may have a boiling-point which is lower than 
either of its constituents, or higher. 

Excess of either constituent in the mixture beyond that forming 
the constant boiling mixture can be separated by fractional distilla- 
tion and it will distil over either before or after the mixture according 
to the boiling-points. Such constant boiling mixtures are mixtures 
of ethyl alcohol and water, methyl alcohol and acetone, benzene and 
alcohol, pyridine and water, water and formic acid, chloroform and 
acetone. They can only be separated by chemical means. 

Fractional Distillation in l/acuo. 

The same apparatus as described above for distillation in vacuo can be 
used for fractional distillation in vacuo, but as each fraction distils the 
apparatus must be disconnected so as to insert a new receiver. To avoid 
releasing the vacuum and disconnecting the apparatus, and to allow fractional 
distillation to proceed continuously several contrivances have been suggested. 
Usually the vapours of liquids distilled in vacuo condense without being cooled 
by water or ice, and an apparatus as in Fig. 5 may be used. The receivers 
are turned into position when necessary. A very simple form is shown in 



PREPARATION OF PURE COMPOUNDS 



Fig. 6 : the apparatus can be rotated on the cork connecting it with the 
distilling flask when a new fraction begins to distil over. 





FIG. 5. 



FIG. 6. 



An apparatus of the type in Fig. 7 is the most convenient. By means of 
the several taps the receiver can be shut off and its vacuum released, whilst 
distillation continues and the fraction collects in the bulb. The fresh 




FIG. 7. 

receiver is exhausted whilst the taps to the bulb and distilling flasks are 
closed ; no great decrease of vacuum occurs as the small receivers are 
rapidly exhausted. 

If necessary the bulb and receiver can be cooled by a stream of cold water, 
or by immersing the receiver in ice or a freezing mixture. 



12 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

C. Steam Distillation. 

Very frequently a separation of a liquid or a solid from a mixture 
can be effected by the process of steam distillation. The liquid, or 
solid, has usually a higher boiling-point than water, but the vapours 
of the liquid and of the water do not interfere with each other. The 
effect of the steam is to reinforce the vapour pressure of the liquid, so 
that the liquid distils with water under atmospheric pressure at a lower 
temperature. 

Steam is generated in a large flask, or tin can, which is provided 
with a cork carrying a long safety tube about 80 cm. long, reaching 
almost to the bottom, and with a delivery tube. The flask is half- 
filled with water. The delivery tube when the steam is ready is con- 
nected to the flask containing the mixture. This flask is placed in a 




sloping position so as to prevent splashing of its contents and mechanical 
carrying over of substance into the condenser. The steam is passed 
into the bottom of the vessel by a tube which is bent so that its end 
lies in a vertical position, and to prevent condensation of the steam 
the flask is also heated. The steam and other vapour reach a long 
condenser by which water and substance are condensed and are col- 
lected in a receiver (Fig. 8). 

The separation of substance and water is effected by filtration if 
solid, by simple separation if liquid, or by extraction with a solvent 
such as ether, chloroform, etc. The solvent is then dried, removed by 
distillation, and the substance or substances obtained by distillation 
or fractional distillation. 



PREPARATION OF PURE COMPOUNDS 13 

EVAPORATION OF LIQUIDS. 

Only aqueous solutions can be evaporated over a flame, and 
the evaporation should be completed over a water-bath to prevent 
charring the substance as it becomes concentrated. Evaporation over 
a flame must be carefully watched to prevent charring and also to avoid 
spurting when the solution begins to concentrate. 

Most organic liquids which are used as solvents are readily inflam- 
mable and must not be brought near a flame. 

When a considerable amount of solvent is present it is removed by 
distillation and in cases where the solvent (ether, acetone, ligroin, etc.) 
is very readily inflammable the distillation must be carried out on- 
a water-bath, heated by a flame specially protected by a gauze, or by 
steam, or electrically. If the bath be heated by a burner, the end of 
the condenser should be placed as far away as possible and a sheet 
of cardboard or asbestos interposed between the burner and receiver. 

Not only is evaporation by distillation absolutely necessary with 
inflammable liquids, but also it is economical. The solvent is recovered 
and after purification can be used again. 

When only small quantities of liquids up to 25 c.c. require evapora- 
tion they are set aside, away from flames, and allowed to evaporate 
spontaneously, or they may be put upon a warm water-bath, with the 
flame extinguished. 

Another common procedure is to evaporate small quantities by 
placing them in a vacuum desiccator and exhausting. The evaporation 
is greatly accelerated if the liquid be previously warmed on a water-bath 
and whilst warm put into the desiccator. 

Evaporation in l/acuo. 

Evaporation in vacno is the most rapid method of concentrating solutions, 
especially if the temperature can be kept at an elevated point. 

Very frequently evaporation of solutions must be carried out at a low tem- 
perature (35-45) to prevent decomposition. This is carried out in the same 
way as distillation in vacua, the distilling flask being kept Jh water at 35-45". 
With large flasks of 2-3 litres capacity and a good vacuum of about 15 mm., 
a litre of water can be distilled off in 2-2^ hours at 35-45. Instead of a distil- 
ling flask an ordinary flask fitted with capillary and bent tube may be used and 
a similar one as receiver. 



I 4 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Frothing During Evaporation in Vacuo. 

Aqueous solutions of extracts of plant or animal matter have a great 
tendency to froth during concentration in vacua. 

The froth can be broken by allowing drops of alcohol to fall upon it 
from a tap funnel inserted into the distilling flask. The alcohol vapour helps 
in the evaporation when the vacuum has been established. 

The liquid to be evaporated can be allowed to flow into the exhausted 
vessel through a spiral tube at such a rate that no large volume is ever present 
in the. flask. 

A contrivance to catch the froth and return it to the flask has been devised 
by Davis and Daish. It has been very useful for evaporating plant extracts. 
It is shown in Fig. 9. 




FIG. 9. From the Journal of Agricultural Science, Vol. V, p. 435 (Cambridge Uni- 
versity Press). 

* 

Any froth which is formed is broken by the piece of copper gauze in B 
and the liquid runs back into the flask A. The vapours are condensed by 
the condenser D and collected in G. The glass piece E is introduced so as 
to allow of the emptying of G : the tap is turned and the screw clamp on 
the pressure-tubing at S closed ; liquid collects in E whilst G is removed, 
emptied, replaced and evacuated. The tube M leads to a manometer, the 
bottles P and H and the valve J serve to regulate inequalities of pressure. 



PREPARATION OF PURE COMPOUNDS 



III. SEPARATION OF SOLID AND LIQUID. 
FILTRATION. 

The mixture of solid and liquid may consist of suspended particles 
consisting of residues of filter paper, etc., which do not require ex- 
amination, or it may consist of matter requiring examination, obtained 
either naturally or by evaporation of the liquid, or it may consist of 
solid in a crystalline form purified by crystallisation. 

Obviously the procedure to adopt is filtration. Filtration is effected 
in several ways depending on the material. 

(a) Fluted or Pleated Filter Paper. 

Suspended matter is removed by filtration through a filter paper 
folded in the ordinary way to fit a funnel, or better and more rapidly 
through a fluted or pleated filter paper which exposes as large a sur- 
face as possible to the liquid. A filter 
paper is folded into quarters in the ordin- 
ary way. Each quarter is then bisected 
by folding towards' the hollow of the 
central fold, and each of these divisions 
is bisected again in such a way that the 
hollows and ridges alternate (Fig. 10). 
More pleats are obtained in the same way 
by bisecting the divisions and folding 
alternately. The paper is thoroughly 
pressed to make the pleats permanent. 

Such a filter paper is used for rapidly 
filtering off a small number of particles 
and also for filtering off solid matter which 
is not crystalline, such as the residues re- 
maining after extracting animal and plant 
tissues with solvents, residues which are not easily filtered by the 
other methods. 

(b) Filter Plate. Buchner Funnel. 

The filtration of crystalline compounds is best effected by means 
of a perforated porcelain plate placed in a funnel or a complete 
funnel of porcelain of this pattern (Buchner or Hirsch funnel). The 
perforations are covered over with a filter paper of the right size and to 
prevent breaking of the paper two thicknesses may be used, or better 
hardened filter paper. The paper is wetted with the liquid and sucked 
down by a vacuum produced by a filter pump (Fig. 1 1 ). 

1 The substance is placed upon the paper and the liquid drained off as 




FIG. 10. 



i6 



PRACTICAL ORGANIC AND BIO-CHEMISTRY 



completely as possible, the solid being pressed down with a spatula 
or flat piece of glass. The solid is washed two or three times with 
solvent which is added in small portions. The whole of the solid must 
be wetted and the solvent drained off completely before more is added. 




FIG. ii. 

(c) By Filter Presses. 

Large quantities of solid matter with small amounts of liquid are separated 
by means of a filter press. The material is pressed either between layers of 
cloth or in a sheet of cloth which can be folded to make a sort of bag. 
Squeezing by hand in the latter method will remove some of the liquid and 
the greater pressure from a press will remove nearly all. The dry residue can 
be taken out, stirred up with solvent and again pressed out. 

The greatest pressure usually attainable is from a Buchner press or a hy- 
draulic press. The solid matter must be in a fairly dry condition and, if moist, 
is mixed with some absorbent, generally siliceous earth or " Kieselguhr ". 
Liquid is squeezed out of this mass by the high pressure. 

Subsequent filtration through a fluted paper is usually necessary after fil- 
tration by a filter press as particles come through the pores in the cloth. 

(d) Through a Layer of Neutral Material. 

Solutions containing colloidal particles are generally most difficult to filter 
as the particles either come through fluted filter paper or soon clog the pores. 

Osborne has used filter paper pulp as a medium for filtering solutions of pro- 
teins. A sufficient quantity of pulp is mixed with the liquid and this is poured 
upon the paper on a Buchner funnel, where it forms a layer exposing a large sur- 
face and prevents the underneath paper from breaking and becoming clogged. 

A layer of siliceous earth upon a Buchner funnel has also been found very 
effective. A sufficient quantity of the siliceous earth is mixed in a small mor- 
tar with the liquid and poured upon the funnel so as to form a layer from i - 2 cm. 
thick, and the liquid is then filtered through this. 

The first portions of filtrate may be cloudy, and must be returned to the 
filter and filtered again. 



PREPARATION OF PURE COMPOUNDS 17 

IV. PURIFICATION OF SOLIDS BY CRYSTALLISATION, 

The majority of solid organic compounds are crystalline, but 
many of the complex solids, such as starch, glycogen, various pro- 
teins, which belong to the class of substances termed colloids, have 
not yet been prepared in a crystalline form and are only known in an 
amorphous state. Others, such as the fats, though obtainable in 
a crystalline form, are mixtures of closely related substances and it 
is extremely difficult, or almost impossible, to separate them into 
individual compounds. 

The separation of a solid in a crystalline form is essential to its 
preparation in .a pure condition. Its recrystallisation, when once 
obtained in a crystalline form, will lead to its preparation in a state 
of purity. 

The criterion of the purity of a crystalline solid compound is its 
melting-point. ^A pure solid organic compound melts sharply at a 
definite temperature. An impure organic compound does not melt 
sharply and it melts at too low a temperature. The knowledge of 
the melting-point of a compound helps in its identification. 

(a) Choice of Solvent for Crystallisation. 

The purification of a solid by crystallisation depends very largely 
upon the choice of a suitable solvent. The best condition for puri- 
fication is very slight solubility in the cold solvent and ready solubility 
in the boiling solvent. A hot saturated solution of the solid will de- 
posit the greater part of the solid in a crystalline condition on cooling, 
and if the solution be allowed to cool slowly the crystals which are 
deposited will be more regular than if the solution be cooled rapidly. 

In order to ascertain the solubility of a substance in a solvent the 
substance must be in a fine state of division. A small quantity of 
the substance is finely powdered in a watch glass with a glass rod, or 
better in a small agate mortar with a pestle. 

A few milligrams of the substance are then placed in a small test 
tube, a few drops of solvent are added and the solid well stirred or 
shaken with it. If the solid is apparently not soluble in this amount 
of solvent more is gradually added, and so it can be determined 
whether the substance is easily soluble, moderately soluble, or in- 
soluble in the cold liquid. 

Substances which are readily soluble in cold solvent are not 
usually recrystallised from this medium since there will be no great 
difference in solubility in the hot and cold liquid, and a large quantity 
of solid will remain in solution even when crystals are obtained. 

2 



18 PRACTICAL ORGANIC AND BlO-CHEMlStRV 



The solvent in those cases where the substance is slightly soluble 
or insoluble is now heated ; if the solid dissolves easily more is 
added until the solution is saturated ; if not, more solvent is added 
so as to bring, if possible, the solid into solution. The solution is 
cooled by holding under running water and it is noticed how much 
of the solid crystallises out. If a considerable quantity separates 
out, the solvent will probably be suitable for recrystallising larger 
quantities. 

Sometimes crystallisation does not occur spontaneously on cooling, 
but it may be started by scratching the sides of the test tube or by 
adding a crystal of the solid. 

The following solvents are most frequently used : - 



(1) water 

(2) alcohol 

(3) acetone 



(4) benzene 

(5) chloroform 

(6) ligroin 



(7) glacial acetic acid 

(8) methyl alcohol 



The following substances cane sugar, oxalic acid, benzoic acid, 
urea and succinic acid may be taken as examples for observing the 
difference in solubility in water and alcohol, and for the choice of 
solvent for crystallisation. 

(b] Recrystallisation. 

If the suitable solvent has been found to be water, or glacial 

acetic acid or a liquid which is not 
inflammable and boils at a fairly 
high temperature, the recrystallisa- 
tion may be carried out in a beaker 
heated over a gauze. 

If the suitable solvent has been 
found to be alcohol, acetone, 
ligroin, benzene liquids which are 
volatile and inflammable the re- 
crystallisation must be carried out 
in a flask to which is attached a 
reflux or inverted condenser, as in 
Fig. 12. 

Solvents boiling below 90 are 
heated on the water-bath, above 
FlG - I2 - I OO over a flame .through a wire 

gauze and with an air condenser (inner tube of condenser or a tube 
about 80 cm. long by "5-1 cm. in diameter} as reflux. 




PREPARATION OF PURE COMPOUNDS 19 

(i) Solution. 

> . The substance to be recrystallised is powdered finely and placed in 
a flask or beaker with a small quantity of solvent. Excess of solvent 
must be avoided as the object is to prepare a hot Saturated solution. 
The solvent is boiled. If, after boiling for some time, a consider- 
able amount of solid remains, more solvent is cautiously added 
(through the condenser) and the boiling continued ; solvent is added until 
the whole of the solid, except insoluble matter, is dissolved. It should 
be noted that the last portions of a solid often only dissolve with 
difficulty and fresh solvent should not be added too soon. 

(ii) Filtration. 

Particles of insoluble impurity are now filtered off by rapidly filter- 
ing the hot solution through a pleated filter paper. Very frequently 
the solution is very concentrated and begins to crystallise immediately 
filtration is commenced. To avoid this a funnel with a very short 
stem or without stem is used, and it is previously heated in an oven or 
by passing through a flame ; the solution is filtered whilst it is still hot. 

When the tendency to crystallise immediately is very pronounced the nitra- 
tion must be carried out through a funnel heated by steam. The funnel 
may be surrounded by coils of metallic piping through which steam from a 
generator is passed, or it may be enclosed in a larger metal funnel with two 
walls between which there is water and from the outer of which there is a 
projection for heating the contents by a flame. Care must be taken that 
inflammable liquids do not become ignited if this form of hot-water funnel be 
used. The water can be raised to boiling and the flame removed. 

If crystallisation of solid should commence during the filtration, the 
funnel and paper are placed over the flask, the paper pierced and the 
particles washed with a little solvent into the flask and the solution 
again boiled up. 

The filtrate is collected in a beaker of such a size that it is not 
filled more than two-thirds. 

Some liquids "creep," and if the vessel be filled -too full or if 
shallow dishes be used the solution will creep over the edge and de- 
posit crusts of impure crystals over the edges. 

If crystals begin to separate before the filtration is completed, it is 
best to heat the filtrate until solution is again effected. To exclude dust 
and prevent evaporation, the beaker is covered with a clock glass with 
its convex side uppermost. Condensed drops of solvent will then run 
towards the side and not drop into the liquid and disturb the formation 
of the crystals. The solution is set aside in a cool place to crystallise. 

Crystallisation may be complete as soon as the solution is cold, or 
it may take several hours, or days. 



20 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

(iii) Collecting of Pure Crystals. 

Any crusts of crystals which may have formed on the sides and 
edges of the vessel by evaporation must be removed before the re- 
mainder of the crystals are collected, as they are impure. They are 
carefully scraped off with a spatula and collected and returned to the 
mother liquor after filtration. 

The pure crystals are collected on a filter plate or on a Buchner 
funnel (p. 16). The vessel is rinsed out with a little solvent which is 
used also for washing the crystals, and washing is done two or three 
times. 

(iv) Drying of Pure Crystals. 

The crystals are left to drain as completely as possible in the 
funnel and are then transferred to either 

(1) several thicknesses of filter paper and the liquid pressed 

out ; 

(2) a piece of unglazed porous plate, carefully dusted before 

use ; 

(3) a watch or clock glass and dried in the air. 

To keep out dust they are covered over by a clock glass or funnel 
which is raised up about 2 cm. on supports so that an air space is 
left for the solvent to evaporate ; this may take 24 hours ; or they 
are dried by placing them in a vacuum desiccator over sulphuric acid, 
soda lime, etc. 

Sometimes the crystals, if placed on a watch glass and when they are 
nearly free from solvent, are dried by putting the glass containing them 
on a boiling water-bath. The crystals in this case should not melt 
below 1 00 or contain sufficient solvent that they dissolve in it on 
warming. They are cooled by placing in a desiccator. 

Some substances, e.g. carbohydrates, are very difficult to dry completely, 
but can be obtained anhydrous by keeping them in vacua at 100 or 130 in 
presence of pho'sphorus pentoxide for a few hours. A convenient form of ap- 
paratus for drying such compounds is shown in Fig. 13. The substance is put 
into a tube or boat and placed in the central vacuum vessel. This is connected 
by a ground joint to a bulb containing P. 2 O 5 in the neck of which there should 
be some glass wool to prevent P 2 O 5 from entering the vacuum tube. It is ex- 
hausted and heated by inserting in a jacket tube, which is kept hot by the 
vapour of a liquid boiled in a flask below and condensed above. 



PREPARATION OF PURE COMPOUNDS 

.CONDENSER 



21 




VAPOUR JACKET y 






AIR 


VACUUM 










J 


s 



AroB=8iNCHES 
CToD=6i " 
E ToD=5i 
BTOF=3 



4-1 

TO FLASK 




FIG. 13. 

(v) Mother Liquor. 

The mother liquor generally contains some dissolved solid, which 
should be recovered. The impure crusts, if any, are added to the 
liquid and the liquid is concentrated by distilling or evaporating (p. 1 3) 
until crystals begin to separate. The solution is poured or filtered into 
a beaker and allowed to cool ; a second crop crystallises out and is 
treated as above. A third and more crops may be obtained on further 
concentration. These are not so pure as the original crop, but may 
be recrystallised and obtained pure. 

(vi) Decolorising Solutions. 

Substances containing tarry or resinous impurities or colouring 
matter cannot sometimes be freed from them by simple recrystallisation. 
During recrystallisation and while the solid is in solution (especially 
aqueous or alcoholic), the solution is boiled for 2-5 minutes or longer 
with a small quantity of blood charcoal which is removed by filtration. 
The first portions of filtrate generally require filtering again through 
the same paper as the finely divided charcoal passes through at first. 
To remove colouring matter from a solution which should be colour- 
less, prolonged boiling with several quantities of charcoal is sometimes 
necessary. 

The purification by crystallisation of about 5 gm. benzoic acid, 
oxalic acid and succinic acid from water and of urea from alcohol serve 
as simple examples of the method of recrystallisation. 



22 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

(c) Crystallisation from a Mixture of Solvents. 

A solid may be too readily soluble in some solvents, but insoluble in other 
solvents, and consequently its recrystallisation from a single solvent may be 
difficult. Crystallisation may be then effected by using a mixture of solvents. 
A concentrated solution is made in the hot solvent and the other solvent is 
added whilst the first is still hot until the mixture becomes turbid, when it is 
allowed to cool. Sometimes it is better to add the second solvent to a strong 
solution until it is turbid, heating until the turbidity has gone, and adding, if 
necessary, more of the first solvent. On cooling crystals may appear. 

Alcohol and water, acetone and water, benzene and ligroin, chloroform 
and ligroin, alcohol and ether are mixtures of solvents frequently used. 

Trials should be made to ascertain the best mixture of solvents. 

(d) Crystallisation by Evaporation. 

Another possibility is that the substance is easily soluble in all solvents. 
Crystallisation is then effected by partial evaporation. The filtered solution is 
placed in a shallow dish, i.e. a crystallising dish ; it is covered with a funnel or 
clock glass which is raised up so as to leave an air space and the dish is pre- 
ferably placed on a clock glass in case the solution creeps, so that the crystals 
can be scraped up without contamination from the bench. Crystals gradually 
form and they are removed before complete evaporation has occurred. Crusts 
are removed and treated as described above ; the crystals are filtered off, 
washed with very little solvent and dried. The mother liquor in this case will 
contain a large amount of solid. 

The evaporation need not be conducted in the air ; it may be hastened 
by placing the solution in a desiccator over sulphuric acid, etc., and under 
reduced pressure, or by evaporation by distilling the solution ; if aqueous, 
evaporation over a flame or on a water-bath. 

(e) Solids Soluble with Difficulty in all Solvents. 

If a solid is almost insoluble in all solvents recrystallisation is effected, after 
preliminary testing, by boiling the solid for some time with the best solvent, 
filtering off the insoluble portion, and evaporating the filtrate to a small 
volume. The crystals which separate are treated in the way described 
above. 

Operations with Small Quantities. 

Filters of small sizes are obtainable to retain the solid. Small filter flasks 
to contain the mother liquor are obtainable in the shape of test tubes, or a 
test tube may be placed inside the filter flask so that the mother liquor collects 
in it. 

When there is too little solid and mother liquor for filtration, the crystals 
and mother liquor are placed upon a piece of porous earthenware or between 
sheets of filter paper, so that the liquid is absorbed. 

Recrystallisation of the solid may be effected in a small test tube, and 
the mother liquor may be dissolved out of the filter paper or earthenware, if 
required. 



PREPARATION OF PURE COMPOUNDS 23 

DETERMINATION OF THE MELTING-POINT. 

A small quantity of the finely powdered substance is introduced 
into a melting-point tube; this is attachedito a thermometer and the 
two are heated together in a bath until the substance is seen to melt. 
The first determination of an unknown substance 'is usually only 
approximate: it is repeated, heating rapidly to within 10 and then 
more slowly. 

A small beaker containing water is used as a bath if the melting- 
point is below 1 00, and the thermometer in a cork is held in the 
centre of it by a clamp. The beaker is heated over a gauze by a 
small flame and the liquid is stirred with a circular glass stirrer. 

A flask of about 50 c.c. capacity with a long neck (10-20 cm.) filled 
about two-thirds with strong sulphuric acid is more generally used. 
The thermometer is secured in a cork into which a notch is cut to 





FIG. 14. 

allow hot air to escape when the flask is heated and to see the gradua- 
tions if the mercury reaches this level. The flask is held by a clamp 
and heated with a flame directly, the burner being inclined at an angle 
and held by the hand so that it is heated round and round and not 
directly in the centre (Fig. 1 4). After frequent use the acid becomes dark 
in colour, but it will become clear again if a tiny crystal of potassium 
nitrate be added. 

Paraffin wax is most generally used when the melting-point of a 
substance is above the boiling-point of sulphuric acid (290). The 
solid substance is introduced and melted until it fills two-thirds of the 
space. It becomes brown after being used several times and must be 
renewed 



24 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

A melting-point tube consists of a capillary of thin glass about 
i mm. in diameter, 5-6 cm. long and closed at one end. It is 
made by heating near one end a dry piece of glass tubing of about 
I cm. bore in a blow-pipe flame until it is red-hot and soft, removing 
it from the flame and pulling it out carefully just when the glass begins 
to harden. A long capillary tube is thus made. Several more such 
lengths can be made from the glass tube if the capillary so made, be 
broken off about 2-3 inches from the remainder of the glass tube. 
The long capillaries are cut into short lengths of about 5-6 cm. by 
scratching at these distances with a file and breaking by bending. 
Short lengths and lengths of too small bore must be rejected. One 
end of each capillary is sealed by holding it in a small flame. The 
tubes so made are preserved in a corked dry test tube. 

The powdered substance is introduced by scooping up solid with 
the open end and making it fall to the other end by gently tapping 
the closed end on the bench. This process is repeated until sufficient 
of the substance to occupy a lengh of 2-5 mm. in the capillary has 
been introduced and shaken down, or pushed down with a fine wire, 
so as to form a compact and continuous layer. 

The filled melting-point tube is attached to the thermometer so that 
the substance is on a level with the bulb. If a bath of water or 
paraffin wax be used the attachment is made with a strip of rubber cut 
from a length of rubber tubing. If a bath of sulphuric acid be used 
the attachment is made by adhesion. The thermometer is wetted with 
acid and if held horizontally the acid runs along it. The melting-point 
tube is wetted with acid by drawing it along the thermometer and it 
will adhere when the surfaces of contact are wet and will not fall off on 
putting it carefully into the bath. 

The pure specimens of benzoic acid, succinic acid and urea may be 
used for determination of the melting-point. 

Corrected Melting-point Determinations. 

Just as in the case of boiling-point determinations a correction should be 
made for the thread of mercury outside the bath. The small thermometers 
of 50 range may be used and attached by platinum wire to the ordinary ther- 
mometer. A corrected thermometer is generally used which has been cali- 
brated against the small ones. 



PREPARATION OF PURE COMPOUNDS 25 



V. SEPARATION OF SOLIDS. 

It is almost impossible to formulate a general scheme for the 
separation of a mixture of solids. The separation depends to a very 
large extent upon whether all the constituents, or whether only one 
constituent, or whether one or more groups of compounds in the 
mixture, is to be investigated ; it depends also upon the nature of 
the constituents in the mixture and of those requiring separation. 

A scheme of extraction with various solvents, such as the following, 
may be adopted for the separation of the constituents in a natural 
product. 

Extraction with Solvents. 

The solvents most commonly used in separating one or a group of 
constituents from plant or animal material are : 

(1) Ether, chloroform, petroleum ether, acetone, methyl alcohol. 

(2) Dilute mineral acid (HC1). 

(3) Dilute alkali sodium carbonate and sodium hydroxide. 

(4) Water or glycerol. 

When solvents such as ether, petroleum ether, etc., which are not 
miscible with water are used as the first solvent, the material is gener- 
ally dried preparatory to their use. 

The extraction of the material can be carried out in several 
ways : 

(1) If ether, etc., be used, the extraction is effected in a Soxhlet 
apparatus (p. 178), or, more simply, by hanging up the material in a 
paper or linen bag in a wide tube so that condensed liquid falls upon 
the material and from the material back to the extracting solvent. 

(2) In a percolator. 

(3) Or simply in a glass or metal vessel. The mass is frequently 
stirred with the solvent and filtered off and the residue pressed out. 

Drying of Material. 

Plant and animal tissues are composed to a very large extent of water, 
which varies from 10-75 P er cent - i n amount, and in order to ensure proper 
contact of solvent, which is immiscible with water, with the material it is 
necessary to dry it before extraction. 

Plant tissues are generally more easily dried than animal tissues ; the 
material is brought into a fine state of division by chopping, or grinding, or 
some other process, by hand or by machinery, and it is exposed to temperatures 
ranging from 40-100, depending on the constituent required. 

Animal tissues which consist mainly of protein are more troublesome to 
obtain in a dry state. They are more difficult to bring into a fine state of 



26 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

division and on simple drying by exposure the surface forms an impenetrable 
skin, preventing evaporation of water. Mincing, chopping, grinding with sand 
and other processes are used to prepare them for drying. 

It is essential that drying be carried out rapidly to prevent metabolic 
changes going on during the process of drying. 

(a) Exposure to Air or Indifferent Gas. 

The finely ground material is spread over a large surface on glass plates, 
or if vegetable on a metal mesh. It can be left exposed to air at the 
ordinary temperature or in a room or box at 40 or higher, or better by 
blowing a current of clean air, heated to 40, over the surface. To prevent 
oxidation of the constituents during drying, the material is placed in a vacuum 
desiccator or suitable chamber through which a current of carbon dioxide or 
nitrogen can be blown instead of air. 

After exposure to air or indifferent gas, more water can generally be 
removed by placing the material in a vacuum desiccator over dehydrating 
agents. 

(&} Treatment with Alcohol, or Acetone. 

Since alcohol and acetone are miscible with water in all proportions, they 
not only help to remove water, but also they disturb the conditions in the 
tissue, which are necessary for metabolic changes, by precipitating proteins, 
etc., and by disturbing the peculiar solubility of fatty and protein material. 
The material is mixed with about an equal bulk of solvent or sufficient to 
cover it completely and the mass is allowed to stand for 12-24 hours. The 
material may be put into boiling solvent and heated for about an hour. 

The solvent is filtered or strained off and the tissue treated again with 
the same solvent, or with ether, etc. Substances are soluble in the mixture of 
alcohol or acetone and water separated from the tissue and this extract 
requires examination. 

(c) Admixture with Neutral Dehydrating Agent. 

Animal tissues after grinding are sometimes mixed with anhydrous 
sodium sulphate, or calcium sulphate, by grinding together equal parts in a 
mortar. The mixture sets after some time to a cake which can be finely 
ground and extracted with ether, acetone, etc. By boiling with alcohol 
proteins are coagulated and the other constituents may be extracted with 
water. 

ISOLATION OF SOLIDS FROM SOLUTION. 

The extracts obtained above, either directly or after concentration 
by evaporation, evaporation in vacua, distillation, etc. (if acid or alkaline 
after neutralisation), or solutions of solid compounds whether natural 
or obtained by chemical reactions between compounds, may be treated 
in several ways in order to separate the solid compound. 

A. Precipitation. 

(i) By adding another solvent. 

(ii) By acidifying. Aqueous or alkaline aqueous extracts may 
contain acids. On acidifying with mineral acid, the acid or acids, if 
they are insoluble or soluble with difficulty in water, will be pre- 
cipitated. 



PREPARATION OF PURE COMPOUNDS 27 

(iii) By making alkaline. Aqueous and acid extracts may contain 
bases ; when made alkaline with sodium carbonate or sodium hydrox- 
ide, the base or bases may be precipitated. 

(iv) If no precipitate has been obtained in (ii.) or (iii.) the solution 
may be submitted to steam distillation. Volatile acids and bases 
distil over. 

(v) Acids may be precipitated from the exactly neutral solution 
as insoluble salts of heavy and other metals by adding lead acetate, 
mercurous nitrate, calcium chloride, etc. 

(vi) Bases may be precipitated by adding to the previously acidi- 
fied solution phosphotungstic acid, or other reagents used for precipitat- 
ing alkaloids from solutions. 

(vii) Bases may be precipitated (especially from alcoholic solution) 
as double salts with mercuric chloride, gold chloride, platinum chloride. 

B. Extraction. 

The acid or alkaline extract may be extracted with an immiscible 
solvent by repeated shaking or by means of an extractor (p. 600). 

C. Fractional Crystallisation. 

The solution on evaporation may deposit crystals. On filtering 
and evaporating further, another crop of crystals may be obtained. 

D. Salting Out. 

Proteins, polysaccharides, soaps and other complex compounds 
separate out when their aqueous solutions are saturated with sodium 
chloride, magnesium, zinc and ammonium sulphates. 

E. Dialysis. 

Mixtures of colloids and crystalloids are separated by dialysis. 

F. By Preparing Suitable Chemical Derivatives. 

Some knowledge of the chemical nature of the compound is 
essential before a derivative can be prepared. The principal reactions 
of the various groups of compounds are given under the separate 
headings. 

Two other procedures are occasionally employed in separating solids : 
(i) Sublimation. On carefully heating a mixture in a basin, one or 

more solids may be volatilised. The vapours are condensed on a cool clock 

glass or funnel and the substance is thus obtained. 

(ii) Sedimentation. A mixture of solids of different specific gravity 

may be separated by shaking with a liquid of suitable density. 

Separation may not be effected by applying any one of the above 
methods. Usually only an incomplete separation is made and further 
separation is carried out by using another method. 



COMPOSITION OF ORGANIC COMPOUNDS. 

A pure carbon compound which has been prepared is analysed, 
i.e. the elements besides carbon contained in it and their amounts are 
determined. 

Carbon compounds may contain the elements hydrogen, oxygen, 
nitrogen, halogens, sulphur, phosphorus, etc., either singly or collectively. 
Usually all the possible elements are not present, but the proteins 
contain carbon, hydrogen, oxygen, nitrogen and sulphur ; some con- 
tain also phosphorus and a. few contain halogens. Proteins belong to 
the most complex of the organic compounds. The method of analysis 
of the compound is varied according to the elements which are present. 
The elementary composition, or detection of the elements, precedes the 
quantitative composition. Since all organic compounds contain carbon 
and most of them contain hydrogen it is not absolutely essential that 
the presence of these elements should be ascertained. Their quantita- 
tive analysis is carried out simultaneously under the same conditions. 

A. ELEMENTARY COMPOSITION. 

DETECTION OF THE ELEMENTS. 

i. Carbon and Hydrogen. 

(a) A small portion of the substance (e.g. cane sugar) is gently 
heated in a test tube or on platinum foil. It melts and chars. The 
charring denotes the presence of carbon. There is a condensation of 
water on the sides of the tube where it is cool ; this denotes the 
presence of hydrogen. 

(&) About 5 grms. of finely powdered cupric oxide are dried 
thoroughly by heating in a small crucible. Whilst still warm l it is 
mixed with a little of the substance (e.g. oxalic acid) and the mixture 
is introduced into a hard glass tube. The end is closed with a cork 
through which passes a glass tube, bent at right angles. This end 
is dipped into a little baryta water contained in a small beaker or test 
tube. On heating the mixture in the hard glass tube, water will 
condense on the cooler parts of the tube presence of hydrogen ; and 
the baryta water will become turbid owing to the formation of barium 
carbonate presence of carbon. 

1 As cupric oxide takes up water on cooling it must be used warm, otherwise it must be 
allowed to cool in a desiccator over sulphuric acid. 

28 



COMPOSITION OF ORGANIC COMPOUNDS 29 

2. Nitrogen. 

(a) As Ammonia. A portion of the substance (e.g. caseinogen) is 
ground up with soda-lime and heated in a dry test tube. Ammonia 
is given off as shown by the smell, by litmus paper and by the 
production of white fumes when a glass rod dipped in hydrochloric 
acid is held over the mouth of the tube (Will and Varrentrapp's 
method). 

The peculiar smell of burning flesh, horn, etc., produced on heat- 
ing such substances alone, also indicates the presence of nitrogen. 

(<5) As Sodium Cyanide. A small piece of metallic sodium is heated 
in a small dry test tube of hard glass until the metal begins to boil ; 
successive minute portions of the substance (dried egg albumin) are 
added. The heating is continued for a short time, the tube is cooled, 
and the lower end of it is broken in a mortar, containing a few drops 
of alcohol ; water is added when effervescence has ceased. The solu- 
tion is transferred to a test tube, warmed and filtered. To the filtrate 
some ferrous sulphate solution (this must be freshly prepared by dis- 
solving a few small crystals in a little water) and caustic soda are 
added and it is boiled for a few minutes. It is cooled and a drop or 
two of ferric chloride and excess of dilute hydrochloric acid are added. 
A precipitate or coloration of Prussian blue indicates the presence of 
nitrogen (Lassaigne's method). 

Note. It is important that the substance be made to come into 
proper contact with the sodium. 

Castellands Modification of this Test. A small quantity of the sub- 
stance is intimately mixed with about ten times its quantity of equal 
parts of magnesium powder and dry sodium carbonate and gently 
heated until the magnesium burns ; it is then heated to redness as 
with the sodium (b). The remainder of process is carried out as 
described above, i.e. breaking the tube in a mortar, etc. 

3. Halogens. 

(a) Beilsteiri s Test. A piece of copper wire is heated in a Bunsen 
flame until the flame is no longer coloured green. A little of the sub- 
stance, e.g. chloroform, is placed on it and it is again heated. Copper 
chloride is formed which colours the flame green. 

(b} Halogen may also be detected by means of sodium employed 
just as in the nitrogen test. The filtered solution is acidified with 
nitric acid, boiled to remove any hydrocyanic acid which will be 
formed if the substance also contains nitrogen, and then treated with 
silver nitrate. 



30 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

The nature of the halogen may be determined by treating a little 
of the acidified solution with chlorine water, and then testing with 
starch solution for iodine, or by extracting with carbon bisulphide or 
chloroform for bromine. 

(V) Heating with Lime. Halogens are best detected by heating 
with quicklime. The substance is finely powdered and mixed inti- 
mately with lime (if liquid, e.g. chloroform, the lime" is moistened with 
the substance) and then heated strongly. When cool, water is added 
and the lime dissolved in nitric acid. On adding silver nitrate, a pre- 
cipitate of silver halide is obtained if halogen be present. 

4. Sulphur. 

(a) As sodium sulphide. A small portion of the substance is 
heated with metallic sodium as described under 2 (fr). The hot tube 
is broken in a little water, the contents are filtered and tested for 
sodium sulphide with (i) lead acetate, (2) sodium nitroprusside. 

($) As sulphate. A small portion of the substance (dried fibrin) 
is fused in a crucible with three times its quantity of fusion mixture 
(2KNO 3 + Na 2 CO 3 }. The mixture is heated cautiously at first round 
the edge and the heating is continued after the fusion until all charred 
particles have vanished. The mass, when cool, is extracted with hot 
water and the filtered solution is tested for sulphates with barium 
chloride in the presence of mineral acid (HC1 or HNO 3 ). 

5. Phosphorus. 

(a) Some caseinogen is fused with fusion mixture as described for 
sulphur, the fused mass is extracted with hot water, and the solution 
is divided into two parts. To the one part is added excess of nitric 
acid and ammonium molybdate : a yellow precipitate on warming 
indicates phosphoric acid ; to the other part excess of ammonia is 
added and phosphates are precipitated with magnesia mixture. 

(ff) A small quantity of caseinogen in a small flask is covered with 
5-10 c.c. of concentrated sulphuric acid and an equal volume of con- 
centrated nitric acid is added. The mixture is heated gently over 
a small flame (in the draught chamber) until the mixture becomes 
colourless. If it becomes brown, it is cooled, more nitric acid is 
added and it is heated again. When it is colourless, it is allowed to 
cool, water and a little ammonium nitrate solution are added and it 
is heated nearly to boiling ; on adding ammonium molybdate solution, 
a yellow colour or precipitate indicates the presence of phosphoric 
acid (Neumann's method). 



COMPOSITION OF ORGANIC COMPOUNDS 31 

6. -Other Elements. 

Amongst natural compounds the mo$t important other elements 
combined with carbon are iron, e.g. iron in haemoglobin, ferrocyanides, 
and magnesium, e.g. magnesium in chlorophyll. Copper is found in 
certain other animal pigments. Silicon is present in certain vegetables, 
e.g. in grasses. Organic silicon, arsenic, antimony and magnesium 
compounds have been prepared in the laboratory. These elements 
are best detected after the organic matter has -been completely re- 
moved by burning either alone or in the presence of an oxidising 
agent (fusion mixture). Thus : 

Detection of Iron in Haemoglobin. A small portion of haemo- 
globin is heated in a crucible with 3-4 times its quantity of fusion 
mixture until all the organic matter has been oxidised. The mass, 
when cold, is dissolved in dilute hydrochloric acid. The solution is 
filtered and the filtrate is tested for ferric salts with (a) ammonium 
thiocyanate and () potassium ferrocyanide. 

B. QUANTITATIVE COMPOSITION. 

ESTIMATION OF THE ELEMENTS. 

i. Carbon and Hydrogen. 

An organic compound on oxidation with copper oxide is con- 
verted into carbon dioxide and water. The amount of each element 
contained in the compound is determined by weighing the carbon 
dioxide and water produced from a known weight of the compound. 




FIG. 15. Combustion Furnace. 

The analysis is carried out in a long tube of hard glass, a com- 
bustion tube, about 80 cm. long, which is heated in a furnace (Fig. 1 5). 



32 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Five-eighths of the length of the tube is filled with coarse copper 
oxide which is kept in position by narrow plugs of oxidised copper 
gauze. Next to the copper oxide there is a small boat (copper or 
porcelain) of suitable size, containing a known weight of the substance, 
and the remaining space is filled by a roll of oxidised copper gauze 
(Fig. 1 6). This end of the tube is connected with a gasometer 



A = Oxidised Roll of Copper Gauze. 

B = Boat. 

C = Coarse Copper Oxide. 

FIG. 16. From Price and Twiss' " Practical Organic Chemistry ". 

containing air (or oxygen) and a current of air freed from carbon 
dioxide and water by passing through potash and sulphuric acid or 
calcium chloride, is passed through the combustion tube so as to*drive 
out the products of the combustion and to help in the oxidation. 
To the other end of the combustion tube are attached absorption tubes 
(Fig. 1 7), of which there are various forms, to collect the carbon dioxide 





Calcium Chloride Tube. Potash Absorption Bulbs. 

FIG. 17. From-Price and Twiss' " Practical Organic Chemistry". 

and water. The first absorption tube, generally of U-shape, contains 
calcium chloride 1 or pumice wetted with concentrated sulphuric acid ; 
the second, generally complex in shape so as to give several surfaces, 
contains caustic potash of 33 per cent, strength; a small tube con- 
taining calcium chloride is attached so as to retain water vapour, which 
may be carried away during the passage of the gases. These tubes are 
weighed before and after the combustion and their increase in weight 
gives the required data. 

1 After filling the tube with calcium chloride dry carbon dioxide must be passed 
through it until its weight remains constant. 



COMPOSITION OF ORGANIC COMPOUNDS 33 

In practice, the combustion tube is filled, as described, with coarse copper 
oxide, which has been heated to redness in a copper basin and allowed to 
cool. The plugs and the roll are made of copper gauze which is rolled round 
a piece of copper wire and heated in a blowpipe flame to oxidise the metallic 
copper and burn away any organic matter. A space is left for the boat. 
The tube is heated in the furnace at a low red heat and a current of dry clean 
air or oxygen is passed through the tube from the gasometer. The carbon 
dioxide and water present in the tube and on the copper oxide are thus 
removed and the copper is completely oxidised to copper oxide. The heating 
of that portion of the combustion tube into which the boat is to be placed is 
discontinued so that this portion cools to room temperature whilst the rest of 
the tube is kept at a red heat. The absorption tubes are filled and weighed. 
About '2 gm. of substance is exactly weighed out into the boat which has been 
heated and cooled in a desiccator. When the end of the combustion tube is 
cool the absorption tubes are attached, that for water next to the tube. From 
the other end the roll is removed with a hooked copper wire, the boat quickly 
introduced and the roll replaced. The tube is closed and the pure air or 
oxygen current passed through at a rate of about 3 bubbles every two 
seconds. The roll is heated commencing at the end farthest from the boat 
and the heating is gradually extended from this point towards the boat and 
the coarse copper oxide until the substance has burnt away and the whole 
tube is heated from end to end. The air or oxygen current is continued for 
about half an hour after the combustion is finished so as to drive out the water 
and carbon dioxide. Any water which condenses on the end of the com- 
bustion tube is driven into the absorption tubes by means of a small flame, 
of hot brick, held under the end of the combustion tube. When the oxida- 
tion is completed, the absorption tubes are removed, allowed to cool for ^ to 
i hour and weighed. 

This method of analysis requires some modification if elements other than 
hydrogen or oxygen are present in the substance. 

(a) Halogens. On combustion, the halogen in an organic compound is 
evolved as hydrogen halide, or as halogen. To prevent its entry into the ab- 
sorption tubes it is combined with silver as silver halide. This is effected by 
putting at the end of the combustion tube a roll of silver gauze. 

(b) Nitrogen. Oxides of nitrogen may be evolved when organic nitrogen- 
ous compounds are analysed. A roll of metallic copper gauze, prepared by 
heating a roll in a blowpipe flame and dropping it into a few c.c. of methyl 
alcohol contained in a test tube (held in a duster) and drying at 100, is intro- 
duced at the end of the combustion tube. Any oxides of nitrogen are thus 
reduced to nitrogen and prevented from being absorbed by the potash. 

(c) Sulphur and Phosphorus. To prevent hydrogen sulphide or hydrogen 
phosphide being formed, the oxidation of the organic compound is effected 
with lead chromate instead of copper oxide and the substance is mixed with 
it instead of being placed in the boat. Lead chromate may replace the 
coarse copper oxide entirely or about half of it. 

Organic phosphorus compounds are very difficult to oxidise completely 
and frequently give results for carbon which are too low by about -5 to i per 
cent. 



34 



PRACTICAL ORGANIC AND BIO-CHEMISTRY 



2. Nitrogen. 

(a) Dumas' Method. On heating an organic compound contain- 
ing nitrogen with copper oxide, its nitrogen is given off as nitrogen. 
The gas given off from a known weight of substance is collected and 
its volume measured, from which value its weight can be calculated. 

The analysis in practice is carried out in a similar way to that 
described for carbon and hydrogen, but the substance is mixed with 
finely powdered copper oxide and introduced into the tube, and a roll 
of reduced copper gauze is placed at the end of the combustion tube 
so as to reduce any oxides of nitrogen, which may be formed, to 
nitrogen. Instead of a current of air, a current of carbon dioxide 
is passed through the tube. The gas is collected in a Schiff s nitro- 
meter over caustic potash which absorbs the carbon dioxide leaving 
the nitrogen (Fig. 1 8). 




FIG. 18. 

There are several ways of passing the carbon dioxide through the 
combustion tube ; it may be evolved from a Kipp apparatus or it may 
be evolved by heating magnesite, either contained in a special tube or 
in the combustion tube, which in this case is sealed at the end. It is 
obvious that before carrying out the analysis all the air must be ex- 
pelled from the apparatus. 



COMPOSITION OF ORGANIC COMPOUNDS 35 

(&) Kjeldahl's Method. The principle of this method consists in 
oxidising the substance with concentrated sulphuric acid ; the nitrogen 
is converted into ammonia. The solution is made alkaline with 
caustic soda and distilled. The ammonia is evolved and is collected 
in excess of standard acid ; on subsequent titration with standard 
alkali the amount evolved is given by difference. 

This method is much simpler to carry out than the Dumas' method, 
but it cannot be employed for all nitrogen- containing compounds. 

Dakin and Dudley l have found that pyrrole and its derivatives, piperidine 
and some of its derivatives give satisfactory results if the heating of the sub- 
stance about '15 gm. with sulphuric acid be continued for at least four 
hours after the solution becomes clear. Pyridine, quinoline, pyrazole and 
their derivatives do not give satisfactory results. 

Of other groups of compounds, nitro-, nitroso-, azo-, diazo-, hydrazo-, 
aminoazo- compounds, also compounds of ni'tric and nitrous acid, i.e. those 
compounds containing nitrogen joined to oxygen or another nitrogen atom, 
give satisfactory results if they are previously reduced with tin. Osazones do 
not give satisfactory results. 

On account of its simplicity this method has found extensive use 
in biological chemistry. The large group of compounds the pro- 
teins all contain nitrogen ; the amount of protein in a solution is 
estimated by determining the nitrogen content (see below) ; and the 
amount of nitrogen in urine is a factor of importance in studying 
metabolism. 

In most laboratories there is an apparatus in which six determina- 
tions can be carried out at the same time as in Fig. 20, p. 37. 

Example: Estimation of Nitrogen in Egg-white Solution. 

A known volume, say 5 c.c., of the egg-white solution is placed 
with a pipette into a clean round-bottom Jena glass flask of 700 c - c - 
capacity. IO or 2O c.c. of pure concentrated sulphuric acid and a crystal 
of copper sulphate, about the size of a pea and weighing about 0-25 gm., 
which helps in the oxidation, are added, (i gm. of potassium sulphate 
is also sometimes added for this purpose, as it raises the temperature.) 
The flask is heated in a fi/me-cupboard until the liquid, which at first 
becomes brown from charring, becomes quite or nearly colourless, a pro- 
cess which takes about an hour. The flask is allowed to cool and is 
half-filled with distilled water. By means of a special distillation tube 
it is connected to a condenser set up in a vertical position as in Fig. 
19, No. I. 

1 J. Biol. Chem., 1914, 17, 275. 



3* 



PRACTICAL ORGANIC AND BIO-CHEMISTRY 



With a pipette a quantity of standard sulphuric acid (5 c.c. of N 
or 50 G.C. 'iN H 2 SO 4 ) are measured out into a clean beaker or flask 
(preferably conical) of about 600 c.c. capacity, and this is placed below 
the condenser so that the end of the condenser just reaches the surface 
of the liquid. It is preferable to add a few drops of indicator, methyl 
orange or alizarin red, 1 before the distillation is commenced in case 
more acid is required than that originally taken ; the change in colour 
of the indicator gives notice of this fact. 

Sometimes the distillation is carried out without using a condenser 
(Fig. 19, No. 2} ; the end of the special distilling tube is then dipped into 
the standard acid. There is in this case usually greater danger of the 
liquid being sucked back into the flask which is being distilled, and 
further the glass is attacked by the ammonia with liberation of alkali, 
which causes inaccuracies in the determination. 





No. i. No. -2. 

FIG. 19. 

The round-bottomed flask is removed, a piece of porous earthenware 
is added and excess of caustic soda solution (50 c.c. of 40 per cent, 
for every 10 c.c. concentrated H 2 SO 4 used) is run in under the dilute 
acid without mixing. The flask is connected again to the condenser 
seeing that all corks fit tightly. The soda and acid are mixed and 
the ammonia is distilled off into the acid. In about half an hour the 
ammonia will have completely passed over into the standard acid. To 

1 Cochineal, congo red and other indicators may also be used, or the titration may be 
effected using sodium iodate and potassium iodide and standard thiosulphate solution 
(P- 563). 



COMPOSITION OF ORGANIC COMPOUNDS 37 




No. i. 




No. 2. 
FIG. 20. Apparatus for estimation of nitrogen by Kjeldahl's method. 



38 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

test if the distillation is finished the flask is lowered and the condensed 
water is allowed to wash out the inside of the condenser tube for about 
two minutes : after this time the distillate is tested with litmus paper 
to ascertain if any more ammonia is being evolved. When finished, 
the outside of the condenser tube is washed with distilled water and 
the contents of the flask are titrated with iN alkali. 

The difference between the amount of standard alkali and standard 
acid gives the amount of ammonia evolved, from which the amount of 
nitrogen can be calculated : 

5 c.c. N H 2 SO 4 = 50 c.c. -iN H 2 SO 4 . 

37*4 c.c. *iN NaOH used. 

i2'6 c.c. 'iN difference. 



12-6 c.c. -iN H 2 SO 4 = 12-6 c.c. -iN NH 3 

= I2'6 c.c. 'iN Nitrogen 
= I2'6 x 0-0014 gm. Nitrogen. 
5 c.c. solution contain 0-01764 

.\ too c.c. ,, 0*3528 

The amount of protein to which this amount of nitrogen corresponds 
is ascertained as follows : 

Proteins contain from i 5 to 16 per cent, of nitrogen. Taking 1 5 -5 as 

the average value, -or 6*45 times the amount of nitrogen gives the 
amount of protein. The factor 6-25 is, however, generally used. 

too c.c. solution thus contain 6-25 x 0*3528 gm. protein = 2*2 gm. 



COMPOSITION OF ORGANIC COMPOUNDS 39 

3. Halogens. 

After completely oxidising all the organic matter to carbon dioxide 
and water, the halogens are present as inorganic compounds and are 
estimated in the usual way. 

The commonest method is to oxidise a known weight of the sub- 
stance in a sealed tube with fuming nitric acid at 200 for several hours, 
a few crystals of silver nitrate being at the same time placed in the 
tube : silver halide is formed and this is washed out of the tube and 
weighed (Carius). 

In practice, about -2 gm. of the substance is weighed out into a narrow 
test tube about 2 in. long. Some silver nitrate crystals are placed in a thick- 
walled combustion tube sealed at one end and covered with 10-20 c.c. of 
concentrated nitric acid, care being taken not to wet the sides of the tube with 
acid. This is done by introducing the acid through a tube with a long capillary. 
The test tube containing the substance is put in avoiding contact of the sub- 
stance with the acid. The open end of the tube is now sealed in the following 
way : a glass rod for a handle is fastened by heating to the side of the tube at 
the open end. The tube is heated near this end in a blow-pipe flame in such 
a way that the walls collapse together ; when it is nearly closed the end is 
drawn out so as to form a capillary 'tube with even and thick walls. The 
capillary is sealed by pulling off the end with the handle attached. The tube 
is wrapped in asbestos paper and carefully placed, capillary outwards, in an 
iron tube which can be closed with a screw cap. The iron jacket and tube 
are placed in a specially constructed furnace in such a position that the capillary 
point faces a wall. Should the tube burst, the contents are then not blown into 
the room. The tube is heated to about 200-220 for 4 or 5 hours. The 
furnace and contents are allowed to cool. The cap of the iron tube is re- 
moved and the capillary point allowed to project a little. The point is heated 
in a flame. When the glass is soft the pressure inside the tube forces an 
opening. Owing to the high pressure inside the tube it is unsafe to open the 
tube in any other way. The capillary is cut off and the contents washed out 
into a beaker. The silver halide is filtered off and weighed by the usual 
method employed in inorganic chemistry. 

Less frequently, halogens are estimated by heating the substance 
in a combustion tube with quicklime. 

A thin layer of quicklime is placed at the closed end of a combustion 
tube. Next to this is put a mixture of the substance (about '2 gm.) with 
quicklime and then another layer of quicklime. The tube is heated in a 
furnace, as in the estimation of carbon and hydrogen, the layers of quicklime 
being heated to redness before the mixture of substance and lime. 

The contents of the tube, after the oxidation, are dissolved in nitric acid 
and the halogen precipitated with silver nitrate. The silver halide is filtered 
off, washed, dried and weighed. 

A convenient method of estimating chlorine is that described by Neumann. 
The substance is oxidised with a mixture of nitric acid and sulphuric acid. 
Hydrochloric acid is evolved, and this is collected in a solution of silver 
nitrate. After boiling the solution for about half an hour to remove hydrogen 
cyanide, which is also formed if nitrogen be present in the substance, the 
silver; chloride is filtered off, washed, dried and weighed. 1 

J. Physiol., vol. 31, p. 65. 



40 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

4- Sulphur. 

This element is most generally estimated by the same method as 
the halogens (Carius) ; sulphuric acid is formed and precipitated as 
barium sulphate. 

It is more convenient to oxidise the substance in a nickel crucible 
with sodium or barium peroxide; the contents are acidified with 
hydrochloric acid and the barium sulphate formed is weighed. Still 
more convenient is the oxidation mixture used for estimating total 
sulphur in urine (see p. 542). 

5. Phosphorus. 

Phosphorus is usually estimated in the same way as sulphur by 
the Carius method, the phosphoric acid formed being precipitated as 
ammonium magnesium phosphate. 

The most rapid and 'convenient method is that of Neumann. 
The substance is oxidised in an open flask with a mixture of nitric 
and sulphuric acids. The phosphoric acid formed is precipitated as 
ammonium phosphomolybdate and this is then estimated by solu- 
tion in excess of ^N caustic soda and subsequent titration with *5N 
sulphuric acid. The difference between - 5N NaOH and ^N H 2 SO 4 
multiplied by I -268 gives the number of milligrams of P 2 O 5 in the 
given weight of substance taken. 1 This method is described on p. 545. 

Micro-Analyses. 

Minute quantities of substance can be analysed by the methods 
devised by Pregl. These methods are difficult to perform and require 
much practice. The full details are given by Pregl in Abderhalden's 
" Handbuch der Biochemischen Arbeitsmethoden," vol. v., part 2, p. 



Folin has also described a method for estimating nitrogen with 
special reference to its estimation in urine and blood. Its technique is 
comparatively simple and is given on p. 558. 

1 See J. Physiol., 1906, 33, 439. 



COMPOSITION OF ORGANIC COMPOUNDS 41 



C. CALCULATION OF RESULTS. 

With the exception of oxygen all the elements present in an 
organic compound are thus estimated. The amount of oxygen is 
found by difference. 

From the figures obtained the percentage composition is calculated, 
i.e. the amount given by 100 grams of substance, thus : 

o'2cog gm. substance gave o 2987 gm. CO 2 and 0-1092 gm. H 2 O. 
0-1887 g m - -1 I 5' 2 c - c - moist N at 16-5 and 767 mm. 

Now, 0-2987 gm. CO 2 = 0-2987 x gm. C = 0-2987 x -?- gm. C = 0-0815 gm. C. 

44 JI 

0-1092 gm. H 2 O = 0-1092 x gm. H = 0-1092 x gm. H = 0-01213 g m - H. 
18 9 

15-2 c.c. moist N at 767 mm. and 16-5 C. = * 5 2 X 753 ^Z? c . c . a t o and 760 mm. 

760 x 289-5 
= 14-2 c.c. 

28 

= 14-2 x gm. 

22.400 6 

= 0-01775 gm. N. 

c ,-, o'oSis x 100 

.-. percentage 01 C = = 40-56. 

0-2009 

H = ' OI2I 3 x I0 = 6-04. 
0*2009 

N . 0-QI775 x IPO = 

0-1887 

O by difference = 44-00. 
Total = 100 oo 

The formula of the compound is obtained by dividing the percent- 
ages by the atomic weights of the elements ; the ratio of the number 
of atoms to each other is then obtained by dividing by the lowest 
value : , 

.-, 40-56 

C t-A. = 3-3^ -r 0-67 = 5. 

H i = 6-04 -f 0-67 = 9. 

N ^i? = 0-67 -f 0-67 = i. 
J 4 

O 11^ = 2-75 -f 0-67 = 4-1. 
16 

The formula of the compound is therefore C 5 H Q NO 4 . 

In any estimation only a difference of o - 2-O - 3 per cent, is allowed 
between the values found and those calculated from the formula. The 
calculated values are 

C = 40-81. diff. = - 0-25. 
H = 6-12. = + 0-08. 

N = 9-52. = - 0-12. 

The analysis was therefore sufficiently accurate. 

* Vapour pressure of water at 16-5 C. = 14-0 mm. .-. pressure on gas = 767 - 14 = 753 



42 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



D. DETERMINATION OF THE MOLECULAR WEIGHT. 

As will be seen later, several organic compounds can have the same 
empirical formula, thus, for instance, lactic acid C 3 H 6 O.j and glucose 
C fi H 12 O have the same empirical formula, namely CH 2 O. 

In order to ascertain which of these formulae is the correct one, a 
molecular weight determination is carried out, i.e. the weight of the 
molecule of the substance compared with that of an atom of hydrogen 
(Avogadro's law). 

The methods employed to determine the molecular weight are of 
two kinds : (a) physical, (&) chemical. 

(a) Physical Methods. 

i. Victor Meyer's Method. Of the physical methods, that by 
Victor Meyer is the most frequently used when the substance can be 
vaporised without decomposition. A known weight of the substance 
is converted into vapour at a temperature 40-50 above its boiling- 
point in a special apparatus. The air previously contained in the 
apparatus is displaced by the vapour, collected in a graduated cylinder 
and its volume measured ; this volume, after making corrections for 

temperature and pressure, corresponds to that 

occupied by the substance. 

Thus, if v c.c. are given by w grammes substance, 

, w x 22.400 
.. 22,400 c.c. are given by 

= mol. wt. 

The apparatus employed is shown in the 
accompanying Fig. 21. A liquid, boiling 40-50 
above the temperature at which the substance is 
volatilised, is boiled in the round bulb of the 
outer vessel. As soon as the temperature is 
constant and no more air escapes from the inner 
vessel by the side tube, the inverted graduated 
cylinder, filled with water, is placed over the end 
of the side tube, the cork is removed and a 
known weight of substance, contained in a 
small glass vessel, is dropped through the open- 
ing into the inner vessel and the cork is quickly 
FIG. 21. replaced. The substance is rapidly vaporised 

and the vapour displaces an equal volume of air, which is driven out 
and collected and measured in the graduated cylinder. 




COMPOSITION OF ORGANIC COMPOUNDS 



43 



2. Raoult- Beckmann Method. Substances dissolved in a liquid 
lower its freezing-point. It was shown by Raoult that the freezing- 
point was lowered the same number of degrees when weights of 
different substances proportional to their molecular weights were dis- 
solved in the same volume of liquid. Each liquid was found to have 
a definite freezing-point. By employing this value as a constant, the 
molecular weight of an unknown substance can be found. It is given 

by the formula 

M = IPO x C x w 

where C is the constant, w the weight of the substance, W the weight 
of the solvent, and d the depression of the freezing-point 

The constants are: water 19 benzene 49 
acetic acid 39 phenol 76 

Conversely by determining the lowering of the freezing-point, the 
amount of salt in a solution can be ascertained, e.g. in serum, urine. 

The apparatus (Fig. 22) devised by Beckmann con- 
sists of the freezing-point tube C with side opening D. 
It is closed by a cork through which a Beckmann ther- 
mometer T and a stirrer S (through a glass tube) pass. 
The Beckmann thermometer is a large thermometer 
graduated usually in T ^Vo P ar ts of a degree and having 
a range of only 5-6 degrees. 1 The tube C is placed in 
a wider tube B which serves as a jacket and prevents too 
rapid cooling. This is fixed in position in a freezing 
mixture of salt and ice in the vessel A by a cork which 
fits the opening in the brass lid L. The brass lid has 
also openings for the passage of a stirrer E and a 
thermometer. In carrying out a determination a known 
weight of solvent is placed in C and its freezing-point is 
taken. The tube is then removed and the solid allowed 
to melt. A known weight of substance is then intro- 
duced through D, dissolved in the liquid and the freez- 
ing-point again determined. 

Several determinations of the freezing-point of the 
solvent and the solvent containing the substance should 
be taken. Whilst the freezing-point is being taken the 
liquid becomes super-cooled. To prevent very great 
super-cooling it is vigorously stirred with the stirrer. 




FIG. 22. (From Find- 
lay's " Practical Phy- 
sical Chemistry ".) 



At the freezing-point the temperature rises and the highest point reached is 
taken as the freezing-point. 

Similarly, a rise in the boiling-point of a solvent, when substances 
are dissolved in it, will give the molecular weight of the substance. 

Micro-Molecular Weight Determinations. 

Micro-molecular weight determinations may be made by Barger's 
method. 2 

1 It is so constructed that mercury can be removed from the thread or introduced into the 
thread from a small bulb at the top. It can thus be used for any liquid. 
'-Trans. Chem. Soc., 1904, 85, 286. 



44 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

(8) Chemical Methods. 

When the substance is an acid or a base the molecular weight can 
be determined by chemical methods. 
(i) In the case of an acid : 

The molecular weight can be calculated from the amount of standard 
alkali required to neutralise, using phenolphthalein as indicator, a known weight 
of the acid, according to the equation 

H acid + NaOH = H 2 O + Na acid. 
e.g. x c.c. -iN NaOH = y gm. of acid. 

.-. 40 gm. NaOH = 4 x y 

x x o - oo4 
= mol. wt. of acid. 

If the acid be dibasic or tribasic, two or three molecules of sodium hy- 
droxide will be required. The presence of such an acid will be indicated by 
titrating the acid using methyl orange, or alizarin red, and phenolphthalein as 
indicators. The acid salt will be neutral to methyl orange or alizarin red, the 
neutral salt to phenolphthalein. The basicity of the acid is definitely ascer- 
tained by the analysis of the salt and the free acid. 

It is most usual to employ the silver salt of an acid. A quantity 
of the salt is prepared by adding silver nitrate to the neutral solu- 
tion of the acid, filtering off the silver salt, washing and drying it. 
A known weight is heated in a crucible and the metallic silver obtained 
is weighed. 

a gm. of silver salt gave b gm. of silver. 
If the acid be monobasic it will contain i atom of silver, 

107-9 gm. of silver will be contained in IO 7 9 x a g m o f s ii ver sa it. 

b 
Since the silver replaces i atom of hydrogen 

? _ 107*9 + i is the mol. wt. of the acid. 



.. - 

b 

If the acid be dibasid it will contain -2 atoms of silver, 

107-9 x 2 gm. of silver will be contained in I0 7 9*2 x J* g m . of silver salt. 

b 
Since the silver replaces 2 atoms of hydrogen 

107*9 x 2 x a , . , .. 

. - - (io7'9 x 21 + 2 is the mol. wt. of the acid. 

B 

The zinc* salt or barium salt is also sometimes employed ; a known 
weight of salt is heated with a drop of concentrated sulphuric acid in 
a crucible ; zinc or barium sulphate is obtained from which the amount 
of barium or zinc is calculated. 



COMPOSITION OF ORGANIC COMPOUNDS 45 

(2) In the case of a base : 

Organic bases form double salts with metallic salts, such as platinum 
chloride, mercuric chloride. On heating the double salt in a crucible, 
a residue of the metal is left. The estimation of the amount of metal 
in a known weight of the compound gives the molecular weight. 

Ammonia and platinum chloride give the compound ammonio- 
platinum chloride, 

(NH 3 . HC1) 2 . PtCi 4 . 

The organic bases form analogous compounds, the base replacing 
the ammonia. Their general formula is therefore 

(B . HC1) 2 . PtCl 4 or B 2 . H 2 PtCl,.. 
The molecular weight of 2. molecules of base is thus 

B, . H 2 PtCl B - H 2 PtCl 6 . 
e.g. x gm. of salt gave y gm. ot platinum. 

.-. 194-8 gm. platinum will be given by - "^ gm. salt B 2 . H 2 PtCl H . 

Now 194*8 gm. platinum are contained in 409-8 gm. H 2 PtCl 6 . 

.-. I 94 1_* - 409-8 is the molecular weight of B 2 . 

y 

194-8 x x 

- 409-8 

Hence molecular weight of i molecule of base is -^ 

The gold salts have the general formula, 

B . HC1 . AuCL, or BHAuCl 4 , 

from which the molecular weight of the base is calculated in a similar 
way. 



PRACTICAL ORGANIC AND BIO-CHEMISTRY 



IDENTIFICATION OF AN ORGANIC COMPOUND. 

Knowing the formula of a pure organic compound from its analysis 
and molecular weight, it has to be identified. The compound may be 
a known or an unknown one. 

To find out if the compound is known reference is made to 
Richter's " Lexicon of Carbon Compounds " l in which the melting- 
points and other constants of the various compounds are given. Cor- 
responding properties identify the substance. 

If the compound be unknown, further analysis is necessary ; it 
must be ascertained to what group of carbon compounds the unknown 
body belongs, whether it is an alcohol, an ester, an acid, a carbo- 
hydrate, an amide, an amine, a protein, etc. With the complex 
natural substances this is a matter of great difficulty, and it may take 
many years before a question is settled ; e.g. tyrosine was discovered 
in 1846 and its constitution only definitely proved in 1882. 

The identification of an unknown substance, or the rapid identifica- 
tion of a known substance, is greatly facilitated by a few preliminary 
tests. If in solution a portion of it should be evaporated to see if there 
is a residue and whether it is solid or liquid. The residue can be 
tested for the elements present, especially nitrogen. If there is no 
residue the solution must be distilled and the boiling-point observed. 

1. Colour. Vegetable colouring matters : if blue, they are changed 
to red by acid and the blue colour is restored, or changed to a green, 
by ammonia ; if yellow, they are changed to brown by . alkali and the 
colour is restored by acid. 

Ferric salts and copper salts are reddish-brown and blue or green 
respectively. 

Many coloured compounds show absorption spectra, such as 
haemoglobin and its derivatives. 

2. Taste. Tasting must be done carefully on account of the 
extremely poisonous nature of some organic compounds. A drop of 
a weak solution in water or alcohol may be used. Acids have a sour 
and astringent taste. Alkaloids and glucosides are bitter. Sugars 
and glycerol are sweet 

1 The most recent compounds are given in the yearly volumes of the Journals of the 
English and Foreign Chemical Societies. 



47 

3. Odour. The odour is sometimes characteristic. 

4. Appearance. The appearance under the microscope gives evi- 
dence of homogeneity or impurity. The microscopical appearance is 
very useful in identifying the different kinds of starch. Many substances 
have a characteristic crystalline structure, e.g. cholesterol,' cystine, 
osazones of carbohydrates, etc. 

5. Effect of Heat. By heating the substance firstly on platinum, 
secondly in a small dry tube many valuable details can be ascertained. 
The odour may be peculiar, the substance may melt, char, decompose, 
sublime, or boil. The melting-point and boiling-point of solids and 
liquids can be observed directly after such a preliminary examination. 

6. Detection of the Elements. By ascertaining whether the 
substance does or does not contain nitrogen, it may be placed in either 
of the following groups : 

Non-nitrogenous. Nitrogenous. 

Hydrocarbons. Cyanogen compounds. 

Alcohols, phenols. Amides. 

Esters, ethers. Amines. 

Aldehydes. Amino acids. 

Ketones. Guanidine compounds. 

Acids and Salts. Purines. 

Fats and cholesterol. Proteins. 
Carbohydrates. 

7. Solubility. 

(a) Alkali salts and salts of bases, 

The lower alcohols, aldehydes, acids, ketones, amides, amines 

The polyhydric alcohols and carbohydrates '*S *o 

Phenols and hydroxy acids 

In general, compounds containing several OH groups 

(jf) Aromatic acids are insoluble or very slightly soluble, but dis- 
solve in boiling water. 

Starch is insoluble and gives an opalescent solution with hot 
water. 

Tyrosine, cystine and uric acid are soluble with difficulty in 
water. 

Fats, higher fatty acids and cholesterol are insoluble in water, but 
soluble in ether. 



48 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

8. Behaviour towards Reagents. 

(a) Reaction of aqueous solution to litmus. 

A marked acid reaction indicates an acid or a phenol ; if there is an 
odour, it may be a volatile fatty acid or a phenol. 

A neutral reaction indicates a salt of an acid or a base ; an al- 
cohol, aldehyde, ketone (note smell), carbohydrate. An ester 
in alcoholic or ethereal solution has also a neutral reaction. 

An alkaline reaction indicates a base, or an acid dissolved in 
excess of alkali. 

() Sodium carbonate: acids insoluble in water, e.g. uric acid, also 
cystine, tyrosine dissolve ; bases insoluble in water do not 
dissolve or if in solution are precipitated. 

(c) Sodium hydroxide: ammonium salts are decomposed with 

evolution of ammonia and bases are liberated from their salts. 
On boiling, amides are decomposed, and esters are hydrolysed. 

(d) Hydrochloric acid : acids insoluble in water do not dissolve or 

if in solution are precipitated; bases insoluble in water dis- 
solve, e.g. tyrosine, cystine, aniline. 

(e) Sulphuric acid. 
(/) Nitric acid. 

(g) Bromine water. 
(K] Permanganate. 

(2) Effect of heating with soda lime. 
The exactly neutral solution may be tested with 
(/) Schijfs reagent for aldehydes. 

(k] Ammoniacal silver nitrate for aldehydes, reducing carbo- 
hydrates, etc. 

(/) Fehling's solution for aldehydes, reducing carbohydrates, etc. 
, (ni) Ferric chloride for phenols (violet or green colour), 
for acetoacetic acid (claret colour), 
for formates, acetates (reddish-brown colour, 

precipitate on boiling), 
for lactates, oxalates (yellow colour). 
() Calcium chloride for oxalates, urates, etc. (insoluble calcium 

salts are precipitated). 

(o] Sodium nitroprusside and sodium hydroxide: 
Acetone gives a red colour, changing to purple with acetic acid. 
Creatinine ,, ared ,, ,, ,, yellow ,, ,, ,, 

Indole ,, a blue-violet ,, blue 

Confirmatory tests must be made after an indication of the nature 
of the substance has been obtained, according to the reactions given 
under the various groups of compounds. 



HYDROCARBONS. 

A. SATURATED. 

i 

The simplest organic compounds are the hydrocarbons, which con- 
sist of carbon united with hydrogen. 

Carbon is a tetravalent element, but, unlike other elements, the 
carbon atom can combine with itself many times, thus 2, 3, 4, 5, 6, 
etc., carbon atoms can be combined together. 

I I I I I I 
_c c c c c - c 

I I I I I I 

The hydrocarbons are the compounds in which the remaining 
valencies of the carbon atoms so joined together are satisfied by 
hydrogen, as for example in 

H H H H H H 

I II III 

H C H H-C C H H C C C H 

I II III 

H H H H H H 

Methane. Ethane. Propane. 

/ 

They form a homologous series of compounds in which the member 
containing i carbon atom more than the preceding one also contains 
2 hydrogen atoms more, i.e. the members differ from one another 
by CH 2 . They possess the general formula C M H 2 w + 2- 

If we continue the process of adding CH 2 to propane, two ways 
are possible : it may be added to one of the end carbon atoms or to 
the middle carbon atom. The two compounds 

H 

H C H 

H H H H H | H CH, 

H C C C C H and H C C C H or CH 3 C CH 3 

I I I I III I 

HHHH HHH H 

Butane. Isobutane or trimethyl-methane. 

are thus obtainable. Continuing the process we obtain 

49 4 



50 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



H-C H 



H H H H H 

H C C C C C H 

I I I I I 
H H H H H 

Pentane. 



H 

H C H 
H | H H 

H C C C C H 

A A AA 



Dimethyl-ethly-methane. 



H H 

I I 



CH :! 



H C C C C H or C 2 H 5 C CH a 

H H H H H 

Dimethyl-ethyl-methane. 



H 



H C H 
H I H 

I I I 

H C C C H or CH S C CH, 

I 
CH, 



CH, 

I 



A i 

H C H 

A 



Tetramethyl-methane. 



Two of these compounds are identical in structure, so that only 
three compounds can be derived from butane and isobutane. 

Two or more compounds which have the same empirical composi- 
tion (C 4 H 10 or C 5 H i2 ), but a different structure as represented by the 
graphic formulae, are therefore possible. Such compounds are known 
as isomers. 

The compounds with a straight chain of carbon atoms are 
termed normal compounds. Those with a branched chain of carbon 
atoms are regarded as derivatives of methane, the radicles CH 3 , C 2 H 5 , 
C 3 H 7 , etc., being termed methyl, ethyl, propyl, etc., which shows 
their origin from the parent hydrocarbon. 

In accordance with this theory an enormous number of hydro- 
carbons are possible ; those containing from I up to 60 atoms of carbon 
in their molecule are actually known. The theory was advanced and 
developed to account for their large number. 

The saturated hydrocarbons are the basis of the nomenclature and 
classification of all the carbon compounds. The carbon compounds 
are classified according as to whether they contain I, 2, 3, or 4, etc., 
carbon atoms in their molecule, i.e. whether they are derived from 
methane, ethane, propane, butane, etc. 

The saturated hydrocarbons are thus distinguished by the suffix ane ; 
the prefix meth means I carbon atom ; eth means 2. carbon atoms ; 
prop means 3 carbon atoms, and so on. 

The majority of the saturated hydrocarbons are natural substances. 
The lower members of the series of the hydrocarbons (up to 4 carbon 
atoms, which are gases) are formed by the dry distillation of diverse 
organic substances and are contained in coal gas. Methane occurs in 



HYDROCARBONS 51 

coal seams, but was originally .called marsh gas, because it was found 
to escape from the water of ponds, where it is now known to be formed 
by the decomposition of cellulose. By a similar process of bacterial 
action it may be produced in the intestines of animals. The middle 
members, containing 5-16 atoms of carbon, are liquids, and are con- 
tained in petroleum, which consists of a mixture of saturated hydro- 
carbons. The higher members are solids. 

Two theories have been advanced to account for their formation. 
According to the first, they are the products of the dry distillation of 
animal remains ; according to the second, they are formed by the action 
of water upon the metallic carbides, of which the interior of the earth is 
supposed to consist. If the former supposition be the correct one, as 
the most recent work tends to show, they become of still greater interest 
in biological chemistry. 

Several fractions are separated by the fractional distillation of the 
natural mineral oil. The following are the principal fractions from 
American petroleum : 

1. Cymogene, B.P. o\ gases which are liquefied by pressure and used for producing 

2. Rhigolene, B.P. i8/ cold by evaporation. 

3. Petroleum ether or naphtha, B.P. 5o-6o, contains chiefly pentane and hexane. 

4. Benzoline or Benzine, B.P. 7o-go, contains chiefly hexane and heptane. 

5. Ligroin or " petrol," B.P. go-i2o~\ .. ,. a , 

6. Petroleum Benzine, B.P. I2o-i 5 oj contain ch.efly heptane and octane. 

7. Paraffin oil or Kerosene, B.P. i5o-3oo, contains chiefly octane to hexadecane. 

8. Vaseline, B.P. above 300, contains chiefly heptadecane to heneicosane (C^H^). 

The fractions may be purified by shaking with concentrated sulphuric 
acid and caustic soda to remove unsaturated hydrocarbons. 

The portions of higher boiling-point are decomposed by overheat- 
ing or by distilling under pressure (cracking process) and yield fractions 
of lower- boiling-point. 

The other natural mineral oils, found in Russia, Roumania, etc., are 
also distilled fractionally. They contain generally less of the lower boil- 
ing fractions and a greater quantity of naphthene hydrocarbons (p. 237). 

Liquid hydrocarbons are also prepared by the distillation of bitu- 
minous shale. 

The higher members, which are solid, remain as distillation residues, 
and are also found in nature, e.g. ozokerit. 

The distillation residues are converted into oil and paraffin wax 
by freezing and pressing ; the liquid portion forms lubricating oil and 
the solid portion paraffin wax. The residues and fractions may be 
purified by treatment with sulphuric acid and caustic soda. 



52 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Examination of a Commercial Specimen of Hydrocarbons by 
Fractional Distillation. 

On distilling 50-100 c.c. of ligroin or kerosene from a small dis- 
tilling flask attached to a condenser and observing the temperature 
indicated by the thermometer, it will be seen that the temperature never 
remains constant for any length of time. The substance is a mixture. 
Several fractions which boil within 10 or 20 ranges of temperature 
can be collected in separate receivers. By redistilling these fractions 
and using a fractionating column (cf. p. 9) a pure product with a 
constant boiling-point can eventually be obtained. 

Properties. 

The saturated hydrocarbons have a peculiar odour. They are in- 
soluble in water, but are soluble in alcohol, ether and other organic 
liquids. 

Inflammability. 

Marsh gas and the other gases burn with a non-luminous flame and 
form explosive mixtures when mixed with a certain proportion of 
oxygen or air. 

The lower members amongst the liquids are also inflammable and 
burn with a more or less luminous flame. E.g. if about 3 c.c. of 
ligroin be placed in a watch glass and a lighted match applied it will 
burn. 

The higher liquid members do not burn until they have been 
warmed. E.g. on applying a lighted match to about 3 c.c. of kerosene 
contained in a watch glass, the flame is extinguished, but if the kerosene 
be warmed on the water-bath to about 40 and a lighted match again 
applied, the vapours of the kerosene will be ignited. 

In a lamp the kerosene rises to the surface of the wick by capil- 
larity, and on applying a light the oil becomes hot and turns into 
inflammable vapour. 

Inertness towards Chemical Reagents. 

The saturated hydrocarbons are very inert substances ; they are not 
acted upon by concentrated acids or alkalies except under special 
conditions, and on account of their stability they are known as the 
paraffins from parum affinis, little affinity. 

E.g. on shaking about I c.c. of ligroin or kerosene with 

(a) concentrated sulphuric acid, 

() concentrated nitric acid, 

(c) potassium permanganate solution, 

(d) bromine dissolved in chloroform, 



HYDROCARBONS 53 

there is no reaction, unless the commercial mixture of hydrocarbons 
contain hydrocarbons belonging to the unsaturated series. 

They are acted upon by the halogens forming substitution products 
(P- 57). 

Synthetical Preparation. 

1. The lower members of the series can be prepared by the action 
of water on certain metallic carbides, e.g. : 

Marsh gas is evolved if about 2 gm. of aluminium carbide in a test 
tube be covered with water. The gas may be collected in an inverted 
test tube and shown to be inflammable. 

2. Saturated hydrocarbons can be prepared by the dry distillation 
of the dry sodium salt (i part) of a fatty acid with soda lime (3 parts). 

E.g. Methane is given off when fused sodium acetate and soda lime 
in the above proportions are heated together in a test tube. The 
evolved gas may be ignited at the mouth of the tube. 
CH 3 COONa + NaOH = CH 4 + Na 2 CO a . 

3. Saturated hydrocarbons are prepared from the correspond-, 
ing halogen derivative by reduction with hydriodic acid, zinc-copper 
couple, zinc and hydrochloric acid, etc. 

C 2 H,I + HI = C 2 H 6 + I., 
C 2 H 5 I + 2H = C 2 H 6 + HI. 

4. The higher members of the series are prepared from the lower 
members by treating the dry halogen derivative (alkyl halide) with zinc 
or with sodium : 

CH 3 I + 2Na + CH 3 I = CH 3 . CH 3 + 2NaI 
2CH,I + Zn = CH, . CH, + Znl,.. 



54 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

B. UNSATURATED. 

In addition to the series of saturated hydrocarbons there are other 
series which contain less hydrogen in the molecule and are represented 
by the general formulae C M H 2w (olefines) and C^H 2W _ 2 (acetylenes). 
The two compounds ethylene or ethene, C 2 H 4 , and acetylene or ethine, 
C 2 H 2 , are the first and typical examples. They are represented by 

the constitutional formulae : 

CH, CH 

I! HI 

CH 2 CH 

Ethylene. Acetylene. 

The four valencies of the carbon atoms are not completely satisfied by 
hydrogen atoms and they are therefore termed the unsaturated hydro- 
carbons. The unsaturated hydrocarbons are given the suffix ene and 
ine respectively. The higher members ai^ derived from the corre- 
sponding saturated hydrocarbons by the loss of two or four hydrogen 

atoms and the^ insertion of a double vor triple bond. Isomers exist 

*- "; / r 

' amongst the higjter members, afld further, compounds are known which 

Maontain two or ttipre tlouble bon'Hs in thtfif molecule, e.g. 

* ., f; C H 

;:c CH=CH 2 
CH/ 

Isoprene. 

It should be noted that the double and the triple bonds do not indi- 
cate greater, but on the contrary lesser, stability. 

(a) OLEFINES. 
Preparation. 

1. The olefines are most usually prepared by abstracting the ele- 
ments of water from alcohols by means of dehydrating agents zinc 
chloride, sulphuric acid, phosphoric acid : 

C 2 H 5 OH = CH 2 =:CH 2 + H 2 0. 
The preparation of ethylene by this method is described on p. 58. 

2. Olefines are prepared by the action of alcoholic potash upon 
the alkyl halide (p. 57). 

C 2 H 5 I + KOH = CH 2 =CH 2 + H 2 O + KI. 

Ethylene may be prepared as follows : 

50 c.c. of a 20 per cent, solution of caustic potash in alcohol is placed 
in a 250 c.c. distilling flask in the neck of which a tap funnel is fastened with 
a cork. The distilling flask is fixed on a stand at an angle so that its neck 
may be attached to an inverted condenser (or its neck bent at an angle). A 
glass tube suitably bent leads from the condenser to a water trough. The 
potash solution is warmed and about 15 c.c. of ethyl iodide are slowly dropped 
in. Ethylene is evolved and potassium iodide is precipitated. When all the 
air has been displaced from the apparatus the gas may be collected in a gas 
cylinder over water. 

Note. Ether is formed in the reaction according to the equation : 
KOC 2 H S + C,H B I = KI + C 2 H fl . O . C 2 H n . 



HYDROCARBONS 55 

Properties. 

The lower members with 2, 3 and 4 atoms of carbon are gases. 
The higher members are liquids and solids. They are lighter than 
water in which they are only slightly soluble. They are soluble in 
alcohol, ether and other organic liquids. They are inflammable and 
burn with a luminous.^moky flame. 

Addition Reactions. 

(1) Hydrogen. When mixed with hydrogen and passed through 
a hot tube over platinum black, or finely divided nickel, they are con- 
verted into saturated hydrocarbons : 

CHg^^Crijj + H 2 = CH 3 CHg. 

The catalyst can be suspended in an inert solvent and a mixture of 
ethylene and hydrogen bubbled through the liquid. 

(2) Halogens. The defines combine with the halogens, chlorine 
and bromine, but less readily with iodine, to form halogen compounds 
containing two atoms of halogen (see p. 57) : 

CH 2 =rCH 2 + Br., = CH 2 Br . CH 2 Br. 

(3) Halogen Acids. The following reaction occurs : 

CH 2 =CH 2 -t- HI = CH 3 .CH 2 I. 

(4) Sulphuric Acid. The alkyl hydrogen sulphate (p. 71) is 
formed by addition : 

CH 2 =CH 2 + H 2 SO 4 = CH 3 . CH 2 . HSO 4 . 

This reaction serves for the separation of saturated and unsaturated 
hydrocarbons. 

(5) Hypochlorous Acid. Chlorhydrins are formed : 

CH 2 =CH 2 + HOCl = CH 2 OH . CH 2 C1 

Ethylene chlorbydrin. 

(6) Potassium Permanganate. The defines are oxidised by dilute 
permanganate : 

CH 2 =CH 2 + H.,O + O = CH 2 OH . CH 2 OH 
Ethylene glycol. 

This reaction may be used for detecting unsaturated compounds in 
a mixture of hydrocarbons. 



56 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

(6) ACETYLENES. 
Preparation. 

Acetylene is formed by the incomplete combustion of other hydro- 
carbons, but is most usually prepared by the action of water upon cal- 
cium carbide : 

CaC 2 + H 2 O = C 2 H 2 + CaO. 

The hydrocarbons of this group are prepare^ by the action of alco- 
holic potash upon halogen compounds in the same way as ethylene : 

CH 2 Br CHBr 

| + KOH = || + KBr + H 2 O 

CH 2 Br CH 2 

Vinyl bromide. 
CHBr CH 

j| + KOH =||| + KBr + H 2 O. 

CH 2 CH 

Properties. 

The lower members are gases, the higher members are liquids. 
Acetylene is soluble in water (i : i) and other organic liquids. Acetone 
dissolves thirty-one times its own volume of the gas at N.T.P. Acety- 
lene burns with a smoky, intensely hot flame which is very luminous ; 
it is consequently employed for illuminating purposes, the burners, 
generally of clay, being designed so that complete combustion is effected. 

Addition Reactions. 

Acetylene and its homologues behave like the defines, but react 
with two molecules : 

(1) Hydrogen. 

C 2 H 2 + H 2 = C 2 H 4 (ethylene) 
C 2 H 4 + H 2 = C 2 H 6 (ethane). 

(2) Halogen Acid. 

CH., 
C 2 H 2 + HC1 = || (vinyl chloride) 

CHC1 

CH 2 CH 3 

[j + HC1 = | (ethylidene chloride). 

CHC1 CHC1 2 

(3) Halogens. 

CH CHBr 

III + Br 2 = || (acetylene dibromide) 

CH CHBr 

CHBr CHBr 2 

+ Br 2 = | (ethane tetrabromide). 

CHBr CHBr 2 

Acetylene and the other members of the series form characteristic 
compounds with copper, silver and other heavy metals. 

Cuprous ace-tylide, C 2 Cu 2 ,and silver acetylide,C 2 Ag 2 , are precipitated 
as amorphous compounds when acetylene is passed through ammoni- 
acal solutions of cuprous chloride or silver nitrate. In the dry state 
these compounds are very explosive ; they are decomposed on treat- 
ment with hydrochloric acid or potassium cyanide yielding acetylene. 
Acetylene may be separated from other hydrocarbons by this property. 



HALOGEN DERIVATIVES OF THE HYDROCARBONS. 

The only chemical property of the saturated hydrocarbons is that 
they are attacked by the halogens yielding halogen substitution deriva- 
tives, one atom of hydrogen being progressively replaced by an atom 
of halogen ; thus, from methane by the action of chlorine, we can 
obtain 

CH 3 C1 CH 2 C1 2 CHC1 3 CC1 4 . 

A mixture of the compounds results and the reaction is slow, so that, 
in practice, these compounds are not prepared from the hydrocarbon, 
but from other compounds. 

The unsaturated hydrocarbons differ from the saturated hydrocar- 
bons in their behaviour to the halogens. They react by addition, thus, 
e.g. ethylene combines with two atoms of bromine, forming the saturated 
compound, ethylene dibromide : 

CH 2 CH 2 Br 

|| +Br 2 = | 
CH 2 CH 2 Br. 

Dihalogen compounds of this type are generally prepared by this 
reaction. 

MONOHALOGEN DERIVATIVES. ALKYL HALIDES. 

Preparation. 

The alkyl halides are prepared from the corresponding alcohol 
by the action of the halogen acid, or by the action of the phosphorus 
halide : 

CH 3 OH + HBr = CH 3 Br + H g O 
3 CH 3 OH + PI 3 = CH 3 I + H 3 P0 3 
CH 3 OH + PC1 5 = CH 3 C1 + POC1 3 + HC1. 

Preparation of Methyl Iodide. 

1 8 gm. of methyl alcohol and 5 gm. of red phosphorus are placed in a 
small flask (250 c.c.) and a reflux condenser is attached to it. 50 gm. of 
iodine are slowly added by detaching the flask from the condenser and rapidly 
refixing. Heat is evolved in the reaction and loss of alcohol and iodide is 
prevented by the condenser. The apparatus and mixture is allowed to stand 
for 12 to 24 hours so that the reaction completes itself. The contents of the 
flask are distilled from a water-bath. The distillate is shaken in a separating 
funnel with dilute caustic soda to remove iodine. and hydriodic acid, and if 
sufficient has been used the lower layer of methyl iodide will be colourless. 
The lower layer of methyl iodide is separated, allowed to stand with a little 
calcium chloride till it is clear and distilled from a water-bath (b.p. 44 . 
About 45 gm. or 75 per cent, of the theoretical yield should be obtained. 

57 



58 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Preparation of Ethyl Bromide. 

A distilling flask of about i litre capacity is closed by a cork and its neck 
attached to a condenser. To the end of the condenser is attached an 
adapter (a bent tube wide at one end and narrow at the other, p. 12) which 
dips under water contained in a conical flask of about 250 c.c. capacity 
cooled by standing in ice. 54 c.c. (100 gm.) of sulphuric acid are mixed 
in the flask with 75 c c. (60 gm.) of absolute alcohol and cooled to the tem- 
perature of the air. 100 gm. of coarsely powdered potassium bromide 
are added to the contents of the flask and the mixture is heated on a 
sand bath or carefully on a gauze. The contents boil and froth up and heavy 
oily drops of ethyl bromide collect under the water in the receiver. If the 
frothing is too great the flask is removed from the source of heat for a minute. 
The heating is continued so long as oily drops distil over. The contents of 
the receiver are placed in a separating funnel and the lower layer collected. 
It is purified by returning to the separating funnel and shaking with a dilute 
solution of sodium carbonate. The ethyl bromide is then shaken with water 
to remove alkali and it is placed in a clean dry distilling flask and left in con- 
tact with calcium chloride till it is clear. The flask is furnished with a ther- 
mometer, attached to a condenser and the ethyl bromide (b.p. 35-40) distilled 
over from a water-bath. About 75 to 80 gm. should be obtained. 

DIHALOGEN DERIVATIVES. 

Methylene chloride, CH 2 C1 2 , is generally prepared by reducing 
chloroform in alcoholic solution with zinc and hydrochloric acid. 

Methylene iodide, CH 2 I 2 , is prepared by reducing iodoform with 
hydriodic acid. 

Methylene bromide, CH 2 Br 2 , is prepared by treating methylene 
iodide with bromine: 

CH 2 I, + 2Br 2 = CH 2 Br 2 + aBrl. 

The numerous isomers of the halogen derivatives of the higher 
hydrocarbons are prepared by various methods, e.g. : 

(a) by addition of halogen to unsaturated hydrocarbons ; 

() by the action of phosphorus pentachloride upon the aldehydes 
and ketones. 

Preparation of Ethylene Dibromide. 

Ethylene is prepared by dropping a mixture of 30 c.c. of absolute alcohol 
and 80 c.c. sulphuric acid from a tap funnel upon a mixture of 124 c.c. of 
alcohol and 1 08 c.c. of concentrated sulphuric acid contained in a 2 litre flask 
and mixed with sand to prevent frothing, the mixture being gently heated until 
a steady stream of gas is evolved. The evolved gas is passed through two wash 
bottles l with safety tubes containing caustic soda solution to remove sulphur 
dioxide into two ordinary wash bottles containing 50 c.c. bromine and i c.c. 
of water and 15 c.c. of bromine and i c.c. of water respectively. These two 
bottles are placed in a basin of water to which ice may be added to prevent 
the contents becoming warm during the reaction. The outlet tube is 
connected to a tower containing soda lime so that bromine vapour does not 
escape into the room. The bromine in the bottles is gradually decolorised 

1 It may be necessary to change these bottles' for fresh ones during the preparation. 



HALOGEN DERIVATIVES OF HYDROCARBONS 59 

and changes into ethylene bromide which may have a straw-yellow colour. 
The heavy liquid product is shaken in a separating funnel with dilute caustic 
soda solution and then with water. It is dried with calcium chloride and 
purified by distillation (b.p. 130-132). About 60 gm. should be obtained. 

Properties of the Monohalogen and Dihalogen Derivatives. 

The monohalogen derivatives are liquids heavier than water in 
which they are insoluble or only slightly soluble. They have a 
peculiar smell and do not burn readily. Their properties are in 
general like those of chloroform (p. 60). 

Chemically the monohalogen derivatives or alkylhalides are very 
reactive substances and readily exchange the atom of halogen with 
other atoms or groups. They are thus largely used for introducing 
alkyl radicles into other compounds, thus : 

1. CH 3 I + 2H = CH 4 + HI (p. 53). -7^-*^*^., 

2. CH 3 I + Zn + CH 3 I = C 2 H 6 + ZnI (p. 53). / ^U^. . 

3. CH 3 I + 2Zn + CH 3 I = CH 3 . Zn . CH 3 + ZnT 2 . 

4. CH 3 I + KOH = CH 3 OH + KI (p. 63). 

aqueous 

5. C 2 H 5 I + KOH = C 2 H 4 +KI + H 2 O (p. 54). 

alcoholic 

6. C,H 5 I + NH 3 = C 2 H 5 NH, + HI (p. 124). 

7. C 2 H 5 I + KCN = C,H 5 CN + KI (p. 158). 

8. C 2 H 5 I + KN0 2 = aH 5 N0 2 + KI. 

9. C 2 H 5 I + KHS = C 9 H 5 HS + KI (p. 78). 

The dihalogen derivatives are very similar to the monohalogen de- 
rivatives in both their physical and chemical properties. Both the 
halogen atoms can be replaced by OH, NH 2 , etc. 

TRIHALOGEN DERIVATIVES. 
CHLOROFORM. 

Preparation. 

100 gm. of fresh bleaching powder are rubbed up in a mortar with 
200 c.c. of water so as to form a paste, the paste is rinsed into a large 
flask of about 1000 c.c. capacity with another 200 c.c. of water ; 25 
c.c. of acetone or alcohol are added and the mixture shaken up thor- 
oughly. The flask is connected by means of a bent tube to a con- 
denser and receiver and gently heated through a wire gauze. As soon 
as the reaction commences, which is shown by the frothing, the flame 
is removed. When the frothing has subsided and the reaction has 
moderated, the contents of the flask are boiled until no more chloro- 
form distils over with the water. The chloroform consists of heavy 
oily drops which sink in water, and it forms the lower layer of the 
distillate. 

The distillate is transferred to a separating funnel and shaken 
with a little dilute caustic soda solution ; the lower layer of chloroform 



60 PRACTICAL ORGANIC AND BIO-CHEMISTRY ' 

form is drawn off into a clean, dry flask and dried by adding anhydrous 
calcium chloride, either by shaking for 5-10 minutes or allowing to 
stand from 12-24 hours, until it is clear. The chloroform is filtered 
into a clean, dry distilling flask and distilled. 

The mechanism of the reaction by which the chloroform is formed 
is probably : 

1. The oxidation of the alcohol to aldehyde (p. 80), 

CH 3 . CH 2 OH + O = CH 3 . CHO + H 2 O. 

2. The chlorinatiori of the aldehyde to chloral, 

CH 3 . CHO + 3C1 2 = CC1 3 . CHO + sHCl. 

3. The decomposition of the chloral to chloroform and calcium 
formate by the calcium hydroxide (p. 87), 

2CC1 3 . CHO + Ca(OH) 2 = 2 CHC1 3 + (HCOO) 2 Ca. 

Purification of Commercial Chloroform. 

Chloroform prepared from alcohol, methylated spirit (methylated 
chloroform) or acetone may contain chlorine, hypochlorous acid or 
hydrochloric acid, aldehyde, etc. 

The specimen is shaken several times with water, the chloroform 
is separated, dried with (i) calcium chloride, (2) phosphorus pentoxide 
and distilled. 

The last traces of alcohol may also be removed by adding slices of 
metallic sodium, allowing to stand for 12-24 hours and then distilling. 

Properties. 

Chloroform is a volatile colourless liquid with a distinct and sharp 
odour and sweetish taste. It boils at 6iand has a sp. gr. of i '483-1 -487. 

Its vapour does not burn, but when mixed with alcohol the com- 
bined vapours burn with a smoky flame edged with green. 

It is soluble in about 200 volumes of cold water (-44 gm. in 
100 c.c.) to which it gives a sweet taste. 

It mixes in all proportions with absolute alcohol, ether, benzene, 
petroleum ether. It is slightly soluble in dilute alcohol' and readily 
dissolves fats, resins, india-rubber, camphor, iodine, bromine. 

Many specimens of commercial chloroform undergo change on 
keeping, especially in the light, and are liable to contain chlorine, 
hypochlorous acid or hydrochloric acid. This decomposition is hin- 
dered by the addition of I per cent, of alcohol. The bottle should 
be kept in the dark. I c.c. of chloroform on evaporation should leave 
no residue and if allowed to evaporate on clean filter paper should leave 
no disagreeable odour. 

Chloroform is decomposed by boiling with aqueous alkali, more 
rapidly in alcoholic solution, into alkali formate and chloride : 
CHC1 3 + 4 NaOH = HCOONa + 3NaCl + 2H 2 O. 



HALOGEN DERIVATIVES OF HYDROCARBONS 61 

A few drops of chloroform are heated with dilute caustic soda. 
The presence of chloride is tested for in a small portion of the solu- 
tion, the remainder is neutralised exactly, if it be still alkaline, and 
heated with mercuric chloride solution. A deposit of mercurous 
chloride and mercury shows the presence of formate. 

Tests for Impurities in Chloroform. 

A quantity of the specimen is shaken up with two volumes of water. 
The water is separated and silver nitrate is added. Pure chloroform 
gives no reaction, but a precipitate of silver chloride indicates the 
presence of chlorides. If, on heating, the precipitate blackens the 
presence of aldehyde or formic acid is indicated. The water should 
not react with blue litmus. 

Chloroform is not soluble in concentrated sulphuric acid. Any 
darkening which occurs on shaking them together is due to the 
presence of aldehyde, methyl alcohol, etc. The presence of alcohol in 
chloroform may be detected by shaking some of the specimen with 
five volumes of water, filtering through a wet paper, and testing for 
alcohol in the filtrate by the iodoform reaction (p. 67). 

Tests for Chloroform. 

(1) A red or yellow precipitate of cuprous oxide is formed on add- 
ing some solution of chloroform in water to Fehling's solution (p. 84) 
and heating. 

(2) Carbylamine Reaction. To the dilute solution of chloroform 
in water is added some alcoholic sodium hydroxide and a drop of 
aniline and the mixture heated. Phenyl isonitrile or carbylamine 
is formed, which has a disgusting smell : 

CHC1 3 + 3KOH + C 6 H 5 NH, = C S H 5 NC + sKCl + 3 H 2 O. 

This reaction is extraordinarily sensitive and will detect one part 
of chloroform in 5000 parts of alcohol. It is also given by bromoform, 
iodoform, chloral, trichloracetic acid and substances which yield chloro- 
form when treated with alkali. 

From liquids, such as blood, it is better to remove the chloroform 
as described under estimation and to test the liquid in the receiver. 

Estimation of Chloroform. 

Hydrochloric acid is formed when chloroform vapour mixed with 
hydrogen is passed through a red hot tube. 

Hydrogen is slowly passed into a flask containing the solution of chloroform 
and the flask is gently heated. The mixed vapours are passed through a 
short, heated combustion tube containing platinum wire gauze or loose 
asbestos and into a receiver containing water. The contents of the receiver 
are titrated with standard alkali or precipitated with silver nitrate. As 
acetylene and hydrogen cyanide may also be present the contents of the 
receiver should be boiled before titrating or precipitating. 

This procedure may be used for detecting and estimating chloroform in 
blood and other liquids which do not contain other chlorinated compounds. 



62 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



IODOFORM. 

Preparation. 

4 gm. of crystallised sodium carbonate are dissolved in 20 c.c. 
of water. 2 c.c. of absolute alcohol and 2 gm. of iodine are added, 
and the solution warmed to about 70 on the water-bath until it 
is decolorised. lodoform separates as a lemon-yellow powder. It 
is filtered off, washed with cold water and dried on an unglazed 
plate. 
_ The melting-point of the preparation serves to prove its identity. 

Properties. 

lodoform is a light yellow, shining crystalline solid with a per- 
sistent unpleasant odour, It has a characteristic microscopic appear- 
ance hexagonal plates, stars, or rosettes, and melts at 119. On 
gently heating it sublimes without change, but on heating strongly it 
is decomposed : violet vapours of iodine are formed and a deposit of 
carbon is left. 

lodoform is nearly insoluble in water (i part in 10,000) and in 
dilute acids and alkalies. It is slightly soluble in alcohol (l part in 
50) but more easily soluble in absolute alcohol (i part in 23). It is 
easily soluble in ether, chloroform and carbon disulphide, but very 
slightly soluble in glycerol, benzene and petroleum ether. In its 
chemical properties iodoform closely resembles chloroform. 

Tests for Impurities in lodoform. 

1. No residue should remain when it is heated in the air. 

2. It should be completely soluble in boiling alcohol, but insoluble in 
brine. 

3. On shaking up with water and filtering, the filtrate should give no re- 
action with barium chloride or silver nitrate. 

4. If picric acid be present as adulterant, it may be detected (a) By testing 
the aqueous extract with potassium cyanide when a reddish-brown coloration 
is produced, (ft) By treating with .caustic soda solution and shaking this 
solution with chloroform. Picric acid remains in the aqueous solution. 
(c) By extracting the acid with dilute sodium carbonate solution, neutralising 
exactly with acetic acid and adding potassium nitrate ; potassium picrate 
is precipitated. 



ALCOHOLS. 

Alcohols are hydrocarbons in which a hydrogen atom (or more in 
the case of the higher members, e.g. glycerol) has been substituted by 
a hydroxyl or OH group. This relationship is shown : 

1. By the action of water and aqueous alkalies upon the halogen 
mono-substituted hydrocarbons : 

CH 3 . Cl + HOH = HC1 + CH 3 . OH. 

2. By the action of phosphorus pentachloride upon the alcohol : 

CH 3 . OH + PC1 5 = CH 3 . Cl + POC1 3 + HC1. 

All alcohols are designated by the suffix -ol,.e.g. methyl alcohol 
or methanol, ethyl alcohol or ethanol. 

Most of the alcohols are natural substances and serve as the start- 
ing point for the preparation of other compounds. 

METHYL ALCOHOL. CH 3 .OH. 
Commercial Methyl Alcohol. 

Preparation. 

Methyl alcohol, together with acetone, acetic acid, methyl acetate 
and other substances is formed in the dry distillation of wood. The 
acid aqueous distillate is known as pyroligneous acid ; on standing 
wood tar separates out. The acid liquid contains 1-2 per cent, of 
methyl alcohol, - i-'5 per cent, of acetone and about 10 per cent, of 
acetic acid. It is distilled until the distillate has a specific gravity of 
9-1. The crude wood spirit so obtained is a greenish-yellow liquid 
with disagreeable odour. It is mixed with about 2 per cent, of lime 
and again distilled. This retains the acetic acid as calcium acetate, 
the neutral substances methyl alcohol, acetone, acetaldehyde, methyl 
acetate passing over. This distillate is wood spirit and contains about 
93 per cent, of methyl alcohol. It is diluted with water to precipitate 
oily impurities and is again treated with lime and distilled. Basic 
impurities are removed by distilling it with -I -'2 per cent, of sul- 
phuric acid and the methyl alcohol boiling at 64-66 is collected. 

Methyl alcohol is also prepared by dry distillation from vinasses 
the mass remaining after fermentation of the residues from the pre- 
paration of beet sugar. 

63 



64 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Pure Methyl Alcohol. 

Commercial methyl alcohol contains acetone. By dissolving 
anhydrous oxalic acid (prepared by heating oxalic acid at 100) in the 
boiling spirit, methyl oxalate is formed ; it separates in crystals on cool- 
ing. The crystals are filtered off, washed free from acetone with water, 
and then decomposed into oxalic acid and methyl alcohol by boiling 
with water or ammonia. Methyl alcohol is obtained on distillation and 
is dehydrated by distilling over quicklime (see under ethyl alcohol). 

Pure methyl alcohol may also be obtained by boiling commercial 
methyl alcohol with anhydrous calcium chloride. Calcium chloride 
crystallises out in combination with methyl alcohol as CaCl 2 + 4CH 3 OH 
from the saturated solution on cooling. The crystals are drained from 
the mother liquor and are decomposed by heating ; methyl alcohol is 
evolved and is collected. 

The acetone may also be removed by passing chlorine into it form- 
ing trichloracetone. The methyl alcohol is separated by fractional 
distillation. 

Properties. 

Methyl ""alcohol is a colourless liquid which boils at 66 and has 
a sp. gr. of 797 at 1 5. It closely resembles ethyl alcohol in its 
properties, but it does not give the iodoform reaction. 

ETHYL ALCOHOL. 

Preparation. 

Ethyl alcohol is obtained by the fermentation of sugar by yeast 
and occurs in all fermented liquids such as wine and beer. It is made 
chiefly from potatoes and cereals, the starch being first converted into 
the sugar, glucose, which is fermented by the yeast and changed into 
alcohol and carbon dioxide. 

1. Rectified Spirit. 

The alcohol produced by fermentation is separated from the fer- 
mented liquor by distillation. The distillate is then fractionally re- 
distilled, or rectified, so as to separate as much water as possible and 
the greater part of the higher alcohols. The product is rectified 
spirit. It contains about 84 per cent, by weight of ethyl alcohol and 
has a sp. gr. of 0-838 at 15. 

2. Methylated Spirit. 

The rectified spirit is denatured and rendered unfit for drinking 
purposes by the addition to it of one-ninth of its volume of wood spirit 
and three-eighths of I per cent, of mineral naphtha or paraffin oil. 

Since 1905 methylated spirit has been obtainable in approved 
scientific institutions free of duty and free from mineral naphtha. 



ALCOHOLS 65 

3. Absolute Alcohol. 

Rectified spirit is filtered through charcoal and fractionally distilled, 
the first portions which contain aldehyde and the last portions which 
contain fusel oil being rejected. The middle fraction is distilled over 
quicklime and commerciaj absolute alcohol is obtained. This contains 
about *5 per cent, of water. Pure alcohol is prepared from this by 
adding the requisite quantity of metallic sodium or calcium and again 
distilling. 

4. Absolute Alcohol from Methylated Spirit. 

Methylated spirit (i litre) is boiled upon a water-bath under a 
reflux or inverted condenser (p. 18) with about 30 gm. of caustic soda 
for one hour in a 2-litre flask. Acetone, aldehyde and other impurities 
are destroyed and the spirit turns brown. The contents of the flask 
are distilled and the distillate collected in another flask of the same 
capacity containing 400-500 gm. of quicklime. The flask is connected 
with a reflux condenser and either allowed to stand for twenty-four 
hours or heated for one hour on a water-bath. The liquid is distilled 
again without pouring off from the flask. The yield of absolute 
alcohol is about 80 per cent., and it contains 2-3 per cent, of water. 
By treating it again with half the previous quantity of quicklime the 
amount of water may be reduced to less than I per cent. The boiling- 
point (76-78) may be determined by distilling 50 c.c. in a small 
apparatus. 

Properties. 

(1) Ethyl alcohol is a colourless, pleasant-smelling liquid with a 
hot taste. It boils at 78 and has a sp. gr. of 79384 at I5'5 
or 60 F. 

(2) It mixes with water in all proportions. Absolute alcohol is 
very hygroscopic and readily absorbs water on exposure to the air. 

On mixing alcohol with water there is an evolution of heat and a 
contraction in bulk. 

The addition of water to methylated spirit produces a cloudiness 
due to the precipitation of the mineral naphtha. 

(3) Alcohol burns with a faint blue non-luminous flame even when 
mixed with considerable amounts of water. 

On mixing 10 c.c. of alcohol with 10 c.c. of water in a measuring 
cylinder the evolution of heat will be noticed, and when the mixture 
is cold the diminution in volume can be measured. By placing the 
mixture in a small basin and applying a light it will be seen whether it 
is or is not inflammable. 

5 



66 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Detection of Water in Absolute Alcohol. 

(a) If the alcohol contains a considerable quantity of water its 
presence will be shown by adding some anhydrous copper sulphate 
which turns blue. 1 

() 0-5 per cent, of water may be detected by adding a crystal of 
potassium permanganate ; the liquid will assume a pink colour. 

(c) Traces of water in absolute alcohol according to Yvon may be 
detected by means of calcium carbide. If water be present, bubbles 
of acetylene are given off and the liquid becomes milky, due to the 
formation of calcium hydroxide. 

Reactions. 

1. Action of Sodium. 

On adding about I gm. of sodium to 20 c.c. of absolute alcohol in 
a small flask there is an evolution of hydrogen just as with water, but 
the reaction is by no means so violent. The gas which is evolved may 
be collected in an inverted test tube and shown to be hydrogen by 
burning. 

When the sodium has dissolved the solution is evaporated to dry- 
ness on the water-bath. A white solid sodium ethoxide remains, 
which is very hygroscopic and is decomposed by water, yielding 
alcohol which can be recognised by its smell and by the iodoform 
test : 

C.,H 5 OH + Na = C 2 H 5 ONa + H 
C~ 2 H 5 ONa + H 2 O = C 2 H 6 OH + NaOH. 

This reaction shows that one of the hydrogen atoms in alcohol is 
replaceable by sodium. 

2. Action of Phosphorus Pentachloride. 

On adding a little phosphorus pentachloride to a small quantity 
of alcohol, a vigorous action occurs and hydrochloric acid fumes are 
evolved. Ethyl chloride and phosphorus oxychloride are the other 
products. The smell of ethyl chloride will be noticed when the 
hydfochloric acid fumes cease to be given off. This reaction shows 
the presence of an hydroxyl or OH group : 

C 2 H 5 OH + PC1 5 = C 2 H 5 GJ + POC1;5 + HC1. 



1 Prepared by gently heating a crystal of copper sulphate in a crucible until it falls to 
powder, 



ALCOHOLS 67 

Tests. 

(1) Smell. Even in dilute solutions alcohol may be detected by 
its smell. 

(2) Oxidation to Acetaldehyde. On warming a little dilute alcohol 
in a test tube with a few drops of potassium dichromate and some 
dilute sulphuric acid the pungent characteristic odour of aldehyde will 
be observed and the solution turns green : 

C 2 H 5 OH + O = CH 3 . CHO + H 2 O. 

(3) Formation of Ethyl Acetate. The fruity odour of ethyl acetate is 
produced when some of the dilute solution is heated with concentrated 
sulphuric acid and a little solid sodium acetate. 

(4) lodoform Reaction (Lieben). About an equal volume of iodine in 
potassium iodide is added to a very dilute solution of alcohol I or 2 
drops in half a test tube full of water and then, drop by drop, 
caustic soda till the mixture is decolorised. On gently warming 
the mixture, iodoform is formed and may be recognised by its 
characteristic smell. A yellow crystalline precipitate will separate if 
the solution of alcohol is not too weak. 

Note. This very sensitive reaction is not characteristic of alcohol 
"as it may be given by aldehyde, acetone, acetic ester and other sub- 
stances which contain the grouping CH 3 C joined to oxygen. 

Alcohol gives the reaction on warming, acetone gives the reaction 
in the cold. 

Estimation of Alcohol in Beer, Wines, Spirits. 

The amount of alcohol in these liquids is ascertained by distilling off the 
alcohol and determining the specific gravity of the distillate. 

(a) 100 c.c. beer are distilled and 80 c.c. distillate are collected. 

(b) 100 c.c. wine + 80 c.c. water and a little tannin are distilled and nearly 100 c.c. 
distillate are collected. 

(c) 50 c.c. spirit + too c.c. water, or 25 c.c. spirit + 150 c.c. water, are distilled and 
nearly 100 c.c. distillate are collected. 

The distillate is made up to 100 c.c. with water, the liquids are. mixed, 
and the sp. gr. at 15 -5 or 60 F. is determined by weighing in a sp. gr. bottle. 
The amount is given by referring to an alcohol specific gravity table for the 
percentage by weight. The amount in the sample is ascertained from the 
formula : 

sp. gr. of distillate x amount of distillate in c.c. x per cent, of alcohol from table 

sp. gr. of sampfe x amount of sample taken 
= percentage of abs. ale. by weight in the sample. 

If the specific gravity of the sample be unknown, it may be calculated from 

wt. of distillate x per cent, of alcohol from table 

wt. of sample taken 
= percentage of abs, ale. by weight in the sample, 



68 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

PROPYL ALCOHOLS. C 3 H 7 OH. 

Normal Propyl Alcohol. CH 3 . CH 2 . CH 2 OH. 

Normal propyl alcohol is formed in the process of alcoholic fermen- 
tation and is contained in fusel oil, from which it is obtained by frac- 
tional distillation. It is present to the extent of about 3 per cent, in 
the fusel oil obtained from potato spirit. 

It is a liquid resembling ethyl alcohol but with a less pleasant 
smell, and burns with a luminous flame. It boils at 97 and has a sp. 
gr. of -807 at 15. 

GHj 

Isopropyl Alcohol. \CHOH. 

CH/ 

Isopropyl alcohol is prepared either by the reduction of acetone 
with sodium amalgam or from isopropyl iodide by boiling it with lead 
hydroxide and water. Isopropyl iodide is prepared by the action of 
phosphorus and iodine upon dilute glycerol. 

It is a liquid resembling normal propyl alcohol, but it boils at 82 
and has a sp. gr. of 792 at 15. 

BUTYL ALCOHOLS. C 4 H 9 OH. 

Four isomers are possible in the case of the butyl alcohols : 



CH 2 OH 


CH 2 OH 


CH 3 


CH. 


1 


1 


1 


1 


CH 2 


CH 


CHOH 


COH 


CH 2 


/\ 
CH 3 CH 3 


CH 2 


/\ 

CH 3 CH 3 


1 




1 




CH 3 




CH 3 




Normal 


Primary 


Normal 


Tertiary 


primary 


isobutyl 


secondary 


butyl 


butyl 


alcohol. 


butyl 


alcohol. 


alcohol. 




alcohol. 





The chief of- these is primary isobutyl alcohol which is formed in 
alcoholic fermentation and is separated by fractional distillation from 
the fusel oil. 

Normal primary butyl alcohol has once been found in fusel oil. 
It is formed by the action of- the Schizomycetes upon glycerol. 
Bacillus butylicus (contained in the excrement of cows) produces 6-8 
per cent, from glycerol and 10 per. cent, from mannitol. Normal 
secondary butyl alcohol and tertiary butyl alcohol are prepared by 
synthetical methods. The butyl alcohols are liquids with the following 
boiling-points and specific gravities at 20 : 

B.P. Sp. Gr. B.P. Sp. Gr. 

Normal primary 117 '810 Primary isobutyl 107 -806 

Normal secondary 100 -8c8 Tertiary 83 -786 

They are not miscible with water in all proportions ; normal 
primary butyl alcohol requires 1 2 parts of water to dissolve it. 



ALCOHOLS 69 

AMYL ALCOHOLS. C 5 H U OH. 

Eight isomers are possible, all of which are known : 

B.P. Sp. Gr. at 20. 

1. Normal primary, CH.; . CH 2 . CH 2 . CH 2 . CH OH 138 -817 

CH 3 

2. Isobutyl carbinol, ^CH . CH 2 . CH,OH 130 '810 

CH 3 / 

CH 3 x 

3. Secondary butyl carbinol, /CH . CH 2 OH 128 -816 

CH 3 CH/ 

CH,, 

4. Tertiary butyl carbinol, X 

(primary) CH s C . CH 2 OH 113- 

CH/ 

5. Methyl propyl carbinol, CH 3 

(secondary) >CHOH 119 

CH 3 .CH 2 .CH/ 

CH 3 

6. Methyl isopropyl carbinol, CH 3X \ C HOH 112^ -819 

(secondary) \ CH / 

CH/ 

CH 3 . CH 2 

7. Diethyl carbinol, > C HOH 117 

(secondary) CH 3 . CH 2 / 



8. Dimethyl ethyl carbinol, CH 3 C . OH 102' 

(tertiary) ^ c ^/ 

Secondary butyl carbinol contains an asymmetric carbon atom (see 
under lactic acid) and consequently exists in a dextro- and a laevo-form. 
Together with primary isobutyl alcohol and propyl alcohol the two 
amyl alcohols, isobutyl carbinol and laevo-secondary butyl carbinol, are 
the principal constituents of fusel oil and they together constitute 
fermentation amyl alcohol. 

The fusel oil from potatoes or cereals contains chiefly isobutyl 
carbinol, secondary butyl carbinol being present only to 13-22 per 
cent. The fusel oil from beet molasses contains 48-58 per cent, of 
secondary butyl carbinol. 

Fermentation amyl alcohol is a strongly refractive liquid which 
boils at 130-131 and is very slightly soluble in water 3*3 volumes 
in 100 volumes of water at 22. Its vapours, on being inhaled, produce 
a peculiar sensation in the head, causing headache, etc. The two 
alcohols cannpt be separated by fractional distillation, but only by 
chemical means. The mixture generally referred to as amyl alcohol is 
frequently used as a solvent. 



;o PRACTICAL ORGANIC AND BIO-CHEMISTRY 

HIGHER ALCOHOLS. 

A hexyl alcohol has been isolated from the fusel oil obtained from grape 
skins. Two primary hexyl alcohols occur as esters : n-primary hexyl alcohol 
in the oil from the seeds of the parsnip, Heracleum giganteum, and 3-methyl- 
pentanol in Roman camomile oil. 

Normal primary heptyl alcohol is prepared by the reduction of oenan- 
thylic aldehyde which is obtained by distilling castor oil. 

Normal primary octyl alcohol occurs in the oil from the fruits of the 
parsnips, Heracleum sphondylium, Heracleum giganteum and Pastinaca sativa. 

Normal nonyl alcohol is prepared by reducing with sodium and alcohol 
methyl heptyl ketone which is contained up to 5 per cent, in oil of rue. 

Normal secondary undecylic alcohol is prepared by reducing methyl 
nonyl ketone, which occurs in camomile in large quantities. 

n-dodecyl alcohol occurs as ester in oil of Cascara sagrada. 

Normal hexadecyl alcohol, or cetyl alcohol, C 16 H 3? OH, the most 
important of the higher alcohols, is easily prepared from spermaceti, in 
which it is present as ester, by hydrolising the ester with alcoholic soda, 
diluting with water, filtering off and recrystallising the cetyl alcohol from 
alcohol. Cetyl alcohol has also been described as being present in the fat 
from an ovarian dermoid cyst. Cetyl alcohol is a white solid which melts at 

5- 

Ceryl alcohol, C 2 rH 55 OH, or more probably C 26 Hr )3 OH, is prepared from 
Chinese wax, and melts at 76-79. 

Myricyl alcohol, C 30 H 61 OH, is best prepared from carnauba wax' 
Beeswax contains this alcohol or the alcohol C 31 H 63 OH. 

Psylla-stearyl alcohol, C 33 H fi8 O, has been obtained from the fat of the 
leaf louse (Psylla alni). 



ESTERS. 

Alcohols are like the bases NaOH, KOH in containing an OH 
group. The bases combine with acids to form salts. Alcohols com- 
bine with acids to form esters. 

An enormous number of esters is possible since any alcohol can be 
combined with any acid, inorganic or organic. The organic acids may 
be grouped into three classes : (a) those insoluble or very insoluble in 
water ; (<) those soluble in water, but volatile with steam ; (c) those 
soluble in water, but not volatile with steam. 

Preparation. 

There are several methods of preparing esters : 

(1) By the action of the acid upon the alcohol in the presence of a 
dehydrating agent, or catalyst. 

(2) By the action of concentrated sulphuric acid upon the sodium 
salt of the acid and the alcohol. 

(3) By the action of the acid chloride, or anhydride, upon the 
alcohol. 

(4) By the action of the alkyl halide upon the silver salt. 

ESTERS OF INORGANIC ACIDS. 

Halogen Acids. 

These compounds are the same as the monosubstituted halogen 
derivatives of the hydrocarbons (p. 57): 

C 2 H 5 OH + HCl = C ? H 5 C1 + H 2 0. 

Amyl Nitrite. 

The calculated quantity of concentrated sulphuric acid is allowed to drop 
slowly upon the calculated quantity of sodium nitrite mixed with the calcu- 
lated quantity of amyl alcohol contained in a flask cooled by a freezing 
mixture. Amyl nitrite floats to the surface as an oil. It is separated, washed 
with water, dried with calcium chloride and distilled. 

Ethyl Sulphuric Acid. Barium and Potassium Ethyl Sulphate. 

IO c.c. of concentrated sulphuric acid are poured carefully into 
and mixed with 20 c.c. of ethyl alcohol ; the mixture becomes hot. It 
is heated on a water-bath under a reflux condenser for -1 hour. On 
cooling it is poured into about 200 c.c. of cold water. The acid 
solution is neutralised to litmus by stirring it up with calcium or 

71 



72 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

barium carbonate. Carbon dioxide is evolved and the excess of 
sulphuric acid is precipitated as insoluble sulphate ; this is filtered off 
after heating on a water-bath for -1 hour. The solution contains 
calcium or barium ethyl sulphate (as shown below under hydrolysis). 

HOx C 2 H 5 CK 

C H 5 OH + \SO 2 = \SO 2 + H 2 O 

HCT H(T 

C 2 H 5 O\ C,H 5 OSO 2 O\ 

2 \S0 2 + BaC0 3 = ">Ba + C0 2 + H 2 O. 

HO' C 2 H S OS0 2 CT 

The clear filtrate is heated on the water-bath and treated with a strong 
solution of potassium carbonate (10 gm.) or potassium oxalate until no further 
precipitate is formed : 

/O SO 2 OC 2 H 5 C 2 H 5 0\ 

Ba/ + K 2 CO. = BaC0 3 + 2 . ^>SO 2 . 

^O S0 2 OC 2 H 5 TSXy 

The barium carbonate is filtered off and the filtrate evaporated to a 
small volume (a drop withdrawn on a glass rod should crystallise on cooling). 
The crystals which form after standing for several hours are filtered off, 
washed with dilute alcohol and dried between sheets of filter paper. The 
mother liquor yields more crystals on further evaporation. The salt is dis- 
solved in boiling alcohol under a reflux condenser, filtered, using a hot- 
water funnel, and allowed to crystallise out. 

ESTERS OF ORGANIC ACID. 

Ethyl Acetate. 

Molecular proportions of glacial acetic acid (50 c.c.) and absolute 
alcohol (50 c.c.) are mixed in a distilling flask, and I per cent, by 
volume of concentrated sulphuric acid (i c.c.) is added. The distilling 
flask is connected to a condenser and receiver and the mixture is dis- 
tilled. A yield of 86*5 per cent, of ester is obtained (Senderens' 
method). Ethyl sulphuric acid appears to be the catalyst :-^r 

C,H 5 HSO 4 + C,H 6 OH = (C 2 H 5 ) 2 S0 4 + H 2 O 
(C 2 H 5 ) 2 S0 4 + CHjCOOH = C 2 H 5 HSO 4 + CH 3 COOC 2 H 5 . 

The distillate which contains water, alcohol and acetic acid is purified 
by shaking it in a separating funnel with strong sodium carbonate 
solution which is added in small quantities until the aqueous por- 
tion shows an alkaline reaction. The aqueous portion is withdrawn 
and the ester shaken with saturated salt or strong calcium chloride 
solution to remove alcohol ; the ester layer is separated and dried by 
contact with solid calcium chloride. It is then distilled from a dry 
flask, the portion passing over when the thermometer reads 74-78 
being collected. 



ESTERS 73 

Ethyl Benzoate. 

20 gm. of benzoic acid are dissolved in 75 c.c. of absolute 
alcohol and I c.c. of concentrated sulphuric acid is added. The flask 
containing the mixture is connected to a reflux condenser and gently 
heated for 1-2 hours over a gauze, a piece of porcelain being added 
tp prevent bumping. The esterification is complete when, on testing 
by pouring a few drops into water, only oil drops and no crystalline 
benzoic acid is seen. The whole is then poured into about 400 c.c. 
of water and the oil is allowed to settle. The water is decanted 
off and the remainder is shaken up with ether in a separating 
funnel. The aqueous layer is withdrawn, the ethereal layer shaken 
with sodium carbonate solution and then with water. It is dried with 
calcium chloride and the ether distilled off over a water-bath. The 
ester is distilled over a flame and the fraction boiling from 210-215 
is collected. Ethyl benzoate boils at 2 1 3. 

Properties. 

Esters are usually liquids having a sweet and fragnant odour ; a few 
are solid. 

Neutral esters of monobasic, dibasic, etc., acids are insoluble in 
water, or only slightly soluble, e.g. ethyl formate and acetate. 

Acid esters of dibasic, etc., acids are soluble in water, e.g. ethyl 
sulphuric acid, ethyl oxalic acid, etc. 

O Neutral esters are soluble in alcohol and ether, acid esters may be 
soluble in alcohol, but are insoluble in ether. 

Esters are comparatively inert substances and are unaffected by cold 
dilute sodium carbonate, sodium hydroxide, hydrochloric acid, sul- 
phuric acid, but there are exceptions, e.g. methyl oxalate, which is decom- 
posed by cold dilute caustic soda. They are acted upon by sodium more 
or less readily (cf. ethers). All esters are hydrolysed by boiling with 
water, acids, or alkalies. The last method of hydrolysis is known 
as saponification. They are thus converted into their constituents, 
namely acid and alcohol. The recognition of these identifies the ester. 
Esters which are of frequent occurrence in animals and plants are 
identified in this way. The hydrolysis is effected by boiling under 
a reflux condenser with aqueous sodium hydroxide, or 80 per cent, 
sulphuric acid. If only the acid is to be identified hydrolysis is 
effected by boiling with alcoholic sodium hydroxide. The alcohol 
is isolated by distilling the alkaline liquid if the alcohol be volatile, by 
extracting the alkaline solution with ether if not volatile, and it is identi- 
fied by the reactions for alcohols. If the alcoholic portion of the ester be 
a phenol or an aromatic alcohol it does not distil and is not extracted 



74 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

from an alkaline solution. The solution must be firstly acidified to 
liberate the phenol (see under phenols). The acid, which is formed 
by hydrolysis with alkali, is liberated by acidifying the cold solution 
with mineral acid sulphuric acid. If insoluble it is filtered off; if 
soluble and volatile with steam, distilled ; if soluble and not volatile, 
it is extracted with ether or precipitated as insoluble calcium or other 
salt. 

HYDROLYSIS OF ESTERS. 
Ethyl Sulphuric Acid. 

Ethyl sulphuric acid is not readily hydrolysed by alkali, but it is 
decomposed by boiling with acid. 

The solution of calcium or barium ethyl sulphate obtained above 
contains one or other of these bases as shown by adding dilute sulphuric 
acid, -the insoluble sulphate being precipitated. 

If a portion of the solution be heated with dilute hydrochloric acid 
for 3-5 minutes, the insoluble sulphate is again precipitated : 
C 2 H 5 o . SO 3 . o 

/Ba + 2H O = 2C.,H,OH + BaSO 4 + H 9 SO 4 . 
C 2 H.O . SO,, . (X 

Ethyl Acetate. 

About 10 c.c. of ethyl acetate are placed in a flask with about 80 c.c. 
of sodium hydroxide, and the mixture is boiled over a gauze under 
a reflux condenser for 20-30 minutes until no more oily drops are 
visible and until the smell of ethyl acetate has disappeared. A piece 
of unglazed porcelain is added with advantage to prevent bumping of 
the liquid during the heating : 

CH 3 COOC 2 H 5 + NaOH = CH 3 COONa + HOC,H a . 

The flask is connected with a condenser and about a quarter of 
the liquid is distilled over. 

This liquid contains the alcohol. It may be identified by the 
tests for ethyl alcohol. 

The alcohol is separated and identified by saturating the solution 
with solid potassium carbonate, collecting the alcohol in a pipette, 
determining its boiling-point and performing other reactions for the 
alcohol. 

The liquid remaining in the flask is acidified with dilute sulphuric 
acid and again distilled as long as the distillate reacts acid to litmus. 

The distillate is neutralised and evaporated down and the acetic 
acid prepared and identified (p. 97). 



ESTERS 75 

Ethyl Benzoate. 

The hydrolysis is effected as described above for ethyl acetate. 

The insoluble acid is more readily prepared by hydrolysing with 
alcoholic soda and then identified : 

5-10 gm. of the ester are placed in a flask and boiled under 
a reflux condenser with excess of caustic soda (1-2 gm.), dissolved 
in 10 c.c. water and 100 c.c. of alcohol, for 10-15 minutes. The 
saponification is continued until a few drops poured into water show 
no oily drops of unchanged ester. 

If any insoluble sodium benzoate separates out, it is dissolved by 
adding a little water through the condenser. 

The solution is poured into an evaporating basin, water added and 
the alcohol evaporated off on the water-bath. On cooling and after 
adding about 25-50 c.c. of water the solution is acidified with dilute 
mineral acid. Benzoic acid is precipitated. It is washed with water, 
recrystallised from hot water, and identified (p. 256). 

Ethyl Oxalate. 

10-20 gm. of ethyl oxalate are hyclrolysed with caustic soda 
solution containing sufficient alkali (6-12 gm.) as described under 
ethyl acetate and the alcohol is distilled off. 

The acid contained in the solution on acidifying with mineral acid 
is not precipitated nor is it volatile with steam. The acid solution 
may be extracted several times with ether, the ethereal solution dis- 
tilled to remove the ether, and the acid which is left identified. 

Oxalic acid is more easily separated as its calcium salt. The acid 
liquid is carefully neutralised with soda and calcium oxalate is precipi- 
tated by adding calcium chloride. The acid is obtained as described 
under oxalic acid (p. 108). 



76 PRACTICAL ORGANIC AND BIO-CHEMISTRY. 

ETHERS. 

Preparation. 

Ethers are prepared either by distilling alcohols with concentrated 
sulphuric acid or by the action of sodium ethoxide upon an alkyl 
halide : 

f C 2 H 5 OH + H 2 S0 4 = C 2 H S HSO 4 + H 2 O 
\C H 5 OH + C 2 H 5 HS0 4 = C 2 H 5 OC 2 H 5 + H 2 SO 4 
CH 3 ONa + CH 3 I = CH 3 OCH 3 + Nal. 

If in the first preparation a different alcohol be used in the second 
reaction, and if in the second preparation the alkoxide and halide con- 
tain different radicles, mixed ethers are formed, e.g. 

CH 3 . . C 2 H,. 

ETHYL ETHER. 

Preparation. 

Ethyl ether is generally prepared by distilling ethyl alcohol with 
sulphuric acid hence its name of sulphuric ether. According to the 
equation the sulphuric acid is combined and again liberated so that it 
should be possible to convert an unlimited quantity of alcohol into 
ether, but bye-products are formed which interfere with the reaction. 
The process is known as the continuous process. 

A distilling flask of about 500 c.c. capacity is fitted with a tap funnel and 
a thermometer, the bulb of which reaches nearly to the bottom of the flask. 
The neck of the flask is connected to a long condenser and the receiver is 
cooled by standing in ice water. A mixture of no c.c. of absolute alcohol 
and 80 c.c. of concentrated sulphuric acid is placed in the flask and heated to 
140-145. At this temperature ether is formed and absolute alcohol is 
dropped in from the tap funnel at the same rate as the liquid distils. The 
preparation is continued until about twice the volume of alcohol originally 
mixed with the sulphuric acid has been added. The distillate consists of 
ether, alcohol, water and sulphurous acid. It is put into a separating funnel 
and shaken with dilute caustic soda. The alkaline layer is withdrawn and 
the upper layer of ether shaken with saturated salt solution, which is also 
withdrawn. The ether is put into a distilling flask, which is loosely corked, and 
dried by being allowed to stand in-contact with calcium chloride for 12- 
24 hours. The flask is connected with a condenser and the ether distilled 
off from a water-bath (b.p.-35). 

Purification of Ethyl Ether. 

The ether obtained above contains traces of alcohol and water. These 
can only be removed by treatment with metallic sodium. The ether is placed 
in a flask, provided with a calcium chloride tube to prevent access of moisture 
and to allow the escape of hydrogen, and several slices of sodium are added. 
When no further effervescence is observed the ether is decanted into a dis- 
tilling flask and distilled from a water-bath. Pure ether of constant boiling- 
point 35 is collected. 



ETHERS 77 

Purification of Commercial Methylated Ether. 

This ether is made by the continuous process from methylated spirit and 
contains water, alcohol and other impurities. The ether may be washed with 
water to remove most of the alcohol. By distilling it over solid caustic 
potash, aldehydic impurities are destroyed. It is dried by standing over 
calcium chloride and then treated with metallic sodium. 

Sometimes, after treatment with sodium, ether is left in contact with 
phosphorus pentoxide and then distilled from the solid dehydrating agent. 

Distillation of Ether. Precautions. 

As ether is very inflammable and exceedingly volatile no flame 
should be in the neighbourhood. Ether should never be distilled 
over a free flame and the most convenient way, if steam or electric 
heaters are not available, is to heat a water-bath, extinguish the flame, 
and immerse the distilling flask containing the ether in the hot water. 

Large quantities of ether should not be distilled from a large flask, 
but a small flask provided with a tap funnel should be employed. As 
the ether distils a fresh quantity can be added without interrupting 
the distillation. The ether should be collected in small receivers and 
transferred to a larger reservoir. 

Properties. 

The first member of the series, dimethyl ether, is a gas. 

Ethyl ether, or simply ether, the chief representative of the group, 
is a very volatile, colourless liquid with a pleasant characteristic smell. 
It boils at 35 and has a sp. gr. of '7195 at 15. It is sparingly 
soluble in water, less soluble in glycerol. It mixes in all proportions 
with alcohol, chloroform, benzene, ligroin, and is largely used as a sol- 
vent for fats, resins, etc. The lower members of ethers of the aliphatic 
series are also volatile liquids, like ethyl ether, which boil at a lower 
temperature than the alcohol from which they are derived. The 
highest members are odourless solids. 

The ethers are inert compounds and are not acted upon by 
phosphorus pentachloride and sodium (distinction from alcohols) , or by 
aqueous or alcoholic potash (distinction from halogen compounds and 
esters). 

The lower members especially those containing methyl and ethyl 
radicles are decomposed by heating with hydriodic acid forming 
alkyl iodides (distinction from hydrocarbons). This reaction is used 
in estimating methoxy CH 3 O and ethoxy C 2 H 5 O groups in organic 
compounds (Zeisel's method). 



78 PRACTICAL ORGANIC AND BIO-CHEMISTRY 
MERCAPTANS AND SULPHIDES. 

The sulphur compounds corresponding to the alcohols, i.e. thio-alcohols, 
are known as mercaptans ; the sulphur compounds corresponding to the ethers, 
i.e. thio-ethers, are known as sulphides, or alkyl sulphides. Bisulphides are 
also known. 

CH 3 . SH C 2 H 5 . S . C 2 H 5 C a H 5 . S S . C 2 H 5 

Methyl mercaptan. Ethyl sulphide. Ethyl disulphide. 

Mercaptans. 

Methyl mercaptan is a product of the putrefaction of proteins. It occurs 
in the urine after a diet of asparagus and gives it the peculiar unpleasant 
odour. 

Preparation. 

Mercaptans are prepared : 

(1) By heating the alcohol with phosphorus pentasulphide : 

5 CH 3 OH + P 2 S 5 = 5 CH 3 SH + P 2 S . 

(2) By heating the alkyl halide or alkyl potassium sulphate with potas- 

sium hydrosulphide : 

CH 3 I + KSH = CH 3 SH + KI 
C 2 H 5 O . SO 2 . ONa + KSH = C 2 H 5 SH + NaKSO 4 . 

About 2-5 c.c. of a saturated solution of sodium ethyl sulphate are made 
alkaline with sodium hydroxide and an equal volume of sodium hydrosulphide 
(33 P er cent.) is added. On warming ethyl mercaptan is formed which is 
recognised by its garlic-like unpleasant odour. 

Properties. 

Methyl mercaptan is a gas, ethyl mercaptan is a colourless liquid boiling 
at 36. The other mercaptans are also liquids which are insoluble in water 
and have a disgusting smell. 

Like the alcohols they react with sodium with evolution of hydrogen : 

2CH 3 SH + Na.j = 2CH :! SNa + H 2 . 

The mercaptans react with mercuric oxide forming crystalline com- 
pounds : 

2O>H g SH + HgO = (C S H S . S) 2 Hg + H 2 O. 

These compounds are termed mercaptides, the name of the group being 
derived from the mercury compounds. On oxidation with nitric acid the 
mercaptans yield sulphonic acids : 

CH 3 SH + 3 = GH a . SO,H. 

The sulphonic acids are isomeric with alkyl hydrogen sulphites. The 
latter compounds are esters and are hydrolysed by alkali ; the sulphonic acids 
are stable. In the sulphonic acids the sulphur atom is joined to carbon, in 
the sulphites it is joined to oxygen : 



o O 

CH s o CH,.O.S< 

CH3 ~ S - \OH 

\OH 

Methyl sulphqnic acid, Methyl hydrogen sulphite, 



MERCAPTANS AND SULPHIDES 79 

Alkyl Sulphides. 

Ethyl sulphide, C 2 H 5 . S . C 2 H 5 , is another product of the putrefaction of 
proteins, being derived from cystine (p. 143). 

Preparation. 

Sulphides are obtained : 

(1) By the action of phosphorus pentasulphide upon ethers : 

5 (C 2 H 5 ) 2 + P 2 S 5 = 5 (C 2 H 5 ) 2 S + P 2 5 . 

(2) By the action of potassium sulphide on an alkyl halide or alkyl 

potassium sulphate : 

2C H B I + K 2 S = 2KI + (C H 5 ) S 
2 C 2 H 5 KS0 4 + K 2 S = 2K 2 S0 4 + (CaHj^S. 

Properties. 

The sulphides are colourless, neutral liquids with very unpleasant smell ; 
ethyl sulphide boils at 91. 

They resemble the ethers in being comparatively stable compounds. On 
oxidation with nitric acid, they are converted into sulphones which are stable 
crystalline compounds : 

(C 2 H B ),S + 2 = (C 2 H 5 ) 2 S0 2 . 

Alkyl Disulphides. 

Bisulphides are formed when mercaptans are exposed to the air : 

2C 2 H 5 SH + O = H 2 O + C 2 H 5 . S S . C 2 H 5 , 
or by the action of iodine upon sodium mercaptides : 

2 C 2 H 5 S . Na + I 3 = 2NaI + C 2 H 5 . S S . C,H 5 . 



ALDEHYDES. 

Aldehydes are the first products of oxidation of primary alcohols, 
e.g. : 

CH 3 CH 3 CH 3 

I -* I / OH - | 

CH 2 OH CH< CHO 

\OH J 

Ethyl alcohol. Hypothetical. Acetaldehyde. 

The hypothetical intermediate compound does not exist; it at 
once loses a molecule of water and is converted into aldehyde. Two 
OH groups cannot exist attached to one carbon atom ; aldehyde is 
formed by loss of water. There are a few exceptions, such as chloral 
hydrate, 

CC1 3 

I / OH 
CH< 

\OH. 

Formaldehyde is formed from methyl alcohol, propyl aldehyde 
from primary propyl alcohol, etc. 

/H 

The group CHO or C/ is characteristic of aldehydes. 

^O 

Preparation. 

When the alcohol is available the aldehyde is usually prepared by 
oxidation ; otherwise it may be prepared by the dry distillation of 
molecular proportions of calcium formate and the calcium salt of the 
corresponding acid (compare ketones). 

FORMALDEHYDE. 

Preparation. 

Formaldehyde is prepared by passing the vapour of methyl alcohol 
mixed with air over heated platinum or copper, or other substances. 
The formaldehyde formed by oxidation is passed into water. 

Formol, or formalin, is a commercial aqueous solution containing 
40 per cent of formaldehyde. 

ACETALDEHYDE. 

Preparation. 

2$ gm. of coarsely powdered potassium bichromate and 100 c.c. of 
water are placed in a distilling flask of 250 c.c. capacity. The flask is 
connected with a condenser and a strong current of cold water is made 
to flow through it. Through a tap funnel, secured in the neck of the 

80 



ALDEHYDES 81 

flask by a well-fitting cork, a mixture of 25 gm. (30 c.c. of absolute 
alcohol and 35 gm. (20 c.c.) of concentrated sulphuric acid is slowly 
added to the contents of the flask which have been gently warmed and 
the flame removed. During .the addition the contents of the flask, 
which darken in colour, are occasionally shaken. A mixture of alde- 
hyde, alcohol and water distils over. When the mixture has been 
added the flask is heated until all the aldehyde (recognised by smell) 
has distilled over. 

The tests for acetaldehyde can be carried out with this distillate 
(P- 84). 

Purification. 

The solution is redistilled through an inverted condenser filled with water 
at 30-35. Water and alcohol are condensed, but the aldehyde passes on. 
The aldehyde vapour is passed through a 100 c.c. pipette into about 30 c.c. 
of pure dry ether contained in a bottle standing in ice. 

Pure ammonia, prepared by gently heating concentrated ammonia solution 
with a small flame and dried by passage through a tower containing quick* 
lime, is passed into the ether until it is saturated. Aldehyde ammonia 
crystallises out. After standing for one hour the ether is decanted off, the 
crystals are drained on a Buchner filter and washed with ether. 

The crystals of aldehyde ammonia are dissolved in an equal weight of 
water, and the solution is distilled with a mixture of 1*5 parts of sulphuric 
acid and 2 parts of water from a water-bath, which is gradually raised to 
boiling. The receiver is cooled in ice. The distillate is dried with calcium 
chloride from which the aldehyde is distilled in a bath at 20 and collected 
in a receiver in ice. The aldehyde must be preserved in a well-stoppered 
bottle. 

Properties of Aldehydes. 

Formaldehyde is a gas at the ordinary temperature easily soluble 
in water and alcohol and with a peculiar pungent smell. 

Acetaldehyde is a colourless liquid having a fruity pungent smell 
and boils at 21. It is easily soluble in water, alcohol and ether. 

The next members of the series of aldehydes are also liquids and 
resemble acetaldehyde very closely in their properties. 

Polymerisation. 

Paraformaldehyde or Paraform. 

On evaporating formalin (about I c.c.) in a watch-glass on a 
water-bath a solid mass of paraformaldehyde is left. 

If a portion of the solid be heated in a test tube dissociation occurs 
at about 100, the mass melts between 153 and 172, a white sublimate 
is formed and formaldehyde is evolved. 



82 PRACTICAL ORGANIC AND BIO-CHKMISTRY 

Paracetaldehyde or Paraldehyde. 

On adding a drop of concentrated sulphuric acid to about I c.c. of 
acetaldehyde, a violent reaction occurs and the liquid becomes hot. Par- 
aldehyde (CH 3 . CHO) 3 separates out as an oil on diluting with water. 
Acetaldehyde is re-formed if the acid aqueous liquid be heated. 

Paraldehyde is a colourless liquid which boils at 124. 

Metaldehyde. 

The polymer, metaldehyde, is formed from acetaldehyde when it is 
treated with hydrochloric acid gas or dilute sulphuric acid at a low 
temperature. 

Aldol Condensation. 

Solutions of formaldehyde and acetaldehyde when kept with dilute 

solutions of lime or potassium carbonate undergo aldol condensation. 

"*** . "**^"^'' i * ^ 

Formaldehyde gives a sweet syrup which contains monosaccharides, 

especially dl-fructose : 

HCHO + HCHO = HCHOH . CHO 
HCHO + HCHO + HCHO = HCHOH . CHOH . CHO, 

etc. 
Acetaldehyde gives aldol : 

CH 3 . CHO + CH 3 . CHO = CH 3 . CHOH . CH 2 . CHO. 

These reactions of aldehyde probably take place in nature. In 
plants, under the action of light and chlorophyll, carbon dioxide is 
reduced to formaldehyde which undergoes aldol condensation into 
sugars. The higher fatty acids are probably formed from acetaldehyde 
in this way in both animals and plants. 
Action of Ammonia. 
Hexamethylene Tetramine. 

Formaldehyde behaves differently to the other aldehydes. 
On adding ammonia gradually to formalin (i c.c. in 5 c.c. water) it 
is absorbed. On now adding excess of ammonia and evaporating on 
the water-bath hexamethylene tetramine, or urotropin, remains as a 
white solid : 

6CH 2 O + 4NH 3 .= (CH 2 ) 6 N 4 + 6H 2 O. 

Hexamethylene tetramine consists of colourless crystals soluble in 
about I '5 parts of hot or cold water and 10 parts of alcohol. It is 
volatilised on heating and it is converted into ammonium sulphate 
and formaldehyde on treatment with strong sulphuric acid. 
Aldehyde Ammonia. 

On passing dry ammonia gas into a dry ethereal solution of 
acetaldehyde, acetaldehyde ammonia is formed : 



/OH 
CH, . CHO + NH, = CH S . CH/ 



I ' LJ I ' I_I / 

X NH 6 



Acetaldehyde ammonia is a white crystalline compound easily 



ALDEHYDES 83 

soluble in water and alcohol, and easily decomposed by acids and 
alkalies. 

On dissolving a little aldehyde ammonia and heating with dilute 
sulphuric acid, aldehyde is given off. Ammonia is also evolved on 
heating with dilute caustic soda. 

Aldehyde Sodium Bisulphite. 

On adding 1-2 c.c. of .a cold saturated solution of sodium bisulphite 
to 5-10 drops of aldehyde and shaking vigorously, aldehyde sodium 
bisulphite crystallises out : 

CH-, . CHO + NaHSO., = CH 3 . CH\ 

X SO 3 NA. 

Aldehyde Cyanhydrin. 

Hydrogen cyanide combines with aldehydes forming cyano- 
hydrins : 

CH 3 . CHO + HCN = CH 3 . CH\ 

N CN. 

In this way another carbon atom can be added to organic com- 
pounds. Compounds containing the CN group are hydrolysed by 
acids or alkalies and converted into the corresponding acid (see cyano- 
gen compounds) : 

/OH OH 

i r _ r<u fW' + NH S . 



CH n .CH\ + 2 H.,O = CH,.CH 



\COOH 



Aldehyde Hydrazone. 

Aldehydes combine with hydrazine and substituted hydrazines, 
especially phenylhydrazine, forming hydrazones. 

The calculated quantities of aldehyde ('5 c.c.), phenylhydrazine 
hydrochloride (-2 gm.) and cryst. sodium acetate (-5 gm.) are dissolved 
in about 10 c.c. of water and warmed; an oil (acetaldehyde phenyl- 
hydrazone) is formed : 

CHj.CHO + H 2 N.NH.C fi H 5 = CH n .CH :JM. NH.C 6 H 5 + H 2 O. 

Aldoxime. 

Aldehydes combine with hydroxylamine forming oximes : 

CH 3 . CHO + H 2 NOH = CH 3 . CH : NOH + H 2 O 

(acet)aldoxime. 

The calculated quantity of hydroxylamine hydrochloride is dissolved in 
water, the equivalent quantity of caustic soda required to liberate the 
hydroxylamine is added and then the calculated quantity of the aldehyde. 
The mixture is shaken and allowed to stand until it no longer reduces 
Fehling's solution. The oxime is extracted with ether, most of the ether dis- 
tilled off, and the concentrated solution poured into a basin. The crystals 
which separate are drained on a porous plate and recrystallised from ligroin. 

6* 



84 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Tests. 

Aldehydes are easily further oxidised into the corresponding fatty 
acids containing the same number of carbon atoms and they conse- 
quently behave as reducing agents. 

Reduction of Metallic Oxides in Alkaline Solution. 

(a) Silver. 

An ammoniacal solution of silver hydroxide is prepared by adding 
dilute ammonia' to silver nitrate ,until the precipitate first formed 
just re-dissolves. Some dilute aldehyde solution is added and the 
mixture is placed in a cold water-bath and heated to the boiling- 
point. A mirror of metallic silver forms on the glass. 

A very sensitive reagent may be prepared by mixing equal volumes of 
10 per cent, silver nitrate and sodium hydroxide and then adding ammonia 
drop by drop till the silver hydroxide dissolves. 

A mirror is formed immediately if the solution contains i per cent, of 
acetaldehyde, in 30 seconds if i per thousand ; a yellow-brown mirror forms 
in 5 minutes if i per 10,000 be present. 

(b] Copper. 

Dilute aldehyde solution reduces Fehling's solution x on warming 
with the formation of cuprous oxide. 

Action of Sodium Hydroxide. 

Except with formaldehyde, benzaldehyde and a few other alde- 
hydes, (taustic soda solution decomposes dilute aldehyde solutions on 
warming*^ Yellow to brownish-red resins which rise to the surface 
aldehyde resin are formed. The liquid has usually a peculiar smell. 
Aldehyde resin is insoluble ir| water but soluble ip ajcnhol and erjier. 

Formaldehyde is converted into methyl alcohol and formic acid. 

Oxidation. 

Aldehydes are converted into the corresponding acid on warm- 
ing their solutions with potassium bichromate and dilute sulphuric 
acid, the solution becoming green. 

The aldehyde may be identified by preparing the acid by oxidation. 

SchifPs Test. 

A solution of magenta, or fuchsin, is decolorised by bubbling 
sulphur dioxide through it. On adding the dilute aldehyde solution 
the purple-red colour returns. 

Numerous other sensitive tests have been described for aldehydes, 
especially formaldehyde. The following one has been used more particularly 
in testing for formaldehyde in distillates from plant leaves, etc. 

1 Fehling's solution consists of copper sulphate, caustic soda and Rochelle salt (sodium 
potassium tartrate). On adding caustic soda to copper sulphate a blue precipitate of 
cupric hydrate Cu(OH) 2 is formed, which turns black on boiling. The presence of the 
Rochelle salt keeps the Cu(OH) 2 in solution forming a deep blue solution. This solution 
does not keep, so that it must be freshly made for each experiment. For this purpose two 
solutions are therefore kept. The one contains the copper sulphate, the other the Rochelle 
salt and caustic soda. When required for use, equal parts of each are mixed together, and 
this forms the reagent. 



ALDEHYDES 85 

Rimini's Test. 

A small quantity (2 drops) of phenylhydrazine is added to the solution, 
then a drop of dilute freshly prepared sodium nitroprusside solution and a 
few drops of sodium hydroxide solution. A deep blue colour forms if form- 
aldehyde be present ; the colour changes through green and brown to red. 

Schryver has modified this test and made it more sensitive : 2 c.c. of 
a freshly prepared and filtered i per cent, solution of phenylhydrazine 
hydrochloride are added to 10 c.c. of the solution of formaldehyde, then i c.c. 
of a 5 per cent, solution of sodium ferricyanide and 5 c.c. Cjf hydrochloric acid ; 
a magenta colour is formed. This test will show the presence of i part of 
formaldehyde in 100,000 to 1,000,000 parts of solution. No colour is given 
by acetaldehyde. 

ESTIMATION. 
Formaldehyde. 

(#) By Converting into Hexamethylenetetramine. 

25 c.c. of normal ammonium hydroxide solution are placed in a 100 c.c. 
strong bottle provided with a rubber stopper. A measured volume of the solution 
(not containing above '5 gm. of formaldehyde) is added. The cork is securely 
fastened by tying and the bottle is submerged in a cold water-bath which 
is then heated to boiling for i hour, the bottle being kept under water the 
whole time. The bottle is cooled, opened and the contents titrated with 
standard acid until the methyl orange which is used as indicator first becomes 
red. 

A series of bottles should be taken containing different amounts, or none, 
of the aldehyde solution. Allowing for the blank each c.c. of normal am- 
monium hydroxide used corresponds to "0601 gm. of formaldehyde. 

The estimation should be carried out in water. The formaldehyde is 
therefore distilled from its original' solution, e.g. milk, plant extracts, and the 
distillate is used. 

(b] By Titrating with Iodine and Sodium Thiosulphate. 

A known volume of the solution (10 c.c.) is mixed with 25 c.c. of 'iN iodine 
solution, and sodium hydroxide is added drop by drop till the liquid becomes 
clear yellow. The flask is closed for 10 minutes, dilute hydrochloric acid is 
added, and the free iodine is titrated with -iN thiosulphate. 

2 atoms of iodine = i molecule of formaldehyde. 

Good results are not given by this method for aldehydes other than form- 
aldehyde. 

Acetaldehyde. 

By Combination with Sulphite. 

The solution of sulphite is prepared by dissolving ,12 '6 gm. of sodium 
sulphite in 400 c.c. of water, adding 100 c.c. of - iN sulphuric acid and diluting 
to 1000 c.c. with the purest ethyl alcohol of 95 per cent. 

The volume of aldehyde solution, not containing more than 2 per cent. 
of aldehyde, is placed in a 100 c.c. measuring flask. A known volume of 
the sulphite solution is added and the mixture dihited to too c.c. with the 
purest 50 per cent, alcohol. A blank with the reagents is carried out simul- 
taneously. 

The flasks are kept at 50 for 4 hours, cooled and titrated with standard 
iodine solution, using starch as indicator. 

Each c.c. of 'iN iodine solution corresponds to -0032 gm. of SO 2 or -0022 
gm. of acetaldehyde. 



86 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

CC1 3 
CHLORAL. | 

CHO. 

Preparation. 

Chloral is prepared by the prolonged action (about 10 days) of dry 
chlorine upon absolute alcohol. The gas is passed into the cold alcohol 
until it is saturated and acquires a sp. gr. of I -400 and the temperature 
is gradually raised to 100. Chloral alcoholate is formed. An equal 
weight of concentrated sulphuric acid is added and the mixture is 
distilled. The fraction passing over between 94 and ioo 9 is collected, 
neutralised with calcium carbonate and again distilled. 

Properties. 

Chloral is a colourless oily liquid with a peculiar penetrating smell, 
having a sp. gr. of i'5O2 at 18. It boils at 97 and is soluble in 
ether and chloroform. 

Metachloral. 

On keeping, or on leaving in contact with moderately concentrated 
sulphuric acid, chloral polymerises to metachloral, a solid which is 
sparingly soluble in boiling water, but insoluble in cold water, alcohol 
and ether. The polymerisation does not occur with pure chloral and 
may be hindered by adding chloroform. On heating to 180 meta- 
chloral is decomposed and chloral distils over. 

Chloral Alcoholate. 

If chloral be mixed with an equivalent quantity of absolute 
alcohol, chloral alcoholate is formed. 

It consists of white crystals which melt at 46 and boil at 1 1 3*5 and 
are readily soluble in chloroform (distinction from chloral hydrate). 

/OH 
CHLORAL HYDRATE. CC1 3 . CH< 

X OH. 

Preparation. 

Equivalent parts of chloral (6. c.c.) and water (i c.c.) are mixed 
together. The mixture becomes hot and solidifies to a mass of crystals 
of chloral hydrate. 

Properties. 

Chloral hydrate is a white crystalline solid, which melts at 50-51. 
It is soluble in I -5 times its weight of water, also in alcohol, ether, 
petroleum ether and carbon disulphide. It is soluble with difficulty 
in cold chloroform. 

Pure chloral hydrate is completely volatile on heating and com- 
mences to boil rapidly at 97-98. 



ALDEHYDES 87 

Reconversion into Chloral. 

About 2 gm. of chloral hydrate are placed in a dry test tube and 
covered with concentrated sulphuric acid and the mixture is warmed 
gently. Chloral is formed and floats to the surface. 

An aqueous solution heated with zinc to 50 and gradually treated with 
dilute acid yields aldehyde and paraldehyde which may be distilled off. 

Tests for Chloral and Chloral Hydrate. 

Aqueous solutions in the cold give no reaction with silver nitrate. 
On adding a few drops of ammonia and boiling, metallic silver is 
deposited. 

Aqueous solutions reduce Fehling's solution on heating. Traces 
of chloral may be detected by the carbylamine reaction for chloro- 
form (p. 6l). 

Decomposition of Chloral by Alkali. 

Chloral or chloral hydrate is .rapidly decomposed by caustic alkali 
with the formation of chloroform and alkali formate : 
CC1 3 CH(OH) 2 + NaOH = CHC1 3 + HCOONa + H 2 O. 

The odour of chloroform is noticed at once on gently warming an 
aqueous solution of chloral with caustic soda. 

Estimation. 

1. By measuring the volume of chloroform. 

25 gm. of chloral hydrate or chloral are placed in a graduated cylinder 
and excess of sodium hydroxide solution (80-100 c.c.) are carefully added. 
The tube is kept well cooled at first on account of the violence of the reaction. 
Afterwards the cylinder is closed and shaken. On standing the liquid be- 
comes clear and separates into two layers. When cold (at 17) the volume of 
the lower layer of chloroform is measured. The volume in c.c. multiplied by 
1-84 gives the number of grams of chloral, or by 2-064 of chloral hydrate, in the 
sample. 

2. By titrating the acid. 

1-2 gm. are dissolved in water and shaken with barium carbonate to 
remove any acid. The carbonate is filtered off and washed. Excess of 
normal caustic soda (100-150 c.c.) is added and the solution titrated with 
normal acid, using litmus as indicator. 

Each c.c. of alkali neutralised = '1475 m - chloral or '1655 gm. chloral 
hydrate. 

Butyric Chloral Hydrate. 

This compound is formed when chlorine is passed into paraldehyde or 
acetaldehyde. It is a white crystalline substance with peculiar fruity flavour 
and melts at 78. 



PRACTICAL ORGANIC AND BIO-CHEMISTRY 



KETONES. 



Ketones are the first products of the oxidation of secondary 

alcohols, e.g. : 

CHj> CHo CHo 

I I / OH I 

CHOH ->C\ -> CO 

X H 



Isopropyl alcohol. Hypothetical. Acetone. 

The same statements apply here as in the case of the formation of 
aldehydes. 

The group >CO is characteristic of ketones. 

Acetone is the first member of the homologous series of ketones 
and the chief representative. 

ACETONE. 
Preparation. 

Acetone is formed in the dry distillation of wood and is separated 
from methyl alcohol by fractional distillation (p. 63). 

Acetone is also prepared by the dry distillation of calcium or 
barium acetate : 

CH 3 . COO, CH 3 v 

>Ca = )CO + CaCO 3 . 

CH 3 . COO/ CH 3 / 

50-100 gm. of dry calcium acetate are placed in a retort or dis- 
tilling flask and at first heated gently, afterwards more strongly, and 
the vapours are passed through a condenser. A brownish liquid col- 
lects in the receiver. It contains acetone, aldehyde and higher ketones. 
The acetone is separated by fractional distillation. 



KETONES 89 

Purification. 

The proper quantity of crude acetone (100 gm. or 125 c.c.) is added to 
the calculated quantity of sodium bisulphite (70 gm.) in saturated solution 
(this should smell of sulphur dioxide, if not, SO 2 is passed into it until it 
smells strongly of the gas), and the mixture is shaken vigorously in a closed 
vessel. Heat is evolved and a mass of crystals, C 3 H 6 O . NaHSO 3 , separates out. 
After standing, the crystals are filtered off on a Buchner funnel and well drained. 
They are placed in a distilling flask and decomposed by adding a solution of 
sodium carbonate (40 gm.). The solution is distilled, preferably using a frac- 
tionating column, until the thermometer reaches 60. 

The distillate is dried with calcium chloride and the acetone distilled off. 

Properties. 

Ketones closely resemble aldehydes in most of their properties, but 
there are several differences. 

Acetone is a colourless, pleasant smelling liquid which boils at 
56 and has a sp. gr. of 797 at 15. It is very volatile and in- 
flammable. It mixes with water, alcohol and ether in all proportions. 
Like alcohol it can be separated from water by saturating the solution 
with potassium carbonate. 

Polymerisation and Condensation. 

Acetone does not polymerise like aldehyde, but when distilled with 
moderately concentrated sulphuric acid it is converted into mesitylene 
(sym. trimethylbenzene). 

Action of Ammonia. 

Acetone does not form simple condensation products with ammonia like 
aldehyde does, but it reacts forming diacetonamine, C 6 H 13 ON, and triaceton- 
amine, C 9 H 17 ON. 

Acetone Sodium Bisulphite. 

On shaking together about I c.c. of acetone and 5 c.c. of a cold 
saturated solution of sodium bisulphite, acetone sodium bisulphite 
crystallises out : 

CH 3 , CH 3 \ /OH 

\CO + NaHS0 3 = /^\ 

CH/ CH 3 / \SO 3 Na. 

Acetone Cyanhydrin. 

Acetone combines with hydrogen cyanide forming the addition 
compound, acetone cyanhydrin : 

CH S , CH SX /on 

\CO + HCN = \C/ 

CH,/ CH / NCN. 



90 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Acetone Phenylhydrazone. 

Acetone combines with hydrazine and substituted hydrazines 
forming hydrazones : 

Acetone phenylhydrazone is formed as an oil when acetone is 
mixed with phenylhydrazine hydrochloride and sodium acetate : 

r^T-T x f^TJ 

UW 3\ ^*a\ 

)CO + H,N . NHC 6 H 5 = )C : N . NHCH, + H O. 

CH 3 / CH 3 / 



Acetoxime. 

Combination occurs between acetone and hydroxylamine when the 
calculated quantities are allowed to react together as described under 
aldehyde (p. 83) : 

CH 3 \ CH 3 \ 

>CO + H 2 NOH = >C : NOH + H 2 O. 

CH 3 / CH 3 / 

I 

J * Tests for Acetone. 

Acetone is more stable than aldehyde and does not behave as a 
reducing agent. 

* * Acetone reduces ^mmoniacal silver nitrate solution on prolonged 
boiling. 

4 * Acetone does not reduce Fehling's solution. 

\ * Acetone does not give a resin when heated with sodium hy- 
droxide. 

* Acetone does not give Schiff's test. 

These four reactions are characteristic only for aldehydes. 
Oxidatiog. 

* Acetone is oxidised on heating with potassium bichromate and 
sulphuric acid and yields acetic and formic acids : 

CH 3 \ 

)CO + 30 = CH 3 COOH + HCOOH. 
CH 3 / 

The constitution of a ketone is determined by identifying the acids 
it yields on oxidation. 

From 2-5 gm. of the ketone are mixed in a flask attached to a reflux 
condenser with 30-50 c.c. water and the calculated quantity of sulphutic acid 
is added. The calculated quantity of finely powdered potassium bichromate is 
added in portions of -5-1 gm. If the oxidation is very energetic, the contents 
should be cooled and kept at 50-60. The flask is finally heated on the water- 
bath for 15 minutes. The acids are then distilled and collected in the receiver 
(see under acids). 



KETONES 91 

lodoform Reaction (Lieben). 

Acetone gives iodoform in the cold ; 3-5 drops of sodium hydrox- 
ide are added to about 2 c.c. of the solution and then, drop by drop 
iodine solution until the liquid is faintly yellow. lodoform separates 
at once. 

If ammonia be used in place of sodium hydroxide and iodine 
solution be added drop by drop, a small black precipitate of nitrogen 
iodide is formed. On standing, or on warming, this disappears and 
iodoform is produced ; this reaction may serve to distinguish acetone 
and alcohol. 

Sodium Nitroprusside Test (Legal}. 

On adding about 5 drops of freshly prepared sodium nitroprusside 
solution to about 5 c.c. of the dilute acetone and about I c.c. of sodium 
hydroxide, a ruby-red colour is produced. This fades to yellow on 
standing. 

If the reaction be repeated and the solution acidified at once with 
acetic acid, a purple-red colour is produced. 

Rothera suggests that the reaction be carried out by adding 3 drops 
of 5 per cent, sodium nitroprusside and 1-2 c.c. of ammonia to the 
dilute acetone and a small quantity of solid ammonium sulphate. A 
permanganate colour slowly develops, reaches a maximum in about 
30 minutes and then fades away. 

Creatinine does not react under these conditions ; a brownish-red 
colour is given by aldehydes. 

Salicylic Aldehyde Test. 

i gm. of solid potassium hydroxide is added to 10 c.c. of the acetone 
solution, and before it dissolves 10 drops of salicylic aldehyde are added. 
On warming to 70 a purple-red contact ring appears. If the potash has dis- 
solved before adding the salicylic aldehyde the liquid becomes yellow, red, 
and finally purple-red. 

Note. The iodoform, nitroprusside and salicylic aldehyde reactions are 
carried out preferably incolourless solutions. The acetone should be separated 
by distillation and the distillate tested. 



92 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Estimation of Acetone. 

1. Acetone is most usually estimated by converting it into iodoform with 
excess of iodine and caustic soda and titrating the excess of iodine with thio- 
sulphate (Messinger's method). 

In the case of wood spirit -5 c.c. are added to 25 c.c. of N sodium 
hydroxide contained in a stoppered bottle of 200 c.c. capacity ; the mixture 
is well shaken and allowed to stand 5-10 minutes. 'aN iodine solution is 
slowly run in from a burette drop by drop, shaking thoroughly till the upper 
portion of the solution on standing for a minute becomes quite clear. A few 
more c.c. of the iodine solution are run in so as to have an excess of about 
25 per cent, and the solution is allowed to stand 10-15 minutes. 25 c.c. of 
N sulphuric acid are added and the iodine which is liberated is titrated with 
iN sodium thiosulphate solution, using starch as indicator. 

i c.c. *iN iodine solution = '00967 gm. acetone. 

A blank experiment should be made as sodium hydroxide may contain 
nitrite. 

Aldehydes and other compounds which react with iodine are included in 
this estimation, if present. 

2. Jolles has suggested the estimation of acetone by conversion into ace- 
tone sodium bisulphite with excess of sodium hydrogen sulphite and titration 
of the excess of sulphite with standard iodine solution. 

3-4 times the excess of the bisulphite solution of known strength is 
added to the acetone solution ; after standing for 30 hours the excess is 
titrated with 'iN iodine solution. 

i mol. of NaHSOjj = 2 atoms of I = i mol. of acetone. 

3. Deniges makes use of an insoluble compound of acetone with mercuric 
sulphate for the estimation of acetone. The reagent is prepared by dissolving 
5 gm. of mercuric oxide in 100 c.c of water to which 20 c.c. of sulphuric 
acid have been added. 

The acetone content of the solution must be not greater than -2 per cent, 
so that strong solutions must be diluted. 25 c.c. of the reagent are added to 
25 c.c. of the solution and the mixture heated on the water-bath for 10 
minutes. The precipitate is filtered off on a weighed filter, washed with not 
more than. 100 c.c. of cold water, dried at 100 and weighed. The amount 
of acetone in the precipitate, 3Hg 5 S 2 O u . 4.C 3 H 6 O, is obtained by multiplying 
by the factor '0609. 

This reaction can be carried out volumetrically by estimating the excess 
or mercury. The mercuric sulphate solution must therefore be of known 
strength. The filtrate and washings from the precipitate are collected and 
made up to 100 c.c. To 20 c.c. of this solution 15 c.c. of ammonia, 50 c.c. 
of water and 10 c.c. potassium cyanide solution (13 gm. per litre) are added. 
The excess of cyanide is estimated by titration with -iN silver nitrate solution, 
using potassium iodide as indicator, until there is a slight permanent precipitate. 

Since acetone is a decomposition product of aceto-acetic acid and 
the two compounds are usually associated in tissues and extracts 
of organs, the estimation of acetone in urine, etc., is combined with the 
estimation of aceto-acetic acid (p. 593). 



THE FATTY ACIDS. 

The fatty acids are the second products of oxidation of the 
primary alcohols, the aldehydes being the intermediate products. 
Secondary alcohols and ketones also give rise to fatty acids on 
oxidation, but the number of carbon atoms in the molecules of the 
fatty acids so formed is less than in the original secondary alcohol. 
Conversely, on reduction fatty acids give aldehydes and primary 
alcohols, thus : 

CH 3 . CH 2 OH ^ CH 3 . CHO ^ CH 3 . COOH. 

The fatty acids are characterised by the presence of the carboxyl 
or COOH group. 

They occur widely distributed in nature, both in the free state and 
in combination with glycerol as the fats. 

Only those acids containing an even number of carbon atoms 
occur in combination as fats, and as far as is known they all have 
a straight chain of carbon atoms. Acids with an uneven number of 
carbon atoms and with branched chains of carbon atoms are also found 
in nature. 

The lower members of the series of the fatty acids up to capric 
acid with 10 carbon atoms are volatile with steam and hence are 
termed the volatile fatty acids. They are separated in this way from 
the higher members which are not volatile with steam. They thus 
form two groups. 

In the following list are given the names of the homologous series of hydrocarbons, 
primary alcohols, aldehydes and fatty acids : 



mber of 


Saturated 


Primary 






on Atoms. 


Hydrocarbon. 


Alcohol 


Aldehyde 


Fatty Acid 




tl*. ' 


CH 2 OH. 


CHO. 


COOH. 


I 


Melhane 


Methyl 


Formaldehyde 


Formic 


2 


Ethane 


Ethyl 


Acetaldehyde 


Acetic 


3 


Propane 


Propyl 


Propionic aldehyde 


Propionic 


4 


Butane 


Butyl 


Butyric 


Butyric 


5 


Pentane 


Amyl 


Valeric 


Valerianic 


6 


Hexane 


Hexyl 


Caproic ,, 


Caproic 


7 


Heptane 


Heptyl 


Oenanthic , 


Oenanthic 


8 


Octane 


Octyl 


Caprylic , 


Caprylic 


9 


Nonane 


Nonyl 


Pelargonic , 


Pelargonic 


10 


Decane 


Decyl 


Capric , 


Capric 


ii 


Undecane 


Undecyl 


Undecylic , 


Undecylic 


12 


Dodecane 


Dodecyl 


Laurie ,, 


Laurie 


13 


Tridecane 





Tridecylic ,, 


Tridecylic 


14 


Tetradecane 


Tetradecyl 


Myristic ,, 


Myristic 


15 


Pentadecane 








Pentadecylic 


16 


Hexadecane 


Cetyl 


Palmitic 


Palmitic 


17 


Heptadecane 





Margaric ,, 


Margaric 


18 


Octadecane 


Octadecyl 


Stearic 


Stearic 


ig 


Nonadecane 











20 


Eicosane 








Arachic 


21 


Heneicosane 











22 


Docosane 








Behenic 


23 


Tricosane 











24 


Tetracosane 








Lignoceric 


25 














26 





Ceryl 





Cerotic 


27 


Heptacosane 











28 














2Q 














3 





Myricyl 





Melissic 



93 



94 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

FORMIC ACID. H . COOH. 

Preparation. 

Formic acid was first prepared by distilling crushed ants with 
water hence its name. The stings of some insects and plants also 
probably contain it. It occurs together with acetic and other 
lower fatty acids in urine. It can be obtained by oxidising methyl 
alcohol with potassium permanganate. It is formed in the decom- 
position of chloroform by alkali (p. 60), by the action of water upon 
hydrogen cyanide (p. I 54), and its alkaline salts are obtained by the 
reaction of carbon monoxide with alkalies : 

CO + KOH = HCOOK. 

It is manufactured by heating glycerol with oxalic acid. It has 
been shown by Chattaway that in this reaction glyceryl acid oxalate 
is formed ; on raising the temperature carbon dioxide is evolved and 
glyceryl monoformin is produced On hydrolysing this ester with a 
further quantity of oxalic acid, formic acid is produced and the acid 
oxalate again formed. There is thus a continuous reaction : 

CH 2 OH CH 2 O OC . COOH 

HOOC | 

CHOH + | = CHOH + H O 

HOOC | 
CH 2 OH CH 2 OH. 

CH 2 O OC . COOH CH 2 O OC . H 

CHOH = CO 2 + CHOH 

CH 2 OH CH..OH. 

CHp OC . H CH 2 O OC . COOH 

COOH 
CHOH + | = HCOOH + CHOH 

COOH 
CH 2 OH CH 2 OH. 

Properties. 

Formic acid is a colourless volatile liquid with pungent odour. 
It has a sp. gr. of 1-221 at 20, freezes at 8-3, and boils at 100. 
It is a very strong acid, about 12 times as strong as acetic acid, and 
produces blisters on the skin and intense irritation. 

It dissolves in water, alcohol and ether, and in general properties 
resembles acetic acid. 

The formates crystallise well and are prepared in the same way 
as acetates (p. 97). The lead and magnesium salts are insoluble in 
alcohol ; the corresponding acetates are soluble. The acids may 
therefore be separated by treating a concentrated solution of these 
salts with alcohol ; the formate is then precipitated. Potassium 
formate is almost insoluble in alcohol and may thus also be separated 
from the acetate, which is soluble. 



THE FATTY ACIDS 95 

Reactions and Detection. 

A solution of formic acid must be exactly neutralised with soda 
or ammonia before the tests can be carried out. Solid formates are 
obtained by evaporating their solutions to dryness. 

(1) On boiling a solution of a formate with dilute sulphuric acid, 
formic acid is evolved. Its pungent odour is only perceptible with 
strong solutions. 

(2) On heating a solid formate with concentrated sulphuric acid, 
carbon monoxide is evolved and it may be ignited at the mouth of 
the test tube. 

(3) Ethyl formate is formed when solid formates are heated with 
alcohol and concentrated sulphuric acid. 

(4) A red solution containing ferric formate is obtained when 
ferric chloride or ferric nitrate is added to a solution of a formate. 
On heating a reddish-brown precipitate of basic ferric formate is 
produced. 

Formic acid differs from acetic acid in its reducing properties 
which are due to the presence of the aldehyde group CHO in its 
molecule. 

(5) In concentrated solution it forms with silver nitrate a white 
crystalline precipitate of silver formate. This precipitate darkens on 
standing owing to reduction to metallic silver. A precipitate is not 
formed in dilute solution, but the solution is reduced on heating with 
separation of metallic silver. The reduction is retarded in the pre- 
sence of ammonia. 

(6) On adding mercuric chloride solution and heating a pre- 
cipitate of mercurous chloride is produced, which, on further heating, 
may be reduced to metallic mercury. 



96 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

ACETIC ACID. CH 3 . COOH. 

Preparation. 

Acetic acid is one of the few products made commercially by 
biological methods, i.e. by the oxidation of dilute alcohol by means of 
the micro-organism Mycoderma aceti, or " mother of vinegar". Me- 
chanical contrivances are used rn order to expose a large surface of 
the alcoholic liquid to the air so that the acetification is as rapid 
as possible. 

Wine, red and white, cider, beer and malt, and sugar prepared 
from starch are the materials from which the vinegar is made. Be- 
sides acetic acid vinegar contains other organic acids, sugar, dextrin 
and colouring matters which were present in the original material. 
The amount of acetic acid in the solution varies from about 3-12 
per cent, the average 'quantity being about 5 per cent. 

A large quantity of acetic acid is produced by the dry distillation 
of wood, the crude material obtained in this way being termed pyro- 
ligneous acid (p. 63). Tarry matter separates out on adding hydro- 
chloric acid to the solution which has been neutralised with lime 
and distilled to remove methyl alcohol and acetone. The clear liquid 
is neutralised and evaporated to dryness and the dry residue heated 
to decompose the empyreumatic products. Comparatively pure acetic 
acid is obtained on distilling the residue with hydrochloric acid. Pure 
acetic acid is prepared by distilling with potassium bichromate, or 
neutralising with soda and distilling the sodium salt, which has been 
heated to destroy tarry matter, with sulphuric or hydrochloric acid. 

Preparation by the Oxidation of Alcohol with Permanganate. 

14 gm. of potassium permanganate are dissolved in about 200 
c.c. of water in a litre flask and 8 c.c. of concentrated sulphuric acid are 
added. The flask is fitted with a reflux condenser and through the 
condenser a mixture of 5 c.c. of alcohol and 50 c.c. of water is slowly 
added. The reaction must be kept moderate and, after all the alcohol 
has been added, the mixture is boiled for about 1 5 minutes. The 
acetic acid is separated by distilling over about three-fourths of the 
liquid. The distillate will contain the acetic acid which may be tested 
for as described on p. 97. 

Properties. 

Acetic acid is a colourless liquid with a characteristic pungent 
smell. The pure acid boils at 119 and distils without decom- 
position; on cooling it crystallises in plates which melt at 17 and 
hence is termed glacial acetic acid ; its sp. gr. at 16-5 is 1-052. 



THE FATTY ACIDS 97 

It is miscible in all proportions with water, alcohol and ether. 
Heat is evolved on adding water to acetic acid and there is a con- 
traction in volume. 

The liquid is not inflammable, but its vapour burns with a blue 
flame. 

Acetic acid is a very corrosive liquid and dissolves oils, resins, 
camphor, gelatin and many metallic salts which are insoluble in 
water. It is a very stable compound and is attacked only with 
difficulty by the most powerful oxidising agents. It is not affected 
by nitric acid or chromic acid. A solution of chromic acid in acetic 
acid is employed for oxidising hydrocarbons. Chlorine converts it 
into chloracetic acids (p. 100). 

As an acid, acetic acid forms salts. Most of the salts are soluble 
in water ; the silver and mercurous salts are sparingly soluble ; the 
sodium and potassium salts are soluble in alcohol. Some of the basic 
salts are insoluble. 

The salts are prepared by boiling the acid with the oxide or 
carbonate of the metal until the solution is neutral, filtering and 
evaporating the solution until crystallisation begins. The metallic 
acetates on being subjected to dry distillation yield acetone. 

Reactions and Detection. 

Free acetic acid may be recognised by its odour. The acid solution 
is exactly neutralised -with sodium hydroxide and then tested. Neutral 
solutions of acetates may be tested directly. Insoluble (basic) acetates 
are converted into sodium acetate by boiling with sodium carbonate, 
filtering off the insoluble carbonate, neutralising and testing the 
filtrate : 

(1) On warming the solution with dilute sulphuric acid the pungent 
odour of acetic acid is evolved. 

(2) On adding ferric nitrate or ferric chloride, the neutral solution 
gives a deep red liquid, which contains ferric acetate. An excess' must 
be avoided. On boiling, the liquid becomes colourless and a brownish- 
red precipitate of basic ferric acetate is produced. 

The cold red liquid is decolourised by adding dilute hydrochloric, 
or sulphuric acid, but not by mercuric chloride solution. 

(3) Concentrated solutions and dry acetates give the smell of ethyl 
acetate on heating with alcohol and concentrated sulphuric acid. 

(4) On mixing a solid acetate with arsenious oxide and heating, 
cacodyl oxide, which has a garlic-like smell, is evolved. Only minute 
quantities should be used as the product is very poisonous. 

4 CH 3 . COONa + As,0 3 = (CH 3 ) As . O . As(CH 3 ) 2 + 2CO 2 + 2Na 2 CO 3 . 

" 7 



98 PRACTICAL ORGANIC AND BIO-CHEMISTRY' 

Propionic Acid. CH 3 . CH 2 . COOH. 

Propionic acid is present with acetic acid in pyroligneous acid ; it is 
found in sweat and is a product of putrefactive fermentation. It is most 
easily prepared by the oxidation of propyl alcohol with potassium bichromate 
and sulphuric acid. 

Propionic acid closely resembles acetic acid in its properties : it is a liquid 
which boils at 140 and has a sp. gr. of '996 at 19. It mixes with water 
in all proportions, and may be separated from solution by adding calcium 
chloride, which causes it to float as an oily layer. 

Butyric Acid. CH 3 . CH 2 . CH 2 . COOH. 

Butyric acid occurs in the free state in various animal and vegetable 
secretions in the form of its glyceride butyrin ; it is always stated to exist 
in butter to the extent of about 6 per cent., but this compound could not be 
separated by Hurtley by the distillation of pure butter in vacua. Since butyric 
acid results from the putrefaction of proteins and amino acids, it seems most 
probable that its occurrence in butter is due to the presence of butter milk 
which has not been removed and which has undergone decomposition. Its 
smell is always obvious in rancid butter. 

Butyric acid is prepared by the butyric fermentation of glucose and 
other carbohydrates in the same way as lactic acid (p. no). The filtered 
solution is evaporated, acidified and distilled. 

Butyric acid is a colourless liquid with a pungent and disagreeable smell. 
Like propionic acid it can be separated from aqueous solution by the addition 
of calcium chloride. 



Isobutyric Acid. >CH . COOH. 

CH/ 

Isobutyric acid is found as the free acid, or as ester, in certain plants. It is 
a product of putrefaction of proteins and would arise from the amino acid, valine. 
It is very like normal butyric acid, but not so offensive in smell. 

Valerianic or Valeric Acids. C 4 H 10 . COOH,. 
Four isomers are possible. The commom valerianic acid is isovaleric 
CH 3 \ 
acid /CH . CH 2 . COOH which occurs in valerian root and various 

CH/ 

other animal and vegetable secretions. It is probably formed by the decom- 
position of leucine. 



Methyl ethyl acetic acid \CH . COOH is also found in nature. 

C 2 H 5 < 

This compound is optically active and would be derived from isoleucine by 
putrefaction. 

Normal valerianic acid, which has been found as a fermentation product, 
presumably of carbohydrate, most likely arises from amino acids in the same 
way as the other acids. 

These acids are liquids with an unpleasant smell and behave in most 
respects like butyric acid. 

Caproic to Myristic Acids. 

The fatty acids with 6, 8 and 10 carbon atoms are found in combination 
in various fats. They are liquids slightly soluble in water. 

The fatty acids with 12 and 14 atoms of carbon are solids of low melting- 
point, 



THE FATTY ACIDS 99 



THE HIGHER FATTY ACIDS. 

The principal higher fatty acids are palmitic and stearic acids with 
1 6 and 18 atoms of carbon in their molecules respectively. These 
acids, together with oleic acid (p. 105), are obtained by the hydrolysis of 
fats. The liquid oleic acid is removed by pressure, and the solid mix- 
ture of palmitic and stearic acids, " stearine," is used for making candles. 

Properties. 

The higher fatty acids are white odourless solids. On heating, they 
melt at a low temperature, and on further heating they boil giving off 
white vapours which condense on the cool parts of the test tube. 

They are insoluble in water, slightly soluble in alcohol and readily 
soluble in ether. The solubility in alcohol may be seen by adding 
some of the alcoholic solution to some alcohol containing a drop of 
dilute caustic soda and a drop of phenolphthalein. The red colour of 
the latter is discharged. 

They dissolve in dilute caustic alkali, aqueous or alcoholic, forming 
solutions of soap. 

Soaps. 

Soaps are the sodium and potassium salts of the higher fatty acids ; 
the former constitute hard soaps, the latter soft soaps. 

(1) Solutions of soap in water have an alkaline reaction to litmus 
owing to partial hydrolysis of the salt. 

(2) On adding excess of mineral acid (H 2 SO 4 ) to a solution of soap 
in water the fatty acids are liberated and form a precipitate which 
floats to the surface. 

(3) On adding calcium chloride or magnesium sulphate to a solu- 
tion of soap in water a curdy precipitate of the calcium or magnesium 
salt is formed just as is obtained with hard water. 

(4) On adding finely powdered sodium chloride to a soap solution 
the soap is salted out as a curdy mass which clings to the side of the 
vessel. 



7* 



ioo PRACTICAL ORGANIC AND BIO-CHEMISTRY 

HALOGEN SUBSTITUTION DERIVATIVES OF THE 
FATTY ACIDS. 

The fatty acids behave like a saturated hydrocarbon towards the 
halogens, especially chlorine and bromine, substitution of hydrogen 
atoms in the chain of carbon atoms (not the COOH group) taking 
place. The most typical compounds are mono-, di- and tri-chloracetic 
acids. 

In the case of the higher fatty acids containing three and more 
carbon atoms several isomers can be formed : 

CH 3 . CHCl . COOH CH 2 C1 . CH 2 . COOH 

o-chloropropionic acid. j8-chloropropionic acid. 

These acids are distinguished by using the Greek letters, that carbon 
atom next to the carboxyl group being called the a-carbon atom, the 
next /3, the next 7, and so on. 

Preparation. 

The chloro-substituted fatty acids are prepared (a) by the action of 
chlorine upon the fatty acid in direct sunlight, or in the presence of 
iodine, or (b) by the action of halogen upon the acid chloride (p. 101), or 
(c) by indirect methods from malonic ester. 

Monochloracetic Acid. . . 

Chlorine is passed into boiling acetic acid, to which a little sulphur 
or iodine has been added : 

CH 3 COOH + ICl., = CH 2 C1 . COOH + IC1 + HC1 
IC1 + C1 2 = IC1 3 . 

Monochloracetic acid is a colourless solid melting at 62 and boil- 
ing at 185-187. It closely resembles acetic acid in its reactions. 

Dichloracetic Acid. 

Dichloracetic acid is usually prepared by heating chloral hydrate 
with potassium cyanide or ferrocyanide : 

CC1 3 . CH(OH) 2 + KCN = CHCL). COOH + HCN + KC1. 
It is a liquid which boils at 190-191. 

Trichloracetic Acid. 

Trichloracetic acid is prepared by oxidising chloral with concen- 
trated nitric acid : 

CC1 3 . CHO + O = CC1 3 . COOH. 

It is a colourless solid melting at 55 and boiling at 195. On boiling 
with alkalies it is converted into chloroform and carbonate : 

CC1 3 . COOH + NaOH = CHCI 3 + NaHCO 3 . 

It forms salts with bases and yields an acid chloride (p. 101) like acetic 
acid. 

The acidity of these acids increases with the number of chlorine 
atoms ; trichloracetic acid is a strong acid almost equal to mineral acids. 



ACID OR ACYL CHLORIDES. 

The fatty acids, like the alcohols, contain a hydroxyl group. Phos- 
phorus pentachloride and phosphorus trichloride act upon the acids 
forming the acid or acyl chloride : 

CH 3 . COOH + PC1 5 = CH 3 . CO . Cl + POC1 3 + HC1 
3CH 3 . COOH + 2PC1 3 = 3CH 3 . CO . Cl + P 2 O 3 + sHCl. 

Preparation of Acetyl Chloride. 

A distilling flask is fitted with a tap funnel and connected with a condenser 
and receiver. If the preparation be not carried out in a fume cupboard, the 
receiver should be connected with a tower containing soda lime to absorb 
hydrochloric acid. 

25 gm. of glacial acetic acid are placed in the distilling flask and 20 gm. 
of phosphorus trichloride are slowly dropped upon it through the tap funnel. 
The flask is warmed upon a water-bath at 40-50 until the hydrochloric 
acid evolution has ceased ; the contents of the flask are then distilled from 
a water-bath. Acetyl chloride, which boils at 55, passes over. 

Properties of Acyl Chlorides. 

Formyl chloride is not known. Acetyl chloride is a liquid, other 
acyl chlorides are liquids or solids. They fume in moist air and under- 
go decomposition into hydrochloric acid and the acid from which they 
are derived. 

Reactions of Acyl Chlorides. 

Acyl chlorides are decomposed by water giving the acid and hydro- 
chloric acid : 

CH 3 COC1 + H 2 = HC1 + CH 3 COOH. 

They react with alcohols giving esters (p. 71). 

They react with ammonia giving amides (p. 129). 

The acyl chlorides, though decomposed by water, are sometimes 
only decomposed slowly and can be used in aqueous or alkaline solu- 
tion for preparing esters or for preparing acyl derivatives of amines. 
The process is known as acetylation, or acylation, or arylation if aro- 
matic acid chlorides be used. An alkaline solution of the alcohol or 
of an amine is shaken with the acyl chloride. The ester or acyl de- 
rivative is generally insoluble and can be filtered off and purified by 
crystallisation. 



102 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



ACID ANHYDRIDES. 

If acid chlorides be allowed to act upon the sodium salt of a fatty 
acid an acid anhydride is formed : 

CH 3 CO . Cl + NaOOC . CH 3 = NaCl + CH 3 . CO O OC . CH 3 . 

The constitution of these compounds is analogous to the ethers ; 
two radicles are united by an oxygen atom. Mixed anhydrides can 
be prepared by using different acyl chlorides and different sodium 
salts of fatty acids : 

r*T-T /~*TJ r^u r*/^\ r*ij r*c\ 

^riov v/iTnv is.n.'A.'U \ ^rio^LJv 

\ \ \ \ 

\f\ ^o ^/"\ ^o 

/ / / /- 

CH/ C 2 H 5 / CH 3 CO/ CH 3 CH 2 CO/ 

Ethers. Anhydrides. 

Preparation of Acetic Anhydride. 

40 gm. of fused sodium acetate are placed in a retort which is connected 
to a condenser and receiver and fitted with a dropping funnel. 30 gm. 
of acetyl chloride are run in slowly and the co.ntents of the flask are kept cold 
by immersion in cold water. The contents of the retort are well stirred and 
distilled. Acetic anhydride, which boils at 139, passes over between 130 
and 140. 

Properties. 

The anhydrides are liquids 'possessing a pungent smell, but do not 
fume in the air. 

Reactions. 

The reactions of the anhydrides are the same as the acyl 
chlorides. 

They are decomposed by water giving the constituent acid. 

They yield esters with alcohols. 

They yield amides with ammonia. 

Like the acyl chlorides they are also used for acylating alcohols 
and compounds containing amino (NH 2 ) groups. The compound is 
boiled under an air condenser with the anhydride for some hours and 
poured into water. The acyl derivative is generally insoluble and is 
recrystallised from a suitable solvent. 



UNSATURATED ALCOHOLS, ALDEHYDES AND 
FATTY ACIDS. 

Allyl alcohol, acrolein and acrylic acid are unsaturated compounds 
and the first members of the series : 



CH^OH 


CHO 


COOH 


C!H" 

II 


AH 

ii 


in 

|| 


II 
CH 2 
Allyl alcohol. 


11 
CH 2 
Acrolein. 


II 

CH 2 

Acrylic acid. 



Amongst the unsaturated acids there are several which occur in 
nature. 

Allyl Alcohol. 

Preparation. 

Allyl alcohol is prepared by distilling glycerol at a temperature of 
about 260 with oxalic acid. As stated on p. 94 the acid oxalic ester 
of glycerol is formed ; on heating it to a high temperature the neutral 
ester is produced and decomposed, yielding allyl alcohol and carbon 
dioxide : 

CH 2 OH CH 2 O CO 

I HOOC | | 

CHOH + | = CHOH COOH + H O 

| HOOC | 

CH 2 OH COOH. 

CH 2 CO CH 2 CO 

CHOH COOH = CHO CO + H 2 
CH 2 OH CH 2 OH. 

CH O CO CH 2 

I I II ' 

CHO CO = CH + 2CO 2 

CH 2 OH CH 2 OH. 

The liquid which distils over between 220 and 260 is collected and 
redistilled, the thermometer being placed in the liquid. Allyl alcohol 
is present in the fraction boiling below 105. It is dehydrated with 
potassium carbonate and again distilled. 

103 

X. 



104 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Properties. 

Allyl alcohol is a colourless neutral liquid with an irritating smell 
and boils at 96-97. It mixes with water, alcohol and ether in all 
proportions. 

It has the properties of a primary alcohol and of an unsaturated 
compound, e.g. it reacts with sodium, forms esters and yields acro- 
lein and acrylic acid on oxidation ; it combines with 2 atoms of 
chlorine or bromine. 

Esters of Allyl Alcohol. 

Allyl iodide. This ester may be prepared from allyl alcohol, but is 
more conveniently prepared from glycerol by the action of phosphorus 
and iodine. The glycerol is probably converted into tri-iodide, which 
decomposes into iodine and allyl iodide. 

It is a colourless liquid boiling at 101 with the odour of garlic. 

Allyl sulphide. Allyl sulphide occurs in garlic and other plants, and 
is obtained by distilling the plant after it has been macerated with water. 

It may be prepared from allyl iodide by heating with potassium 
sulphide in alcoholic solution (cf. p. 79). 

It is a colourless, oily liquid boiling at 140 with the smell of garlic 
and hence is termed oil of garlic. 

Allyl isothiocyanate is a constituent of black mustard seeds and is 
termed oil of mustard. 



Acrolein. 

Preparation. 

Acrolein is prepared by distilling glycerol (i part) with potassium 
hydrogen sulphate (2 parts) : 

CH 2 CWH . CHO 

CHOH =. CH + 2 H 2 O 

I I 

CH 2 OH VCH 2 

The smell of acrolein is noticed on heating a few drops of glycerol in a 
dry test tube with anhydrous phosphoric acid or acid potassium sulphate. 

Properties. 

Acrolein is a colourless liquid boiling at 52, with a most irritating 
and peculiar odour ; it affects the eyes, producing tears, and it forms 
sores upon the skin. 

It has the reactions of an aldehyde, but does not combine with 
sodium bisulphite, and also the reactions of unsaturatedjcompounds. 



UNSATURATED ALCOHOLS 105 

Unsaturated Fatty Acids. 

These acids contain in their molecule one or more pairs of their 
carbon atoms linked together by a double bond. Unsaturated acids 
containing a triple bond are also known. 

Acrylic acid is the simplest and first member of the homologous 
series of Unsaturated acids containing one double bond. It was first 
obtained by the oxidation of acrolein with silver oxide, but is more 
readily prepared by the reactions described for obtaining unsaturated 
hydrocarbons (p. 54) from /3-bromopropionic acid. 

Acrylic acid is a liquid with a pungent smell and boils at 140. 

The next member is crotonic acid, CH 3 . CH=CH . COOH. 

Crotonic acid is a solid which melts at 72. 

Oleic acid, which contains 1 8 carbon atoms and the double bond 
in the middle of the chain, is present in combination with glycerol in 
animal and vegetable fats from which it is prepared (p. 99). 

Oleic acid at the ordinary temperature is a colourless, oily liquid 
of sp. gr. -900 at 1 1 '8 with neither smell nor taste. It oxidises very 
readily in the air, becoming brown, acid in reaction and rancid in 
smell. It can be frozen to a white crystalline solid which melts at 14. 
It cannot be distilled at the ordinary temperature, but at 10 mm. pres- 
sure it distils at 223 and it is volatile with superheated steam. 

Linoleic acid also contains 1 8 carbon atoms, but two double bonds. 
It is contained in linseed and other oils. 

Linoleic acid resembles oleic acid, but is more readily oxidised by 
the oxygen of the air. It is owing to its presence and that of other 
more unsaturated acids in linseed, cotton seed and rape seed oils that 
these oils possess the property of forming the so-called "drying oils". 
Oxygen is absorbed and transparent resinous substances are formed. 

The salts of the unsaturated fatty acids are more soluble than those 
of the Saturated fatty acids. The lead and mercury salts of oleic acid 
are soluble in ether and are used for separating the mixture of acids 
obtained from fats. 

Owing to the presence of the double bonds the unsaturated acids 
combine by addition with the halogens, halogen acids, etc., and reduce 
permanganate solution becoming oxidised ; thus 

If a solution of oleic acid in chloroform be treated with bromine 
dissolved in chloroform, or iodine dissolved in chloroform containing 
also mercuric chloride, the colour of the halogen is discharged until 
the acid is completely saturated by absorption of the halogen. 

If a solution of sodium oleate be poured into a solution of potassium 
permanganate, the colour of the permanganate disappears and man- 
ganese dioxide separates out. 



HYDROXY-, KETO- AND DIBASIC ACIDS. 

In the previous sections compounds having only a single function 
of either alcohol, aldehyde, or acid have been considered. In com- 
pounds of carbon containing two or more atoms of carbon in their 
molecule the replacement of hydrogen atoms by other atoms or groups 
can occur in several of the atoms, and compounds will result which 
have multiple functions. They may be alcohol and acid, ketone and 
acid, etc., at the same-time. The properties of such compounds are 
the sum of the properties possessed by the particular groups contained 
in the molecule. Numerous natural compounds are included amongst 
the large number of compounds which are theoretically possible : most, 
if not all, of these have been prepared in the laboratory. 

COMPOUNDS CONTAINING TWO CARBON ATOMS. 

The variety of the compounds is most easily seen in the series of 
compounds which are derived from ethane : 

COOH 



CH 3 

"CH 3 
Ethane. 


CH 2 OH 

CH 2 OH 
Glycol. 


CHO 

CH 2 OH 
Glycollic 
aldehyde. 


COOH 

CH 2 OH 
Glycollic 
acid. 


CHO 

CHO 
Glyoxal. 


COOH 

CHO 

Glyoxylic 
acid. 



COOH 
Oxalic 
acid. 

Glycollic Aldehyde. 

Glyeollic aldehyde is prepared by oxidising glycol with hydrogen 
peroxide in the presence of ferrous sulphate, or by hydrolysingibromacet- 
aldehyde with baryta. 

It is a sweet crystalline substance having the properties of an alde- 
hyde. It is the first representative of the group of carbohydrates. 



106 



HYDROXY-, KETO- AND DIBASIC ACIDS 107 

Glycollic Acid. 

Glycollic acid is prepared by boiling potassium chloracetate under 
a reflux condenser with water : 

CH 2 C1 . COOK + H 2 O = CH 2 OH . COOH + KC1. 

The solution is evaporated in vacuo to dryness and the glycollic 
acid extracted from the residue with acetone. 

It is present in unripe fruit and was first obtained from glycine. 
Glycollic acid is a deliquescent crystalline solid which melts about 80. 
It is a monobasic acid and at the same time a primary alcohol, and 
consequently has the properties of both of these types of compounds. 

Glyoxal. 

Glyoxal can be prepared by oxidising acetaldehyde with nitric 
acid at the ordinary temperature and is isolated as its bisulphite 
compound. 

It is an amorphous solid, or when not quite free from water, a 
syrup. It has all the properties of an aldehyde. 

Glyoxylic Acid. 

Preparation. 

Glyoxylic acid is prepared most conveniently by the reduction ot 
oxalic acid ; sodium amalgam was most frequently employed until 
Benedict suggested the use of magnesium. 

About I gm. of powdered magnesium is placed in a small flask 
and just covered with distilled water; 25 c.c. of saturated oxalic acid 
solution are slowly added. The reaction proceeds rapidly with libera- 
tion of heat and the flask should be cooled with water. The insoluble 
magnesium oxalate, which is formed, is filtered off and the glyoxylic 
acid is obtained by evaporation in vacuo. 

Properties. 

Glyoxylic acid is a syrup, very soluble in water. It gives the re- 
actions of an acid and of an aldehyde. 

The solution, prepared above, may be tested for aldehyde by 
Schiff s reaction, ammoniacal silver nitrate and other reactions. It is 
used in testing for proteins, the above solution being acidified with 
acetic acid and made up to 100 c.c. with distilled water. 



io8 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Oxalic Acid. 

Oxalic acid occurs naturally in many plants e.g. sorrel, rhubarb 
deposited in the cells as calcium oxalate. Small quantities of oxalic 
acid are present in normal urine, from -O2-'I2 gm. in 24 hours. 
It arises most probably from the carbohydrate of the diet. An in- 
creased output follows the consumption of rhubarb and other vege- 
tables which contain oxalic acid, and occasionally an increased output 
occurs in certain diseases, e.g. in diabetes. Calculi of calcium oxalate 
are sometimes found in the bladder and kidneys. 

Preparation. 

Oxalic acid is formed by the oxidation of numerous organic com- 
pounds, acetic acid and sugar. It is made commercially by the oxidation 
of the cellulose of sawdust with air and caustic alkali. A mixture of 
caustic potash and caustic soda is made into a paste with sawdust 
and heated in open vessels to about 240. The mass is extracted 
with cold water ; the potash dissolves leaving sodium oxalate which 
is only slightly soluble. By boiling the sodium oxalate with milk of 
lime, insoluble calcium oxalate is formed. This is washed and de- 
composed with sulphuric acid, and the oxalic acid isolated from the 
solution by crystallisation. 

The alkali salts of oxalic acid are made commercially by heating 
alkali formates. The reaction proceeds most easily in the presence 
of small amounts of alkali at 280 under diminished pressure, or at 
400 in .absence of air : 

aHCOONa = H 2 + COONa 
COONa. 

Properties. 

Oxalic acid crystallises from water in colourless prisms containing 
2 molecules of water of crystallisation (m.p. ioi'5). On heating to 
1 00, it loses the water, becomes opaque and forms a white powder 
which melts at 189 

It is easily soluble in alcohol, but only slightly soluble in ether. 
It is insoluble in chloroform, benzene and petroleum ether. 

Reactions. 

(1) On heating on platinum, nickel, or a crucible lid, oxalic acid 
is volatilised without charring. 

(2) No charring occurs on heating oxalic acid with concentrated 
sulphuric acid, but it is decomposed yielding carbon monoxide and 
carbon dioxide : 

COOH . COOH = C0 2 + CO + H,O. 



HYDROXY-, KETO- AND DIBASIC ACIDS 109 

The gases may be passed into lime or baryta water; barium 
carbonate is precipitated and the carbon monoxide may be ignited. 

(3) On warming a solution of oxalic acid with dilute sulphuric 
acid and potassium permanganate, it is oxidised with liberation of 
carbon dioxide and the permanganate is decolorised : 

COOH . COOH + O = 2CO 2 + H 2 0. 

(4) Calcium oxalate is precipitated when calcium chloride is added 
to a solution of a neutral oxalate. It is insoluble in acetic acid, but 
soluble in mineral acids. 

(5) Mercurous nitrate gives a precipitate of mercurous oxalate even 
in very dilute solutions of neutral oxalates. 

Detection. 

Oxalic acid is precipitated from a neutral solution, or a solution 
acidified with acetic acid as calcium oxalate. The crystalline form of 
the precipitate (p. 565) is very characteristic. The calcium oxalate is 
filtered off and can be identified by heating it or by the action of 
permanganate in sulphuric acid solution. 

Estimation. 

The solution, e.g. urine, is treated with calcium chloride and ammonia 
and evaporated to a small bulk. The precipitate which consists of calcium 
phosphate, sulphate and oxalate is separated by filtration and washed with 
water. It is dissolved in a small quantity of dilute hydrochloric acid (30 c.c.) 
and extracted from aqueous solution by extracting in an extractor (see p. 600) 
with ether. The ether is evaporated off, the residue dissolved in water, 
neutralis.d with ammonia, acidified with acetic acid, and precipitated with 
calcium chloride. The calcium oxalate is filtered off, ignited, and weighed 
as CaO, or dissolved in dilute sulphuric acid and titrated with standard per- 
manganate. 



no PRACTICAL ORGANIC AND BIO-CHEMISTRY 



COMPOUNDS CONTAINING THREE CARBON ATOMS. 

The number of compounds which can be derived from propane are 
more numerous than those from ethane, since the extra carbon atom 
introduces further possible combinations and permutations. The 
principal compounds are : 



CH 2 OH 


CH 3 


CH 3 


COOH 


COOH 


CH 2 


CHOH 


i 


CH 2 


CHOH 


COOH 


COOH 


COOH 


COOH 


COOH 


/3-hydroxy- 


a hydroxy- 


Pyruvic 


Malonic 


Tartronic 


propionic 


propionic 


acid. 


acid. 


acid. 


acid. 


acid. 










Lactic acid. 









The first two compounds are isomers ; in their nomenclature the 
position of the hydroxyl group is indicated by the Greek letters a and /3, 
the lettering or numbering being commenced at the carbon atom next 
to the COOH group, which stands at the end of the chain (cf. p. 100). 

Lactic Acid. 

Lactic is formed by the fermentation of sugar by lactic acid 
bacteria ; hence its presence in milk when it turns sour. Lactic acid 
is contained in muscle, especially after activity, and other organs of 
the animal body. 

Preparation. 

Lactic acid has been prepared synthetically by several methods, 
but its usual method of preparation is by fermentation, i.e. by bio- 
logical means. 

To a solution of 50 gm. of cane sugar in 500 c.c. of water 20 gm. 
of chalk or zinc carbonate and 20-30 c.c. of sour milk (which con- 
tains lactic acid bacteria) are added and the mixture is kept in a warm 
place, or better in an incubator at 37, for 6-8 days and occasionally 
shaken. The chalk or zinc carbonate is added to neutralise the lactic 
acid which hinders the growth of the bacteria. Calcium or zinc lactate 
is formed. 

The solution is boiled to kill bacteria, filtered, and evaporated 
on the water-bath till crystallisation commences and allowed to cool. 
The lactate is filtered off, pressed between sheets of filter paper and 
recrystallised from hot water. The acid is obtained from the salt by 
decomposition with sulphuric acid, extraction of the liquid with ether, 
and removal of the ether by distillation. 



HYDROXY-, KETO- AND DIBASIC ACIDS m 

Properties. 

Lactic acid is a syrupy liquid having a sp. gr. of I -248 at 1 5. 
It is decomposed on distillation at ordinary atmospheric pressure, 
but at a pressure of *5-i mm. it distils at about 85 and then sets 
to a hygroscopic crystalline solid melting at 18. It is soluble in 
water, alcohol or ether, and is only very slightly volatile with steam. 

Lactic acid is the simplest compound which exhibits the pheno- 
menon of circular polarisation. Most natural compounds exhibit this 
phenomenon. Circular polarisation is the property of rotating a ray 
of polarised light to either the right or the left. 

According to its source lactic acid may be either dextrorotatory, or 
laevorotatory, or inactive. Thus lactic acid from muscle sarcolactic 
acid is dextrorotatory. Certain bacteria produce laevorotatory 
lactic acid. Fermentation lactic acid is inactive. 

The examination of natural substances which exhibit circular 
polarisation has shown that they all contain one or more asymmetric 
carbon atoms, i.e. carbon atoms combined with four different groups. 
By representing the carbon atom as a regular tetrahedron and placing 
the different groups at the four apices, Van't Hoff and Le Bel have 
given us an explanation of the phenomenon. Adopting any arrange- 
ment of the groups round the tetrahedron, a reverse arrangement 
is represented by its image in a mirror. If therefore one variety is 
represented by the first arrangement, the mirror image of the figure 
represents the opposite variety. Projected on to a plane surface the 
following formulae are then obtained : 



HO.C.H H.C.OH 

I I 

COOH 9 COOH. 

One figure will represent laevo or /-lactic acid, the other figure 
dextro or ^-lactic acid. A mixture of the two in equal quantities re- 
presents inactive or ^/-lactic acid. This can be proved by the separa- 
tion of inactive lactic acid into its constituent d- and /-forms by the 
fractional crystallisation of its strychnine salt. 

These optical isomers are known as stereoisomers and the pheno- 
menon as stereoisomerism. 

Lactic acid has the chemical properties of a secondary alcohol and 
of an acid. The most characteristic salt of lactic acid is the zinc salt. 
This is prepared by boiling a solution of lactic acid for some time with 
excess of zinc carbonate and filtering whilst hot. On cooling, zinc 
lactate crystallises out, if the solution be sufficiently concentrated. 



ii2 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Detection. 

(1) Lactic acid in concentrated solution is decomposed by heating 
with concentrated sulphuric acid with the formation of carbon mon- 
oxide ; the gas may be ignited at the mouth of the test tube : 

CH 3 . CHOH . COOH = CH 3 . CHO + H 2 O + CO. 

Weak solutions of lactic acid are neutralised with sodium carbonate 
and evaporated to a small volume before heating with sulphuric acid. 

(2) The formation of acetaldehyde may be detected by Deniges method. 
The dilute solution of lactic acid is heated for 2 minutes in a boiling water- 
bath with 10 times its volume of concentrated sulphuric acid. The liquid is 
cooled, and 2 or 3 drops of a 5 per cent, solution of guaiacol in alcohol are 
added. On mixing, a rose-red colour formed from the aldehyde and guaiacol 
is produced which increases in intensity on standing. 

(3) Lactic acid forms a soluble deep yellow ferric salt. If a dilute 
solution of ferric chloride (scarcely coloured) be treated with a few 
drops of a dilute solution of lactic acid, the colour becomes yellow. 

Other hydroxy acids and oxalic acid also give a similar colour. 

(4) Uffelmanris Test. Uffelmann's reagent a I or 2 per cent, 
solution of phenol treated with ferric chloride solution till of a dis- 
tinctly violet colour is changed to yellow on the addition of lactic 
acid. 

Note. Mineral acids decolorise the reagent ; other organic acids 
also give a yellow or brownish colour. 

(5) Lactic acid gives the iodoform reaction (p. 67). 

(6) Thiophene Test. A few drops of a I percent solution of lactic 
acid in alcohol are added to 5 c.c. of concentrated sulphuric acid 
containing 3 drops of saturated copper sulphate solution and heated 
in a boiling water-bath for 5 minutes. Two drops of a ~2 per cent, 
alcoholic solution of thiophene are added to the cooled solution, and 
on again warming a cherry-red colour is formed (Hopkins]. 

These tests for lactic acid are not easily observed in extracts of 
organs, etc., which contain lactic acid. The lactic acid should be ex- 
tracted with ether, the ethereal solution evaporated and the residue 
then tested for lactic acid. 

Estimation. 

Lactic acid is sometimes estimated by converting it into its zinc salt by 

boiling the solution with excess of zinc carbonate, filtering, evaporating and 

weighing the zinc lactate which crystallises out. It is more usually estimated 

by oxidising it with permanganate in acid solution ; acetaldehyde is formed : 

CH 3 . CHOH . COOH + O = CH S . CHO + CO 2 + H 2 O. 

The acetaldehyde is distilled off during the oxidation, collected and 
determined by treatment with excess of standard potassium bisulphite solution, 
the excess of which is estimated by titration with standard iodine solution 
(see p. 85). 

The estimation of lactic acid in tissues is described on p. 590. 



HYDROXY-, KETO- AND DIBASIC ACIDS 113 

Pyruvic Acid. 

The work of recent years shows that pyruvic acid and other 
ketonic acids are very probably intermediate products in the catabolism 
of fatty acids and proteins. Pyruvic acid may be a stage in the 
transformation of sugar into alcohol and carbon dioxide and into 
lactic acid : 

f T T f*TT fT T f*T-T 

I II I 

CHOH *- CO -> CHO -> CH 2 OH 

I I +* 

COOH COOH CO 2 . 

Preparation. 

Pyruvic acid is usually prepared from tartaric acid. A mixture of 500 gm. 
of tartaric acid and 780 gm. of potassium bisulphate is distilled from a 2 li. 
copper retort provided with a condenser ; the receiver is cooled with ice. 
The distillate is redistilled in vacua using a fractionating column. A yield of 
about 60 per cent, is obtained. 

Properties. 

Pyruvic acid is a liquid which freezes at 9 and boils at 168 under atmos- 
pheric pressure, or at 59-60 at 12 mm. pressure. It smells very like acetic 
acid. It has the properties of an acid and of a ketone and forms a character- 
istic hydrazone. 

Test. 

Hurtley has described the following delicate test for pyruvic acid. The 
test depends upon the formation of a red colour on oxidising the phenyl- 
hydrazone of pyruvic acid. The reaction is positive at a dilution of i in 
10,000 and can be obtained at a dilution of i in 100,000. 

10 c.c. of pyruvic acid (i per cent.) are treated with 10 c.c. of phenyl- 
hydrazine hydrochloride solution (6'4i gm. in 500 c.c. acid = 5 gm. pyruvic 
acid) ; about 2 c.c. of persulphuric acid (25 gm. K 2 S 2 O 8 are ground up with 
50 gm. H 2 SO 4 and left for i hour, then poured upon ice and diluted to 500 
c.c.) are added avoiding an excess and then 5 c.c. concentrated hydrochloric 
acid. A red colour develops. 

Malonic Acid. 

This acid is found in beetroot as its calcium salt. It was origin- 
ally prepared by the oxidation of malic acid with potassium bi- 
chromate and sulphuric acid, but is usually made by the cyanide 
synthesis from .chloracetic acid. 

Potassium chloracetate is boiled with potassium cyanide; the product is 
hydrolysed with hydrochloric acid and the solution evaporated to dryness. 
The residue is extracted with ether, and the malonic acid obtained by distil- 
lation of the ether. 

Malonic acid is a colourless, crystalline solid which melts at 
132, and is readily soluble in water, alcohol and ether. On heat- 
ing to 140-150 it loses carbon dioxide and is converted into acetic 
acid : 



1 1 4 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

COOH 

CH 3 
CH, = C0 2 + | 

COOH. 
COOH 

The diethyl ester of malonic acid is a compound much used for 
the synthesis of other organic compounds. 

COMPOUNDS CONTAINING FOUR CARBON ATOMS. 

The vegetable acids, malic 'and tartaric, and the two acids, ft- 
hydroxybutyric and acetylacetic, which are found in urine in cases of 
diabetes are the chief biological representatives of this group of com- 
pounds. Succinic acid is the dibasic acid of this series ; malic acid is 
monohydroxy-succinic acid ; tartaric acid is dihydroxy-sticcinic acid : 



COOH 


COOH 


COOH 


CH 3 


CH. 


CH 2 


CHOH 


CHOH 


1 
CHOH 


<Jo 


1 


1 


I 


I 


1 


CH 2 


CH 2 


CHOH 


CH 2 


CH 2 


COOH 


COOH 


COOH 


COOH 


COC 


Succinic 


Malic 


Tartaric 


/8-hydroxy- 


Ace 


acid. 


acid. 


acid. 


butyric acid. 


acetic 



Succinic Acid. 

Succinic acid occurs in certain lignites and fossils, in lettuce, unripe 
grapes and other fruits. It has also been found in animal tissues 
and meat extracts. 

Succinic acid is obtained by the dry distillation of amber. The 
aqueous distillate is filtered whilst hot to separate oil, and on cooling 
crystals of succinic acid are deposited. They may be purified by boiling 
with nitric acid and recrystallisation. 

Succinic acid is usually prepared by the fermentation by yeast of 
calcium malate or ammonium tartrate. It is formed by the reduction 
of these compounds. 

Succinic acid crystallises in colourless prisms or plates which melt 
at 182. On heating, it emits suffocating fumes; at higher tempera- 
tures it boils and gives a sublimate of succinic anhydride. 

It is not readily soluble in cold' water, but dissolves readily in 
alcohol and sparingly in ether. It is insoluble in chloroform and 
benzene. 



HYDROXY-, KETO- AND DIBASIC ACIDS 115 

Malic Acid. 

Malic acid is contained in apples, pears and other fruits. It is 
usually prepared from rhubarb stalks or unripe mountain ash berries. 
The juice is boiled with milk of lime ; the neutral calcium salt is 
precipitated. The salt is recrystallised from dilute nitric acid and the 
acid salt is so obtained. It is decomposed with the calculated quantity 
of sulphuric acid, the liquid is filtered from calcium sulphate and 
evaporated. Malic acid crystallises out. 

Malic acid crystallises in groups of colourless 4 or 6-sided prisms. 
It is deliquescent and readily soluble in water, alcohol and ether. 

On heating to about 180, it melts and loses water yielding 
fumaric and maleic acids. 

Malic acid is optically active and contains one asymmetric carbon 
atom. The natural form is /-malic acid. 

The salts of malic acid resemble those of citric, oxalic and 
tartaric acids. Calcium malate is not precipitated in the cold. On 
boiling in neutral and concentrated solution calcium malate is pre- 
cipitated ; alcohol precipitates calcium malate from dilute aqueous 
solution. A mixture of oxalic, tartaric, citric and malic acids may 
thus be separated. The oxalate and tartrate are precipitated from 
dilute solution in the cold ; on boiling the filtrate calcium citrate is 
precipitated, and on adding 2 volumes of alcohol to the filtrate 
calcium malate is thrown down. 

Tartaric Acid. 

Tartaric acid occurs in certain plant juices ; its only important 
source is grape juice. During fermentation a deposit forms on the 
bottom lees and a crystalline crust on the sides tartar or argol of 
the cask. The argol consists mainly of potassium hydrogen tartrate and 
calcium tartrate. Their precipitation is due to their insolubility in 
the alcohol as it is produced. If the crude argol be boiled with water 
and filtered and the solution crystallised, cream of tartar separates out, 
the term cream of tartar having arisen from the fact that the salt 
collects in crusts on the surface during the evaporation. 

Preparation. 

Tartaric acid is prepared from tartar by dissolving it in water and 
neutralising with lime. Insoluble calcium tartrate is thrown down, and 
from the solution, which still contains tartaric acid, insoluble calcium 
tartrate is precipitated by adding calcium sulphate or calcium chloride. 
The insoluble calcium salt is decomposed with sulphuric acid and tar- 
taric acid isolated from the solution by crystallisation. 



n6 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Properties. 

Tartaric acid crystallises in large hemihedral monoclinic prisms 
which are colourless and transparent. It melts at 167-170 and is easily 
soluble in water and alcohol, but insoluble in ether. 

Tartaric acid is optically active ; the ordinary tartaric acid is dextro- 
rotatory having [a] D = 13'! for a 15 per cent, solution and = 14*7 
for a 2 per cent, solution. A laevo-tartaric acid and two inactive forms 
of tartaric acid also exist. Tartaric acid contains two asymmetric 
carbon atoms in its molecule and to these carbon atoms the same groups 
are attached. If the arrangement of these groups round each carbon 
atom is the same, an optically active form will result ; but if different an 
inactive form will result ; in the former case both asymmetric carbon 
atoms are rotating to the right or to the left ; in the latter case the 
one carbon atom rotates in one direction as much as the other carbon 
atom rotates in the other direction and they neutralise one another. 
Internal compensation occurs. 

This inactive acid is known as meso-tartaric acid and is produced 
by prolonged heating of dextrorotatory tartaric acid to 165 with a 
small quantity of water. 

The other inactive form is a mixture of the dextro and laevo forms 
in equal proportions. As a mixture it can be separated into its two 
constituents. It occurs with ^/-tartaric in crude tartars. 

Reactions. 

(1) On heating, tartaric acid melts and chars giving off an odour 
resembling that of burnt sugar. 

(2) Tartaric acid chars almost immediately when it is heated with 
concentrated sulphuric acid. 

(3) A white precipitate of silver tartrate is formed on adding 
silver nitrate to a neutral solution of a tartrate. The precipitate 
dissolves in ammonia and when this solution is slowly warmed a 
silver mirror is formed on the sides of the vessel. 

(4) On adding calcium chloride to a cold solution of a neutral tartrate 
(sodium potassium tartrate) a white precipitate of calcium tartrate is 
formed. This precipitate, after filtering and washing, is soluble in 
acetic acid and caustic soda (free from carbonate) ; on boiling the 
solution in the latter, it is reprecipitated (distinction from calcium 
oxalate). 

(5) A precipitate of potassium hydrogen tartrate is formed on 
adding potassium chloride and acetic acid to a not too dilute 
solution of a tartrate. 

This reaction is used in estimating tartaric acid. 



HYDROXY-, KETO- AND DIBASIC ACIDS 117 

(6) The presence of tartaric acid or tartrates in a solution prevents 
the precipitation of metallic hydroxides by caustic soda. 

(a) No precipitate is formed if caustic soda be added to ferric 
chloride solution containing some tartrate solution, but a yellow- 
brown solution results. 

() A dark-blue solution results if caustic soda be added to copper 
sulphate solution containing a tartrate. 

This property is used in the preparation of Fehling's solution. 

Citric Acid. 

Citric acid is another hydroxy acid, which occurs in the free state 
in the juices of many plants. Small quantities are present in milk. 

Preparation. 

About 5 -5 per cent, of citric acid is obtainable from good lemons ; 
about i per cent, from unripe gooseberries. It is usually extracted 
from lemons, limes and bergamot. The hot liquid is neutralised with 
calcium carbonate, and the calcium citrate so obtained is decomposed 
by sulphuric acid in equivalent amount. The solution on evaporation 
gives citric acid. 

Citric acid contains 6 atoms of carbon in its molecule, 5 being 
in a normal straight chain. Its formula is 

CH 2 . COOH 
C(OH).COOH 
CH 2 .COOH 

which shows that it is a tribasic acid. It closely resembles tartaric 
acid, but there are many points of difference. 
Properties. 

Citric acid is obtainable either as a crystalline powder, or in trans- 
parent colourless prisms having the formula C 6 H 8 O 7 . H 2 O. It has a 
strong acid taste, is very easily soluble in water, and is also soluble in 
dilute and absolute alcohol ; it is almost insoluble in ether, chloro- 
form, petroleum ether and benzene. 
Reactions. 

(1) On heating, citric acid loses water becoming anhydrous, melts 
and decomposes, giving off acid fumes of aconitic acid. 

(2) When heated with concentrated sulphuric acid, citric acid 
chars slowly. 

(3) Silver citrate is precipitated on adding silver nitrate to a 
neutral solution of citric acid or a citrate. The precipitate dissolves 
in ammonia, but the solution on warming is not reduced and does 
not form a silver mirror. 



n8 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

(4) Calcium citrate is not precipitated when calcium chloride is 
added to a cold neutral solution of a citrate. This salt is less soluble in 
hot water and hence on boiling the solution calcium citrate is pre- 
cipitated ; it dissolves again as the solution cools. 

(5) Citric acid does not form an insoluble acid potassium salt when 
its solutions are treated with potassium chloride and acetic acid. 

Aceto-Acetic Acid or Acetylacetic Acid. 

Ethyl aceto-acetate was prepared in 1863 by Geuther by acting 
upon ethyl acetate with sodium and acidifying the product with acetic 
acid. An oil resulted ; it was separated and purified by distillation. 

Ethyl aceto-acetate is a liquid with a fruity smell which boils at 
182. It is largely used in synthesis in organic chemistry, like malonic 
ester. 

Aceto-acetic acid can be prepared from the ester by hydrolysis. 
It is a very unstable, strongly acid, hygroscopic syrup which decom- 
poses on warming into acetone and carbon dioxide. Solutions of 
aceto-acetic acid are readily decomposed on distilling with dilute acid 
or alkali. 

Traces of aceto-acetic acid occur in normal urine 2-4 mg. in 
24 hours. The amount is increased in starvation, on a diet of protein, 
and on a diet of fat i.e. whenever there is a shortage of carbohy- 
drate. The excretion of aceto-acetic acid is lessened if carbohydrate 
be added to the food. 

The origin of the aceto-acetic acid in the urine appears to be 
partly from the protein of the food, but mainly from the fat. The 
work of Knoop and of Dakin has shown that the oxidation of the 
fatty acids takes place at the /3-carbon atom ; the long chains are 
broken down with the loss of 2 carbon atoms at a time. This accounts 
in part for the occurrence of those fatty acids in nature containing 
an even number of carbon atoms. Butyric acid, if present as such 
or formed by the oxidation of higher fatty acids by /3-oxidation, is 
oxidised and converted into aceto-acetic acid or /3-hydroxybutyric 
acid : 

CH, . CH, . CH, . COOH 



CH, . CO . CR, . COOH - CH, . CHOH . CH 2 . COOH. 



HYDROXY-, KETO- AND DIBASIC ACIDS 119 

Aceto-acetic acid is converted by reduction into /3-hydroxy- 
butyric acid, and vice versa, /3-hydroxybutyric acid is converted by 
oxidation into aceto-acetic acid. It seems most likely that aceto- 
acetic acid, the keto acid, is the chief product of the oxidation of 
butyric acid. 

The formation of aceto-acetic acid in the organism from fat when 
carbohydrate is .withheld from the food explains its formation in 
diabetes. Here the organism has lost its power of utilising the carbo- 
hydrate in the food, or its power of utilising carbohydrate is greatly 
diminished. 

Aceto-acetic acid is very unstable and is readily converted into 
acetone and carbon dioxide. This decomposition occurs spontaneously 
in normal urine. 

The three closely related substances, /3-hydroxybutyric acid, 
aceto-acetic acid and acetone are generally referred to in medical 
literature as the acetone bodies. There is no basis for the older 
statement that mild cases of diabetes excrete only acetone, that severer 
cases excrete acetone and aceto-acetic acid, and that still severer cases 
excrete /3-hydroxybutyric acid in addition. It seems to have been 
due to a misinterpretation of the tests and to the inadequacy of our 
methods of estimating these substances. 

The occurrence of acetone in the breath of diabetics can be ac- 
counted for by the difference in the blood circulation. This is slow 
through the systemic system and lung capillaries through which the 
blood passes before it goes to the kidney. Venous blood is more 
acid than arterial blood, so that the conditions for the decomposition 
of aceto-acetic acid are most favourable. Acetone is very volatile 
and if decomposition occurs during the passage of the blood through 
the lungs it would pass into the expired air. 1 

Tests. 

The various tests for aceto-acetic acid were reviewed and sum- 
marised by W. H. Hurtley in 1913 in the " Lancet " for 26th April, and 
the following preparation of a dilute solution of sodium aceto-acetate 
was described : 

13 gm. of pure ethyl aceto-acetate are treated with 100 c.c.' of 
normal caustic soda and diluted to 500 c.c. The ester is almost 
completely hydrolysed by allowing this solution to stand for 44*5 
hours. A solution containing I gm. of aceto-acetic acid is obtained 
by diluting it to 5000 c.c., or better, by diluting 49*2 c.c. of this solu- 
tion to i litre. This solution is conveniently used for the tests. 

the excellent account by Kennaway in the "Guy's Hospital Reports," Vpl, 



120 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

(1) Gerhard? s Ferric Chloride Test. Dilute ferric chloride solution, 
added drop by drop, to about 10 c.c. of the solution gives a claret- 
red colour. 

The delicacy of this reaction is very nearly i in 100,000. 

In applying this test to urine a precipitate of ferric phosphate is 
formed. Ferric chloride is added so long as a precipitate is produced 
and the ferric phosphate filtered off. On adding ferric chloride to the 
filtrate the claret colour appears if aceto-acetic acid be present. 

The delicacy of the reaction with urine is less than given above 
on account of the presence of other substances which give a red 
colour with the iron salt. 

There is also the disadvantage in testing urine that aromatic 
compounds salicylates, antipyrine which are excreted after their 
administration, also give a violet colour with ferric chloride. 

To avoid confusion the urine is shaken with benzene or chloroform 
which removes salicylic acid. The urine is then acidified with sul- 
phuric acid and shaken with ether which extracts the aceto-acetic 
acid. The ethereal solution on shaking with dilute ferric chloride 
solution will give the claret colour. The colour disappears on stand- 
ing for 12-24 hours. Thiocyanic acid is also extracted by ether, 
but the colour of ferric thiocyanate is permanent. 

The statement that the colour if produced by aceto-acetic acid 
disappears on boiling is, according to Kennaway, not exactly true. 
The colour in either case becomes paler and redder, and in the case of 
aceto-acetic acid a reddish flocculent precipitate appears on boiling. 

(2) LegaFs Sodium Nitroprusside Test. On adding 3 drops of 
a freshly prepared 5 per cent, solution of sodium nitroprusside to 
about 10 c.c. of the solution and rendering alkaline with a few drops 
of caustic soda, a deep red colour is formed. The colour changes 
to magenta on acidifying with acetic acid. 

When applied to urine it should be remembered that creatinine 
gives a similar colour reaction (p. 1.71). 

(3) Rothercis Sodium Nitroprusside Test. 10 c.c. of the solution 
are saturated with ammonium sulphate by adding 5 gm. of the 
crystals ; 3 drops of 5 per cent, sodium nitroprusside and 2 c.c. 
of strong ammonia are then added. A fine permanganate colour is 
produced. 

i part of aceto-acetic acid in 100,000 gives the reaction in 2 
minutes, I part in 400,000 in 5 minutes. 

This reaction was described as characteristic for acetone but was 
shown by Hurtley to be far more delicate for aceto-acetic acid. 



HYDROXY-, KETO- AND DIBASIC ACIDS 121 

(4) Arnold's Test. This test depends upon the formation of a colouring 
matter from aceto-acetic acid and diazotised para-amino-acetophenone : 

CH 3 . CO . C 6 H 4 NH 2 . HC1 + HNO, = CH 3 . CO . C 6 H 4 . N : N . Cl + 2H. 2 O 

xCO. LH 3 

3 . CO . C 6 H 4 . N : NCI + CH 3 . CO . CH 2 . COOH = CH 3 . CO . C 6 H 4 . N : N . CH\ + HC1 

\COOH 

Two solutions are required : 

(a) i gm. of para-amino-acetophenone dissolved in a little water with 
the aid of 2 c.c. of concentrated hydrochloric acid and made up to 100 c.c. 

(li) i gm. of sodium nitrite dissolved in water and made up to 100 c.c. 

2 volumes of (a) and i volume of (fr) are mixed, an equal volume of the 
solution is added to the mixture and i or 2 drops of strong ammonia. A 
large excess of concentrated hydrochloric acid (15 c.c.) is added to a portion 
of the above (2 c.c.). A fine purple colour is produced. 

In testing urine, Arnold recommends filtering it through animal charcoal 
before applying the test. The delicacy is increased and more urine can be 
added to the mixture of (a) and (b} than if unfiltered urine be used. 

The sensitiveness of the test is increased according to Lipliawsky if, after 
adding the excess of hydrochloric acid, 3 c.c. of chloroform and 2-4 drops 
of ferric chloride be added and the mixture carefully shaken so as to avoid an 
emulsion. The purple colour is taken up by the chloroform forming a very 
stable solution. 

The delicacy of the test is i in 40,000. The reaction is also given by 
ethyl aceto-acetate, but not by other substances, and can therefore be directly 
applied to the urine of patients who have taken salicylic acid preparations, 
antipyrine, etc. 

(5) Rieglers Absorption of Iodine Test. This test depends upon the forma- 
tion of iodo-aceto-acetic acid, a colourless substance : 

CH 3 . CO . CH 2 . COOH + L 2 = CH 3 . CO . CHI . COOH + HI. 

The conditions necessary for this test are that the solution must be acid 
and that iodine must be present. 

10 c.c. of the solution, or of urine, are acidified with 5 drops of 30 per 
cent, acetic acid and 5 drops of iodine solution are added. This mixture 
is shaken with 2 or 3 c.c. of chloroform. No colour appears if aceto-acetic 
acid be present. 

The amount of aceto-acetic acid present in the solution can be gauged 
from the amount of iodine solution which is required to be added so as to 
form an excess over that required to combine with the aceto-acetic acid and 
so as to colour the chloroform violet. 

Ondrejovich has modified the test as follows : 

5 c.c. of the solution, or urine, are acidified with 5 drops of 50 per cent, 
acetic acid and i drop of i in 2000 solution of methylene blue added. The 
liquid should be distinctly blue. The liquid turns red on adding cautiously 
2 to 4 drops of tincture of iodine or iodine solution, and the red colour 
turns blue or green at a speed depending on the amount of aceto-acetic 
acid present. The green colour is restored if the solution has been made too 
acid. 

Indican also absorbs iodine, but its amount in urine is so small that it 
may be neglected. No other substance seems to show this reaction. 



122 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

(6) Hartley's Test. The reaction involved in this test is the conversion of 
aceto-acetic acid into isonitroso-acetone by nitrous acid. This compound 
forms salts ; the ferrous salt has a purple colour : 

CH 3 . CO . CH 2 . COOH ~> CH 3 . CO . CH : NOH - (CH 3 . CO . CH : NO) 2 Fe 
Aceto-acetic acid. Isonitroso-acetone. Ferrous salt. 

2 - 5 c.c. of concentrated hydrochloric acid and i c.c. of a i per cent, solu- 
tion of sodium nitrite are added to 10 c.c. of the solution, or of urine, and 
allowed to stand for 2 minutes. 15 c.c. of strong ammonia and 5 c.c. of 
a 10 per cent, solution of ferrous sulphate or of ferrous chloride (2 gm. iron 
in 100 c.c.) are added; the mixture is shaken and poured into a 50 c.c. 
Nessler glass where it is allowed to stand undisturbed. It is inadvisable to 
filter. A violet or purple colour is slowly produced at a speed depend- 
ing on the amount of aceto-acetic acid present. The colour deepens for several 
hours after its first appearance. 

The reaction appears at a dilution of i in 50,000. Ethyl aceto-acetate 
gives a blue colour when more nitrite than prescribed above is used ; acetone 
does not react. 

The reaction is very useful for gauging the amount of aceto-acetic acid in 
a solution or in urine. 

By diluting a i per cent, solution of sodium aceto-acetate prepared as de- 
scribed on p. 119 so that it contains '025, '02, '15 and - oi gm. of aceto- 
acetic acid in 100 c.c. and applying the test to these four solutions at the same 
time as it is applied to the unknown, and allowing them to stand 4 or 5 
hours the initially turbid liquids become clear and the unknown can be placed 
in position amongst the standards. Solutions, or samples of urine, containing 
more than the standard amounts must be diluted 10 or 20 times, and the test 
repeated. 

Estimation. 

The estimation of aceto-acetic acid is based upon its decomposition into 
acetone and carbon dioxide by heating with dilute acid. The acetone is 
separated by distillation and determined by converting it into iodoform with 
excess of -iN iodine solution and caustic soda, the excess of iodine being found 
by titration with - iN sodium thiosulphate solution : 

CH 3 . CO . CH <2 . COOH = CH 3 . CO . CH 3 + CO., 

I 2 + 2NaOH = NaOI + Nal + H 2 O 
/ CH 3 . CO . CH 3 + 3 NaOI = CH 3 . CO . CI 3 + 3 NaOH 
| CH 3 . CO . CI, + NaOH = CHI 3 + CH 3 . COONa 

I 2 + 2Na 2 S 2 O 3 = 2NaI + Na,S 4 O 6 . 

The estimation of aceto-acetic acid in urine and tissues is described on 
P- 593- 



HYDROXY,- KETO- AND DIBASIC ACIDS 123 

y3-Hydroxybutyric Acid. 

/3-Hydroxybutyric acid is readily prepared by the reduction of aceto- 
acetic acid. In this way it probably arises in the animal body. 
/3-hydroxybutyric acid contains an asymmetric carbon atom and can 
therefore occur in three forms dextro, laevo and inactive. The 
laevo form is present in urine in diabetes and under the conditions 
detailed under aceto-acetic acid. The dextro form is produced by 
the reduction of aceto-acetic acid by yeast. The inactive form is pro- 
duced by chemical reducing agents. 

/3-hydroxybutyric acid is usually obtained as a colourless syrup, 
but has been prepared in colourless crystalline plates by Magnus 
Levy; these sinter at 47-48 '5 and melt at 49-50. It is very hygro- 
scopic. 

It is easily soluble in water, alcohol, ether, ethyl acetate and 
acetone but not in benzene and petroleum ether. 

The rotation of /-/3-hydroxybutyric acid is [a\~ = - 24-12 at 
temperatures between 17 and 22 and in concentration less than 
12 per cent. 

Several salts have been prepared in a crystalline condition, e.g. 
silver, calcium, zinc. 

Detection. 

/3-hydroxybutyric acid has no reactions like aceto-acetic acid. Its 
detection is therefore indirect. 

(1) It is converted into <z-crotonic acid by loss of water either 
by heating alone or more readily by heating with dilute sulphuric 

acid : 

CH 3 . CHOH . CH 2 . COOH = CH 3 . CH=CH . COOH + H 2 O. 

, Urine is mixed with an equal volume of concentrated sulphuric acid and 
distilled ; during the process the volume is kept constant by adding water to 
the contents of the distilling flask through a tap funnel. Crotonic acid some- 
times separates out from the first portions of the distillate, but usually the 
distillate is extracted with ether. The ether is allowed to evaporate spontan- 
eously ; the crystals of crotonic acid which separate are pressed out on a 
porous plate and their melting-point determined. Crotonic acid melts at 7 1 -7 2. 
The melting-point must be ascertained since benzoic acid also passes into the 
distillate. They may be purified by solution in ether and precipitation by 
petrol ether. 

(2) By oxidation with potassium bichromate and sulphuric acid 
or with hydrogen peroxide and ferrous sulphate it is converted into 
aceto-acetic acid and then into acetone by decomposition of the aceto- 
acetic acid (see under estimation). 

The detection of hydro xybutyric acid is carried out most certainly 
by isolation of the substance by extracting it with ether in the same 
way as it is estimated (p. 597). 



AMINES. 

The amines are compounds which are derived from ammonia by 
the replacement of one, two, or three of its hydrogen atoms by alkyl 

groups, e.g. 

CH 3 NH 2 (C 2 H 5 ) 2 : NH (CH 3 ) 3 : N 

(Mono)methylamine. Diethylamine. Trimethylamine. 

Their relationship to the hydrocarbons is shown by their method 
of preparation from the alkyl halides. Primary amines may be re- 
garded as derived from hydrocarbons in which a hydrogen atom has 
been replaced by the amino (NH 2 ) group, or as derived from alcohols 
in which the hydroxyl group has been replaced by the amino group. 
Numerous amines occur in nature ; they are products of decomposi- 
tion of the amino acids, which lose carbon dioxide during putrefac- 
tion. 

Preparation. 

When an alkyl halide is treated with alcoholic ammonia, the 
halogen atom is replaced by the NH 2 group. This new compound 
again reacts with the alkyl halide, and the reaction continues until all 
the hydrogen atoms of ammonia are substituted by alkyl groups : 

CH 3 C1 + NH 3 = HCl + CH 3 . NH 2 
CH 3 C1 + CH 3 . NH 2 = HCl + CH 3 . NH . CH 3 
CH 3 C1 + (CH 3 ) a : NH = HCl + (CH 3 ) 2 : N.CH 3 . 

A mixture of the three compounds is obtained. 

The three compounds are termed respectively a primary, a 
secondary, and a tertiary amine according as I, 2, and 3 of the 
hydrogen atoms in ammonia are replaced by alkyl groups. If the 
alkyl groups are the same, they are known as simple amines, if 
different, as in methylethylamine, they are known as mixed amines. 

Primary amines are characterised by the presence of the amino 
("NH 2 ) group ; secondary amines are characterised by the presence of 
the imino (:NH) group ; tertiary amines by | N completely substituted 
by alkyl groups. 

Primary amines can also be prepared : 

(1) By the hydrolysis of isocyanates : 

CH 3 . CH 2 . N . CO + 2 NaOH = CH 3 . CH 2 . NH 2 + Na^O.,. 
Primary amines were first prepared by this reaction by Wurtz in 
1849; 

(2) By the reduction of nitriles (p. 158): 

CH 3 . CN + 2H 2 = CH 3 . CH 2 . NH 2 . 

This reaction serves for passing from a lower to a higher series. 

124 



AMINES 125 

(3) by the reduction of oximes : 

CH 3 . CH : NOH + 2 H 2 = CH 3 . CH 2 . NH 2 + H 2 O ; 

(4) by the action of bromine on an amide : 

CH 3 . CO . NH 2 + Br 2 = CH 3 . CO . NHBr + HBr, 
Acetobromamide 

and the decomposition of the bromamide by heating with excess of 
sodium hydroxide. Hydrobromic acid is removed and an isocyanate 
is formed : 

CH 3 . CO . NHBr + NaOH = CH 3 . N . CO + NaBr + H 3 O 

Methyl 
isocyanate. 

the isocyanate on hydrolysis gives the primary amine : 
CH 3 . N . CO + 2NaOH = CH 3 . NH 2 + Na 2 CO 3 . 

This reaction serves for passing from a higher series of compounds 
to a lower one containing one atom of carbon less in the molecule. 

Diamines. 

Dihalogen derivatives yield diamines in a similar manner : 

CH 2 Br CH^.NHo 

| + 2 NH 3 = | " + 2HBr 

CH 2 Br CH 9 .NH 2 

Ethylene 

diamine. 

The two compounds, putrescine and cadaverine, are products of the 
putrefaction of the corresponding diamino acids, ornithine and lysine : 
CHo.NHU CH 2 .NH 2 

I I 

CH 2 CH 2 

CH 2 CH, 

CH 2 .NH 2 CH 2 

CH 2 . NH 2 
Putrescine. Cadaverine. 

The isolation of amines from natural sources involves a com- 
plicated process of extraction, precipitation, etc., depending on the 
formation of double salts with mercuric chloride, gold chloride, 
platinic chloride, etc. Full 'details can be found in Barger's Mono- 
graph " The Simple Natural Bases ". 

Properties. 

The lower members, whether primary, secondary, or tertiary, are 
gases, the next members are liquids, the highest members are solids. 
They have a characteristic and peculiar smell, which is pungent like 
ammonia and " fishy " in the lower members. The lower members are 
easily soluble in water and closely resemble ammonia. 

Like ammonia they are strong bases which turn red litmus blue and 
unite with acids to form salts. These salts are frequently deliquescent, 
and generally soluble in water, sometimes in alcohol, chloroform and 



126 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

other organic solvents. The bases are liberated on adding sodium 
hydroxide to their solution. The salts yield double salts with gold, 
platinum chloride, etc. ; these are of use for determining the molecular 
weight of the base (p. 45). 

Reactions of a Primary Amine (Methylamine). 

(1) On treating a solution of methylamine hydrochloride, or about 
2 gm. of the solid, with sodium hydroxide, the base is liberated : 

CH 3 . NH 2 . HCl + NaOH = CH 3 . NH 2 + NaCl + H 2 O. 

Methylamine resembles ammonia in odour, but is " fishy," turns 
red litmus blue, and fumes with vapours of hydrochloric acid. It 
differs from ammonia in being combustible. 

(2) A double salt is formed on adding an alcoholic solution of gold or 
platinum chloride to an alcoholic solution of methylamine hydrochloride. 

(3) Action of Nitrous Acid. On adding dilute hydrochloric acid and 
a few drops of sodium nitrite solution to a solution of methylamine 
hydrochloride, there is an evolution of nitrogen (the gas extinguishes 
a glowing splinter) and methyl alcohol is formed : 

CH 3 . NH 2 . HCl + NaNO 2 = CH 3 OH + N 2 + NaCl + H 2 O. 
This reaction is characteristic of primary amines ; they are converted 
into the corresponding alcohol. 

(4) Carbylamine Reaction. On warming a solution of methylamine 
hydrochloride with one drop of chloroform and alcoholic sodium 
hydroxide, methylcarbylamine, or isocyanide, is formed : 

CH 3 . NH 2 + CHC1 3 + aNaOH = CH 3 . N i C + aNaCl + 3H 2 O. 

(5) Reaction with Acetyl Chloride. Primary amines react with acetyl 
chloride, benzoyl chloride, etc., to form substituted amides : 

CH 3 . NH 2 + CH 3 COC1 = HCl + CH 3 . CO . NH . CH 3 . 
These compounds are crystalline and serve for identifying the amine. 

Reactions of a Secondary Amine (Dimethylamine). 

(1) Dimethylamine is liberated from a solution of dimethylamine 
hydrochloride on treatment with sodium hydroxide : 

(CH 3 ) 2 : NH . HCl + NaOH = (CH 3 ) 2 : NH + NaCl + H 2 O. 
It is a gas with an ammoniacal and fishy odour and resembles 
methylamine. 

(2) A double salt is formed with alcoholic solutions of the heavy metals. 

(3) Action of Nitrous Acid. On adding dilute hydrochloric acid 
and some nitrite to a solution of dimethylamine hydrochloride, there 
is no evolution of nitrogen, but dimethylnitrosamine is formed : 

(CH 3 ) 2 : NH . HCl + NaNO 2 = (CH 3 ) 2 : N . NO + NaCl + H 2 O. 

Dimethylnitrosamine is a yellowish oil which is volatile with 
steam and can thus be separated. 

It is reconverted into the secondary amine by the action of con- 
centrated hydrochloric acid. 



AMINES 127 

(4) Carbylamine Reaction. Dimethylamine and secondary amines 
do not yield isocyanides with chloroform and alcoholic potash. 

(5) Reaction with Acetyl Chloride. Secondary amines form with 
acetyl chloride, etc., crystalline substituted amides which are useful for 
purposes of identification : 

CHgCOCl + HN(CH 3 ) 2 = HC1 + CH 3 CO N(CH 3 ) 2 . 

Reactions of a Tertiary Amine (Trimethylamine). 

(1) Trimethylamine is evolved on adding excess of alkali to a 
solution of its hydrochloride : 

(CH 3 ) 3 ;N : HCl + NaOH = (CH 3 ) 3 : N + NaCl + H 2 O. 

It is a gas like the primary and secondary amines with a fishy 
ammoniacal odour and is combustible. 

(2) Double salts are formed on adding alcoholic solutions of gold 
chloride, etc., to an alcoholic solution of trimethylamine hydrochloride. 

(3) Action of Nitrous Acid. Trimethylamine does not react with 
nitrous acid. 

(4) Carbylamine Reaction. This reaction is not given by trimethyl- 
amine and tertiary amines. 

(5) Reaction with Acetyl Chloride. Tertiary amines do not react. 

(6) Quaternary Ammonium Salts. Tertiary amines combine with 
alkyl iodides to form a quaternary ammonium salt : 

/ CH 3 

(CH 3 ) 2 ; N + CH 3 I = (CH 3 ) 3 : N<\ 

In this compound the halogen atom can be replaced by the 

hydroxyl group : 

,CH 3 /CH S 

(CH 3 ) 3 : N< + AgOH = Agl + (CH 3 ) 3 : N/ 

\I \OH. 

Choline. 

Choline, or hydroxy-ethyl-trimethyl-ammonium hydroxide is a 
trimethylamine derivative of ethyl alcohol, 

CH 2 OH 

CH 2 -N:(CH 3 ) 3 

OH. 

It occurs in the free state in most animal tissues and is widely distri- 
buted in plants. It is a constituent of lecithin, which is present in 
egg yolk, nervous tissue, and in seeds of plants from which it arises by 
hydrolysis. 

Preparation. 

Choline is usually prepared from egg yolk. The lecithin is extracted with 
ether or alcohol. The residue left on evaporation of the solvent is hydrolysed 
by boiling with baryta. The barium soaps (p. 99), which are formed, are 
filtered off and the solution evaporated to dryness. The choline is extracted 



128 PRACTICAL ORGANIC AND BIO-CHEMISTRY . 

from the dry residue with alcohol and precipitated as double salt with mercuric 
or platinum chloride, from which its hydrochloride is obtained by decomposi- 
tion with hydrogen sulphide. 

Synthetically choline has been prepared by the action of trimethylamine 
upon ethylene oxide (glycol anhydride, p. 1 73) : 

CH 2 . CH 2 OH 

CH 2 / CH 2 -N(CH 3 ) 3 

Properties. OH. 

Choline is a hygroscopic crystalline mass which has an alkaline 
reaction. It forms salts with acids such as choline hydrochloride, 

CH 2 OH 

CH a NICH,,), 

Cl 
and these salts form double salts with the salts of heavy metals. 

Choline has no peculiar reactions and must be isolated and 
analysed in the 'form of its salts for its identification. It is decomposed 
by boiling with alkalies yielding trimethylamine and glycol. 

Esters of Choline. 

As an alcohol choline forms esters with acids : 

(1) Acetyl choline, CH.,O OC.CH 3 

CH 2 N(CH 3 ) 3 

OH, 
has been found in ergot. 

(2) Palmityl choline, CH 2 O OC . C 15 H 31 

CH 2 . N(CH 3 ) 3 

OH, 

has also been prepared. It is easily soluble in water and readily 
hydrolysed by alkali. It has a powerful haemolytic action (Fourneau 
and Page). 

(3) Choline nitrite, CH 2 O NO and choline nitrate, CH 2 O NO 2 

CHa^-NCCH^g CKj - N(CH 3 ) 3 

OH, . OH 

resemble the toxic constituents of the toad-stool (Amanita agarica). 
The nitrite is formed by the action of nitric acid on choline and was 
formerly supposed to be the aldehyde (pseudomuscarine), 

CHO 

CH 2 . N(CH 3 ) 3 

OH. 

The ethyl ether of choiine resembles the action of natural mus- 
carine still more closely (Ewins). 

Esters of choline have marked pharmacological actions. They 
have only recently been isolated and prepared and will probably 
account for many characteristic physiological actions. 



AMIDES. 

The amides are derivatives of fatty acids, dibasic acids, etc., in 
which the hydroxyl of the carboxyl group or groups has been replaced 
by an amino (NH 2 ) group. They may also be regarded as derived 
from ammonia by the replacement of one of the hydrogen atoms by 
an acid radicle, e.g. : 

^/NH,, COOH CO.NH 2 

H.CO.NH 2 CO I I 

\NH., CO.NH 2 CO.NH 2 

Formamide. Carbamide Oxamic Oxamide. 

or acid, 
urea. 

Amides are found in nature both in animals and plants : in plants 
asparagine, the monamide of aspartic acid (p. 1-8), is perhaps the 
most common, in animals urea. Urea is formed by the liver (and 
other organs) from ammonia and carbon dioxide. It circulates in 
the blood and is excreted by the kidney. Urine contains about 
2 per cent, or 30 gm. per day. 

The amides resemble the primary amines in containing an amino 
(NH 2 ) group, but differ from them in other respects. 

Preparation. 

Amides are prepared by several methods : 

(r) by the distillation of the ammonium salt of an acid : 
CH 3 . COONH 4 = H 2 O + CH 3 . CO . NH 2 

(2] by the action of ammonia upon an acid chloride : 

CO + 2NH 3 = CO + 2HC1. 

(3) by the action of ammonia upon an acid anhydride : 

CH 3 .CO 

>0 + 2 NH 3 = CH 3 . CO . NH 2 + CH 3 . COONH 4 
CH 3 . CO/ 

(4) by the action of ammonia upon an ester : 



COOC,H 5 CO . NH 2 

+ 2NH 3 = | + 2 C,H S OH. 

OOCH CO . NH 



C 



Properties. 

The amides, except formamide which is liquid, are white crystalline 
solids generally easily soluble in water and alcohol. The substitution 
of a hydrogen atom in ammonia by acid radicles decreases its basic 
character ; the amides are consequently neutral in reaction, but they 
are weak bases and form salts only with strong acids. They are un- 
stable compounds and are readily decomposed into their constituent 
acid and ammonia by boiling with acid or alkali. 

129 9 



130 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Acetamide. 

Preparation. 

Acetamide is usually prepared by distilling ammonium acetate. About 
50 gm. are melted in a basin and poured into a distilling flask attached to a 
wide air condenser. Water, acetic acid and ammonia first pass over on heating, 
but at about 180 acetamide commences to distil. It solidifies in the receiver. 
A better yield is obtained if, previous to the distillation, the ammonium 
acetate be heated in a sealed tube to 200 for 5 hours. 

. Acetamide is more easily prepared by mixing together 5 c.c. of 
ethyl acetate and 5 c.c. of strong ammonia (sp. gr. -880) in a small 
flask, closing the flask and allowing it to stand in a warm place for 
some time. The two layers gradually form a single homogeneous 
solution. The liquid is fractionally distilled : ammonia, alcohol and 
water pass over first and subsequently acetamide, which solidifies. 

Properties and Reactions. 

Acetamide is a white crystalline solid which melts at 82 and boils 
at 222. It is easily soluble in water, alcohol and ether. It forms a 
salt with hydrochloric acid when the gas is passed into its solution in 
ether ; the salt is decomposed by water. 

(1) On boiling a solution of acetamide (about 50 c.c.) with an 
excess of caustic soda, ammonia is evolved, recognisable by its smell 
and action on red litmus paper. The boiling is continued until 
ammonia is no longer produced, and the solution is tested for acetic 
acid by carefully neutralising and adding ferric chloride (p. 97). 

(2) Acetamide is also hydrolysed by heating with acid in the same 
way; the ammonia may be detected by adding excess of magnesium 
oxide and boiling ; the acid by neutralising and testing with ferric 
chloride : 

CH 3 . CO . NH 2 + H 2 O = CH 3 . COOH + NH 3 . 

(3) On adding dilute hydrochloric acid and a few drops of sodium 
nitrite solution to a solution of acetamide, an effervescence of nitrogen 

occurs : 

CH 3 .CO . NH 2 + HNO 3 = CH 3 . COOH + H 2 O + N 2 . 

Acetic acid is formed and may -be detected in the usual manner. 
This reaction is typical of all organic compounds containing the 
NH 2 group (compare primary amines and amino acids). 

Oxamide. 

Preparation. 

Oxamide is precipitated on adding excess of ammonia to ethyl oxal- 
ate (i c.c.). The precipitate is filtered off, washed with water and dried. 

Properties and Reactions. 

Oxamide is a white solid almost insoluble in water. 

On boiling it for some time with caustic soda, it gradually dissolves 
forming sodium oxalate and at the same time ammonia is evolved. 



AMIDES 131 

Carbamide or Urea. 

Preparation. 

(a] By synthesis from ammonium cyanate. 

A solution of approximately equal parts of ammonium sulphate 
and potassium cyanate (p. 160) are boiled together and evaporated to 
dryness on the water-bath. The dry residue is extracted with alcohol, 
which dissolves the urea leaving potassium sulphate. Urea is left as a 
residue on evaporating the alcoholic solution on a water-bath. It is 
crystallised from alcohol : 

(NH 4 ) 2 SO 4 + 2KOCN = 2NH 4 OCN + K,SO. 
NH 4 OCN = CO(NH 2 ) 2 . 

(b] By other synthetical methods. 

Urea is formed by the methods described for the preparation of 
amides. 

(c] From urine. 

(i) About 25 or 50 c.c. of urine are evaporated to dryness on the 
water-bath. The dry residue is treated with about 10 c.c. of acetone 
allowing the solvent to boil on a hot water-bath. The acetone is 
poured off into a clean vessel and allowed to evaporate (not in the 
neighbourhood of a flame). Urea crystallises out in long silky needles 
and is recrystallised from alcohol. A yield of about I gm. per 50 c.c. 
of urine should be obtained. 

(ii) 100 c.c. of urine are evaporated to a syrupy consistency (to 
about ) and thoroughly cooled by immersion in cold water. Excess 
of concentrated nitric acid is added to the cold solution, the solution 
being kept cold during the addition and stirred vigorously. Urea 
nitrate is precipitated in crystalline form. The crystals are filtered off 
through glass wool, or asbestos, and freed as much as possible from 
mother liquor by pressing between sheets of paper, or the crystals are 
placed on a porous plate and drained from mother liquor. About 4 
gm. should be obtained corresponding to about 2 gm. of urea (163 gm. 
nitrate contain 60 gm. urea). Urea is prepared from the nitrate 
by mixing it with excess of barium carbonate and adding a little 
alcohol to form a paste; carbon dioxide is evolved, barium nitrate and 
urea are formed. The paste is extracted with hot alcohol, the alcohol 
filtered from barium carbonate and evaporated. Urea separates in 
needles and is recrystallised from alcohol. 

(iii) Urea may also be prepared from urine by precipitating it as 
urea oxalate by adding I gm. of oxalic acid to every 10 c.c. urine; 
the yield is aboqt I per cent. (Roaf). 

9* 



132 PRACTICAL ORGANIC AND BIO-CHEMISTRY 




Properties and Reactions. 

Urea is a white solid which crystallises from water in long prisms 

(Fig. 23). It melts at 132 and is 
easily soluble in water, alcohol, 
acetone, but not in ether or chloro- 
form. 

(i) As an amide it is a weak 
base and forms salts with strong 
acids. 

(a) Urea nitrate. If a few cry- 
stals of urea be dissolved in water 
on a watch glass and one or two 
drops of concentrated nitric acid 
be added, crystals of urea nitrate, 
are formed. These are seen to 
FIG. 23. Urea. (After Funke.) consist of rhombic six-sided plate- 

lets, often imbricated (like tiles) when examined under the microscope 
(Fig. 24). 

COfNHJ, + HNO 3 = CO(NH 2 ) 2 . HNO S . 

* (b} Urea oxalate. If a saturated solution of oxalic acid be used 
instead of nitric acid, crystals of urea oxalate are formed. Under the 
microscope they are seen to consist of short rhombic prisms (Fig. 25). 

COOH COOH 

2 CO(NH 2 ) 2 + | . [CO.(NH g )J 2 . 




COOH 



COOH 





FIG. 24. Urea nitrate. FIG. 25. Urea'oxalate. 

(After Funke.) 



AMIDES 133 

(2) Action of Sodium Hydroxide. Urea is decomposed with evolu- 
tion of ammonia by boiling its solution with excess of caustic soda : 

CO(NH 2 ) 2 + 2H,O = CO 2 + H 2 O + 2NH 3 . 

(3) Action of Nitrous Acid. On adding dilute hydrochloric acid and 
a few drops of sodium nitrite solution to some urea solution, an 
effervescence of nitrogen and carbon dioxide takes place : 

CO(NH 2 ) 2 + 2HNO 2 = CO., + 3H 2 O + aN 2 . 

(4) Action of Hypobromite. Urea is decomposed into carbon dioxide 
and nitrogen on adding sodium hypobromite to its solution : 



CO(NH 2 ), + 3NaOBr = CO. + N 2 + aNaBr + H,O 
CO, + 2NaOH = Na 2 C0 3 + H 4 O. 

Note. An effervescence also occurs when sodium hypobromite is 
added to ammonium chloride. 

(5) Biuret. On heating some urea in a test tube it melts; on 
continuation of the heating the mass becomes solid, white and opaque : 
ammonia is* evolved and a ring of sublimed cyanuric acid may be 
formed on the cooler parts of the test tube. The white residue consists 
mainly of biuret, but contains also cyanuric acid ; on treating the mass 
with water the biuret dissolves. On pouring off the water and testing 
it with caustic soda and I or 2 drops of dilute copper sulphate (i per 
cent.) a pink colour is formed (biuret reaction). 

The residue dissolves in dilute ammonia ; if barium chloride be added 
to a portion of this solution a white precipitate of barium cyanurate is 
formed ; on adding copper sulphate to another portion an amethyst- 
coloured precipitate of copper cyanurate is formed : 
NH 2 H 2 N NH 

/ \ / \ 

CO CO = CO CO + NH 3 

\ / I I 

NH 2 H 2 N NH 2 NH, 

Biuret. 

NH, H,N NH 



I + 3 NH 3 



CO CO CO CO 

NH 2 NH 2 NH NH 

NH, NH, 



CO 

Cyanuric 

'co' acid - 



(6) By adding dilute mercuric nitrate solution (i per cent), urea 
is precipitated from the solution as a white compound having the com- 
position CO(NH 2 ) 2 Hg(N0 3 ) 2 . HgO. 



134 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



rs 

A 



Estimation. 

Urea is hydrolysed by water, acids, or alkalies into carbonic acid 
and ammonia ; it is decomposed by nitrous acid or alkaline hypobrom- 
ite into carbon dioxide and nitrogen. These reactions are made use 
of in order to determine the amount of urea in a given solution. 

(i) Hypobromite Method. , 

The most simple and rapid method is the decomposition by alkaline 
hypobromite ; the carbon dioxide is absorbed by the 
alkali and the evolved nitrogen is collected and 
measured. This method is the one suggested by 
Hiifner and most frequently employed in medical 
practice. The estimation is generally carried out in 
the apparatus designed by Dupre and knowft as 
Dupre's ureometer (Fig. 26). 

The estimation is carried out as follows : 
25 c.c. of freshly-prepared hypobromite solution * 
are placed in the bottle of about 120 c.c. capacity. 
5 c.c. of urea solution are measured out with a 
pipette into a small tube which is placed in the 
bottle, taking great care not to upset the solution 
into the hypobromite. The bottle is closed with 
an india-rubber stopper and placed in cold water 
to cool. Through the india-rubber stopper a glass T-piece passes. 
One end of this is connected by rubber tubing to a graduated burette 
which is placed in a jar of water. The rubber tubing is of such a length 
that the burette can be lifted out of the water without stretching. The 
other end of the T-piece is closed by a piece of rubber tubing and a small 
clip. The burette is filled with water by opening the screw clip ; and it 
is raised or lowered until the water stands at the uppermost graduation 
and at the same level outside and inside. The clip is closed and leak- 
age in the system is tested for by raising arid lowering the burette in 
the water for at least a minute and then seeing whether the level of 
the water returns to the top graduation when the water inside and 
outside the tube are again made to stand at the same level. - When 
the system has been tested to see that it is air-tight the analysis can 
be commenced. The reading of the top graduation is noted. The 
bottle is tilted so as to upset the urea solution in the little tube into 
the hypobromite solution and it is thoroughly washed out with the latter. 
Nitrogen is rapidly evolved and displaces the water in the burette. 

1 100 gm. caustic soda are dissolved in 250 c.c. water and to the cold liquid 25 c.c. 
bromine are added. 




AMIDES 135 

The bottle must now be brought to its original temperature by placing 
it for a few minutes in a fresh supply of cold water. As soon as it is 
cool, the burette is raised till the level of the water is the same inside 
and outside and the level is read. The difference in the readings gives 
the volume of nitrogen evolved. By making the levels inside and 
outside the burette the same, this volume is measured at the atmos- 
pheric pressure. The temperature of the water and the barometric 
pressure and the tension of aqueous vapour at that temperature are 
ascertained and the volume of nitrogen is corrected to the volume at 
o and 760 mm. by the formula : 

V x 273 x (B - T)- 

(273 + t) x 760 

whefeV = volume of gas evolved, B = barometric pressure, T = tension 
of aqueous vapour (p. 608). 

The amount of urea corresponding to this volume is given by the 
equation : 

CO(NH 2 ) 2 + 3 NaBrO + aNaOH = 3 NaBr + N 2 + Na 2 CO 3 + 3H 2 O. 

60 gm. 22-4 litres ( = 28 gm.) 

i gm. 373 c.c. 

from which the amount of urea in 5 c.c. of the solution is calculated ; 
hence the amount in loo or 1000 c.c. 

Actually, however, only 354 c.c. nitrogen are evolved by I gm. of 
urea so that the method is not quite accurate ; this should be allowed 
for. 

(2) By Hydrolytic Methods. 

The most accurate methods of estimating urea are by hydrolysis. 
Urea is rapidly hydrolysed by alkali, but more slowJy by hydrochloric 
acid. 

(i) By Acid. 

The hydrolysis by acid proceeds rapidly and is complete in 
about i hour if the hydrolysis be effected at a temperature of about 
150-160 as was shown by Folin. This method has been particularly 
useful in the analysis of urine and is described on p. 55 3. 



136 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



(ii) By Urease. 

Since the discovery of the enzyme, urease, in the soy bean which 
converts urea into ammonia and carbon dioxide, this hydrolytic agent 
has displaced hydrochloric acid on account of simplicity. An apparatus 
consisting of a wash bottle, a gas cylinder or other tall vessel, and 
a receiving bottle is required (Fig. 27). The gas cylinder is fitted 
with a cork carrying a long tube reaching to the bottom and a short 

tube ; the receiving bottle is 
fitted with a cork carrying a 
tube with a bulb blown at its end 
and pierced with several small 
holes. The long tube in the gas 
cylinder is connected by rubber 
tubing to the wash bottle and 
the short tube to the bulb tube 
of the receiving bottle. Air is 
drawn as rapidly as possible 
through the apparatus by suc- 
tion with a pump attached to the 
short tube of the receiving bottle. 
The wash bottle contains 
sulphuric acid to remove am- 
monia from the air drawn 
through the vessels. The re- 
ceiving bottle contains a known 
volume (25 or 50 c.c.) of 'iN sulphuric acid coloured with a few 
drops of alizarin 'red or methyl orange solution. 5 c.c. of the solu- 
tion of urea (1-2 per cent.), 25 c.c. of water, and about 2 c.c. of kerosene 
or liquid paraffin and O'5 to I gm. of powdered soy bean are placed in 
the gas cylinder, and air is drawn through the apparatus for - to I hour 
depending on the amount of urea present. The gas cylinder should be 
placed in warm water at 40 or in a bath kept at 40 to hasten the 
decomposition of the urea. At the end of this time the parts of the 
apparatus are disjointed, I gm. of anhydrous sodium carbonate put into 
the gas cylinder so as to liberate ammonia which may be retained as 
ammonium salt, the connections are again made and the ammonia 
drawn into the receiving bottle by air suction for another half-hour. 
The excess of -iN acid in the bottle is titrated with *lN alkali. The 
amount of urea is calculated from the equation : 
CO(NH 2 )., + H 2 = CO 2 + 2NH g 




FIG. 27. 



60 
30 
i c.c. of 'iN alkali 



34 



0-003 gm. urea. 



THE AMINO ACIDS. 

The amino acids are derivatives of the fatty acids, or__of the dibasic 
acids, in which one or more of the hydrogen atoms in the chain have 
been replaced by the NH 2 group. They are both amines and acids in 
their chemical nature. 

These compounds can at the same time contain hydroxyl (OH) 
groups or thio (SH) groups in their molecule and further aromatic and 
other radicles can be substituted for hydrogen atoms in the chain. 
The main characteristic is the presence of an amino group and a car- 
boxyl group. 

They form an excessively important group of compounds, since 
they are constituents of the proteins, in which they are combined to- 
gether in various proportions (see p. 361). Amino acids have also been 
isolated from extracts of animal and vegetable tissues. The following 
have been definitely identified as constituents of proteins from which 
they are obtained by hydrolysis : 

A. Monoaminomonocarboxylic Acids. 
Glycine, or glycocoll, or amino-acetic acid : 

CH 2 (NH 2 ) . COOH. 
Alanine, or a-aminopropionic acid : 

CH 3 . CH(NH 2 ). COOH. 
Serine, or /3-hydroxy-a-aminopropionic acid : 

CH 2 OH . CH(NH 2 ) . COOH. 

Cysteine, or /3-thio-a-aminopropionic acid, formed by the decom- 
position of cystine : 

CH 2 SH . CH(NH 2 ) . COOH. 

Cystine, or dicysteine, or- di-(/3-thio-a-amiriopropionic acid) :-r 

HOOC . CH(NH,) . CH, . S S . CH 2 . CH(NH 2 ) . COOH. 
Phenylalanine, or /3-phenyl-a-aminopropionic acid : 

C 6 H 5 . CH 2 . CH(NH 2 ) . COOH. 
Tyrosine, or /3-parahydroxyphenyl-a-aminopropionic acid : 

HO . C 6 H 4 . CH 2 . CH(NH 2 ) . COOH. 
Histidine, or /3-iminazole-a-aminopropionic acid : 

CH 



HN N 

I I J 
HC = C CH 2 . CH(NH 2 ) . COOH. 

137 



138 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Tryptophan, or /3-indole-a-aminopropionic acid : 

C 8 H 6 N . CH 2 . CH(NH 2 ) . COOH. 
Valine, or a-aminoisovalerianic acid : 



CH 



CH . CH(NH 2 ) . COOH. 



Leucine, or a-aminoisocaproic acid : 

)CH . CH 2 . CH(NH 2 ) . COOH. 
CH/ 

Isoleucine, or /3-methyl-/3-ethyl-a-aminopropionic acid : 

CH 3 

>CH . CH(NH 2 ) . COOH. 
C 2 H 5 / 

B. Monoaminodicarboxylic Acids. 

Aspartic acid, or aminosuccinic acid : 

HOOC . CH 2 . CH(NH 2 ) . COOH. 
Glutamic acid, or a-aminoglutaric acid : 

HOOC . CH 2 . CH., . CH(NH 2 ) . COOH. 

C. Diaminomonocarboxylic Acids. 

Ornithine, or a-, S-diaminovalerianic acid, formed by the decom- 
position of arginine (p. 165) : 

CH 2 (NH 2 ) . CH 2 . CH 2 . CH(NH 2 ) . COOH. 

Lysine, or a-, e-diaminocaproic acid : 

CH 2 (NH 2 ) . CH 2 . CH 2 . CH 2 . CH(NH 2 ) . COOH. 

It should be noted that all these amino acids are a-amino deriva- 
tives of acids, i.e. derivatives in which a hydrogen atom in the a-carbon 
atom next to the COOH group has been substituted. 

In addition to these compounds the substances, proline and hydro xy- 
proline are found as constituents of the protein molecule (pp. 304, 306). 

Preparation. 

The amino acids are prepared by two general methods : 

(i) By the action of ammonia upon the corresponding halogen 
derivative : 

CH 2 C1 CH 2 . NH 2 

I + NH S = HC1 + | 



COOH 

Chloracetic acid. Aminoacetic acid. 



THE AMINO ACIDS 



139 



(2) By the addition of hydrogen cyanide and ammonia to aldehydes 
and the subsequent hydrolysis of the aminolpyanohydrin : 



CH 
| 
C 



CH 



NH = 



HO 



HC 



\ 
X 



OH 



CH 



CH 



HCN 



|/ 
= HC 



NH 



+ H,,0 



CH 3 
HC NH 2 

CN 



2H 



CH 

,0 = HC 
i( 



NH, + NH, 



OOH 

Alanine. 



These reactions cannot always be used In the synthesis of 
amino acids since the halogen derivative or aldehyde is sometimes 
difficult to obtain. These amino acids have therefore been prepared by 
indirect methods. A full account of the syntheses is given in the 
author's "Chemical Constitution of the Proteins,'.' Part II., 3rd edition. 
The amino acid is not always prepared most easily by synthesis, but 
by the hydrolysis of proteins, e.g. tyrosine, cystine, etc. 

Properties. 

The amino acids in a pure state are white crystalline substances 
having characteristic forms. Glycine and leucine crystals are shown in 
Figs. 28 and 29. 




FIG. 28. Glycine. FIG. 29. Leucine. 

(From Funke's " Atlas of Physiological Chemistry ".) 

They are usually easily soluble in water (cystine and tyrosine are 
exceptions) but insoluble in alcohol (proline and hydroxyproline are 
exceptions) and ether. They have generally high melting-points and 
decompose when they melt, losing carbon dioxide. 



140 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

If only one of each of the amino or acid groups be present in 
the molecule the substance is neutral, the basic group neutralising the 
acid group, but when either two amino groups or two acid groups with 
only one of the other groups are present, the substance is basic or acidic 
in character. 

The monoaminomonocarboxylic acids, which have a neutral reac- 
tion, possess a sweet taste, which is different for the different substances, 
and serves (with experienced workers) as a useful guide for defining 
the particular amino acid present in a solution. 

Except glycine, the amino acids are optically active;. some are 
dextrorotatory, others laevorotatory. An inactive mixture is formed 
by synthesis. In all cases it has been separated into its stereoisomers. 

Reactions. 

The reactions. of glycine are typical of the reactions of all the 
amino acids, but glycine does not give certain reactions which are 
characteristic of cystine (p. 143), tyrosine(p. 267), tryptophan (p. 348), 
the amino acids which contain thio or aromatic groups in their molecules. 

Reactions of Glycine. 
A. As Acid. 

(1) Glycine forms salt with bases: 

CH 2 .NH 2 NH 2 NH 2 

COONa CH 2 . COO Cu OOC . CH 2 

The copper salt is the most characteristic salt of amino acids ; it has 
a somewhat deep blue colour and generally crystallises well, so that it 
serves for the isolation of amino acids and for their identification by a 
determination of the copper content. 

On boiling a solution of glycine with excess of copper hydrate 
or copper carbonate, filtering off the excess and evaporating the solu- 
tion until it crystallises and then allowing to cool, the blue copper salt 
of glycine crystallises out. 

The depth of colour of the copper salt of glycine can be seen by 
adding a few drops of copper sulphate solution to a solution of glycine. 
The shade of colour is different to that of copper sulphate and in 
comparison deeper. 

(2) Glycine forms esters with alcohols : 

CH 2 .NH 2 CHo.NH, 

+ H 2 0. 
COOH + C 2 H 5 OH = COOC 2 H 5 

These esters are prepared by passing dry hydrogen chloride into a 
suspension of the amino acid in absolute alcohol. The amino acid is 
converted into its hydrochloride, dissolves and is esterified : 



THE AMINO ACIDS 141 

CH 2 .NH.,.HC1 
COOC 2 H 5 . 

The ester is obtained by evaporating off the alcohol, making alkaline 
with soda and extracting the ester with ether. The esters of amino 
acids are generally oily liquids having an alkaline reaction. They can 
be distilled in vacua. The complex mixture of amino acids which 
results on the hydrolysis of proteins is separated by fractional distil- 
lation of the esters in vacuo. 

B. As Amine. 

(1) Glycine forms salts with acids, e.g. : 

CH 2 .NH 2 . HCl 
COOH. 

These salts are generally crystalline and very easily soluble in water 
and are acid in reaction. 

Glutamic acid hydrochloride is soluble with difficulty in concentrated 
hydrochloric acid and is separated from a mixture of amino acids in 
this way. 

The amino acid is obtained from the salt by neutralising with soda, 
or other alkali, e.g. silver hydroxide, and crystallisation. 

(2) Ammonia is not evolved on boiling an amino acid with sodium 
hydroxide (compare acid amides). 

On boiling a solution of glycine with caustic soda, no evolution 
of ammonia can be detected by smell, litmus paper, etc. 

(3) Like amines, amino acids are decomposed by nitrous acid. On 
adding dilute hydrochloric acid and a few drops of sodium nitrite 
solution to a solution of glycine, there is an evolution of nitrogen : 

CH .NH., CH,.OH 

+ HNO = | + N, + H O. 

COOH COOH 

The corresponding hydroxy acid is formed. 

(4) On treating with acid chlorides, the amino acids yield substi- 
tuted amides : 

CH. .NH, CH 2 .NH.OC.CH 3 

+ CH 3 COC1 = | 
COOH COOH 

CH n .NH 2 CH 2 .NH.OC.C 6 H 5 

| + C 6 H 5 COC1 = | 

COOH COOH 

Benzoyl glycine 

or 
hippuric acid. 

The benzoyl derivatives are readily formed by shaking the amino 
acid in solution in sodium bicarbonate with benzoyl chloride. 



142 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



Hippuric Acid. C 6 H 5 CO NH . CH 2 . COOH. 
Hippuric acid, or benzoyl glycine, is formed synthetically by the 
kidney when benzoic acid is injected into the blood-stream of animals. 
It is a compound which is normally present in the urine of animals, 
especially herbivora. Benzoic acid is formed from the aromatic sub- 
stances of the food and is converted into hippuric acid during its 
excretion. 

Preparation from Urine. 

Hippuric acid is readily obtained from herbivorous urine by adding 
I c.c. of concentrated hydrochloric acid and 12 gm. of ammonium 
sulphate to every 25 c.c. The compound commences to crystallise 
out in about 5 minutes and the crystallisation is complete in 10- 

15 minutes. The crystals are 
filtered off and dried. They are 
generally more or less pigmented 
and are purified by recrystallisation 
from boiling water with the addi- 
tion of a small quantity of animal 
charcoal. On filtering and cooling 
the filtrate, hippuric acid crystal- 
lises out in the form of needles 
(Fig. 30). 

Properties and Reactions. 
Hippuric acid is a white crys- 
talline substance which melts at 
1 87*5. It is soluble with difficulty 
in cold water, but readily in hot water, in alcohol and ethyl acetate. 
It is only slightly soluble in ether and chloroform and insoluble in 
petroleum ether (this distinguishes it from benzoic acid). 

(1) On heating, hippuric acid melts; the mass on further heating 
turns reddish-brown, due to the decomposition of the glycine, and a 
smell of bitter almonds is produced. A sublimate of benzoic acid is 
also formed. If this be dissolved in dilute sodium carbonate solution 
and the solution be acidified with dilute hydrochloric acid, benzoic acid 
is precipitated. The crystals have a different appearance under the 
microscope to hippuric acid and give the reactions for benzoic acid 
(p. 256). 

(2) Ammonia is given off on heating hippuric acid with soda lime. 

(3) Hippuric acid is hydrolysed into benzoic acid and glycine by 
boiling with concentrated hydrochloric acid : 

C 6 H 5 CO NH . CH 2 . COOH + H 8 O = C 6 H 5 COOH + H 3 N . CH S . COOH, 




FIG. 30. Hippuric acid. (After Funke.) 



The benzoic acid crystallises out on cooling the solution and is 
separated by filtration. 

The presence of glycine in the solution may be shown by adding 
a slight excess of ammonia, boiling the solution till neutral, and adding 
a few drops of copper sulphate solution ; the deep blue colour char- 
acteristic of the copper salt of glycine is formed. 

(4) On adding ferric chloride to a neutral solution of hippuric acid, 
a reddish-brown precipitate is formed ; this is soluble in hydrochloric 
acid and the solution will deposit crystals of hippuric acid. 

Cystine. 

The amino acid, cystine, is present in greatest amount in some of 
those proteins belonging to the group of sclero-proteins, namely, the 
keratins. It has been found in the liver and other organs and occasion- 
ally forms concretions in the bladder and deposits in the urine 
(cystinuria) . The amount excreted in these conditions is small, but 
from 0-5- 1 gm. have been recorded per diem. 

Preparation. 

Cystine is prepared by the hydrolysis of keratins. Folin has 
shown that cystine can be readily obtained by the hydrolysis of wool. 

Wool is hydrolysed into its constituent amino acids by boiling 
it under a reflux condenser with concentrated hydrochloric acid for 
about five hours in the proportion of 50 gm. wool to 100 c.c. acid. 

Solid sodium acetate is slowly added to the hot solution until 
the reaction of the solution is no longer acid to congo red. A dark 
precipitate containing the cystine comes down on cooling. The 
precipitate is filtered off when the solution is cold and washed with 
cold water. It is dissolved in 5 per 
cent, hydrochloric acid, filtered from 
tarry matter and the solution is 
boiled with animal charcoal till it is 
colourless. The solution is filtered 
whilst hot and hot sodium acetate 
solution added until it is neutral 
to congo red. Cystine crystallises 
out in the typical hexagonal plates 
on cooling. 

Properties. 

Cystine crystallises in colour- 
less hexagonal plates or prisms (Fig. 
31). It is almost insoluble in water, FIG. 3 i.-Cystine. (After Funke.) 

alcohol and ether. It dissolves in dilute acids and in ammonia and 




144 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

in solutions of caustic alkali and alkali carbonates. It crystallises from 
its solution in ammonia in the typical hexagonal plates when the 
solution is allowed to evaporate. This is the most convenient way 
to obtain crystals for microscopic examination and identification by 
crystalline form. 

If cystine be heated on platinum foil it burns with a bluish-green 
flame without melting. 

The presence of sulphur in the molecule of cystine affords a means 
of readily distinguishing it from other amino acids. The sulphur 
is held in loose combination and is partially evolved as hydrogen 
sulphide on boiling with alkali : 

On dissolving some cystine in caustic alkali, or on adding caustic 
alkali to a solution of cystine, to which a drop of lead acetate solution 
has been added and boiling, a brownish or black precipitate of lead 
sulphide is formed. 



Taurine. 

Taurine, or aminoethyl sulphonic acid, is an amino acid containing 
sulphuric acid instead of carboxyl as the acid group. It has not been 
found as a constituent of proteins, but is probably derived from cystine 
or cysteine, which is converted by oxidation in the animal body into 

taurine : 

CH 2 .SH CH 2 .SO 3 H CH 2 .S0 3 H 

CH.NH 2 ->CH.NH 2 -> CH 2 . NH 2 + CO 2 

COOH COOH 

Cysteine. Cysteic Acid. Taurine. 

Taurine has been isolated from the lungs and kidneys of oxen and 
from the muscles of invertebrates. In combination with cholalic acid 
as taurocholic acid it is present in bile. 

Preparation. 

Taurine is most easily prepared from ox bile by boiling it for some hours with 
dilute hydrochloric acid. The filtrate from the insoluble anhydrides of the bile 
acids is concentrated on the water-bath to a small volume and filtered whilst 
hot from sodium chloride, etc. The solution is evaporated to dryness and 
the residue dissolved in 5 per cent, hydrochloric acid. It is precipitated 
from solution by adding 10 volumes of 95 per cent, alcohol. The crystals are 
purified by solution in acid, precipitation by alcohol and recrystallisation from 
water. 



THE AMINO ACIDS 



145 



Properties. 

Taurine crystallises in colourless four or six-sided prisms (Fig. 32). It is 
soluble in 15-16 parts of cold water, 
moreieasily in hot water. It is insoluble 
in absolute alcohol and ether, but is 
slightly soluble in cold dilute alcohol, 
more easily in hot. 

It is decomposed on boiling with 
caustic alkali yielding acetic and sul- 
phurous acids. 

It is identified by its crystalline form, 
sulphur content, and the formation of 
a white insoluble compound when its 
solution is boiled with freshly precipi- 
tated mercuric oxide. It is not precipi- 
tated by metallic salts. 

Estimation of Amino Acids. 

(i) By Titration. 

As the amino acids are neutral 
in reaction, they cannot be titrated by means of standard alkali like 
ordinary acids. They combine, however, with formaldehyde and 
yield an acid which can be titrated in this way : 

CH, . NH~^ x CH 2 . N=CH 

| ' f+ OH P . H*= I ' 
COOH V. X COOH 




FIG. 32. Taurine. (After Funke.) 



+ H O. 



The process of estimation is carried out as follows : 
10 c.c. of commercial formalin are diluted with two volumes of 
water and neutralised with -iN alkali, using 6 to 8 drops of phenol- 
phthalein in the solution as indicator. This neutralised formalin is 
added to 20 or 25 c.c. of the amino acid solution measured out with 
a pipette into a small flask or beaker. The pink colour disappears. 
iN sodium hydroxide solution is run in from a burette until a dis- 
tinct pink colour again appears. The number of c.c. of alkali used is 
noted. 

If the amino acid be known its amount in the solution can be 
calculated : 

CH 2 . (NH 2 ) . COOH NaOH 

"- -v + ' = CH 2 . (NH 2 ) . COONa + H 2 0. 

75 40 . 

i c.c. 'iN NaOH = i c.c. 'iN glycine = 0-0075 gin. glycine. 

If the amino acid be unknown, or if a mixture of amino acids be 
present, the amount is best expressed in terms of - iN acid, or in terms 
of nitrogen. . 



10 



146 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



(2) By estimation of the animo nitrogen. 

Van Slyke has shown that amino acids can be readily estimated by the 
measurement of the volume of nitrogen evolved by the action of nitrous acid 
according to the equation : 



CH 2 .NH 2 
COOH 



HN0 = 



CH 2 OH 
| ' 
COOH 



H 2 0. 



Half of the volume of nitrogen evolved corresponds to the amount of 
nitrogen in the amino acid. 

The apparatus required is shown in Figs. 33 and 34. 




cl-' 



FIG-. 33. 
From the J: Biological Chemistry, 1912, 12, 278. 

It consists of the deaminising bulb D, of 40-45 c.c. capacity, to which are 
fused (i) a tap d for purposes of emptying it ; (2) a cylindrical vessel, A, of about 
35 c.c. capacity with a mark at 7 c.c. and tap a ; (3) a 10 c.c. burette, B. The 
glass tubing is strong walled and of 3 mm. diameter, and the bores of the 
taps should also be 3 mm. The connection between D and B should be 
at least 8 mm. inner diameter so as to allow free circulation. The bulb 



THE AMINO ACIDS 



is connected through a 3-way tap c with a large gas burette, F, of 150 c.c. 
capacity, the upper portion holding 40 c.c. and graduated in tenths. The gas 
burette is connected to a Hempel pipette of special shape (Fig. 34). The 




FIG. 34. 

whole apparatus is arranged as in Fig. 34, so that the bulb D and Hempel 
pipette can be fastened by a specially bent hook to a wheel driven by a 
water or electric motor. The wheel should make 300-500 revolutions per 
minute. 



148 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

In the original arrangement the parts of the apparatus are suspended by wires from 
clamps on a metal stand. The whole apparatus is thus moveable instead of being per- 
manently fixed as in Fig. 34. 

By this means the bulb and pipette can be shaken mechanically so as to 
hasten the reaction and facilitate the absorption of the nitric oxide evolved 
by the action of acid upon the nitrite. For convenience, rubber tubing is 
fastened to the tap of the bulb D, the burette B and the tap c, and arranged 
so that the liquids can be passed straight into the sink. It is also con- 
venient to have a small handle h on the driving wheel so as to shake the vessels 
for short periods by hand. 

The manipulation is carried out in three stages : 

(1) Displacement of the air in the apparatus. 

The gas burette F is filled with water, the air being allowed to escape 
through c. 

One bulb of the Hempel pipette is filled with a solution of alkaline per- 
manganate. 1 By lowering the levelling bulb of the burette the air in the 
Hempel pipette is drawn into the gas burette until permanganate just reaches 
the tap. This air is driven out through c and water fills the gas burette 
and connection as far as c. The tap c is turned so as to shut off this part 
of the apparatus and to be in connection with D and the exit tube. 7 c.c. 
of -glacial acetic acid are put into A and run into D. About 30 c.c. of 
sodium nitrite solution (30 per cent.) are put into A and run into D. 
Sufficient nitrite solution should be used so that excess stands in A above the 
tap. The tap c is closed by a quarter \urn, and the bulb is shaken for a few 
seconds. The nitric oxide which is evolved drives the liquid into A. By 
opening the tap c to the exit, the liquid from A again fills the bulb D and 
the gas passes out at c. The tap c is again closed and the shaking repeated. 
The gas is let out and the shaking again repeated. In thi,s way the air is 
driven out of the bulb D. Finally, a space of about 20 c.c. should be left in the 
bulb by shaking and driving the liquid into A ; the tap a is closed. 

Tap c is opened to connect D and F. 

(2) Decomposition of the amino substance. 

10 c.c. or less of the solution to be analysed is placed in the graduated 
burette B ; any excess can be run off. The desired volume is run into D. 
The burette B need not be graduated ; the desired amount can be introduced 
with a pipette into B, run into D, and B washed with a little water, which is 
also run into D. 

D is shaken for 3-5 minutes. Only in a few cases is longer shaking 
necessary. If frothing occurs, as in the case of hydrolysed proteins, B can be 
washed out and a little capryl alcohol introduced through B. 2 If the re- 
action takes some time to complete, it is allowed to stand for the required 
time and finally shaken for 2 minutes. 

The gas given off passes into the burette F. The residual gas in the bulb 
is driven into the burette F by opening the tap a and filling D with solution 
from A as far as the tap c. 

(3) Absorption of nitric oxide and measurement of the nitrogen. 

The tap of the burette F is turned so as to connect it with the Hempel 
pipette. The gas is driven into the pipette by raising the levelling tube. The 
Hempel pipette is shaken gently for 1-2 minutes, and the gas returned to 
the burette and measured. It is driven over into the permanganate again and 
returned to the burette. If no change in volume has occurred the volume is 

1 50 gm. KMnO 4 + 25 gm. KOH per 1000 c.c. 

2 In later forms of apparatus another tube is sealed to D for introducing the capryl 
alcohol. 



THE AMINO ACIDS 149 

recorded at the temperature and barometric pressure. The weight of nitro- 
gen corresponding to the amount of nitrogen in the amino substance is cal- 
culated or may be taken from the table compiled by Van Slyke and reproduced 
on p. 605. 

In this table the allowance has been made for the evolution of only half 
of the volume. The weights are therefore those for the amino nitrogen. 

During the measurement of the gas the bulb D can be emptied and washed. 
A fresh solution of permanganate is not required for every analysis ; sufficient 
is present in the Hempel pipette for 10-12 estimations. 

This pattern of apparatus has many advantages over the older pattern. 
Some 6-8 estimations can be made in an hour. 

Micro Apparatus. 

Van Slyke l has also described a smaller form of apparatus for use when 
only small quantities of material are available, such as in the analysis of the 
amino acids in blood, tissues, etc. I ts_ dimensions ar.e,l: 

(1) the gas burette : 10 c.c., the upper part, of 2 mm. diameter, 
measuring 2 c.c., and graduated in ^ c.c., the lower part wider and graduated 
in ^Q c.c. ; a gas burette holding a total volume of 20 or 30 c.c. is more 
advantageous as frequently more than 10 c.c. of nitric oxide is evolved ; 

(2) the deaminising bulb: 11-12 c.c. ; 

(3) the burette : 2 c.c. 

The quantities of reagents required are 10 c.c. of sodium nitrite solution 
and 2 '5 c.c. of acetic acid for which the correction for impurities amounts to 
06-' 1 2 c.c. 

It is not necessary to have a smaller Hempel pipette for permanganate. 
With the micro apparatus the reagent in it lasts for a considerable time. 

The solution to be analysed may be introduced into the burette with a 
pipette and the burette is washed with 6 or 7 drops of water. 

The error in the estimation need not exceed '005 mg. when 2 c.c. or 
less gas is measured ; with more gas '01 mg. 

With the micro apparatus one-fifth of the amount required for the 
macro apparatus can be analysed. Not only is there an advantage 
economically with reagents, but also the apparatus is less fragile. 

o - 5 mg. of amino acid can be analysed with an accuracy of i per 
cent. It is slightly more rapid : at 15-20 4 minutes' shaking suffice, at 
20-25 3 minutes, above 25 2-2*5 niinutes. 

It is essential that the burettes be accurate and the stopcocks be tight. 
The burettes are tested by weighing the water they deliver ; the stopcocks by 
submitting them to a pressure of a column of water i metre high. 

The apparatus may be cleaned with a mixture of potassium bichromate 
and sulphuric acid. 

A still smaller form of apparatus may be used. 2 See 3rd edition " The 
Chemical Constitution of the Proteins," Part I. 

1 J. Biol. Chem., 1913, 16, 121. "Ibid., 1915, 23, 408. 



BETAINES. 



Betaine, the first member of the series of trimethylamine derivatives of 
fatty acids, or trimethylamine acetic acid, has been known since 1863 and 
was given its name by Scheibler in 1866, who isolated it from the sap of the 
sugar beet (Beta vulgaris) and from molasses. 

Betaine also occurs in a large number of plants and has been isolated 
from shrimp extract and extracts of other invertebrates ; -05 per cent, has 
been separated from ox kidney. 

Betaine, and the other members of the group, when dried at 100 lose a 
molecule of water. Their .constitution is most probably that of the an- 
hydride : 

CH 2 - N(CH 3 ) 3 CH 2 -N(CH 3 ) 3 

COOH OH CO O 

Betaine. 



Other members of the group are : 



y-w-butyro-betaine. 
CH 2 -N(CH 3 ) 3 

CH 2 



CO O 



Trimethylhistidine. 
CH NH 



Carnitine or Novaine, or 
o-hydroxy-y-butyro-betaine. 
CH 2 N(CH 3 ) 3 



CHOH 



CO O 

Ergothioneine, or 
Thiolhistidine-betaine. 
CH NH V 



CH 2 

CH-N(CH 3 ) 3 
CO O 

Stachydrine or 
Proline-betaine. 
CH 2 CH 2 

CH 2 CH CO 

VLJ 



CH_N(CH 3 ) 3 
CO O 

Betonicine and Turicine or 
hydroxyproline-betaine. 
CHOH CH., 

CH CH CO 



N 



CH, CH, 



/ \ 
CH 3 CH, 



150 



BETAINES 




Hypaphorine or 
Tryptophan-betaine. 

CH 2 -CH'N(CH 3 ) 3 
CO-0 



Trigonelline or 
Nicotinic acid-betaine. 




CH 3 



The betaines can be prepared by the methylation of the amino acids with 
methyl iodide and alkali but are usually obtained by separation from the 
natural sources. 

They are colourless crystalline solids and have a feebly basic character 
forming salts with mineral acids, e.g. betaine hydrochloride, and double salts 
with salts of the heavy metals. 

A full account of these compounds is given in Barger's " The Simpler 
Natural Bases ". 



CYANOGEN COMPOUNDS. 

CN 
(i) Cyanogen. | 

CN. 

Cyanogen, as it contains only the two elements carbon antf nitro- 
gen, is the simplest carbon compound containing nitrogen. It is 
present in the gases of the blast furnace and is formed on passing 
electric sparks between carbon poles in an atmosphere of nitrogen, 
and on treating ammonium oxalate or oxamide (p. 1 30) with dehy- 
drating agents, e.g. phosphorus pentoxide : 

COONH 4 CONH 2 CN 

I -> I =2H 2 0+ | 

COONH 4 CONH 2 CN. 

Preparation. 

Cyanogen is most readily prepared by heating mercuric cyanide : 

CN 
Hg(CN) 2 = | + Hg. 

CN 

On heating a small quantity of mercuric cyanide in a dry test 
tube, white fumes are given off which condense on the cooler parts 
of the tube. On igniting the gas at the open end it will be observed 
to burn with its characteristic pink flame. 

Cyanogen is also prepared by heating a concentrated solution of 
I part of potassium cyanide with 2 parts of copper sulphate dissolved 
in 4 parts of water. A yellow precipitate of cupric cyanide, Cu(CN) 2 , 
is first formed and this decomposes into cyanogen and cuprous 
cyanide, CuCN : 

4 KCN + 2CuSO 4 = Cu 2 (CN) 2 + (CN) 2 + 2K 2 SO 4 . 

Properties. 

Cyanogen is a colourless gas with a peculiar pungent smell and 
has been condensed to a liquid. It burns with a pink flame forming 
carbon dioxide and nitrogen. It is easily soluble in water and 
alcohol and is intensely poisonous. 

152 



CYANOGEN COMPOUNDS 153 

Reactions. 

Cyanogen and the other compounds of this group resemble the 
halogens very closely in their properties, thus on passing cyanogen 
into alkali it is converted into alkali cyanide and cyanate : 

(CN) 2 + 2KOH = KCN + KOCN + H 2 O 
C1 2 + 2KOH = KC1 + KOC1 + H 2 O. 

Cyanogen is converted by hydrolysis, on boiling with acids, into 
oxalic acid and ammonia : 

CN COOH 

| * 4 H 2 0= I +aNH 3 . 

CN COOH 

The compounds of this group which contain the CN radicle are 
termed nitriles, because on hydrolysis with acids they are converted 
into the corresponding acid : cyanogen is the nitrile of oxalic acid or 
oxalonitrile. 

(2) Hydrogen Cyanide, or Prussic Acid. HCN. 

Hydrogen cyanide is present in numerous plants, e.g. in laurel 
leaves, bitter almonds, cherry and peach kernels, usually ift combina- 
tion with glucose and benzaldehyde as the glucoside amygdalin (p. 215), 
which is hydrolysed by acids, or by enzymes in the plant, into its con- 
stituents. It is formed by the oxidation of glycine and other amino 
acids ; it may thus be produced in animals and plants : the latter prob- 
ably then combine it with glucose to form a glucoside, the former 
apparently combine it with sulphur to form sulphocyanide or thio- 
cyanate, which is present in saliva and other secretions. 

Preparation. 

A dilute solution of hydrogen cyanide is obtained by distilling 
potassium ferrocyanide, or a cyanide, with dilute sulphuric acid : 

2 K 4 Fe(CN) 6 + 3 H. 2 S0 4 = 6HCN + K 2 FeFe(CN) 6 + 3 K 2 SO, 

Potassium 
ferrous ferrocyanide. 

A small flask is connected with a condenser by means of a bent 
tube ; the open end of the condenser is dipped into water in a 
test tube containing a drop of strong caustic soda. 10 c.c. of a 
cold saturated solution of potassium ferrocyanide and 20 c.c. of 20 
per cent, sulphuric acid are put in the flask. On heating, hydrogen 
cyanide distils over and is converted into sodium cyanide. Its 
presence is tested for by converting it into Prussian blue by heating 
in alkaline solution with a ferrous salt, acidifying and adding a drop 
of ferric chloride. The distillation should be carried out in the fume 
cupboard. 



154 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Note. Carbon monoxide is obtained when anhydrous potassium ferro- 
cyanide is heated with concentrated sulphuric acid. 

Hydrogen cyanide is also obtained by dehydrating ammonium 
formate, or formamide, with phosphorus pentoxide. 

Pure anhydrous hydrogen cyanide is prepared by distilling potas- 
sium cyanide with moderately concentrated sulphuric acid, passing 
the gas over anhydrous calcium chloride and collecting the distillate 
in a receiver cooled by ice. 

Properties. 

Pure hydrogen cyanide is a colourless, mobile liquid having a 
specific gravity of '697 at 18. It becomes a crystalline solid at - 15 
and it boils at 26' 5. It has a peculiar smell, resembling that of oil 
of bitter almonds and is intensely poisonous. It burns with a violet 
flame and is easily soluble in water and alcohol. 

It is a very weak acid and turns blue litmus only a faint red. 

Reactions. 

Aqueou's solutions of hydrogen cyanide are unstable and slowly 
undergo decomposition into ammonium formate : 
HCN + aH,O = HCOONH 4 . 

The pure acid is also rapidly decomposed by concentrated hydro- 
chloric acid. Formamide is first formed and this passes into formic 
acid and ammonium chloride : 

HCN + H 2 O = HCONH 2 
HCONH 2 + HC1 + H 2 O = HCOOH + NH 4 C1. 

Hydrogen cyanide is thus the nitrile of formic acid. 

The salts of hydrocyanic acid are decomposed in the same way on 
boiling their aqueous solutions : 

If about 20 c.c.- of a I per cent, potassium cyanide solution be 
boiled for some time it is converted into ammonia and potassium 
formate. The ammonia is readily detected by its action on red 
litmus paper and the formate may be detected by testing the solution, 
after the ammonia has been given off, with (i) ferric chloride and with 
(2) mercuric chloride, as on p. 95. 

Hydrogen cyanide in alcoholic solution is reduced by sodium to 
methylamine : 

HCN + 2H = CH,.NH 2 . 



CYANOGEN COMPOUNDS 155 

Metallic Cyanides. 

Hydrogen cyanide resembles hydrochloric acid in behaviour, form- 
ing salts with alkalies and metallic hydroxides. The alkaline salts 
crystallise like sodium and potassium chloride ; the silver salt is white 
and insoluble in water and acids, but soluble in ammonia. Silver 
cyanide, unlike silver chloride, is decomposed by boiling with mineral 
acids forming hydrogen cyanide. 

The chief salt is potassium cyanide which is used extensively for 
extracting gold and in electroplating. 

Preparation of Potassium Cyanide. 

Potassium cyanide is prepared by fusing potassium ferrocyanide : 

K 4 Fe(CN) 6 = 4 KCN + N 2 + FeC 2 . 

On heating about I gm. of potassium ferrocyanide in a crucible to 
redness, allowing to cool, extracting the mass with water and filtering, 
the solution will be found to contain potassium cyanide as shown by 
the test on p. 156. 

In this reaction the whole of the nitrogen of the ferrocyanide is not 
obtained as cyanide ; if potassium ferrocyanide be fused with potassium 
carbonate a mixture of cyanate and cyanide is formed : 

K 4 Fe(CN) 6 + K 2 CO 3 = sKCN + KOCN + CO 5 + Fe. 

If potassium ferrocyanide be fused with sodium, a mixture of potassium 
and sodium cyanides results : 

K 4 Fe(CN) 6 + 2 Na = 4 KCN + 2NaCN + Fe. 

The cyanides dissolve in water leaving the iron and are obtained by 
evaporation. 

Large quantities of cyanide are now prepared by two other methods : 

(1) by heating sodium with charcoal in a current of ammonia at 400. 
Sodamide is formed and converted into sodium cyanamide (p. 159) : 

2NH 3 + Na., = aNaNH 2 + H 2 
2NaNH 3 + C = Na 2 CN 2 ~+ 2H 2 . 

On raising the temperature to 800 the sodium cyanamide and charcoal 
react, forming sodium cyanide : 

Na,CISL + C = 2NaCN. 

Sodium cyanide is formed directly at 800 : 
NaNH 2 + C = NaCN + H 2 . 

(2) by heating beet-sugar molasses to 1000. At this temperature the 
trimethylamine is decomposed into hydrogen cyanide and methane : 

, (CH 3 ) 3 N = HCN + 2CH 4 . 

Sodium cyanide is prepared by passing the gases into sodium hydrate and 
evaporating the solution. 

Metallic gold dissolves in potassium cyanide solution in the presence of 
air or other oxidising agent : 

2Au + 4KCN + H 2 O + O = 2KAu(CN) 2 + 2KOH, 
forming a double cyanide from which the gold is obtained by electrolysis. 



156 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Double Cyanides. 

The alkali cyanides dissolve the insoluble cyanides of silver, gold 
and other heavy metals forming the double cyanides : 

KCN + AgCN = KAg(CN) 2 . 

On adding a few drops of a I per cent, solution of potassium 
cyanide to a few drops of silver nitrate solution, a white precipitate of 
silver cyanide is formed : 

KCN + AgNO 3 = AgCN + KNO 3 . 

On adding more potassium cyanide solution, the precipitate dis- 
solves forming the double salt. The double salt is decomposed with 
the formation of silver cyanide by adding dilute nitric acid : 
KAg(CN) 8 + HNO 3 = KN0 3 + HCN + AgCN. 

The double cyanides are extensively used in electroplating; on 
electrolysis the compound is decomposed with the formation of potas- 
sium and Ag(CN) 2 ions at the cathode and anode respectively. The 
double cyanide is reduced at the cathode, the silver being deposited. 
Silver is used as the anode and is dissolved by the Ag(CN) 2 ions 
forming 2AgCN, which is soluble in potassium cyanide giving the 
double cyanide. The reactions are thus : 

KAg(CN) 2 -> K + Ag(CN) 2 Ag + Ag(CN) 2 -^ 2 AgCN 

K + KAg(CN) 2 -> aKCN + Ag AgCN + KCN -> KAg(CN) 2 . 

Tests for Cyanides. 

(1) The smell of hydrogen cyanide, either before or after acidifying 
the solution with dilute nitric acid and warming, is an indication of 
the presence of a cyanide. 

(2) The formation of silver cyanide, by adding silver nitrate to a 
solution acidified with nitric acid, or better by holding a drop of silver 
nitrate on a glass rod in the vapour of the solution in (i), also in- 
dicates the presence of a cyanide. 

(3) The formation of Prussian blue by boiling the solution with a 
ferrous salt and alkali, acidifying and then adding a drop of ferric 
chloride is characteristic. 

(4) The formation of ferric thiocyanate by adding to the solution 
a drop of ammonium sulphide, evaporating to dryness, acidifying with 
hydrochloric acid and adding a drop of ferric chloride is the most 
delicate way of detecting a cyanide. 

If a cyanide be present with other organic substances, e.g. in 
stomach contents, etc., in cases of poisoning, the material is acidified 
with a non-volative organic acid, such as tartaric acid, and distilled. 
The distillate is tested for hydrogen cyanide as above. 



CYANOGEN COMPOUNDS 157 

Complex Cyanides. 

The cyanides of sodium and potassium are converted into ferro- 
cyanides on boiling their solutions with ferrous salts in alkaline solution. 
This reaction is made use of in testing for nitrogen in organic com- 
pounds. 

Potassium Ferrocyanide. K 4 Fe(CN) 6 . 

Preparation. 

Potassium ferrocyanide is prepared by fusing together protein re- 
sidues, such as blood, or horn, or leather with scrap iron and potassium 
carbonate. The fused mass is extracted with water and the yellow 
solution which results is evaporated down until it crystallises. 

Potassium ferrocyanide is also prepared from the hydrogen cyanide, 
which is a bye-product in the manufacture of coal gas. The hydrogen 
cyanide is absorbed by iron oxide in the "purifiers". By boiling this 
material with lime, calcium ferrocyanide is formed ; potassium ferro- 
cyanide is obtained by treating it with potassium carbonate. Some- 
times the hydrogen cyanide is converted into .sodium ferrocyanide by 
passing it into an alkaline solution of ferrous salts. 

Properties and Reactions. 

Potassium ferrocyanide forms large yellow crystals (yellow 
prussiate of potash) which are easily soluble in water. 

(1) On adding concentrated hydrochloric acid to a saturated solu- 
tion of potassium ferrocyanide, hydroferrocyanic acid is thrown down 
as a white precipitate. It turns blue on filtering owing to decomposi- 
tion and oxidation. 

(2) On adding a drop of ferric chloride solution to a solution of 
potassium ferrocyanide, a blue precipitate (Prussian blue) is formed : 

3 K 4 Fe(CN) 6 + 4 FeCl 3 = Fe 4 [Fe(CN) 6 ] 3 + i 2 KCI. 

(3) Other metals also form insoluble ferrocyanides if their solutions be 
added to a solution of potassium ferrocyanide. 

Zinc ferrocyanide is white, copper ferrocyanide is reddish-brown, uranium 
ferrocyanide is brown. 

Sodium Nitroprusside. Na 2 Fe(CN) 5 . NO. 

Potassium ferrocyanide is converted into nitroprusside by the action of 
moderately concentrated nitric acid ; potassium nitrate crystallises out and 
is removed. The solution on neutralisation with sodium carbonate yields 
sodium nitroprusside on evaporation. 

Sodium nitroprusside, Na<}Fe(CN) 5 . NO + 2H 2 O, forms beautiful red 
rhombic prisms which are easily soluble in water ; the solution is a sensitive 
reagent for sulphides, acetone, etc. 



158 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Potassium Ferricyanide. K 3 Fe(CN) 6 . 

Preparation. 

Potassium ferricyanide is formed by the oxidation of potassium 
ferrocyanide by means of chlorine or bromine : 

On adding a slight excess of bromine water to some potassium 
ferrocyanide solution and boiling off the excess of bromine the colour 
changes from yellow to brown-red, and on evaporation of the solu- 
tion red crystals of potassium ferricyanide are obtained. 

Properties and Reactions. 

Potassium ferricyanide, or red prussiate of potash, is a red 
crystalline substance soluble in water, giving a reddish-yellow solution. 

(1) On adding ferric chloride to its solution it turns dark brown. 

(2) On adding a solution of a ferrous salt to its solution, a deep- 
blue precipitate (Turnbull's blue) is formed : 

2 K 3 Fe(CN) 6 + 3 FeS0 4 = Fe 3 [Fe(CN) 8 ] 2 + 3K 2 SO 4 . 

(3) In alkaline solution it is decomposed into potassium ferro- 
cyanide and oxygen ; it acts therefore as an oxidising agent : 

2K 3 Fe(CN) 6 + aKOH = 2 K 4 Fe(CN) 6 + H 2 O + O. 

Thus, if some litharge be added to a solution of potassium 
ferricyanide rendered alkaline with sodium hydroxide and warmed, it 
becomes brown owing to the formation of lead peroxide, PbO 2 . The 
solution may be tested for ferrocyanide by filtering, acidifying and 
adding ferric chloride which gives a precipitate of Prussian blue. 

Alkyl Cyanides Nitriles. 

Preparation. 

When potassium cyanide is treated with an alkyl iodide (methyl, 
ethyl, etc., iodide) the alkyl cyanide is obtained : 

KCN + CH 3 I = KI + CH 3 CN. 

These compounds are also formed by treating the amides of the 
corresponding acid with phosphorus pentoxide : 
CHCONH' = HO + CHCN. 



Properties and Reactions. 

The lower members of the series are liquids with peculiar smell, and 
are more or less soluble in water ; the higher members are solids. 

Like hydrogen cyanide they are hydrolysed by acids into the cor- 
responding acid : 

CH 3 CN + 2H 2 O = CH 3 COOH + NH 3 . 

On reduction they are converted into amines : 
CHCN + 2H 2 = CH . CH 2 . NH.,. 




CYANOGEN COMPOUNDS i$g 

Alkyl Isocyanides. 

If silver cyanide be treated with alkyl iodides, isocyanides, com- 
pounds isomeric with the above, are formed : 

CH 3 I + AgCN = CH 3 NC + Agl. 

It would thus appear that silver cyanide has a different structure 
to potassium cyanide or that in the reaction a rearrangement occurs : 

KCN - KNC -> AgNC. 
These compounds are also formed by heating primary amines (p. 6l) 

with chloroform and potash : 

CH 3 NH 3 + CHC1 3 + 3KOH = CH..NC + 3KC1 + 3H 2 O. 

They are liquids with an abominable smell : on hydrolysis they 
give the corresponding amine and formic acid : 

CH 3 NC + 2H 2 O = CH 3 NH a + HCOOH. 

Cyanogen Chloride. C1CN. 

If mercuric cyanide, potassium cyanide, or hydrogen cyanide be treated 
with chlorine, cyanogen chloride is obtained : 

KCN + CL, = C1CN + KC1. 

This compound, which is a liquid, polymerises on standing into solid 
cyanuric chloride, C1 3 C 3 N 3 . Potash converts liquid cyanogen chloride into 
potassium cyanate, and solid cyanuric chloride into potassium cyanurate : 
C1CN + 2KOH = KOCN + KC1 + H 2 O. 
C1 3 C 3 N 3 + 6KOH = K 3 3 C 3 N 3 + 3 KC1 + 3H 2 O. 

Cyanamide. NH 2 . CN. 

Cyanamide is formed on passing cyanogen chloride into an ethereal 
or aqueous solution of ammonia : 

C1CN + NH, = NH 2 . CN + HC1. 
It is more readily obtained by the action of mercuric oxide on 

thiourea : 

,NH 2 

CS/ + HgO = HgS + H 2 O + NH 2 . CN. 

\NH 2 

Calcium Cyanamide is manufactured by passing nitrogen over calcium 
carbide at 1000. 

CaC 2 + N 2 = CaCN 2 + C. 

Cyanamide is prepared from this salt by decomposition with aluminium 
sulphate, filtration, evaporation in vacua and crystallisation from ether. 

Sodium cyanamide is prepared as described on p. 155. 

Cyanamide forms colourless hygroscopic crystals, easily soluble 
in water, alcohol and ether, and melting at 40. It forms salts with 
strong acids and also with bases. The salts with acids are decom- 
posed by water. The calcium salt is frequently employed as an arti- 
ficial manure. 

Cyanamide is readily hydrolysed by the action of acids forming urea : 

/NH 2 
NH .CN + H 2 O = C0( 

\NH 2 . 

By the action of hydrogen sulphide it is converted into thiourea, 
and by ammonia it is converted into guanidine. 



160 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Cyanamide is used in the synthesis of creatine and arginine (p. 165). 
These compounds behave like cyanamide in giving urea on hydrolysis. 

Cyanic Acid. HOCN. 

Cyanic acid is formed by distilling cyanuric acid. 

A small quantity of cyanuric acid is placed in a small bulb blown upon 
the end of a glass tube and the glass tubing is bent at an angle. The end of 
the glass tube leads into a test tube surrounded by a freezing mixture. On 
heating, the cyanuric acid is decomposed and cyanic acid collects in the test 
tube as a liquid. 

Cyanic acid is only stable below o and is a mobile, volatile liquid with 
a strong acid reaction and with a smell resembling that of glacial acetic acid. 
It produces blisters upon the skin. 

Pure cyanic acid, on exposure to the air, polymerises to cyanuric acid, 
with explosiveness ; a small quantity, such as prepared above, polymerises with 
a cracking noise. 

It is an extremely unstable substance ; its aqueous solution above o de- 
composes giving carbon dioxide and ammonia : 

HOCN + H 2 O = CO 2 + NH 3 . 

It dissolves in alcohol forming the ethyl ester of allophanic acid : 
H 2 N . CO . NH . COOC,H 5 . 

Its salts are more stable than the free acid. 

Potassium Cyanate, KOCN. 

Potassium cyanate is formed by the oxidation of potassium cyanide 
by a variety of oxidising agents, air, lead oxide, potassium perman- 
ganate, , or sodium hypochlorite. 

It may be prepared from potassium cyanide as follows : 

About i gm. of potassium cyanide is heated in a crucible in a fume 
cupboard until it melts. Lead oxide is added in small quantities to 
the fused mass so long as visible reduction occurs. The mass, when 
cold, is extracted with water ; potassium cyanate crystallises out on 
evaporation. 

It may also be conveniently prepared from potassium ferrocyanide : a 
mixture of 4 parts of potassium ferrocyanide and 3 parts of potassium 
bichromate are carefully heated in an iron dish, avoiding the formation of 
ammonia. The potassium cyanate is extracted with water. 

Potassium cyanate forms shining leaflets or quadratic plates. It 
dissolves readily in cold water, with difficulty in hot alcohol. 

In aqueous solution it is unstable and decomposes forming ammonia 
and potassium carbonate. 

This decomposition can be seen with the solution prepared above ; 
carbon dioxide is evolved on adding sulphuric acid, and the presence 
of ammonia may be shown by making alkaline, warming and testing 
with red litmus. 



CYANOGEN COMPOUNDS 161 

Ammonium Cyanate. 

Ammonium cyanate is prepared by bringing cyanic acid into contact with 
dry ammonia. 

It is a white crystalline powder soluble in water ; the aqueous solution 
on evaporation yields urea (p. 131). 

The salts of cyanic acid with the heavy metals are insoluble and are 
formed from potassium cyanate by double decomposition. 

Cyanuric Acid. H 3 O 3 C 3 N 3 . 
Cyanuric acid has probably the constitution, 

C. OH 



N N 

I 
HO.C C.OH, 

\/ 

N 

and is prepared from urea : 

(1) by heating it (p. 133); 

(2) by passing chlorine over it at 130-140 ; 

(3) by heating it with a solution of carbonyl chloride in toluene at 190-230.. 

3 CO(NH 2 ) 2 = 3 NH :; + H.jOsC.iN,. 

Cyanuric acid crystallises from water with 2 molecules of water of 
crystallisation in large rhombic prisms. It is soluble in 40 parts of cold 
water, more easily in hot water and alcohol. It is decomposed by boiling 
with acids into carbon dioxide and ammonia. 

It is a tribasic acid forming soluble salts with the alkali metals and in- 
soluble salts with the heavy metals ; the copper salt is violet in colour. 

Alkyl Isocyanates and Alkyl Cyanates. 

Alky] isocyanates are formed on heating silver cyanate with an alkyl 
iodide : 

AgNCO + CH 3 I = CH 3 NCO + Agl. 

These compounds are also obtained when potassium cyanate is distilled 
with alkyl potassium sulphate : 

KOCN + CH 3 . SO, . OK = CH 3 . NCO + KO . SO. 2 . OK. 

A rearrangement of the atoms of the molecule has occurred on heating. 

The alkyl isocyanates are volatile liquids with a strong, disagreeable, 
suffocating odour. They boil without decomposition and are soluble in ether. 
They polymerise on standing forming isocyanuric esters. 

Their structure is shown by their conversion into amines by the action of 
potash : 

CH 3 . NCO + H 2 = CH 3 . NH 2 +-CO 2 . 

The alkyl cyanates are obtained by the action of cyanogen chloride on 
sodium alkoxide : 

C1CN + NaOCH 3 = CH 3 OCN + NaCl. 

They are colourless ethereal liquids, which at once polymerise to cyanur- 
ates. 

Alkyl cyanurates are obtained from sodium alkoxide and cyanuric chloride. 

II 



162 PRACTICAL OR'GANIC AND BIO-CHEMISTRY 

Thiocyanic Acid. HSCN. 

Thiocyanic acid, or sulphocyanic acid, has long been known to be 
pfesent in the form of its salts in saliva and it has also been found 
in other secretions of the animal body. The amount is always very 
small. 

Thiocyanic acid is obtained by distilling its potassium salt with 
dilute sulphuric acid, or by the action of dry hydrogen sulphide upon 
mercuric thiocyanate. 

Thiocyanic acid is a gas and, like cyanic acid, is easily condensed 
in a freezing mixture to a liquid which has a penetrating and acrid 
odour and is soluble in water and alcohol. It is an unstable substance ; 
on removal from the freezing mixture it polymerises to a yellow amor- 
phous body. It forms soluble salts with the alkali metals and insoluble 
salts with the heavy metals. 

Potassium Thiocyanate. 

Potassium thiocyanate is readily prepared from potassium cyanide 
by evaporating its solution with flowers of sulphur or ammonium 
sulphide : thus : 

10 c.c. of a i per cent, solution of potassium cyanide are boiled for 
some minutes with flowers of sulphur and filtered. The presence of 
potassium thiocyanate is shown by the red colour which is formed on 
the addition of a drop of ferric chloride solution. 

Potassium thiocyanate crystallises from alcohol in long colourless 
prisms, which deliquesce in the air. 

Sodium thiocyanate is also deliquescent. 

Ammonium thiocyanate is prepared in a similar manner to the 
potassium salt. It is a product obtained in the manufacture of coal 
gas from ammonium salts, hydrogen cyanide and sulphur. 

It is usually prepared by the action of carbon bisulphide upon 
alcoholic ammonia, or ammonia under pressure. Ammonium thio- 
carbamate is formed, and this is decomposed by steam into ammonium 
cyanate and hydrogen sulphide : 



aNH 3 + CS 2 = rX 



NH 2 

\S.NH 4 



CS( = H 2 S + NH 4 SCN. 

\S.NH 4 

Ammonium thiocyanate crystallises in prisms which are easily soluble 
in water and alcohol. 
Metallic Thiocyanates. 

(1) Ferric thiocyanate is formed on adding ferric chloride solution 
to a soluble thiocyanate ; ferric thiocyanate is soluble and has an 
intense red colour and is used in detecting thiocyanates. 

(2) Silver thiocyanate is thrown down as a white curdy precipitate 
on adding silver nitrate to a solution of a thiocyanate. 



CYANOGEN COMPOUNDS 163 

Alkyl Thiocyanates and Alkyl Isothiocyanates. 

Allyl isothiocyanate occurs in combination in the glucoside, sinigrin, of 
mustard seed. The allyl thiocyanate is formed on contact with water, the 
glucoside being decomposed by the enzyme, myrosin. To the allyl thio- 
cyanate is due the pungent smell and taste of mustard. 

Alkyl thiocyanates are prepared by the action of alkyl halides upon 
potassium thiocyanate : 

KSCN + C 2 H 5 I = C 2 H 5 SCN + KI. 

Alkyl isothiocyanates, or mustard oils, are obtained by heating alkyl 
thiocyanates, isomeric change occurring : 

C 2 H 5 SCN -> C 2 H 5 NCS. 

They are also prepared by the action of primary amines on carbon bisul- 
phide in alcoholic or ethereal solution, and then heating the aqueous solution 
of the thiocarbamate so formed with mercury chloride or ferric chloride : 

/NH . C 2 H 5 
CS + 2C 2 H 5 .NH 2 = CSC 

\SH.NH_. C H 5 
NH . CH 



/ 
/ 

SH.NH 2 .CH 



CS = H 2 S + NH 2 . C 2 H 5 + C 2 H 5 NCS. 



^ 

The alkyl isothiocyanate distils over with steam. 

The alkyl thiocyanates are oily liquids, insoluble in water, possessing a 
garlic-like smell. 

The alkyl isothiocyanates are pungent smelling liquids, the odour of which 
provokes tears. They are generally called mustard oils on account of the 
occurrence of allyl isothiocyanate in mustard. They boil at a lower tempera- 
ture than the isomeric alkyl thiocyanates and are almost insoluble in water. 

Constitution of Alkyl Thiocyanates and Isothiocyanates. 

In the thiocyanates the alkyl group is joined to the sulphur atom ; in the 
isothiocyanates it is attached to the nitrogen atom as is shown by the following 
reactions : 

(1) Thiocyanates on reduction give the primary amine and a mercaptan : 

C 2 H 5 SCN + 3 H 2 = C 2 H 5 SH + CH 3 NH 2 . 

(2) Thiocyanates on oxidation give a sulphonic acid : 

C 2 H 5 SCN + O -> C 2 H 5 S0 3 H. 

(3) Thiocyanates on treatment with alcoholic potash give alcohol and 
potassium thiocyanate : 

C 2 H 5 SCN + KOH = KSCN + C 2 H B OH. 

These reactions point to the attachment of the alkyl group to the sulphur 
atom. 

(1) Isothiocyanates on heating with hydrochloric acid yield amines : 

C 2 H 5 NCS + 2H 2 O = C 2 H 5 NH 2 + CO S + H 2 S. 

(2) Isothiocyanates on reduction yield a primary amine and thioform- 
aldehyde : 

C 2 H 5 NCS + 2H 2 = C 2 H 5 NH 2 + HCSH. 

(3) Isothiocyanates are converted into isocyanates by boiling their solu- 
tion in alcohol with mercuric oxide or chloride : 

C 2 H 5 NCS + HgO = C 2 H B NCO + HgS. 
The alkyl group is thus attached to the nitrogen atom. 

ii* 



1 64 PRACTICAL ORGANIC AND BIO-CHEMISTRY 
GUANIDINE AND ITS DERIVATIVES. 

Guanidine was first obtained by the oxidation of guanine (p. 294), and is 
also a product of the oxidation of arginine (p. 165) with permanganate. Its 
formation by the oxidation of arginine explains its formation in the oxidation 
of proteins, which contain arginine. Guanidine has been found in self-digested 
solutions of pancreas and in extracts of vetch seedlings and in the sap of the 
beet. 

Guanidine is formed by the action of ammonia upon ortho-carbonic ester 
in a manner similar to the preparation of urea from ethyl carbonate or car- 
bonic ester (p. 129) : 



OCo Hr, /NH, 




NH 



X OC H 



Ortho-carbonic Hypothetical. Guanidine. 

ester. 

It is generally prepared by the action of ammonia upon cyanamide : 

NH 2 

H 2 N . C=N + NH 3 = C=NH 

NH 2 . 

In practice, this reaction is most conveniently accomplished by heating 
ammonium thiocyanate to 180-190. Thiourea and cyanamide are formed. 
The cyanamide reacts with ammonium thiocyanate to yield guanidine thio- 
cyanate : 



NH 4 SCN. 

^NH 2 .CN 
* NH 2 

NH 4 SCN + NH 2 CN = C^NH 

NH 2 . HSCN. 

Guanidine is thus urea in which the O atom has been replaced by the 
=NH (imino) group. 

Guanidine is a deliquescent crystalline substance, easily soluble in water and 
alcohol. It is a strong base ; its solutions have an alkaline reaction and absorb 
carbon dioxide from the air forming guanidine carbonate, (CH 6 N 3 ) 2 . H 2 CO S , 
which is soluble in water, but not in alcohol. Guanidine also forms salts 
with other acids ; the n : trate, CH 5 N 3 . HNO 3 , is not easily soluble and consists 
of large plates which melt at 214. The chief salt is the picrate which melts 
at 315 and is very insoluble in cold water. This salt is used for the isolation 
of guanidine from solution and for its estimation. Double salts are formed 
with gold chloride and cadmium chloride. 

Guanidine is hydrolysed by alkalies into urea and ammonia : 

NH 2 NH 

/ / " 

C=NH + H O = C NH 2 + NH,. 

X NH. X 



GUANIDINE AND ITS DERIVATIVES 165 

Methyl Guanidine and Dimethyl Guanidine. 

/NH . CH :i /NH . CH 3 

C NH C NH 

^NH 2 ^NH.CH.,. 

Methyl guanidine has been isolated from meat and meat extracts, in which 
about -i per cent, is present. It has also been isolated from urine. 
Dimethyl guanidine has been isolated from urine. 
Methyl guanidine is probably derived from creatine. 

NH, NH 2 

I I 

Arginine. NH c NH . CH 2 . CH 2 . CH 2 . CH . COOH. 

Arginine, or S-guanidine-tf-aminovalerianic acid is a constituent 
of the protein molecule ; in some proteins the protamines it forms 
over 80 per cent, of the molecule (p. 432). It was discovered in ex- 
tracts of seedlings. 

Preparation. 

Arginine is most easily prepared by the hydrolysis of edestin, the 
protein of hemp seed, 1 or from seedlings of the yellow lupin. 2 Its con- 
stitution was proved by its synthesis from cyanamide and ornithine : 
NH 2 NH 2 

CN + H 2 N CH 2 CH 2 CH 2 CH COOH = 
NH 2 NH 2 

NH C NH CH 2 CH 2 CH 2 CH COOH. 

Properties and Reactions. 

Arginine is a white crystalline substance, easily soluble in water, 
but insoluble in alcohol. It melts with decomposition at 207 '5. It 
is a strong base and absorbs carbon dioxide from the air forming 
arginine carbonate. It also forms salts with other acids, of which the 
nitrate is the principal one. 

Natural arginine is optically active, the synthetical product is 
optically inactive but has been separated into its stereoisomers. 

Like guanidine it is hydrolysed by alkalies into urea and ornithine : 
NH 2 NH 2 

NH;=C NH . CH 2 . CH 2 . CH 2 . CH . COOH + H 2 O = 

NH 2 NH 2 

' ! I 

NH 2 C O + NH 2 . CH 2 . CH 2 . CH 2 . CH . COOH. 

This hydrolysis of arginine occurs in the liver by the action of the 
enzyme arginase. A portion of the urea in the urine is probably 
derived in this way. 

iSee Ber., 1905, 38, 4187. 

2 See Zeit. Physiol. Chem., 1902, 35, 314. 



1 66 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

NH 2 

Creatine. NH=C N CH 2 .cooH. 

CH 3 

Creatine, or methyl guanidine acetic acid, is a constituent of all 
vertebrate muscle and is most abundant in voluntary muscle. The 
creatine content of the muscle of any particular species is remarkably con- 
stant ; thus -5 per cent, in rabbit's, '45 per cent, in cat's, -39 per cent, 
in man's, "37 per cent, in dog's muscle. It is not present normally in 
human urine, but appears under certain conditions, e.g. when carbo- 
hydrates are absent from the food, in diabetes and other diseases. It is 
present in the urine of infants and children and in that of women after 
menstruation, and during and after pregnancy. Creatine is normally 
present in bird's urine. 

Preparation. 

Creatine is more readily prepared from meat or from urine than by 
synthesis. 

(1) From meat. 

Finely minced meat is extracted several times with hot water. The 
aqueous solution is boiled to remove coagulable proteins and filtered. The 
filtrate is treated with lead acetate so long as a precipitate is formed and again 
filtered. Excess of lead is removed from the solution by means of hydrogen 
sulphide and the filtrate from lead sulphide is evaporated down to a small 
volume. Creatine crystallises out as the solution stands. It is filtered off 
and washed with 88 per cent, alcohol. 

(2) From urine. 

A solution containing chiefly creatinine is obtained from urine as described 
on p. 169. Folin 1 has given the following method of preparing creatine 
from it : 

The solution is evaporated to dryness on the water-bath and the residue 
dissolved in 15-20 parts of boiling water. Twice as much 95 per cent, 
alcohol is added .to this boiling solution. Nearly the whole of the creatine 
separates out in a few hours ; it is filtered off after standing in a cool place for 
about 1 2- 1 6 hours and washed with dilute alcohol (i part water, 2 parts 
alcohol). 

The filtrate and washings are placed in a large flask and kept in a water- 
bath at 80-90 for a week. The creatinine is converted into creatine ; the 
conversion of the creatinine can be controlled by removing samples and esti- 
mating it (p. 171), but the weight of the flask and its contents must be known. 

The solution is evaporated to dryness ; the residue is dissolved in boiling 
water so as to make a 10 per cent, solution of the creatinine still present and 
2 volumes of alcohol added as above. This procedure can be continued 
until nearly the whole of the creatinine is converted into creatine. About 
30 per cent, of material is lost, but a yield of creatine equal in weight to that 
of the creatinine is obtained. 

J J. Biol. Chem., 1914, 17, 463. 






GUANIDINE AND ITS DERIVATIVES 



S. R. Benedict l gives a simpler method for preparing creatine from 
creatinine zinc chloride : 

100 gm. of creatinine zinc chloride are heated to boiling in a large basin 
with 700 c.c. of water; 150 gm. of pure powdered calcium hydrate are added 
and the mixture is stirred and boiled for twenty minutes. The hot mixture is 
filtered and the residue washed with hot water. The filtrate is treated for a 
few minutes with hydrogen sulphide to remove zinc and filtered through a 
folded paper. It is acidified with about 5 c.c. of glacial acetic acid and 
rapidly boiled down to about 200 c.c. The solution is allowed to cool and 
kept at o. The crystals of creatine are filtered off, washed with cold water 
and alcohol, and dried. The filtrate is kept to recover unchanged creatinine. 
The creatine is recrystallised by solution in seven times its weight of boiling 
water, which is allowed to cool slowly and stand for some hours at o. It is 
filtered off, washed with alcohol and ether, and dried in the air for thirty 
minutes. The creatine contains water of crystallisation which is lost by ex- 
posure to the air. It may be dehydrated by heating for some hours at 95. 
The yield is about 18 gm. 

About 50 per cent, of the creatinine is not converted in this process, but 
it is not advisable to boil longer with the lime as loss of creatinine occurs. 

The creatinine is recovered by diluting the solution with alcohol and 
treating with about 50 c.c. of a 30 per cent, alcoholic solution of zinc chloride. 

Creatine has been synthesised from cyanamide and sarcosine, or 
methyl-glcyine : 

NH 2 NH 2 

C ==N + HN . CH 2 . COOH = NH=C N CH 2 . COOH. 
CH 3 CH 3 

Properties and Reactions. 

Creatine separates from water in colourless, transparent, hard 
rhombic prisms (Fig 35), con- 
taining one molecule of water of 
crystallisation, which is given off 
at 1 00, the specimen becoming 
opaque. It has a peculiar bitter 
taste, is easily soluble in hot water, 
less so in cold (i in 74), almost 
insoluble in alcohol and insol- 
uble in ether. Its solution has 
a neutral reaction. It forms 
salts with acids which are very 
unstable. 

It is hydrolysed by alkalies 
into urea and sarcosine and may, 




FIG. 35. Creatine. (After Funke.) 



therefore, like arginine, contribute to the quantity of urea in urine : 



J J. Biol. Chem., 1914, 18, 186. 



168 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

NH 2 NH 2 

NH == C N CH 8 . COOH + H 2 O = NH 2 C = O + HN . CH . COOH. 

I I 

CH 3 CH :! 

It is converted by boiling with acids into creatinine. 

Conversion of Creatine into Creatinine. Estimation of Creatine. 

It has long been known that creatine on heating with acids is converted 
into its anhydride creatinine : 



NH 2 NH . 

HN = C N CH 2 COOH = H 2 O + HN = C N CH 2 ! CO. 
CH 3 CH 3 

The reverse action takes place when creatinine solutions are heated to 
boiling, or on boiling with alkalies. 

The solution containing creatine of a concentration of about cri per cent, 
is heated on a water- bath in a flask covered with a watch glass with an equal 
volume of normal hydrochloric acid for 3-4 hours, or the aqueous solution 
is heated in a flask covered with tin-foil in an autoclave at 130-140 for half 
an hour exclusive of the time taken in heating up and cooling the autoclave. 

Benedict finds it most convenient to boil down to dryness the solution of 
creatine to which has been added an equal volume of N hydrochloric acid, 
the final evaporation being done on a water-bath. 

Solid creatine on heating at 130-140 for 3 hours in an autoclave is 
also converted into creatinine. The mixture of creatine and creatinine ob- 
tained in the above preparation from urine may be converted into creatinine 
by adding one to two drops of water for every gram of substance and heating 
in an autoclave for 3 hours at 130-140. 

The estimation is effected as is described for creatinine (p. 1 7 1) by neutral- 
ising, adding picric acid and soda and comparing the colour against a 
standard. 



GUANIDINE AND ITS DERIVATIVES 169 

Creatinine. 

Creatinine is present in all mammalian urines. The amount of 
creatinine in human urine varies from about 075-1 -5 gm. per diem. 
It is not present in muscle or it is present only in traces. It has been 
found in wheat, rye and other crops and has been isolated from 
cultivated soil. 

Creatinine is present in meat extracts. Its presence is probably 
due to the action of the acids of muscle extracts upon creatine during 
evaporation. 

Preparation. 

Creatinine is obtained from creatine by boiling with acids. It is 
most conveniently prepared from human urine by Folin's method and 
it is advantageous to use as large quantities of fresh, not ammoniacal, 
urine as possible. 

60-80 gm. of picric acid (i.e. 6-8 gms. per litre of urine) are dissolved 
in 400 c.c. of hot alcohol and are added with stirring to 8 litres of urine. 
The double picrate of creatinine and potassium is precipitated. After the 
mixture has stood for 12-24 hours the liquid is decanted or syphoned off 
and the precipitate drained and washed with cold water on a Buchner funnel. 

100 gm. of dry potassium carbonate and 750 c.c. of water are added 
to 500 gm. of dry picrate, the mixture is thoroughly stirred for 10 minutes 
and allowed to stand for 1-2 hours. The precipitate is filtered off on 
a Buchner funnel and the sediment washed two or three times with small 
quantities of water. The nitrate containing the creatinine is transferred to 
a large jar and 100 c.c. of 99 per cent, acetic acid (i c.c. per gm. of carbonate) 
are added in such a way that it drops upon the foam which is formed and 
breaks it up. The acid solution which is wine-red in colour is treated with 
one-fourth of its volume of a concentrated alcoholic zinc chloride solution. 1 
A large precipitate of creatinine zinc chloride is formed at once, if sufficient 
zinc chloride has been added, and is filtered off. 

This double salt is decomposed by lead hydroxide which must be freshly 
precipitated and is prepared from lead nitrate. 4-5 gm. of lead nitrate 
per gm. of creatinine zinc salt a're dissolved in 7-8 parts of cold water and 
precipitated by adding 2 c.c. of strong ammonia per gm. of nitrate. The 
lead hydroxide settles rapidly, the liquid is syphoned off, and the hydroxide 
washed three times with large quantities of water. 

The creatinine zinc chloride is placed in 30 parts of water previously 
heated to boiling and contained in a flask filled not more than two-thirds, and 
again heated to boiling so as to dissolve as large a portion as possible. The 
lead hydroxide in suspension in water is added in portions of about one-fifth 
of the requisite quantity and the mixture is boiled after each addition. The 
solution is boiled for half to one hour after all the hydroxide has been added 
so that the precipitate becomes granular, cooled and filtered. The filtrate 
should be clear, but if not clear may be treated with hydrogen sulphide and 
again filtered. The filtrate is freed from lead by hydrogen sulphide and eva- 
porated to dryness. The dry residue consists of creatinine and creatine. It 

1 One part of zinc chloride dissolves in about one part of alcohol ; a sediment of zinc 
hydroxide may be brought into solution by adding a few drops of hydrochloric acid. 



i;o PRACTICAL ORGANIC AND BIO-CHEMISTRY 

is converted into creatinine by heating with 1-2 drops of water per gm. of 
solid in an autoclave at 130-140 for 3 hours. A little boiling water is 
poured over the residue and alcohol added. Pure creatinine (98-100 per 
cent.) is obtained. 

S. R. Benedict 1 finds the following procedure for preparing creatinine 
from urine more convenient than that described by Folin. At least 10 litres 
of urine should be used. 

18 gm. of picric acid per litre of urine are dissolved in about 50 c.c. of 
hot alcohol and added with stirring to the urine, which must be fresh. The 
potassium creatinine picrate which is precipitated is filtered off after about 
12 hours upon a Buchner funnel and washed once or twice with, saturated picric 
acid solution. The dry, or nearly dry, picrate is decomposed by stirring it in 
a mortar with concentrated hydrochloric acid (60 c.c. per 100 gm. picrate) for 
3-5 minutes. The picric acid is filtered off by suction through a hardened 
filter paper and washed once or twice with a small quantity of water. The 
filtrate is transferred to a flask and neutralised with excess of magnesium 
oxide (commercial, heavy) which is added in small quantities at a time whilst 
the liquid is kept cold with water. The liquid turns bright yellow when it 
is neutral and litmus may be used to test the reaction. The mixture is 
filtered by suction and the residue is washed once or twice with a little 
water. The filtrate is acidified with a few c.c. of glacial acetic acid (paying 
no attention to a precipitate which may form), diluted with 4 volumes of 
alcohol and filtered after about 15 minutes from a small precipitate (chiefly 
calcium sulphate). The filtrate is treated with a 30 per cent, alcoholic 
solution of zinc chloride, using 3-4 c.c. per litre of urine employed, stirred well 
and allowed to stand for 12 hours. The precipitate of creatinine zinc chloride 
is filtered off and washed once, with water, 50 per cent, alcohol and 95 per 
cent, alcohol. A nearly white product is obtained in a yield of i'S-i'8 
gm. per litre of urine. 

Though a 10 per cent, loss is incurred, the creatinine zinc chloride must 
be recrystallised : 10 gm. of the compound are treated with 100 c.c. of water, 
about 60 c.c. of N sulphuric acid are added and the mixture is heated till a 
clear solution is obtained; 4 gm. of animal charcoal are added and the 
boiling is continued for i minute. The solution is filtered and the residue 
is washed with water. The filtrate is transferred to a beaker and treated with 
3 c.c. of zinc chloride solution and 7 gm. of potassium acetate dissolved in a 
little water. After 10 minutes the solution is diluted with an equal volume of 
alcohol and allowed to stand in a cool place. The crystals which separate 
contain some potassium sulphate ; this is removed by stirring them up twice 
with their weight of water, washing with water and alcohol. 8*5-9 gm. 
of recrystallised salt are obtained. 

Creatinine is prepared from the recrystallised zinc chloride compound by 
placing the powdered substance in a dry flask and treating it with seven times 
its weight of concentrated aqueous ammonia and warming slightly until a clear 
solution is obtained, avoiding loss of ammonia as far as possible. The flask 
is stoppered and kept at o. Pure creatinine crystallises out in a yield of 
60-80 per cent. If it be slightly brown, it may be recrystallised from boiling 
alcohol, or by dissolving it in five times its weight of ammonia as described 
above. 

J J. Biol. Chem., 1914,8, 1813. 



GUANIDINE AND ITS DERIVATIVES 



171 



Properties. 

Creatinine separates from hot saturated solutions in colourless, 
shining prisms (Fig. 36), from cold saturated solutions in plates or 
prisms containing 2H 2 O. It has 
a caustic taste and its solutions 
react slightly alkaline. It is sol- 
uble in 1 1 -5 parts of cold water, 
more easily in hot water, in 625 
parts of cold absolute alcohol and 
more easily in hot alcohol. 

It behaves as a strong alkali 
displacing ammonia from its salts. 
It forms salts with acids and double 
salts with salts of the heavy metals, 
of which the zinc chloride double 
salt is the most characteristic. 

Reactions. 




FIG. 36. Creatinine. (After Funke.) 



Creatinine has two reactions by which its presence in a solution 
can be detected, e.g. in urine : 

(1) Sodium Nitroprusside Reaction (Weyl}. 

A few drops of a dilute freshly prepared solution of sodium nitro- 
prusside are added to a small quantity of the solution (urine) and dilute 
sodium hydroxide is added drop by drop. The solution becomes 
red in colour and in a short time changes to yellow. If the yellow 
solution be acidified with glacial acetic acid and heated, the solution 
becomes green and a deposit of Prussian blue forms on standing. 

Note. Acetone gives a similar colour reaction, but the colour 
changes to purple on acidifying. It is advisable to remove acetone, if 
present, by boiling the solution before testing for creatinine. 

(2) Picric Acid Reaction (Jaffi?'}. 

To the solution containing creatinine (urine) some saturated picric 
acid solution is added and the mixture made alkaline with sodium 
hydrate. The solution becomes deep orange in colour which remains 
permanent for some hours. 

One part of creatinine in 5000 can be detected by this reaction. 

Note. Aldehyde, acetone and other compounds also reduce picric 
acid in the cold ; glucose, fructose, urea, etc., reduce it on warming 
(Chapman). 1 The picric acid is converted into picramic acid, amino- 
dinitrophenol and diamino-nitrophenol. 



1 Analyst, 1909. 



172 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Estimation of Creatinine. 

Folin has shown that creatinine can be accurately estimated by means of 
Jaffe's reaction. The orange-red colour produced is matched in a colori- 
meter against the colour of ^N solution of potassium bichromate, or better 
against a solution of creatinine or creatinine zinc chloride which is treated 
with the same amount of picric acid and caustic soda (see pp. 555, 562). 

The Biological Relationship of Creatine and Creatinine. 

From the chemical point of view the presence of creatinine in 
urine would be explained by its formation from the creatine in muscle 
by an enzyme in the animal body. An enzyme which can convert 
creatine into creatinine has been described as being present in the liver. 
The physiological experiments do not bear out this relationship. The 
daily amount of creatinine in urine is constant in amount and is de- 
rived mainly from the tissues, but very small amounts come also from 
the food. The addition of creatine to the food does not increase the 
amount of creatinine in urine. If the amount of creatine eaten be 
i gm. it is not excreted as such or as creatinine ; if more than I gm. be 
eaten, the excess over I gm. is excreted as creatine. The amount of 
creatinine eliminated is also related to the muscular condition ; less is 
eliminated at rest, more at work. 

Folin has suggested the following explanation : 

Creatine is a normal constituent of the living muscle. At death 
the muscle substance breaks down giving creatine. 'Normally the 
muscle substance during its life processes gives rise to creatinine. 
During fasting, in fevers, etc., the normal breakdown is accompanied 
by the breakdown into creatine. Creatine, taken as food, is absorbed 
into the muscle and the excess is eliminated. The presence of traces 
of creatine in urine, or of creatinine in muscle, arise chemically by the 
action of acids or alkalies. 



DI-, TRI- AND POLYHYDRIC ALCOHOLS. 

In compounds containing two or more carbon atoms in their mole- 
cule not only can one of the hydrogen atoms be replaced by OH 
groups, but also 2, 3, 4, etc., so long as two OH groups are not 
attached to I carbon atom (see aldehydes). Thus : ., 



CH 2 OH 


CH 2 OH 


CH 2 OH 


CH 2 OH 


CH 2 OH 


CH 2 OH 


CHOH 


CHOH 


CHOH 


CHOH 


Glycol. 


| 


1 


1 


| 




CH 2 OH 


CHOH 


CHOH 


CHOH etc. 




Glycerol. 


CH 2 OH 


CHOH 


CHOH 






Erythritol. 


CH 2 OH 


CHOH 








Adonitol. 


| 










CH 2 OH 










Mannitol. 



These compounds have the properties of primary and secondary 
alcohols. 

Glycol. 

Glycol is the first member of the series of polyhydric alcohols, and 
is prepared from ethylene dibromide : 

CH 2 Br CH,OH 

| " + 2KOH = 2 KBr + | 
CH 2 Br CH 2 OH. 

Glycol is a colourless syrupy liquid with a sweet taste ; it boils 
at 198 and has a sp. gr. of 1-1297 at o. It is miscible with water 
and alcohol in all proportions, but is very slightly soluble in ether. It 
is very hygroscopic and takes up water from the atmosphere forming 
a hydrate C 2 H 6 O 2 . 2H 2 O. 

Glycerol. 

Glycerol, a trihydric alcohol, is the chief member of the series. It 
occurs in nature in the free state, but mainly in combination with fatty 
acids in the form of esters the fats or glycerides from which it is pre- 
pared by hydrolysis (p. 177). 

Glycerol is a thick, colourless, very hygroscopic liquid without 
smell but with a sweet taste. It boils and distils under atmospheric 
pressure at 290 but undergoes slight' decomposition ; in vacuo it can 
be distilled without decomposition. If kept at o for some time it 
crystallises and the crystals melt at 17. It has a sp. gr. of 1-265 
at 15. 

173 



174 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

It mixes in all proportions with water and alcohol but is insoluble 
in ether and chloroform. 

Glycerol dissolves alkalies and many inorganic salts ; its presence 
in a solution prevents the precipitation of cupric hydrate by alkalies. 
This behaviour is common to other compounds which contain 
several OH groups in their molecule, such as tartaric acid and the 
sugars. 

Tests. 

(1) On account of its oily appearance it may be mistaken for fat 
On moistening a piece of paper with a drop of glycerol, the paper be- 
comes marked as with a grease spot, but on washing the paper with 
water and drying the spot is removed. 

(2) On heating a few drops of glycerol in a dry test tube with 
potassium bisulphate or anhydrous phosphoric acid the pungent odour 
of acrolein (p. 104) is noticed : 

CH 2 OH . CHOH . CH 2 OH = CH 2 : CH . CHO + 2H 2 O. 

(3) On adding an aqueous solution of glycerol (about 20 percent.) 
to a 5 per cent, solution of borax, to which sufficient phenolphthalein 
solution has been added to produce a distinct red colour, the red colour 
is discharged, but on boiling it returns if excess of glycerol has not 
been used. This is known as Dunstan's test. 

Any polyhydric alcohol may give this reaction. Ammonium salts 
also decolorise the solution, but the colour does not return on 
heating. 

Several polyhydric alcohols, containing 4 carbon atoms, erythritols, exist. 
They differ from one another in their stereochemistry (see tartaric acid). 
The inactive, internally compensated, compound occurs naturally. 

Adonitol is the natural pentahydric alcohol : the other stereoisomeric 
forms are obtained by the reduction of the pentoses. 

Mannitol. 

The hexahydric alcohols can exist in ten stereoisomeric forms, eight 

of which are known. They are closely related to the monosaccharides 

from which they are obtained on reduction and to which they give rise 

on oxidation. Mannitol and sorbitol are found in nature, the others 

lave been prepared from the hexoses. 

Mannitol is obtained from manna, the juice of the manna ash, by 
extracting it with hot water or hot dilute alcohol and crystallising the 
solution. It crystallises from water in prisms, from alcohol in silky 
needles and melts at 165-166. It requires 6 parts of water for 
solution ; it is very slightly soluble in cold alcohol and almost in- 
soluble in ether. 



FATS AND OILS. WAXES. LECITHINS OR 

LIPINS. 

Fats and oils are present as reserve food material in most animal 
and vegetable tissues. They are the esters of the higher fatty acids, 
especially palmitic, stearic and oleic acids, with glycerol. All these 
glyceryl esters have similar names in which the name of the fatty acid 
describes the nature of the fat, e.g. butyrin, caproin, palmitin, olein. 

CH 2 0-OC . C 17 H 35 

CHO OC . C 17 H 35 

CH 2 0-OC . C 17 H 35 
Stearin. 

There is no chemical distinction between oils and fats ; the solid esters 
are fats, the liquid esters are oils. The consistency of a fat or oil de- 
pends upon the nature of its constituents. Beef and mutton tallow 
which are hard solids contain chiefly palmitin and stearin. Lard and 
human fat which are soft solids contain more olein. Palm oil consists 
principally of palmitin. The vegetable oils, such as olive, cotton seed 
and linseed oils contain chiefly olein or other esters of unsaturated acids. 

Fats are economically of great value as food, as illuminating agents, 
as lubricating agents, for soap making and for other purposes. 

Waxes, which in appearance somewhat resemble fats, are chemically 
very different. They are esters of the higher alcohols, cetyl alcohol, 
CjgHggOH, myristic alcohol, C 3o H 6l OH, and cholesterol, C 27 H 45 OH, with 
the higher fatty acids, e.g. carnauba wax contains ceryl and myricyl 
alcohols and cerotic and carnaubic acids ; wool wax or lanolin contains 
cholesterol ; spermaceti is the palmitic acid ester of cetyl alcohol. 

Lecithins are present in all animal and vegetable cells and accom- 
pany the fat, but are intimately associated with the life processes, ex- 
isting in loose combination with protein. Lecithin has the composition 
and properties of a fat, but contains in addition phosphoric acid and 
choline (p. 127), 
CH 3 OC . R CH 2 OC . R 

CHO OC . R' CHO OC . R' 



,OCU 2 . CH 2 . N(CH 3 ) 3 OH ,O . CH 2 . CH 2 . NH 2 

CH 2 OP( CH 2 O OP( 

\OH \OH 

Lecithin. Kephalin ? 

Kephalin has a similar composition but contains amino-ethyl alcohol 
in the place of choline. These two lipins are generally found to- 
gether in most tissues and are difficult to separate. They both 
.contain a saturated acid and an unsaturated acid. About 10 per cent. 
of lecithin is present in egg yolk ; liver and blood contain about 2 per 
cent., vegetable tissues from '25 to 1-5 per cent 

175 



176 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

The substance, sphingomyelin, is also found in tissues, but in larger 
quantities in brain substance. It contains phosphorus like lecithin and 
kephalin, but not glycerol. Its constituents are two fatty acids, 
choline, sphingosine and phosphoric acid. These three substances 
form the group of phosphatides. Sphingosine is an unsaturated com- 
pound and contains two hydroxyl groups : 

C 12 H 25 . CH = CH . CHOH . CHOH . CH 2 . NH 2 . 

Brain tissue contains sphingomyelin and the two cerebrosides, 
phrenosin and kerasin. This mixture constitutes protagon. The 
cerebrosides do not contain phosphorus. They are composed of the 
base sphingosine, galactose and a fatty acid, the sphingosine in com- 
bination with the galactose in the form of a glucoside and in com- 
bination with the fatty acid in the form of an acid amide : 

galactose spbingosine phrenosinic acid (C25H 5 oO3). galactose sphingosine lignoceric acid 



phrenosin. kerasin. 

These compounds have also been isolated from other organs. A full 
account of these substances is given in Maclean's monograph. 

Properties. 

Fats and oils, waxes and lecithin have solubilities like the fatty 
acids. They are not soluble in water and are not soluble in dilute 
cold sodium hydroxide. 

On warming, the fats melt and become oils. They have fairly 
definite melting-points. Fats and oils are not" easily soluble in 
alcohol, but dissolve readily in ether, ligroin, carbon disulphMe, etc. 

Lecithin is easily soluble in cold absolute alcohol, in ether and 
other solvents. It is nearly insoluble in acetone, and may be pre- 
cipitated from a concentrated ethereal solution by the addition of 
acetone. Kephalin is not soluble in alcohol. The cerebrosides dis- 
solve -in hot alcohol, acetone, benzene, but not in the cold solvent. 
They are insoluble in ether. 

Extraction of Fats, etc. 

The fats are contained in animal and vegetable tissues mixed with 
protein and carbohydrate. Three methods are in use for their separa- 
tion. The oldest and simplest method consists in melting out the fat 
from the tissue by simply placing it in a suitable receptacle of muslin 
or cloth in a warm room ; the fat melts and runs out leaving the re- 
mainder of the tissue behind. The most modern method consists in 
pressing out the fat in a hydraulic press ; this is the method most fre- 
quently employed for obtaining the oils from vegetable seeds. The third 
method consists in extracting the fat by means 1 of suitable solvents, 
such as benzine, carbon disulphide. Alcohol and ether are not usually 
employed for this purpose, but they are generally used if an estimation 
of fat in a tissue be required. For food the fat or oil is pressed out ; 
for soap making and other purposes the residue is extracted. 



FATS AND OILS. WAXES. LECITHINS 177 

Composition. Hydrolysis. 

Fats are hydrolysed into their constituent fatty acids and glycerol 
by boiling with water, treatment with steam, and by boiling with acids 
and alkalies. This latter process of decomposing fats and esters is 
known as saponification and is a special form of hydrolysis ; it was first 
used in the manufacture of soap, hence the term. 

Fats undergo the process of hydrolysis during digestion. They are 
decomposed by the enzyme lipase in the pancreatic juice and hydrolysed 
into their constituents, fatty acids arid glycerol. Emulsification occurs 
in the intestine, where the reaction is alkaline, during the process of 
the hydrolysis. 

(a) Butter. 

A small quantity (2 gm.) of butter is heated with excess of alcoholic 
sodium hydroxide until a clear yellow solution is obtanied. No oil 
drops should be seen on pouring the solution into water. The aqueous 
solution is heated to expel alcohol, acidified with dilute sulphuric acid 
and again heated. The smell of butyric and other volatile fatty acids 
is noticed. They are obtained by distilling the acid solution. The 
higher fatty acids are also present, but do not distil and remain as an 
oily layer on the surface of the hot liquid. 

(b} Olive Oil. 

If a little olive oil be dissolved in twice its quantity of ether and 
5 times the volume of 2 per cent, alcoholic sodium hydroxide be 
added, and the mixture be allowed to stand in a corked vessel, it 
gradually solidifies and forms a jelly. Complete saponification has oc- 
curred and soap has been formed. The jelly dissolves in water and 
the soap solution will give (a) a precipitate of fatty acids on acidifying 
with sulphuric acid and (7>) a precipitate of the calcium soap on adding 
calcium chloride solution. 

(c) Lard. 

About 5 gm. of lard are boiled with 25 c.c. of 10 per cent, alcoholic 
sodium hydroxide under a reflux condenser for 5-10 minutes to 
saponify the fat. 25-50 c.c. of water are added ; if the saponification 
is complete no oil drops should be seen ; if it be incomplete, the saponi- 
fication is continued by adding alcoholic soda and again boiling. The 
liquid is poured into an evaporating basin and the alcohol evaporated 
on a water-bath. The solution is acidified with sulphuric acid ; the 
fatty acids are precipitated and are filtered off through a wet paper and 
washed free from acid with water. The filtrate contains the glycerol 
which is detected as described below. 

12 



i;8 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



The presence of fatty acid in the precipitate is shown : 

(1) By dissolving a small portion in ether and adding the solution 
to alcohol containing a drop of phenolphthalein and a few drops of 
dilute sodium hydroxide. The red colour disappears. 

(2) By dissolving another portion in dilute sodium hydroxide. A 
soap lather is formed on shaking it up with warm water. 

The soap is salted out by adding sodium chloride and rises to the 
surface. 

A precipitate of calcium soap is formed on adding calcium chloride. 

(3) On heating with acid potassium sulphate there is no smell of 
acrolein, if the precipitate has been washed free from glycerol. 

The presence of glycerol in the filtrate is shown by neutralising it 
and evaporating it to a syrup on the water-bath. The 
syrup is mixed with alcohol which precipitates the 
sodium sulphate. The alcoholic solution is poured off 
and evaporated, and the residue tested for glycerol by 
heating it with acid potassium sulphate, when acrolein 
is formed. 

Estimation of Fats, etc. 

In the estimation of fat, the tissue must first be 
dried : this is effected by mixing a known weight of 
the material with clean dry sand or other suitable ab- 
sorbing medium and then heating for 1-2 hours in a 
steam oven. In the case of milk, it is most convenient 
to absorb a known weight (or volume) in clean fat-free 
filter paper, which is made into a small roll, and to dry 
this. The dried material is then placed in a paper 
thimble of suitable size and this is extracted with ether 
for 2-3 hours in a Soxhlet apparatus, which allows of 
a continual extraction for that time without constant 
attention. The Soxhlet apparatus (Fig. 37) consists 
of (l) a small dry flask, the weight of which has been 
accurately determined, (2) a special extracting tube 
into which the thimble and material is placed, (3) a short 
condenser. The extracting tube is composed of a wide 
piece of glass tubing like a test tube fused at its closed 
end to a narrower piece of glass tubing which is cut off 
at an angle at its other extremity. Just below the join of these pieces 
of tubing a glass side tube is fused into the narrower piece ; its other 
end is fused into the wider piece at the upper end. At the base of the 
wide tube, on the other side of the apparatus, one end of a narrow 




<? 



FIG. 37. 



FATS AND OILS. WAXES. LECITHINS 



179 



syphon tube is attached ; its other end is fused to . the narrow piece 
through which it passes and opens just above the angular extremity. 
The narrow end of this tube is fastened into the flask ; the condenser 
is attached to the wider end. Ether is placed in the flask which is 
gently heated. The volatilised ether passes through the side tube and 
reaches the condenser. The condensed drops fall upon the thimble 
and cover it. When completely covered the ether is syphoned off 
and returns to the flask and the process is repeated. It remains to 
distil off the ether from the flask, dry at 100 and weigh. The differ- 
ence in weight gives the amount of fat in the known weight of tissue. 
This method gives comparatively good results ; other substances 
besides fats are extracted from the tissue and some of the fat, present 
inside the cells, is not extracted. It is now more usual to estimate the 
fat as fatty acid, see page 60 1. 

Analysis. 

The natural fats consist of a mixture of the glyceryl esters of the saturated 
fatty acids, butyric, caproic, palmitic and stearic, of the unsaturated fatty acids, 
oleic, linoleic and others and also of hydroxy fatty acids. Free fatty acids are 
present in small quantities and increase in amount as the fat is kept. The 
various fats and oils have a fairly constant composition so that by determining 
the amounts of the various constituents it can be identified. The following 
six analyses are usually made : 

(1) the acid value, i.e. the amount of potassium hydroxide in mgm. re- 
quired to neutralise the free fatty acid in i gm. of fat ; 

(2) the saponification value, i.e. the amount of potassium hydroxide in 
mgm. required to saponify i gm. of the fat ; 

(3) the iodine value, i.e. the amount of iodine in gm. absorbed by 100 gm. 
of the fat ; 

(4) the Reichert-Meissl or Reichert-Wollny value, i.e. the amount of 
potassium hydroxide in c.c. of -iN required to neutralise the volatile fatty 
acids in 5 gm. of the fat ; 

(5) the Hehner value, i.e. the amount of non-volatile and insoluble fatty 
acids (and unsaponifiable matter) present in 5 gm. of the fat ; 

(6) the acetyl value, i.e. the amount of potassium hydroxide required to 
combine with the acetic acid in i gm. of fat, which has been acetylated. 

The values of some of the commoner fats are given in the accompanying 
table : 



Fat. 


Saponification 
value. 


Iodine 
value. 


Reichert- 
Wollny 
value. 


Acetyl 
value. 


Butter . 


220-233 


26-50 


26-33 


2-8-6 


Lard 


I95-5 


46-70 


68 


2-6 


Tallow . 


IQ2-20O 


35-46 


0'5 


2-7-8-6 


Olive oil 


185-196 


79-88 


0-6 


10-6 


Linseed oil 


192-195 


173-201 





4 -o 


Cotton seed oi 


IQ3-I95 


108-110 







Coco-nut fat 


246-260 


8-10 


6-6-7-0 


I- 12 


Palm-nut fat 


242-250 


13-17 


5-6-8 


2-8-5 



i8o PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Determination of the Acid Value. 

A weighed amount of fat, from 3 or 5 to 10 gm., is dissolved in neutral 
alcohol l or a mixture of alcohol and ether, a few drops of phenolphthalein 
are added and the solution is titrated with 'iN or ^N potassium hydroxide 
until it is pink in colour. The pink colour should be permanent for about 
2 minutes : after this time it usually disappears. 

The acid value depends on the purity and age of the fat, i.e. on the 
amount of hydrolysis and oxidation that has taken place. . 

Determination of the Saponification Value. 

The fat is saponified with an approximately ^N solution of alcoholic 
potassium hydroxide, standardised against ^N hydrochloric acid. 

The alcoholic potassium hydroxide is prepared by dissolving 28 gm. of 
pure potassium hydroxide in a little water and diluting to 1000 c.c. with 
alcohol or purified methylated spirit ; after 24 hours the solution is filtered 
into a litre bottle, which is closed by a rubber stopper carrying a 25 c.c. 
pipette. The pipette is closed by a piece of rubber tubing and a glass rod. 

In measuring out the volume it is convenient to allow the liquid to run out 
of the pipette without touching the sides of the vessel and then to let three 
drops fall from the end. 

A weighed amount of fat (1-5 to 2 gm.) is heated in a 200 c.c. flask on a 
water-bath under a reflux condenser with 25 c.c. of the alcoholic potash solu- 
tion for 30 minutes. At the same time a blank experiment (i.e. the same 
experiment without the fat) is carried out. The contents of the flasks are 
heated so that they boil gently and are shaken from time to time. 

When the saponification is complete and the fat has dissolved giving a 
clear solution, i c.c. of phenolphthalein solution is added and the mixture is 
titrated with ^N hydrochloric acid. 

The difference between the values found in the blank experiment and the 
one with fat is the volume of ^N alkali required to saponify the fat. Hence 
the amount in mgm. required to saponify i gm. of fat can be calculated. 

Determination of the Iodine Value. 

There are two methods of determining the iodine value, (a) Hubl's, (fr) 
Wijs. The process is the same in the two methods, the difference being in 
the iodine solution. Hiibl used a mixture of iodine and mercuric chloride, 
Wijs iodine trichloride. 

The following reagents are required : 

(i) Iodine solution. 

(a) Hubl's. Equal volumes, e.g. 30 c.c. of a solution of 25 gm. of iodine 
in 500 c.c. of pure 95 per cent, alcohol and of a solution (filtered if necessary) 
of 30 gm. of mercuric chloride in 500 c.c. of pure 95 per cent, alcohol are 
mixed 12-24 hours before use. The mixture should not be used if it has 
been prepared longer than 24 hours. 

(b] Wijs. 9'4 gm. of iodine trichloride are dissolved in 200 c.c. of glacial 
acetic acid contained in a 300 c.c. flask heated on the water-bath and closed 
with a cork carrying a calcium chloride tube; at the same time 7*2 gm. of 
finely powdered iodine are dissolved in glacial acetic acid in a similar way. 
The two solutions are poured into a 1000 c.c. flask and any undissolved 
iodine dissolved in a fresh portion of acetic acid. The mixed solutions are 
cooled and made up to 1000 c.c. 

This solution may also be made by dissolving 13 gm. of iodine in 1000 c.c. 
of glacial acetic acid and passing washed and dry chlorine through the 
solution. A colour change occurs at the point when iodine trichloride is 
formed. In preparing the solution, the iodine content of the solution is de- 
termined before the passage of the chlorine ; the chlorine is passed into the 
solution until the iodine content is doubled (Lewkowitch). 

1 Methylated spirit which has been kept in contact with sodium hydroxide and distilled 
may be used. If it should contain acid it may be neutralised with -iN potassium hydroxide 
until it shows a faint pink colour to phenolphthalein, a few drops of which are added as 
indicator. 



FATS AND OILS. WAXES. LECITHINS 



181 



(2) -iN thiosulphate solution. This is prepared by dissolving 24-823 gm. 
of the pure salt in 1000 c.c. of water or by dissolving 25 gm. of the salt in 1000 
c.c. and standardising after 24 hours against potassium bichromate as follows : 

20 c.c. of a bichromate solution containing 3-8657 gm. in 1000 c.c. are 
placed in a bottle containing 10 c.c. of the potassium iodide solution. 5 c.c. 
of concentrated hydrochloric acid are added. The brown solution is titrated 
with the thiosulphate solution, using starch solution as indicator. 

Since 20 c.c. of the bichromate solution yields o - 2 gm. iodine, i c.c. of 

*2 

thiosulphate = gm. of iodine, if x c.c. are required in the titration. 
oc 

(3) 10 per cent, potassium iodide solution. The quantity of iodate pre- 
sent must be taken into consideration. 

(4) i per cent, starch solution. 

(5) Pure chloroform or carbon tetrachloride. Their purity is tested by 
adding 10 c.c. of iodine solution and titrating after 2-3 hours. The value 
should be same as for 10 c.c. iodine solution. 

Pure glacial acetic acid (purified by recrystallisation). It should not give 
a green colour on heating with bichromate and sulphuric acid after prolonged 
standing. 

Procedure : 

15 to '18 gm. of marine animal oil, '2 to -3 gm. of semi-drying oil, '3 to 
4 gm. of non-drying oil, or - 8 to i'o gm. of solid fat is placed in a 500-800 
c.c. bottle with well-fitting stopper and dissolved in 10 c.c. of chloroform or 
carbon tetrachloride. 1 25 or 50 c.c. of iodine solution are added, the pipette 
being allowed to drain for 2 or 3 drops after it has emptied. Iodine solution 
and solvent must give a clear solution on shaking, otherwise more solvent is 
added. The mixture is kept in a dark place for 6-8 hours in the case of fat 
and non-drying oils, for 8-10 hours in the case of semi-drying oils or for 12-18 
hours in the case of marine animal oils. The mixture must contain 3 times the 
amount of iodine necessary and should be deep brown after 2 hours. More 
iodine solution is added, if the colour be paler. 15-20 c.c. of potassium iodide 
solution and 400 c.c. of water are added and the iodine titrated with - iN 
thiosulphate. If a red precipitate should form, more potassium iodide must 
be added. 25 c.c. of the iodine solution are titrated previously or subsequently. 
The difference gives the amount of iodine absorbed. The result is calculated : 

. ,. TVTTM.' } .-. iodine used = 16 

e.g. 25 c.c. iodine solution = 35-5 c.c. -iN Thio | 

0-983 gm. fat + 25 c.c. iodine solution = 18-6 c.c. -iN Thio j 

= ' 



= 



100 gm. fat = 2i'8 gm. iodine. 

Determination of the Reichert-Wollny Value. 

The standard apparatus for this estimation is shown in Fig. 38. 

A. 



c.c. -iN 
x . Q - 

j O( y ne 




FIG. 38. 

1 Glacial acetic acid in the case of oxidised oils not completely soluble in carbon tetra- 
chloride. 



182 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

5 gm. of the fat are weighed out into a flask with a flat bottom of 
about 250 c.c. capacity and with a neck 2 cm. wide and 7-8 cm. long. 2 c.c. 
of a solution of 98 per cent, caustic soda in an equal weight of water, 
which is protected from carbonic acid, and 10 c.c. of 92 per cent, alcohol are 
added. The contents are heated on a water-bath under a reflux condenser 
for 15 minutes; the alcohol is removed by warming the 'flask for about 30 
minutes without a condenser ; 100 c.c. of boiling water, freed from carbon 
dioxide by boiling for at least 10 minutes, are added and the soap dissolved. 
The flask is connected to a condenser by a bent tube 7 mm. in diameter and 
15 cm. in length from the cork of the flask. The condenser is 8 mm. in 
diameter and 35 cm. long. The contents of the flask are acidified with 40 
c.c. of N sulphuric acid, a piece of porcelain added, and distilled from an 
asbestos board 12 cm. in diameter containing an opening 5 cm. in diameter. 
The heating is begun cautiously so as to melt the fatty acids and then at such 
a rate that no c.c. distil over in about 30 minutes. The distillate is collected 
in a measuring flask of 1 10 c.c. capacity. The distillate is mixed and 100 c.c. 
are titrated with -iN alkali using 0*5 c.c. of i percent, phenolphthalein solu- 
tion as indicator. 

Determination of the Hehner Value. 

3-4 gm. of the fat are weighed out in a porcelain basin 13 cm. in 
diameter; 50 c.c. of alcohol and 1-2 gm. of potassium hydroxide are 
added. The mixture is heated on a water-bath with constant stirring till 
saponification is complete and until a clear solution is obtained. If a drop of 
water be added and no turbidity be produced, the saponification is complete. 
The solution is evaporated till it becomes pasty and 100 to 150 c.c. of water 
are added. It is acidified with sulphuric acid and warmed till the fatty acids 
form an oily layer on the surface. The fatty acids are filtered off on to a 
weighed filtered paper 10 cm. in diameter. This should have a texture so 
that it prevents fatty acid from passing through it and it is half filled with hot 
water and kept at this level with hot water during the filtration. The fatty 
acids are washed till the washings no longer react acid. The filter and its 
contents are dried at 100 for two hours, cooled and weighed. 

Determination of the Acetyl Value. 

10 gm. of the fat are boiled with twice the weight of acetic anhydride in 
a round flask under a reflux air condenser for two hours. The solution is 
poured into about 500 c.c. of hot water contained in a beaker and boiled for 
half an hour, whilst a slow current of carbon dioxide is passed through it to 
prevent bumping. On standing it separates into two layers ; the aqueous layer 
is syphoned off and the remaining oil washed three times with water, so as 
to remove acetic acid, which may be tested for by its acid reaction. The 
acetylated fat is collected on a filter paper and dried at 1 00. 

2-5 gm. of the acetylated product are saponified with -iN alcoholic 
potash as described under determination of the saponification value. The 
solution is evaporated to expel the alcohol and the residue dissolved in water. 
The same volume of 'iN acid as of alkali used in the saponification is added 
and the solution warmed. The aqueous solution is syphoned off through a wet 
filter and the fatty acids washed with hot water till all the acid is removed. 
The filtrate and washings are titrated with 'iN alkali using phenolphthalein 
as indicator. Soluble fatty acids, if present in the fat, must be separately 
determined in the same way and their amount deducted from the value 
obtained. 



THE CARBOHYDRATES. 

The very large group of compounds termed the carbohydrates, or 
sugars, are compounds of the nature of alcohols, primary and secondary, 
and at the same time aldehyde or ketone. Their empirical formula 
shows that they consist esssentially of rarhnn and water, C n (H^Q) 
hence their name though substances other than carbohydrates, e.g. 
formaldehyde, CH 2 O, acetic acid, C 2 H 4 O 2 , and lactic acid, C 3 H 6 O 3 , also 
possess the same empirical formula, and some carbohydrates have em- 
pirical formulae in which the ratio of the elements H : O is not 2:1, 
e.g. the methyl pentose, rhamnose C 6 H 12 O 5 . 

This group of compounds contains simple and complex members. 
The simple members contain 2, 3, 4, 5, 6, 7, 8, 9 carbon atoms in 
their molecule, the chief physiological representatives being the 
members with 6 carbon atoms and in a lesser degree those with 5 
atoms of carbon. It was formerly supposed that only those mem- 
bers containing 6 atoms of carbon belonged to the class of sugars, 
and it is convenienttp term the six carbon atom representatives 
the sugars, whilst the whole group is termed the carbohydrates. 

The complex members consist of combinations together in an an- 
hydride form of 2, 3, 4 and more of the simple units, generally of 
those containing 6 carbon atoms and also of those with 5 carbon 
atoms. Accordingly asthey contain 2, 3, etc., simple units incom- 
bination they are termed disaccharides, trisaccharides, or polysac- 
criarides, the simple unit being termed a monosaccharide. _A11 the 
complex members are converted into their constituent single units by 
hydrolysis with acids. The members of the carbohydrate group are 
distinguished by the suffix -ose, but this suffix is not applied to some 
of the complex compounds. 

The carbohydrates are especially abundant in plants ; the amount 
present in animals is by comparison very small. The complex carbo- 
hydrates 'form the structural basis of plants and are deposited in 
various parts as reserve material and as food-stuffs for the young 
plant. Both the complex reserve material and the simple carbohy- 
drates are the chief food-stuff of animals. 

The physical properties, appearance, solubility, taste, etc., of the 
various carbohydrates is very different and no proper classification can 
be based upon their properties, but they are classified according to 
their complexity : 



1 84 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Monosaccharides. 
Diose. Glycollic aldehyde. 

Trioses. Glyceric aldehyde, dihydroxyacetone. 
Tetroses. Erythreose, threose. 
Pentoses. Arabinose, xylose, ribose, etc. 
Hexoses. Glucose, mannose, galactpse. etc. 

In this group it is convenient to include d-g\u- 
cosamine or aminoglucose. 

Fructose, sorbose, etc. 
Heptoses, etc. 

Disaccharides. 

Sucrose, maltose, lactose. 

Trisaccharides. 

Raffinose. 
Tetrasaccharides. 

Stachyose. 
Polysaccharides. 

Starch, cellulose, dextrin, glycogen, inulin. 
Gums, pectins, pentosans, mannosans/etc. 

THE MONOSACCHARIDES. 
( Diose. 

CH 2 OH, 
Glycollic aldehyde, | which contains a primary alcohol 

CHO 

group and an aldehyde group, is the first member of the series. 
It is derived, like all the members of the carbohydrate group, 
by oxidation of the corresponding dihydric alcohol, glycol. 
Trioses. 

Glyceric aldehyde and dihydroxyacetone are obtained from gly- 
CH 2 OH CH 2 OH 

CHOH CO 

CHO CH 2 OH 

cerol by oxidation with sodium hypobromite or hydrogen peroxide in 
the presence of ferrous sulphate. They contain respectively an aldehyde 
and ketone group and they are the first examples of an isomeric 
aldose and a ketose. Glyceric aldehyde contains an asymmetric carbon 
atom, but the two stereoisomers, d- and /-glycerose, have not yet been 
separated. 



THE CARBOHYDRATES 185 

Tetroses. 

The tetroses contain 4 atoms of carbon. An aldose or a ketose 
are theoretically possible and several stereoisomers, e.g. the aldotetroses, 
d- and /-erythreose, threose. 

Pentoses. 

Five carbon atoms are present in the molecule of a pentose, 
and isomers, an aldose and 2 ketoses, are possible. Three asym- 
metric carbon atoms are present : 2 3 or 8 stereoisomeric aldoses can 
exist. All but one are known, but all do not occur in nature. 

^/-ribose is contained in the nucleic acid of plants from which it is 
obtained by hydrolysis. 

/-arabinpse is contained in the polysaccharides cherry gum, gum 
arabic, peach gum. It is obtained by the hydrolysis of these substances 
with dilute sulphuric acid. Arabinose crystallises in prisms, has a 
sweet taste, is dextrorotatory, though termed /-arabinose on account of 
its stereochemical relation to glucose ; it melts at 160. 

Xylose is obtained by the hydrolysis of wood gum or xylane, straw, 
and various forms of cellulose. It is optically inactive and melts at 
144-145. 

A pentose, as yet not definitely identified, but probably. arabinose 
or ribose, is excreted in the urine in certain diseases. 

Methyl Pentoses. 

Rhamnose, C 6 H 12 O 5 , is obtained by the hydrolysis of the glucosides, 
quercftrin, xanthorhamnin, and some saponins. Rhamnose crystallises 
with a molecule of water, C 6 H 14 O 6 , and was formerly regarded as a 
hexahydric alcohol isodulcitol. It melts at 93. 

Fucose in seaweed, chinovose in chinovin, and other methyl pen- 
toses have also been prepared. 

Hexoses. 

The hexoses contain 6 atoms of carbon ; two isomeric ketoses and 
one aldose are possible. Four asymmetric carbon atoms are p'resent 
in the molecule of an aldohexose ; 2 4 or 1 6 stereoisomers are possible 
but only three are found in nature. Most of the other stereoisomers 
have been prepared in the laboratory by Emil Fischer. Two 
stereoisomeric ketoses are also found in nature. The formulae of the 
natural hexoses are : 



1 86 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



CH 2 OH 
H . C . OH 


H . 


CH 2 OH 
C.OH 


H. 


CH 2 OH 
C.OH 


CH 2 OH 

I 

H .C.OH 


CH 2 OH 
HO.C.H 


H.C.OH 


H. 


C.OH 


HO. 


4.H 


H.C.OH 


H.C.OH 


HO . C . H 


HO, 


|H 


HO, 


,C.H 


HO . C .' H 


HO.C.H 


HC.OH 


HO 


.C.H 


H 


.C.OH 


CO 


' CQ 


CHO 

<f-glucose. 


CHO 
d-mannose. 


CHO 
d-galactose. 


CH 2 OH 
J-fructose. 


CH 2 OH 
(f-sorbose. 



Constitution of the Aldoses (Glucose). 

Analysis and molecular weight determinations show that glucose has the 
empirical formula C fi H 10 O^. The 6 atoms of carbon are joined together 
in a straight chain as is shown by its reduction with hydriodic acid into normal 
hexyl iodide. The stability of the compound and . the formation of esters 
with 5 molecules of acid show the presence of five OH groups attached to 
different carbon atoms. The remaining group is an aldehyde group as shown 
by the aldehyde reactions and the formation of an acid, gluconic acid, by 
oxidation. Further oxidation of glucose gives a dibasic acid by the oxidation 
of the primary alcohol group. 

Glycuronic acid, another oxidation product of glucose, is formed in the 
animal body; in the formation of this compound the primary alcohol group 
of glucose is oxidised whilst the aldehyde group is unchanged. Glycuronic 
acid is formed under certain conditions, e.g. after the administration of 
chloral hydrate. Chloral, and also other compounds, apparently combine 
with the aldehyde group of glucose ; the alcohol group is then oxidised in 
the body and the combination product (paired glycuronic acid) is excreted in 
the urine. The best source of glycuronic acid is euxanthic acid, the calcium 
and magnesium salt of which constitutes the yellow pigment, Indian 
yellow. This is obtained from the urine of cows that have been fed on 
mango leaves. Glycuronic acid is prepared by hydrolysis of the Indian 
yellow, or other combination product. We have therefore 

CH OH CH 2 OH COOH COOH 

I I I I 

(CHOH) 4 (CHOH) 4 (CHOH) 4 (CHOH) 4 

CHO COOH COOH CHO 

Glucose. Gluconic acid. Saccharic acid. Glycuronic acid. 

Constitution of the Ketoses (Fructose). 

The 6 carbon atoms in fructose are present in a straight chain. It can 
be reduced t& normal hexyl iodide. Five hydroxyl groups are present. On 
oxidation fructose breaks down giving trihydroxy butyric acid and glycollic 
acid, which shows the presence of a ketone group and in the position shown 
in the formula. Further proof of the position of the ketone group is given 
by the formation of a cyanohydrin which yields an acid. This acid on 
reduction gives methylbutylacetic acid. 

Glucose and the other hexoses are represented above as hydroxy 
aldehydes, but this constitution does not entirely explain all their 
chemical and physical properties. 

(i) A freshly prepared solution of glucose shows a higher rotatory 
power than a solution which has been kept for some hours (Muta- 
rotation). Tanret has isolated from glucose solutions, under certain 



THE CARBOHYDRATES 



187 



conditions, two compounds, one of which, a-glucose, has a high initial 
rotatory power of 110, the other, /3-glucose, a low one of 19. On 
being kept in solution both compounds give a solution of the same 
rotatory power of 52*5. 

(2) Two isomeric methyl etc. derivatives (glucosides) are also ob- 
tainable from glucose. 

(3) Glucose in its chemical behaviour is also less active than is 
expected. 

These properties are most satisfactorily explained by assuming that 
glucose has a <y-lactone formula, i.e. a formula in which four carbon 
atoms and an oxygen atom are included in a ring. It is derived from 
the hypothetical aldehydrol : 



CH a OH 

CHOH 

CHOH 

CHOH 

CHOH 

CHO 



CH 2 OH 

CHOH 

CHOH 

CHOH 
I 
CHOH 

I / 
CH< 



CH 2 OH 
CHOH 




CHOH, 
CHOH 



Aldehyde. Aldehydrol. 7-lactone. 

Under these conditions the carbon atom to which the aldehyde group 
was attached becomes asymmetric. Two stereoisomeric forms are 
therefore possible. These represent a- and /3-glucose : 
CH..OH CH 2 OH 

H-C-OH 





Each form in solution will change into the aldehydrol form and 
then into the other form. A solution of constant rotatory power will 
contain an equilibrium mixture of a- and /3-glucose depending on the 
concentration. The solution will give the reactions of an aldehyde as 
it will contain a small quantity of aldehydrol (aldehyde). The two 
derivatives are obtained from the a- and /3-forms. 



1 88 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Glucose, Grape Sugar, or Dextrose. 

Glucose occurs in the seeds, leaves and other parts of plants and together 
with fructose in sweet fruits and honey. It is present to the extent of about 
i per cent, in the blood of animals and in other organs of the animal body. 
It is formed by the hydrolysis of cane sugar and other polysaccharides which 
contain it. 

Small quantities of glucose are most conveniently prepared from cane 
sugar as follows : 40 gin. of powdered cane sugar are added to a mixture of 
5 c.c. of concentrated hydrochloric acid and 120 c.c. of 90 per cent, alcohol 
heated to 45-50. The mixture is kept at this temperature for 2 hours with 
occasional stirring and allowed to cool. Glucose crystallises out on cooling, 
more rapidly after adding a crystal of anhydrous glucose which helps the 
crystallisation. The crystals are filtered off, washed with alcohol and re- 
crystallised from a mixture of 2 parts of alcohol and i of water. 

Commercially, glucose is prepared from the starch of potato, maize, 
etc. The starch is hydrolysed by heating in copper vessels with dilute 
sulphuric acid under 3 atmospheres pressure. . The solution is neutralised 
with chalk, the calcium sulphate filtered off and the filtrate heated with 
animal charcoal to decolorise it. It is evaporated in vacua to a syrup and 
allowed to stand. The glucose crystallises in a cake of small crystals, which 
are purified by crystallisation from dilute alcohol. 

Glucose crystallises from alcohol, or concentrated aqueous solutions at 
30, in needles which are anhydrous. It crystallises from cold water in the 
form of plates of the composition C 6 H 12 O 6 . H 2 O. It is easily soluble in 
water, very slightly soluble in absolute alcohol, but more soluble in methyl 
alcohol. It is insoluble in ether. Glucose and other carbohydrates are difficult 
to prepare free from moisture. This can only be effected by heating them 
in vacuo at 70-110 in a vessel connected to phosphorus pentoxide (see p. 21). 

Fructose, Fruit Sugar, or Laevulose. 

Fructose occurs with glucose in fruits, honey, etc. It is most easily 
prepared from the polysaccharide inulin by boiling it with 5 parts of "5 per 
cent, sulphuric acid, or dilute oxalic acid, for i hour. The acid is removed 
with barium carbonate and the solution is treated with charcoal and evaporated 
to a syrup. The syrup is dissolved in alcohol from which fructose slowly 
crystallises out. 

On a large scale fructose is made from cane sugar. The solution of cane 
sugar which has been hydrolysed by dilute acid is neutralised and treated with 
milk of lime. An insoluble calcium compound of fructose is formed; this is 
filtered off, decomposed with carbon dioxide and the fructose obtained as above. 

Fructose crystallises from alcohol in the form of rhombic crystals ; it 
crystallises from water in needles of the composition 2C 6 H 12 O 6 . H 2 O. It is 
soluble in hot absolute alcohol and can thus be separated from other sugars. 

Mannose. 

Mannose does not occur as such in nature, but is widely distributed 
as the polysaccharide mannan. It can be obtained by the oxidation of 
mannitol, but is usually prepared by the hydrolysis of the mannan contained 
in ivory-nut, which is used in making buttons. The material is hydrolysed by 
heating it on a water-bath for 6 hours with twice its weight of 6 per cent, 
hydrochloric acid. The insoluble matter is removed by filtration and the 
solution is decolorised by heating with animal charcoal, neutralised and 
treated with phenylhydrazine acetate. Mannose phenylhydrazone is formed 
from which the sugar is obtained by decomposing it with cold concentrated 
hydrochloric acid. Mannose is a hard colourless solid, which deliquesces, is 
easily soluble in water, slightly soluble in alcohol and insoluble in ether. 



THE CARBOHYDRATES i89. 

Galactose. 

Galactose occurs in combination with glucose in milk-sugar, or lactose, 
in some gums and seaweeds as the polysaccharide galactan and in some 
glucosides, e g. xanthorhamnin and saponin of plants, the cerebrosides of the 
brain and nervous tissue of animals. 

It is prepared from lactose by boiling it with 4 times its weight of 2 per 
cent, sulphuric acid for 6 hours. The solution is concentrated and allowed 
to crystallise. The crude galactose is recrystallised by dissolving it in four- 
fifths of its weight of water and adding 2 volumes of 93 per cent, alcohol. 
Galactose consists of very small hexagonal crystals which melt at 168. 

d-Glucosamine. 

The hydrochloride of glucosamine or aminoglucose was obtained in 1878 
from chitin, the organic constituent of the shells of the lobster. It has since 
been obtained from the organic material of the shells of other arthropods. 
It is a constituent of fungus cellulose and has been prepared from various 
glucoproteins (p. 468). These conjugated proteins contain glucosamine or a 
polysaccharide composed of glucosamine as their carbohydrate moiety. 

d-Glucosamine was synthesised by Fischer and Leuchs from d-arabinose 
and was shown to have the formula, 

H I 
CH 2 OH C C C C ^CHOH 




Preparation. 

Chitin (p. 203), or lobster or crab shells, which have been treated with dilute 
hydrochloric acid to decompose the calcium carbonate and washed with 
water, are gently boiled with concentrated hydrochloric acid for 3-4 hours. 
The solution is evaporated until it crystallises and allowed to cool. The dark 
brown crystals of glucosamine hydrochloride are filtered off, dissolved in water 
and the solution evaporated until it crystallises. Glucosamine may be ob- 
tained from the hydrochloride by the action of diethylamine or sodium 
methylate. 

Properties. 

Glucosamine is a very unstable base and is known chiefly in the form of 
its hydrochloride. 

Glucosamine hydrochloride forms large colourless glistening crystals which 
are soluble in water. 

It cannot be converted directly into glucose; by the action of nitrous 
acid it is converted into chitose which Fischer and Andreae have shown to be 
a furfurane derivative of the formula, 

HO . CH CH . OH 
CH,OH . CH CH . OH 



Glucosamine is hence frequently termed chitosamine. 

It resembles glucose closely in its properties : it forms a pentacetyl de- 
rivative, an oxime and a phenyl-hydrazone and yields an osazone identical 
with glucosazone. 

Its solutions behave like those of glucose as a reducing agent to Fehling's 
solution, etc. 



190 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

PROPERTIES AND REACTIONS OF THE MONO- 
SACCHARIDES. 

The properties and reactions of the monosaccharides are very 
similar, the differences between the individual members being only in 
certain peculiarities. The reactions of glucose may be taken as typical 
of the reactions of all monosaccharides. 

A. GLUCOSE. 

(1) Formation of Esters. 

Glucose is converted by acids, acid anhydrides and acid chlorides 
into esters. 

Pentabenzoyl glucose is precipitated when a solution of glucose 
is shaken with benzoyl chloride and excess of sodium hydroxide : 
C 6 H 7 O(OH) 5 + 5C 6 H 5 . CO . Cl = C 6 H 7 O(O . OC . C 6 H f ) 5 + sHCl. 

(2) Formation of Compounds with Metallic Hydroxides. 

Glucose forms compounds with metallic hydroxides which are analagous 
to the alkoxides. 

(a) If copper sulphate and sodium hydroxide be added to a solution of 
glucose in the proportions of 

C 6 H 12 O fi : 5CuS0 4 . sH 2 O : uNaOH, 

the glucose is precipitated almost completely from solutions as a voluminous 
blue compound. 

(b} On adding basic lead acetate and caustic soda, or ammonia, to a 
solution of glucose in the proportions of 



the glucose is precipitated completely as an insoluble white compound. 

The precipitation is not complete unless these quantities are nearly pro- 
portional. Glucose is most conveniently removed from solution by this means. 

(3) Reduction. 

When glucose is reduced with sodium amalgam it is converted into the 
hexahydric alcohol, sorbitol. 

(4) Oxidation. 

When oxidised by bromine water glucose is converted into gluconic acid ; 
when oxidised by nitric acid it is converted into saccharic acid. 

(5) Action of Alkali. Glucose is acted upon by sodium hydroxide 
at 37. The rotation of the solution diminishes and its acidity in- 
creases. It passes over into fructose and mannose. Carbonates have 
a slighter action and ammonia of the same concentration is almost 
without action. 

Moores Test. On boiling a solution of glucose with sodium 
hydroxide it turns yellow, then dark brown, and smells of caramel. The 
smell becomes more distinct on acidifying the solution with dilute 
sulphuric acid and its colour becomes lighter. Lactic acid and other 
acids are formed. 



THE CARBOHYDRATES 191 

(6) Action of Concentrated Hydrochloric Acid. If a solution of 
glucose be boiled for some time with an equal volume of concentrated 
hydrochloric acid, the solution becomes brown and "humus" sub- 
stances, which are black, separate out. The chief product of the action 
of hydrochloric acid on glucose is laevulinic acid or acetylpropionic 
acid, CH 3 .CO. CH 2 .CH 2 .COOH. 

(7) Reduction of Metallic Oxides in Alkaline Solution. 

(a) Silver. On adding a solution of glucose to some ammoniacal 
silver nitrate solution (prepared by adding dilute ammonia to silver 
nitrate until the precipitate first formed is just redissolved) and warming 
in the water-bath, a mirror of metallic silver gradually forms. 

[b] Copper. 

(i) Trommers Test. On making a solution of glucose alkaline 
with sodium hydroxide and adding copper sulphate, drop by drop, 
shaking after each addition, the solution becomes deep blue. The 
addition of excess of copper sulphate causes the precipitation of cupric 
hydrate, i.e. it is no longer dissolved by the glucose solution. The 
addition of a few small crystals of Rochelle salt will redissolve the 
precipitate (see Fehling's test). On heating the clear blue solution 
nearly to boiling a yellowish-red precipitate of cuprous-oxide is formed. 

(ii) Fehling's Test. On adding some glucose solution to equal 
quantities of Fehling's solution (a) CuSO 4) () NaOH + NaK Tart, 
and heating to boiling, cuprous oxide is precipitated. 

It should be noted that ammonium salts modify the reaction ; the 
cuprous oxide is not precipitated, but the blue colour of the solution 
becomes less intense and may disappear. 

(iii) Benedict's Test. As glucose is destroyed by the action of 
sodium hydroxide the reaction is more sensitive if sodium carbonate be 
employed in its place. If sodium citrate be substituted for Rochelle 
salt a permanent solution (Benedict's qualitative reagent, p. 613) is 
obtained. 

On adding 5 to 10 drops of glucose solution to about 5 c.c. of 
the reagent and boiling vigorously for 2 or 3 minutes, it becomes 
turbid with a red, yellow, or green precipitate which fills up the 
solution depending on the amount of glucose. If the amount of 
glucose be very small a precipitate is only observed on allowing the 
solution to cool. 

The test is sensitive to -08 per cent, of glucose. 

(iv) Barfoecfs Test. Glucose also reduces cupric hydrate in acetic 
acid solution. If some glucose solution be added, drop by drop, to 
some Barfoed's reagent which is kept boiling during the addition, red 
cuprous oxide is precipitated. 



192 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

This test is given by glucose and other monosaccharides, but not 
by lactose and maltose. It may be used for distinguishing between 
glucose and the disaccharides, but the reagent must be freshly prepared, 
otherwise maltose and lactose will also reduce it. 

(V) Bismuth. 

Boettger's Test. On boiling some glucose solution with a few crystals 
of bismuth subnitrate and twice the quantity of sodium carbonate, the bismuth 
hydrate first formed becomes reduced to metallic bismuth ; the precipitate 
becomes grey or black in colour. 

Ny lander's Test. If 5 parts of glucose solution be boiled with i part 
of Nylander's reagent (p. 613) for 2-5 minutes, reduction occurs and a black 
precipitate settles out on cooling. 

These reactions are particularly useful for detecting small quantities of 
glucose in urine. The uric acid and creatinine in urine also reduce Fehling's 
solution, but not Nylander's solution. 

(8) Reduction of Dye-stuffs. 

(i) On adding picric acid and caustic soda to a solution of glucose and 
warming, a blood- red colour is formed due to the formation of picramic acid. 

(ii) On warming a dilute solution of sodium sulphindigotate, made alkaline 
with sodium carbonate, with some glucose solution, the blue colour changes to 
green, purple-red and finally yellow. The blue colour returns on cooling and 
shaking the solution with air. 

(iii) If some glucose solution be added to about 5 c.c. of a solution of 
safranin and the mixture boiled, the opaque red colour changes to light 
yellow. 

(9) Formation of Osazones. The reaction of glucose and other 
reducing sugars with phenylhydrazine is very characteristic, as it serves 
for the identification of the different carbohydrates. Glucose reacts 
with phenylhydrazine in acetic acid solution in two stages ; the phenyl- 
hydrazone is first formed : 

CH 2 OH . CHOH . CHOH . CHOH . CHOH . CHO + H 2 N . NH . C 6 H 5 = 
H 2 O + CH 2 OH . CHOH . CHOH . CHOH . CHOH . CH : N . NH . C 6 H 5 . 

This is a colourless compound soluble in water. 

The secondary alcohol group next to the aldehyde group is oxidised 
by excess of the reagent to the ketone group, which reacts with phenyl- 
hydrazine and an osazone is formed : 

CH 2 OH . CHOH . CHOH . CHOH . CHOH . CH : N . NH . C 6 H 5 + H 2 N . NH . C 6 H 5 = 
CH 2 OH . CHOH . CHOH . CHOH . CO . CH : N . NH . C 6 H 5 + NH 3 + H 2 N . C B H S 

CH 2 OH . CHOH . CHOH . CHOH . CO . CH : N . NH . C B H 5 + H 9 N . NH . C 6 H 5 = 
CH 2 OH . CHOH . CHOH . CHOH . C( : N . NH . C 6 H 5 ) . CH : N . NH . C 6 H 5 + H 2 O. 

On adding equal quantities of phenylhydrazine and glacial acetic 
acid (5 to 10 drops of each), or-f-^part of phenylhydrazine hy-drocMorieteT 
and_-2-part? of sodiurrTacetSfe-to about 20 c.c. of glucose solution and 
warming in a boiling water-bath for half to one hour, a yellow crystalline 
mass of phenylglucosazone is formed. The solution is allowed to cool, 
the crystals are filtered off and examined under a microscope. They 
consist of long needles arranged in sheaves as in Fig. 39. 



THE CARBOHYDRATES 



193 



(10) Fermentation Glucose is fermented by yeast into alcohol 
and carbon dioxide. If a little fresh yeast be rubbed up with some 
glucose solution and a 
test tube be filled with the 
mixture and inverted in 
warm water in a crucible 
at 25, it will be seen that 
after a short time bubbles 
rise to the top and dis- 
place the liquid. In about 
24 hours most of the glu- 
cose will have disappeared 
and alcohol can be de- 
tected in the liquid. 

(i i) Molisch's Test. 
On adding a drop of a- 
naphthol solution to about 
5 c.c. of glucose solution 
and running about 5 c.c. of 
concentrated sulphuric acid below it, a purple ring appears at the surfaces 
of contact, either at once or after a short time. The two liquids may 
be mixed but the mixture must be kept cold by holding under running 
water. The whole liquid becomes reddish-violet. An examination of the 
coloured solution with a spectroscope will show an absorption band 
between D and E, whilst the violet end is totally absorbed (cf. p. 478). 
<w-Hydroxymethyl furfural, which gives the pigment with a-naphthol, 
H . C C . H 

II II 
CH,OH . C C . CHO 




FIG. 39. Glucosazone. 



is formed. 

This reaction is the most general one for all carbohydrates. 

(12) Rotation. Glucose in solution is dextrorotatory when 
examined with a polarimeter, and shows mutarotation the initial high 
rotatory power decreases and becomes constant in about 24 hours, or on 
boiling, or on adding a drop of ammonia. 

(13) Hydrogen Cyanide. Glucose combines with hydrogen 
cyanide forming a cyanohydrin, which yields an acid containing seven 
carbon atoms on hydrolysis. This acid (or its ^-lactone or anhydride) 
on reduction yields an heptose. Octoses and nonoses have been pre- 
pared by continuing the addition of hydrogen cyanide to the heptose 
and octose. 

13 



194 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

(14) Hydroxylamine. Glucose reacts with hydroxylamine to 
form an oxime. The oxime loses water on heating with concentrated 
sodium hydroxide giving the nitrile of gluconic acid. On further heat- 
ing hydrogen cyanide is split off and a pentose is formed. 

A pentose is more easily obtained by oxidising gluconic acid with 
hydrogen peroxide in the presence of ferrous salts. 

B. FRUCTOSE. 

Fructose gives all the reactions given by glucose, but the following 
differences should be noted : 

(3) Reduction. Fructose on reduction with sodium amalgam 
gives a mixture of sorbitol and mannitol. 

(4) Oxidation. sFructose on oxidation gives trihydroxybutyric 
acid and glycollic acid. 

(6) Action of Concentrated Hydrochloric Acid. On boiling a 
solution of fructose with concentrated hydrochloric acid, the solution 
generally becomes red or red-brown before it ultimately turns dark 
brown. 

(7) Reduction of Metallic Oxides in Alkaline Solution. Al- 
though fructose is a ketose, it nevertheless reduces metallic oxides in 
alkaline solution. This is due to the terminal CO.CH 2 OH group 
which is easily oxidisable. Though acetone does not reduce metallic 
oxides, monohydroxyacetone CH 3 . CO . CH 2 OH does, as it contains 
the above grouping. 

(9) 'Formation of Osazones. Fructose gives a phenylosazone 
identical with phenylglucosazone, as can be seen when the crystals are 
examined under the microscope, melting-point, analysis, etc. 

(10) Fermentation. Fructose ferments more rapidly than glucose 
with yeast. 

(12) Rotation. Fructose solutions are laevorotatory. Laevo- 
rotatory fructose is known as ^-fructose on account of its stereo- 
chemical relationship to glucose ; the asymmetry of glucose is the basis 
of the stereochemical configuration of all carbohydrates. 

Special Test. SelivanofPs Test. On adding a few crystals of 
resorcinol to a mixture of equal parts of concentrated hydrochloric 
acid and water and a very small quantity of fructose solution and 
heating, the solution becomes red in colour and deposits a brownish-red 
precipitate, which dissolves in alcohol giving a red solution. 

&)-Hydroxymethylfurfural is formed by the action of the acid 
upon fructose and combines with the resorcinol giving the red pig- 
ment, 



THE CARBOHYDRATES 195 

C. GALACTOSE. 

Galactose gives the same reactions as glucose. It differs from glucose in 
the following particulars : 

(3) Reduction. Galactose on reduction with sodium amalgam gives the 
hexahydric alcohol, dulcitol. 

(4) Oxidation. Galactose on oxidation gives galactonic acid. On further 
oxidation with nitric acid it yields mucic acid. 

(9) Formation of Osazones. Galactose gives a different phenylosazone. 

(10) Fermentation. Galactose is fermented by yeast, but much less 
rapidly than glucose. 

(12) Rotation. Galactose has a higher dextrorotatory power than glucose. 

D. MANNOSE. 

Mannose differs in the following particulars from glucose : 

(3) Reduction. Mannitol is formed" by reduction with sodium amalgam. 

(4) Oxidation. It yields mannonic acid and saccharic acid on 
oxidation. 

(9) Formation of Hydrazone and Osazone. Mannose forms a 
phenylhydrazone which is soluble with difficulty in water. It forms the same 
phenylosazone as glucose. 

(12) Rotation. Mannose is dextrorotatory, but has a different rotatory 
power. 

E. PENTOSES. 

The pentoses give most of the reactions given by glucose but with 
the following differences : 

(3) Reduction. They give pentahydric alcohols. 

(4) Oxidation They give acids containing five carbon atoms. 

(6) Action of Concentrated Hydrochloric Acid. The pentoses 
on boiling with hydrochloric acid yield furfural, which is volatile with 
steam and may be detected with aniline acetate. 

If a solution containing pentose, 1 or pentosan, e.g. gum arabic 
solution, be boiled with about half its volume of hydrochloric acid and 
if a piece of filter paper moistened with aniline acetate solution (pre- 
pared from equal parts of aniline, glacial acetic acid and water) be held 
in the vapour escaping from the vessel after most of the hydrochloric 
acid has been evolved, a bright crimson colour will be formed. 

(7) Reduction of Metallic Oxides in Alkaline Solution. Pen- 
toses reduce Fehling's solution on warming for some time. 

(9) Formation of Osazones. The pentoses form phenylosazones 
with phenylhydrazine in acetic acid solution. They differ from 
phenylglucosazone in melting-point, analysis, etc. 

fio) Fermentation. Pentoses are not fermented by yeast. 

(12) Rotation. Pentoses are dextrorotatory, or inactive. 

1 A solution containing arabinose may be readily prepared by boiling 5 gm. of gum 
arabic in 100 c.c. water with 10 c.c. of concentrated hydrochloric acid for 5 minutes and. 
then neutralising with alkali, 

13 * 



I 9 6' PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Special Tests. 

(1) Phloroglucinol Reaction. On adding an equal volume of 
concentrated hydrochloric acid and a small quantity of phloroglucinol 
to a solution of a pentose and heating the mixture in a boiling water- 
bath, it gradually becomes cherry red in colour and turbid and a pre- 
cipitate is formed. The precipitate is dissolved by amyl alcohol, if 
the cold solution be shaken up with this solvent ; the amyl alcohol 
solution will show on examination with a spectroscope an absorption 
band between D and E. 

The formation of a precipitate is not itself sufficient evidence for 
the presence of a pentose, since a precipitate may be formed on heating 
other substances with acid and phlorogucinol. 

(2) Orcinol Reaction (Tollens). If a mixture of equal parts of con- 
centrated hydrochloric acid and pentose, or pentosan solution, be heated 
with a little orcinol, the solution becomes red, then violet, finally blue or 
blue-green with the separation of a precipitate. The appearance of 
the green colour may be hastened by adding a drop of ferric chloride to 
a portion of the solution. The remainder of the solution on being shaken 
with amyl alcohol imparts a bluish-red -colour to the amyl alcohol, 
which gradually becomes green. The solution on examination with 
a spectroscope shows an absorption band between C and D but near D. 

BiaVs Modification. On adding Bial's reagent (p. 614) drop by 
drop to about 5 c.c. of a boiling solution of a pentose, a bright green 
colour is produced. This is soluble in amyl alcohol, as above. 

F. GLYCURONIC ACID. 

Glycuronic acid resembles the pentoses in its reactions. 

A solution of glycuronic acid may be prepared by boiling a smally quantity 
of Indian yellow with dilute hydrochloric acid, cooling, filtering off the 
euxanthone and neutralising the solution. 

(1) It reduces Fehling's solution. 

(2) It gives the phloroglucinol and orcinol reactions. 

(3) It does not ferment. 

(4) The free acid is dextrorotatory, but when combined with euxanthone 
or other compounds it is laevorotatory. 



THE CARBOHYDRATES 



197 



THE DISACCHARIDES. 

The disaccharides consist of two units of monosaccharide combined 
together with loss of water. Theoretically any two monosaccharides 
can be thus combined, but actually only a few disaccharides are known. 
Most of these are natural compounds, but some have been obtained by 
synthesis. Their composition is shown by hydrolysis by acids or 
enzymes when they are converted into their constituents, e.g. in the 
case of a bihexose : 

CitjHjaOjj + H 2 O = C 6 H 12 O 6 + C 6 H 12 O 6 . 

The natural disaccharides are of two kinds, those which reduce 
Fehling's solution and those which do not, and they are usually class- 
fied accordingly : 

Non-Reducing. Reducing. 

Lactose fGlucose 

or 

Milk Sugar [Galactose 

fGlucose 

\ Galactose 

fGlucose 

\Glucose 



Sucrose 


"Glucose 


or 




Saccharose, 




or 




Cane Sugar 


Fructose 


~ , , ("Glucose 
1 rehalose { ^ , 
[Glucose 



Melibiose 



Maltose 



( Glucose " 
Isolactose lGalactose 



lt ( Glucose 
Isomaltose { Glucose 



. ,. f Glucose 
Gentiobiose \ ^, 

( Glucose 

^ ,, , . f Glucose 
Cellobiose ( Glucose 

Turanose | luc se 
Fructose 



Vicianose 



f Glucose 



\ Arabinose 
Gluco- ) Glucose 
xylose \ Xylose 



Galacto- f Galactose 
arabinose 1 Arabinose 



It is not yet definitely known how the two units are combined to- 
gether. In the non-reducing members the two functional aldehyde or 
ketone groupings will be combined ; in the reducing members the 
aldehyde or ketone grouping of the one unit will be united to one of 
the hydroxyl groups of the other unit. The following are the prob- 
able formula for 

Cane Sugar 
CH 2 OH C ' (CHOH)., CH CH 2 OH fructose residue 

/ 

CH (CHOH) 2 ' CH CHQH CH 2 OH glucose residue 



Trehalose 

CHoOH - CHOH - CH <CHOH) 
CHoOH CHOH CH (CHOHJ 2 ^GH / 
*O 








198 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

3faltos 
CH 2 OH CHOH-CH- (CHOH)., CH-O -CH 3 - CHOH- CH (CHOH) L ,-CHOH 



-o- 

Lactose 

CH 2 OH-CHOH-CH-(CHOH) 2 -CH-0-CHo CHOH-CH (CHOH) ./CHOH 

or 
Q CH 2 OH 

CH 2 OH CHOH CH (CHOH) 2 -CH-0 CH CH (CHOH) 2 ^CHOH 

galactose residue glucose residue 

The large number of possible disaccharides may be partly due to 
the possibility of combination with the several hydroxyl groups and 
partly to the possibility of the combination of a- or ft- forms of the 
constituents, thus 

a a, ft a, 



CANE SUGAR. 

Cane sugar is very widely distributed in the vegetable kingdom : 20 per 
cent, is present in the juice of the sugar cane, 10-20 per cent, in beetroot; 
smaller quantities are present in the maple and" birch and sweet fruits contain 
cane sugar together with glucose and fructose, which are probably derived 
from it by hydrolysis; 5-12 percent, of cane sugar is present in bananas, 
apricots, strawberries and pineapple. The mixture of glucose and fructose in 
honey is probably the result of the hydrolysis of cane sugar of the flowers by 
the formic acid secreted by the bees. 

Preparation. 

Cane sugar is prepared mainly from the cane and beet, though other 
plants, e.g. maple, palm, are used as sources of cane sugar. The manufacture 
in all cases is very similar. The juice of the cane, prepared by crushing the 
cane and pressing out, or the aqueous extract of beet, prepared by diffusion 
in a series of vessels, is treated with milk of lime to neutralise acids and boiled 
to precipitate proteins. The solution is treated with carbon dioxide to remove 
the last traces of calcium and with sulphur dioxide to decolorise it. It is 
again boiled and filtered and evaporated in. vacua until it crystallises. The 
residue, termed molasses, which does not readily crystallise yields more cane 
sugar on treatment of the boiling solution with lime or strontia, by which 
means an insoluble calcium or strontium saccharate is formed. The solid is 
separated and decomposed with carbon dioxide and the solution yields cane 
sugar on evaporation. Cane sugar molasses are most frequently fermented 
and converted into rum. 

Properties. 

Cane sugar in contrast to other sugars crystallises extremely readily and 
forms colourless monoclinic crystals easily soluble in water and only slightly 
soluble in alcohol. A saturated solution contains 66 per cent, of cane sugar. 
It melts on heating to about 160 to a glassy mass termed barley sugar which 
gradually crystallises again. If it be further heated to about 200 it is changed 
into a brown substance, caramel, which does not crystallise. 



THE CARBOHYDRATES 199 

Reactions. 

Cane sugar differs considerably from glucose in many of its 
reactions. 

(1) Formation of Esters. Cane sugar forms esters with eight 
hydroxyl groups. 

(2) Formation of Compounds with Metallic Hydroxides. Cane sugar 
forms insoluble compounds with lime, strontia, lead hydroxide, etc., 
more easily than glucose; this property as mentioned above is made 
use of in its commercial preparation. 

(5) Action of Alkali. Cane sugar, since it contains no aldehyde or 
ketone group, is not acted upon by alkali and does not give Moore's 
test. 

(6) Action of Hydrochloric Acid. Cane sugar is easily hydrolysed 
by boiling with dilute hydrochloric acid into glucose and fructose. 
Concentrated hydrochloric acid has the same action upon it as upon 
fructose (and glucose). 

(7) Reduction of Metallic Oxides in Alkaline Solution. Cane sugar 
does not reduce Fehling's solution, etc. Cane sugar, after hydrolysis 
by boiling with dilute acid and neutralisation of the acid with sodium 
hydroxide, reduces Fehling's solution, etc. 

(9) Formation of Osazones. Cane sugar does not form a phenyl- 
osazone. After hydrolysis by acids into glucose and fructose, phenyl- 
glucosazone is formed. 

(10) Fermentation. Cane sugar is fermented by yeast, but before 
fermentation into carbon dioxide and alcohol it is converted by 
hydrolysis by the enzyme, invertase, in the yeast into glucose and 
fructose. 

(11) MoliscJis Test. Cane sugar gives Molisch's reaction. 

(12) Rotation. Cane sugar is dextrorotatory. After hydrolysis by 
acids the mixture of glucose and fructose in equal parts shows laevo- 
rotation due to the laevo-rotation of fructose being greater than the 
dextro-rotation of glucose. Owing r to the change of rotation, or in- 
version, the mixture of glucose and fructose obtained from cane sugar 
is generally spoken of as invert sugar. 

Cane sugar gives Selivanoff's reaction since it contains fructose. 



2oo PRACTICAL ORGANIC AND BIO-CHEMISTRY 



LACTOSE. 

Lactose, or milk sugar, occurs in the milk of all animals, but has 
not been found in plants ; about 4 per cent, is present in cow's milk, 
from 6-8 per cent, in human milk. 

Lactose is prepared and manufactured from whey. The whey is 
evaparated and the lactose crystallises out ; it is purified by recrystalli- 
sation from water. 

Lactose forms a white crystalline powder, soluble in water, but in- 
soluble in alcohol. In taste it is less sweet than glucose or cane sugar. 

Reactions. 

Lactose resembles glucose in its reactions. 

(l) Formation of Esters. Lactose forms esters with eight hydroxyl 
groups. , 

(4) Oxidation. On oxidation of lactose with nitric acid, a mixture 
of mucic and saccharic acids is formed. 

(5) Action of Alkali. Lactose gives Moore's test. 

(6) Action of Hydrochloric Acid. Lactose is hydrolysed by boil- 
ing with dilute acid into a mixture of glucose and galactose. Strong 

acid has the same effect 
as upon glucose. 

(7) Reduction of Me- 
tallic Hydroxides in Alki- 
line Solution. The re- 
ducing power of lactose 
is less than that of glu- 
cose. (See under esti- 
mation.) After hydrolysis 
by acids the mixture of 
glucose and galactose has 
a greater reducing power 
than lactose. 

Lactose does not re- 
duce Barfoed's reagent. 

(g') Formation of Osa- 




FIG. 40. Lactosazone. 



zone. Lactose forms an osazone with phenylhydrazine in acetic acid 
solution in the same way as glucose. ^ Lactosazone is soluble in boil- 
ing water ; the compound separates as the solution cools. Its crystal- 
line form (Fig. 40) is different from that of glucose and it is thus most 
easily distinguished from glucosazone and also maltosazone. 

(10) Fermentation. Lactose is not fermented by yeast. 

(11) MoliscKs Test. Lactose gives Molisch's reaction. 

(12) Rotation. Lactose is dextrorotatory. After hydrolysis by 
acids into a mixture of glucose and galactose the rotatory power of 
the solution is greater than before. 






THE CARBOHYDRATES 



201 



MALTOSE. 

Maltose is found in plants and is formed in considerable quantities 
from starch during the germination of barley and other cereals. The 
polysaccharide is hydrolysed by the enzyme, diastase, in the grain 
into a mixture of maltose and dextrin : 

(C 6 H 10 5 ) n + H,0 = C^H^On + (C 6 H 10 5 ) n _ 2 . 

Maltose is also formed by the careful hydrolysis of starch by acids, 
and also from glycogen by the action of diastase. 

Diastase prepared from barley (30 gm.), (see p. 399), is added to 30 gm. 
of starch or soluble starch in 3000 c.c. of water. The mixture is kept at 
50 for 3 hours and then for 12 hours at room temperature. 60 per cent, 
of maltose is formed. The solution is filtered, evaporated to a thin syrup 
and poured into 95 per cent, alcohol. The precipitate of dextrin is re- 
moved and the alcohol distilled from the solution. Maltose separates out 
on standing. It is purified by dissolving in a little water, pouring into boiling 
alcohol, filtering, removing the alcohol and allowing to crystallise (Baker and 
Day, Brit. Assoc. Report, 1908, Sect. B., 671). 

Maltose is readily soluble in water from which it crystallises in white needles 
of the composition C^H^gOn . H 2 O. 

Reactions. 

Maltose resembles glucose in its reactions more closely than lactose. 

(l) Formation of Esters. It forms esters with eight hydroxyl groups. 

(5) Action of Alkali. Maltose gives Moore's test. 

(6) Action of Hydrochloric Acid. Maltose is hydrolysed by boil- 
ing with dilute hydrochloric acid into two molecules of glucose. 
Concentrated hydrochloric 

acid has the same action 
upon it as upon glucose. 

(7) Reduction of Me- 
tallic Hydroxides in Alka- 
line Solution. -- Maltose 
reduces Fehling's solution 
etc., but its reducing power 
is less than that of glucose. 
After hydrolysis by acids 
the reducing power of the 
solution is greater than 
before hydrolysis. 

Maltose does not re- 
duce Barfoed's reagent. 

(9) Formation of Osa- 
zone.. Maltose behaves like lactose in forming an osazone with 
phenylhydrazine in acetic acid solution ; it is soluble in boiling 




FIG. 41. Maltosazone. 



202 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

water and crystallises out as the hot solution cools. In its appear- 
ance maltosazone is different to glucosazone and lactosazone (Fig. 41) ; 
also in its melting-point, etc. 

(10) Fermentation. Maltose is fermented by yeast, being con- 
verted by the enzyme maltase in the yeast into glucose, which yields 
alcohol and carbon dioxide. 

(11) MoliscJis Test. Maltose gives Molisch's reaction. 

(12) Rotation. Maltose has a high rotation. The rotatory power 
of a solution of maltose diminishes when the maltose is hydrolysed by 
acid. 

Other Disaccharides. 

Trehalose occurs in certain fungi. 

Isomaltose was obtained by Fischer by the action of strong acids on 
glucose. Its formation from starch, together with maltose, by the action of 
diastase has not been definitely proved. 

Gentiobiose is obtained from the trisaccharide gentianose by hydrolysis 
with acids or by the enzyme invertase. 

Cellobiose has been prepared from cellulose. 

Melibiose is a product of the hydrolysis of the trisaccharide raffinose. 

Turanose is prepared from melicitose. 

Vicianose is present in the glucoside vicianin, obtained from the seeds 

of the vetch (Vicia augustifolia). 

i 

TRISACCHARIDES AND TETRASACCHARIDES. 

The number of known compounds in this group is small. They are 

Mannotriose. 
glucose galactose galactose. 

Rhamninose. 
galactose rhamnose rhamnose. 

Raffinose. 
fructose glucose galactose. 

Gentianose. 
fructose glucose glucose. 

Melicitose. 
glucose fructose glucose ? 

Stachyose. 
fructose glucose galactose galactose. 

Further details of these carbohydrates are given in Armstrong's monograph, 
" The Simple Carbohydrates and Glucosides ". 



THE CARBOHYDRATES 



203 



CHITIN AND CHONDROITIN. 

These polysaccharides which contain glucosamine as monosaccharide unit 
appear to be tetrasaccharides. 

Chitin. 

Chitin is composed of four glucosamine units or of three glucosamine 
units and one glucose unit, the amino groups of the glucosamine being acety- 
lated. It has been represented as having the formula 

CHA CHOH-CH-CHOH-CHNHCOCHs-CH 



CH-CHNHCOCH3-CHOH-CH-CHOH-CH 2 



CH-CHNHCOCHs CHOH-CH-CHOH-CH 2 




CHoCHOH-CH-CHOH-CHNHCOCH 3 -CH 



but this formula requires confirmation. 

Chitin is prepared from the shells of lobsters or crabs ; they are freed from 
meat, etc., mechanically, or by treatment with dilute sodium hydroxide and 
washing with water, dried and powdered. The powder is treated with dilute 
hydrochloric acid, water, boiling alcohol, water, boiling alcohol and ether. 

The powder as obtained above is colourless ; if the whole of small animals 
such as cockchafers be used the chitin consists of the skeletal structure of the 
animal. It is insoluble in water and other solvents, but is decomposed by 
concentrated hydrochloric acid yielding glucosamine hydrochloride (p. 189) and 
acetic acid. Three or four molecules of acetic acid are given by chitin. 

Chondroitin. 

Chondroitin or chondroitin sulphuric acid, is contained in cartilage 
either as such or in combination with protein, as a glucoprotein, i.e. as a 
chondroprotein. The work of Levene and La Forge l shows that it is a 
tetrasaccharide consisting of two chondrosamine units and two glycuronic 
acid units, the amino groups of the chondrosamine units being acetylated 
and its primary alcohol groups esterified with sulphuric acid. They gave it 
the formula : 



CO 

OH H NH H 

HO-SO^O-CH, C C C C C- 
H H\OH H / 



-() 



H OH H H 

HOOC C C C C C 

H OK, 



o 



, H OIT ., 

CH 3 HOOC-C C C C C 

CO j/l H OH H H 

OH II JsTH H 

HO-SO,.O-CH 2 C C C C C O 

H H\OH H / 



l ]. Biol. Chem., 1913, 15, 69, 155. 



204 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Chondroitin on hydrolysis with hydrochloric acid yields chondrosin. The 
sulphuric acid and acetic acid are split off and the molecule ruptured between 
the glycuronic acid molecules. Chondrosin is a glucosidic combination of 
chondrosamine and glycuronic acid. 

Preparation of the Barium Salt of Chondroitin and Chondroitin 
Sulphuric Acid. 

The nasal septa of cattle, freed from bone and other extraneous material, 
are ground up in a meat chopper. 5 kilo, portions are allowed to stand for 2 
days with 10 litres of a 2 per cent, solution of potassium hydroxide. The solu- 
tion is strained off through a cloth, the insoluble matter treated with 5 litres 
of potash solution and washed with water. The solutions are united, acidified 
with acetic acid and concentrated on a water-bath with excess of barium 
carbonate to about half the volume. The clear liquid is poured off andMhe 
residue filtered on a folded filter. The liquid and filtrate are acidified with 
acetic acid and evaporated to about 2 litres with barium carbonate. The solid 
matter, consisting of protein and barium carbonate, is separated from the clear 
yellow liquid by centrifugalisation and dropped into 8 volumes of glacial acetic 
acid and stirred with a mechanical stirrer. The acid potassium salt is precipi- 
tated, filtered off, washed with glacial acetic acid, alcohol and ether. 200 
gm. of this product, which may give a slight biuret reaction, are dissolved in 
10 litres of water, the solution is mechanically stirred and basic lead acetate 
added so long as a precipitate is formed. The lead salt is filtered off, washed 
with water by grinding in a mortar, suspended in 5 litres of water, to which 
100 gm. of barium acetate and 50 c.c. of acetic acid have been added, and 
decomposed with hydrogen sulphide. The lead sulphide is filtered off and 
the slightly turbid solution is precipitated by adding one-third of its volume 
of alcohol. The barium salt thus obtained is filtered off and washed with 50 
per cent, alcohol, 95 per cent.. alcohol, absolute alcohol and ether. It forms 
a white powder and is a mixture of the barium salt of chondroitin and chon- 
droitin sulphuric acid. 

Preparation of Chondrosin. 

... 50 gm. of the barium salt of chondroitin sulphuric acid are dissolved in 
150 c.c. of equal parts of concentrated hydrochloric acid and water and heated 
for an hour on the water-bath. Barium sulphate begins to separate out at once, 
the solution after i hour showing the maximum reduction of Fehling's solu- 
tion. The filtered solution is evaporated in vacua to a thick syrup, the syrup 
is dissolved in 40 c.c. of hot water and poured into 500 c.c. of absolute 
alcohol ; partial precipitation of chondrosin hydrochloride takes place. 2 
volumes of absolute ether are added after 12-16 hours and the precipitate is 
filtered off and washed with absolute ether. About 27 gm., dried over cal- 
cium chloride for 2 days, are obtained. It may be purified by solution in 
water and reprecipitated with alcohol and ether. It is a colourless powder, 
which is not hygroscopic if properly washed. 



, 



THE CARBOHYDRATES 205 

THE POLYSACCHARIDES. 

The polysaccharides are substances of high molecular weight. The 
size of their molecule is unknown, but it is composed of a large 
number of monosaccharide units. Their empirical formula is usually 
represented by (C 6 H 10 O 5 ) n , but many polysaccharides contain pentose 
units as well as hexose units and may consist entirely of pentose units 
(C 5 H 8 O 4 ) n . They may be classified into the following groups : 
Hexosans. Glucosans : Starch, dextrin, glycogen, cellulose. 
Fructosans : Inulin. 
Mannans. 
Galactans. 

Pentosans. Gums, pectins. 
Hexosan-Pentosans. Lignocellulose, hemicellulose. 

STARCH. 

Starch is present in various parts of plants and has been found in 
green leaves, fruits, seeds, tubers, etc. The amount of starch present 
in the seeds of cereals varies from 50-70 per cent, of the dry weight ; 
potatoes contain from 15-30 per cent. It occurs in definite 
granules starch grains which are made up of concentric layers 
around a hilum. When examined under a microscope these granules 
are seen to be of different forms. The source of starch grains can thus 
be ascertained from their microscopic structure. 

Preparation. 

Starch is prepared from wheat, rice, maize, potatoes, etc., by 
mechanical processes. The material is disintregrated by crushing, 
washed with water and passed through sieves. The starch in suspen- 
sion passes through and is allowed to settle. The water is drained off 
and the starch grains are dried. 

Starch may be purified by making a I per cent, suspension in water, 
freezing and allowing to melt The starch is left as a residue whilst 
the liquid contains the impurities. The operation is repeated four or 
five times. 

Properties. 

Starch grains form a white powder which is insoluble in cold water. 
If boiled with water the granules swell and burst forming an opale- 
scent solution, termed starch paste. Such a solution is most con- 
veniently made by rubbing starch grains into a cream with water and 
pouring the cream into boiling water and boiling for some minutes. 
The paste so formed varies in consistency with the amount of starch. 
Dilute solutions from 1-4 per .cent are limpid, but stronger solutions 
set into opaque white jellies. 



206 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Soluble Starch. 

Starch grains consist of at least two substances. The French workers 
Maquenne and Roux term them amylocellulose or amylose, the chief con- 
stituent, and amylopectin. Amylose (granulose of previous workers) is par- 
tially soluble in boiling water, but completely soluble in boiling water under 
pressure. On cooling, the insoluble portion is again obtained by " reversion ". 
The one seems to be a polymer of the other. Amylopectin is a gum-like 
substance which swells up without dissolving in water. The gelatinisation of 
starch paste is said to be due to the amylopectin. 

Fernbach states that soluble starch can be obtained from potato starch 
by pouring a 1-2 per cent, suspension into a large excess of acetone and 
shaking vigorously. A flocculent precipitate is formed, which, if filtered off, 
ground up with acetone in a mortar and dried in vacuo, dissolves in cold 
water. y i 

Starch grains treated with dilute hydrochloric acid of about i o per cent. J 
for 24 hours do not lose their external appearance, but they become soluble 
in hot water without forming a paste. Alcohol precipitates from the solution 
a white powder, soluble in water, termed soluble starch (Brown and Morris). 

Soluble starch is most readily prepared by allowing 500 gm. of starch to 
stand in contact with 1000 c.c. of dilute hydrochloric acid of sp. gr. 1*037 for 
7 days, stirring the mixture daily, pouring off the acid, washing the residue 
free from acid with water by decantation, the last portions containing a trace 
of ammonia, and drying it by exposure to the air. The dry product, ground 
up in a mortar and rubbed through a fine hair sieve, is soluble in warm water 
giving a clear solution (Lintner). 

Another method of preparing soluble starch is to treat 400 gm. of potato 
starch with 2300 c.c. of water and 80 c.c. of N HC1 in a flask in boiling 
water for 1*5 hours. The solution is cooled to 50, made ammoniacal and 
800 c.c. of alcohol added. The solution is strained through muslin, and 
whilst warm, poured into 4000 c.c. of alcohol. After 48 hours the precipi- 
tate is filtered off, washed with alcohol and spread out to dry. 



Reactions. 

The following reactions are given by starch paste or a solution of 
soluble starch. 

(1) Action of Alcohol. 

Starch is precipitated completely by adding an equal volume of 
alcohol. 

(2) Action of Iodine. 

If a few drops of iodine solution be added to a starch solution, a 
dark blue colour appears. On heating the solution, the colour disap- 
pears, but appears again on cooling. 

The blue colour is discharged on adding 1-2 drops of caustic 
soda. The colour reappears on neutralising with dilute hydrochloric 
acid. 

(3) Basic Lead Acetate. 

On adding basic lead acetate to starch solutions, the starch is pi 
cipitated. 



THE CARBOHYDRATES 207 

(4) Ammonium Sulphate. 

Starch is precipitated from solution by adding an equal volume of 
saturated ammonium sulphate solution, i.e. by half saturation with this 
salt 

(5) Fehling's Solution. 

Starch solutions do not reduce Fehling's solution. 

(6) Hydrolysis. 

Starch is easily hydrolysed into glucose by boiling its solution with 
dilute sulphuric acid for a few minutes. The presence of glucose can 
be shown by neutralising with soda and testing with Fehling's solution. 

(7) Rotation. 

Solutions of soluble starch have a high dextrorotatory power. 

DEXTRINS. 

Dextrins are glucosans which are intermediate in complexity be- 
tween starch and maltose. They have been found in plants, but are 
usually obtained by the hydrolysis of starch by the diastase in malt 
extract. 

The existence of a large number of dextrins has been supposed, 
but only two can be easily distinguished erythrodextrin, which gives 
a reddish-brown colour with iodine, and achroodextrin, which gives no 
colour with iodine. Erythrodextrin is probably a mixture of achroo- 
dextrin with a small amount of starch (Ost). 

Baker * has described a dextrin, termed tf-amylodextrin, which 
results from the action of the diastase of ungerminated barley upon 
starch paste, or soluble starch. It gives a blue colour with iodine. 

Preparation. 

The dextrins are prepared by the action of malt extract at 55 or 
by the diastase of ungerminated barley at 45-56 upon starch paste 
or soluble starch. The starch is converted by the malt diastase in 2-3 
hours into a mixture of 80 per cent, maltose and 20 per cent, dextrin, 
by the barley diastase into a mixture of 60-65 percent, of maltose and 
35-40 per cent, of dextrin. In the former case the reaction can be 
followed by testing portions of the solution at intervals with iodine ; 
the blue coloration disappears passing through a stage at which a red- 
dish-brown colour is observed. 

The prolonged action of malt extract slowly converts the dextrin 
into maltose. Maquerme and Roux consider that the maltose is 
derived from amylose and the dextrin from amylopectin. 

The solution containing the products of hydrolysis is concentrated 
and the dextrin is precipitated by pouring it into alcohol. The precipi- 
tate is dissolved in water and reprecipitated with alcohol. 

Commercial dextrin is prepared by heating starch at 180-200, 
until it has a pale brown colour. If the starch be previously treated 
with acid, it is heated at a lower temperature. 
1 J. Chem. Soc., 1902, 8l, 1177. 



2o8 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Properties. 

Commercial dextrin obtained by heating starch is a yellow-brown 
powder. 

The dextrin obtained by the hydrolysis of starch by malt extract 
is a white powder resembling starch. It is composed chiefly of achroo- 
dextrin and is soluble in water giving a sticky solution. The solution 
has a faint sweet taste and a peculiar smell. 

The dextrin obtained by the action of barley diastase is similar in 
appearance. 

Reactions. 

(1) Action of Alcohol. 

Dextrin is insoluble in alcohol and is precipitated from its solution 
in water by excess of alcohol. 

(2) Action of Iodine. 

Commercial dextrin generally gives a reddish-brown coloration 
on treating with 1-2 drops of iodine solution; this is due to the 
presence of erythrodextrin. The coloration disappears on heating 
and reappears on cooling. 

The dextrin produced by malt diastase gives no colour with iodine. 
That produced by barley diastase gives a blue colour. 

(3) Basic Lead Acetate. 

Dextrin is not precipitated from solution by basic lead acetate. 

(4) Ammonium Sulphate. 

Dextrin is not precipitated by half saturation with ammonium 
sulphate. 

(5) Fehling's Solution. 

The dextrins reduce Fehling's solution slightly. 

(6) Reduction of Dye-stuffs. 

Solutions of dextrin produce a red colour on warming with picric acid 
and sodium hydroxide. 

(7) Hydrolysis. 

Dextrin is easily hydrolysed' into glucose by boiling its solution 
with dilute sulphuric acid for a few minutes. The presence of glucose 
is shown by neutralising the solution with soda and testing with 
Fehling's solution. 

(8) Action of Alkali. 

On warming a solution of dextrin with alkali it becomes yellow or 
yellow-brown in colour. 

(9) Fermentation. 

Dextrin is not fermented by yeast. 

(10) Rotation. 

Dextrin has a high dextrorotatory power, 



THE CARBOHYDRATES 269 



GLYCOGEN. 

Glycogen is present as reserve food material in the organs of 
animals, but is also found in plants. In plants it is present in largest 
amount in yeast, as much as 30 per cent, of the dry weight having 
been recorded. In animals glycogen exists in greatest amount in the 
liver, usually from 1-4 per cent, but 12-16 per cent, have been 
obtained and 20 per cent, from frog's liver. Glycogen is found in 
other organs of animals, especially muscle. Heart muscle always 
seems to contain a quantity of glycogen. Oysters and other molluscs 
contain considerable quantities of glycogen. 

Preparation. 
From Liver. 

In order to obtain as large an amount of glycogen as possible the animal 
should be fed on a diet containing carbohydrate. The livers of rabbits con- 
tain a considerable quantity if they have been fed on carrots 5 or 6 hours 
previously. The animal is killed by bleeding, the liver removed and washed 
out with saline solution. It is broken up into small pieces and thrown into 
boiling water acidified with acetic acid. The proteins of the liver are thus 
coagulated and the enzyme which converts glycogen into glucose is destroyed. 
The pieces of liver are ground up finely in a mortar and extracted with 
boiling water. The extracts are combined (the remainder of the proteins 
precipitated by adding an equal volume of 10 per cent, trichloracetic acid), 
and the opalescent solution precipitated by adding an equal volume of alcohol. 
The precipitate is redissolved in water and reprecipitated with alcohol. The 
precipitate is dried by treating with alcohol several times, then with ether and 
placing in a desiccator over sulphuric acid. 

Pfluger's method of preparing glycogen yields a purer preparation : 
The finely broken up liver is stirred up with water and 60 per cent, 
potassium hydroxide so that it contains 15 percent. KOH and heated for 
2 hours on the water-bath. The solution is filtered and mixed with an equal 
volume of alcohol. The glycogen is precipitated and washed with a mixture 
of i part of 1 5 per cent. KOH and 2 parts of alcohol. It may be redissolved 
and reprecipitated by alcohol. It is then washed with alcohol and ether and 
dried. 

From Yeast. 

Yeast is ground up with sand to rupture the cells, extracted with boiling 
water, and the filtered solution precipitated by adding an equal volume of 
alcohol. The precipitate is collected, washed with 50 per cent, alcohol and 
heated for 2 hours on a boiling water-bath with a solution of 60 per cent, 
potassium hydroxide to dissolve proteins. The liquid is cooled, poured into 
an equal volume of water, filtered and precipitated by adding two volumes of 
alcohol. The precipitate of glycogen is washed with alcohol containing KOH 
and then with alcohol. It is purified by solution in water, neutralisation of 
the alkali with acetic acid, and precipitation with an equal volume of alcohol. 
This procedure is carried out several times. The product contains yeast gum. 
This is removed by dissolving in water and saturating the solution with am- 
monium sulphate. The glycogen is precipitated and washed with ammonium 

14 



2io PRACTICAL ORGANIC AND BIO-CHEMISTRY 

sulphate, again dissolved and again precipitated. Finally it is dissolved in 
water, the ammonium sulphate removed by dialysis, and the glycogen pre- 
cipitated by alcohol. It is washed with alcohol and ether and dried. 

Properties. 

Pure glycogen is a white amorphous powder, soluble in cold 
water forming an opalescent solution, which is very characteristic. 
Reactions. 

(1) Action of Alcohol. 

Glycogen is precipitated from solution by adding an equal volume 
of alcohol. The precipitation does not occur if the solution does not 
contain some salts ; a small quantity, '05 gm. of sodium chloride, is 
required to precipitate a I per cent, solution of glycogen with two 
volumes of absolute alcohol. 

(2) Action of Iodine. 

Solutions of glycogen give a reddish-brown colour on treatment 
with i to 2 drops of iodine solution. The coloration disappears on 
heating and reappears on cooling. 

(3) Basic Lead Acetate. 

Solutions of glycogen are precipitated by basic lead acetate. 

(4) Ammonium Sulphate. 

Solutions of glycogen are not precipitated by half saturation with 
ammonium sulphate, but the glycogen is precipitated by complete 
saturation of the solution with ammonium sulphate crystals. 

(5) Fehlings Solution. 

Glycogen does not reduce Fehling's solution. 

(6) Hydrolysis. 

Glycogen is converted into glucose by hydrolysis. Solutions of 
glycogen, boiled with dilute acid and neutralised, reduce Fehling's 
solution. 

(7) Action of Alkali. 

Glycogen is not acted upon by alkali. 

(8) Fermentation. 

Glycogen is not fermented by yeast. 

(9) Rotation. 

Glycogen has a high dextrorotatory power. 



THE CARBOHYDRATES 211 

CELLULOSE. 

The cell walls of plants, which are elaborated by the protoplasm, are sup- 
posed to consist primarily of the substance termed cellulose, but during growth 
the plant forms other substances which are encrusted in the cellulose, so that 
the material of the cell wall consists of a mixture (or compound) of cellulose 
and other substances. The materials which are encrusted in the cellulose are 
lignin l or lignone forming lignocellulose in wood, straw, etc., pectins and 
gummy substances forming pectocellulose, fatty substances forming adipo- 
cellulose.. The material which contains most cellulose is the fibre of the 
cotton plant, hemp and flax. Pure cellulose is generally made from these 
products. Wood contains 50-60 per cent, of cellulose; straw contains a 
similar amount, but silica is alsT present. 

Cellulose is also found in the animal kingdom ; the tunicin in the cell 
walls of tunicates is said to be identical with cellulose. 

Preparation of Cellulose from Cotton. 

Since cellulose is very resistant to most chemicals pure cellulose is pre- 
pared from cotton fibre by the following treatment : 

The fibre is boiled with 1-2 per cent, caustic potash and washed with 
water. Pectins are thus removed. The fibre is treated with bromine or 
chlorine at the ordinary temperature. The lignin or lignone is destroyed and 
dissolves. The fibre is then treated with sodium sulphate, carbonate or 
hydrate. The residue is washed with water and dried. 

Preparation of Paper Cellulose. 

Linen rags or cotton waste are cleaned, cut up and boiled under pressure, 
firstly with dilute sodium carbonate and secondly dilute caustic soda so as to 
disintegrate them. The material is bleached with chlorine, washed free from 
the halogen, treated with resin, soap and alum, and spread'out in thin layers 
to dry. The fibres thus become felted together. Wooa is disintegrated 
and the lignin dissolved out by treatment with calcium bisulphite. The 
residue is treated as above with soap, etc. 

Paper of an inferior quality is made from wood which has not been 
treated ; it gives the reactions for pentose if a solution of aniline acetate be 
poured upon it or if it be treated with a 1-2 per cent, solution of phloro- 
glucinol in alcohol and dilute hydrochloric acid. On exposure to light such 
paper becomes yellow. 

Properties. 

Pure cellulose is a white substance which is hygroscopic and. absorbs 
about 10 per cent, of water. The water is removed by heating it to 100. 

Solubility. 

It is insoluble in water and all ordinary solvents, but it is decomposed by 
water* under pressure. 

gelatinous in a solution of zinc chloride and finally dissolves. 
On stirring i part of cellulose with 6 parts of zinc chloride in 10 parts of 
water at 60, it gelatinises after some time; on raising the temperature by 
placing it in a boiling water-bath the cellulose gradually dissolves. 

Cellulose dissolves rapidly in a cold solution of zinc chloride in twice its 
weight of hydrochloric acid. 

Cellulose dissolves in a solution of ammoniacal cupric oxide (Schweitzer's 
reagent). On adding acid, it is precipitated. 

1 Lignin appears to contain aromatic substances and pentosans. 

14* 



212 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

One variety of artificial silk is prepared by dissolving mercerised cotton 
in Schweitzer's reagent (see p. 616), and running it in a thin stream into dilute 
sulphuric acid. A thread of cellulose is thus precipitated. 

Reactions. 

(1) Action of Alkali. 

Dilute solutions (1-2 per cent.) of caustic soda even at 100 have no 
action upon cellulose. More concentrated solutions (10 per cent.) cause the 
fibres to swell and become cylindrical and destroy the central canal. The 
appearance becomes glossy. This property was used by Mercer for treating 
cotton to make it appear like silk. 

Cellulose treated with 15 per cent, alkali reacts with carbon disulphide, 
forming a thiocarbonate. This substance decomposes in the air giving 
carbon disulphide and cellulose. The solution, if forced through fine openings 
and allowed to come into the air, forms continuous threads of artificial silk. 

(2) Action of Acids. 

Dilute sulphuric acid converts cellulose into hydrocellulose. 

Dilute nitric acid (sp. gr. 1*25) at 80 converts cellulose into oxycellulose 
which reduces Fehling's solution. 

Concentrated sulphuric acid dissolves cellulose. On diluting the solution, 
a gelatinous compound is precipitated. This substance is called amyloid, as it 
gives a blue colour with iodine, like starch. Parchment paper is made by 
treating paper with 2 parts of sulphuric acid and i part of water and then 
washing the acid away with water. 

(3) Formation of Esters. 

(a) Nitric acid. 

Concentrated nitric acid, or a mixture of this acid with sulphuric acid, con- 
verts cellulose into nitric acid esters. Collodion is a mixture of the tri- and 
tetra-nitrates dissolved in a mixture of equal parts of alcohol and ether. 
Celluloid is a mixture of the tri- and tetra-nitrates with camphor. 

Gun cotton, or pyroxylin, is cellulose hexanitrate and is prepared by treating 
cotton waste (freed from fats by treating with alkali) with a mixture of i part 
nitric acid and 3 parts sulphuric acid. The product, which has still the 
original appearance, is washed with water, moulded and dried. It is con- 
verted into smokeless powder by dissolving in acetone or ethyl acetate and 
evaporating the solution. When mixed with nitroglycerine and other sub- 
stances it forms blasting gelatin, cordite, etc. 

Artificial india-rubber is a product prepared by mixing together tri- and 
tetra-nitrocellulose with castor-oil. The inflammability of this material is 
eliminated by treating it with alkali. 

(b) Acetic acid. 

Cellulose acetates are obtained on treating cellulose with glacial acetic 
acid and acetic anhydride in the presence of concentrated sulphuric acid. 
These compounds are insoluble in water, but soluble in organic solvents. 
A solution of tetra-acetyl cellulose in acetone on evaporation yields artificial 
gutta-percha. 

A white precipitate is formed when a solution of cellulose acetaj 
glacial acetic acid is poured into alcohol. This solid does not melt, oBT 
bums without leaving an ash. It forms " solid spirit ". 

It is also used to make artificial silk. 

(4) Hydrolysis. 

Cellulose is dissolved by concentrated sulphuric acid, which hydrolyses it 
to glucose. 



THE CARBOHYDRATES 213 

Inulin. 

Inulin occurs in the sap of a number of plants and is most abundant in 
the tubers of the dahlia (10-12 per cent.) and artichoke. 

Inulin is prepared from dahlia tubers by crushing and pressing out the 
juice ; the residue yields more inulin if boiled up with water and chalk. 
The two solutions are combined, boiled with chalk to neutralise acids, 
filtered and treated with lead acetate as long as a precipitate is formed. The 
filtered solution is treated with hydrogen sulphide, filtered from lead sulphide 
and evaporated to half its volume. An equal volume of alcohol is added 
and the precipitate of inulin filtered off after 1-2 days. It may be purified 
by dissolving in water, warming the solution with animal charcoal, filtering 
and reprecipitating with alcohol. The precipitate is washed with alcohol and 
ether and dried in a desiccator over sulphuric acid. 

Inulin forms a white powder with a sphserocrystalline appearance. It has 
no taste. It swells up and dissolves in hot water giving a clear solution. 

Reactions. 

(1) Action of Alcohol. Inulin is insoluble in alcohol and is precipitated 
from solution by adding an equal volume of alcohol. 

(2) Action of Iodine. Solutions of inulin give a brownish coloration with 
iodine. The iodine solution used must be very weak and it is advisable to 
carry out a control test, i.e. adding the same amount of iodine to an equal 
volume of water. 

(3) Basic Lead Acetate. Inulin solutions are precipitated by basic lead 
acetate. 

(4) Fehlings Solution. Inulin does*Mt reduce Fehling's solution. 

(5) Hydrolysis. Inulin is very aa5^nydrolyse i by mineral acids and 
converted into fructose. The hydrolysed solution, after neutralisation, gives 
the reactions for fructose. 

(6) Rotation. Inulin has laevorotation. 

Mannans. Galactans. Hemicellulose, etc. 

Polysaccharides different from those previously described occur in the 
seeds of numerous plants. They have resemblances to cellulose, but differ 
from cellulose in dissolving in dilute -alkali, in being hydrolysed by dilute 
mineral acids and in yielding other monosaccharides as well as glucose. They 
are soluble in Schweitzer's reagent after treatment for a short time with dilute 
hydrochloric acid. They form a very indefinite group of substances and re- 
quire further investigation. 

Gums. Pectins. Mucilages. 

The gums, pectins and mucilages are complex polysaccharides containing 
both hexose and pentose units. The gums appear to be carbohydrates com- 
bined with acids ; some are completely soluble, others are partially soluble in 
water and others only swell up with water. 

Mucilages are very widely distributed in plants and form a slimy liquid 
with water. 

Pectins are contained in fruits, turnips, etc. The gelatinisation of boiled 
fruit extracts is probably due to the presence of pectin. 

Schryver and Haynes * showed that these plant materials contained the 
acid substance, pectinogen, which is soluble in water. Pectinogen is teadily 
changed into another acid substance, pectin, by dilute alkali. Mineral acids 
precipitate pectin as a gel from the alkaline solution ; calcium chloride gives a 
gelatinous precipitate of the calcium salt. Pectin has the composition QfH^Oie 
and contains a pentose group. Pectinogen is extracted from the pressed resi- 
due of the plants by warm 0*5 per cent, ammonium oxalate solution, 
1 Piochem. J., 1916, 10, 539- 



214 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



GLUCOSIDES. 

In addition to the carbohydrates there also occur in nature a large 
number of compounds which contain glucose, more rarely other sugars, 
e.g. galactose, rhamnose and disaccharides, combined with other organic 
compounds, especially those belonging to the aromatic series. These 
are the glucosides. Glucosides have also been prepared in the labora- 
tory from glucose, mannose, maltose, etc. Two isomers are generally 
thus obtained, termed the a- and /3-glucosides. The chief of the syn- 
thetical glucosides are the a- and /3-methyl glucosides which are pre- 
pared by the action of hydrochloric acid upon a solution of glucose 
in methyl alcohol. They are^tekived from a- and /3-glucose by the 
replacement of the hydrogen atom* Tn the hydroxyl group attached to 
the carbon atom which possesses aldeydic properties : 
CH OH CH.,OH 





HO -OH 
H-OOH 

H C^OCH S CH 3 C-H 

o-methyl glucoside. 0-methyl glucoside. 

These two glucosides, besides having different physical properties, be- 
have differently towards the enzymes, maltase and emulsin. Maltase 
hydrolyses the a-glucoside, but not the /3-glucoside, emulsin hydrolyses 
the /3-glucoside, but not the a-glucoside. The natural glucosides are, 
in general, hydrolysed only by emulsin and would be derivatives of 
/3-glucose, i.e. /3-glucosides. 

The three best-known glucosides are probably 

,0 . C 6 H n 5 /CN /O . C 6 H U 5 

C 6 H 4 \ C 6 H 5 .CHy C 6 H 4 x 

\CH 2 OH \0 . C 12 H 21 O ao \OH 

Salicin. ' Amygdalin. Arbutin. 

Salicin is a combination of glucose with saligenin or salicylic alcohol. 



THE CARBOHYDRATES 215 

Amygdalin is a combination of 2 molecules of glucose, hydrogen 
cyanide and benzaldehyde. 

Arbutin is a combination of glucose with hydroquinone (p. 261). 

The composition of glucosides is ascertained by identification of 
their products of hydrolysis. 

Preparation. 

The quantity of glucoside present in plants is usually small. Since 
enzymes are present which hydrolyse the glucoside, it is advantageous 
to destroy the enzyme by heating with water or alcohol before extract- 
ing the glucoside. The glucoside is usually isolated by extracting 
the material with water, alcohol, ethyl acetate or other organic solvent, 
concentrating the extract and crystallising out the glucoside. In 
some cases the extract requires purification so that no general scheme 
can be given for isolating glucosides. 

Properties. 

The glucosides are usually white crystalline substances, soluble in 
water and having a bitter taste; They are soluble in some organic 
solvents, but generally insoluble in ether. 

Reactions of Salicin. 

(1) Salicin does not reduce Fehling's solution. 

(2) Salicin solutions are hydrolysed by boiling with dilute sul- 
phuric acid into glucose and salicylic alcohol. The solution, after 
neutralisation with soda, reduces Fehling's solution. 

Reactions of Amygdalin. 

(1) Solutions of amygdalin do not reduce Fehling's solution. 

(2) Solutions of amygdalin are hydrolysed by boiling with dilute 
nitric acid into benzaldehyde, hydrogen cyanide and glucose. The 
solution smells of benzaldehyde and hydrogen cyanide. The presence 
of hydrogen cyanide may be shown by testing with silver nitrate ; the 
presence of glucose by neutralising with soda and testing with Fehling's 
solution. 

Other glucosides are known which also contain hydrogen cyanide. 
They are generally referred to as cyanogenetic glucosides. Their 
presence in leaves may be detected by chewing a small piece of the 
material, or better by introducing the bruised material and a drop of 
chloroform into a small test tube, hanging a piece of picric acid test 
paper l in it and closing it with a cork. Hydrogen cyanide is slowly 
evolved and it colours the test paper orange red. 

1 This is prepared by dipping strips of filter paper into a i per cent, solution of picric 
acid, drying them, wetting them with a 10 per cent, solution of sodium carbonate and 
again drying. 



ESTIMATION OF CARBOHYDRATES. 

The methods of estimating carbohydrates depend ultimately on 
the methods of estimating glucose. Though at first sight the estima- 
tion of glucose may appear as a comparatively easy task, yet on 
examination of the literature few subjects seem to have been more 
worked at than this simple problem. Over thirty methods have 
been devised by the most distinguished chemists and new ones are 
continually being described and advocated. 

Three of the properties of glucose (and other carbohydrates) are 
most usually made use of for its estimation : 

A. Its optical activity, by means of the polarimeter. 

B. Its aldehyde character, by the reduction of metallic salts, 
especially copper. 

C. Its fermentation, by yeast. 

Each of these methods has its own particular advantages, which 
depend mainly upon its convenience, ease of manipulation, rapidity of 
completion, and desired accuracy. 

A. ESTIMATION BY MEANS OF THE POLARIMETER. 
i. The Construction of a Polarimeter. 

In an ordinary ray of light the. vibrations of the waves take place 
in all planes perpendicular to the direction of its propagation. If 
such a ray of light be passed through a crystal of Iceland- or calc-spar 
and an object be observed through the crystal, two images will be seen. 
The ray of light has been split into two rays, one of which has been 
more refracted than the other. The more refracted, or ordinary, ray 
travels through the crystal just as it would travel through glass and 
obeys the laws of refraction. The less refracted, or extraordinary, ray 
does not obey the ordinary laws of refraction, and it shows a movable 
image when the crystal is rotated. Both of these rays in their passage 
through the crystal have been polarised in two directions at right 

216 



ESTIMATION OF CARBOHYDRATES 217 

angles to each other : i.e. the vibrations of each ray which are trans- 
mitted are now only in one plane. 

By employing a rhombohedron of Iceland-spar, cutting it across 
through its obtuse angles, polishing the cut surfaces, cementing 
together these cut surfaces with Canada balsam, and blackening the 
longer sides, a prism is obtained. On passing light through this prism, 
the ordinary ray is totally reflected by the cut surfaces and absorbed 
by the blackened side, whilst the extraordinary ray passes through and 
emerges in a direction parallel to the source of the light. Such a 
prism is termed, after its discoverer, a Nicol prism. 

In a polarimeter two Nicol prisms, mounted in line with one another, 
are employed. The first is fixed, the second is capable of being 
rotated. Light is passed through the first prism (the polariser) and 
reaches the second prism (the analyser). If this second prism be ex- 
actly parallel to the first, the beam of light will also pass through it ; 
if it be not exactly parallel but inclined at an angle, less light will pass 
through it ; if the second prism be at right angles to the first, or 
crossed, the light is entirely cut off. 

By interposing between the prisms, set parallel to one another, a 
solution of an optically active substance, the amount of the light is 
diminished, but it can be brought to its original intensity by rotating 
the analysing prism. The amount of rotation necessary to effect this 
corresponds with the power of rotation of the solution. As the an- 
alysing prism is mounted on a graduated circle, the number of degrees 
rotated can be measured. This is the rotatory power of the solu- 
tion. 

The determination of equal illumination of light in such an instru- 
ment before and after its passage through an optically active solution 
is very difficult and the readings are erroneous. Several devices have 
been adopted to overcome this difficulty, the simplest being that of 
Laurent. Laurent placed behind the polariser a quartz plate of special 
thickness and of such a size that it covered half the field. This quartz 
plate divides the ray of light passing through it into two rays, one of 
which is retarded by half a wave length and therefore reversed in direc- 
tion, whilst the other is unaffected. The resultant ray formed on 
emergence by their fusion will be vibrating in a plane at an angle to the 
original plane, i.e. the polarised light passing through the quartz plate 
is rotated through a certain angle. Thus, if AO be the original plane 
before passage through the quartz plate, it is resolved into AC and AD. 
Supposing AD is retarded and reversed, then the components AC and 



218 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

AD 1 will form the resultant plane AR. The angle CAO = angle 
CAR. 

R C O 




A 

Two beams of polarised light at an angle to one another will there- 
fore reach the analyser. If the analyser be set parallel to the beam 
AO arriving from the uncovered portion, this half of the field will appear 
light and the other half will appear dark. If it be set parallel to the 
beam AR coming from the covered portion, this half of the field will 
appear light and the other half dark. By adjusting the analyser a 
position will be found where the two halves will appear equally illu- 
minated. This position is the zero point. 

The two halves of the field are illuminated by component portions 
of the two beams. At the zero point the two prisms are almost in a 
crossed position. The instrument is most sensitive under these con- 
ditions, but the amount of light is at a minimum. 

In other polarimeters, such as Lippich's, a prism which has the 
same effect as a quartz plate is placed in the centre of the field. The 
centre and sides of the field appear dark or light. 

In determinations with such polarimeters monochromatic light 
must be used ; for convenience, sodium light is generally used, and in 
this case a cell containing potassium bichromate is introduced in front 
of the polariser to cut off blue rays ; green light from a mercury lamp 
is sometimes used. 

A polarimeter (Figs. 42, 43) will thus consist of a bichromate cell, a 
polarising prism, a quartz plate over half the field, a trough to take the 
solution to be examined, an analysing prism mounted in a movable 



! D 



die' 

FIG. 42. 

L = source of light. A = lens to render rays of light parallel. B = polarising prism. 

C, C' = quartz plate. O = observation tube. D = analysing prism. E, F = telescope. 

(From Findlay's " Practical Physical Chemistry ".) 

circle graduated in degrees. There is, in addition, a telescope to focus the 
edge of the quartz plate and a double vernier on each side of the circle 
in which the analyser is mounted. This vernier is fixed and graduated 
in fractions of a degree, or in minutes. 



ESTIMATION OF CARBOHYDRATES 



219 




F.G. 43- 

At S, lens and bichromate cell. At P, polarising prism. At h, lever to rotate polarising 
prism. At A, analysing prism which can be rotated by a screw. At F, telescope with 
eye-piece. K = graduated scale. n,n' = fixed verniers. T = screw for rotating gradu- 
ated scale. / = magnifying lens to read scale and verniers. 

2. The Observation Tube. 

The solution of the substance is placed in a special observation 
tube (Fig. 44). These tubes are generally 0-5, I, 2, 2-2 decimetres 
long ; they are made of glass of the exact length ; the ends are closed 
by cover glasses held in place by a screw cap and rubber washer. Very 
small tubes for use with small amounts of solution are also made. 

These tubes are thoroughly dried by pushing a plug of filter paper 
through them, or thoroughly washed by rinsing several times with the 




FIG. 44. 

solution under examination. The cover glasses must be dry and 
without serious scratches. One end of the tube is closed by a cover 
glass, brass cap and washer, and the solution is filtered into it at the 
other end until a meniscus just projects above the opening. A short 
time is given to allow air-bubbles to rise. The other cover glass is 
slid horizontally over the end of the tube so that it pushes off the 
excess of liquid and exactly covers the end leaving no air-bubbles 



220 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

underneath it and no liquid on its upper surface. The brass cap and 
washer is then screwed down over it. The brass caps must not press 
too tightly on the glass covers. 

3. Reading the Polarimeter. 

At the point of equal illumination of the two halves of the field 
the zero of the circular scale coincides, or very nearly coincides, with 
the zero of the vernier. The exact position must be determined. 
When an optically active substance is placed between the prisms and 
equal illumination of the two fields restored, the circular scale will 
have moved in a clockwise direction ( = dextrorotation), or in a counter 
clockwise direction ( = laevorotation), from the vernier. The distance 
apart of the two zeros measured on the circular scale gives the amount 
of rotation in degrees ; the fraction, or minutes, more is given by the 
vernier scale. 

Several observations of the zero point of the instrument and then 
several of the solution must always be made. The mean of each is 
taken and the difference gives the rotation. 

4. Estimation. 

As the rotatory powers of all the common optically active com- 
pounds have now been determined, use can be made of these values to 
determine the strength of an unknown solution. These values are 
expressed as specific rotatory power, i.e. the rotation of I gm. of sub- 
stance in I c.c. of liquid examined in a layer I decimetre (10 cm.) 
long, i.e. it is the rotatory power of a 100 per cent, solution. This 
has not actually been carried out, but it has been calculated from the 
rotations of exactly known strengths of solution. The symbol [<z] D 
is used to express this value, the D standing for sodium light. The 
rotation varies with the temperature of the solution and is also 
recorded ; this reading is included in the symbol, thus [a]*. Rotations 
are generally measured at 20, but may be taken at other temperatures. 

The following are the values for the principal sugars in solutions 
containing about 10 per cent : 

Mannose = + 14-2 Lactose = + 52-5 Glycogen = + 196-6 

Glucose = + 527 Maltose = + 138 Dextrin = + 195 

Fructose = - 93 Sucrose = + 66-5 Starch = + 199 

Galactose = + 83 Raffinose = + 104 

The strength of the solution is then given by the formula : 

r n , 

HD = 



a X IOO 



c x I 

in which [o] D = specific rotation. 
a = observed rotation. 
c = concentration. 
I = length of tube in decimetres. 



ESTIMATION OF CARBOHYDRATES 221 



B. ESTIMATION BY REDUCTION OF COPPER SALTS. 

This method of estimating glucose is the one most frequently 
used and is the one which is the most varied in manipulation. The 
variations may be divided into the following groups : 

L Complete reduction of cupric to cuprous salt. 

Methods of Fehling-Soxhlet ; Pavy ; Gerrard ; Benedict. 

II. Incomplete reduction of cupric to cuprous salt. 

(a) Gravimetric by the estimation of the precipitated cuprous oxide. 
Methods of Maercher ; Allihn ; Kjeldahl ; Brown, Morris and 

Millar ; Pfliiger. 

(b) Volumetric. 

(i) Direct by the estimation of the precipitated cuprous oxide. 
Methods of Mohr-Bertrand ; Caven and Hill ; Sidersky ; Bang's 
second method. 

(ii) Indirect by the estimation of the residual cupric salt. 
Methods of Lehmann-Maquenne ; Bang's first method. 

(i) Fehling-Soxhlet Method. 

Barreswil in 1 844 was the first to use this property as a means of estimat- 
ing glucose, his reagent consisting of an alkaline solution of neutral potassium 
tartrate and coppei* sulphate. Fehling, in 1849 and 1858, established this re- 
duction process of estimating glucose by showing that the ratio of glucose to 
cupric oxide was 1:5. He used Rochelle salt, as suggested by Bodeker, in 
place of neutral potassium tartrate. It was found later that the ratio of i : 5 
was not exact and that it varied with the concentration, alkalinity and time of 
boiling. These difficulties were overcome in 1880 by Soxhlet, who showed 
that accurate results could be obtained if (i) the copper solution had always 
the same concentration and (2) the sugar solution under examination had a 
concentration of about i per cent. Soxhlet, following suggestions by Krause 
and Staedeler and by Graeger, employed two solutions, which were mixed in 
equal volumes immediately before use. Such solutions were found to keep well, 
whereas, if mixed, the titer changed and the solution reduced itself on boiling. 

The method as described by Fehling with Soxhlet's modifications is the 
simplest and the one most commonly used at the present time for a rapid 
and very fairly accurate estimation of glucose. 

It is as follows : 



222 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

(a) Preliminary Rough Estimation. 

10 c.c. of Fehling's solution (i.e. 5 c.c. of each) are measured out 
with a pipette into a porcelain basin or small flask, diluted with about 
40 c.c. of water and raised to the boiling-point. 

The sugar solution is run in from a burette, i c.c. at a time, whilst 
the Fehling's solution is kept gently boiling the whole time. 

The reduction must be allowed to complete itself before adding a fresh 
quantity of the sugar solution. 

It is noted when the blue colour of the solution has entirely dis- 
appeared. The solution may become slightly yellow, due to the action 
of the alkali of the Fehling's solution upon excess of the sugar solution. 

An idea of how much sugar is present in the solution is thus ob- 
tained. 

(b} Dilution or Concentration of the Sugar Solution. 

Since the method is only accurate if the concentration of the 
sugar is between 0-5 and I per cent, the sugar solution must be diluted 
or concentrated. 

It is best to have as nearly as possible 10 c.c. of the diluted or con- 
centrated sugar solution = 10 c.c. of Fehling's solution. If less than 
10 c.c. of the glucose solution have been used, a known volume of the 
solution is diluted ; if more, a known volume is concentrated to a smaller 
volume, e.g. 100 c.c. to 30 c.c. 

Suppose 3 c.c. of the sugar solution were sufficient ; then 3 c.c. 
should be diluted to 10 c.c. 

It is more convenient to dilute a larger quantity : 30 c.c. are 
measured out with a pipette into a 100 c.c. measuring flask, the flask is 
filled to the mark with water and the contents are mixed : or 30 c.c. 
are measured into a dry flask and 70 c.c. of water are added with 
a clean pipette. 

The burette is carefully rinsed out with the diluted sugar solution 
and the final titration carried out. 



ESTIMATION OF CARBOHYDRATES 223 

(c) Final Titration. -\\ 

10 c.c. of Fehling's solution are diluted as before and the diluted 
glucose solution carefully added to the boiling liquid. It is advisable 
to run in at once a little less than the amount required to decolorise 
the solution entirely (say 8 c.c.), and then to add cautiously cri to 0*2 
c.c. at a time until there is complete decolorisation, always allowing time 
for the reduction to occur. This final titration should be repeated 
running in practically all the glucose solution necessary at one time, 
and then completing with cri c.c. at a time. Suppose icn c.c. were 
insufficient, but 10-3 c.c. too much, as seen by a faint yellow colora- 
tion of the solution, then 10*2 c.c. is the proper value. 

Soxhlet carried out altogether 5 or 6 titrations, adding more or 
less than the exact amount of glucose solution at once, and thus deter- 
mined the limits of too much and too little until they approached one 
another and differed by only cri c.c. 

(d) The Determination of the End Point. 

The great difficulty of the estimation is the determination of the 
end point, i.e. when the blue colour is completely discharged. The 
eye by itself is not very sensitive, but the first trace of yellow in the 
solution can generally be seen. When the sugar solution is added cri 
c.c. at a time, this amount, or O'2 c.c., can be deducted, depending on 
the observer's judgment. 

Lavalle has suggested that the dilution of the Fehling's solution be done 
with caustic soda solution instead of with water. The cuprous oxide either 
settles better or stays in solution, depending on the amount used ; but the 
result is not so accurate in the presence of excess of caustic soda. 

(e) Use of Indicators. 

(i) Potassium Ferrocyanide. 

Both Fehling and Soxhlet used indicators to determine the end 
point. A small quantity of the solution is removed and filtered if 
necessary, or a drop may be taken and tested by adding some acetic 
acid to acidify it and potassium ferrocyanide. A brown coloration or 
precipitate of copper ferrocyanide shows that copper is still present in 
the solution. No^colour is formed when the reduction is complete. 

In the case of the estimation of glucose in urine the indicator cannot 
be used, since the ammonia which is formed dissolves some of the 
cuprous oxide and a colour is given with the ferrocyanide. 



224 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

(2) Starch and Potassium Iodide. 

This indicator was suggested hr 1903 by Harrison (see p. 614). Its 
use depends upon the liberation of iodine by cupric salts and it will show 
the presence of copper sulphate in a dilution of i in 20,000. 

A drop of the titration solution is added to i c.c. of the indicator 
acidified with 10 drops of acetic acid. A red or blue colour is shown if 
cupric salt be present ; no colour is given when reduction is complete. 

(3) Ferrous Thiocyanate. 

A more suitable and convenient indicator and one easy to prepare 
is that described by Ling and Rendle in 1905 (see p. 614). It consists 
of an acid solution of ammonium thiocyanate and ferrous ammoniu 
sulphate. On treatment with a cupric salt, the ferrous salt is oxidised 
to ferric which reacts with the thiocyanate giving the bright-red ferric 
thiocyanate. 

This indicator is recommended in making the sugar estimations in 
malting processes. 

In practice, the titration is carried out in a small flask in prefer- 
ence to a basin, and on nearing the end point at which the blue 
colour is discharged drops are taken out and placed against one of a 
series of drops of indicator upon a glass plate on a white surface. The 
reduction of the Fehling's solution is complete when a red coloration is 
no longer produced. 

(4) Reduced Phenolphthalein. 

Carletti suggested this indicator in 1913 (see p. 614). 

If cupric salt be still present in the titration solution when a drop is added 
to a drop of the reagent + 2-3 drops of 10 per cent, potassium cyanide 
solution, a red colour is produced. 

(/) Calculation of the Result. 

Knowing the dilution,^ the amount of sugar in the original solution 
can be calculated. 

10 c.c. Fehling's solution = 0-05 gm. glucose, 

.'. io'2 c.c. diluted sugar solution = 0-05 gm. glucose. 

Now 100 c.c. diluted sugar solution contain 30 c.c. orig nal sugar solution, 

30 x 10*2 
.. io - 2 c.c. diluted sugar solution contain c.c. original sugar solution, 

30 X IO'2 

.-. - c.c. original solution = 0-05 gm. glucose, 

100 x 100 V '05 
.-. 100 c.c. original sugar solution = 

30 x 10-2 

= 1-6 per cent. 

The values of Fehling's solution for other monosaccharides are 
almost the same as glucose, thus 10 c.c. = '05 gm. glucose = -051 1 gm. 
galactose = '05144 gm. fructose = '0431 gm. mannose. 



ESTIMATION OF CARBOHYDRATES 225 

(ii) Pavy's Method. 

Owing to the difficulty experienced in determining the end point in 
Fehling's method, especially in the case of the estimation of glucose 
in liquids, such as urine, owing to the formation of ammonia, which 
prevents the precipitation of cuprous oxide (cf. p. 191), the use of 
ammonium salts was introduced by Monier. A practical method 
for estimating glucose was worked out by Pavy. 

Procedure. 

50 c.c. of Pavy's solution (see p. 615) are measured with a burette 
into a 200 c.c. conical flask. The flask is closed by a cork with two 
holes ; through one of these the end of the burette containing the 
sugar solution is passed and through the other an escape tube to carry 
off steam and ammonia. To prevent the ammonia fumes coming into 
the air, Pavy fitted to the escape tube a U-tube containing pumice and 
sulphuric acid, but it is most convenient to fit a valve as described 
by Allen. This consists of a short length of rubber tubing closed at 
its end by a piece of glass rod and cut near the end with a V-shaped 
slit. This arrangement is preferable to the valve described in 1904 
by Kumagawa and Suto. The end of the valve is placed in a dilute 
solution of sulphuric acid, which is renewed when the acid is neutralised. 

The solution is boiled to drive out the air, which readily oxidises 
ammoniacal cuprous solutions, and the sugar solution (o p 5 to I c.c. at 
a time) is gradually run in until the blue colour is discharged, the 
solution being kept boiling throughout to exclude air. Sufficient 
time must be allowed for the reduction to take place, as it is slower 
than with Fehling's solution. The valve prevents any liquid being 
sucked back, if the sugar solution be run in so quickly that boiling is 
stopped. 

Just as in Fehling's method, the sugar solutioji must be of such a 
strength that 10 c.c. = 50 c.c. of Pavy's solution. The titration must 
be carried out rapidly and must.be completed within three minutes, 
otherwise the ammonia is all evolved before the titration is completed 
and cuprous oxide is deposited. The boiling must not be interrupted 
and the sugar solution must be run in at such a rate that the solution 
is kept boiling the whole time. The final estimation should be made 
by running in rather less ('5 c.c.) than the amount required and finishing 
off more slowly. The minimal amount which is found to reduce the 
Pavy's solution completely, when added at one time, is the exact volume 
of the solution required. 

The amount of sugar in the solution is calculated from 
10 c.c. Pavy's solution = 0-005 g m - glucose. 

The method has been compared against other methods by Kinoshita 
who finds it very accurate. 

15 



226 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

(iii) Gerrard's Method. 

In 1892 Gerrard found that potassium cyanide was an effective agent for 
dissolving cuprous oxide and prevented its precipitation from Fehling's solution 
when reduced by glucose. This observation led to a simple method for 
estimating glucose. It was improved by Allen and described by him as the 
best method. The method has an advantage over Pavy's method in the 
absence of the ammonia vapour and in that the reoxidation of the cuprous 
oxide is slower. 

On adding potassium cyanide to Fehling's solution it is decolorised, the 
colourless double salt of copper and potassium cyanide being formed : 
CuS0 4 + 4 KCN = CuCN 2) 2 KCN + K 2 SO 4 . 

If excess of Fehling's solution above that capable of being decolorised 
be added the blue colour remains, and when boiled with glucose this amount 
is reduced without the precipitation of cuprous oxide. 

Allen described the following procedure : 

10 c.c. of Fehling's solution are diluted with 40 c.c. of water and heated 
to boiling in a porcelain basin. An approximately 5 per cent, solution of 
potassium cyanide is run into the boiling liquid from a burette until it is just 
decolorised, excess being carefully avoided. Another 10 c.c. of Fehling's 
solution are added and the sugar solution of about 0*5 per cent, strength run 
in slowly until the blue colour vanishes. Only the last portion of the Fehling 
solution is reduced by the glucose so that as in Fehling's method 10 c.c. = 
o'o5 gm. glucose. 

(iv) S. G. Benedict's Method. 

In 1907 S. G. Benedict introduced yet another method for the 
direct volumetric estimation of glucose on account of the difficulties 
and inconveniences of the other methods. In 1910 he published im- 
provements in his method and it seems as if this will be the one most 
generally used. 

If potassium thiocyanate be added to Fehling's solution, the 
cuprous oxide is not precipitated on reduction, but if carbonate be 
used instead of caustic alkali, a white precipitate of cuprous thiocyan- 
ate is formed. The method depends upon the precipitation of the re- 
duced copper as cuprous thiocyanate and decolorisation of the solution. 

Procedure. 

25 c.c. of the reagent (see p. 613) are measured with a pipette into 
a porcelain basin, 25-30 cm. in diameter ; 10-20 gm. of cryst. sodium 
carbonate (or 5-10 gm. anhydrous sodium carbonate) and a small 
quantity of pumice, or a piece of porous earthenware, are added. The 
solution is boiled vigorously over a free flame and the sugar solution is 
run in rapidly till a heavy white precipitate is produced and the blue 
colour begins perceptibly to diminish. The sugar solution is then 
run in more slowly with constant vigorous boiling of the reagent until 
the blue colour has entirely disappeared. An interval of 30 seconds 
between the additions of sugar solution (drop by drop) towards the 
end should be given, and water may be added to replace that lost by 
evaporation. The sugar solution should be of 0-5-1 per cent, as in 
Fehling's method. 

The calculation of the result is from 

25 c.c. reagent = 0-05 gm. glucose or -053 gm. fructose. 



ESTIMATION OF CARBOHYDRATES 227 

II. (a) The Gravimetric Estimation. 

No attempt seems to have been made to estimate sugars by determining 
the weight of cuprous oxide precipitated until 1878 when experiments were 
made by Maercker, who found that accurate results were obtained if the re- 
duction were carried out under definite conditions. His procedure was to 
boil for 20 minutes an excess of Fehling's solution with a sugar solution of 
about o'i per cent., the total volume of the solution being kept constant ; the 
cuprous oxide was filtered off rapidly through filter paper, washed free from 
alkali with boiling water and reduced in a current of hydrogen to metallic 
copper which was weighed. The possible errors were unreduced cuprous 
oxide in the filter paper and absorption of hydrogen by the reduced copper. 

Allihn repeated these experiments in 1880; instead of filter paper he 
used an asbestos pad in a glass tube, which was designed by Soxhlet, and 
boiled the Fehling solution for 2 minutes. The accuracy of Maercker's results 
was confirmed and the method has since been known as Allihn's method. 
Salomon in 1881 again stated that the method was accurate if the solution 
contained about o'i per cent, of sugar. Kjeldahl in 1895 studied the 
method and stated that the main error was introduced by reoxidation on the 
surface of the liquid during the boiling rather than by reoxidation during 
filtration. He recommended passing a current of hydrogen or coal gas through 
the liquid during the period in which it was heated. 

H. T. Brown, Morris and Millar published in 1897 the results of a very 
extended study of the methods of estimating sugars in which they pointed out 
that the chief considerations to be attended to were : (i) the use of Fehling's 
solution of constant composition, (2) the maintenance of the same degree of 
dilution in all experiments, (3) the precipitation of an amount of copper which 
shall fall between certain limits, and (4) an invariable method of determination, 
both as regards mode and time of heating. 

Their process is carried out as follows : 

50 c.c. of freshly mixed Fehling's solution are placed in a beaker of about 
250 c.c. capacity and having a diameter of 7 '5 cm. This is placed in a boil- 
ing water-bath ; when the solution has attained the temperature of the 
boiling water, an accurately weighed or measured volume of the sugar solution 
is added and the volume made up to 100 c.c. with boiling distilled water. 
The beaker is covered with a clock glass and the heating continued for 
exactly 1 2 minutes. The precipitated cuprous oxide is rapidly filtered through 
a Soxhlet tube with asbestos pad, washed first by decantation with hot water, 
then with water, alcohol and ether, and finally dried. The dry cuprous 
oxide is reduced to metallic copper by gentle heating in a stream of hydrogen 
and weighed ; or it is oxidised by heating to cupric oxide and weighed. 

A correction must be made for the reduction which takes place when 
the Fehling's solution is heated alone. The amount of sugar corresponding 
to the copper is given in the table on p. 605. 

These figures were confirmed by Davis and Daish in 1913 who also drew 
attention to other particulars, (a) The asbestos used in filtering. This should 
be treated for 30 minutes with boiling 20. per cent, sodium hydroxide and 
washed thoroughly with water, (b) The filtering through a Gooch crucible 
instead of a Soxhlet tube, (c] The oxidation of the cuprous oxide to cupric 
oxide by heating the crucible in a protecting crucible for 30 minutes with 
a inch Teclu burner until the weight is constant. The conversion into 
cupric oxide seems particularly favourable, if sugar estimations in plant ex- 
tracts be required. 

Pfliiger has also examined the gravimetric estimation of glucose and has 
found that the principal error in Allihn's procedure is the time of boiling ; 

15* 



228 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

2 minutes were insufficient, whereas 30 minutes sufficed if the heating were 
carried out in a boiling water-bath and not over a flame. He has also shown 
that the estimation was equally accurate when the precipitate was weighed as 
cuprous oxide, cupric oxide or metallic copper. He considered that the 
estimation as cuprous oxide was the most accurate for the estimation of small 
quantities of glucose. 

Pfliiger has published a table of the corresponding quantities of cuprous 
oxide and glucose. 1 

The corresponding quantity of glucose can be obtained from the table on 
p. 603, if the amount of Cu2O be multiplied by -8883 or 1-1117 respectively 
to give the corresponding amount of Cu or CuO. 

Davis and Daish's preference for the estimation as cupric oxide in the 
case of plant extracts is probably to be accounted for by incomplete re- 
moval of other compounds carried down with the cuprous oxide and re- 
moved by heating. 



II. (b) Volumetric Estimation of the Precipitated Cuprous Oxide. 

(i) Mohr-Bertrand Method. 

The volumetric estimation of the cuprous oxide formed by the reduction of 
alkaline cupric sulphate solution seems to have been first carried out in 1873 
by Mohr, who based his process upon one of Schwarz's methods (1852), namely 
that of dissolving the cuprous oxide in an acid solution of ferric chloride and 
estimating the amount of ferrous chloride so formed. Mohr dissolved the 
cuprous oxide in an acid solution of ferric sulphate and titrated the ferrous 
salt with permanganate. 

This process has been recommended by several workers, amongst whom 
may be mentioned Sonntag, Wood and Berry, and Bertrand. The last 
author has made very thorough experiments with this method and has 
published tables giving the amounts of glucose, lactose and maltose corre- 
sponding to the amount of reduced copper. The method is now generally 
referred to as Bertrand's method. The solutions required are given on 
p. 613. 

Procedure. 

20 c.c. of the sugar solution, which should contain about *i per cent, 
and preferably a little less, are placed in a conical flask of 125-150 c.c. 
capacity ; 20 c.c. of the copper solution A-and 20 c.c. of the alkaline solution 
B are added, and the mixture heated to boiling over a flame and kept gently 
boiling for exactly 3 minutes. If a smaller volume of sugar solution be used, 
water is added so that the total volume is 60 c.c. The flask is removed from 

1 Pfliiger's Archiv, 1903, 96, 105. 



ESTIMATION OF CARBOHYDRATES 229 

the flame and allowed to stand for about ^ minute so that the cuprous oxide 
settles. 

The liquid is filtered by suction through an asbestos filter of a special 
pattern. It is a glass tube about 14 cm. long constricted near the centre ; the 
upper portion is about 6 cm. long and 1 7 mm. wide ; the lower portion about 
8 cm. long with a conical bulb just below the upper portion. The upper 
part contains the asbestos coarse fibres being put near the constriction, less 
coarse particles above, and at the top very fine particles. The whole thick- 
ness of asbestos is about i cm., the upper portion being from 1-2 mm. thick. 
A filter flask of about 150 c.c. capacity is usually taken to receive the liquid. 

In filtering, as little as possible of the cuprous oxide is allowed to come 
upon the filter so as to prevent the formation of a compact mass, which 
subsequently dissolves with difficulty. The cuprous oxide is washed with a 
little hot water, allowed to settle, and the water poured off through the filter. 
It is not necessary to wash the precipitate absolutely free from alkali and 
Rochelle salt, but this is advisable. The cuprous oxide in the flask is 
treated with 5, 10, or 20 c.c. of the ferric sulphate solution; it dissolves 
giving a green solution. This is poured upon the filter; the particles of 
cuprous oxide dissolve more quickly if the top layer be stirred up with a glass 
rod. The liquid is received in the filter flask which has been rinsed out, or 
more conveniently in another clean filter flask. A few more drops of the 
ferric solution may be used if necessary. The conical flask is washed out 
with water and the washings passed through the filter. 

The solution is titrated with the permanganate solution, the colour change 
from green to rose with i drop excess of permanganate being noted ; another 
drop usually shows an intense rose colour. The duration of the operation is 
15-20 minutes. 

After repeated use the upper layer of asbestos becomes dark in colour ; 
it is removed, dried and calcined, and returned to its position. 

Calculation of the Results. 

The result is calculated from the equations : 

Cu 2 O + Fe 2 (SO 4 ) s + H 2 SO 4 = 2CuSO 4 + 2FeSO 4 + H 2 O 

ioFeSO 4 + 2KMnO 4 + 8H 2 SO 4 = 5Fe 2 (SO 4 ) 3 + K,SO 4 + 2MnSO 4 + 8H 2 O 

whence 2 atoms of Cu = f molecule KMnO 4 

or 63-6 gm. Cu = 31-6 gm. KMnO 4 

or 2 - oi2 gm. Cu = i gm. KMnO 4 

i c.c. of Bertrand's permanganate = -005 gm. KMnO 4 or -01006 gm. Cu 
i c.c. of g iN permanganate = 0-00636 gm, Cu. 

The standardisation of the permanganate against ammonium oxalate 
C 2 H 2 O 4 , 2NH a + H 2 O is carried out by Bertrand by dissolving '250 gm. 
in about 50-100 c.c. water, adding 1-2 c.c. of sulphuric acid, heating to 60- 
80 and titrating with the permanganate, of which about 22 c.c. will be 
required. 

5C 2 H 2 O 4 + 2KMnO 4 + 3H 3 SO 4 = ioCO 2 + 2MnSO 4 + K 2 SO 4 + 8H 2 O 
whence i mol. oxalic acid or am. oxalate (mol. wt. = 142'!) = 2 atoms of Cu. 

O *2 * 6 X 2 

The weight of oxalate multiplied by -= or 0*895 gives the amount 

142'! 

of copper corresponding to the number of c.c. of permanganate used, from 
which the value for i c.c. permanganate can be calculated. 

The values for glucose are given in a table compiled by Bertrand (see 
p. 606). 



230 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Bang's Second Method. 

Bang 1 has found that if the reduction of cupric sulphate be carried outj 
in the presence of thiocyanate or chloride in carbonate solution, cuprous 
thiocyanate or cuprous chloride is formed and is not precipitated, i.e. that in the 
presence of excess of potassium thiocyanate or chloride the reversible reaction 

Cu 2 Cl 2 + K 2 CO 3 ^ Cu 2 O + 2KC1 + CO a 

proceeds only in the right-hand direction. A cupric solution reduced by 
glucose will contain cuprous chloride. Bang estimates the reduced copper 
salt by titration in alkaline carbonate solution with standard iodine solution : 

CuCl + I + K 2 CO 3 = CuCO 3 + KC1 + KI. 

The solution which becomes colourless in the reduction again becomes blue 
on oxidation, but if small amounts are used the colour is pale blue, and when 
the iodine titration is carried out using starch as indicator, the deep blue 
colour formed indicating excess of iodine is easily seen. 

The preparation of the reagents is given on p. 613. 

Procedure. 

Owing to the ready oxidation special precautions have to be taken 
during the reduction by the glucose and the titration with the iodine 
solution : o'i or o'2 c.c. (or more) of the glucose solution are put into 
a 100 c.c. Jena glass flask with a straight neck and no rim, and 55 c.c. of the 
alkaline copper solution are added. 

The flask is fitted with a rubber tube 4-5 cm. long and 3 mm. thick, 
leaving about 2 cm. projecting, and boiled for 3 minutes. Just before the 
expiration of this time a spring clip 2 is put over the rubber and closed at 
the end of the time of boiling. The flask is rapidly cooled under running 
water. The clip is removed and the titration effected with 'iN, or 'oiN, 
or -04N iodine, 3 after adding 0-5-1 c.c. of the starch solution. 4 During 
the titration the flask is only shaken gently to prevent access of air, but it is 
advisable and preferable to pass into it a current of carbon dioxide by means 
of a bent tube which can be fastened by a band to the flask. The amount 
of iodine solution is proportional to the amount of glucose present. 

The number of c.c. used divided by 270 gives the amount of glucose in 
gm. 5 

o'4 c.c. should be deducted; this amount is generally absorbed by the 
alkaline copper solution. 

The factor for -iN iodine solution is '285, for -O4N iodine solution 0-7 
(-28 x 2-5). 

1 Biochem. Zs., 1913, 49, i. 

2 A special clip is made by Mekaniker Hill, Lund, Denmark. 

3 The latter are prepared by diluting a ! iN solution with boiled out water and can be 
kept for three months in the dark without undergoing alteration. 

The 'oiN solution is conveniently prepared from iodide and iodate : 

KIO 3 + sKI + 6HC1 = 6KC1 + 3H 2 O + 61 ; 

an equivalent quantity of iodine to hydrogen chloride is formed. It is made by pouring 
i c.c. of 2 per cent, potassium iodate solution into a 100 c.c. measuring flask, adding 2 to 
2-5 gm. of potassium iodide and 10 c.c. of -iN hydrochloric acid and filling up to the mark 
with boiled out water. 

4 i per cent, solution of soluble starch in saturated potassium chloride solution. It 
keeps indefinitely. 

5 26-5 c.c. -oiN iodine solution = 10 mg. glucose; 
or 2-67 c.c. =i mg. 



ESTIMATION OF CARBOHYDRATES 231 

Caven and Hill's Method. 

Caven and Hill in 1897 and 1898 suggested that the cuprous oxide should 
be dissolved in an acid solution of permanganate (i part H 2 SO 4 , 3 parts H 2 O 
and - 2N KMnO 4 standardised against oxalic acid) and the titration of the 
permanganate used in the oxidation with oxalic acid. In the actual process 
it was preferred to add excess of oxalic acid and to titrate back with per- 
manganate. 

The amount of cuprous oxide was obtained by multiplying the oxygen 

value of the permanganate by 8*91 ( "^ j and the glucose by using the 
factor -5045. 

Sidersky's Method. 

This method has been used by sugar experts chiefly in France. The 
cuprous oxide is dissolved in excess of standard sulphuric acid and the excess 
determined by titrating with ^N ammonia. It is carried out as follows : 

The cuprous oxide is filtered off and washed free from alkali. It is 
dissolved in 25 c.c. N sulphuric acid, a few crystals of potassium chlorate 
being added and the reagent heated till solution is effected. An equivalent 
quantity of standard ammonia is added ; a blue colour is obtained and 
N sulphuric acid is run in till the colour is of a permanent greenish tint. 
The copper sulphate is thus the indicator. 

The amount of glucose is calculated as follows : 
i c.c. N H 2 SO 4 = '0318 gm. copper. 

The amount of invert sugar is given by multiplying by '3546. 



II. (b) (ii) Estimation of Residual Cupric Salt. 

Numerous suggestions have been put forward for estimating the excess of 
copper sulphate which is not converted into cuprous oxide by reduction by 
glucose : thus 

1 i) Weill and also Pellet suggested its titration with zinc chloride. 

(2) Maumene suggested its titration with sodium sulphide. 

(3) Lehmann in 1897, Riegler in 1898, Maquenne in 1899, and Schoorl 
in 1899 suggested its titration with potassium iodide and thiosulphate. 

(4) Volh^rd suggested its reduction with sulphur dioxide and precipita- 
tion as Cuprous thiocyanate with excess of ammonium thiocyanate, and the 
estimation of the thiocyanate with silver solution. 

(5) Bang suggested its titration with hydroxylamine. 

Of these methods that of Lehmann or Maquenne and of Bang are the 
simplest and are frequently used. 



232 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

The Lehmann-Maquenne Method. 

The principle of this method depends upon the liberation of iodine 
on adding potassium iodide to copper sulphate, and the estimation of the 
iodine by means of sodium thiosulphate : 

2CuSO 4 + 4 KI = Cu 2 I 2 + I 2 + 2K 2 S0 4 
I 2 + 2Na,S 2 O.j = Na,S 4 O 6 + 2NaI. 

.Lehmann's procedure was to make up the Fehling solution to a definite 
volume after its reduction by glucose, and to take out an aliquot part for' the 
titration after the cuprous oxide had settled. Riegler filtered off the cuprous 
oxide and titrated the filtrate and washings. Maquenne and Schoorl titrate 
the cupric salt without removing the cuprous oxide. 

The conditions for the reduction of Fehling's solution by glucose 
and by lactose have been standardised by JPeters (1912) and by Cole, 1 who 
described also a simple asbestos filter made out of a calcium chloride tube 
and a simple way of boiling the Fehling solution for exactly 2 minutes. 

The procedure of Maquenne is the following : 

10 c.c. of each of the two Fehling's solutions are measured into a 250 c.c. 
conical flask and 10-40 c.c. of the sugar solution 2 are added; the total 
volume of the solution for the reduction must be 60 c.c. and water is added 
if necessary. The solution is boiled for exactly 2 minutes, and cooled under 
running water. 20 c.c. of 20 per cent, potassium iodide and 20 c.c. of 
25 per cent, sulphuric acid are added and the solution is titrated with *iN 
thiosulphate solution (24*83 gm. per litre), using i c.c. of i per cent, starch 
solution as indicator. 

A blank determination of Fehling's solution gives the amount of 
thiosulphate solution (27*8 c.c.) required for 20 c.c. of Fehling's solution and 
from this figure is deducted the number of c.c. required in the titration. 

The following table gives the corresponding amounts of glucose : 

Thiosulphate. Glucose. Thiosulphate. Glucose. Thiosulphate. Glucose, 
c.c. mg. c.c. mg. c.c. mg. 

1 3-1 II 35-7 21 70-7 

2 6'2 12 39*1 22 74*5 

3 9*3 13 42*5 23 78-5 

4 12-5 14 45*9 24 82*5 

5 I5'7 15 49'3 25 86*5 

6 19-0 16 52-8 26 90-6 

7 22-3 17 56-3 27 94-8 

8 25-6 18 59-8 28 97-9 

9 28-9 19 63-3 29 loo-o 
10 32*3 20 66*9 30 

Bang's First Method. 

In this method the reduction of the copper solution is effected in 
potassium carbonate solution to which potassium thiocyanate is added. 
Cuprous thiocyanate is formed and remains in solution. Excess of cupric 
salt is estimated by hydroxylamine solution. Bang has published a card giving 
the reagents, the procedure, and a table of corresponding values of glucose 
and hydroxylamine. This method has been superseded by his other method 
(p. 230). 

1 Tables for converting the copper figures into glucose and lactose are given in this 
paper, Biochemical Journal, 1914, 8, 134. 

2 The sugar solution added must contain less than ! gm. glucose. 



ESTIMATION! OF CARBOHYDRATES 



233 




FIG. 45. 



C. ESTIMATION BY FERMENTATION. 

Sugar is sometimes estimated by fermentation with yeast in an Einhorn 
fermentation tube. This is a U-shaped tube, one limb of which 
is closed and the other expanded into a bulb (Fig. 45). 

The closed limb of these tubes is filled with the sugar solu- 
tion to which some yeast has been added. Mercury is placed 
at the bend. The carbon dioxide evolved collects in the 
closed limb and drives down the solution into the other and 
wider limb. The narrow limb is graduated in percentages of 
glucose, so that the amount of sugar can be directly read off. 

This fermentation method is not very accurate and is con- 
sequently not often used for estimating sugar. 

Another "method of estimation is by taking the specific gravity of the 
solution before and after fermentation. 

The most accurate results by fermentation are obtained with Lohnstein's 
apparatus (Fig. 46). ^_ 

A U-shaped tube is employed. The straight limb is left 
open, but the bulb is closed, after filling, with a stopper. The 
straight limb is narrower at the base than at the upper end, 
and upon the end rests a wooden scale graduated in percent- 
ages of glucose ; the graduations on one side are for working 
at room temperature, on the other side at 37. 

A definite weight of mercury is placed in the bend; it 
almost fills the bulb and reaches up the narrow part of the 
limb to about the zero mark on the scale. Upon the surface 
of the mercury in the bulb is placed 0-5 c.c. of glucose solution 
and 1-2 drops of a suspension of yeast in water. The stopper 
(carefully greased) is inserted. It is perforated with a small 
hole which coincides with a similar hole at the neck of the 
bulb. With the two openings together so that air can enter or 
be displaced the apparatus is tilted so as to put the mercury 
in the limb at the zero point of the scale. The stopper is 
then turned to close the apparatus and it is set aside for the 
fermentation to proceed for 12-24 hours at room temperature 
or for 3-4 hours at 37. To prevent the stopper being forced 
out by the pressure of the gas it is covered with a weight. 

The percentage of glucose is read off on the scale when 
the fermentation has ended and the apparatus has returned 
to room temperature, if the fermentation took place at 37. 
Lohnstein shows that a more accurate result is obtained if the 
readings are taken at 37 and 20 and if the percentage is calculated from an 
equation. 

The apparatus should be cleansed immediately after use ; the opening on 
the stopper is slowly made to coincide with the opening of the bulb. The 
mercury level takes the original position. The liquid is removed with a small 
pad of cotton wool, and the surface of the mercury washed with a little 
water, which is drained away by fresh cotton wool. 



* 




FIG. 46. 



234 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

ESTIMATION OF PENTOSES. 

Like the hexoses, the pentoses reduce Fehling's solution and can be 
estimated in the same way, if they are present in the solution alone ; generally 
a mixture of pentose and other carbohydrates is present ; under these con- 
ditions the pentose can be estimated by converting it into furfural (p. 303) 
by distillation with hydrochloric acid, and combining the furfural with 
phloroghicinol and weighing the compound. 

The estimation is usually carried out in the following way : 

A quantity of material is taken which will yield an amount of phloroglucide 
varying from '03 but not exceeding '3 gm. It is distilled from a flask, which 
is provided with a tap funnel and connecting tube with a trap leading to a con- 
denser, with 100 c.c. of 12 per cent, hydrochloric acid at such a rate that 30 c.c. 
pass over in 10 minutes. The distillate is filtered through a small filter 
paper as it is collected. When 30 c.c. have distilled over, 30,0.0. of dilute 
acid are passed through the tap funnel into the flask, and the distillation 
continued for another 10 minutes. This addition is continued 12 times or 
until about 360 c.c. of distillate have been collected. 1 To the whole distillate 
about double the amount of pure phloroglucinol 2 required dissolved in 1 2 per 
cent, hydrochloric acid is added and the mixture well stirred. The solution 
turns yellow, then green, and a precipitate at first green in colour, but ulti- 
mately black, separates out. The volume is made up to 400 c.c. and after 
standing for 12 hours the precipitate is collected on a weighed filter, washed 
with 150 c.c. of water, dried for 4 hours at 100, cooled and weighed. 
The amount of furfural is (a + '0052) x 0*5185 
,, pentose is (a + '0052) x 1*0075 
,, pentosan is (a + '0052) x 0-8866 

where a is the amount of phloroglucide and '0052 is the quantity of phloro- 
glucide not precipitated but remaining in solution under the above conditions. 

Methyl pentoses behave in the same way. The phloroglucide they pro- 
duce is soluble in alcohol. The alcohol soluble portion of the precipitate 
is returned as methyl pentose. 

Note. Cunningham and Doree 3 have shown that &>-hydroxy- methyl furfur- 
aldehyde is formed by the action of acids upon hexoses, starch and celluloses 
in amounts varying from 1-2 per cent. It is slowly formed and does not 
interfere in the estimation of pentosans, if aniline acetate be used as indicator. 
It causes inaccuracies in the estimation of methyl pentosans. 

Flohil 4 showed that the furfural in the distillate could be estimated by 
means of Fehling's solution, either gravimetrically or by the iodometric method. 
Eynon and Lane 5 thoroughly tested this method and a further improvement 
was introduced by Baker and Hulton. 6 The furfural-containing distillate is 
not collected after it ceases to react with aniline acetate containing sufficient 
sodium acetate, a period of 5 minutes being given to decide upon the negative 
reaction. Usually all the furfural has passed over in 200-300 c.c. of distillate. 
10 c.c. of distillate are titrated with ^N alkali. 50-75 c.c. in a 250 c.c. 
conical flask are neutralised with saturated sodium hydroxide, the solution 
being kept cold, 20 c.c. of Fehling's solution are added, the mixture diluted 
to 100 c.c and heated under a reflux in a boiling water-bath for 40 minutes. 
The cuprous oxide is estimated gravimetrically. A blank experiment with an 
equivalent quantity of salt is also carried out ; the amount of cuprous oxide 
from 2-4 mgm. is deducted. 

3 mgm. Cu = i mgm. furfural. 

1 The completion of the distillation may be tested by means of aniline acetate; a drop 
of reagent and a drop of distillate are placed side by side on a piece of filter paper ; if no 
red colour appears the distillation is complete. 

2 The purity of the phloroglucinol may be tested for by dissolving a small quantity in 
a few drops of acetic anhydride, heating almost to boiling and adding a few drops of con- 
centrated sulphuric acid. A violet colour shows the presence of diresorcinol. 

3 Biochem. J., 1914, 8, 438. 4 Chem. Weekblad, 1910, 7, 1057. 
6 Analyst, 1912, 37, 41. 6 Ibid., 1916, 41, 294. 



ESTIMATION OF CARBOHYDRATES 235 

ESTIMATION OF DISACCHARIDES. 

A. Cane Sugar. 

Cane sugar is estimated by taking the reducing power of the solu- 
tion after hydrolysis by acid ; fructose and glucose have very nearly 
the same reducing power. 

A known volume of the cane sugar solution (say 40 c.c.) is hydro- 
lysed by warming on the water-bath for 5-10 minutes with 5 c.c. 
of dilute hydrochloric acid. The solution is cooled, neutralised with 
5 c.c. of caustic soda, and made up to a definite volume in a measuring 
flask (say 100 c.c.), rinsing out the flask with the water necessary to 
make up the 100 c.c. The amount of reducing sugar is then estimated 
by Fehling's, Pavy's or any of the other methods previously described. 
The sugar solution should be of a strength so that 10 c.c. = 10 c.c. 
Fehling's or 50 c.c. Pavy's solution, i.e. contain about the equivalent of 
5 per cent, of glucose. 

The percentage of cane sugar is calculated from the equation : 

C 12 H 22 O n + H .0 = 2C 6 H 12 6 

342 360 

0*047 '5 = I0 c - c - Fehling's solution. 

B. Lactose and Maltose. 

Lactose and maltose are estimated in the same way as glucose by 
Fehling's or other methods, but their reducing power is less than that 
of glucose. Consequently, since Fehling's solution is a standard 
for glucose, a factor has to be employed in order to obtain the equi- 
valent value for these disaccharides. The factor is obtained by de- 
termining the reducing power before and after hydrolysing them into 
monosaccharides by acid, thus : 

A definite volume of the lactose solution is taken and diluted, 
as previously done for glucose, with water so that 10 c.c. reduce 
10 c.c. Fehling's solution (or 50 c.c. Pavy solution). The value is de- 
termined exactly. 

Exactly the same quantity of the lactose solution is hydrolysed 
by boiling for 3-4 hours in a flask with one-tenth of its volume of 
dilute sulphuric acid ; water must be added to replace that lost on 
boiling, or the hydrolysis is carried out by heating under a reflux 




236 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

condenser. The solution is cooled, neutralised with soda, and made up 
to the same volume as the non-hydrolysed lactose solution with water 
as above. 

est to make up the solutions in a measuring flask, e.g. 25 c.c. 
lactose solution are diluted to 100 c.c. ; 25 c.c. lactose solution are 
hydrolysed by acid, neutralised, and washed into the 100 c.c. measur- 
ing flask. 

The reducing value of the hydrolysed lactose solution is taken ex- 
actly. 

~. .. reducing power of lactose in c.c. 10 

The factor is - - = . 

reducing power of hydrolysed lactose in c.c. 7 

The ratio of the reducing powers of glucose : lactose : maltose are 
as I : 074 : o'62. 

The percentage of lactose may be calculated as follows : 

x c.c. = 10 c.c. Fehling's solution. 

7'4 

.-. x x c.c. = 0-05 em. lactose. 
10 

10 
or x c.c. = o - o5 x gm. lactose. 

7'4 

IO IOO 

Hence 100 c.c. = 0-05 x x gm. lactose. 

7'4 x 

The weights of the disaccharides which will reduce completely 10 
c.c. of Fehling's solution are : 

cane sugar 0-0475 gm. 
maltose 0-0807 
lactose 0-0678 ,, 

ESTIMATION OF POLYSACCHARIDES. 

All polysaccharides are estimated in the same way as cane sugar, 
i.e. by taking the reducing value of a known weight or volume of the 
solution after hydrolysis by acid, i.e. in terms of glucose : 

CHO + HO = CHO. 



CARBOCYCLIC COMPOUNDS. 



In addition to the series of aliphatic compounds, series of carbon 
compounds are known in which the atoms of carbon three to nine 
are linked together in a ring. Amongst them we find saturated and 
unsaturated compounds. They do not form such a large group as the 
aliphatic compounds except those which contain six atoms of carbon. 
These form a special group by themselves known as the aromatic 
and hydroaromatic compounds. 

Only a few, excluding the aromatic compounds, are found in 
nature, the majority being synthetical products. They are termed 
polymethylenes or cyclo-paraffins, cyclo-olefines and cyclo-diolefmes. 
Thus in the case of the hydrocarbons, we have : 



CH 



Trimethylene 
or cyclopropane. 



CH 2 CH 2 

CH 2 CH 2 
Tetramethylene 
or cyclobutane. 



,CH 2 CH 



CH 2 CH 

1 II 

CH 2 CH 
Cyclobutene. 



CH 2 

CHg C 

Pentamethj 
or cyclopen 


:H 2 

lene 
tane. 


CH 2 

CH 2 CH 
Cyclopentene. 



'(-f Ho OHn 



CH 



Cyclopentadiene. 



CH 



CH, 



CH, CH 



X CH, CH 2 CH 2 
Heptamethylene 
or cycloheptane 
(suberane). 



CH 2 

Hexamethylene 
or hexahydrobenzene. 



Some of these hydrocarbons, chiefly alkyl derivatives of penta- 
methylene and hexamethylene, are present in Galician and Russian 
petroleum, and are known as naphthenes. They resemble the paraffins 
in their properties. 

If hydrogen atoms be substituted by hydro xyl groups, ketonic 
groups, carboxylic groups, such as in : 

CH 2 CH 2 CH 2 

CO 

/ 

.j Crrlg C1~1 2 
Suberone, 

a series of compounds is formed which have the properties of aliphatic 
compounds into which they can be easily transformed. 

237 



AROMATIC COMPOUNDS. 

Aromatic compounds are mostly contained in the fragrant and 
peculiar smelling oils, resins, etc., which are present in the flowers, leaves 
and other parts of plants and which often oo'ze out when the bark is 
broken. Though only a few aromatic compounds are actually found in 
animals, yet they are essential to their life. Turpentine, india-rubber, 
tannin, oil of lemon, oil of cloves, cinnamon and numerous plant pig- 
ments are aromatic compounds. They derive their name from their 
origin in these sweet-smelling natural substances. 

Another great source of aromatic compounds is coal tar, which is 
a complex mixture, but contains benzene, the parent substance from 
which all the other compounds can be derived. 

In some respects the aromatic resemble the fatty or aliphatic 
compounds, but in other respects they are totally different. They 
differ by having a greater carbon content and in being more resistant 
to oxidation, reduction, etc. The more complex substances can 
be oxidised and converted into simpler substances, but these simpler 
substances are resistant and are found to possess a stable nucleus com- 
posed of six atoms of carbon. This nucleus is not easily oxidised to 
compounds containing fewer carbon atoms, but on oxidation it is con- 
verted into carbon dioxide and water. The presence of a nucleus of 
six carbon atoms is thus a characteristic of aromatic compounds. 

Another characteristic is the formation of nitro compounds and 
sulphonic acid derivatives by the. direct action of nitric and sulphuric 
acids. 

The Structure of Aromatic Compounds. 

Our representation of the structure of the aromatic compounds is 
based upon the theory of Kekule, which was published in 1865. 

Benzene, C 6 H 6 , is the simplest aromatic compound containing six 
carbon atoms. Its properties do not correspond to the properties of 
an unsaturated hydrocarbon, such as : 

CH=C CH 2 CH a C=CH 

or 

CH 3 C=C CH 2 C=CH, 
238 



AROMATIC COMPOUNDS 239 

but point to the presence of six CH groups and a symmetrical arrange- 
ment of the six carbon and six hydrogen atoms in the molecule. A 
structure in which the six carbon atoms are united in a closed chain or 
ring and joined by alternate single and double bonds, a hydrogen atom 
being united to each carbon atom, satisfies the tetravalency of the 
carbon atom and it gives a symmetrical structure, thus : 

H 
C 



HC CH 

I II 
HC CH 

V 

C 
H 

The symmetry of the molecule can be represented by a regular 
hexagon at each angle of which there is a CH group ; thus : 

CH 




CH 

This structure represents benzene as an unsaturated compound. In 
some respects benzene behaves like an olefine, but not in all respects, 
e.g. it forms addition compounds with the halogens and with hydrogen, 
but not with the halogen acids and sulphuric acid, nor does it decolorise 
permanganate in the cold. 

It explains the formation of derivatives by the substitution of 
hydrogen atoms by other elements or groups, and on account of its 
symmetry explains the formation of one monosubstitution derivative, 
three disubstitution derivatives, and so on. 

The alternate linking of the carbon atoms by double and single 
bonds would point to the existence of two isomeric monosubstitution 
derivatives. To overcome this difficulty Kekule assumed that there 
was a continual alternation between the double and single bonds, and 
that the double bonds are not like the double bonds in unsaturated 
aliphatic compounds. 

The closed chain or ring structure has been accepted by all 
chemists, but some chemists have suggested a different figure such as 
a prism. This does not explain the substitution deriva- 
tives as well as the centric formula proposed by Arm- 
strong, in which the extra bonds to make the carbon 
atoms tetravalent simply point to the centre of the 
figure, and come into play only under certain conditions, 
e.g. amongst the hydroaromatic compounds. 




240 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

For simplification, the benzene nucleus is generally represented 
as a regular hexagon, at each angle of which the existence 
of a carbon atom and a hydrogen atom is recognised. 

The substitution products are then indicated by introducing only 
the particular radicle or radicles. Further, the carbon Cl 
atoms are generally numbered in order. The compound 
is i -chloro-3 -nitrobenzene. 




BENZENE AND ITS MONOSUBSTITUTION 

DERIVATIVES. 
Benzene. 

Benzene was discovered by Faraday in 1825 in illuminating gas 
prepared from oil, before the introduction of coal gas. The gases were 
condensed to a liquid from which benzene was isolated by distillation. 
Mitscherlich prepared benzene in 1834 by distilling benzoic acid with 
lime and in 1845 i* was found in coal tar by Hofmann. Berthelot 
obtained benzene by passing acetylene through a red-hot tube. 

Preparation. 

Benzene is obtained from coal tar. Coal tar consists of a complex 
mixture of aromatic compounds which are acid, basic and neutral in 
character. It also contains unsaturated paraffins. The gases obtained 
by the destructive distillation of coal are passed through vertical con- 
densers connected to a trough/ The condensed liquids collect in 
the trough and are drawn off. A ton of coal yields from: 10-20 
gallons of tar. 

Distillation of Coal Tar. 

Coal tar is distilled in wrought-iron stills and the distillate is 
collected in the following fractions : 

(l) Light oil, or crude naphtha, which distils up to 150 and con- 
tains benzene, toluene, xylene. It forms 3-5 per cent. 



AROMATIC COMPOUNDS 241 

(2) Middle oil, or carbolic oil, which distils from 150-210 and 
contains naphthalene and carbolic acid. It forms 8-10 per cent. 

(3) Heavy oil, or creosote oil, which distils from 210-270 and 
forms 8-10 per cent. 

(4) Anthracene oil, which distils from 270-400 and contains 
anthracene. It is coloured green and forms 16-20 per cent. 

Pitch remains in the still. 

The terms light oil, middle oil, heavy oil denote the specific gravity 
of the distillate with regard to water. A sample is run into water 
during the distillation ; if it floats it is light oil, if it sinks it is 
heavy oil. The middle oil passes over as soon as light oil no longer 
distils over. 

Benzene and its homologues are prepared from the light oil. The 
fraction is shaken with strong sulphuric acid which removes basic sub- 
stances, such as aniline, pyridine, and also unsaturated hydrocarbons. 
It is next shaken with sodium hydroxide which removes acid sub- 
stances, carbolic acid and the sulphuric acid. It is washed by 
shaking with water and fractionally distilled. Pure benzene is isolated 
from the first fraction by careful fractional distillation. 

Properties. 

Benzene is an oily colourless liquid with peculiar smell and boils 
at 80. It solidifies on cooling and the crystals melt at 5 '4. Its 
specific gravity is -874 at 20. It is inflammable and burns with 
a smoky and luminous flame. It is insoluble in water, but mixes with 
alcohol and ether. Benzene is a good solvent for resins, fats, etc., and 
is frequently used for extraction. It dissolves iodine, phosphorus, 
sulphur, etc. 

Benzene is called benzole commercially, the terminal e being added 
to show that it does not possess a hydroxyl (OH) group ; the suffix 
ol, as previously mentioned, is used to designate alcohols. 

Chlorobenzene, C ( ,H 5 C1. Brorhobenzene, C 6 H 5 Br. lodobenzene, 
QH 5 I. 

Like the aliphatic hydrocarbons benzene is acted upon by the 
halogens, chlorine and bromine, and converted into substitution deriva- 
tives. This reaction proceeds most easily in the presence of a halogen 
carrier, such as iodine (iodine chloride is formed which chlorinates more 
vigorously) : 

/\ 

+ Br, = HBr 



16 




242 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

This reaction can be observed by adding a few drops of bromine to 
a few c.c. of benzene in a test tube. The evolution of hydrobromic 
acid is slow. If a piece of aluminium mercury couple or some iron 
filings be added to a few c.c. of benzene in another test tube and then 
a few drops of bromine, the reaction is more rapid. 

Benzene is not acted upon by iodine, but iodobenzene has been 
prepared by heating benzene with iodine and iodic acid : 
5 C 6 H 6 + 4 I + HI0 3 = 5 C 6 H B I + 3H 2 0. 

Iodobenzene is obtained through the diazo reaction (p. 249). 

Properties. 

These halogen derivatives are colourless liquids with a faint but not 
disagreeable smell. They boil without decomposition, are insoluble in 
water but soluble in organic solvents. 

Reactions. 

These compounds are more stable than the corresponding aliphatic 
halogen compounds. The halogen atom is not replaceable by OH 
groups or other groups. 

Nitrobenzene. C 6 H 5 NO 2 . 

Benzene is not acted upon by dilute nitric acid, but it is converted 
into nitrobenzene by the action of concentrated nitric acid (nitration) : 



+ HN0 3 = H 2 + , . 

This reaction is one of the principal reactions which benzene and 
its derivatives undergo and in which aromatic compounds differ from 
aliphatic compounds. 

Preparation. 

3 or 4 drops of benzene are added to a mixture of 2 c.c. of con- 
centrated sulphuric acid and I c.c. of concentrated nitric acid and 
warmed. Nitrobenzene is formed and is recognised by its smell of 
bitter almonds, which remains after removing the excess of acid by 
alkali. 

On a larger scale nitrobenzene may be prepared as follows : 
100 gm. of concentrated nitric acid are added with shaking to 150 gm. of 
concentrated sulphuric acid in a 500 c.c. flask and cooled by placing under 
running water. 50 gm. of benzene are added in portions of 2 c.c. to the 
cold mixture of acids ; after each addition the mixture is thoroughly shaken. 
There is an energetic reaction and the contents of the flask must be kept below 
50 by immersing it in cold water. The addition of benzene should take at 
least half an hour. The reaction is completed by heating the contents of the flask 
under an air condenser in a water-bath at 60. The nitrobenzene floats as an 
oil on the surface. It is separated from the acid by means of a tap funnel 
and shaken with water. The heavier layer of nitrobenzene is separated, 
washed with excess of sodium carbonate solution to remove acid and again 





AROMATIC COMPOUNDS 243 

with water. It is dried by being shaken with calcium chloride, filtered through 
glass wool and distilled, using an air condenser, the fraction boiling from 204- 
208 being collected. A small residue of dinitrobenzene may remain in the 
flask. 

Properties. 

Nitrobenzene is a pale yellow liquid which possesses an odour of 
bitter almonds. It boils at 205 and can be frozen to a solid which 
melts at 3. It is frequently used for scenting soap but chiefly in the 
preparation of aniline and benzidine : it is sometimes used as a 
solvent. 

Benzene Sulphonic Acid. C 6 H 5 . SO 3 H. 

Benzene slowly dissolves in warm concentrated sulphuric acid and 
is converted into benzene sulphonic acid (sulphoriation) : 

HO 
HO/ 

v 

Preparation. 

2 c.c. of benzene are mixed with 7 c.c. of concentrated sulphuric 
acid and carefully heated with constant shaking. The benzene which 
at first floats on the surface gradually dissolves. A clear solution 
is obtained on pouring a portion of the cooled mixture into water. 
Sodium benzene sulphonate separates if some be poured into a satu- 
rated solution of sodium chloride. 

On a larger scale the benzene and sulphuric acid are carefully heated 
under a reflux condenser with constant stirring. The reaction product is 
poured into water (or salt solution if the sodium salt be required) and the 
solution neutralised with calcium carbonate. The calcium sulphate is filtered 
off and the filtrate is evaporated until the calcium salt crystallises. The other 
salts or the free acid are prepared from the calcium salt by double decomposi- 
tion with potassium carbonate, etc., or sulphuric acid. 

Properties. 

Benzene sulphonic acid is a hygroscopic solid which is readily 
soluble in water and melts at 50. The solution is strongly acid. 

It forms salts with the metallic carbonates, or oxides. These salts 
generally crystallise well. 

On heating, the sulphonic acid is decomposed. The sulphonic acid 
group may be removed by heating it in a sealed tube with concentrated 
hydrochloric acid or with strong sulphuric acid in a current of steam ; 
the hydrocarbon is regenerated : 

C 6 H 5 S0 3 H + H 2 = C 6 H 6 + H 2 S0 4 . 



16 



244 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Benzene Sulphonyl Chloride. 

Benzene sulphonic acid or its salts are converted by the action of phos- 
phorus pentachloride into benzene sulphonyl chloride : 

C 6 H S . SO 2 OH + PC1 5 = HC1 + POC1 3 + C 6 H 5 SO 2 C1. 

The two substances are heated on a water-bath till hydrochloric acid is no 
longer evolved. The product is poured into water and the sulphonyl chloride 
extracted with ether. It is purified by distillation in vacua. 

Benzene sulphonyl chloride is a white solid which melts at 14 and boils 
at 1 1 6. It has a pungent odour and is not rapidly decomposed by water. 

By the action of ammonium carbonate upon benzene sulphonyl chloride 
benzene sulphonamide is formed : 

C 6 H 5 SO 2 C1 + NH 3 = C 6 H 5 S0 2 NH 2 + HC1. 

Benzene sulphonyl chloride also reacts with aniline and primary amines, 
secondary amines, but not with tertiary amings : 

C 6 H 5 S0 2 C1 + H 2 N .C B H 5 = C 6 H 5 .SO 2 .HN. C 6 H 5 + HC1 

Benzene sulphonanilide. 

With alcohols it 'forms esters : 

C 6 H 5 SO,C1 + C 2 H 5 OH = C 6 H 5 SO 2 . OC 2 H 5 + HC1. 

Phenol. C 6 H 5 OH. 

Phenol is obtained by fusing benzene sulphonic acid with caustic 
potash. 

0" 
OH 
+ KHSO 3 . 

It is also obtained by the decomposition of diazobenzene (p. 249). 

Preparation. 

Phenol is contained in coal tar and is present in the middle oil 
fraction. This fraction on cooling deposits crystals of naphthalene, 
which are filtered off and pressed out. The oil is shaken with caustic 
soda which dissolves the phenol ; the alkaline layer is separated and 
treated with sulphuric acid. Phenol separates out as an oil : it is 
washed with water and distilled. The distillate is separated into pure 
crystalline phenol and impure liquid phenol. 

Properties. 

Phenol crystallises in colourless prisms which are deliquescent and 
turn pink on contact with air and light. It melts at 42 and boils at 
1 82. It has a characteristic smell, is very poisonous and has a marked 
caustic action upon the skin. It is not easily soluble in water (I part 
in 15 parts of water), but it dissolves in alcohol and other organic sol- 
vents. It is volatile with steam. 

Phenol in its constitution is a tertiary alcohol ; on oxidation it is 
broken down and gives a variety of products. It differs from aliphatic 
compounds containing hydroxyl groups in having acid properties. It 
reacts with caustic alkalies but not with carbonates and forms salts 
which are obtained by evaporating the solution, e.g. potassium phenate. 






AROMATIC COMPOUNDS 245 

These salts are stable to water, but are decomposed by carbon dioxide. 
It has acid properties and hence is usually termed carbolic acid. 

As an alcohol it will form esters, bu.t owing to its acid character 
the esters are not easily formed by the direct action of the acid. They 
are prepared by the action of the acid chloride, or anhydride, upon the 
phenol or its potassium salt : 

f\ O . OC . CH 3 + HCl. 
CH 3 COC1 = ! 

Phenyl sulphuric acid is present in mammalian urine. 
'Phenol also forms ethers; these are prepared by the action of an 
alkyl iodide upon potassium phenate : 

X\OCH 3 
+ CH 3 I = ! + KI. 

These ethers resemble the aliphatic ethers, but also show the 
typical aromatic reactions with nitric acid, etc. 
Reactions and Tests. 

(1) A violet coloration is formed on adding a few drops of ferric 
chloride solution to a solution of phenol in water. 

(2) Phenol is readily brominated. On adding bromine water 
gradually to s,ome phenol solution, there is first a cloudiness due to 
mono- and dibromophenol which are characterised by a very penetrating 
smell. The further addition of bromine water produces a precipitate 
of tribromophenol in yellowish-white needles or flakes. Tribromo- 
phenol is formed directly with very dilute solutions of phenol. 

(3) Phenol is also readily nitrated. On adding concentrated nitric 
acid to a solution of phenol and warming, a yellow colour is produced. 
On cooling and making alkaline with ammonia, the colour becomes 

orange. Picric acid is formed : 

C 6 H 5 OH + 3HN0 3 = C 6 H 2 (N0 2 ) 3 OH + 3 H 2 O. 

(4) A deep red coloration is produced on adding Millon's reagent 
to a solution of phenol and warming. 

Detection of Phenol in Urine (Roaf). 

i c.c. of concentrated hydrochloric acid is added to 10 c.c. of horse's 
urine and the mixture is boiled for two minutes. The phenyl-sulphuric 
acid is hydrolysed. The solution is cooled and extracted with ether. 
The ethereal layer is separated and the ether evaporated. The residue 
dissolved in water will give the reaction with Millon's reagent. 

It is better to distil the urine with dilute sulphuric acid sufficient 
concentrated acid being added to make the mixture contain 5 per cent. 
and collect about a quarter of the volume. The phenol can be 
tested for in the distillate. 




246 PRACTICAL ORGANIC AND BIO-CHEMISTRY - 

Aminobenzene or Aniline. C 6 H 5 . NH 2 . 

Nitrobenzene is converted into aniline by the action of reducing 
agents in acid solution : 

( ]NH 2 
+ 3 H 2 = I I + 2 H 2 0. 

Preparation. 

To 3 or 4 drops of nitrobenzene about 4 gm. of granulated 
tin and 3-4 c.c. of concentrated hydrochloric acid are added. The 
mixture is warmed to start the reaction and it is kept warm until the 
reaction ceases and until the smell of nitrobenzene is no longer per- 
ceptible. An excess of caustic soda is added and the alkaline solution 
is extracted with ether. The ether is allowed to evaporate in a basin 
and the residue is tested for aniline by treating it with bleaching 
powder solution ; a purple colour appears which becomes dirty red. 

On a larger scale 20 gm. of nitrobenzene and 40 gm. of granulated tin are 
placed in a litre flask and warmed on a water-bath for a few minutes. The 
flask is removed from the bath and fitted with an air condenser. 80 c.c. 
of concentrated hydrochloric acid are added in portions of 5 c c. during the 
course of half an hour. If the mixture react violently, it is cooled in water. 
The reaction is completed by heating the flask on a boiling water-bath for 
about one hour. If the smell of nitrobenzene be still observed more hydro- 
chloric acid may be added and the heating continued until it vanishes. 

The double salt (C 6 H 5 . NH 2 . HC1) 2 . SnCl 4 separates out if the product 
be allowed to cool; it is diluted with a 100 c.c. of water and immediately 
decomposed by carefully adding 65 gm. of caustic soda dissolved in a 100 c.c. 
of water. Heat is developed on neutralising and stannic hydrate is precipi- 
tated ; this dissolves in excess of caustic soda and there results a dirty liquid 
containing aniline floating on the surface. The aniline is separated by distilla- 
tion in steam (p. 1 2). The distillate is collected so long as drops of aniline pass 
over. The aniline is extracted by shaking it with ether and the ethereal solution 
is dried with solid caustic soda. The ether is distilled off from a water-bath 
and the residue is 1 distilled over a flame. Aniline passes over at 182-184 
as a pale yellow liquid. 

Properties. 

Aniline is a pale yellow oily liquid which boils at 1 82 and has a 
peculiar odour. It gradually turns brown on exposure to light and 
air. 

Aniline is soluble with difficulty in water, but easily in alcohol and 
ether. 

The solubility of aniline in water can be readily seen by placing 
3 or 4 drops in a test tube full of water and shaking vigorously. The 
oily drops will be no longer visible. 

Aniline is a weak base and forms salts with acids from which it is 
liberated by alkalies, thus : 



AROMATIC COMPOUNDS 247 

About i c.c. of aniline is placed in about 10 c.c. of dilute hydro- 
chloric acid. On shaking the aniline dissolves. On making alkaline 
with about 10 c.c. of caustic soda the aniline separates in oily drops. 

The salt of the aniline is obtained by evaporating its solution in 
the corresponding amount of acid until it crystallises. 

Reactions. 

(1) Aniline is readily brominated : 

A solution of aniline in water is prepared as above and bromine 
water is added ; a pinkish precipitate, which becomes grey-green, of 
tribromaniline is formed : 

C 6 H 5 NH 2 + 3 Br 2 = C 6 H 2 Br 3 . NH 2 + 3 HBr. 

(2) In aqueous solution (above) it is readily oxidised by bleaching 
powder, giving a violet coloration. 

(3) It turns black when it is oxidised with potassium bichromate 
and dilute sulphuric acid. 

(4) A blue colour is formed if a drop of aniline be mixed with 
2 or 3 drops of strong sulphuric acid and the paste so formed stirred 
with a few drops of potassium bichromate solution. 

(5) It gives the carbylamine reaction with chloroform and alcoholic 
potash (p. 61). 

Acetanilide. C 6 H 5 . NH . OC . CH 3 . 

Aniline is acylated by treatment with acetyl chloride, glacial acetic 
acid or acetic anhydride : 

C 6 H 5 . NH 2 + HOOC . CH 3 = H 2 O + C 6 H 5 . NH OC . CH 3 . 

Preparation. 

2 c.c. of aniline are boiled under a reflux air condenser with 4 c.c. of 
glacial acetic acid for an hour. The mixture is poured into water. 
Acetanilide is precipitated and is recrystallised from boiling water. 

Properties. 

Acetanilide is a white crystalline solid which melts at 114 and is 
used in medicine under the name of antifebrin. It is readily decomposed 
by boiling with acids or alkalies : 

C 6 H 6 NH OC . CH 3 + H 2 O = C 6 H 5 . NH 2 + HOOC . CH 3 . 

Thus, on boiling about 2 gm. of acetanilide with about 5 c.c. of 
concentrated hydrochloric acid for a few minutes and pouring the solu- 
tion into water, a clear solution is obtained. On adding excess of caustic 
soda, the aniline is precipitated and may be extracted with ether and 
tested for as above. 



248 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Alkyl Anilines. 

Aniline as a primary amine will react with one or two molecules of an 
alkyl halide to form alkyl anilities : 

C 6 H 5 . NH 2 + CH 3 I = C 6 H g . NH . CH 3 + HI 
C 6 H 5 . NH . CH 3 + CH 3 I = C 6 H 5 . N . (CH 3 ). + HI. 

These compounds are readily obtained by heating aniline with the alcohol 
and hydrochloric acid at 200 to 250 : 

C 6 H S NH 2 . HC1 + CH 3 OH = C 6 H 5 NH . CH 3 HC1 + H 2 O 
C 6 H 5 NH 2 . HC1 + 2 CH 3 OH = C 6 H 5 . N(CH 3 ) 2 HC1 + 2H 2 O. 

The methyl anilines are stronger bases than aniline, as they are more like 
the aliphatic amines. They may be regarded as phenylmethylamine and 
phenyldimethylamine. 

Methyl aniline is a colourless oily liquid which boils at 192. As a 
secondary amine it gives a nitrosamine with nitrous acid. 

SX \NH + O:N.OH = >N . NO + H 2 O. 

CH 3 / CH 3 / 

Phenyl-methyl-nitrosamine is a yellow oil which gives Liebermann's 
nitroso reaction. 

Dimethylaniline is a colourless oil which boils at 192; it is largely used 
in the dye industry. 

Dialkylanilines, such as dimethylaniline, react with nitrous acid giving 
nitroso compounds : in these compounds the reaction takes place with the 
hydrogen atom in the para position (p. 260) in the benzene nucleus: 

/NO 

C 6 H 5 . N(CH 3 ) 2 + ONOH = C 6 H 4 ( 

\N(CH 3 ) 2 + H 2 0. 

Diphenylamine cannot be prepared by heating aniline with bromoben- 
zene, but is obtained by heating aniline hydrochloride with aniline in a closed 
vessel at 240. 

C B H 6 NH 2 . HC1 + C 6 H 5 NH 2 = ' 5 ^>NH + NH 4 C1. 

Diphenylamine is a crystalline solid melting at 54 and boiling at 310. 
It is a very weak base, its salts being decomposed by water; it is almost 
insoluble in dilute acids. It dissolves in concentrated sulphuric acid. This 
solution on the addition of a trace of nitric acid gives a deep blue coloration 
and serves for detecting nitrates. Diphenylamine owing to the acid char- 
acter of the phenyl groups reacts with potassium giving potassium diphenyl- 
amine, 

5 \NK. 
CH 5 / 

Triphenylamine. (C 6 H 5 ) 3 N. 

This compound is prepared by heating potassium diphenylamine with 
bromobenzene at 300. It is a colourless crystalline solid melting at 127. It 
does not form salts with acids. 



AROMATIC COMPOUNDS 249 

Diazobenzene. C 6 H 5 . N : N . OH or C 6 H 5 . N( ; N) . OH. 

Aniline is a primary amine. Primary amines of the aliphatic 
series are converted by the action of nitrous acid into the correspond- 
ing alcohol, but diazobenzene is formed by the action of nitrous acid 
upon aniline : 

/\ 

1NH, 




The reaction is carried out at about o. 

Properties. 

Diazobenzene behaves like a strong base (NH 4 OH) and is known 
only in the form of its salts. When liberated from a solution of its 
salts it is precipitated as a yellow oil which is very unstable and 
decomposes with explosion. It forms crystalline salts with mineral 
acids, which are also explosive. The nitrate explodes violently if 
gently struck ; the other salts explode on heating. These salts are 
easily soluble in water, less soluble in alcohol and insoluble in ether. 
They are generally called diazonium salts. 

Reactions. 

(1) An aqueous solution of a diazonium salt is decomposed on 
boiling. Nitrogen is evolved and phenol is formed : 

C 6 H 5 . N 2 C1 + H 2 O = C 6 H 5 OH + N 2 + HC1. 

Thus : 

A few drops of sodium nitrite solution are added to a dilute 
solution of aniline in hydrochloric acid. Diazobenzene chloride is 
formed. On warming the solution, it is decomposed with evolution 
of nitrogen and the smell of phenol (carbolic acid) becomes noticeable. 

(2) On boiling a diazobenzene salt with absolute alcohol, nitrogen 
is evolved and benzene is formed, reduction occurring : 

C 6 H 5 N 2 C1 + Ho = C 6 H 6 + N 2 + HC1. 

(3) A precipitate of diazobenzene perbromide is formed on adding 
bromine dissolved in potassium bromide to a solution of diazoben- 
zene chloride ; on boiling with alcohol, nitrogen is evolved and 
bromobenzene is formed : 

C 6 H 5 N 2 C1 + Br 2 + KBr = KC1 + C 6 H g NBr . NBr 2 
C 6 H" 5 NBr NBr 2 = C 6 H s Br + N a + Br 2- 

(4) lodobenzene is formed if potassium iodide be added to a 
solution of diazobenzene chloride and the solution warmed : 

C 6 H 5 N 2 C1 + KI = KC1 + N 2 + C 6 H 5 I. 



250 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

(5) Sandmeyer's Reactions. 

On adding a solution of cuprous chloride in hydrochloric acid, 
or ,, cuprous bromide in hydrobromic acid, 
or ,, ,, ,, cuprous cyanide in potassium cyanide, 
to a solution of diazobenzene chloride and warming, nitrogen is 
evolved and chlorobenzene, bromobenzene or cyanobenzene (phenyl- 

cyanide) is formed : 

C 6 H 5 N 2 C1 + CuCN = C 6 H 5 CN + N 2 + CuCl. 

In practice these reactions are carried out by mixing aniline with a slight 
excess of hydrochloric acid, cooling in ice and adding the calculated quantity 
of sodium nitrite solution, as shown by testing with potassium iodide-starch 
paper. The aqueous solution is warmed, or potassium iodide is added, or it 
is poured into the solution of the cuprous salt and warmed. The product 
can generally be isolated by steam distillation. 

Phenylhydrazine. C 6 H 5 . NH . NH 2 . 

Phenylhydrazine is obtained by reducing diazonium chloride with 

stannous chloride : 

C 6 H 5 . N a . Cl + 2H 2 = C 6 H 5 NH . NH 2 . HC1. 

Its constitution is proved by its conversion into aniline and 
ammonia by reduction with zinc and hydrochloric acid : 
C 6 H 5 NH . NH 2 + H 2 = C 6 H 5 NH 2 + NH 3 . 

Preparation. 

A molecular proportion of aniline (9^3 gm.) is dissolved in about 10 times 
the calculated quantity of concentrated hydrochloric acid (200 c.c.), thoroughly 
cooled in ice and diazotised by adding the calculated quantity of sodium 
nitrite (6-9 gm.). As soon as excess of nitrite is present, as indicated by 
starch-iodide paper, rather more than the calculated quantity of stannous 
chloride (45 gm.) dissolved in the proper amount of concentrated hydrochloric 
acid (100 c.c.) is slowly added. Phenylhydrazine hydrochloride separates out. 
It is filtered off by suction and washed with concentrated hydrochloric acid. 
It is dissolved in water and decomposed with excess of caustic soda ; the oil 
is extracted with ether, the ethereal solution dried with solid potash, the ether 
distilled off and the base distilled in vacua. 

Properties. 

Phenylhydrazine consists of colourless prisms which melt at 23 
and boil at 241 with slight decomposition. It dissolves slightly in 
cold water and easily in alcohol and ether. As a strong base it forms 
salts with acids ; the hydrochloride crystallises in needles and is 
easily soluble in warm water. 

Reactions. 

(1) Phenylhydrazine and its salts reduce Fehling's solution. 

(2) It is converted into benzene on heating its solutions with 
copper sulphate or ferric chloride. 

(3) It combines with aldehydes, ketones and carbohydrates to 
form hydrazones and osazones. The hydrazone is decomposed by con- 
centrated hydrochloric acid, and on reduction gives an amine and aniline. 



AROMATIC COMPOUNDS 251 

Toluene. C 6 H 5 . CH 3 . 

Toluene, or methylbenzene, or phenyl methane, is present in coal 
tar and is contained with benzene in the first fraction on fractionally 
distilling the tar. It is separated from benzene by fractional distillation. 

Toluene is also obtained from balsam of tolu, or from toluic acid 
by distillation with soda lime a reaction analogous to the preparation 
of methane from sodium acetate : 

/CH 3 

C 6 H 4 ( = C 6 H 5 .CH 3 + C0 2 . 

\COOH 

Toluene can be prepared from benzene by either of the following 
two reactions : 

(1) Fittig's Reaction. A mixture of bromobenzene and methyl 
bromide is heated with sodium ; 

C 6 H 5 Br + Naa + CH 3 Br = C 6 H 5 . CH 3 + 2NaBr. 

(2) Friedel and Graffs Reaction. Benzene is heated with 
methyl iodide in the presence of aluminium chloride : 

C 6 H 6 + CH 3 I = C 6 H 5 .CH 3 + HI. 

In this reaction a compound of benzene and aluminium chloride 
is probably first formed and this compound reacts with the alkyl 
halide : 

C 6 H 6 + A1 2 C1 B = C 6 H 5 . A1 2 C1 5 + HC1 
C 6 H 5 A1 2 C1 5 + CH 3 C1 = A1 2 C1 6 + C fi H 5 . CH 3 . 

Dry benzene is treated under a reflux condenser with a third of its weight 
of aluminium chloride and the alkyl chloride is slowly added. The benzene 
may be mixed with a neutral solvent such as ether or petroleum ether. The 
mixture is heated on a water-bath until halogen acid is no longer evolved. 
The mixture is allowed to cool and water added to dissolve the aluminium 
chloride ; the layer of benzene and ether is separated, dried with calcium 
chloride, the ether distilled off and the residue distilled. 

Properties. 

Toluene, an oily colourless liquid with characteristic smell, boils 
at 110 and has a sp. gr. of -882 at o. It burns with a smoky 
luminous flame, is insoluble in water, but soluble in organic solvents. 
It is known commercially as toluole. Toluene closely resembles ben- 
zene in its properties in forming nitro- and other derivatives, but it 
differs from benzene in being also an aliphatic compound. Toluene 
is the first instance of an aromatic compound containing a side chain. 
It is this side chain which gives toluene the properties of an aliphatic 
compound. (See benzyl chloride, alcohol, benzaldehyde, etc.) 

On oxidation with dilute nitric acid and other oxidising agents, 
the nucleus remains intact but the side chain is oxidised to a carboxyl 

group : 

C 6 H 5 . CH 3 + 30 = C 6 H S . COOH + H 2 O. 



252 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Ethylbenzene, C 6 H 5 . C 2 H 5 , and other Homologues of Benzene. 

Ethylbenzene is also contained in coal tar and can be prepared 
from benzene by the reactions given under toluene. It is a liquid 
like toluene, but boils at 1 34 and is isomeric with the xylenes (p. 260). 

The other homologues, propyl, butyl, etc., benzene can be prepared 
in the same way. 

These homologues of benzene contain both a benzene nucleus and 
an aliphatic radicle or side chain. They behave as aromatic compounds 
by forming nitro and sulphonic acid derivatives. They behave as 
aliphatic compounds in forming halogen derivatives in which the 
halogen atom can be replaced by OH and other groups. On oxida- 
tion the side chain undergoes shortening until ulimately benzoic acid 
is formed : 

C 6 H S . CH 2 . CH 2 . CH 3 -> C 6 H 5 . CH 2 . CH 2 . COOH -> C 6 H 5 . CH 2 . COOH -> 

Styrene or Phenylethylene, CgH^ . CH = CH 2 t is an aromatic hydro- 
carbon containing an unsaturated group in the side chain. It is obtained by 
heating cinnamic acid. 

Benzyl Chloride, QH 6 . CH 2 C1. 

Benzyl chloride is the chief representative of an aromatic compound 
in which a halogen atom is present in the side chain. Like aliphatic 
compounds it can be obtained by the action of phosphorus pentachloride 
upon the corresponding alcohol benzyl alcohol C 6 H 5 . CH 2 OH. The 
radicle C 6 H 5 . CH 2 is termed benzyl in order to distinguish it from 
the radicle C 6 H 5 which is termed phenyl. 

Preparation. 

Benzyl chloride is prepared by passing a stream of dry chlorine 
into toluene, heated under a reflux condenser, until the increase in 
weight corresponding to the equation has been reached. The product 
is then separated and purified by distillation. The reaction takes 
place most readily if the flask be exposed to sunlight. The following 
reaction takes place : 

C 6 H 5 . CH 3 + C1 2 = C 6 H 5 . CH 2 C1 + HC1. 

The procedure is quite different to that used in the preparation of 
bromobenzene and chlorotoluene (p. 241). 

Properties. 

Benzyl chloride is a colourless liquid which boils at 176. It has 
an unpleasant smell, is insoluble in water, but soluble in alcohol, ether 
and benzene. 

It is nitrated, sulphonated, etc., by nitric or sulphuric acid, but 
in its other reactions it resembles ethyl chloride. It is mainly used 
for the preparation of benzaldehyde. 



AROMATIC COMPOUNDS 253 

Benzal Chloride, C 6 H 5 . CHC1,. 

This compound is prepared by the further action of chlorine upon 
boiling toluene, until chlorine corresponding to the equation, 

C 6 H 5 . CH 3 + 2CJ 2 = C 6 H 5 CHCI 2 + 2HC1, 
has been absorbed. 

It can be obtained by the action of phosphorus pentachloride upon 
benzaldehyde : 

C 6 H 5 .CHO + PC1 5 = C 6 H 5 CHC1 2 + POC1 3 . 

Benzal chloride is a colourless liquid of boiling-point 206 ; it is 
also used for making benzaldehyde. 

Benzotrichloride, C 6 H 5 . CC1 3 , or Phenylchloroform. 

By the further action of chlorine upon toluene, benzotrichloride is 
formed : 

C 6 H 5 CH 3 + 3 C1 2 = C 6 H 8 CC1 3 + 3 HC1. 

It is a liquid which boils at 213 and is converted into benzoic acid by 
boiling with water. 

Benzyl Alcohol, C 6 H 5 . CH 2 OH. 

Benzyl alcohol occurs as such and also as ester with benzoic and 
cinnamic acids in the resin storax, in balsam of Tolu and balsam of 
Peru. 

It is the chief type of an aromatic alcohol in which the 
hydroxyl group is present in the side chain (compare phenol). 

Preparation. 

As an alcohol it may be obtained by reducing the corresponding 
aldehyde, benzaldehyde : 

C 6 H 5 . CHO + H 2 = C 6 H 5 . CH 2 OH, 

or by the action of water and aqueous alkalies upon benzyl chloride : 
C 6 H 5 CH,C1 + H 2 = HC1 + C 6 H 5 .CH 2 OH. 

Benzyl alcohol is usually obtained by the action of aqueous po- 
tassium hydroxide upon benzaldehyde : 

2C 6 H 5 CHO + KOH = C 6 H 6 CH 2 OH + C 6 H 5 COOK. 

Benzaldehyde is shaken up with four times the amount of potash dissolved 
in about an equal weight of water. The emulsion which is formed is allowed 
to stand for twenty- four hours. On the addition of water, the potassium ben- 
zoate dissolves ; the solution is extracted with ether, the ether dried and the 
benzyl alcohol obtained by distillation. 

Properties. 

Benzyl alcohol is a colourless liquid of boiling-point 206 ; it is 
not easily soluble in water, but dissolves in alcohol and ether. It 
behaves like ethyl alcohol with sodium and phosphorus pentachloride. 
It forms esters with acids or acid anhydrides, etc., e.g. benzyl bromide, 
benzyl acetate. 



254 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Benzaldehyde. C 6 H 5 . CHO. 

Benzaldehyde was originally isolated from bitter almonds and 
called oil of bitter almonds. The almonds contain the glucoside, amyg- 
dalin, which is hydrolysed by the enzyme, emulsin, into glucose ben- 
zaldehyde and prussic acid. It is the aldehyde of benzyl alcohol from 
which it may be obtained by oxidation with nitric acid. 

It may be obtained by distilling calcium benzoate with calcium 
formate in the same way as aliphatic aldehydes. 

Preparation. 

(1) Benzaldehyde is prepared from benzal chloride by boiling it 
with dilute sulphuric acid or lime water under pressure : 

C 6 H 8 . CHC1 2 - C 6 H E CH(OH) 2 -> C 6 H 5 . CHO. 

(2) Or it is prepared by boiling benzyl chloride with lead nitrate 
or copper nitrate. Benzyl alcohol is probably first formed and is oxi- 
dised to the aldehyde : 

C 6 H 5 . CH 2 C1 -> C 6 H 5 CH 2 OH -> C 6 H 5 . CHO. 

Molecular proportions of benzyl chloride (12 '6 gm.) and copper nitrate 
(20 gm.), dissolved in about the same weight of water (25 c.c.), are- boiled for 
6-8 hours under a reflux condenser, whilst a current of carbon dioxide is 
passed through the mixture to expel oxides of nitrogen and to avoid further 
oxidation. When the oil contains no chlorine or only traces as shown 
by testing it, after washing with water, with silver nitrate and nitric acid, the 
oil is extracted with ether and the ethereal extract is shaken with saturated 
sodium bisulphite solution. The crystalline bisulphite compound is filtered off 
and washed with ether. The benzaldehyde is obtained by decomposing it with 
dilute sulphuric acid, extracting with ether, drying and distilling. 

(3) It is prepared by Friedel and Craft's reaction from benzene, a mixture 
of carbon monoxide and chlorine being passed into the benzene. Formyl- 
chloride is apparently formed which reacts as follows : 

C 6 H 6 + H . CO . Cl = C 6 H 5 . CHO + HC1. 

Properties. 

Benzaldehyde is a colourless liquid with a strong smell of bitter 
almonds. It boils at 179 and has a sp. gr. of 1*05 at 15. It is very 
slightly soluble in water, but dissolves in alcohol and ether. It is used 
extensively for flavouring purposes: 

Reactions. 

In most reactions benzaldehyde resembles the aliphatic aldehydes: 

(1) It is easily oxidised; by exposure to air crystals of benzoic 
acid gradually separate ; 

C 6 H 5 . CHO + O = C 6 H 5 . COOH. 

(2) On reduction it yields benzyl alcohol (p. 253). 

(3) It yields benzal chloride with phosphorus pentachloride. 

(4) It gives an oxime with hydroxylamine. 



AROMATIC COMPOUNDS 255 

* (5) It gives a hydrazone'with phenylhydrazine. , 

* (6) It combines with sodium bisulphite. 
(7) It combines with hydrogen cyanide. 

In the following reactions benzaldehyde and other aromatic alde- 
hydes, which have the aldehyde group attached to the benzene nucleus, 
differ from aliphatic aldehydes ; 

* (i) It does not reduce Fehling's solution or ammoniacal silver 
solutions. 

(2) It does not polymerise. 

(3) I* gives a mixture of alcohol and acid on treating with potash. 

(4) It is converted into benzoin on shaking with an alcoholic solu- 
tion of potassium cyanide : 

C 6 H S . CHO + C 6 H 5 . CHO = C 6 H 5 . CO . CHOH . C 6 H 5 . 

Benzoin is a complex ketonic alcohol formed by the condensation of 
two molecules of benzaldehyde. 

(5) It condenses with acetone and aniline on shaking up the two sub- 
stances with a few drops of caustic soda : 

C 6 H 5 . CHO + CH 3 . CO . CH 3 = C 6 H 5 . CH = CH 2 . CO . CH 3 + H 2 O. 

As an aromatic compound benzaldehyde forms nitro, sulphonic acid 
derivatives, etc. 

If nitrobenzaldehyde in acetone solution be mixed with a few drops 
of dilute caustic soda, a precipitate of indigo-blue (p. 343) slowly forms : 

/NO 2 / NH \ / NH \ 

aC 6 H / + aCH, . CO . CH 3 = C fi H 4 ( >C = C/ >C 6 H 4 + 2H 2 O + 2 CH 3 . COOH . 

\CHO \C(K \CO/ 

Benzoic Acid, C 6 H 5 . COOH. 

Benzoic acid occurs in gum benzoin and other resins such as 
balsam of Peru. In gum benzoin it is present chiefly as the ester, 
benzyl benzoate. 

Preparation. 

(1) Benzoic acid is readily obtained by subliming gum benzoin. 
Gum benzoin is heated on an iron tray or porcelain basin, the tray 
being covered with a cone of filter paper or a funnel. The resin 
melts and the benzoic acid which volatilises condenses on the cone. 
It is recrystallised from water. 

(2) Benzoic acid is made commercially by oxidising benzyl chloride 
with 60 per cent, nitric acid. 

(3) It is also prepared by heating the calcium salt of phthalic 
acid. 

(4) It can be prepared by the hydrolysis of the nitrile, phenyl 
cyanide. 

(5) As previously mentioned, it results from the oxidation of 
aromatic compounds possessing a side chain. 



256 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Properties. 

Benzoic acid forms glistening crystals which melt at 121 -5 and 
boil at 249 

On heating, it melts and gives off white vapours with characteristic 
smell and suffocating effect upon the throat ; the vapours condense as 
a crystalline sublimate. 

It dissolves easily in hot water and crystallises out on cooling ; it is 
only slightly soluble in cold water (i part in 400). 

It dissolves in alcohol and ether and other organic solvents. 

It forms salts with alkalies, dissolving in caustic alkalies and 
alkali carbonates, in lime water, etc. ; on acidifying these solutions, it is 
precipitated. 

A neutral solution gives a precipitate of a pale brown colour with 
ferric chloride. 

It is easily nitrated : nitrobenzoic acid is formed on evaporating a 
little benzoic acid in a porcelain basin with nitric acid. 

It is converted into benzene by heating with soda lime. 

It forms esters, e.g. ethylbenzoate (p. 73) is formed if benzoic 
acid be heated with a little alcohol and a few drops of concentrated 
sulphuric acid. The ester has a peculiar aromatic odour and boils at 
213. 

Benzoyl Chloride, C 6 H 5 . CO . Cl. 

This compound is formed by the action of phosphorus penta- 
chloride upon benzoic acid : 

C 6 H 5 . COOH + PC1 5 = C 6 H 5 COC1 + POC1 3 + HC1. 

Benzoic acid and a slight excess of phosphorus pentachloride are placed 
in a distilling flask ; the reaction proceeds at the ordinary temperature and 
the vapours of hydrogen chloride are passed into soda. As soon as the 
reaction is complete the contents are distilled in a fume cupboard ; phos- 
phorus oxychloride passes over at 107, benzoyl chloride at about 198. It is 
purified by redistillation. 

Benzoyl chloride is a colourless oily liquid with a peculiar pungent smell. 
It is slowly decomposed by water into benzoic acid and more readily by 
alcohol into ethyl benzoate. 

Benzoic Anhydride, (C f) H 5 . CO) 2 O. 

The anhydride of benzoic acid is prepared like other anhydrides by the 
action of benzoyl chloride upon sodium benzoate : 

C 6 H S COC1 + C 6 H 5 COONa = C 6 H 5 . CO . O . CO . C 6 H 5 + NaCl. 

It is a colourless crystalline substance melting at 42 and resembles acetic 
anhydride. 



AROMATIC COMPOUNDS 257 

Benzamide, C 6 H 5 . CO . NH 2 . 

Benzamide is prepared by either of the reactions : 

(ij Benzoyl chloride and ammonia : 

C 6 H 5 COC1 + NH 3 = C 6 H S . CO. NH 2 + HC1. 

Benzoyl chloride is mixed with a slight excess of dry ammonium carbonate 
in a mortar until the smell of benzoyl chloride vanishes. Cold water is added 
to dissolve the ammonium salts ; the insoluble benzamide is crystallised from 
hot water. 

(2) Ammonia and ethyl benzoate. 

Benzamide is a colourless crystalline solid melting at 130, easily soluble 
in hot water, but soluble with difficulty in cold. It is decomposed by boiling 
with acids, or alkalies, into benzoic acid and ammonia. 

Benzonitrile, or Phenyl Cyanide, C 6 H 5 . CN. 
Benzonitrile is prepared : 

(i) by fusing potassium benzene sulphonate with potassium cyanide or 
ferrocyanide : 

C 6 H 5 SO 3 K + KCN = C 6 H 5 CN + K 2 SO 3 . 

(2) by Sandmeyer's reaction from aniline ; the aniline is diazotised and 
the solution heated with cuprous cyanide (p. 250). 

Benzonitrile is a colourless oil with a smell resembling that of nitroben- 
zene. It boils at 191 and resembles the aliphatic nitriles in its reactions : 

(1) hydrolysis : 

C 6 H 5 CN + 2HoO = C 6 H 5 COOH + NH 3 , 

(2) reduction : 

C 6 H 5 CN + 4 H = C B H 5 . CH 2 . NH 2 
Benzylamine. 

Benzylamine, C 6 H 5 . CH 2 . NH 2 . 

Benzylamine is an aromatic a mine in which the amino group is present 
in the side chain (compare aniline). It is prepared like the aliphatic 
primary amines (p. 124) : 

(1) ammonia upon benzyl chloride ; 

(2) bromine and potash upon the amide of phenylacetic acid ; 

(3) reduction of the nitrile or oxime. 

It is a colourless oily liquid boiling at 187 with pungent smell. It is 
a strong base like the aliphatic primary amines and with similar properties. 

Dibenzylamine, (C 6 H 6 .CH 2 ) 2 NH. Tribenzylamine, (C 6 H 5 .CH 2 \,N. 

Again, these compounds resemble the secondary and tertiary aliphatic 
amines. They are obtained by heating benzylamine with benzylchloride. 

The three amines are formed when benzylchloride is heated with 
ammonia. 

They have the typical aromatic reactions, as well as the aliphatic ones. 



258 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Acetophenone, C 6 H 5 . CO . CH 3 . 

Acetophenone is an example of an aromatic ketone. It is formed by 
distilling calcium benzoate with calcium acetate. 

Acetophenone is most readily prepared by slowly dropping i molecular 
proportion of acetyl chloride upon i molecule of benzene containing in 
suspension i molecule of aluminium chloride and cooled with ice. When the 
evolution of hydrochloric acid is over, ice-cold water is very carefully added. 
The solution is extracted with ether, the ethereal extract dried and distilled. 
Acetophenone passes over between 194 and 200 : 

C 6 H 6 + CH 3 COC1 = C 6 H 5 . CO . CH 3 + HC1. 

This reaction is a general one for preparing aromatic ketones. 

Acetophenone is a crystalline solid melting at 20-5 and boiling at 202. 
It is soluble in water and alcohol. It is sometimes used as a hypnotic under 
the term hypnone. It closely resembles acetone and the aliphatic ketones in 
giving a secondary alcohol on reduction, benzoic acid and acetic acid on 
oxidation and in forming an oxime and a phenylhydrazone. It resembles 
benzene in forming nitro- and other derivatives. 

Benzophenone, C 6 H 5 . CO . C 6 H 5 . 

Benzophenone is obtained by heating calcium benzoate and is prepared 
by the action of benzoyl chloride upon benzene in the presence of aluminium 
chloride. 

It is a crystalline solid melting at 48-49 and resembles acetophenone. 
It yields diphenylmethane on reduction with zinc dust. 

Phenylacetic Acid, C 6 H 5 . CH 2 . COOH. 

This acid containing a carboxyl group in the side chain is formed 
in the putrefaction of proteins, arising from phenylalanine. 
Synthetically it is obtained from benzyl chloride : 

C 6 H 5 . CH 2 C1 -> C 6 H 5 . CH 2 . CN -> C 6 H E . CH 2 . COOH. 

Molecular proportions of benzyl chloride and potassium cyanide are 
boiled in dilute alcoholic solution for 3-4 hours. The benzyl cyanide is isolated 
by fractional distillation (220-335 fraction) and hydrolysed by boiling with 
dilute sulphuric acid. The phenylacetic acid is purified by crystallisation. 

Phenylacetic acid crystallises in colourless shining plates which melt at 
76-5 and boil at 262. It has a characteristic smell. 

Phenaceturic Acid, C 6 H 5 . CH 2 . CO NH . CH 2 . COOH. 

Phenylacetic acid, which is formed in the large intestine in small amounts, 
on absorption into the body, or when it. is injected into the blood, is excreted 
as phenaceturic acid, i.e. in combination with glycine. It thus resembles 
benzoic acid, which is excreted as hippuric acid (p. 142). 

It is a colourless crystalline substance melting at 143, is soluble with 
difficulty in water, but easily in alcohol. It is hydrolysed by boiling with 
acids into phenylacetic acid and glycine. 

Phenylpropionic Acid, C 6 H 5 . CH 2 . CH 2 . COOH. 

Phenylpropionic acid accompanies phenylacetic acid amongst the putre- 
faction products of proteins. 

It is most conveniently prepared by reducing cinnamic acid : 

C 8 H 5 . CH = CH . COOH + H,, = C 6 H 8 . CH 2 . CH 2 . COOH. 
It is a colourless crystalline substance melting at 47 and boiling at 280. 



AROMATIC COMPOUNDS 259 

Cinnamic Acid, C 6 H 5 . CH=CH . COOH. 

Cinnamic acid is the chief representative of an aromatic compound con- 
taining an unsaturated acid as the side chain. It occurs in greatest amount 
in storax, the resin of Styrax offirinalis. 

It is prepared from storax by warming it with dilute sodium hydroxide ; 
the filtered alkaline solution is acidified with hydrochloric acid. Cinnamic 
acid is precipitated and purified by crystallisation from water. 

Cinnamic acid is usually prepared by synthesis by Perkin's reaction : 
C 6 H 5 . CHO + H S C . COONa = C 6 H 5 . CH=CH . COONa + H 2 O. 

Molecular proportions of benzaldehyde and sodium acetate are heated 
in an oil-bath under a reflux condenser with 3-4 parts of acetic anhydride 
for 8-10 hours. The mixture is poured into water and unchanged benzalde- 
hyde is removed by steam distillation ; the residue is treated with caustic 
soda, filtered from oily impurities and acidified with concentrated hydrochloric 
acid. Cinnamic acid is precipitated and purified by crystallisation from water. 

Cinnamic acid crystallises in needles and melts at 133. It is soluble 
with difficulty in cold water, more easily in hot water. It dissolves in alcohol, 
ether and other organic solvents. As an aromatic compound it forms nitro 
derivatives. As an unsaturated aliphatic compound cinnamic acid forms 
addition compounds with bromine and halogen acids. It is converted by 
reduction into phenylpropionic acid or hydrocinnamic acid. It is readily 
oxidised in the cold by permanganate, a solution of cinnamic acid in alkali 
decolorising permanganate immediately. 

Cinnamic Aldehyde, C 6 H 5 . CH=CH . CHO. 

Cinnamic aldehyde is the chief constituent of oil of cinnamon from 
which it may be obtained by treatment with sodium bisulphite. 

It is a liquid which boils at 247 and has the peculiar and characteristic 
odour of cinnamon. It resembles the aliphatic aldehydes in properties. 

Phenylalanine, C 6 H 5 . CH 2 . CH . (NH 2 )COOH. 

Phenylalanine was first isolated from an extract of growing seed- 
lings and subsequently recognised as a constituent of proteins ; it is 
the constituent which gives rise to phenylpropionic and phenylacetic 
acids during putrefaction. It also gives rise to phenylethylamine, 
and most probably cinnamic acid is also derived from it. In the de- 
composition either carbon dioxide or ammonia is lost and then the 
side chain is oxidised : 
C 6 H 5 . CH 2 . CH 2 . COOH <- C B H 5 . CH 2 . CH(NH 2 )COOH _> C 6 H 5 . CH 2 . CH 2 . NH 2 

I 
C 6 H S . CH 2 . COOH -> C 6 H 5 . COOH. 

Phenylalanine is isolated with some difficulty from the complicated mix- 
ture of amino acids resulting from proteins; full details are given in "The 
Chemical Constitution of Proteins," Part I. 

It has also been prepared by synthesis (see " The Chemical Constitution 
of Proteins," 2nd ed. Part I., or 3rd ed. Part II.). 

Phenylalanine crystallises in glistening platelets which melt at 
275-280. The natural substance is laevorotatory. It has aromatic 
properties and the properties of an amino acid. 

17* 



2 6o PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Mandelic Acid, C 6 H 5 . CHOH . COOH. 

Mandelic acid is formed when amygdalin is hydrolysed by boiling with 
acids ; it is present in amygdalin as the nitrile of mandelic acid : 

,0 . C 12 H 22 U ,OH 

C 6 H 5 . CH( + 2 H 2 = C 6 H 5 . CH< + 2C 6 H 12 6 

\CN X CN 



C 6 H 5 . CH + 2 H 2 = C 6 H S . CHOH . COOH + NH 3 . 

XN 

It is prepared synthetically from benzaldehyde ; the benzaldehyde is 
converted into the cyanohydrin and this is hydrolysed. 

Mandelic acid is a colourless crystalline solid which melts at 133. It 
is soluble in water, ether and other organic solvents. The natural acid is 
optically active and laevorotatory ; the synthetical acid has been separated into 
its stereoisomers by the usual methods. 

Mandelic acid closely resembles lactic acid in its properties, as it contains 
the OH group in the side chain. It differs from salicylic acid and other aro- 
matic acids which contain the OH group attached to the benzene nucleus. 



DISUBSTITUTION PRODUCTS OF BENZENE. 

The disubstitution products of benzene exist in three forms : 





The number of derivatives is very large. They can be divided into 
those in which the substituting groups are the same and those in which 
they are different. 



Dimethylbenzenes or Xylenes, C 6 H 4 <' 

X CH 3 . 

These three compounds are present in coal tar and are contained in the 
benzene fraction from which they are prepared by fractional distillation ; 
m-xylene exists in largest amount. The fraction in which they are present 
boils at 136-141. Their boiling-points are so close that they cannot be 
separated by fractional distillation ; their separation depends on the formation 
of nitro- and sulphonic acid derivatives. 

They closely resemble benzene and toluene and are obtained from these 
compounds by the same methods as toluene is prepared from benzene. A 
different isomer is formed under different conditions. 

They yield nitro- and other derivatives, and on oxidaiton are converted 
into methyl benzoic acids and into phthalic acids. 



AROMATIC COMPOUNDS 261 

X N0 2 
Dinitrobenzenes, C 6 H 4 <; 

X N0 2 . 

m-Dinitrobenzene is obtained by nitrating benzene with sulphuric acid 
and nitric acid and heating. It is a yellow crystalline solid melting at 90. 

o- and p- Dinitrobenzenes are formed in small quantities at the same 
time. They are colourless solids melting at 118 and 173 respectively. 

NH 2 
Diaminobenzenes, or Phenylenediamines, C 6 H 4 <' 

X NH 2 . 

Like aniline they are obtained by reducing the dinitrobenzenes. 
They also resemble aniline. m-Phenylenediamine melts at 63. It gives a 
deep yellow colour with nitrites and is used for detecting nitrites in small 
quantities. 



Benzene Disulphonic Acids, C 6 H 4 < 



,SO 3 H 



The m-compound is formed by heating benzene with two molecular 
proportions of sulphuric acid. 



Dihydroxy- Benzenes, C 6 H 4 < 

X OH. 

The three isomeric dihydroxy benzenes are natural compounds 
and are termed : 

Catechol, Hydroquinone, 

or or 

pyrocatechin Resorcinol quinol 

OH OH OH 




Ortho. Meta. 

Catechol occurs in catechu, a resin obtained from Acacia catechu, 
and was first obtained from this source. It is prepared by fusing 
o-phenolsulphonic acid with potash, or by reducing guaiacol with 
hydriodic acid : 

/OCH 3 /OH 

C 6 H/ + HI = CH 3 I + C 6 H 4 ( 

\OH \OH. 

It is a colourless crystalline solid melting at 104. 

Resorcinol is obtained by fusing benzene-m-disul phonic acid and 
the other disulphonic acids with potash at higher temperatures. 

It forms colourless crystals which melt at 110 and are easily 
soluble in water, alcohol, ether. It is used largely for making eosin 
and other dyes. 

Quinol is formed by the hydrolysis of the glucoside, arbutin, by 
boiling with water. It is 'usually prepared by reducing quinone 
with sulphurous acid. It is a colourless crystalline solid melting at 
169 and very easily soluble in water. 



262 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Quinone. 

Quinol is easily oxidised by ferric chloride to quinone, but quinone 
is usually prepared by oxidising aniline with potassium bichromate 
and sulphuric acid. 

It is a yellow crystalline solid with a peculiar smell and melts 
at 1 1 6. It is not very soluble in water, but dissolves in alcohol and 
ether. It is volatile in steam. 

Quinone in some respects behaves as a diketone, but in other 
respects as an aromatic compound : it is, generally represented by the 
O 
II in which there are two pairs of double bonds. On re- 

c I ii II duction the centric formula is formed, but on oxidation 
tormula II II 

\/ it breaks down at the double linkings ; it combines 

II with 2 or 4 atoms of bromine. 
O 

Reactions of Catechol, Resorcinol, Quinol. , 

Aqueous solutions behave as follows : 

(1) With FeCl 3 : Catechol gives a green coloration ; this colour 
changes to violet, then to red on adding sodium carbonate or ammonia. 
Resorcinol gives a deep violet coloration. Quinol on boiling with ferric 
chloride yields quinone with its peculiar irritating smell. 

(2) With bromine water : Resorcinol gives a crystalline precipitate 
of tribromoresorcinol. 

(3) Catechol and quinol reduce ammoniacal silver nitrate. 

(4) Catechol and quinol reduce Fehling's solution. 

(5) Solutions of catechol and quinol, made alkaline with caustic 
soda, turn brown, firstly at the surface but on shaking throughout the 
whole solution. This is due to absorption of oxygen and oxidation. 

(6) With Millon's reagent : Quinol gives a yellow colour and then 
a yellow precipitate which becomes red on heating. 

Guaiacol and Veratrol. 

OCH 3 OCH 3 These compounds are methyl deriva- 

0/\ tive's of catechol and are contained in 

j | OCH 3 natural substances. Guaiacol, like o- 
L ) hydroxy-phenols, gives a coloration with 

ferric chloride ; veratrol gives no colora- 
tion. 



AROMATIC COMPOUNDS 263 

Phthalic Acids. 

The three phthalic acids, or benzene dicarboxylic acids, 
COOH COOH COOH 

AcOOH A 

S J S^^/COOH 

COOH 

Ortho, or Meta, or Para, or 

phthalic acid, isophthalic acid, terephthalic acid, 

are obtained by oxidising the xylenes with nitric acid, or the toluic 
(methyl benzoic) acids with permanganate. Phthalic acid results from the 
oxidation of naphthalene. . 

They are crystalline solids resembling benzoic acid. Phthalic acid on 
heating is converted into phthalic anhydride, 

rV\o, 

IJco/ 

a substance used in making fluorescein, eosin and other dyes. 



Nitrotoluenes, C (3 H 4 <' 

X N0 2 . 

The o- and p-compounds are obtained by nitrating toluene ; the m-com- 
pound is obtained by indirect methods. They are all solids. 



Aminotoluenes, or Toluidines, C 6 H 4 \ 

' NH 2 . 

o- and p-Toluidine are obtained by reducing o- and p-nitrotoluene ; 
o-toluidine is an oil, p-toluidine is a crystalline solid. The m-compound is 
obtained in a similar way and is an oil. 

They yield diazonium salts with nitrous acid and behave generally like 
aniline. 



Nitranilines, C 6 H 4 \ 

X NH 2 . 

m-Nitraniline is obtained by the partial reduction of m-dinitrobenzene 
with alcoholic ammonium sulphide. The o- and p-nitraniline cannot be ob- 
tained by nitrating aniline, but are obtained by nitrating acetanilide and 
saponifying the nitro-acetanilides. 

xCH 3 
Toluene-Sulphonic Acids, C fl H 4 <T 

X SCXH. 



The o- and p-compounds result by sulphonating toluene, the o-compound 
being the chief product. They yield the cresols on fusion with potash. 

Sulphanilic Acid, C 6 H 4 < 



X SO 3 H. 



The p-compound is obtained by heating aniline sulphate at 200 for 
some hours. 

It is a colourless crystalline solid easily soluble in hot water, very little 
in cold. It does not behave as a base, but the amino group can be diazotised. 
It is used largely in making dyes. 



264 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

,CH 3 
Cresols, C 6 H 4 <( 

X OH. 

The three cresols are contained in the acid fraction of coal tar with 
phenol. Their separation is difficult to effect and they are prepared 
from the toluidines, or toluene sulphonic acids, by the methods given 
under aniline and diazonium salts and phenol. 

p-Cresol occurs in urine in combination with sulphuric acid and is 
isolated together with phenol by the methods given on p. 245. It is 
also a product of the putrefaction of proteins and arises from the 
amino acid, tyrosine. The cresols are crystalline solids; o-cresol 
melts at 31, m-cresol at 5, p-cresol at 36. They resemble 
phenol very closely in properties. 

As phenols they react 

(1) with ferric chloride ; 

(2) with bromine water ; 

(3) with nitric acid ; 

(4) with Millon's reagent. 



Toluic Acids, C 6 H/ 

X COOH. 

These three acids result by oxidising the xylenes with dilute nitric acid. 
The o- and p-acids are most readily prepared from the toluidines by Sand- 
meyer's reaction with cuprous cyanide (p. 250). 
They are solids resembling benzoic acid. 

NH 2 
Anthranilic Acids, C 6 HX 

X COOH. 

The o-acid was first obtained by oxidising indigo and is a colourless 
crystalline solid melting at 144; it loses carbon dioxide on heating and 
yields aniline. 

/SO 3 H 
Sulpho- Benzoic Acids, C 6 H 4 <^ 

X COOH. 

The o-acid is of interest as saccharin is prepared from it. o-Sulpho- 
benzoic acid is prepared by oxidising o-toluene-sulphonic acid. The am- 
monium salt on heating loses ammonia and gives the imide, saccharin : 
/CH 3 /COOH /COONH 4 /CO \ 

Cu / .. p TJ / C VS 'S ^PH/ NMH 

6 H 4\ ->^6"4\ -> U bW 4 , -C 6 H' ,JNhl 

X S0 3 H \S0 3 H X S0 3 NH 4 X SO/ 

Saccharin. 
/C0\ 
- C 6 H 4 / \N . Na 

Sodium Salt. 

Saccharin is a white crystalline solid melting at 224 and it is only slightly 
soluble in water. It forms a sodium salt, which dissolves easily in cold 
water. The sodium salt, containing 2H 2 O and crystallising in large plates, 
is generally used as sweetening agent. The sweetness of saccharin is about 
500 times greater than that of cane sugar. 



AROMATIC COMPOUNDS 265 

/OH 
Saligenin, or Salicylic Alcohol, C 6 H 4 ^ 

CH 2 OH. 

Saligenin occurs as the glucoside, salicin, in the bark of the willow 
tree. The glucoside, on hydrolysis, gives glucose and saligenin. 

It is generally prepared by reducing salicylic aldehyde with 
sodium amalgam and dilute alcohol. 

Saligenin is a crystalline solid which melts at 82 and is easily 
soluble in water. It is o-hydroxy-benzyl alcohol. 

As it contains a phenolic group in the a-position it gives a blue- 
violet colour with ferric chloride. It forms alkali salts with alkaline 
hydroxides and behaves like a phenol. It also behaves like a primary 
aliphatic alcohol and is converted on oxidation into salicylic aldehyde 
and salicylic acid. 

/OH 
Salicylic Aldehyde, C 6 H 4 < 

X CHO. 

o-Salicylic aldehyde is found in certain volatile oils from plants. 

It can be prepared by oxidising saligenin with potassium bichro- 
mate and sulphuric acid. 

It is generally prepared by Reimer's reaction : a mixture of phenol, 
chloroform and caustic potash is heated under a reflux condenser : 

C 6 H 5 OH + CHCI 3 + 4 KOH = C 6 H 4 (OK)CHO + sKCl + 3H,O. 
The solution is acidified after distilling off the chloroform and distilled 
with steam ; phenol and o-salicylic aldehyde pass over. The distillate 
is extracted with ether and the aldehyde converted into the bisulphite 
compound ; this is decomposed with sodium carbonate, the salicylalde- 
hyde extracted with ether and distilled. 

p-Hydroxybenzaldehyde is also formed in the reaction, but is not 
volatile with steam. 

Salicylic aldehyde is an oily liquid boiling at 196 with characteristic 
aromatic smell : it gives a violet colour with ferric chloride. 

/OH 
Salicylic Acids, C 6 H 4 ( 

\COOH. 

The chief of the hydroxy-benzoic acids is the o-compound or sali- 
cylic acid, which occurs in the flowers of Spircza ulmaria and in the 
form of its methyl ester in oil of winter green. 

Salicylic acid was formerly prepared (i) by the hydrolysis of oil of 
winter green ; (2) by oxidising salicylic alcohol ; (3) by the action of 
nitrous acid on anthranilic acid. 



266 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

It is now prepared almost entirely from phenol : Sodium phenate 
is heated in carbon dioxide ; sodium phenyl carbonate is formed : 

C 6 H 5 ONa + CO 2 = C 6 H 5 . O . COONa. 
On heating, this is changed into sodium salicylate : 



2 C 6 H 5 . O . COONa = C 6 H 5 OH + C 6 H 4 

X COONa; 
half the phenol used is recovered. 

If the sodium phenyl carbonate be heated under pressure at 120- 
140, it yields the acid salt of salicylic acid : 

OH 
C 6 H 5 . O . COONa = C 6 H 4 / 

\COONa. 

Salicylic acid forms colourless needles which melt at 155. It is 
not easily soluble in cold water, but dissolves readily in hot water, alco- 
hol, ether and other organic solvents. It is an antiseptic, like phenol, 
and is used for preserving food-stuffs and also largely in medicine, 
more frequently in the form of its derivatives, aspirin and salol. 

Salicylic acid behaves as an acid and as a phenol; it dissolves in 
caustic alkali forming a salt with the carboxyl and phenolic groups ; 
in alkali carbonates forming a salt only with the carboxyl group. 

Reactions. 

(1) On heating it melts and sublimes, but on further heating it loses 
carbon dioxide and yields phenol. 

The formation of phenol takes place more readily on heating with 
soda lime. 

(2) As it is a phenol it gives the reactions : 

(a) With ferric chloride a violet colour. This is discharged by 
mineral acids, but not by acetic acid. It may thus be distinguished from 
phenol. 

Note. Only the hydroxy acid containing the OH group in the 
ortho position gives a violet colour with ferric chloride. 
The m- and p-compounds do not give colours with ferric 
chloride. 

() With bromine water a yellowish-white precipitate of dibromo- 
and tribromosalicylic acids. 

(c) With nitric acid a yellow colour intensified on making the 
solution alkaline with ammonia. 

(d] With Millon's reagent a red colour on heating. 



AROMATIC COMPOUNDS 267 

Aspirin is acetyl-salicylic acid. It is prepared by heating salicylic 
acid with acetyl chloride or acetic anhydride : 

OH ,Q.OC.CH 3 

C 6 H / + CH 3 COC1 = HC1 + C 6 H 4 S 

\COOH ^-COOH. 

On hydrolysis by acids or alkalies, it yields acetic and salicylic acids. 

Salol is phenyl salicylate. It is prepared by heating a mixture of 
sodium phenate and sodium salicylate with phosphorus oxychloride : 

xOH .OH 

2C 6 H 4 <^ + 2 C 6 H 5 ONa + POC1 3 = aNaCl + NaPO 3 + 2C 6 H 4 <^ 

X COONa X COOC 6 H 5 . 

On hydrolysis, it yields phenol and salicylic acid. 

If the hydrolysis of aspirin and salol be effected with caustic soda 
the sodium salts are obtained ; on acidifying with sulphuric acid, sali- 
cylic acid is precipitated and the acetic acid or phenol can be isolated 
by steam distillation. 

/OH 
Tyrosine, C fi H/ 

' X CH 2 .CH(NH 2 ).COOH. 

Tyrosine or p-hydroxyphenylalanine is a constituent of proteins 
from which it was first obtained by Liebig in 1846 who fused cheese 
(TU/JO?) with, caustic potash. It has since been isolated from the pro- 
ducts of hydrolysis of most proteins. It is found in the liver and 
other organs in certain diseases in considerable quantities ; in minute 
amounts it is present in all tissues. 

Preparation. 

The best yield of tyrosine is obtained from silk ; silk is hydrolysed by boiling 
with concentrated hydrochloric acid for 5 or 6 hours, the solution is evaporated 
to remove hydrochloric acid, the greater part of the remainder is removed as 
cuprous chloride by adding cuprous oxide, and on neutralising the solution 
tyrosine separates out. It may also be obtained by hydrolysing silk and 
other proteins with six times their amount of 30 per cent, sulphuric acid, 
removing the sulphuric acid with baryta and concentrating the solution. 
Tyrosine separates out and is recrystallised. 

It is most easy to prepare tyrosine by the tryptic digestion of caseinogen. 
100-500 gm. of caseinogen are dissolved in 2-10 litres of -4 per cent, sodium 
carbonate, 1-2 gm. of dried pancreas (trypsin) are added and 1-2 per cent, of 
toluene or chloroform are shaken up with the solution to prevent putrefac- 
tion. The solution is kept at 35 for 7-10 days. It becomes cloudy with the 
separation of tyrosine which gradually settles out. The filtrate gives the 
reactions for tyrosine and on evaporation and on cooling deposits a further 
quantity. Almost pure tyrosine may be obtained from the first deposit by 
dissolving it in -iN hydrochloric acid, boiling with charcoal to decolorise it 
and exactly neutralising the clear solution with ammonia. Pure tyrosine may 
be obtained from the second deposit by the same treatment repeated two or 
three times. 



268 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



Properties. 

Tyrosine is a colourless crystalline solid, which is soluble with 
difficulty in cold water, more easily in hot. It dissolves readily in 
dilute acids or alkalies from which it separates on neutralising the 
solution. 

If a small quantity of tyrosine be dissolved in a drop of ammonia 
on a glass slide and the ammonia be allowed to evaporate, the tyrosine 

crystallises out in charac- 
teristic bunches of fine 
needles (Fig. 47). It is 
insoluble in alcohol and 
ether. 
Reactions and Tests. 

(1) Its crystalline form 
is very characteristic. 

(2) It gives a yellow 
colour on heating with 
nitric acid ; this becomes 
orange on making alkaline 
with ammonia. 

(3) It gives a red 
colour, even in extreme 
dilution, on heating with 

Millon's reagent. 

(4) Pirias test. 3 drops of concentrated sulphuric acid are put 
on a little tyrosine in a dry test tube and it is placed in the boiling 
water-bath for half an hour. The red liquid is diluted with 10 c.c. of 
water and neutralised with barium carbonate. The filtrate (from 
BaSO 4 ) on evaporation* to a small volume gives a violet colour with 
2-3 drops of ferric chloride, showing the presence of phenol. 

(5) When boiled with copper carbonate it gives a blue copper salt 
like aliphatic amino acids. 

(6) Mbrner's test. A solution of tyrosine gives a green colour on boiling 
with a solution of formalin in sulphuric acid (i vol. formalin, 45 vols. water, 
55 vols. cone. H 2 SO 4 ). 

(7) A wine-red colour is formed if tyrosine be added to 3 or 4 drops of 
formalin in 5 c.c. of concentrated sulphuric acid. The liquid becomes green 
on adding double the volume of glacial acetic acid and boiling (Denigks). 




AROMATIC COMPOUNDS 269 

Tyramine. 

OH Tyramine or p-hydroxyphenylethylamine is a base 

formed from tyrosine by putrefaction. It occurs in ergot 
of which it is one of the active principles. 

The decomposition of tyrosine by putrefaction is ex- 
actly similar to that of phenylalanine and takes place in the 
following stages : 




/OH /OH /OH 

CTT / - p tr / $. f XJ / > 

6"4\ ^6 n 4\ ^"tv 

\CH 2 .CH 2 .COOH X CH 2 .CH(NH 2 )COOH \CH 2 .CH 2 .NH a 

p-hydroxyphenyl propionic Tyrosine. Tyramine. 

acid. 

I 

/OH /OH 

C 6 H 4 ( C 6 H 4 <- C B H 5 .OH. 

X CH 2 .COOH X CH 3 

p-hydroxyphenyl acetic p-cresol. Phenol, 

acid. 



The two following other disubstitution products of benzene occur 
in nature : 

(1) Cymene, or methylisopropyl benzene, which occurs in numer- 

ous essential oils (p. 312) and can be obtained by 
heating camphor with phosphorus pentoxide, by 
heating turpentine with concentrated sulphuric acid 
and by reducing carvacrol and thymol with phos- 
phorus pentasulphide. 
CH g CH 3 . jt j s a colourless liquid which boils at 175-176 

and has a sp. gr. of -8722 at o. It yields p-toluic acid and terephthalic 

acid on oxidation. 

(2) Anethole, or p-methoxyphenylpropylene, is the principal 

OC^TT 

/^ 3 constituent of oil of aniseed. It yields p-methoxy- 

benzoic acid, or anisic acid, on oxidation with 
chromic acid, but anisic aldehyde or p-methoxy- 

benzaldehyde on oxidation with bichromate and 
CH 

|| sulphuric acid. Anisic aldehyde on reduction 

^ with sodium amalgam and alcohol is converted 

CH, into anisic alcohol. 



270 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

TRISUBSTITUTION DERIVATIVES OF BENZENE. 

Trisubstitution derivatives of benzene exist in the three forms : 






Jx 

X 

Vicinal. Asymmetric. Symmetric. 

and the number of isomers is very large. Mention can only be made 
of those which occur naturally, or are prepared from natural sources. 
The trihydric phenols, 

OH OH OH 






OH 

OH 
Pyrogallol. Hydroxyquinol. Phloroglucinol. 

Pyrogallol, or pyrogallic acid, is obtained by heating gallic acid 
at 210 until carbon dioxide is no longer evolved : 
C 6 H 2 (OH) 3 . COOH t C0 2 + C 6 H 3 (OH) 3 . 

It is a colourless crystalline solid, which melts at 1 1 5. It is easily 
soluble in water, but less in alcohol and ether. It will be noticed 
that the solubility of phenols in water increases with the number of 
hydroxyl groups. 

Hydroxyquinol is obtained by fusing quinol with potash. It dis- 
solves easily in water and melts at 140. 

Phloroglucinol results from the fusion of numerous resins with 
caustic potash. It is prepared by fusing resorcinol with caustic potash. 

Phloroglucinol is a crystalline solid containing two molecules of 
water and melting at 218. It is very soluble in CH s 

water and is also soluble in alcohol and ether. In OC ( Nco 
most respects it behaves as a trihydroxyphenol, but H c \ Jen 
it forms an oxime and probably has also a ketonic co 

structure. 

Reactions. 

(1) With ferric chloride, pyrogallbl gives a deep-blue coloration; 
hydroxyquinol gives a greenish-brown colour which changes to blue, 
on adding sodium carbonate and then to red ; phloroglucinol gives 
a blue violet coloration. 

(2) In alkaline solution in contact with air, they absorb oxygen and 
the solution becomes brown. Pyrogallol is consequently used for ab- 
sorbing oxygen. 

(3) They all reduce Fehling's solution and ammoniacal silver 
nitrate. 



AROMATIC COMPOUNDS 271 

Orcinol is dihydroxy-toluene. Thymol and carvacrol are deri- 
vatives of cymene : 

CH, CH 3 

HOV JOH k /OH 






CH 3 CH g 
Orcinol. Thymol. Carvacrol. 

Protocatechuic acid, or catechol-carboxylic acid, results from the 
fusion of numerous resins, e.g. gum benzoin, 
catechin resin, with caustic potash. It is pre- 
pared by heating catechol with water and am- 
monium carbonate to 140. 

COOH It is a colourless crystalline solid melting 

at 199 and dissolves readily in water. 

With ferric chloride its solution gives a green colour which changes to 
blue and then to red on the addition of very dilute sodium carbonate. With 
ferrous sulphate a violet colour is given. It is precipitated from solution by 
lead acetate. 

Veratric acid, the dimethyl ether of protocatechuic acid, occurs in the 
seeds of Veratrum sabadilla, together with veratrine. 

Vanillin is obtained from coniferyl alcohol by oxidation with chromic 
acid. Vanillin is the sweet-smelling constituent of the vanilla bean. 

Coniferyl alcohol occurs in the glucoside, coniferin, and is obtained from 
it by hydrolysis. 

OCH 3 OH OH 

OCH, ( 10CH S /NoCBL 




0' 



COOH CHO 

CH = CH . CH 2 OH 
Veratric acid. Vanillin. Coniferyl alcohol. 

Homogentisic acid, or quinol-acetic acid, is found in the urine in 

O J-T 

the rare disorder known as alkaptonuria. 
^]CH 2 .COOH Its presence is first shown by the urine 

turning brown and black on standing, or by 

its reducing power. 
It is a white crystalline solid melting at 146-147. 
As a derivative of hydroquinone it reacts with 

(1) ferric chloride ; 

(2) Fehling's solution or ammoniacal silver nitrate ; 

(3) turns black in the air in alkaline solution. 



272 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Adrenaline is a derivative of catechol and is the active principle 

9*" 1 of the adrenal gland from which it is prepared as 

f \OH well as by synthesis. 

\^J The natural substance is laevorotatory : the syn- 

| thetical is inactive : the dextrorotatory form has only 

CHOH .. t . , . . . '. 

I a very slight pharmacological action in comparison 

CH 2 .NH(CH 3 ). w jth the laevorotatory or natural form. 

Preparation. 

Abel's method is probably the most convenient one for preparing adrena- 
line. To minced suprarenal glands in a series of flasks is added with 
thorough shaking an equal weight of 3-5 per cent, trichloracetic acid in 
alcohol. After 12 hours the mass is filtered. The filtrate is concentrated 
to about one-fiftieth and again filtered. Concentrated ammonia is added to 
the filtrate until the liquid just smells perceptibly of ammonia. Adrenaline is 
precipitated, filtered off, washed with water, alcohol and ether. A yield of 
about '2 per cent, is obtained. A further -i per cent, can be obtained by 
extracting the mass again with trichloracetic acid. It is recrystallised by 
solution in alcohol containing oxalic acid and precipitation by ammonia. 

Properties. 

Adrenaline is a colourless crystalline solid melting at 211-212. It is not 
easily soluble in cold water, but more readily in hot water and is not soluble 
in most organic solvents. 

It is a strong base and dissolves in mineral acids ; as a phenol it dissolves 
in caustic alkalies, but not in carbonates or ammonia. Its aqueous solutions 
are not stable, but turn pink in the air. 

Reactions. 

(1) Ferric chloride in neutral or faintly acid solution gives a green colora- 
tion which on the careful addition of very dilute alkali changes to violet, red- 
violet and red. 

(2) Oxidising agents and air produce a pink colour : potassium persulphate 
added up to *i per cent, to the solution of adrenaline gives a colour at a 
dilution of i in 5,000,000 (Ewins). 

(3) Adrenaline gives an intense blue colour with Folin's phosphotungstic 
acid reagent for uric acid (see p. 557). One part in 3,000,000 parts of water 
gives a reaction with this reagent. 

A full account of adrenaline is given in Barger's " Simpler Natural Bases ". 

Estimation. 

The estimation of adrenaline in the suprarenal gland is most easily 
effected by means of the colour reaction with phosphotungstic acid as shown 
by Folin, Cannon and Denis in 1913. 

The weighed gland is ground up in a mortar with sand and -iN hydro- 
chloric acid and rinsed into a conical flask. 15 c.c. of 'iN acid and 45 c.c. 
of water are used for every 2 gm. of gland. The solution is raised to boiling ; 
there is no coagulation, but on adding 5 c.c. of 10 per cent, sodium acetate 
solution for every 15 c.c. hydrochloric acid and again heating to boiling, 
coagulation of the protein occurs. The mixture except the sand is transferred 
to a 100 c.c. measuring flask and diluted to the mark. The solution is 
filtered or centrifuged. 5 c.c. of the filtrate are pipetted into a 100 c.c. 






AROMATIC COMPOUNDS 273 

measuring flask and at the same time i c.c. of the standard uric acid 
solution (below) into another 100 c.c. flask. To each are added 2 c.c. of the 
uric acid reagent and 20 c.c. of saturated sodium carbonate solution. They are 
allowed to stand for 2 to 3 minutes, diluted to the mark, mixed, and the colours 
compared in a Duboscq colorimeter, the uric acid tint being placed at 20 mm. 

Adrenaline gives exactly 3 times the colour that uric acid does : the 
readings in the colorimeter are proportional 

The uric acid standard contains i mgm. uric acid dissolved in i c.c. of 
4 per cent, lithium carbonate solution and is prepared by dissolving 250 
mgm. in 25-50 c.c. of water + 25 c.c. Li 2 CO 3 solution by shaking for an 
hour and diluting to 250 c.c. in a measuring flask. The other standard 
solution which is permanent may also be used (see p. 557). 

TETRASUBSTITUTION PRODUCTS OF BENZENE. 

Picric acid, which is so easily formed from phenol, is trinitrophenol. 

N0 




OH 

It is a tetrasubstitution product of benzene. 

Theoretically a large number of tetrasubstitution produces of ben- 
zene are capable of existence, but the number of natural compounds 
which are included here is very small. 

Gallic Acid. 

OH 

Gallic acid is present in gall nuts, tea and 

other plants. 
It "is prepared by the hydrolysis of tannin. 

It crystallises in silky needles which melt at 22O. It is not very 
soluble in cold water, but readily in hot water. It dissolves in alkalies 
and the solution turns brown in the air. It resembles pyrogallol in its 
reactions with ferric chloride, Fehl ing's solution, etc. 

It does not precipitate gelatin and is not precipitated by lead acetate. 
Tannic Acid or Digallic Acid. 

This acid, the anhydride of 

Of \ OH gallic acid, occurs in gall nuts, 
OH HOOC\ /OH sumach and other kinds of bark. 
OH It may be prepared by heating 

gallic acid with phosphorus oxychloride. Its constitution is not 
definitely known and it is sometimes referred to as tannin, but the 
synthetical product obtained from gallic acid has not the same pro- 
perties as natural tannin which contains digallic acid in its constitution. 
The natural product is here referred to as tannin, the synthetical as 
digallic acid. 

Two other digallic acids are known. 

18 



TANNINS. 

The various kinds of tannin which can be extracted from gall nuts, 
sumach, pomegranate, oak bark, kino, etc., seem to belong to two main groups 
according to the reactions which they give with ferric chloride, bromine 
water, etc, They appear to contain a pyrogallol nucleus, or a catechol 
nucleus, thus : 

Pyrogallol variety Catechol variety 

Ferric salts . . Dark blue Greenish-black. 

Bromine water . No precipitate Yellow or brown precipitate. 

Leather . Produce a " bloom " No bloom. 

Cone. H 2 SO 4 Dark red ring at junction of liquids. 

Some of the tannins of the catechol variety, gambier and cutch, appear 
to contain a phloroglucinol nucleus, since a pine shaving moistened with an 
extract and treated with concentrated hydrochloric acid gives a red or purple 
stain which is characteristic of phloroglucinol. 

Constitution. 

As yet the constitution of the various tannins has not been definitely- 
determined, but it appears from the work of Emil Fischer and his pupils that 
the tannins are esters of glucose with gallic acid, digallic acid and other 
complex phenolic acids. 

Fischer and his pupils have definitely shown that the tannin from 
Aleppo, Chinese and other galls contains 7-8 per cent, of glucose. The 
tannin was carefully purified; the purified product was hydrolysed by 
dilute sulphuric acid and the glucose identified and estimated. If the 
former supposition that tannins were glucosides were correct, a larger 
amount of glucose would result. The yield of glucose corresponds very 
closely to that which would be obtained if tannin were the pentadigalloyl 
ester of glucose. This constitution, though not absolutely proved, has been 
made extremely probable by synthesis. Fischer has prepared esters of glucose 
with a series of these phenolic acids and they possess the main properties of 
the tannins. 

Tannins would therefore be constituted thus : 



/h 

y c: 

Nv CHO -t 

<CHO 1 

where t represents a complex acid like tannic acid or digallic acid. 

These compounds correspond to the fats in which glycerol is the basis 
and various fatty acids are combined with it. 

274 



TANNINS 275 

This constitution not only allows for a large number of varieties of tannins 
where t is the same throughout the molecule, but also where t is different. 1 

In addition to the interest attached to the syntheses of a tannin there is 
another interest. Some of the synthetical tannins possess very high molecular 
weights; one of them has a higher molecular weight, 2051, than any other 
known synthetical compound, and has largely exceeded the figure of 1213, 
which is the molecular weight of the synthetical octadecapeptide (p. 363). 

Oak-gall Tannin. 

Tannin is prepared from finely powdered gall nuts by extraction with a 
mixture of 1 2 parts of ether and 3 parts of alcohol ; 1 2 parts of water are 
added and the mixture thoroughly shaken. The lower aqueous layer is 
separated and evaporated. The tannin may be purified by boiling with char- 
coal. Or the powdered gall nut is heated under a reflux condenser with a 
mixture of 30 parts of ether, 5 parts of water and 2 parts of alcohol. 
The lowest layer contains about 30 per cent, of tannin which is obtained 
as above. 

Tannin is an amorphous powder ; the pseudo-crystalline appearance of 
some kinds of tannin results from the method of its preparation, a syrupy 
solution being drawn up into threads, which are dried and broken. It is 
almost colourless when pure. It dissolves easily in water and its solution is 
acid in reaction to litmus and has an astringent taste. It is soluble in alcohol 
and glycerol, but very little soluble in ether, chloroform, ligroin. It is pre- 
cipitated from solution by hydrochloric acid and sulphuric acid ; it is soluble 
in alkalies and the alkaline solution turns brown on exposure to the air. 

It is decomposed on heating at 160-2 15* into pyrogallol, gallic acid and 
other products. 

Reactions. 

(1) With ferrous sulphate containing no ferric salt there is no change, 
but on exposing to the air the solution darkens. 

(2) With ferric chloride a blue-black colour or precipitate is formed (ink). 

(3) It is precipitated from solution by gelatin, hide powder (leather and 
other proteins). 

(4) It precipitates alkaloids from solution. 

(5) Dilute iodine solution gives a pink colour. 

(6) Potassium cyanide gives a reddish-brown colour, which changes to 
brown ; on shaking with air the red tint appears again. 

(7) Lime water gives a grey precipitate. 

(8) It is precipitated by lead acetate or by lead nitrate. 

1 A summary of the researches on tannins is given in the " Ber. deutsch. Chem. Ges.," 
, 4<5. 3253- 



18 



HETEROCYCLIC COMPOUNDS. 

Numerous compounds exist containing rings or nuclei composed 
of carbon atoms and atoms of other elements, especially oxygen, 
sulphur and nitrogen. These ring compounds are grouped together as 
the heterocyclic compounds. 

Some of the heterocyclic compounds are closely connected with the 
aliphatic series of compounds, e.g. the anhydrides of dibasic acids, such 
as succinic anhydride ; the ^-lactones and other lactones ; the imides 
from ammonium salts of dibasic acids, such as succinimide ; the 
polymers of the aldehydes, such as trioxymethylene, paraldehyde ; 
and of cyanic acid, namely, cyanuric acid. Creatinine may also be 
placed in this group. In these compounds the ring is formed easily 
and it is easily ruptured. They are therefore usually considered as 
aliphatic compounds. Other heterocyclic compounds possess a more 
stable ring and they resemble the aromatic substances very closely in 
their properties. They include pyridine, quinoline and their deriv- 
atives, the alkaloids. 

Intermediately between these two classes there are other heterocyclic 
ring compounds which do not possess the chief properties of aromatic 
compounds in forming nitro- and sulphonic acid derivatives, but they 
possess a ring which is comparatively stable and is not easily broken 
down. In this group are included the cyclic ureides, pyrimidine and 
purine and their derivatives, pyrrole, thiophene, furfurane. 

As in the carbocyclic compounds the rings containing 5 atoms 
and 6 atoms are the most stable. 



276 



UREIDES. 

In the same way as ammonia forms amides with acids so also 
does urea form ureides. Ureides are therefore derivatives of urea with 
acid radicles. 

A. UREIDES OF MONOBASIC ACIDS, e.g. acetyl urea. 

These are obtained by the action of acid chlorides, or acid anhydrides, 
upon urea : 

CH 3 . CO . Cl + H 2 N . CO . NH 2 = HC1 + CH 3 . CO NH . CO . NH 2 . 

They are solid compounds. Acetyl urea forms long silky needles which 
melt at 214 and which are not easily soluble in cold water or alcohol. 
They are neutral in reaction and do not form salts with acids. 

Like amides they are easily decomposed by hydrolysis, especially by 
alkalies and are converted into their constituents : 

CH 3 . CO NH . CO . NH 2 + H 2 O = CH 3 . COOH + H 2 N . CO . NH 2 . 

The urea may be decomposed into ammonia and carbon dioxide. 
Diacetyl urea, CH 3 . CO NH . CO . NH CO . CH 3 , is formed by the 
action of carbonyl chloride upon acetamide. 

B. UREIDES OF HYDROXY AND ALDEHYDE ACIDS. 

Two classes of ureides are formed with these acids, an open chain 
compound such as hydantoic acid, and a closed chain compound (a 
cyclic ureide) such as hydantoin. Both these compounds are ureides 
of glycollic acid : 

CH, HN CH HN 



CO HN 
COOH NH, 

Hydantoic acid or Hydantoin. 

glycoluric acid. 

Hydantoic Acid. 

Hydantoic acid is obtained from hydantoin by boiling it with baryta 
water. It may be synthesised from glycine sulphate and potassium cyanate, 
a reaction analogous to the synthesis of urea : 
CH,.NH,. HOCN CH 2 .NH 

\ 
CO 

COOH COOH NH 2 . 

Hydantoic acid is a white solid easily soluble in water and alcohol. It 
is converted into glycine, carbon dioxide and ammonia on heating with 
hydriodic acid. 

277 



278 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Hydantoin. 

Hydantoin is obtained by heating allantoine and alloxanic acid, two oxida- 
tion products of uric acid, with hydriodic acid. It is prepared synthetically 
by heating bromacetyl urea with alcoholic ammonia : 
JSfH . OC . CH 2 Br ,NH OC 

CO = CO 

\ V + HBr - 

X NH 2 \NH CH 2 

It is a white crystalline solid which melts at 216. 

Allantoine. 

Allantoine is a combination of 2 molecules of urea with glyoxylic 
acid, and is derived from the hypothetical dihydroxy-acetic acid : 



i, 



HO CH OH NH CH HN 

:OOH co co 

I I 

NH 2 CO HN 
Allantoine. 



Allantoine was first found in the allantoic fluid of calves ; it has 
since been found in the urine of various animals, ox, dog, monkey, 
rabbit, sheep, and also in various organs of animals. It has been 
found in plants. Allantoine is an oxidation product of uric acid, and 
arises in those animals in which allantoine is present in the urine by 
the oxidation of uric acid. 

Preparation. 

(1) By the Oxidation of Uric Acid. 

too gm. of uric acid are suspended in 1500-2000 c.c. of water 
and dissolved by the careful addition of caustic soda in small quantities 
at a time. The alkaline liquid is treated with a concentrated solution of 62 
gm. of potassium permanganate and well stirred. Manganese dioxide is 
formed and the permanganate decolorised. This takes place rapidly and is 
complete in about an hour ; it must be tested for by filtering a sample if 
necessary. As soon as the solution is decolorised, it is filtered from manganese 
dioxide, acidified with acetic acid and evaporated (best in vacua) until it 
crystallises. The crystals are recrystallised from hot water. 

(2) From the Urine of Dogs, etc. 

The urine is treated with a concentrated solution of phosphotungstic acid 
to remove pigments, etc. The excess of phosphotungstic acid is removed 
with lead carbonate and excess of lead with hydrogen sulphide and then the 
hydrogen sulphide is removed. Chlorides are removed with silver acetate, 
excess of silver with hydrogen sulphide and the ^hydrogen sulphide by a 
current of air. The allantoine is precipitated by adding a -5 per cent, 
solution of mercuric acetate in sodium acetate. The precipitate is filtered 
off, washed and decomposed with hydrogen sulphide. Allantoine crystallises 
out on evaporation of the solution. 



UREIDES 279 

Properties. 

Allantoine forms shining colourless prisms which have no taste or smell 
and are neutral in reaction to litmus. It is not easily soluble in cold water 
(i in 1 60 parts) or cold alcohol but easily soluble in hot water and hot 
alcohol ; on heating it turns brown at 220 and melts with decomposition at 
231. 

Allantoine forms compounds with metals : it is precipitated by ammoniacal 
silver solutions : the precipitate is soluble in ammonia; it is also precipitated 
by salts of lead, copper and mercury (see above). It reduces Fehling's 
solution on prolonged boiling. 

Allantoine is decomposed by hydrolysis with acids or alkalies, giving urea 
(or ammonia and carbon dioxide) and acetic acid and oxalic acid. It is also 
decomposed by hypobromite solution with evolution of nitrogen. 

Tests and Identification. 

In order to identify allantoine it must be isolated from solution, either as 
such or as its silver compound, and analysed ; the silver compound contains 
4 '73 P er cent, of silver. The presence of glyoxylic acid may be shown (i) 
by boiling with alkali and testing the solution by adding some dilute indole 
solution and pouring sulphuric acid under the mixture ; a red ring is formed 
at the junction of the liquids. (2) By boiling with about 15 percent, soda 
for 1-2 minutes, cooling, acidifying with acetic acid and testing for oxalic 
acid with calcium chloride. 



C. UREIDES OF DIBASIC ACIDS. 

Some of these ureides were first obtained byi the oxidation of uric 
acid and were the fundamental substances from which its constitution 
was determined. Others were prepared synthetically in the study of 
these ureides and in the attempts to prepare uric acid synthetically. 

Parabanic Acid and Oxaluric Acid. 

CO NH, CO NH, 

)CO X CO NH 2 . 

CO NH/ COOH 

Parabanic acid, or Oxalylurea, is formed on oxidising uric acid 
with nitric acid: It is prepared synthetically by the action of phosphorus 
oxychloride upon a mixture of urea and oxalic acid. 

Parabanic acid is a white crystalline substance, which is soluble in water 
and alcohol. 

Oxaluric acid is obtained by the action of bromine upon parabanic 
acid and by the action of water upon salts of parabanic acid. 

Oxaluric acid is a crystalline powder soluble in water with difficulty and 
is present in small quantities in urine. 

Both parabanic acid and oxaluric acid are decomposed by hydrolysis by 
boiling with water, acids, or alkalies into urea and oxalic acid. 



280 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



A series of ureides is derived from acids containing three carbon 
atoms in their molecule : 



CO NH 

CH 2 CO 

1 1 
ro NH 


CO NH 
CHOH CO 
CO NH 


Malonyl urea, 
or 
barbituric acid. 

ro NH 


Tartronyl urea, 
or 
dial uric acid. 

CO NH 


CH . NO, CO 

1 1 


CH . NH 2 CO 



CO- 
CO 
CO- 



-NH 
CO 
-NH 



Mesoxalyl urea, 

or 
alloxan. 



-NH 



CO 

C:NOH 
CO 



-NH 

i 

-NH 



CO 

Nitromalonyl urea, Aminomalonyl urea, Isonitrosomalonyl urea, 

or or or 

dilituric acid. uramil. violuric acid. 



CO 

C(C 2 H 5 ) 2 



co- 



-NH 

CO 

-NH 



Diethylbarbituric acid, 

or 
veronal. 



CO N H 

CH.NHCO.NH 2 CO 

CO NH 

Carbamidomalonyl urea, 

or 
pseudo-uric acid. 



Uric acid, the principal ureide biologically, is derived from a 
hypothetical acid, C(OH) 2 = C(OH) COOH, containing 3 carbon 
atoms and 2 molecules of urea ; it has the formula : 

NH C=o 

CO C NH\ 

I II >CO. 

NH C NH/ 

Alloxan. 

Alloxan is the most interesting of this series of compounds as it 
can be transformed into the others ; it forms the central point in our 
knowledge of the constitution of uric acid and other purines (p. 286). 

Preparation. 

Alloxan is produced by the careful oxidation of uric acid with nitric acid, 
bromine or chlorine ; also by the oxidation of xanthine. * 

It is easy to prepare alloxantin from uric acid and to prepare alloxan 
from alloxantin. 

Alloxantin may be prepared as follows : 10 gm. of uric acid are covered 
with 20 c.c. of water and 20 gm. of concentrated hydrochloric acid and 
heated to 35 ; 2-5 gm. of powdered potassium chlorate are gradually added 
with continuous stirring. The uric acid dissolves and a pale yellow liquid 
results. This is diluted with abeut 75 c.c. of water, allowed to stand and 
filtered. The filtrate, which contains alloxan, is saturated with hydrogen 
sulphide and allowed to stand for 12-16 hours. Alloxantin mixed with sulphur 
separates out ; it is filtered off and washed with water. It is separated from 
sulphur by solution in a small quantity of boiling water from which it separates 
on cooling in colourless crystals. These crystals are sometimes tinged with 
pink. 



UREIDES 281 

Alloxantin is readily reduced or oxidised. 

Alloxan is prepared : 3 gm. of finely powdered alloxantin are mixed with 
3 gm. of concentrated nitric acid and 7 gm. fuming nitric acid (sp. gr. 1-5). 
Slow oxidation occurs on standing and large crystals of alloxan are formed. 
The oxidation is complete in about 2 days and is shown by the complete 
solubility of the crystals in water. The crystals are placed on a porous plate 
to drain off the nitric acid and dried in the air. They are recrystallised from 
water. 

Properties. 

Alloxan is a white crystalline substance which separates from water in 
long shining rhombic prisms containing 4 molecules of water of crystallisation ; 
on exposure to air, the crystals effloresce and lose 3 molecules of water ; the 
last molecule of water is driven off by heating to 150. 

It is easily soluble in water ; the solution has an acid reaction and dis- 
agreeable taste and it slowly turns the skin a purple red. A deep indigo 
blue colour is formed when ferrous sulphate is added to its solution. 

If a few drops of its solution in water be evaporated to dryness and the 
reddish residue be treated with ammonia, it turns purple. 

Reactions. 

(1) Alloxantin is formed by the action of reducing agents upon alloxan 
in the cold : 

NH CO , CO NH 

I I /\ I I 
CO C C CO 

II II 

NH CO CO NH 

Alloxantin. 

(2) Dialuric acid is formed by the action of reducing agents on alloxan 
on warming. 

(3) Parabanic acid and carbon dioxide are formed by the oxidation of 
alloxan with boiling dilute nitric acid. 

(4) Barbituric acid is obtained from alloxantin by the action of con- 
centrated sulphuric acid. 

(5) Dilituric acid is formed by the action of fuming nitric acid upon 
alloxantin, or by the oxidation of violuric acid. 

(6) Violuric acid is formed by the action of potassium nitrite upon 
alloxantin, or by the action of hydroxylamine upon alloxan. 

(7) Uramil is formed by the reduction of dilituric acid and violuric 
acid. 

These ureides are white crystalline substances which are easily soluble 
in water; uramil is only slightly soluble and becomes red on exposure to 
the air. 



PYRIMIDINES. 

The cyclic ureides derived from urea and acids containing three 
carbon atoms (p. 280) are heterocyclic compounds. Those cyclic 
ureides which are derived from urea and the unsaturated acids, acrylic 
acid, methylacrylic acid, crotonic acid, form the group of compounds 
termed the pyrimidines. They have the structure : 

1 N C 6 

I I 

2 C C 5 

I II 

3 N-C 4 

To this group belong the three compounds, thymine, uracil and 
cytosftie which are constituents of nucleic acid : 

N = CH HN CO N = C . NH 2 HN CO 

II II II I I 

HC CH OC C.CH 3 OC CH ' OC CH 

II II | II | II I II 

N CH HN CH HN CH HN CH 

Pyrimidine. Thymine, or Cytosine, or Uracil, or 

5-methyluracil, 6-amino- 2, 6-dioxy- 

or 2-oxy- pyrimidine. 

5-methyl, 2, 6- pyrimidine. 
dioxypyrimidine. 

The ring contained in these compounds is a portion of the ring 
structure of the purines and it was first supposed that cytosine and uracil 
were decomposition products of adenine and guanine, but it has been 
definitely proved that these compounds are not secondary products 
and that they are part of the molecule of nucleic acid (p. 299). 

Preparation. 

These three compounds have been prepared by synthesis and their con- 
stitution established (see W. Jones' monograph on Nucleic Acid). They are 
more readily prepared from nucleic acid. Plant nucleic acid yields uracil and 
cytosine ; animal nucleic acid yields thymine and cytosine. The following 
method is given by W. Jones : 

50 gm. of nucleic acid are hydro lysed by heating in an autoclave with 
250 c.c. of 25 per cent, sulphuric acid for 5 hours at 150-160. The solution 
is diluted with water to 1000 c.c. and hot saturated baryta is added in excess 
to remove phosphoric and sulphuric acids. The excess of baryta is removed 
with carbon dioxide. The yellow solution is evaporated to about 400 c.c. and 
faintly acidified with nitric acid. The purines, which are precipitated, are 
filtered off. Silver nitrate is added to precipitate the remainder of the purines. 
To the clear yellow filtrate silver nitrate is added in small portions until a 
test portion gives a yellow coloration with a drop of baryta on a watch glass. 
The solution is made faintly alkaline with baryta and the precipitate of pyrimi- 
dine silver compounds is filtered off. The subsequent treatment depends on 
whether thymine and cytosine are being prepared from animal nucleic acid or 
uracil and cytosine from plant nucleic acid. 

252 



PYRIMIDINES 283 

(a) Thymine and Cytosine. 

The precipitate is suspended in hot water and decomposed with hydro- 
chloric acid. Traces of silver chloride which remain in solution are removed 
by hydrogen sulphide and the filtrate from silver sulphide is evaporated in 
vacua at 60 to a small volume. Thymine separates out during evaporation 
and on cooling. It is filtered off and recrystallised from hot water containing 
animal charcoal. 

The filtrate containing cytosine is carefully evaporated to dryness to re- 
move hydrochloric acid and dissolved in a small amount of water. Some 
thymine may remain undissolved. The solution contains cytosine hydro- 
chloride from which the picrate or platintchloride may be made. Cytosine is 
obtained by treating the concentrated solution with ammonia ; it crystallises 
out and is purified by recrystallisation from water. 

(fr) Uracil and Cytosine. 

The precipitate of pyrimidine silver compounds is suspended in hot water 
and decomposed with hydrogen sulphide. If barium be present, it is quanti- 
tatively removed with sulphuric acid and the solution is concentrated. The 
cytosine is precipitated . by slowly adding a hot saturated solution of picric 
acid. The cytosine picrate is recrystallised from water, dissolved in 5 per 
cent, hydrochloric acid and separated from picric acid by extracting the solu- 
tion with ether. The solution of cytosine hydrochloride yields cytosine as above. 

The uracil is obtained from the filtrate by acidifying with sulphuric acid, 
extracting the picric acid with ether, removing the sulphuric acid with baryta, 
evaporating to a small volume and allowing it to crystallise out. The im- 
pure crusts of crystals are recrystallised firstly from hot water containing charcoal 
and then from 5 per cent, sulphuric acid. 

Properties. 

Thymine generally crystallises from water in rosettes of small platelets, 
sometimes in the form of needles. 

It is soluble with difficulty in cold water '4 parts in 100 parts of water at 
25 but easily in hot water. It is slightly soluble in alcohol. It sublimes if 
carefully heated and melts when heated in a capillary at 32 1 with decomposition. 

It does not form salts with acids, but a potassium salt has been prepared. 

It combines with silver nitrate forming a compound which is precipitated 
by ammonia or baryta ; the compound is soluble in excess of ammonia, but 
not of baryta. 

It is precipitated by mercuric nitrate, but not by phosphotungstic acid. 

It is identified by its melting-point, sublimation and analysis. 

Cytosine crystallises in colourless glistening plates containing iH 2 O which 
is given off at 100. It decomposes at 320-325. It is soluble with difficulty in 
water i part in 1 29 parts of water at 25. Cytosine forms s'alts with acids ; the 
chief salt is the picrate which turns brown on heating at 255 and melts at 270. 

It forms also double salts with platinum chloride, etc. 

It behaves like thymine towards silver nitrate and ammonia or baryta. 

It is slowly precipitated by mercuric sulphate and also by phosphotung- 
stic acid. 

It is converted into uracil by the action of nitrous acid. 

Uracil forms a white crystalline powder consisting of needles arranged in 
clusters. It partially sublimes on heating and gives off red vapours. 

It is soluble with difficulty in cold water, more easily in hot and is almost 
insoluble in alcohol and ether. 

It behaves like thymine towards silver nitrate and ammonia or baryta. It 
is precipitated by mercuric nitrate, but not by phosphotungistic acid. 



GLYOXALINE OR IMINAZOLE DERIVATIVES. 

Histidine, histamine and urocanic acid are compounds which 
contain the heterocyclic glyoxaline or iminazole ring made up of 
2 nitrogen atoms and 3 carbon atoms : 

C N x 

II ^C 
/ ' 
C N/ 

This ring structure is also present in the purine ring which is a 
combination of the pyrimidine and iminazole rings. 

Histidine is /3-iminazole-a-aminopropionic acid. 

Histamine is /8-iminazole-ethylamine. 

Urocanic acid is /3-iminazole-acrylic acid. 

HC NH HC NH HC NH 



\ 



CH 



\ 



CH 



\ 



CH 



C N C N C N 

CH 2 CH 2 CH 

I I II 

CH.NH 2 CH 2 .NH 2 CH 

COOH COOH 

Histidine. Histamine. Urocanic acid. 

Histidine is a constituent of proteins and is contained in greatest 
amount in the protein haemoglobin. 

Histamine is a product of the putrefaction of histidine (or proteins). 
It is a constituent of ergot and is present in putrified meat, etc. It 
has a marked physiological action upon the sympathetic nervous 
system. 

Urocanic acid has been isolated twice from the urine of dogs and 
also from a trypsin digest of protein. 

Histidine. 

Preparation. 

Histidine can be readily prepared from blood as follows : 
2 parts of ox blood are added in portions to i part of concentrated 
hydrochloric acid contained in a large round-bottom flask and heated to boil- 
ing for 10 hours. The solution is evaporated to remove most of the hydro- 
chloric acid, nearly neutralised and filtered. The yellow filtrate is made 
alkaline with sodium carbonate and boiled until ammonia is no longer evolved, 
again filtered and precipitated with mercuric chloride solution, the reaction 

284 



GLYOXALINE OR IMINAZOLE DERIVATIVES 285 

being kept slightly alkaline with sodium carbonate. The precipitate is filtered 
off, washed and dissolved in a minimum of dilute hydrochloric acid. The 
filtered solution is considerably diluted, some mercuric chloride added and 
precipitated with sodium carbonate. The precipitate is filtered off, washed, 
suspended in water and decomposed with hydrogen sulphide. The filtrate 
from mercuric sulphide on evaporation yields histidine monochloride. 10 
litres of blood give from 70-90 gm. (Knoop, 1907). 

Properties. 

Histidine forms small platelets which melt at 253 with decomposition. 
It is easily soluble in water, very slightly soluble in alcohol and insoluble in 
ether. 

Its solution is alkaline in reaction ; it forms salts with acids and also 
double salts with gold chloride, etc. 

It is precipitated by silver nitrate and ammonia or baryta water, by mer- 
curic sulphate in sulphuric acid solution and by phosphotungistic acid. 

Reactions. 

(1) A solution of histidine treated with sodium hydroxide and a trace 
of copper sulphate and heated gives a violet colour, which changes to red 
(compare biuret reaction). 

(2) A solution of histidine made alkaline with sodium carbonate and 
treated with 3-5 c.c. of a fresh alkaline (Na 2 CO 3 ) solution of about -05 gm. 
of diazobenzene sulphonic acid l gives on standing a deep cherry-red colour, 
which changes to orange on acidifying (Pauly). 

The delicacy of this reaction is i in 20,000 ; with i in 100,000 a pale red 
colour is produced. 

Tyrosine gives a similar colour with the reagent and must consequently 
be absent from the solution. 

A modification of the reaction by which histidine can be distinguished 
from tyrosine was devised by Totani. 2 

(3) An acid aqueous solution of histidine is treated with bromine water 
until it has a permanent yellow colour. On heating, the yellow colour dis- 
appears, but the solution becomes gradually red until it assumes a deep wine- 
red colour and a black amorphous precipitate settles out. This reaction is 
positive in a dilution of i in 1000. 

Pilocarpine. 

This basic substance or alkaloid (p. 351) contains an iminazole 
ring. Its formula is probably the following : 

C,H, . CH CH CH 9 C W 



CO O- 



-CH 2 H . C 



1 This is prepared by stirring 2 gm. of finely powdered sulphanilic acid into a paste 
with 3 c.c. of water and 2 c.c. of cone, hydrochloric acid. The paste is treated, within a 
period of i minute, with a fresh solution of i gm. of potassium nitrite in i to 2 c.c. of 
water, the mixture being cooled after each addition with cold water. Most of the sulphanilic 
acid rapidly dissolves and a thick white crystalline precipitate of diazobenzene sulphonic 
acid forms. It is filtered off after a few minutes and washed with a little water. 

2 Biochem. J., 1915, 9, 385. 



PURINES. 



Uric acid, xanthine, hypoxanthine, guanine, adenine, caffeine, theo- 
bromine and others are classed together in the special group of com- 
pounds known as the purines. Not only are these compounds found 
associated in nature in both animals and plants, but also they are 
chemically very closely related. They yield alloxan, or dimethyl- 
alloxan, on oxidation, and have many other similar reactions. 

All these compounds have been synthesised by Emil Fischer and 
their exact chemical relationship to one another established. The result 
of these investigations has shown that they are all derived from the 
compound purine, which stands in the same kind of relationship to 
them as a hydrocarbon does to an alcohol, an amine, etc. Thus : 



Purine 

Hypoxanthine = monoxypurine 



Xanthine = dioxypurine 

Uric acid = trioxypurine 

Adenine = aminopurine 

Guanine = amino-oxypurine 

Theobomine = dimethyl dioxypurine 

Theophyllin = dimethyl dioxypurine 

Caffeine = trimethyl dioxypurine 

The compounds have the heterocyclic ring structure in which the 
atoms are numbered in the following order : 

*N 6 C 



C 5 H 4 N 4 

C 5 H 4 N 4 

C 5 H 4 N 4 2 

C 5 H 4 N 4 3 

C 5 H 3 N 4 .NH 2 

C S H 3 N 4 .O.NH 2 

C 5 H 2 N 4 2 (CH 3 ) 2 

C 5 H 2 N 4 2 (CH 3 ) 2 

C 5 HN 4 2 (CH 3 ) :t . 



5C 7 N 



\ 



C 8 . 



> 

3N_C N 9 / 

The formulae for the various compounds are ; 

HN C=0 HN C=0 

NH HC C NH 0=C C NH 



N=CH 

HC C- 
n^ Vx 



CH 



N C N 
Purine. 



HN C=O 

1 I 
O= C C NH 

C=0 

HN C NH 
Uric acid, 

or 
2, 6, 8-trioxypurine. 



H 



N C N 
Hypoxanthine, 

or 
6-monoxypurine. 

N=C NH 2 

HC C NH 

\ 



CH 



HN C N 



CH 



Xanthine, 

or 
2, 6-dioxypurine. 

CH 3 .N C=0 

O=C C NH 
\ 



N C N 
Adenine, 

or 

6-aminopurine. 
286 




CH 



CH 3 . N C N 
Theophylline, 

or 
i, 3-dimethylxanthine. 



PURINES 



287 



HN C=O 

O =C C N . 



CH 3 
CH 



CH 3 . N C N 
Theobromine, 

or 
3, y-dimethylxanthine. 



HN C=O 
H N C C 



NH 
CH 



HN C N 
Guanine, 

or 
2-amino, 6-oxypurine. 



CH 3 .N C=O 
O=C C N. 



CH 3 
CH 



CH 3 . N C N 
Caffeine, 

or 
I, 3, 7-trimethylxanthine. 



It will be noticed that the complex or ring of atoms is the same in 
all these compounds, but that according as the compounds contain 
oxygen atoms attached to a carbon atom the double bond is changed 
in position so as to keep the nitrogen and carbon atoms trivalent and 
tetravalent respectively. The double bond between the carbon atoms 
4 and 5 is the same throughout. 

URIC ACID. 

Scheele discovered uric acid in urinary calculi in 1776 and also 
isolated it from urine. He made a careful investigation of its properties 
and reactions, most of which are still used at the present time for its 
identification. It was called lithic acid or ouric acid by Fourcroy in 
1793, who showed that it contained urea. It was discovered in guano 
in 1805 and in bird's excrement in 1815, and later it was shown 
to be the chief constituent of snakes' excrement. Prout about this 
time showed that uric acid on oxidation with nitric acid gave alloxan. 
It was first analysed in 1834 by Liebig and Wohler and found to pos- 
sess the empirical formula C 5 H 4 N 4 O 3 . These workers by oxidising uric 
acid with lead peroxide obtained allantoine, which had been previously 
found in the allantoic fluid of calves. Other ureides were obtained 
by Strecker and by Baeyer who established their constitution. 

The formation of alloxan and urea, and of allantoine, by the oxida- 
tion of uric acid, showed that uric acid contained 2 molecules of urea 
and that it contained the structures of alloxan and allantoine in its 

molecule ; 

N C 

C C C N\ 

I I I ^r 

/c. 

N C C N/ 

* Two formulas were put forward to represent the constitution of 

uric acid : 

HN CO 



HN C NH 



L 



-CO 



OC 



HN C NH- 
Formula 

of 
Medicus. 



CO 



io 



NH C NH 

Formula 

of 
Fittig. 



288 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

The formula proposed by Medicus was ultimately proved to be the 
correct one by the synthesis of uric acid by Fischer. 

The previous syntheses of uric acid by Horbaczewski (i) by fusing 
together glycocoll and urea, (2) by combining trichlorlactamide with 

urea : 

NH, CC1, H,N HN C HN, 



I I I I 

CO + CHOH + CO - OC 



>CO 

C HN/ 



I III 

CO. 



NH 2 CO.NH 2 H 2 N HN CO 

did not definitely prove its constitution. The synthesis by Behrend 
and Roosen from aceto-acetic acid and the following synthesis com- 
menced by Baeyer and completed by Fischer proves the constitution 
of uric acid : 

(1) Malonyl urea or barbituric acid is obtained by heating urea and 
malonic acid with phosphorus oxychloride. 

(2) Nitrous acid converts malonyl urea into oximidomesoxalyl urea 
or violuric acid. 

(3) Aminomalonyl urea or uramil is obtained by reducing violuric 
acid. 

(4) Potassium cyanate converts uramil by rearrangement, as in 
the formation of urea, into pseudo-uric acid. 

(5) Pseudo-uric acid loses water on heating with fused oxalic acid, 
or boiling with hydrochloric acid, and is changed into uric acid. 
COOH NH CO NH CO NH CO NH CO 

CH 2 -CO CH 2 ->CO C=NOH-CO CH.NH 2 -CO CH . NH 2 . HOCN-> 

COOH NH CO NH CO NH CO NH CO 

Malonic Barbituric Violuric Uramil. 
acid. acid. acid. 

NH CO NH CO 

II I i 

CO CH.NH.CO. NH 2 -> CO C NH\ 

I I U >0. 

NH CO NH C NH/ 
Pseudo-uric Uric 

acid. acid. 

Preparation. 

(i) From Snakes* Excrement, or Guano. 

5 to i gm. of snakes' excrement (or guano) is powdered, suspended 
in 100 c.c. of water, heated nearly to boiling and dissolved by adding 
dilute sodium hydroxide. The solution is heated until ammonia from 
ammonium salts and urea is no longer evolved and filtered from 
insoluble material (sand, etc.). Excess of dilute hydrochloric acid is 
added to the filtrate. Uric acid is precipitated ; it is filtered off when 
the solution has cooled and washed free from acid with water. The 
product is generally almost pure, but may be purified by dissolving 



PURINKS 



289 



in sodium hydroxide and reprecipitating with acid. It is dried in the 
air or at 100. 

(2) From Human Urine. 

A twenty- four hours' quantity of human urine contains from 0*5 to 
I '5 gm. of uric acid, sometimes as much as 2'O or 2*5 gm. 

(a) 500 c.c. of urine are treated with 50 c.c. of concentrated hydro- 
chloric acid and allowed to stand in a cool place for twenty-four hours. Pig- 
mented crystals of uric acid slowly separate out and adhere to the sides of the 
vessel. 

Microscopic examination of the crystals shows that they consist of ir- 
regular, much pigmented crystals, generally arranged in sheaves (Figs. 48, 80, 
P- 565)- 

(&} 100 c.c. of urine are saturated with crystals of ammonium chlor- 
ide (27 gm. necessary) and I or 2 drops of strong ammonia are added. 
A gelatinous precipitate of ammonium hydrogen urate is formed. This 
is filtered off after about fifteen minutes. It can be shown to contain 
uric acid by testing a small portion by the murexide test. The uric 
acid is obtained from the precipitate by dissolving it in the smallest 
quantity of hot water containing a drop of sodium hydroxide, filter- 
ing, if necessary, and acidifying with a drop of concentrated hydro- 
chloric acid. Uric acid crystallises out on cooling if too much water 
has not been used in dissolving the ammonium urate. 

Properties. 

Pure uric acid is a colourless crystalline powder, but as obtained 
from solutions containing urinary pigments it is generally more or 
less pigmented ; the pigment is difficult to remove by treatment with 
animal charcoal. The crystals 
usually consist of rhombic plates 
or prisms, but various shapes are 
observed depending on the rate 
of its crystallisation from solution. 
These are shown in Fig. 48. 

Uric acid has no taste or smell. 
It is only slightly soluble in water 
i part in 39,500 parts of water 
at 1 8, i part in about 1900 parts 
of hot water. It is insoluble in 
alcohol and ether, but soluble in 
glycerol. It is soluble in solutions 

of the borates, phosphates, carbon- FlG - 48Uric acid. (After Funke.) 
ates and acetates of the alkali metals, with the formation ot acid 
salts of these acids and of uric acid. It dissolves in concentrated 
sulphuric acid from which it is precipitated by the addition of water. 

19 




290 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



Uric acid is a weak acid and forms two series of salts, neutral and 
acid salts. The formation of salts can be explained either by the 
tautomeric formula in which hydroxyl groups attached to the carbon 

N=C . OH 

I I 
HO . C C NH 

I I >' OH 

N C N 

atoms are present, evidence of which is shown by the action of phos- 
phorus trichloride upon uric acid, which gives trichloropurine, or by 
assuming that the acid character of the CO groups influences the pro- 
perties of the hydrogen atoms attached to the N atoms so that they 
are acidic in character and replaceable by metals. 

The neutral salts, such as C 5 H 2 Na 2 N 4 O3, are comparatively easily 
soluble in water and are obtained on dissolving uric acid in alkali 
hydroxides or in hot solutions of the carbonates. Lithium urate is 
the most soluble salt of uric acid. 

Uric acid is insoluble in cold solutions of the carbonates and may 
thus be separated from other acids such as benzoic acid. 

The acid salts, such as C 5 H 3 NaN 4 O 3 , are soluble with difficulty in 
water. They are obtained on passing carbon dioxide into a solution 
of the neutral salt. 

One part of acid sodium urate is soluble in 1 1 oo-i 200 parts of cold 
water and 125 parts of hot water; I part of acid potassium urate is 





FIG. 49. Sodium urate. 
(After Funke.) 



FIG. 50. Ammonium urate. 
(After Funke.) 



soluble in 800 parts of cold water and in 70 parts of hot water ; 
I part of acid ammonium urate is soluble in 1600 parts of cold water, 
more easily in hot water and is insoluble in ammonium chloride solution. 



PURINES 291 

A double compound of uric acid and acid sodium urate, C 5 H 4 N 4 O 3 
+ C 5 H 3 NaN 4 O5, is said to be deposited in gouty joints and cartilages. 

All the salts are decomposed by acetic acid or hydrochloric acid 
with the gradual separation of uric acid. 

Reactions. 

(1) On heating, uric acid is decomposed with the formation of urea, 
ammonium carbonate, cyanuric acid and hydrogen cyanide and a 
charred mass remains. 

(2) Uric acid is decomposed on heating with solid potassium 
hydroxide with the formation of ammonia and potassium cyanide ; the 
presence of potassium cyanide may be shown by extracting the residue 
with water and testing for cyanides (p. I 56). 

(3) Uric acid chars on heating with concentrated sulphuric acid. 
()~Murexide Test. On evaporating a small quantity of uric 

acid or an urate to dryness with dilute nitric acid, a yellow or yellowish- 
red residue is left. On adding to it a drop of ammonia with a glass 
rod, the colour changes to purple ; ammonium purpurate or murex-ide 
is formed. A drop of caustic soda gives a blue-violet colour. 

(5) Schiff's Test. A solution of uric acid in sodium carbonate 
solution reduces silver nitrate. This is best observed by pouring some 
of the urate solution upon a filter paper moistened with silver nitrate. 
A black stain of metallic silver results. 

(6) Fehlings Solution. A white precipitate of copper urate is 
formed when a solution of uric acid is added to Fehling's solution and 
warmed. On boiling for some time, the solution is reduced with the 
formation of cuprous oxide. 

Note. On this account urine on prolonged boiling reduces Feh- 
ling's solution and the reduction may be wrongly attributed to small 
amounts of glucose (see under pathological urines). 

(7) If a small quantity of uric acid be carefully heated with dilute nitric 
acid just to effervescence and the excess of acid be carefully evaporated- so as 
to avoid coloration, a blue colour results on the addition of 2-3 drops of con- 
centrated sulphuric acid and a few drops of commercial benzene (containing 
thiophene). The colour changes to brown on evaporation of the benzene, 
but returns on again adding benzene (Deniges). 

(8) Dilute solutions of uric acid are completely precipitated by the addition 
of ammoniacal silver nitrate and magnesia mixture. Silver magnesium urate 
is formed. 

(9) Dilute solutions of uric acid are precipitated by adding copper sulphate 
and sodium bisulphite. On boiling, cuprous urate is formed. 

These two reactions (8) and (9) are used in the precipitation of uric acid 
and the other purines from urine and extracts of tissues. 

(10) Very dilute solutions of uric acid containing -5 mg. give an intense 
blue colour with a specially prepared phosphotungstic acid reagent (p. 557), 
(Folin). 

19* 



292 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Estimation of Uric Acid. 

A. Uric acid is readily oxidised by potassium permanganate and 
converted into allantoine and other products of its oxidation. Use 
is made of this reaction for its estimation. From solutions, such as 
urine, in which other organic substances are present, the uric acid 
must be precipitated before it can be estimated (p. 554). 

O5N potassium permanganate (i -581 gm. per litre) is commonly 
used in the process. 

The permanganate solution is placed in^a burette with a glass tap 
(rubber cannot be used as it is attacked by permanganate). The level 
of the liquid in the burette is most conveniently read by holding a 
lighted match behind it. 

100 c.c. of a solution of uric acid, or the same volume of a solution 
containing a weighed quantity of uric acid, or solid urate (-5-1 gm. 
dissolved in water containing soda and diluted to i litre), are placed 
in a flask or beaker, 20 c.c. of concentrated sulphuric acid are added, 
the - solutions are well mixed and the permanganate is run in whilst 
the solution is hot. 

At first every drop of permanganate is decolorised before it 
diffuses through the liquid. The end point is reached as soon as a 
drop produces a pink flush throughout the liquid 1 . This pink colour 
disappears on standing ; another drop of permanganate will again 
produce a pink flush. This can be continued for some time so that 
the first pink flush must be carefully looked for. 
Calculation : 

i c.c. of -O5N KMnO 4 corresponds to 0-00375 gm. uric acid. 

. . x c.c. correspond to x x -00375 S m - i n IO cc - solution. 

B. By means of the colour reaction with phosphotungstic acid Folin has 
shown that quantities of uric acid amounting to i mg. in i c.c. can be ac- 
curately estimated. This method is particularly useful for estimating uric 
acid in blood and can be used for estimating uric acid in small quantities of 
urine (pp. 565, 580). 



PURINES 293 

Xanthine. 

Xanthine was discovered in an urinary calculus by Marcet in 
1817 and in 1859 Scherer found it in meat and pancreas. It has 
since been shown to be present in other animal organs and to be 
widely distributed in plants. 

Its constitution was indicated by its products of oxidation, alloxan 
and urea ; Fischer proved it by synthesis. 

Preparation. 

Xanthine is most easily prepared by the action of nitrous acid upon guan- 
ine. 

Its preparation from extracts of tissues involves a comp'icated process of 
separation from other purines (p. 584). 

Properties. 

Xanthine is a colourless powder which assumes a waxy appearance on rub- 
bing. It separates slowly from its solution in alkali on the addition of acetic 
acid in the form of colourless nodules consisting of microscopic rhombic 
shining platelets containing i molecule of water of crystallisation. 

It is very slightly soluble in water i part in 14,151 parts at 16, i part 
in 1300-1500 parts at 100. It is insoluble in alcohol and ether. It is 
easily soluble in caustic alkalies and in 2 per cent, ammonia. On evapora- 
tion of the ammoniacal solution, xanthine separates in groups of platelets. 
It is soluble with difficulty in cold dilute nitric and hydrochloric acids, but 
more easily on warming. It dissolves with difficulty in 5 per cent, sul- 
phuric acid even on boiling. Xanth'ine hydrochloride and other salts are 
decomposed by water. 

Ammoniacal silver nitrate precipitates it from solution as C 5 H 4 N 4 O 2 . 
Ag 2 O ; the precipitate is soluble in nitric acid and from this solution 
xanthine silver nitrate, C 5 H 4 N 4 O2 . AgNO 3 , slowly separates in aggregates of 
tiny needles. This compound is soluble with difficulty in nitric acid. 

Reactions. 

(1) On evaporation with nitric acid, xanthine leaves a yellow residue, 
which is coloured red by caustic soda and becomes purple on heating. 

(2) On boiling a small quantity of xanthine with chlorine water, or with 
dilute hydrochloric acid and a small crystal of potassium chlorate, and evapor- 
ating the solution to dryness, a white or pale yellow residue is left. On bring- 
ing this into contact with ammonia vapour under a glass cover, it changes to 
a rose red colour (murexide). 

(3) On adding a specimen of xanthine to a little bleaching powder in caustic 
soda in a watch glass, the surface of the specimen becomes dark green, then 
brown ; the colour ultimately disappears. 

(4) A solution of xanthine in dilute sodium hydroxide gives a red colour 
on the addition of diazobenzene sulphonic acid (see p. 285). 

(5) Xanthine is precipitated, like uric acid, by silver nitrate and ammonia 
and by copper sulphate and sodium bisulphite. 



294 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Hypoxanthine. 

Hypoxanthine, like xanthine, occurs widely distributed in the 
tissues of animals and plants. 

Its constitution has been proved by synthesis by Emil Fischer. 
Preparation. 

It is prepared most easily by the action of nitrous acid upon adenine. 
Its isolation from tissues necessitates a complicated process of separation from 
other purine bases (p. 584). 

Properties. 

Hypoxanthine forms colourless microscopic crystals, soluble with difficulty 
in water; i part is soluble in 1400 parts of water at 19 and in 70 parts of 
boiling water. It is practically insoluble in alcohol. 

It is soluble in dilute acids and alkalies and in ammonia and forms salts 
with acids, bases and other salts, which crystallise readily. 

The hydrochloride C 5 H 4 N 4 O . HC1 + H 2 O and other salts with acids 
decompose on recrystallisation from water. 

The nitrate C 5 H 4 N 4 O . HNO 3 + H 2 O is insoluble in nitric acid. Platino- 
chlorides, picrates and other salts are known. 

Reactions. 

Hypoxanthine differs from xanthine in not giving reactions with nitric 
acid and chlorine water, but the reaction with diazobenzene sulphonic acid is 
positive if an excess of alkali be avoided. 

It is precipitated from solution by ammoniacal silver nitrate and by copper 
sulphate and sodium bisulphite. 



Guanine. 

Guanine is also found widely distributed in the tissues of animals 
and plants and is a constituent of nucleic acid. It is the chief con- 
stituent of the excrement of spiders and is found in Peru guano in 
small quantities. It is deposited in the muscles and joints of pigs in 
certain cases of illness and it occurs in fish scales and other epidermal 
structures of fishes. 

Fischer has proved its constitution by synthesis. 

Preparation. 

From Guano. 

The material is extracted with boiling dilute lime water and then with 
sodium carbonate solution as long as the extracts are coloured. On acidify- 
ing the extracts with acetic acid, uric acid and guanine separate out. This 
mixture is boiled with dilute hydrochloric acid and made alkaline with am- 
monia ; the guanine is precipitated. 



PURINES 295 

From Nucleic Acid. (Jones.) 

50 gm. of nucleic acid are hydrolysed by heating in a boiling water-bath 
under a reflux condenser for 2 hours with 200 c.c. of 10 per cent, sulphuric 
acid. The hot solution is treated with concentrated ammonia until it is 
neutral, the ammonia being added slowly as soon as the precipitation of 
guanine begins. An excess of ammonia is added until 2 per cent, is present. 

The guanine which is precipitated in a granular form is filtered off and 
washed with ammonia. It is suspended in boiling water and dissolved by 
adding a minimum of 20 per cent, sulphuric acid. The solution is decolorised 
by boiling with charcoal (the decolorisation does not occur readily if excess of 
sulphuric acid has been used) and the guanine precipitated by adding am- 
monia as above ; it is filtered off, dried at 40 and dissolved in 20-25 
times its weight of boiling 5 per cent, hydrochloric acid. Pure guanine 
hydrochloride crystallises out on cooling. It is filtered off, washed with dilute 
hydrochloric acid and dried in the air. 

Pure guanine is prepared from the hydrochloride by dissolving it in boiling 
i per cent, hydrochloric acid and adding ammonia. 

Adenine is prepared from the ammoniacal filtrate and washings after 
filtering off guanine which may deposit on standing (see under Adenine). 

From Tissue Extracts. 

It is precipitated by ammoniacal silver nitrate or copper sulphate and 
sodium bisulphite and separated from other purine bases as described on 
p. 584. 

Properties. 

Guanine forms a colourless, generally amorphous, powder. It is insol- 
uble in water, alcohol, ether and soluble with difficulty in ammonia. It is 
easily soluble in all mineral acids and alkalies. 

The sulphate (C 6 H 5 N 5 O) 2 . H 2 SO 4 . 2H 2 O and other salts are generally 
decomposed by water. 

The metaphosphate C 5 H 5 N 5 O . HPO 3 . ^cH 2 O is precipitated on adding 
metaphosphoric acid to its solution ; it is stable to water and is soluble with 
difficulty in water and dilute acids. 

Guanine forms a very insoluble picrate, C 5 H 5 N 5 O . C 6 H 3 N 3 Ov + H 2 O. 
The picrates of xanthine and hypoxanthine are more soluble. 

Its compound with silver nitrate C 5 H 5 N 5 O . AgNO 3 is almost insoluble in 
cold nitric acid, but more soluble in hot, from which it crystallises on cool- 
ing. 

Potassium ferricyanide gives prismatic yellow-brown crystals with dilute 
solutions of guanine. 

Reactions. 

Guanine on evaporation with dilute nitric acid leaves a brownish- red residue, 
which becomes bluish-violet on heating. The combinations with picric acid 
and potassium ferricyanide distinguish it from xanthine and hypoxanthine ; 
its behaviour towards metaphosphoric acid from hypoxanthine and adenine, 



296 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Adenine. 

Adenine was first obtained by Kossel from the pancreas, but has 
since been obtained from other organs and from plants. Its constitu- 
tion was proved by synthesis (Emil Fischer). 

Preparation. 

Adenine may be prepared from extracts of tea, or from nucleic acid, or 
from tissue extracts (p. 584). 

From Nucleic Acid. 

The ammoniacal filtrate free from guanine is acidified with 20 per 
cent, sulphuric acid, heated to boiling and treated with 10 per cent, copper 
sulphate solution as long as a white precipitate is formed and until yellow 
cuprous oxide begins to be formed, (Usually sodium bisulphite solution is 
added with the copper sulphate solution, but this is not necessary when the 
solution contains pentose.) The mixture is boiled for several minutes and 
filtered. The precipitate is washed with boiling water, suspended in hot 
water and decomposed with hydrogen sulphide. The filtrate from the 
copper sulphide is evaporated to dryness and the residue of adenine is 
crystallised from hot 5 per cent, sulphuric acid arid decolorised with char- 
coal, if necessary. Pure adenine sulphate is obtained. 

Properties. 

Adenine forms long colourless needles with 3 molecules of water of crystal- 
lisation, or whetstone-like crystals ; the former become opaque on exposure 
to air and if heated in insufficient water become opaque at 53. It sublimes 
at 220 and at 250 partially decomposes. It melts if heated rapidly in a 
capillary tube at 360-365, becoming brown before reaching the melting-point, 
at which temperature it decomposes. 

It is soluble with difficulty in cold water, i part in 1086 parts, but it is 
more easily soluble in hot water. The solution has a neutral reaction. It 
dissolves in alkalies, mineral acids and acetic acid and is precipitated on 
neutralising the solutions. It is more easily soluble in ammonia than guanine, 
but less so than hypoxanthine. 

It forms salts with acids and other salts ; the double salt with gold chloride 
helps to distinguish it from the other nuclein bases. The picrate is very in- 
soluble and serves for isolating it from solution and separating it from hypo- 
xanthine. 

Reactions. 

Adenine gives no reaction on evaporation with nitric acid or with chlorine 
water. The reaction with diazobenzeriesulphonic acid is positive if excess of 
alkali be avoided. 

Adenine behaves in a characteristic manner on heating with zinc and 
hydrochloric acid on the water-bath. The solution turns purple-red ; if 
filtered, made strongly alkaline with caustic soda and allowed to stand or 
shaken with air, it turns ruby-red and then brownish-red. Guanine does not 
give this reaction, but hypoxanthine gives the colours, though fainter. 



PURINES 297 

Caffeine. Theophylline. Theobromine. 

These three compounds are not found in animals but are fairly 
widely distributed in plants. Caffeine and theobromine are the active 
constituents of tea, coffee and cocoa : they produce a stimulating effect 
on the central nervous system and act as powerful diuretics. 

Caffeine. 

Caffeine is present to the extent of -8-1 7 per cent, in coffee beans, 
i-'8 per cent, in cocoa beans, 1-2 per cent, in kola nuts, 2-5 per cent, 
in tea leaves; 2*5-5 P er cent, is present in guarana, the roasted fruit 
of Paullinia which is eaten in South America. 

Preparation. 

Caffeine is readily prepared from tea leaves by boiling about 250 gm. 
with 500 c.c. of water for 15 minutes; the solution is filtered through cloth 
into a basin and the leaves boiled again with 250 c.c. water and again filtered 
off. The odour is due to a small quantity of essential oil and the solution con- 
tains protein and tannin, which gives it the brown colour. The proteins and 
tannin are removed by adding basic lead acetate as long as a precipitate is 
formed. The precipitate is filtered off and washed with water and the solution 
is treated with sulphuric acid or hydrogen sulphide to remove lead. The lead 
sulphate or sulphide is filtered off and the solution evaporated with some 
charcoal until its volume is 250-300 c.c. It is filtered from charcoal and 
when cold extracted two or three times with chloroform. The chloroform is 
distilled off and the caffeine, which remains, is recrystallised from boiling 
water containing animal charcoal. From 1-2 gm. are obtained. 

Properties. 

Caffeine forms long silky needles containing one molecule of water of 
crystallisation which it loses at 100. It melts at 233 and has a bitter taste. 
It forms salts with mineral salts which are decomposed by water. 

Caffeine, on evaporation with chlorine water, leaves a reddish-brown 
residue which becomes purple when treated with ammonia. 

Theobromine. 

Theobromine is present to the extent of 1*5-2 -4 per cent, in cocoa 
beans ; smaller amounts are present in kola nuts and tea leaves ; it is not 
present in coffee beans. 

Theobromine forms a crystalline powder which has a bitter taste, is 
soluble with difficulty in hot water and alcohol but is easily soluble in am- 
monia. It forms salts with mineral acids, which are decomposed by water, 
and with silver nitrate and other metallic salts. 

Theophylline. 

Theophylline was discovered by Kossel in 1888 in extracts of tea and has 
been synthesised by Emil Fischer. 

It forms a white powder which melts at 264. 

Paraxanthine or i, y-dimethylxanthine^ have been isolated from human 



n 

Heteroxanthine or 7-methylxanthine | urine. They are products 
i-methylxanthine j formed f 

Epiguanine or y-methylguanine J organism. 



298 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

The Biological Relationship of the Purines. 

Adenine and guanine are constituents of the nucleic acid of 
animals and plants. Whilst still in combination in the molecule of 
nucleic acid they may be acted upon by enzymes in the tissues and 
converted respectively into hypoxanthine and xanthine. Nucleic acid 
on decomposition in the tissues will yield adenine and guanine, or 
hypoxanthine and xanthine. Adenine and guanine are acted upon 
by the enzymes, adenase and guanase, in the tissues and converted 
into hypoxanthine and xanthine. These enzymes are present in 
most organs, but not in all organs ; sometimes an organ contains 
both enzymes, sometimes only one enzyme, sometimes neither 
enzyme. Hypoxanthine is oxidised by the tissues to xanthine and 
xanthine >is oxidised to uric acid (xanthine oxidase). In some 
animals, but not in man, uric acid is oxidised to allantoine (uricase). 
The changes may be briefly represented as follows : 

Nucleic acid 

I 




guanine 

^ t 
hypoxanthine ^- xanthine > uric acid 

V 

allantoine 

The mechanism of these transformations has been difficult to eluci- 
date and much confusion as to the origin of uric acid has existed. 
A full account of the work is given in W. Jones' monograph on 
" Nucleic Acids ". 



NUCLEIC ACIDS. 

The first chemical examination of cell nuclei was made in 1868 
by F. Miescher. Pus cells were digested with artificial gastric juice ; the 
protoplasm dissolved and a residue consisting of the more resistant 
nuclei was left as an insoluble grey powder. This dissolved in dilute 
sodium carbonate and was precipitated by dilute acetic acid. It was 
found to contain phosphoric acid and to give the colour tests for 
proteins. It was named nuclein. 

Eight years later Miescher examined the spermatozoa of Rhine 
salmon. He found that they consisted almost entirely of a salt com- 
posed of the base protamine (a protein) and an organic acid which he 
termed nucleic acid ; this contained phosphorus. 

Nucleins were prepared from other tissues, yeast, red blood corpuscles, 
etc., by other workers. They were analysed most carefully by Kossel 
and his pupils to whom our knowledge of the constitution of nucleic 
acid is almost entirely due. Kossel found that nucleins and also 
nucleic acid, for which a method of preparation from thymus and other 
organs was devised by Kossel and Neumann, on hydrolysis by acids 
gave rise to the purine bases, guanine, xanthine, hypoxanthine and 
adenine. The two bases, guanine and adenine, have since been shown 
to be the only ones present in nucleic acid. In addition to the two 
purine bases three other bases, the three pyrimidine bases, thymine, 
uracil and cytosine, have been shown to be present in nucleic acid, and 
besides these compounds there is also present a carbohydrate, a hexose 
or a pentose. These compounds are obtained from animal or plant 
nucleic acids. Nucleic acids consist of a carbohydrate, phosphoric 
acid, two purine bases and two pyrimidine bases, as expressed in the 
following scheme : 

Animal Nucleic Acid. Plant Nucleic Acid. 

Phosphoric acid. Phosphoric acid. 

Hexose (laevulinic acid). Pentose = ^/-ribose. 

Guanine. Guanine. 

Adenine. Adenine. 

Cytosine. Cytosine. 

Thymine. Uracil. 

Plant nucleic acid differs from animal nucleic acid in the nature of 
the carbohydrate constituent and in the nature of one of the pyrimidine 
constituents. 

It appears that all animal nucleic acids are the same and that all 
plant nucleic acids are the same. 

The constitution of the nucleic acids has not yet been definitely 

399 



3oo PRACTICAL ORGANIC AND BIO-CHEMISTRY 



ascertained, but the following formulae have been provisionally 
assigned : 

Animal Nucleic Acid. 
HO, HO 

-C H H 10 O 4 guanine group. 



Plant Nucleic Acid} 



O = P O 



\ 







HO, 



O = P O C fi H 8 O 2 thymine group. 



O 

H0 \ 

O = POC K H 8 O 2 



O 



cytosine group. 



O = P O C 8 H 7 O 2 guanine group. 

HO/ 

O 



O = POC 5 H 6 O cytosine group. 
HO/ 

O 

HO, 
O = P O C 5 H 6 O uracil group. 

HO/ 

O 
HO, 



O = P O C 6 H 10 O 4 adenine group. 
HO/ 



O = PO C S H 7 O 2 adenine group. 
HO/ 



The combination of phosphoric acid with carbohydrate in each 
case has been proved by the isolation of a carbohydrate ester of phos- 
phoric acid, and the combination of purine base with carbohydrate by 
the isolation of such a compound. This compound is a glucoside and 
glucosides of carbohydrate and purine have been synthesised by Emil 
Fischer. There is as yet no evidence as to how the groups are com- 
bined together. 

Each of the groupings phosphoric acid + carbohydrate + purine 
or pyrimidine base is termed a mononucleotide. Nucleic acid is thus 
a tetranucleotide. The carbohydrate and purine base or pyrimidine 
combination is known as a nucleoside. 

a-N ucleoproteins. 

If aqueous extracts of various organs be made and these extracts 
be acidified with acetic acid, a precipitate consisting of protein and 
nucleic acid is formed. It has been termed nucleoprotein. 

These a-nucleoproteins must be regarded as salts or of combinations 
of nucleic acid and protein ; as salts on account of the occurrence 
of protamine nucleate in spermatozoa, their easy separation, and on 
account of the property of nucleic acid of precipitating protein from 
solution when a solution of sodium nucleate in a solution of protein 
is acidified. 

/3-Nucleoproteins. Mononucleotides. 

If organs, especially the pancreas, be suspended in water, the sus- 
pension raised to the boiling-point and filtered from the coagulum of 
protein which is formed, a clear yellow liquid is obtaind. If this 
1 Jones and Read, J. Biol. Chem., 1917, 31, 40. 



NUCLEIC ACIDS 301 

liquid be acidified with acetic acid a precipitate is formed. This 
precipitate on purification does not contain protein. It is a mono- 
nucleotide, termed guanylic acid, and consists of guanine, pentose 
and phosphoric acid. Another mononucleotide, inosinic acid, has 
been prepared from meat extract. It consists of xanthine, pentose 
and phosphoric acid, and is identical with vernine, a mononucleotide 
prepared from plants. 

These /3-nucleoproteins of animals have thus the constitution of 
plant nucleic acids. They are not constituents of the nuclei of 
animal cells, but have been ingested by the tissue from vegetable food. 

The work of the various investigators upon nucleic acid is given 
by Walter Jones in his monograph on Nucleic Acid. 

Preparation of Nucleic Acid from Thymus, etc. 

There are various methods for preparing nucleic acid, but the following 
given by W. Jones is most convenient : 

i kilo, of thymus, freed from fat, connective tissue, etc., and finely minced, 
is added in small portions to 2 litres of boiling water containing 33 gm. of 
sodium hydroxide and 100 gm. of sodium acetate. The material dissolves 
giving a pale brown solution : lumps are removed and brought into solution 
separately by heating over a flame. The solution is heated in a boiling water- 
bath for 2 hours with occasional stirring. It is diluted with one-third of 
its volume of water and acidified to litmus with 50 per cent, acetic acid; 
about 100 c.c. are required. The proper acidity must be obtained so as to 
ensure rapid filtration ; it can be attained by adding acetic acid or sodium 
hydroxide as may be required. The solution is heated to boiling and filtered 
through a hot-water funnel. It will gelatinise on cooling. The solution 
and washings are evaporated to 750 c.c. and poured slowly into i litre of 95 
per cent, alcohol. Sodium nucleate mixed with phosphates is precipitated. 
After 1 2- 1 6 hours the liquid is decanted away and the precipitate pressed 
out, washed with 80 per cent, alcohol and 90 per cent, alcohol, the final parts 
of the washings being pressed out. The precipitate is placed in a flask with 300 
c.c. of water and heated on a water-bath. The phosphates collect leaving a 
clear liquid. To facilitate filtration through a hot- water funnel 10 c.c. of 20 
per cent, sodium hydroxide are added. The filtrate is acidified with acetic 
acid and poured into 75 c.c. of 95 per cent, alcohol. The liquid is decanted 
and the precipitate washed as before. It is ground up in a mortar with one 
or two changes of absolute alcohol a greater amount of washing . causes 
emulsification and it crumbles to a fine powder, which is filtered off and 
washed with alcohol and dried in a desiccator. About 33 gm. of sodium 
nucleate are obtained from i kilo, of thymus. 

Nucleic acid can be obtained by pouring the solution into about 3 volumes 
of alcohol containing 2 c.c. of concentrated hydrochloric acid per 100 c.c. 

Properties. 

Nucleic acid is a white powder, insoluble in alcohol and ether, but soluble 
in alkalies and ammonia ; it is insoluble in water forming a slimy mass. It is 
not precipitated from dilute alkaline aqueous solution by acetic acid, but it is 
precipitated by mineral acids. Acetic acid will precipitate nucleic acid from 
concentrated solutions in the presence of small amounts of alkaline acetates. 

The sodium salt dissolves in water ; a 4 per cent, solution gelatinises. The 
solutions are optically active. The phosphorus and nitrogen content serve to 
characterise nucleic acid, but proper characterisation is only possible by an 
analysis of its decomposition products. 



302 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Preparation of Plant Nucleic Acids. 

These nucleic acids are most simply prepared by the method of Clarke 
and Schryver. 1 The material is boiled with alcohol to render the proteins 
insoluble and extracted with i o per cent, sodium chloride solution ; on acidify- 
ing the extract, the nucleic acid is precipitated. In the case of wheat embryos 
it is advantageous to digest away the starch previous to the extraction. 

(i) Preparation of Yeast Nucleic Acid. 

30 Ibs. of freshly pressed yeast are mixed with excess of 95 per cent, 
alcohol and allowed to stand for 24 hours. The alcohol is filtered off on a 
Buchner funnel and the solid dried in the air. 

The air-dried solid, in portions of i kilo., is boiled for 2 hours with 95 
per cent, alcohol, filtered off, pressed, dried in an air current at 37 and ground 
to a fine powder, i kilo, of this material is extracted for 4-5 days with 10 
litres of 10 per cent, sodium chloride solution at 60-80, the mass being raised 
to this temperature daily and allowed to cool slowly. The extract is strained 
through muslin and the residue pressed out. The extract is filtered clear 
through paper and acidified with dilute hydrochloric acid (i : i) which is added 
with vigorous stirring. Nucleic acid separates and settles out as a hard cake. 
After 2 hours the liquid is syphoned off; the crude nucleic acid is washed 
with 50 per cent, alcohol till free from chlorine, left under 95 per cent, alcohol 
for 24 hours, washed with absolute alcohol and ether. A fine light brown 
powder in a yield of 1*5 per cent, is obtained. The crude nucleic acid (20 gm.) 
is purified by dissolving in 10 per cent, sodium acetate solution (500 c.c.), 
filtering from insoluble matter, adding i oo c.c. of alcohol and acidifying with 
hydrochloric acid. A white mass separates ; it is washed with 50 per cent, 
alcohol, 95 per cent, alcohol, ether and dried in vacuo. This preparation 
contains 9*3 percent, of phosphorus and 16*4 per cent, of nitrogen. Further 
purification is effected by dissolving 5 gm. in 200 c.c. of water and 100 c.c. of 
o'i per cent, caustic potash and precipitating the clear solution with 20 c.c. 
of '5N hydrochloric acid. An equal volume of alcohol is added, the pre- 
cipitate of nucleic acid is filtered off, washed with alcohol and ether and dried 
in vacuo. It contains 9-6 per cent, of phosphorus and 16-4 per cent, of 
nitrogen. 

(ii) Preparation of Wheat Nucleic Acid (Tritico-nucleic acid}. 

i kilo, of fresh wheat embryos is boiled for 2 hours with 3 litres of 95 per 
cent, alcohol, filtered and pressed free from the liquid and dried in the air. 
The air-dried material is put in small portions at a time in 10 litres of water 
and heated for 2 hours in a boiling water-bath to gelatinise the starch. The 
thick paste so formed is transferred to two flasks and cooled to 40 and to each 
are added 2-5 gm. of takadiastose. The liquids are covered with a layer of 
toluene and fermentation allowed to continue till starch can no longer be de- 
tected. The liquid is boiled, cooled, sodium chloride added so as to make a 
10 per cent, solution of salt, and heated daily for 4-5 days to 60-80. The 
solution is filtered through paper pulp and pressed out from the residue. 100 
c.c. of hydrochloric acid (i : i) are added to the clear filtrate. The precipitate 
of crude nucleic acid is washed free from chlorine with 50 per cent, alcohol, 
left under 95 per cent, alcohol, washed with absolute alcohol and ether and 
dried in vacuo. Yield = 7 gm. 

Purification of the nucleic acid is carried out by solution in 400 c.c. of 
0*1 per cent, caustic potash, precipitation with hydrochloric acid, washing 
with alcohol and ether and drying in the air. This product contains 8-9 per 
cent, of phosphorus and 16-3 per cent, of nitrogen. 

1 Biochem. J., 1917, II. 



FURFURANE, OR FURANE, AND ITS DERIVATIVES 303 



FURFURANE, OR FURANE, AND ITS DERIVATIVES. 

Furfurane and its derivatives contain a five-membered ring made up of 
4 carbon atoms and i oxygen atom. 

The origin, main interest and importance of these compounds is in the 
fact that they are formed from carbohydrates by dry distillation, or by distillation 
with acids. The following compounds are so obtained : 

HC(0) (0)CH HC CH HC CH HC CH 



HC() ( a )CH 



HC C.CH 2 OH HC C.CHO CH 3 . C C . CHO 




O 

Furfur- 
alcohol. 



HC CH 



P i 
Furfur- 
aldehyde, 
furfural 
(furfurol). 
HC CH 



O 

a', or co-methyl- 
furfural. 



HC C.COOH CH,OH C C.CHO 



O 

Pyromucic 
acid. 



or w-hydroxymethyl 
furfural. 



Furfurane, C 4 H 4 O, is obtained by the distillation of the barium salt of 
pyromucic acid and is contained in the tar from pinewood. Furfurane is a 
liquid with a peculiar smell. It boils at 32 and is insoluble in water. It is 
reduced to tetrahydrofurfurane when passed over zinc dust, or nickel dust, 
heated to 1 70. It reacts violently with concentrated hydrochloric acid form- 
ing a brown amorphous substance, and gives a purplish colour reaction with 
sulphuric acid and isatin or phenanthraquinone. 

Furfuralcohol, C 4 H 3 O . CH 2 OH, is formed from furfuraldehyde by re- 
duction, or by the action of caustic soda. It is present in the oil from roasted 
coffee. It is a colourless liquid which boils at 171 and is easily soluble in 
water. In solution it rapidly resinifies. It gives a blue-green colour to a 
pinewood shaving moistened with hydrochloric acid. 

Furfuraldehyde, C 4 H 3 O . CHO. 

Furfuraldehvde or fnrfnrg.1 is the chief compound of the group and is 
formed from bran and other carbohydrates by distillation with dilute sulphuric 
acid. It is formed quantitatively by the distillation of pentoses with dilute 
acid and therefore serves in their estimation (p. 234) : 

O 



. 

! 



CH a OH CHOH.CHO HC C.CHO 

I I = 3H 2 + || H 

CHOH CHOH HC CH 

It is a colourless liquid, which boils at 162 and has a peculiar aromatic 
smell. It turns brown in the air, is easily soluble in alcohol, but only slightly 
soluble in water. 

It has all the properties of an aromatic aldehyde forming an oxime, a 
hydrazone, etc. By alkali it is converted into a mixture of alcohol and alde- 
hyde. It condenses with numerous other compounds. It gives colour re- 
actions with a-naphthol and other phenols which serve in testing for 
carbohydrates (p. 193). 

a-Methyl-furfuraldehyde, C 4 H 2 O . CH 3 . CHO, is formed by the 
distillation of methyl pentoses with hydrochloric acid and resembles furfural. 

Hydroxymethyl-furfuraldehyde, C 4 H 2 O . CH 2 OH . CHO, is formed 
in small quantities from hexoses, especially ketoses, by the action of concen- 
trated acids. It resembles furfural. Molisch's reaction for carbohydrates 
(p. 193) is due to this substance. 

Pyromucic Acid, C 4 H 3 O . COOH, is obtained by the dry distillation of 
mucic acid or by the oxidation of furfural. It is a liquid which boils at 134. 



304 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



THIOPHENE AND ITS DERIVATIVES. 

Thiophene, C 4 H 4 S, was discovered in the benzene from coal tar from which 
it was obtained by extraction with concentrated sulphuric acid. Its constitu- 
tion has been shown to be 

HC CH 

II II 
HC CH 

v 

S 

a heterocyclic compound containing 4 carbon atoms and a sulphur atom. 

Thiophene resembles benzene in its reactions more closely than furfurane 
and pyrrole, but as yet neither thiophene nor its derivatives have been obtained 
from natural substances. 



PYRROLE AND ITS DERIVATIVES. 

Pyrrole and its derivatives are closely connected with the proteins 
and with the two respiratory pigments, chlorophyll and haemoglobin. 
A pyrrole derivative is also present in the molecule of nicotine, and 
cocaine contains a pyrrole nucleus. The heterocyclic ring in pyrrole 
consists of 4 carbon atoms and I nitrogen atom. We have to con- 
sider the following compounds in order to understand the constitution 
of the proteins and the complex chlorophyll and haemoglobin mole- 
cules : 



1 


HQ, CH H 2 C CH 

- II . 11 | II 
HC CH* H,C CH 
\ / " \ / 


H 2 C CH 2 
H 2 C CH, 




\/ \/ 
NH NH 


NH 




Pyrrole. Pyrroline. 


Pyrrolidine. 




CH 3 .C C.C 9 H, CH 3 .C C.C 2 H 5 

II !l II II 


H 2 C CH 2 

I 1 




CH,.C C.H H.C C.CH, 

\/ \x 


H 2 C CH.COOH 




NH NH 


NH 




Isohaemopyrrole Kryptopyrrole 
o, ^-dimethyl, o-methyl, 
3-ethyl-pyrrole. /3-methyl- 0-ethyl 
pyrrole. 


a-pyrrolidine- 
carboxylic acid 
or 
proline. 


CH 
CH 


3 .C C.C.H, CH 3 .C C CH 2 .CH 2 .COOH HO . CH CH 2 

II II II 1! II 
S .C C.CH, CH^.C CH H 2 C CH . COOH 

\ / \ / \ / 




\/ \/ 

NH NH 


\/ 
NH 


3 


Phyllopyrrole Isophonopyrrole- 
or , carboxylic acid 
a, o-dimethyl, o-methyl-3-ethyl- 
methyl-)3-ethyl pyrrole- 
pyrrole. j8-propionic acid. 


Hydroxyproline 
or 
3-hydroxy-o-pyrrolidine 
carboxylic acid. 



PYRROLE AND ITS DERIVATIVES 305 

Pyrrole. C 4 H 5 N. 

Pyrrole was found in coal tar in 1834 and in bone oil in 1858 and 
is usually obtained from bone oil. 

Preparation. 

The oil which is obtained by the dry distillation of bones contains 
pyridine and basic substances, aromatic hydrocarbons, pyrrole and its 
homologues, but consists mainly of the nitriles of fatty acids. The basic 
substances are removed by agitation with dilute acid, the nitriles are 
hydrolysed by boiling with alkali and the oil which remains is fractionally 
distilled. The fraction passing over between 115 and 130 contains the 
pyrrole. By boiling with solid caustic potash it is converted into solid 
potassium pyrrole, QH 4 NK, which is filtered off and decomposed by water. 
The pyrrole is then isolated by distillation. 

Fat-free bone gelatin is said to give a distillate consisting mainly of 
pyrrole and its homologues. 

It is probably formed from the proline and hydroxyproline contained in 
the protein, but may also arise by the dry distillation of glutamic acid. 

It has been synthesised by passing acetylene and ammonia through red- 
hot tubes, by the dry distillation of the ammonium salt of mucic acid and by 
the reduction of succinimide by distillation over zinc dust :-^- 
CH 2 CO. CH^CHs 

>NH + 2H., = | >NH + 2H 2 O. 

CH 2 CO/ CH=CH/ 

Properties. 

Pyrrole is a colourless liquid smelling like chloroform but turning 
brown in the air. It boils at 131, is very slightly soluble in water, 
but is easily soluble in alcohol and ether. 

Pyrrole is a secondary amine and has a slight basic character ; it 
dissolves slowly in dilute acids and is converted into a resin by strong 
acids. Its solution in dilute acids on warming deposits a red precipi- 
tate termed pyrrole red. 

It gives a fiery red colour with a pine shaving moistened with 
hydrochloric acid. Hence its name from irvppos. 

As a secondary amine pyrrole forms a nitroso compound with 
sodium ethoxide and amyl nitrite. 

Potassium dissolves in pyrrole with evolution of hydrogen. The 
combination of pyrrole with potassium to form solid potassium pyrrole 
is probably due to the acid influence of the CH groups. 

The pyrrole ring is easily ruptured.' Succinyl dialdoxime is formed by 
the action of hydroxylamine upon pyrrole. a-substituted pyrroles yield 
ketoximes, /2-substituted pyrroles yield aldoximes from which dibasic acids can 
be obtained. This reaction serves for determining the position of substituting 
groups. 

Pyrrole reacts violently with halogens ; but derivatives are obtained by 
using dilute solutions. Tetraiodopyrrole, which is prepared by the action of 
iodine on pyrrole in the presence of alkali, forms yellow-brown prisms which 
melt at 140. Under the name of iodol it is used as an antiseptic and has 
an advantage over iodoform in possessing no smell. 

2O 



3 o6 PRACTICAL ORGANIC AND BIO-CHEMiSTRY 

Pyrroline and Pyrrolidine. 

Pyrrole is easily reduced by zinc and acetic acid, or by electrolysis, 
to pyrroline ; it is converted into pyrrolidine by hydriodic acid, or 
by passing pyrrole and hydrogen over nickel dust heated to 190. 

Pyrrole is a liquid boiling at 91 and has an ammoniacal smell. 
It is a strong base and forms stable salts with acids. 

Pyrrolidine is a liquid which has a smell resembling pepper. It 
boils at 87 and like pyrroline is a strong base. 

Proline and Hydroxyproline. 

These compounds are constituents of proteins. They result from 
the hydrolysis of proteins by acids or alkalies. A complex process 
of separation is required to isolate them from proteins (see " Chemical 
Constitution of the Proteins "). They differ from other units of the 
protein molecule by being easily soluble in alcohol (cf. glycine, p. 1 39). 

Alkyl Derivatives of Pyrrole. 

Derivatives of pyrrole are easily prepared from potassium pyrrole. 
Potassium pyrrole reacts with alkyl halides, acid chlorides, etc., to form 
derivatives in which the substituting group is attached to the nitrogen 
atom : 

L/ri ^zrC/ri v L/H. ^^^t^rr ^ 

I >NK + CH 3 I = | >N . CH 3 + KI. 

CH CH/ CH=CH/ 

On heating, these compounds undergo rearrangement ; the sub- 
stituting group changes its position and attaches itself to a carbon 
atom. 

Isohaemopyrrole, kryptopyrrole, phyllopyrrole and other alkyl 
pyrroles are formed by the reduction of haemin, chlorophylland bile 
pigments. A mixture is obtained from which the individual compounds 
are separated. 



PYKIDINE AND ITS DERIVATIVES 307 

PYRIDINE AND ITS DERIVATIVES. 

The six-membered heterocyclic ring compounds, containing 5 
atoms of carbon and I atom of nitrogen, of which pyridine is the 
simplest member and from which all the other compounds of the group 
can be derived, resemble the benzene compounds very closely. The 
simpler members are present in coal tar and bone oil and are formed 
by the oxidation of the complex alkaloids which occur in plants. 

The Structure of Pyridine. 

The empirical formula of pyridine, CsH^N, points to its not being an open 
chain compound. For reasons similar to those which led to the adoption of 
a closed ring structure for the constitution of benzene and from the great 
similarity which pyridine has to benzene in its reactions the following ring 
structure has been assigned to pyridine : 

OH 

$' Hi 

m 

This structure shows that pyridine is a tertiary base, that three isomeric 
monosubstitution and six disubstitution derivatives can be derived from it ; 
in general it expresses all the facts known about pyridine and its derivatives. 

Pyridine. 

Pyridine was first obtained from bone oil, but is contained in coal 
tar from which it is usually prepared. 

Preparation. 

The acid solution, or liquor, which results in the purification of benzene 
and its homologues from coal tar is treated with sodium hydroxide ; the 
basic substances separate out as oils. This oil consists of a mixture of 
pyridine, its homologues, quinoline and other substances. These constituents 
can be separated by repeated fractional distillation, but the pure compounds 
are finally isolated by the fractional crystallisation of their salts. The bases 
are liberated from the salt by alkali and purified by distillation. 

Bone oil is extracted with sulphuric acid ; the bases are separated by 
sodium hydroxide, distilled and purified as described above. 

Properties. 

Pyridine is a colourless liquid which boils at 1 1 5 and has a specific 
gravity of 1-003 at - -It has a pungent characteristic and disagree- 
able odour and mixes with water in all proportions. 

It is a strong base, which can turn red litmus blue, and forms salts 
with acids. 

Pyridine is a tertiary amine as shown by its negative behaviour 
to nitrous acid and the fact that it combines with alkyl halides to 
form pyridine alkyl halides, such as pyridine methiodide C 5 H 5 N . CH 3 I. 

On heating, the methyl group attached to the N atom changes its 
position and a-alkyl pyridines are formed, 



308 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

This reaction may be used as a test for pyridine : 

If a few drops of pyridine be heated with a few drops of methyl 
iodide, a violent reaction takes place and pyridine methiodide is formed. 
On adding a small quantity of solid potash and again heating, the com- 
pound is decomposed and the disagreeable smell of methyl pyridine 
hydroxide will be noticed. 

Pyridine is not oxidised by nitric acid, or chromic acid, and it is only 
slowly attacked by the halogens and sulphuric acid forming substitution 
products. 

Piperidine. C 5 H 10 NH. 

Piperidine is a constituent of the alkaloid piperine from which it is 
obtained by hydrolysis with alkali. 

It is formed by the reduction of pyridine with sodium and alcohol, 
and it is converted into pyridine by heating with concentrated sulphuric 

acid at 300 : 

CH 2 

H 2 C/\CH 2 

^ 1 I 

H.CV^CH, 

N NH. 

Piperidine is also formed by the distillation of pentamethylene 
diamine : 

HN 2 . CH 2 . CH, . CH 2 . CH, . CH, . NH 2 = NH 3 + CH 2 \NH. 

\/"^T_T /"*TT 

v/ rT 2 ^ rl 2 

Preparation. 

Powdered pepper is extracted with alcohol ; the extract is evaporated to 
dryness and the residue is distilled with soda. The alkaline distillate is 
neutralised with hydrochloric acid and evaporated to dryness. The residue 
which consists of ammonium chloride and piperidine hydrochloride is treated 
with hot alcohol. The solution containing the piperidine hydrochloride is 
evaporated and distilled with soda and the oil which passes over is purified 
by distillation. 

Properties. 

Piperidine is a colourless liquid which boils at 106. It has the 
pungent smell of pepper and mixes with water. It is a strong base 
and a secondary amine, forming nitrosopiperidine with nitrous acid. 



PYRIDINE AND ITS DERIVATIVES 309 

Homologues of Pyridine. 

The following homologues of pyridine, mixed with pyridine, are contained 
in the basic fraction of coal tar and bone oil and are separated by fractional 
distillation as stated under pyridine. 

I. The monomethyl pyridines or picolines : 

CH S 

A 

CH 3 

N N N 

a-methylpyridine j8-methylpyridine y-methylpyridine 

or or or 

a-picoline. /3-picoline. 7-picoline. 

II. The Dimethyl pyridines or lutidines. 

III. The trimethyl pyridines or collidines. 
They closely resemble pyridine in properties. 

Pyridine Carboxylic Acids. 

The side chain of the methyl pyridines on oxidation is converted into 
carboxyl and the pyridine carboxylic acids are obtained : 

COOH 




^COOH 
I COOH 



N N N 

Picolinic acid. Nicotinic acid Isonicotinic acid. 

These compounds are white crystalline solids soluble in water. They 
possess basic properties and acidic properties forming salts with acids and 
bases. 

Nicotinic Acid is formed by the oxidation of nicotine (p. 353). This 
substance therefore contains a substituting group in the /^-position of the ring. 

Picolinic Acid gives a red coloration with ferrous sulphate. This 
reaction is given by all acids derived from pyridine containing a carboxyl group 
in the a-position. 

Quinolinic Acid is formed by the oxidation of quinoline with per- 
manganate. . Quinolinic acid is a dibasic acid having the constitution : 

'jCOOH 
JCOOH 



It is a crystalline solid, soluble with difficulty in water. It gives an 
orange coloration with ferrous sulphate which shows that one carboxyl 
group is in the a-position. It is converted into nicotinic acid when it is 
heated to 190. These reactions show the position of the carboxyl groups. 



HYDRO-AROMATIC COMPOUNDS. 

Benzene, its homologues and derivatives, though they form a special 
group of compounds with special properties, behave nevertheless in 
some respects like unsaturated compounds. They can be reduced 
under certain conditions and they will combine by addition with the 
halogens on exposure to sunlight. The reduced compounds are 
known as the hydro-aromatic compounds, and the halogen addition 
compounds are regarded as derived by substitution from reduced ben- 
zene. The aromatic compounds are most easily reduced by the method 
of Sabatier, which consists in passing their vapour mixed with hydrogen 
over nickel dust heated to about 170. 

Benzene gives three reduction products : 

CH 2 CH CH 




"H 2 CH CH 

Hexahydrobenzene, Tetrahydrobenzene, Dihydrobenzene, 

or or or 

Hexamethylene, or Cyclohexene. Cyclohexadiene. 

Cyclohexane. 

These compounds correspond to the carbocyclic compounds con- 
taining 3, 4, 5, etc., atoms of carbon (p. 237), and have properties like 
the aliphatic compounds. 

The number of compounds in this group is not very large, but the 
natural compounds, the terpenes, inositols, cholesterol, cholalic acid are 
hydro-aromatic compounds. 

THE INOSITOLS. 

The small number of natural compounds included under this heading 
are hydroxy-derivatives of hexahydrobenzene. 

Quercitol, or cyclohexanepentol, is found in acorns and in the leaves 
CH, of Chamaerops humilts, a variety of palm. It is a 

HOHC( //X NcHOH c l ur l ess solid which melts at 235 and is dextro- 
rotatory, [a] D = + 24-1 6. A laevorotatory quercitol has 
| HC ( S ^/ | CH been found in the leaves of Gymnema sylvestre. It 

CHOH melts at 174 and has [a] D = - 73'9- 

Inositol. Several varieties of inositol have been found in nature : 

an inactive form, 2 active forms and a racemic form. 

CHOH Seven inactive forms are theoretically capable of exist- 

HOHC/^CHOH ence - i- Inositol is found ;n heart-muscle and other 

animal organs, but is present in larger amounts in unripe 

' HCl \ v / CHOH beans and peas. It is present in the free state and also 

CHOH in combination with phosphoric acid as ester in the 

husks of various cereals. The calcium and magnesium 

salt of this acid is termed Phytin. d-Inositol is obtained by the reduction of 
pinitol with hydriodic acid. I- Inositol is obtained from quebrachitol by 
reduction. 

Scyllitol, an inactive inositol, is present in the organs of various 
elasmobranch fish the dog fish, skate and shark. 

310 



INOSITOLS 311 

Cocositol has been isolated from the leaves of cocos and closely 
resembles /-inositol. 

Pinitollis monomethyl ^-inositol. 
Quebrachitol is monomethyl /-inositol. 
/-Inositol. 
Preparation. 

(1) From phytin. Phytin is extracted from unripe peas or husks of 
cereals with dilute hydrochloric acid. The acid extract is either neutralised 
or made alkaline with ammonia, or calcium chloride is added and it is made 
alkaline with ammonia. The calcium-magnesium or calcium salt of phytic 
acid, or phytin, is precipitated. It is redissolved in acid and reprecipitated, 
filtered off and dried. 

Phytin is hydrolysed by heating in a sealed tube with dilute sulphuric acid 
to about 150 for several hours. On removing the acids with baryta and 
evaporating to a small volume and adding alcohol, the inositol is precipitated. 

It is recrystallised from a mixture of water and alcohol. 

(2) From muscle. Muscle is extracted with water ; the aqueous extract 
is boiled to coagulate proteins and the filtrate treated with lead acetate to 
remove the remaining proteins. The clear solution is precipitated with basic 
lead acetate. The precipitate is suspended in water and decomposed with 
hydrogen sulphide and the solution, filtered from lead sulphide, is concen- 
trated. On adding from 2-4 volumes of alcohol and filtering rapidly from 
any amorphous precipitate and allowing to stand, inositol crystallises out, or 
ether may be added until a turbidity appears and the mixture again allowed 
to stand. The crystals are recrystallised from water to which from 2-4 
parts of alcohol are added. 

Properties. 

/-inositol separates in large colourless crystals (Fig. 51) or, if impure, in 
bunches of small crystals, and contains 
two molecules of water of crystallisation 
which are given off at 110. The an- 
hydrous substance melts at 225. It is 
soluble in water (i part in 7^5 parts at 
20) and the solution has a sweet taste. 
On account of this property and its 
empirical composition it was formerly 
called muscle sugar. It does not re- 
duce Fehling's solution, nor has it any 
of the properties of a carbohydrate. 

Reactions. 

(1) ShereSs Test. A rose- red 
colour is formed on evaporating a small 
quantity with nitric acid in a procelain 
basin nearly to dryness, adding am- 
monia and a few drops of calcium 

chloride and again evaporating to dry- FlG - 51- Inositol. (After Funke.) 

ness. 

(2) SeideVs Test. If in the above reaction strontium acetate be used 
instead of calcium chloride, a green colour and a violet precipitate are formed. 

(3) Gallois' Test. On evaporating a solution of inositol nearly to dryness, 
adding a drop of mercuric nitrate and again evaporating to dryness, a yellowish 
residue is left. This turns a dark rose-red on warming ; the colour disappears 
on cooling. 




312 PRACTICAL ORGANIC AND BIO-CHEMISTRY. . 

THE TERPENE GROUP. 

Nearly all parts of plants contain volatile substances with a highly 
characteristic and pleasant smell. These substances are the essential 
oils, e.g., oil of turpentine, oil of lemons, etc. The various kinds of 
camphor, which are crystalline solids, the resins and india-rubber are 
closely related substances. 

They are prepared from plants by steam distillation, by pressing, 
or by extraction with organic solvents. Besides their use in per- 
fumery, in making essences, they are used in the preparation of oil 
paints, varnishes, etc. Several are used in medicine. 

The essential oils are generally complex mixtures, the main constitu- 
ent imparting the characteristic properties ; several essential oils may 
contain the same constituent and yet differ in smell on account of the 
presence of different highly odoriferous substances. Oil of turpentine 
exists in the greatest quantity. It flows from the stems of pine trees 
when incisions are made in the surface and consists of solids dissolved 
in the liquid. Crude oil of turpentine is separated by steam distillation, 
the solids remaining behind and constituting colophony or resin. 

The chief constituent of oil of turpentine is pinene. Limonene is 
present in oil of lemon. They are colourless, very refractive liquids 
boiling between 150 and 180. Camphene is solid. They are insol- 
uble in water, but soluble in most organic liquids. They are good 
solvents, dissolving resin, caoutchouc, iodine, phosphorus and sulphur. 
The majority are optically active ; sometimes both the dextro and 
laevo forms are found in nature, and the inactive mixture of some of 
them has been prepared. 

Reactions. 

(1) They easily polymerise to form resinous substances. 

(2) Oh exposure to air or oxygen, they are oxidised and yield 
resins. 

(3) On oxidation with permanganate, etc., they are converted into 
benzene derivatives. 

(4) On treatment with ozone, they form ozonides. 

(5) On reduction, they are converted into hydroterpenes. 

(6) They combine with bromine and halogen acids to form addi- 
tion compounds, which are frequently crystalline solids. 

(7) They react with nitrosyl chloride, NOC1. 

Constitution of the Members of the Terpene Group. 

Most of the members of the terpene group are unsaturated hydrocarbons 
of the formula C 10 H 16 ; others, such as camphor, are alcoholic or ketonic 
derivatives and possess the empirical formulae C 10 H 16 O, C 10 H 18 O, C 10 H 20 O. 

Though most of the hydrocarbons have the empirical formula C 10 H 16 
several have the formulae C 15 H., 4 , C 20 H 32 and (C 6 H 8 ) n . The unsaturated 
hydrocarbon isoprene, C 5 H 8 , has the same percentage composition and is 
obtained by the distillation of caoutchouc ; it polymerises to a hydrocarbon 
C 10 H ]6 and can be made to polymerise to caoutchouc, the constituent of 
rubber. The group may therefore be divided into 



THE TERPENE GROUP 



3*3 



(1) Hemiterpenes C 5 H 8 

(2) Terpenes C 10 H 16 

(3) Sesquiterpenes C 15 H 24 



(4) Diterpenes C 20 H 32 

(5) Polyterpenes (C 5 H 8 ) n 

and their derivatives. 



The structure of most of the compounds has been established and many 
of the natural ones have been prepared by synthesis. 
A few are open chain compounds, namely : 

Myrcene \C=CH . CH 2 . CH 2 . C^ 

or 
PT-i r*Tj 

2 \ / ^f*2 

^C CH 2 . CH 2 . CH 2 . C" 
CH/ \CH=CH 2 

C* TT O T.T 

\*>rln . v^rlo 

Ocimene ^C=CH . CH, . CH=C/ 



Citronellol 



Geraniol 



Linalool 



Citral geraniol 



CH 2 

CH 3 
CH 3 

CH 3 
CH 3 

CH 3 
CH., 



CH, . CH . CH 



\CH.,.CH OH 



\ 



CH 2 .CH 2 .C/ 

^CH.CHoOH 

XCH 3 
CH . CH .C CH=CH n 



.CH, 



C=CH . CH 2 . CH . C 



Geraniol makes up about 90 per cent, of Indian geranium oil. Citral is 
present in oil of lemons, orange, etc., and is obtained by oxidising geraniol. 
Linalool is present in lavender oil and bergamot oil. Terpin (p. 315) is 
formed by shaking it with acids. 

Most are hydro-aromatic compounds derived from cymene and are most 
conveniently regarded as unsaturated hydrocarbons derived from hexahydro- 
cymene or menthane, 



and possessing 
the skeleton 





H 



CH 3 

Menthane. 




From the saturated menthane six isomeric menthenes containing one 
double bond may be derived : 









314 



PRACTICAL ORGANIC AND BIO-CHEMISTRY 



and from these a larger number of isomers, menthadienes, containing two 
double bonds, e.g. 







Further, instead of forming the second double bond between two adjacent 
carbon atoms union may be formed between the carbon atoms 8 and 3, 8 and 
2, 8 and i, thus 








The chief terpenes and their oxygen derivatives are classified in several 
groups and have been found to possess the following structural formulae : 

Menthane Group. 
CH 




CH 3 

Carvomenthol. 



CH S 

Carvomenthone. 



THE TERPENE GROUP 



315 



GEL 




HO 




CH 




trans-Terpin. cis-Terpin. Cineol, eucalyptol. 

Menthane is not a natural product. 

Menthol is the chief constituent of peppermint oil and can be made by 
reducing menthone. 

Menthone is present in Japanese, American and Russian peppermint oil, 
It exists in two optically active forms. 

Terpin is formed by the action of dilute acids upon the terpenes in tur- 
pentine and upon other terpenes. 

Cineol is found in eucalyptus oil and is the anhydride of cis-terpin. 

Terpin easily forms a crystalline hydrate which melts at 117. 

Menthene Group, C 10 H 18 . 




Pulegone. 



Dihydrocarvone. 



316 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



" Liquid " terpmeol is made from terpin hydrate by treatment with dilute 
sulphuric acid and is used largely in perfumery. Terpinenol is found in 
cardamom and majoram oil. Dihydrocarveol has been detected in oil of 
kummel. Pulegone is contained in the essential oils of Mentha pulegium and 
Hedeoma pulegio'ides and dihydrocarvone has been found in kummel oil. 



CH 




HoC 



HC 



d and Mimonene, 
d, /-limonene 

or 
dipentene. 

CHg 




o-Terpinene 

CH n CH 3 




H 



CH 



CH 3 

a-Phellandrene. 



Menthadtene Group. 
CH 3 CH 3 



HC 



HC 



CH 3 

Terpinolene, 
not natural. 



H., 





)8-Terpinene, 
not natural. 



CH 3 




HoC 



CH 2 

3- Phellandrene. 

CH 3 JCH 2 




CH 3 

Carvone. 



CH 



CH, 




HC 



2, 4-A-Menthadiene, 
not natural. 




CH 3 
y-Terpinene. 



CH 3 




CH 3 



CH, 



Sylvestrene. 



THE TERPENE GROUP 



317 



</-Limonene is found in numerous essential oils, in that of lemon, ber- 
gamot, kummel, dill, celery. 

/-Limonene is present in pine-needle oil and Russian peppermint oil. 

Dipentene, or d, /-limonene, is present in Russian and Swedish turpentine 
which has been heated to a high temperature. It is also formed by heating 
other terpenes. It is contained in the distillate from rubber, having been 
formed by the polymerisation of isoprene. 

a and y-Terpinenes are found in cardamom oil and other oils. They 
have a smell of lemons. 

Phellandrene is present in fennel oil. Carvone is present in oil of 
kummel and oil of dill. 

Sylvestrene is contained in Swedish and Russian turpentine and pine- 
needle oil. It is dextrorotatory and also has a smell of lemons. 

P inane Group. 
H CH 





CH 3 

d and /-Pinene. 




CH 2 OH 

Myrtenol 

Camphane Group. 
CH CH 



CH 2 
CHOH 



HoC 




Borneol or 
Borneo camphor. 



CH, 



Camphor or 
Japan camphor. 



It 




II 




H 

CH 

Camphene. Bornylene. 

</-Pinene is the chief constituent of American, Algerian and Greek tur- 
pentine, /-pinene of French and Spanish turpentine ; both are prepared by 
fractional distillation. 

Camphene is a solid terpene and is known in its optically active forms. 
It is present in numerous essential oils ginger, Siberian pine-needle, 
camphor. Bornylene is not a natural terpene, but borneol occurs as d- 
borneol in Dryobalanops Camphora, which is grown in Borneo and Sumatra, 
as /-borneol in the oil of Blumea balsamifera. As ester with fatty acids it 
occurs in pine-needle oil. It is very like Japan camphor, but has also a 
peppermint smell. 

^/-Camphor, or Japan camphor, occurs in the camphor tree, Cin- 
namomum camphora, and is obtained by distillation and sublimation. It is 
made artificially from the pinene in turpentine. Pinene is converted into 



318 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



borneol or isoborneol and oxidised with permanganate, ozone, or nitric acid. 
It is a colourless, transparent, tough mass, which crystallises from alcohol and 
is very volatile, and is used in making celluloid and smokeless gunpowder. 

Sabinane Group. 

CH 3 CH 3 




CH 2 

Sabinene. 




CH 3 

a-Thujene. 




CH 3 

3-Thujene. 



CH, 




CH, 




CH 3 
Sabinol. Thujone. 

These terpenes are present in various essential oils in small quantities. 

It seems most likely that all the terpenes are made by the condensation 
of isoprene by the action of acids in the plant juices, and it is most remarkable 
that so many different isomers can be formed from the unsaturated isoprene. 
The method of formation of isoprene in plants is unknown, but it may arise 
by removal of carbon dioxide and ammonia from leucine and isoleucine. 

Caryophyllene, santalene, santalol belong to the sesquiterpene group. 
They are present in the various essential oils, are yellowish, viscous liquids 
boiling between 250-280 with slight but not pleasant smell, and they easily 
change into resins. 

The diterpenes and polyterpenes are also yellow viscous liquids boiling 
above 300 and not easily volatile with steam. They are found in balsams 
and resins. 

The resins occur in the plant oils and are also formed from the terpenes 
by oxidation in the air. Their solutions in the terpenes are generally called 
balsams ; the solid resins are amorphous shining substances. They con- 
sist of a mixture of resin acids and dissolve in alkalies from which they are 
precipitated by acids. They yield various aromatic compounds by fusion 
with potash and on reduction yield benzene, naphthalene, etc. 

Caoutchouc, the constituent of rubber, is particularly important industrially. 
The substance which forms caoutchouc can be extracted by ether from the 
plant juice, and on exposure to light or by action of acids it polymerises to 
rubber. Pure caoutchouc is soluble in benzene, carbon disulphide, chloro- 
form, etc. It is acted upon by ozone giving a diozonide. 

It can take up sulphur by kneading with sulphur or by treating with sulpur 
dissolved in sulphur chloride and this combination constitutes rubber, ebonite. 

The colour of rubber depends on whether lead oxide, antimony oxide, etc., 
has been used in the vulcanising process. 



CHOLESTEROLS 319 

THE CHOLESTEROLS. 

Cholesterol. 

Cholesterol was discovered in bile and has been found in the bile 
of all animals with one exception. It has since been shown to be 
present in small quantities in blood and the tissues of man and 
animals ; in somewhat large quantities it is present in bone marrow 
and nervous tissue. It is very seldom found in urine and, when 
found, only in the smallest quantities. Crystalline deposits of choles- 
terol are found in pathological effusions, in pus and in diseased arteries. 
Gallstones usually consist almost entirely of cholesterol. Not only is 
cholesterol present as such, but also in the form of its esters with 
the higher fatty acids. Lanolin, or wool fat, is composed mainly of 
esters of cholesterol. 

Constitution. 

Cholesterol has been shown to contain an OH group and to be 
a secondary alcohol by oxidation to a ketone. It contains one un- 
saturated bond and an isoamyl group. It belongs to the group of 
terpenes and its formula according to Windaus is the following : 

CH,\ 

)CH . CH 2 . CH 2 C U H 17 

CH 3 / / \ 

CH CH 

/\ /\ 

H C CH CH . CH, 

I I 

H C CH CH 

"\/ II 

CHOH CH 2 . 

The structure of C n H 17 has still to be determined. 

Preparation. 

(1) From Gallstones. 

Cholesterol is. most easily prepared from gallstones. The powdered 
stone is extracted with a mixture of ether and alcohol. The filtered 
solution on evaporation leaves a residue of cholesterol. It is purified 
by boiling with alcoholic potash, the solution is evaporated to dryness 
and the residue extracted with ether. The crystals obtained after 
evaporation of the ether are recrystallised from alcohol. 

(2) From Brain. 

Sheep's brain is thoroughly ground up with some sand and about 
3 parts of plaster of Paris. This mass when it has become hard is 
powdered and covered with acetone. Dried sheep's brain is extracted 
with acetone. The acetone is filtered off and the mass again treated 
with acetone. On distilling off the acetone, crystals of cholesterol 
remain. They rnay be purified as above. 




320 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

(3) From Tissues. 

The tissue is dried by spreading out in thin layers on plates, by mixing 
with alcohol and evaporating, or by mixing with an absorbent material, such 
as sand or siliceous earth. The dry mass is extracted with ether in a Soxhlet 
extractor. The ethereal extract is mixed with excess of alcoholic potassium 
hydroxide to saponify the fats and esters, any soap which is formed is filtered 
off and the ethereal solution distilled to remove ether. The residue is dis- 
solved in water and extracted with ether. The ethereal solution containing 
the cholesterol is evaporated and the cholesterol recrystallised. 

Properties. 

Cholesterol forms a white crystalline solid melting at 147. It 
crystallises in needles from ether, benzene, etc. ; in characteristic four- 
sided plates with a notched angle and 
containing I molecule of water of 
crystallisation (Fig. 5 2) from aqueous 
alcohol. It is insoluble in water 
^ and soluble with difficulty in cold 

f alcohol. In hot alcohol, ether, ace- 
tone, chloroform and other organic 
i 

solvents it is readily soluble. 

The unsaturated character of 
cholesterol is shown by the forma- 
tion of addition compounds with the 
halogens and halogen acids. 

As an alcohol, cholesterol forms 

FIG. 52. Cholesterol. (After Funke.) r~. 

esters. I he acetate and benzoate 

are very characteristic and serve to distinguish cholesterol from phy- 
tosterols (p. 322). 

Cholesteryl Acetate. A small quantity of dry cholesterol is boiled 
with 2 or 3 c.c. of acetic anhydride for I or 2 minutes. The acetic 
anhydride is evaporated, or the solution is poured into water. The 
residue, or precipitate, is crystallised from dilute alcohol. It melts 
at 1 1 4. 

Cholesteryl Benzoate. Cholesterol is boiled with benzoyl 
chloride for a few minutes and the solution poured into alcohol. The 
precipitate of cholesteryl benzoate is recrystallised from hot alcohol. 
This compound melts at 145 to a turbid liquid, which becomes clear 
at 178-180; on cooling it exhibits a play of colours, of which blue 
is the most marked. 

Esters of cholesterol with palmitic, oleic and other acids have also 
been prepared. 




CHOLESTEROLS 321 

Tests. 

(1) Crystalline form of crystals separated from alcohol. 

(2) On running a drop of sulphuric acid (5 vols. of cone, acid to 

1 vol. of water) upon some crystals on a glass slide, covered with a 
cover slip, the crystals become red. A drop of iodine solution placed 
against the cover slip and brought into contact, with the crystals 
by drawing it through with filter paper, changes the colour of the 
crystals at the points of contact to violet, blue and black. 

(3) Salkowskfs Reaction. A small quantity of dry cholesterol 
is dissolved in a little chloroform in a dry test tube. An equal 
volume of concentrated sulphuric acid is added and the liquids mixed. 
The chloroform rises to the surface coloured at first red, then purple, 
and the sulphuric acid is yellow and shows a green fluorescence. If 
the chloroform be poured into a basin it becomes blue, green and yellow. 
It is decolorised if water be added, but the colour returns on adding 
strong sulphuric acid. The colour is only stable in the presence of 
acid. If the sulphuric acid be diluted with glacial acetic acid it be- 
comes red, but still shows a green fluorescence. 

(4) Liebermanns Reaction. A little cholesterol is dissolved in about 

2 c.c. of chloroform in a dry test tube, 2 or 3 drops of acetic anhydride 
are added and drop by 'drop concentrated sulphuric acid. A red colour, 
which becomes blue and finally bluish-green, is formed. 

(5) Tschugaiejfs. Reaction. A little cholesterol is dissolved in glacial 
acetic acid, excess of acetyl chloride and a piece of fused zinc chloride are 
added and the mixture warmed for 5 minutes. The solution becomes red. 
with a green fluorescence. 

(6) Neuberg's Reaction. If to some cholesterol dissolved in 2 or 3 c.c. 
of absolute alcohol a trace of rhamnose (or a solution of methyl furfural) be 
added and sulphuric acid be run under the solution, a red ring is formed at 
the junction ; on mixing and keeping the mixture cold, the red colour diffuses 
throughout the fluid. 

Isocholesterol. C 27 H 46 O. 

Isocholesterol has been found together with cholesterol in lanolin. 

Coprosterol. C^H^O. 

Coprosterol has been found in human faeces ; it is probably formed by 
the reduction of cholesterol in the large intestine. 

H ippocoprosterol. 

Two compounds, C 27 H 54 O and C 27 H 52 O, have been isolated from horses' 
manure and are probably reduction products of phytosterol. 

Spongosterol. C 19 H 32 O ? 

This compound has been isolated from sponges and is very like cholesterol 
in appearance but melts at T 19-120. 

21 



322 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Phytostsrols. C 27 H 46 O. 

Compounds very similar to cholesterol have been prepared from plants 
and have been termed phytosterols. They are probably mixtures of isomeric 
compounds, that from the Calabar bean having been shown to be a mixture of 
sitosterol, C 27 H 45 OH, and stigmasterol, C 30 H 47 OH. They are mostly contained 
in the fat of plant seeds. 

They are prepared from the vegetable fat of the seeds by saponification 
with alcoholic potash ; the alcohol is evaporated and the residue dissolved 
in water. The aqueous solution is extracted with ether, and this extract 
on evaporation yields the phytosterol, which is recrystallised from alcohol. 

The phytosterols crystallise like cholesterol, but the crystals are usually 
six-sided. The melting-point of the phytosterols varies from 135-144^ 
but is usually' 135-137. Insolubility they resemble cholesterol and form 
acetates and benzoates. The acetates melt at 125-137. They can only be 
distinguished from cholesterol by their crystalline form and the melting-point 
of the acetates. 

BILE ACIDS. 

The bile of animals contains the sodium salts of glycocholic and 
taurocholic acids, glycocholeic and taurocholeic acids. These acids 
are decomposed by boiling the bile with sodium hydroxide and yield 
glycine or taurine and cholalic, choleic and deoxycholic acids. 
Choleic acid, according to Wieland and Sorge, 1 is a combination of 
8 mols. of deoxycholeic acid with I mol. of fatty acid (palmitic or 
stearic). They have prepared similar combinations of deoxycholeic 
acid with acetic and other fatty acids. 

Cholalic Acid or Cholic Acid. C 24 H 40 O 5 . 

'COOH Though the constitution of cholalic acid is 

^_ -CHOH unknown, its properties and reactions seem to 
20 3\~~ CH 2 OH show that it should be included amongst the 
^CH 2 OH hydro-aromatic compounds. Its products of oxi- 
dation and their properties show that it is a trihydroxy monobasic acid. 
Dehydrocholalic Acid. C 24 H 34 O 5 . 

COQH This acid is obtained by the oxidation of cholalic 

ac 'd with chromic acid in glacial acetic acid. It crys- 
fHO tallises from alcohol in needles melting at 231-232; it 
is soluble with difficulty in cold water and cold alcohol, 
is dextrorotatory and has a bitter taste. It forms esters, 
yields a trioxime with hydroxylamine and contains i keto and 2 aldehyde 
groups. 

Bilianic Acid, C 24 H 34 O 8 , and Isobilianic Acid. 

..COOH These acids are formed by the oxidation of cholalic 

/ COOH ac id or dehydrocholalic acid with potassium perman- 
^19 H 31 ^-_ COOH g anate in alkaline solution. 

VSs, ro Bilianic acid is soluble with difficulty in cold water, 

\ and crystallises from alcohol in sparkling crystals. It is 

a tribasic acid containing two ketonic groups. 
Cilianic Acid. C 20 H 28 O 8 . 

This is another tribasic acid obtained by the oxidation of cholalic acid. 
1 Zeitschr. physiol. Chem., 1916, 97, i. 





BILE ACIDS 323 

Preparation of Cholic Acid, Choleic Acid and Deoxycholic Acid. 

The most recent methods of preparing these acids from bile are described 
by Pregl and Buchtala, 1 by Schryver 2 and by Mair. 3 

Schryver's Method. 

2-5 litres of bile are heated in an iron vessel under a reflux condenser for 30 
hours with 170 gm. of sodium hydroxide dissolved in 300 c.c. of water. Two 
volumes of water are added and the hot solution acidified with excess of 
hydrochloric acid, the mixture being vigorously stirred after each small ad- 
dition of acid. The crude acids separate out as a thick oil, which forms a 
pasty mass on -cooling ; a small quantity separates in a granular form. After 
standing for 1 2 hours, the acids are filtered off, washed free from mineral acid 
by kneading with water, dried on a water-bath and powdered. They are 
dissolved in dilute ammonia, the solution is diluted so as not to contain more 
than 5 per cent, of the salts and boiled for 10 minutes with animal charcoal 
to remove some of the pigment. The acids are reprecipitated with hydro- 
chloric acid, washed, dried in vacua over calcium chloride and soda lime, the 
mass being removed from time to time to powder the surface lumps and to 
expose the under layers. The mass is readily powdered as more water is 
removed and is finally obtained in a granular form. It is added to boiling 
acetone till the solvent is saturated, the solution is filtered and allowed to 
cool. The crystals so formed are filtered off and washed with cold acetone. 
The filtrate and washings on concentration yield further crops of crystals. A 
syrupy mother liquor finally results from which only a few crystals separate on 
prolonged standing. Over 80 per cent, of the acids having a light greenish 
tinge are thus obtained. 

The separation of the three acids depends on the different behaviour 
of their magnesium salts. The crude product is suspended in alcohol, the 
alcohol warmed and caustic soda added from a burette till the solution is just 
alkaline to phenolphthalein which is added to the liquid. The alcohol is 
evaporated and the sodium salts dissolved in water so that they form a i per 
cent, solution. The alkaline solution is filtered, dilute acetic acid added till 
the red colour disappears and treated with J^ of its volume of 20 per cent, 
magnesium chloride solution. On heating the clear solution on a water-bath, 
a bulky crystalline precipitate gradually separates ; sometimes it becomes 
almost a paste. It is heated for i hour and allowed to cool. 

The precipitate of magnesium choleate and deoxycholate (with not more 
than \ of its total of cholic acid) is filtered off by suction and the mother 
liquor removed as far as possible (below). The acids are regenerated from 
the magnesium salts by rubbing in mortar with excess of dilute hydrochloric 
acid ; they become granular on standing, are easily filtered off, washed free 
from acid and dried in vacuo. They are dissolved in alcohol with the addition 
of caustic soda as before, the solution is neutralised, diluted so as to contain 
about 2 per cent, of salts and treated with about \ of its volume of 20 per 
cent, barium chloride solution. The precipitated barium salts become 
granular and crystalline on standing for 16 hours. The liquid is syphoned 
off on a Buchner filter and the solid added. It is washed by grinding in a 
mortar several times with water. This insoluble barium salt consists of 
barium choleate from which almost pure choleic acid is obtained by stirring 
up in mortar with dilute hydrochloric acid. 

The filtrate and washings are acidified, the precipitate of deoxycholic and 

1 Zeitschr. physiol. Chern., 1911, 74, 198. 2 J. Physiol., 1912, 44, 265. 

3 Biochem J., 1917, II, n. 

21* 



324 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

cholic acids filtered off, washed, converted as before into their sodium salts and 
the hot solution treated with 20 per cent, magnesium chloride. The precipitate 
consists mainly of magnesium deoxycholate ; a further quantity is obtained on 
concentrating the solution and allowing to cool. 

The mother liquor (above) containing magnesium cholate is concentrated 
to about one-fifth, crystals of magnesium deoxycholeate are filtered off, and 
the cholic acid precipitated by adding acid. It is at first resinous, but 
becomes crystalline on standing. 

The crude yellowish crystalline mass of cholic acid is washed, dried in 
vacua and recrystallised from hot alcohol. Further crops of crystals are 
yielded by concentration of the mother liquor. The remainder is separated 
by warming the solution with caustic soda, to hydrolyse ethyl cholate, 
evaporating off the alcohol, dissolving the residue in water and acidifying. 
The precipitated acid is dried, converted into its sodium salt as in the pre- 
paration and treated with barium chloride to remove choleate and deoxycholate. 
The acid is reprecipitated and crystallised from alcohol. The cholalic acid in 
the last mother liquors is isolated by converting into the sodium salt, evaporat- 
ing off the alcohol and acidifying in the presence of ether. 90 per cent, of 
the crude acid is obtained in a crystalline form melting at 197. It is gener- 
ally slightly yellow. 

Choleic acid is obtained from the barium sa-lt by grinding with dilute 
hydrochloric acid. It is almost pure and melts (after drying at 1 10) at 184. 
Small quantities are recrystallised from acetone, or large quantities from 
alcohol ; in the latter case, the mother liquors are boiled with caustic soda, 
the alcohol evaporated, the residue dissolved in water and hydrochloric acid 
added. The precipitated acid is washed, dried and recrystallised from 
acetone. 

Deoxycholic acid is precipitated on grinding the magnesium salt with acid. 
It is filtered off, washed and recrystallised from acetone. Pure deoxycholic 
acid is prepared by crystallisation from glacial acetic acid. 225 gm. of 
cholic acid, 75 gm. of choleic acid and 40 gm. of deoxycholic acid are obtained 
from i o litres of bile. 

Hair's Method. 

This method is designed to prepare deoxycholic acid. 

240 gm. of sodium hydroxide are dissolved by stirring and heating in 
4 litres of ox bile. The alkaline solution is boiled gently for 20 hours in an iron 
digester and its volume reduced by evaporation to 2-3 litres. The hot solution 
is neutralised to phenolphthalein by adding about 350 c.c. of cone, hydro- 
chloric acid. A flocculent precipitate of silica, amounting to 1-2 gm. per 
1000 c.c. of bile is thrown down and filtered off after cooling. The filtrate 
is acidified to litmus by adding about 50 c.c. of glacial acetic acid. The bile 
acids separate as a fluid crystalline mass, which is at first white, but rapidly 
absorbs pigment ; by gentle rotation of the flask, the acids can be made to 
adhere in a single mass from which the liquid is decanted. The pasty mass 
retains acetic acid tenaciously and is squeezed to remove the liquid. It is put 
into a i litre flask and dissolved by heating in about 600 c.c. of glacial acetic 
acid. The solution is poured into a beaker and allowed to stand for 2-3 days. 
The crystals which separate and consist of deoxycholic acid, cholalic acid and 
some fatty acid are filtered off and washed free from pigment with 60 per 
cent, acetic acid. They are dissolved in about 300 c.c. of hot glacial acetic 
acid, filtered from silica (?) and the solution allowed to stand. The crystals, 
mainly deoxycholic acid, are separated, dried between porous tiles and 
dissolved in about 750 c.c. of 60 per cent, acetic acid. The hot solution is 



BILE ACIDS 325 

poured into a beaker and the small quantity of oily substance separated after 
cooling. The crystals are filtered off, washed with 60 per cent, acetic acid 
and water and dried at 100. The product melts at 170-175. A yield of 
32 gm. melting at 172-173 and 42 gm. melting at 170 was obtained from 
4 litres of bile. 

Deoxycholic acid can be prepared in a similar way from commercial 
sodium taurocholate by boiling 500 gm. with 3 litres of water and 200 gm. of 
caustic soda. A yield of 25 gm. melting at 173-174 results. 

Cholic acid is prepared from the mother liquors by distilling off the acetic 
acid in vacua, treating the residue with hot alcohol and making alkaline to 
phenolphthalein with caustic soda ; the alcohol is evaporated, the sodium salts 
dissolved in water and the cholic acid precipitated with hydrochloric acid. 
The fluid crystalline mass is squeezed to remove mother liquor and digested 
with hot water to remove acetic acid. A hard cake smelling of acetic acid 
results on cooling ; it is broken up, powdered, dried in vacua over caustic 
soda and subsequently at 100. It is recrystallised from 2 volumes of ab- 
solute alcohol. 50 gm. of cholic acid and 7 gm. of fatty acid were obtained 
from 4 litres of bile. Schryver's method gives a, bigger yield of cholic acid. 

Properties of Cholalic Add. 

Cholalic acid is soluble in water, i part in 4000 cold, i part in 750 hot ; it 
is more soluble in alcohol, i part in 20. It dissolves in alkalies and in alkaline 
carbonates with evolution of carbon dioxide. It crystallises from dilute aqueous 
solutions or from dilute acetic acid in tetrahedra containing i molecule of water 
and from alcohol containing i molecule of alcohol, which is given off at 130. 
It has a sweet-bitter taste and the anhydrous substance melts at 195. 

Cholalic acid is dextrorotatory. 

It gives off aromatic vapours when heated and is stable to alkali. The 
alkaline salts are easily soluble in water, less so in alcohol ; they crystallise 
on evaporating the alcoholic solution or on precipitating it with ether. The 
lead and barium salts are precipitated on adding lead acetate or barium chloride 
to a not too dilute solution. The barium salt crystallises in glistening needles 
arranged in rosettes and is easily soluble in alcohol and hot water. 

On heating to high temperatures or with dehydrating agents, e.g. glacial 
acetic acid it is converted into anhydrides dyslysin, choloidic acid 
which are not crystalline and are reconverted into cholalic acid by boiling 
with alcoholic potash. On oxidation cholalic acid is converted into dehydro- 
cholalic acid, bilianic acid, isobilianic acid. Another acid, cholicamphoric 
acid, C 10 H 16 O 4 , has also been obtained. It is not easily reduced, but an acid, 
cholylic acid, C 2 4H 40 O 2 , has been obtained. 

Reactions and Tests of Cholalic Acid. 

(1) The crystalline form, dextrorotation and the formation of aromatic 
substances on heating are an indication o the presence of cholalic acid. 

(2) Mylius' Reaction. If 0-2 gm. of cholalic acid be dissolved in 0-5 gm. 
of alcohol and i c c. of 'iN iodine solution be added and the solution 
gradually diluted with water, the. brown liquid sets to a mass of microscopic 
needles with a metallic lustre and blue appearance. 

(3) Cholalic acid dissolves in concentrated sulphuric acid, giving a reddish - 
yellow solution with a green fluorescence. 

(4) Pettenkofer' s Reaction. On adding concentrated sulphuric, acid drop 
by drop to an aqueous solution of cholalic acid in which a tiny crystal of cane 
sugar has been dissolved and keeping the temperature below 70, the cholalic 



326 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

acid is precipitated but redissolves giving a cherry-red, then a purple solution, 
which becomes bluer in shade on keeping. The purple solution shows an 
absorption band between D and E. 

Reactions (3) and (4) are given by other compounds and are not dis- 
tinctive for cholalic acid. 

Properties of Choleic Acid. C 24 H 40 O 4 ? 

Choleic acid crysatllises from alcohol in bundles of flat needles which 
melt at 185-190. It is soluble with difficulty in water, more easily in ether 
and readily in absolute alcohol, but less so than cholalic acid. It is dextro- 
rotatory. On oxidation it yields dehydrocholeic acid, cholanic acid. 

Properties of Deoxycholic Acid. C 24 H 40 O 4 . 

It melts at 172-173 and has [ajo 20 + 57 '02. 

Glycocholic Acid. C 26 H 43 O 6 N. 

C 23 H 39s CO Glycocholic acid is present in ox and 

NH CHjj.cooH. human bile (7-9 per cent), but not in that 
of carnivora. It is converted by hydrolysis with acids into cholalic 
acid and glycine, and has been synthesised. 

Preparation. 

(1) Huf tier's Method. 

Fresh ox bile is covered with a layer of ether in a measuring cylinder and 
treated with concentrated hydrochloric acid (2 c.c. to 40 c.c. bile). The 
turbidity at first formed becomes crystalline. The ether is poured off, the 
mass stirred with water, well shaken, filtered and washed with cold water till 
the washings are colourless. The precipitate is purified as given in Ham- 
marsten's method. 

(2) Plattners Method. 

Bile is evaporated to a syrup and extracted with alcohol. The solution is 
decolorised by boiling with charcoal, some of the alcohol is distilled off and 
the concentrated alcoholic solution is precipitated with ether. The precipitate 
becomes crystalline on standing from a few hours to some days and consists 
of sodium glycocholate, glycocholeate and taurocholate (Plattner's crystallised 
bile). The crystals are dissolved in water, some ether and then dilute 
sulphuric acid are added until there is a permanent cloudiness. A mass of 
fine shining needles fills the solution. They are filtered off and washed free 
from acid with water. The mixture of acids is separated by Hammarsten's 
method. 



BILE ACIDS 



327 



(3) Hammarstens Method. 

Ox bile is evaporated to a syrup, the syrup extracted with alcohol and 
the alcoholic extract evaporated. The residue is dissolved in water and pre- 
cipitated with lead acetate. The precipitate is decomposed by boiling with 
sodium carbonate, the solution evaporated to dryness and extracted with 
alcohol. The alcoholic solution is evaporated, the residue is dissolved in 
water and i boiled with charcoal to decolorise. The solution is mixed with 
ether and precipitated with hydrochloric acid according to Hii finer' s method. 
The crystals are washed free from hydrochloric acid. 

The mixture of acids contained in this precipitate is separated as follows : 

Glycocholic acid is separated by boiling out the precipitate with water ; it 
separates out on cooling and is recrystallised from water. 

The residue is dissolved in very dilute alkali and the neutral solution 
treated with barium chloride. The sticky mass of barium salts is dissolved 
in boiling water and treated with sodium carbonate. The filtrate is evaporated 
and extracted with alcohol. The alcoholic extract is evaporated, the residue 
dissolved in water, the solution decolorised by boiling with charcoal and 
precipitated in dilute solution with hydrochloric acid and ether as above. 
The glycocholeic acid is precipitated and is purified by repeating the process 

Properties. 

Glycocholic acid crystallises in needles (Fig. 53) which are soluble with 
difficulty in cold water (i part in 300), 
more easily in hot water (i part in 120), 
very easily soluble in alcohol and in 
alkalies, almost insoluble in ether. The 
alcoholic solution on the addition of 
water becomes turbid and deposits 
crystals. The alkaline solution is pre- 
cipitated by acids. On heating in a 
capillary tube, glycocholic acid softens 
at 133 and melts at 152. It has a 
sweet-bitter taste and is dextrorotatory. 
The alkaline salts are obtained by 
evaporation of the alcoholic solution ; 
they dissolve fats and cholesterol and 
give precipitates with lead acetate, ferric 
chloride, silver nitrate. The lead salt 
is soluble in hot alcohol and separates 
as a powder on cooling. The barium 
salt is easily soluble in water. 

On boiling with water, glycocholic acid changes into paraglycocholic acid 
which crystallises in platelets melting at 183-188. It is reconverted into 
glycocholic acid by crystallisation from alcohol. 

It dissolves in concentrated sulphuric acid and on warming cholonic 
acid, C 26 H 45 O 5 N, separates out. This substance is also formed by boiling 
glycocholic acid with concentrated hydrochloric acid. Its barium salt is 
insoluble in water. 

Glycocholic acid is distinguished by its solubility, taste and rotation. It 
gives the fluorescent reaction and Pettenkofer's reaction, but not Mylius' 
reaction described under cholalic acid. 




FIG. 53. Glycocholic acid. (After Funke.) 



328 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Glycocholeic Acid. C-j ti N 43 O 5 N or C 2 7H 45 O 5 N. 

This acid accompanies glycocholic acid in bile. 

It is prepared as described under glycocholic acid. 

Glycocholeic acid crystallises in prisms or bunches of fine needles which 
melt at 175-176. It has a bitter taste, is almost insoluble in cold and boiling 
water, but is easily soluble in alcohol. 

Its alkaline salts are soluble in water, but not so easily as those of glyco- 
cholic acid. They are precipitated by calcium, barium and magnesium salts. 
The barium salt is precipitated as a sticky mass, but crystallises from water in 
bunches of needles. 

It yields glycine and choleic acid on hydrolysis by acids or by alkalies. 

Glycocholeic acid is distinguished from glycocholic acid by melting-point, 
rotation and insolubility, also by the insolubility of its barium salt, other- 
wise it resembles glycocholic acid. 

Glycohyocholic Acids. 

Two acids, a-glycohyocholic acid and /3-glycohyocholic acid, have been 
prepared from pig's bile. They differ slightly in properties from glycocholic 
acid and have been seldom investigated. 

Fellinic Acid. C 23 H 4 oO 3 . 

Fellinic acid, together with cholalic acid, has been obtained from human 
bile. 

Chenocholic acid, C 2 7H 44 O 4 , Ursocholeic acid, C 18 H 28 O 4 ? and Lithofellic 
acid, C 20 H 36 O 4 , are other acids obtained from different biles. 

Taurocholic Acid. C 2fi H 45 O 7 NS. 

C H O CO Taurocholic acid accompanies glycocholic 

acid in ox bile. It is present in fish bile and 

WN O W 

snake's bile. Dog's bile and the bile of car- 

/^TT O(~) "LJ 

nivora do not contain glycocholic acid. In all 
cases it is present as the sodium salt. 

Preparation. 

Hammarsten gives the following preparation of taurocholic acid from cod's 
bile and dog's bile. 

(a) From Cod's Bile. 

The bile is precipitated with ferric chloride, the filtrate is treated with 
sodium carbonate to remove iron, the solution from the ferric carbonate 
is nearly neutralised and saturated with sodium chloride. The precipitate of 
taurocholate is washed with saturated salt solution, dissolved in water and 
again precipitated with salt. The taurocholate is dissolved in water and 
separated from sodium chloride by repeated evaporation and solution in 
alcohol. Taurocholic acid is obtained from the sodium salt by rubbing with 
alcohol containing about 2 per cent, of sulphuric acid, shaking up, filter- 
ing from sodium sulphate and adding ether. The flocculent precipitate is 
separated, dissolved in absolute alcohol and again thrown down with ether. 
The clear solution of the precipitate in alcohol is treated with a few drops of 
water and then with ether till it is turbid. An amorphous precipitate, if 
formed, is rapidly filtered off and more ether is added. The acid commences 
to crystallise out after some time and when the amount ceases to increase 
ether may be again added. This is continued until no further crystals are 
obtained. 



BILE ACIDS 329 

(fr) From Dogs Bile. 

Dog's bile is treated with alcohol to remove mucin and the alcoholic 
filtrate is evaporated to dryness. The residue is dissolved in absolute 
alcohol and carefully treated with ether. The precipitate is pressed out and 
dissolved in water. The solution is fractionally precipitated with ferric 
chloride, the reaction being kept neutral by adding sodium carbonate, and 
diluted with water. This is continued so long as the precipitates on decom- 
posing with sodium carbonate give a solution having a bitter taste. Tauro- 
choleic acid is obtained from the precipitate. The filtrate is neutralised, 
and evaporated ; the residue is then extracted with alcohol : evaporation and 
extraction are repeated several times. The free acid is obtained from the 
salt as described under cod's bile. 

Properties. 

Taurocholic acid is easily soluble in water, the amorphous form more 
easily than the crystalline. It dissolves easily in alcohol, but is insoluble in 
ether, benzene, chloroform. It has a sweet, very slightly bitter, taste and 
is dextrorotatory. It yields taurine and cholalic acid on hydrolysis. 

The amorphous form separates on adding ether to the alcoholic solution ; 
the crystalline form on adding ether to the alcoholic solution containing 
a drop of water. The crystals contain i molecule of water. Taurocholic 
acid on heating turns yellow or brown at 100, sinters at 140, begins to de- 
compose at 1 60 and melts at 180 to a brown liquid. 

The sodium salt is soluble in water, less so in cold alcohol ; from hot 
alcohol it separates in large flakes if the solution does not set as a jelly. It 
can be obtained as crystals by adding ether to the alcoholic solution. 

The sodium salt is not precipitated by lead acetate or ferric chloride. 

It resembles the other bile acids in giving the fluorescent and Pettenkofer's 
reactions, but is distinguished by its taste, solubility and sulphur content. 

Taurocholeic Acid. 

Taurocholeic acid accompanies taurocholic acid in dog's bile. 

The precipitates obtained in the preparation of taurocholic acid with ferric 
chloride and sodium carbonate are decomposed with sodium carbonate ; the 
filtrate is evaporated and the residue extracted with hot alcohol. The 
alcoholic solution is evaporated and extracted with cold alcohol. Extraction 
and evaporation are repeated until a clear solution results. The acid is 
obtained by treating this solution with alcohol containing hydrochloric acid, 
the sodium chloride is filtered off and the solution precipitated with ether. 

Taurocholeic acid is readily soluble in alcohol, but insoluble in ether, 
chloroform and benzene. It is amorphous and deliquescent and has a bitter 
taste. 



COMPLEX AROMATIC COMPOUNDS. 

In addition to benzene and its substitution products, compounds 
in which only one benzene ring is present, numerous compounds are 
known which contain two or more benzene nuclei. These nuclei may 
be joined by means of one or more carbon atoms, or they may be 
joined together directly. 

Diphenylmethane. 

a^x^x^ Diphenylmethane is the first and chief 

2 I example of a complex aromatic compound in 

I which two benzene rings are united by a 

^ s \/ / ^ carbon atom. 

It is obtained by heating benzene with benzyl chloride in the presence of 
aluminium chloride : 

Ce^CHoCl + C 6 H 6 = HC1 + C 6 H 5 . CH 2 . C 6 H 5 . 

Diphenylmethane is a crystalline solid, which melts at 26-5. It closely 
resembles benzene in forming nitro and other derivatives. On oxidation with 
chromic acid it yields benzophenone, C 6 H 5 . CO . C 6 H 5 . 

Triphenylmethane. 

Triphenylmethane contains three ben- 
zene nuclei joined to one carbon atom, 
and is obtained by heating benzal chloride 
with benzene in the presence of aluminium 
chloride : 

C 6 H a . CHC1 2 + 2 C 6 H 6 = 2 HC1 + C 6 H B . CH<^ 

or by heating benzene with chloroform in the presence of aluminium 
chloride : 




3 C 6 H 6 + CHC1 3 = 3 HC1 + C 6 H 5 . CH 



\C R H,. 



Triphenylmethane is a colourless solid, which melts at 92 and boils at 
358. It is not easily soluble in cold alcohol, but dissolves easily in ether 
and benzene. 



330 





COMPLEX AROMATIC COMPOUNDS 331 

It yields nitro compounds, amino compounds and other derivatives. 
These derivatives form the group of rosaniline dyes (p. 335). 

When oxidised with chromic acid it is converted intc triphenylcarbinol 
(C 6 H ) 3 . C . OH. 

Diphenyl. 

Diphenyl is the first instance of aromatic com- 
pounds in which the benzene rings are directly 
joined together by a single bond. It is prepared 
by treating an ethereal solution of bromobenzene 
with sodium : 

C 6 H 5 Br + Na 2 + C,H B Br = aNaBr + C 6 H 5 -C 6 H 5 , 
or it is formed when benzene vapour is passed through a red-hot tube. 

It is a colourless solid melting at 71 and boiling at 254; it closely re- 
sembles benzene in its reactions. On oxidation it yields benzoic acid. 

Azoxybenzene. 

When nitrobenzene is reduced with alkaline reducing 
agents, such as sodium and alcohol, it yields azoxybenzene. 
Azoxybenzene is a yellow crystalline solid melting at 36. 
It is insoluble in water, but soluble in alcohol, ether and 
organic solvents. 

Azobenzene. 

Azobenzene is formed when azoxybenzene is carefully 
N = N ( ^j distilled with three parts of iron filings. 

It is a brilliant red crystalline solid which melts at 68 
and distils at 293. It is not soluble in water, but dis- 
solves in organic solvents. 

Hydrazobenzene. 

OWhen azobenzene is reduced with alkaline reagents, 
ammonium sulphide, or zinc and caustic soda, it is 
converted into hydrazobenzene. 
Hydrazobenzene forms colourless crystals which 
melt at 131. It is reduced by zinc dust and acetic acid to aniline. 

Benzidine. 

Benzidine, or diamino - diphenyl, is 
formed when hydrazobenzene is treated 
with concentrated hydrochloric acid ; intra- 
molecular rearrangement takes p*lace. 

It may be obtained by treating azobenzene with tin and concentrated 
hydrochloric acid. 

Benzidine forms colourless crystals which melt at 128. 
It is a base like aniline, forming salts with acids. The sulphate is very 
insoluble and is used for estimating sulphates. 

It is diazotised by nitrous acid and, like aniline, is used largely in the 
preparation of dyes. 

Naphthalene. 

Naphthalene, the hydrocarbon which is present in 
coal tar in the largest quantity, is present in the second 
fraction, boiling from 170-230, when the tar is 
ft l 4 J\ 4 JjS fractionally distilled. 





332 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



The second fraction on cooling deposits crystals of naphthalene. These 
are separated from phenols, etc., by pressure. The impure mass is shaken 
with caustic soda to remove the remainder of the phenols, washed and 
warmed with sulphuric acid, which sulphonates the impurities, dissolving them. 
The naphthalene is obtained by distillation or sublimation. 

Naphthalene is a colourless solid crystallising in shining plates which 
melt at 79 and boil at 218. It has a characteristic smell and is extremely 
volatile. All the naphthalene formed by the distillation of coal is not condensed 
and a portion reaches the gas mains and gas pipes ; in cold weather the 
naphthalene crystallises out and may cause blocking of the pipes. It does 
not dissolve in water, but it dissolves easily in hot alcohol, ether and other 
organic solvents. It combines with picric acid, like other complex hydro- 
carbons, to form a yellow crystalline solid which melts at 149. 

When naphthalene is oxidised by dilute nitric acid or chromic acid, it 
yields o-phthalic acid. On nitration it yields a-nitronaphthalene ; oramino- 
naphthalene is formed by the reduction of the nitro compound. Nitro- 
naphthalene on oxidation gives nitrophthalic acid: aminonaphthalene gives 
phthalic acid. The formation of phthalic acid shows the presence of one 
benzene ring with substituting groups in the o-position. The same is shown 
by the oxidation of nitronaphthalene, but the oxidation of aminonaphthalene 
shows that a different benzene ring is oxidised to that .which is oxidised in 
the case of nitronaphthalene. Naphthalene would thus contain two benzene 
rings joined together in the ortho-position as represented by the formula. 
This constitution is proved by synthesis and emphasised by the similarity in 
behaviour of naphthalene to benzene. 

Derivatives of Naphthalene. 

Naphthalene resembles benzene in forming nitro, sulphonic acid, amino- 
derivatives. Two monosubstitution derivatives can be formed, the d and the 
ft forms. In some cases both are formed, in other cases only one is formed 
The accompanying formulae show how these derivatives are prepared from 
naphthalene : 



sulphonic acid ^T^ ^ 
*fe 

1%t 




KOH o 

t 160 




'NH 2 

/?-naphthylamine 
c j 




a naphthaquinone 



in 

acetic acid 
naphthalene ^2 

a -nitronaphthalene 

I s 
x\ 

diazotise 



SO 3 H 

a -naphthalene 
sulphonic acid 




Ct-naphthol 



NH 2 

a -naphthylamine 




COMPLEX AROMATIC COMPOUNDS 333 

The naphthalene sulphonic acids are crystalline hygroscopic solids. 

a-naphthol is crystalline, melts at 95 and boils at 280; it resembles 
phenol in smell, is only slightly soluble in water, but dissolves easily in 
alcohol and ether. It gives a violet flocculent precipitate with ferric chloride. 

/3-naphthol is a crystalline solid, melting at 138. It forms nitro deriva- 
tives like phenol, many of which are used as dyes. 

a-naphthylamine is a colourless, crystalline solid melting at 50 and 
boiling at 300. It has an unpleasant smell and turns red on exposure to 
air ; on oxidation it is converted into a-naphthaquinone. It gives a blue 
precipitate with ferric chloride. 

/?-naphthylamine is a colourless crystalline solid, melting at 112 and 
boiling at 294. It has no smell and gives no precipitate with ferric chloride. 

It gives phthalic acid on oxidation. 

a-nitronaphthalene is yellow, melts at 61 and boils at 304. 

/3-nitronaphthalene, prepared by indirect methods, melts at 79. 

The chief derivatives of naphthalene are the naphthol sulphonic acids and 
naphthylamine sulphonic acids of which 14 isomers of each are possible. 

Naphthalene and its substitution products are extensively used in the 
preparation of dyes. Apparently no natural compound contains a naphtha- 
lene ring. 

Anthracene. 

a y a The cn ' e f interest attaching to anthracene 

is that it is the. parent hydrocarbon from which 
the red dye of madder root is derived. 

Anthracene is a constituent of coal tar and 
is isolated like naphthalene from the fraction 
boiling above 270. 
Its constitution has been arrived at by its resemblance in properties to 
benzene and naphthalene and by its synthesis by treating tetrabromo-ethane 
with benzene in presence of aluminium chloride : 

BrCHBr / CH \ 

CG**,) + | + C fi H 6 = 4HBr + C (i H 4 ^ /C fi H 4 . 

BrCHBr \CH/ 

It is a colourless crystalline solid with a blue fluorescence, melting at 
2 1 3 and boiling at 351. It is insoluble in water, very slightly soluble in 
alcohol and ether, but easily soluble in benzene. 

Anthracene resembles benzene and naphthalene fairly closely, but it is 
oxidised by nitric acid and converted into anthraquinone. 

Anthraquinone. 

Anthraquinone is prepared by oxidising anthra- 
cene with sodium bichromate and sulphuric acid 
and is the chief derivative of anthracene. 

It consists of pale yellow needles which melt at 
285. It resembles the aromatic ketones, e.g. benzo- 
phenone, rather than quinone and is a very stable 
compound. It is used in making alizarin and other dyes. 

Alizarin. 

Alizarin occurs in madder root as the 

UJd. 

glucoside termed ruberythric acid. This 
glucoside on hydrolysis by enzymes, or by 
acids, gives two molecules of glucose and 
alizarin, the " Turkey red " dye. Since its 

synthesis by Graebe and Liebermann who determiued its constitution, 

alizarin is made entirely from anthraquinone. 





334 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



Anthraquinone is converted by sulphonation with sulphuric acid at 
250, or by heating with fuming sulphuric acid at 160, into anthraquinone- 
/3-monosulphonic acid. The solution is diluted with water, unchanged 
anthraquinone is filtered off and the solution neutralised with soda. Sodium 
anthraquinone-/?-monosulphonate crystallises out and is purified by crystallisa- 
tion. It is heated with caustic soda and a small quantity of potassium 
chlorate ; the sodium salt of alizarin is obtained from which alizarin is pre- 
cipitated on acidifying : 

co co 

H a S0 4 = C 6 H 4 < C 6 H 3 . SO 3 H + H 2 O 



64 6 

/ C \ / C \ 

CgH/ /C 6 H 3 . SO 3 Na + 3 NaOH + O = C 6 H 4 < )C 6 H 2 (ONa) + Na SO 3 + 2H,O. 

\CO/ \CO/ 

Alizarin forms dark red prisms melting at 290. It is almost in- 
soluble in water, slightly soluble in alcohol. 

As a phenol it forms salts with alkalies ; the solution in caustic 
soda has a violet colour ; the salts with the divalent and trivalent 
metals are insoluble and of various colours ; these salts are the dyes 
which colour the fabrics. 



Purpurin. 
CO 



OH 




OH 



Purpurin or I, 2, 4-trihydroxy-anthra- 
quinone is present in madder root with 
alizarin and is made by oxidising alizarin 
CO OH with manganese dioxide and sulphuric acid. 

It forms dark red needles which melt at 253; in dyeing it gives 
yellower shades than alizarin. 

Anthropurpurin and Flavopurpurin 

CO OH CO OH 

>H 





CO 

are two other anthraquinone derivatives used as dyes. It may be noted that 
only those derivatives containing two hydroxy groups in the i, 2 position 
form dyes. 

Mention may finally be made of the following complex aromatic com- 
pounds : 



CH, 



Fluorene. 





Phenanthrene. 



Chrysene, 



Picene. 



COMPLEX AROMATIC COMPOUNDS 335 

DYES. 

Most of the dyes in common use are derivatives of the complex aromatic 
compounds. They may be divided into the following groups : 

(1) Nitro compounds. (3) Azo compounds. 

(2) Triphenylmethane compounds : (4) Phenols. 

(a) basic. (5) Indigo (p. 345). 

(b] acidic. 

Nitro Compounds. 

The nitro compounds are yellow dyes, mainly used for dyeing , silk and 
wool, and on this account, since cotton is the chief material which requires 
dyeing, are few in number. 

Picric acid dyes silk and wool, but not cotton, as can be easily verified 
by dipping silk or wool and cotton into picric acid solution, removing and 
washing. The silk or wool is dyed, the cotton is not dyed. 

Martius yellow, dinitro-a-naphthol, is the chief nitro compound used 
for dyeing silk and wool. The commercial substance is the sodium salt. 

Naphthol yellow is the sulphonic acid of Martius yellow. The 
potassium salt is the commercial dye. 

Triphenyl Methane Compounds. 

Malachite green is prepared by heating a mixture of benzaldehyde 
and dimethylaniline with zinc chloride : 



C 6 H 5 . CHO + 2C 6 H 6 . N(CH 3 ) 2 = C 6 H 5 . CH + 2 H,O. 

\C 6 H 5 N(CH 3 ) 2 

It dyes silk and wool a bluish-green ; but cotton only after mordanting 

(P- 337)- 

Brilliant green is prepared in the same way using diethylaniline : 

,C 6 H s H(C t H s ) s 

C 6 H 5 . CHO + 2C 6 H S N(C 2 H 5 ) 2 = C fi H 5 . CH( + 2 H 2 O. 

\C 6 H 5 N(C 2 H S ) 2 

Acid green is prepared from benzaldehyde and ethylbenzylaniline : 

/ C 6 H S N(C 2 H S )C 7 H 7 

C 6 H 5 CHO + C 6 H 5 N(C 2 H 5 )C 7 H 7 = C 6 H 5 . CH/ + 2 H 2 O. 

\C 6 H 5 N(C 2 H S )C 7 H 7 

It dyes silk and wool in an acid solution, but is not used for dyeing cotton. 
Pararosaniline is prepared by oxidising a mixture of /-toluidine and 
aniline with arsenic acid or nitrobenzene. Probably the/-toluidine is first 
oxidised to the aldehyde : 

,C 6 H 4 .NH 2 
H 2 N . C 6 H 4 . CHO + 2 C 6 H 5 NH = H 2 N . C 6 H 4 . CH/ " + 2 H O. 

\C 6 H 4 .NH 2 
On further oxidation it gives the carbinol : 

/C 6 H 4 . NH 2 
H 2 N . C 6 H 4 . C^ 

I \C 6 H 4 .NH., 
OH 

Rosaniline, Fuchsine or Magenta. 

This compound is prepared by oxidising a mixture of /-toluidine, 
<?-toluidine and aniline as above : 

I r H NM /C 6 H 4 NH 2 

H 3 N . C 6 H 4 . CHO + { H 2 N C !? r ;S 4 H2 CH 3 } = ' C 6 H 4 . CH/ + 2 H 2 O 

. 

H 2 N . c 6 H 4 . c<; 

I^ 
OH 



336 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



Pararosaniline and rosaniline are reddish- blue dyes. 

These compounds on heating with methyl iodide become methylated. 
The colour becomes bluer. It is still more blue when ethyl iodide is used, 
and pure blue when phenyl groups are introduced, e.g. aniline blue. 

C 6 H 4 .NH.C 6 H S 
C 6 H 5 .NH.C 6 H 4 .C/ 

| \C 6 H 3 (CH 3 )NH . C 6 H 5 
OH 

Phenolphthalein is prepared by condensing together phenol and 
phthalic anhydride : 

C 6 H 4 OH 

CO C C 6 H 4 OH 



CO 



2C 6 H 5 OH = C 6 H 4 \/0 
CO 



Fluorescein is obtained by condensing resorcinol with phthalic an- 
hydride. 

Eosin is obtained by condensing dibromo-resorcinol with phthalic 
anhydride. It is a tetrabromofluorescein. 

Krythrosin is obtained by condensing di-iodoresorc'inol with phthalic 
anhydride. It is tetra-iodo-fluorescein. 

These dyes have a magnificent greenish fluorescence and are mostly used 
for dyeing silk. Eosin is used as red ink, phenolphthalein is the well-known 
indicator. The phenol acid is colourless, the alkaline salt is coloured. 

Constitution of Triphenylmethane Dyes. The Theory of Colour. 

An examination of the coloured aromatic compounds has shown that 
they contain particular groupings, e.g. the nitro-group, the azo-group. 
Quinones are also coloured compounds. The triphenylmethane derivatives 
are neither nitro-compounds nor azo-compounds ; they are believed to have 
a quinonoid structure : 

C 6 H 5 -C(OH)-C 6 H4-N(CH 3 ) 2 C 6 H 5 -C-C 6 H 4 -N(CH 3 ) 2 





H Cl 

Colourless leuco base. 



HO-C 6 H 4 -C-C 6 H 4 -CO 




Cl-N'.(CHe 



L 3'2 
Coloured base. 



HO-aH 4 -C-CH/COONa 



OH 

Colourless anhydride 

of 
phenolphthalein. 




Coloured sodium salt, 



COMPLEX AROMATIC COMPOUNDS 337 

Malachite green, as prepared above, is a colourless crystalline solid and 
is known as the leuco base; on oxidation it is converted into the green 

compound 

/C.H 4 . N(CH 3 ) 3 /C 8 H 4 . N(CH 3 ) 3 

C 8 H 4 . CH + O = C 6 H 4 . C . OH 

\CH 4 . N(CH 3 ) 3 \C 6 H 4 . N(CH,) S 

which is known as the colour base. 

The colour bases of the rosanilines are formed directly ; on reduction they 
give the leuco base. 

These colour bases form salts with acids, e.g. 

CagH-jgNaO + HC1 = C,,H K N,C1 + H 2 O. 

Azodyes. 

Diazonium salts are prepared by treating aniline and other aromatic 
amines with nitrous acid. Besides yielding phenol on boiling and giving 
various derivatives by the Sandmeyer reaction these diazonium salts have the 
property of combining with amines and phenols : 

C 6 H 5 . N : N . OH + C 6 H 5 . NH 2 = C 6 H 5 . N : N C 6 H 4 . NH 2 + H 2 O 
C 6 H 5 . N : N . OH + C 6 H 5 . OH = C 6 H 5 . N : N C 6 H 4 . OH + H 2 O. 

By means of this reaction an enormous number of dyes can be prepared 
which are either basic (aniline component) or acidic (phenol component). 
The following are examples : 

Helianthin from diazotised sulphanilic acid and dimethylaniline. 

Chrysoidin from diazotised aniline and w-phenylenediamine. 

Bismarck brown from diazotised w-phenylenediamine and w-phenyl- 
enediamine. 

Resorcin yellow from diazotised sulphanilic acid and resorcinol. 

Congo red from diazotised benzidine and naphthionic acid (naphthyl- 
amine sulphonic acid). 

The free acid is blue in colour, the salts are red. 

The Process of Dyeing. 

There are many coloured substances amongst the aromatic compounds 
which are not dyes, e.g. dinitrobenzene is yellow, azobenzene is red, but the 
chief essential that a coloured substance should be a dye is that it should 
form an insoluble compound, which cannot be washed out by water, upon 
the fabric or material to be dyed. It will be noticed that the dyes above 
mentioned are either basic or acidic substances and are thus capable of form- 
ing salts with alkalies or with acids. 

Silk and wool are proteins and are compounds of the nature of amino 
acids, i.e. they are both basic and acidic in their properties. Most dyes 
combine with them and give insoluble salts. Cotton is a carbohydrate and 
forms no compounds with acids and bases. Cotton can, however, be dyed by 
mordanting, i.e. impregnating the fabric with an acid such as tannic acid, 
or a base such as alumina, ferric oxide, etc. Basic dyes form insoluble 
tannates, acidic dyes form insoluble salts or lakes. In calico printing the 
pattern is marked out with the mordant in a thick solution, such as gum, to 
prevent it from spreading. 

The formation of insoluble salts upon the fabric is the chief method of 
producing insoluble deposits upon the fibre, but most probably the following 
method occurs at the same time. The dyes are all compounds of high mole- 
cular weight and form colloidal solutions (suspensions of the finest particles). 
The fabric, whether it consists of silk, wool or cotton, is a colloid. The pro- 
cess of precipitation of colloids from solution by means of electrolytes, especi- 
ally those with trivalent ions, and the process of mutual precipitation of two 
colloids will also be concerned in the fixing of the dye upon the fabric. 

22 



THE ANTHOXANTHINS. 



The yellow plant pigments usually called flavones and xahthones are 
most conveniently termed the anthoxanthins, the term used in 1835 by 
Marquart and again suggested by Willstatter in 1913 on account of their 
similarity to the anthocyans. 

The anthoxanthins are derived from the complex benzene-y-pyrone 
nucleus. The y-pyrone nucleus is the heterocyclic six-membered ring con- 
taining an oxygen atom and a keto group in the para- or y- 
position to the oxygen atom. The keto group in this ring 
does not behave in all respects like a ketone group ; it forms 
no derivative with hydroxylamine. The oxygen atom is also 
peculiar in its properties ; it is basic and combines with mineral 
acids to form salts, the oxygen atom becoming quadrivalent. 

Flavone and xanthone are phenyl derivatives of benzo-y- 
pyrone. The yellow pigments are hydroxy derivatives of: 






CO 

Flavonol Xanthone. 

or 
hydroxyflavone. 

The constitution of the anthoxanthins has been shown to be the follow- 
ing : 

HO 





OH CO 

Chrysin 

or 
i, 3-dihydroxyflavone. 

o 



OH 'CO 

Apigenin 

or 

i, 3, 4/-trihydroxy- 
flavone. 




OH CO 

Luteolin 
or 

i, 3- 3', 4'- 

tetrahydroxy- 

flavone. 




HO 




r, 3-dihydroxyflavonol. 



OH 



HO 



Caempferol 

or 

i, 3, 4'-trihydroxy- 
flavonol. 



CO 

Fisetin 

or 

3, 3', 4'-trihydroxy- 
flavonol. 



338 



ANTHOXANTHINS 



339 



HO 




HO 



HO 




OH 



OH CO 



i, 3, 3', 4'-tetrahydroxy- 
flavonol. 



Morin 

or 

i. 3, 2', 4'- 

tetrahydroxy- 

flavonol. 




OH 



CO 

Myricetin 
or 

i, 3. 3', 4', 5'- 
pentahy droxy-flavonol . 



OH CO 

Euxanthone 

or 

2, 3'-dihydroxy- 
xanthone. 




OH 



OH CO 

Gentisin 

or 

4-methoxy- 
2, 3'-dihydroxy- 
xanthone. 



These pigments thus contain the simple aromatic compounds, benzene, 
phenol, catechol, resorcinol or pyrogallol combined with benzo-y-pyrone. 
On decomposition they yield polyhydroxybenzoic acids (protocatechuic, 
hydroxybenzoic, or resorcylic) and the above phenols. Their formulae have 
been arrived at by the study of these decomposition products and have been 
proved by synthesis. The details of the products of decomposition and the 
synthesis are given in the larger text-books of organic chemistry. 
Chrysin occurs in various species of poplar and mallows. 
Apigenin occurs in parsley and celery in the form of the glucoside, apiin. 
Luteolin is the yellow colouring matter of weld or dyer's broom. 
Galangin is found in the root of Galanga. 

Caempferol is contained in Galanga root and combined with rhamnose 
as the glucoside, caempferitrin, in Java indigo. 

Fisetin is present in Fiset wood (Rhus cotinus] and Quebracho wood 
(Quebracho Colorado). 

Quercitin is found with fisetin and also as glucoside, with rhamnose, in 
oak bark (Quercus tinctoria). It is present in the flowers of the horse- 
chestnut and in the skin of onions. 

Morin is a constituent of yellow wood (Morus tinctoria}. 
Myricetin is the yellow pigment in the bark of Myrica nagi. 
Euxanthone is formed when cattle are fed with mango leaves and is 
excreted in combination with glycuronic acid as euxanthic acid (Indian 
yellow). 

Gentisin is the yellow pigment present in Gentiana lutea. 

These pigments are yellow crystalline solids, very slightly soluble in 

water. They dissolve in acids giving yellow or yellowish-red solutions ; 

they also dissolve in alkalies giving solutions which are yellow or reddish. 

Some of them give a dull green or red-brown coloration with ferric chloride. 



THE ANTHOCYANS. 

The red, violet and blue pigments which are present in the blossoms, in 
many fruits and in some leaves of plants, and which can be extracted with 
water or aqueous alcohol, are grouped together as the Anthocyans. Accord- 
ing to Molisch they occur not only dissolved in the juices of the cell, but also 
in amorphous and crystalline forms. Molisch obtained crystals by allowing 
aqueous or acetic acid extracts to evaporate slowly under the cover slip of a 
microscope slide. The colour of the aqueous solution of these pigments fades 
on standing from which it would appear that the pigments are very unstable. 

The chemical investigation of these pigments has shown that they are 
glucosides and that the pigment is related to that of the group 
of anthoxanthin pigments. The study of these pigments /^\/\ 
was taken up by Willstatter and his pupils in 1913 and | [ | c 
is still in progress. They have shown that these pigments J CH 

are glucosides and that they are derivatives of the complex \X\X 
benzo-pyrylium nucleus. This nucleus differs from the 
benzo-y-pyrone nucleus in having a CH group in the place of the CO group. 

The properties of this group differ markedly from those of the benzo- 
pyrone group. The oxygen atom in the pyrone ring can O-C1 

become quadrivalent and form salts with acids ; it is feebly 
basic ; the salts are unstable and are decomposed by water. 
The oxygen atom in the benzopyrylium ring can also be quad- 
rivalent and form salts with acids. It is strongly basic ; the 
salts are stable and not easily decomposed by water. 

The benzene ring contains hydroxyl groups; their phenolic character 
allows of the formation of salts with alkali. These properties explain the 
existence of the red, violet and blue colours ; the red is the acid salt, the blue is 
the potassium or .metallic salt and the violet is the anhydride of the pigment ; 
thus the colour is due to the quinonoid structure of the molecule. The 

KO 0-C1 
HO O-C1 







apparent instability of the compounds from the disappearance of the colour 
of the solutions is not confirmed. The colour returns on acidifying or on 
evaporating the solution. The pigment apparently undergoes an isomerisation. 

More convenient methods of preparation than the older one (involving 
precipitation by lead acetate) have been discovered by Willstatter and his 
pupils ; they depend on the formation of oxonium salts which are soluble 
with difficulty. 

The skins of blue grapes, whortle berries and Althcea rosa are ex- 
tracted with cold glacial acetic acid. The extract is precipitated with ether. 
The syrupy precipitate is washed with ether and dissolved in a warm aqueous 
solution of picric acid. The picrate crystallises out on cooling. A solution 
of the hydrochloride is obtained by treating the picrate with methyl alcohol 
containing hydrochloric acid and is precipitated with a mixture of ether and 
petroleum ether. The chloride is crystallised from aqueous alcohol con- 
taining hydrochloric acid. 

The isolation of the anthocyans from other plants depends on the in- 
solubility of the chlorides. These salts are generally soluble in water or 
dilute acid, but soluble with difficulty in 7-15 per cent, hydrochloric acid. 

34 



ANTHOCYANS 



The anthocyans are glucosides and are converted by hydrolysis with acids 
into glucose and anthocyanidins. The anthocyanidins are pigments like the 
anthocyans, but they are soluble in amyl alcohol. The glucosidic character 
of the anthocyans can be easily demonstrated : 

1-2 gm. of blossom are rubbed with 5-10 parts of sand and a few c.c. 
of dilute sulphuric acid and a small quantity of alcohol ; some talc is added 
to help filtration. The mass is shaken with amyl alcohol. The emulsion 
which is formed may be broken by pouring upon a wet filter and then pour- 
ing the material upon a dry filter. On washing the amyl alcohol with water or 
sodium acetate solution it will not be coloured or only faintly. Another 
portion of the blossom may be heated for half an hour with the acid and 
treated in the same way. The amyl alcohol will be pigmented. 

The same can be observed with '05-1 gm. of anthocyan, but the 
filtration is not necessary. 

The following anthocyans have been investigated : 

(1) Cornflower and rose 

cyanin -> 2 x glucose + cyanidin. 

(2) Cranberry 

idain - galactose + cyanidin. 

(3) Blue grapes 

oenin -> glucose + oenidin. 

(4) Whortle berry and Althcea rosea 
myrtillin.-^ glucose + myrtillidin. 

(5) Larkspur 

delphinin -> 2 mol. glucose + 2 mol. /-oxybenzoic acid + delphinidin 

(6) Pelargonium 

pelargonin-> 2 x glucose + pelargonidin. 

The anthocyanidins are closely related to the anthoxanthins : 
Cyanidin is isomeric with luteolin, caempferol and fisetin. 
Pelargonidin is isomeric with apigenin and galangin. 
Delphinidin is isomeric with quercetin and morin. 

Cyanidin on heating with alkali gives phlorogjucinol and protocatechuic acid. 
Pelargonidin on heating with alkali gives phloroglucinol and p-hydroxy- 

benzoic acid. 

Delphinidin on heating with alkali gives phloroglucinol and gallic acid. 
The constitution of these anthocyanidins is probably : 



OH 




Cl 

l 

o 



OH 



HO 




OH 



I'OH 



)U 



Cyanidin. 
Cl 



HO 




HO 



OH OH 

Delphinidin. Oenidin. 

Though these compounds are so closely related to .the anthoxanthins, the 
transformation of the one group into the other is not easily effected, but 
quercitin on reduction has been converted into cyanidin. It is very likely 
that -the two groups of compounds are converted into one another by oxidative 
and reducing enzymes. 



INDOLE AND ITS DERIVATIVES. 

Indigo, tryptophan, scatole, indole are natural substances which 
contain the heterocyclic indole ring the complex nucleus made up of 
a benzene ring and a pyrrole ring : 




The compounds containing this ring have the properties of benzene 
and of pyrrole. 

Indole. 

Indole was first obtained by the reduction of indigo by distillation 
with zinc dust and also in the same way from other products obtained 
from indigo. It was identified as one of the products of the putre- 
faction of protein, together with scatole, and it is present in animal 
excrement. It is a constituent of coal tar and is isolated from the 
fraction of basic character which distils between 240 and 260. Its 
constitution has been shown to be : 

CH 



HCf 

H'cL JIG 

CH NH 

Preparation. 

Indole is most easily prepared by the putrefaction of meat or other pro- 
teins, i kilo, of meat is minced and mixed with about 4000 c.c. water ; 2 gm. 
of potassium phosphate, 5 gm. of magnesium sulphate and about 30 gm. of 
crystallised sodium carbonate and a piece of putrid meat are added. The 
vessel is closed and connected with another flask containing lead acetate and 
the two vessels are placed in a warm place at about 35. Bubbles of gas are 
evolved containing hydrogen sulphide, mercaptan, etc. ; these are absorbed 
by the lead acetate. 

The mixture is kept for about 5 or 6 days, acidified with acetic acid and 
distilled. The distillate, which contains indole and scatole and phenols, is 
made alkaline with soda and again distilled. Such a solution gives the 
reactions of indole. The distillate is acidified with hydrochloric acid and 
treated with picric acid. The precipitate is distilled with ammonia and this 
distillate is extracted with ether. The ethereal solution on evaporation gives 
a mixture of indole and scatole. The mixture is dissolved in absolute 
alcohol and treated with 8-10 volumes of water. The scatole is precipi- 
tated. The two compounds thus separated are purified by crystallisation 
from aqueous alcohol or ligroin. 

342 



INDOLE AND ITS DERIVATIVES' 343 

Properties. 

Indole crystallises in glistening platelets which melt at 52. It is 
volatile in steam and these vapours -have a peculiar and unpleasant 
smell. It is fairly soluble in alcohol, ether, chloroform, benzene, 
ligroin. 

It is a weak base, a secondary amine, and combines with strong 
acids to form salts. 

Reactions. 

(1) A pine shaving moistened with hydrochloric acid and intro- 
duced into an alcoholic solution of indole becomes red. 

(2) On the addition of a few drops of nitric acid and, drop by drop, 
a few drops of very dilute potassium nitrite solution ('I per cent.) to 
a solution of indole, the solution becomes red and in strong solutions 
a precipitate of nitrosoindole is formed. (Baeyer.) 

(3) On adding half the volume of a 2 per cent, alcoholic solution 
of p-dimethylaminobenzaldehyde to a solution of indole and, drop by 
drop, 25 per cent, hydrochloric acid until a red colour appears, the 
further addition of a few drops of 0*5 per cent, sodium nitrite solution 
gives a dark red colour. (Ehrlich.) 

(4) On adding sodium nitroprusside solution to indole solution 
until it is of a yellow colour and then a few drops of caustic soda a 
deep violet-blue colour is obtained ; the addition of acetic acid changes 
it to pure blue. (Legal.) 

(5) If under a solution of indole treated with glyoxylic acid a 
layer of concentrated sulphuric acid be run, a red colour is produced at 
the point of contact. (Hopkins.) This reaction is sensitive to I in 
500,000. 

(6) If formaldehyde be used instead of glyoxylic acid a similar 
colour is produced. This reaction is sensitive to I in 700,000 
(Kondo.) 




344 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Scatole or /3-Methyl Indole. 

Scatole was first isolated from faeces and re- 
cognised as -a constituent of the intestinal con- 
tents of man and animals. Later it was obtained 
by the fusion of proteins with alkali and isolated 
from the products of putrefaction. It is also a 
product of the reduction of indigo with zinc dust. 
The preparation of scatole is given under indole. 
Scatole crystallises in colourless platelets melting at 95 and boil- 
ing at 265-266. It has a pungent faecal smell. It dissolves in 
water, but less readily than indole, but is more easily volatile in steam 
than indole. It dissolves in alcohol, ether, benzene, chloroform. 

It is a weak base and combines with acids to form salts; the 
hydrochloride is easily soluble in alcohol, but insoluble in water and 
ether. 

Reactions. 

(1) It dissolves in concentrated hydrochloric acid giving a violet 
coloured solution. A purple-red is formed on warming its solution in 
sulphuric acid. 

(2) With nitric acid and sodium nitrite it gives a white turbidity 
(compare indole). 

(3) With p-dimethylaminobenzaldehyde solution it gives a blue- 
violet colour which turns blue with sodium nitrite (compare indole). 

* (4) With sodium nitroprusside and soda it gives a yellow colour, 
which turns violet on heating for a few minutes with half its volume 
of glacial acetic acid. 

* (5) The glyoxylic acid reaction is rose-red in colour. 

* (6) The formaldehyde reaction is yellow or brown, but red if a 
trace of a ferric salt be present. 

(7) The pine shaving reaction is negative, but if a pine shaving 
dipped in an alcoholic solution of scatole be placed in cold concentrated 
hydrochloric acid it becomes cherry-red, changing after a little while 
to a dark violet. 

Indoxyl and Indican. 

Indoxyl occurs in various species of the in- 
digofera and in woad, Isatis tinctoria, probably 
in combination with glucose as the glucoside, indi- 
can. The glucoside is hydrolysed by enzymes in 
the plant leaves and converted into indoxyl, which 
undergoes oxidation to indigo blue. Indoxyl occurs in human and 
mammalin urine in combination with sulphuric acid and glycuronic 
acid as ester. It is hydrolysed by acid and oxidised to indigo blue. 




INDOLE AND ITS DERIVATIVES 345 

Indoxyl consists of yellow crystals which dissolve in water with a 
green fluorescence, also in alcohol, ether, acetone. It melts at 85. 
Dilute acids convert it into a red substance and an unpleasant smell 
is produced. In alkaline solution it oxidises in the air to indigo. 

Detection in Urine. 

Indoxyl is detected by conversion into indigo blue : 

(1) An equal volume of concentrated hydrochloric acid is added 
to 10 c.c. of urine and 2 or 3 c.c. of chloroform. A very dilute solu- 
tion of bleaching powder is added, drop by drop, and the solution is 
inverted after the addition of each drop. The chloroform becomes 
bluish-violet owing to the formation of indigo blue. Excess of bleach- 
ing powder must be avoided as the indigo blue undergoes further oxi- 
dation to colourless compounds. 

(2) The further oxidation is to a large extent avoided by using 
a fresh solution of ferric chloride in concentrated hydrochloric acid as 
the oxidising agent (Obermayer's reagent). An equal volume of this 
reagent and a few c.c. of chloroform are added to the urine. On mixing 
thoroughly for 1-2 minutes by inverting the liquids, the chloroform 
becomes blue. 

(3) Salkowski recommends the use of copper sulphate as the oxidising 
agent to prevent further oxidation. An equal volume of hydrochloric acid, 
i c.c. of copper sulphate solution and a few c.c. of chloroform are added to 
10 c.c. of urine and the mixture shaken carefully as above. 

(4) The following other oxidising agents may be employed : 

One drop of a 10 per cent, solution of potassium persulphate to 5 or 6 
c.c. of urine. 

One drop of a 3 per cent, solution of potassium chlorate to 10 c.c. of 
urine. 

Indigo Blue or Indigotin. 

Indigo blue is formed by the oxidation of indoxyl in alkaline solu- 
tion on exposure to the air. Two molecules of indoxyl combine in 
this reaction : 





NH 



Indigo blue is a dark blue powder and shows a metallic coppery 
lustre on rubbing. It sublimes giving copper-red glistening prisms. 
It is insoluble in water, alcohol, ether, dilute acids and alkalies, and has 
neither smell nor taste. It dissolves in aniline and molten paraffin 
with a purple-red colour, also in turpentine from which it crystallises. 



346 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



NH 



C.OH HOC 




Owing to its insolubility it is converted into indigo white or into 
indigotin sulphonic acid so as to be used as a dye. 

Indigo blue has been synthesised by various methods. The 
synthetic product is cheaper than the natural and is gradually displac- 
ing the natural product as a dye. 

Indigo White. 

The insolubility of indigo blue 
renders it useless as such for dyeing 
purposes. On reduction by zinc 
dust and alkali, hydrosulphite, or 
by electrolysis it is converted into indigo white. In air this solution 
reoxidises and forms indigo blue. 

In dyeing the indigo blue is reduced, the cloth soaked in the solu- 
tion and exposed to the air. Insoluble indigo blue is deposited on the 
fibres. 

Indigo white can be precipitated from solution in absence of air 
as white crystals which dissolve in alcohol, ether and alkalies with a 
yellow colour. 

Indirubin or Indigo Red and Isoindigotin. 

Natural indigo blue is generally associated with small quantities of indigo 
red. This is formed by a combination of i molecule of indoxyl with i 
molecule of the isomeric oxindole : 





Combination occurs between the a and ft carbon atoms. If combination 
occurs between the two ft carbon atoms, isoindigotin is formed. 



I ndole-/?- Acetic Acid. 

This compound was first found amongst 
the putrefactive decomposition products of 




OCH./COOH 



proteins. It is often present in urine and is 
found particularly in cases of intestinal disorder. 
It crystallises in platelets melting at 164 and is soluble with 

difficulty in water, but easily in alcohol and ether. On heating it 

decomposes into carbon dioxide and scatole. 



INDOLE AND ITS DERIVATIVES 347 

Reactions. 

(1) On adding a few drops of pure nitric acid and drop by drop a 2 per 
cent, solution of potassium nitrite to a solution of indole-acetic acid, a cherry- 
red colour is formed, followed by a turbidity and separation of a red pigment. 

(2) A purple-red colour and precipitate is formed when an equal volume 
of concentrated hydrochloric acid and a few drops of a 1-2 per cent, solu- 
tion of bleaching powder are added to its solution. 

(3) A violet colour is formed (before and after boiling) if an equal volume 
of concentrated hydrochloric acid and a few drops of ferric chloride solution 
be added to a solution of indole-acetic acid. 

(4) A red colour is formed with p-dimethylaminobenzaldehyde (see 
under indole). 

The Urorosein Reaction of Urine. 

This reaction consists in the formation of a red pigment when 
concentrated hydrochloric acid and a drop or two of sodium nitrite 
solution is added to urine. Stale urines give this reaction on the 
addition of hydrochloric acid only-, as nitrites are formed by bacterial 
decomposition of other constituents in the urine. The colour disap- 
pears on adding alkali but reappears on acidifying. 

Urorosein is insoluble in ether and chloroform, but dissolves in 
alcohol and amyl alcohol with a red colour. The amyl alcoholic 
solution shows an absorption band in the green, between D and E, but 
nearer D. Urorosein is most probably nitrosoindole-acetic acid. 

Indole-/?-Propionic Acid. 

Indole propionic acid has also been shown 
to be a putrefactive decomposition product of 
' H 2 'CH,-C proteins. 

It crystallises in prisms or irregular plates, 

VTT is slightly soluble in water, but easily soluble 

in alcohol and ether. 

Reactions. 

(1) Indole-/3-propionic acid forms a nitroso compound with potassium 
nitrite. In concentrated solution on the addition of concentrated potassium 
nitrite and acetic acid, a yellow crystalline mass may be obtained. 

(2) An aqueous solution gives a white turbidity with ferric chloride which 
becomes red on heating. 

Indole-ethylamine. 

Indole- ethylamine is another substance 

|OCH 2 'CH 2 'NH 2 which is formed in the putrefaction of proteins 
and of tryptophan. It is one of the amines 
which have a marked physiological action, but 
is not so marked in its action as p-hydroxy- 
phenyl-ethylamine or iminazolyl-ethylamine. 






348 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Tryptophan. 

Tryptophan was shown to 

CH(NH 2 ) COOH be a constituent of proteins by 
Hopkins and Cole in 1902, who 
isolated it from the mixture of 
amino acids which results from 
the digestion of proteins with the enzyme, trypsin. Its discovery gave 
the clue to the well-known Adamkiewicz reaction of proteins and the 
proteinochrome reaction of tryptic digests. 
Preparation. 

i kilo, of caseinogen is dissolved in about 8 litres of -8 per cent, crystallised 
sodium carbonate solution and digested with 2-4 gm. of a trypsin prepara- 
tion for 5-6 days at 37 in the presence of toluene or chloroform as an 
antiseptic. The digestion is allowed to proceed until a sample of 5-10 c.c. 
withdrawn at intervals gives a maximum colour reaction with bromine water 
after acidifying with acetic acid. The digestion is stopped by heating the 
solution to 80. The insoluble portion, consisting of undigested proteins, 
calcium phosphate and tyrosine, is filtered off and the clear filtrate, better after 
concentration in vacua to a volume of about i litre and filtration from tyro- 
sine, is acidified with sulphuric acid until it contains 5 per cent. The acid 
solution is precipitated with a 10 per cent, solution of mercuric sulphate in 
5 per cent, sulphuric acid. After 12 hours the precipitate, which contains 
tryptophan, tyrosine and cystine, is filtered off and washed with 5 per cent, 
sulphuric acid to remove tyrosine until the washings show only a faint reaction 
with Millons' reagent for tyrosine. 

The precipitate is suspended in water, warmed and decomposed with 
hydrogen sulphide. The filtrate from mercuric sulphide is freed from hydrogen 
sulphide by a current of air, acidified to 5 per cent, with sulphuric acid 
and again precipitated with the acid mercuric sulphate solution. The 
cystine comes down first and is removed ; the tryptophan is thrown out on 
the addition of more mercuric sulphate. The precipitate is washed with 5 
per cent, sulphuric acid, decomposed with hydrogen sulphide and the solution, 
freed from sulphuric acid by baryta, is evaporated in vacuo to a small volume. 
Tryptophan separates out and is recrystallised from a mixture of water and 
alcohol containing animal charcoal. The yield of tryptophan is from 5-10 gm. 

Properties. 

Tryptophan crystallises in cplourless glistening platelets which 
are not easily soluble in cold water, but readily in hot. It is 
insoluble in absolute alcohol and ether. It dissolves easily in hot 
pyridine, less easily in cold. On heating in a capillary tube it 
changes colour at 220, becomes brown at 240 and melts at 252. 
If heated quickly it turns yellow at 260 and melts at 289. Tryp- 
tophan is a weak base and forms salts with acids. As an amino 
acid it forms salts and forms acyl derivatives with acid chlorides, some 
of which serve for its isolation and characterisation. 



INDOLE AND ITS DERIVATIVES 



349 



Reactions. 

Tryptophan, even when mixed with other amino acids, is readily 
recognised in solution by the following reactions : 

(1) Bromine water reaction. About 5 c.c. of the solution are 
acidified with acetic acid and bromine water is added drop by drop ; 
a reddish-violet colour appears. This gradually deepens, but disap- 
pears if too much bromine water be added, giving a yellow solution. 
When the maximum reddish-violet colour is obtained, the solution is 
shaken with 2 or 3 c.c. of amyl alcohol ; the amyl alcohol dissolves 
the pigment and separates coloured reddish-violet. 

(2) Glyoxylic acid reaction. A small quantity of glyoxylic acid 
solution is added to about 5 c.c. of the solution and concentrated 
sulphuric acid is run under its surface ; at the point of junction a 
reddish-violet ring appears and on gently mixing the two liquids the 
colour spreads throughout the mixture. 

(3) On mixing a tryptophan solution with an alcoholic solution of benzal- 
dehyde and running underneath it concentrated sulphuric acid containing 
ferric sulphate, a blue colour is formed at the junction. 

(4) Using formaldehyde instead of benzaldehyde, the colour at the junc- 
tion of the liquids is blue-violet. 

(5) With p-dimethylaminobenzaldehyde and concentrated hydrochloric 
acid a red colour is formed (see under indole). 

(6) A pine shaving wetted with hydrochloric acid, washed with water, 
dipped into a concentrated solution of tryptophan and dried, becomes purple 
in colour. 

The Biological Relationship of the Indole Derivatives. 

Indole and scatole have long been known to be putrefactive pro- 
ducts of protein, the other compounds were found later. The discovery 
of tryptophan and the determination of its constitution has shown 
that all these compounds are derived from it ; in many cases the direct 
conversion of tryptophan in putrefaction has been carried out. The 
stages in the decomposition of tryptophan are similar to those, which 
tyrosine undergoes, namely : 




,C-CH-CH-NH 



NH 

Indole ethylamine. 
I 




N.H 



c- CH,-CH(NH,ICOOH 



Tryptophan. 





OCH 2 -COOH 

CH 
[ 

Indole acetic acid. 




C-CH,-CH;COOH 
CH 

Indole propionic acid. 
CH 



Scatole. 



350 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



Indole is oxidised and converted into indoxyl which is combined 
with sulphuric acid (or glucose) to form indican. Indican on hydrolysis 
yields indoxyl, which is oxidised in the air or by oxidising agents to 
indigo blue. 




Indican. 




NH 

Indigo blue. 



QUINOLINE AND ISOQUINOLINE. 

These compounds have the empirical formula C 9 H 7 N and are pre- 
sent in coal tar and bone oil. Their constitution is expressed by the 
formulae : 





N 

Quinoline. Isoquinoline. 

The presence of the pyridine ring in these compounds is shown by 
their oxidation. Quinoline gives quinolinic acid : isoquinoline gives 
A 7-pyridine dicarboxylic acid or cinchomeronic acid and phthalic 
acid. The formulae of both compounds have been proved by synthesis. 

Preparation. 

Quinoline is usually prepared by synthesis. Isoquinoline is pre- 
pared from the fraction of coal tar or bone oil which distils between 
236 and 243. The bases are converted into sulphates and fraction- 
ally crystallised from alcohol. The sulphate is decomposed by potash 
and the base distilled. 

Properties. 

Quinoline is a colourless oily liquid which boils at 239 and has a 
specific gravity of I '095 at 20 It has a peculiar and pleasant smell 
and is only slightly soluble in water. Isoquinoline is a colourless 
solid which melts at 23, boils at 241 and closely resembles quinoline. 

Both compounds are tertiary amines and form salts with acids. 
They are stable ring compounds resembling naphthalene and pyridine. 



THE ALKALOIDS. 

The term vegetable alkaloids was originally applied to the group 
of basic substances (hence the name alkaloid), which were found in 
plants, to distinguish them from basic isubstances (amines, formerly 
ptomaines or toxines) found in animals, formed mainly by putrefaction. 
The term alkaloid is now applied only to the basic substances occurring 
in plants which contain in their constitution either a pyridine, quinoline, 
isoquinoline, or pyrrole or pyrrolidine ring, or several rings. They are 
classified according to the ring into the following groups : 

I. Pyridine group : 

Piperine, coniine, trigonelline (p. 151), nicotine. 

II. Pyrrolidine group : 

Hygrine, stachydrine (p. 150). 

III. Tropane group : 

Atropine, hyoscyamine, hyoscine, cocaine. 

IV. Quinoline group : 

Quinine, cinchonine, strychnine, brucine. 

V. Isoquinoline group : 

Narcotine, narceine, morphine, codeine, papaverine, 
berberine. 

The constitution of most of the alkaloids is known, but some are 
still under investigation. The details of the work upon the elucida- 
tion of their constitution are very complex. Only the formulae of the 
chief compounds can therefore be given so as to show their relation- 
ship to pyridine and the other nuclei. 

The alkaloids generally occur in plants in the form of salts with 
organic acids, such as citric, tartaric, malic, oxalic, succinic. They 
are liberated from the salts by means of alkali and can frequently be 
extracted from the alkaline solution by means of chloroform, ether 
and other organic solvents. Most of the alkaloids are solid compounds ; 
confine, nicotine and a few others are liquid. They generally con- 
tain oxygen in their composition, but again there are exceptions. 

351 



352 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Most alkaloids give precipitates with the following reagents : 

(1) tannic acid ; 

(2) picric acid ; 

(3) iodine in potassium iodide ; 

(4) mercuric iodide in potassium iodide ; 

(5) phosphotungstic acid and phosphomolybdic acid. 

These reagents, termed alkaloidal reagents, also give precipitates 
with amines and other bases and with proteins. They cannot in 
consequence be considered as specific for the alkaloids, but they are 
sometimes useful for isolating and detecting alkaloids. 

Piperine. 

Piperine is found in the fruit of the various varieties of pepper ; about 8 
or 9 per cent, is present in black pepper. 

Powdered pepper is warmed with lime water for 15-20 minutes and the 
mixture is evaporated to dryness. The dry residue is extracted with ether. 
The ethereal solution on distillation leaves piperirie, which is purified by 
crystallisation from alcohol. 

Piperine is a white solid which melts at 128. It is almost insoluble in 
water. It behaves as a weak base and dissolves in concentrated sulphuric acid 
giving a deep red solution. 

Constitution. 

On distillation with alcoholic potash, piperine is converted into piperidine 
and piperic acid. Piperine is the amide of piperidine and piperic acid. It 
has been synthesised by the action of piperic acid chloride on piperidine. 
/CH 2 CH 2 

CHo ^N . OC . CH :=CH . CH= CH f ] 



Piperine. 
/CH 2 . CH 2 

CH \NH + HOOC . CH CH . CH=CH 

\ / 

^CH^ . CH 2 

Piperidine. Piperic acid or 

3, 4 methylene dihydroxycinnamenyl- 
acrylic acid. 

Coniine. C 8 H lT N. 

Coniine is the alkaloid which is present in the seeds of hemlock. 
It is prepared therefrom by distillation with sodium hydroxide. 

Coniine is a colourless liquid which boils at 167. It has a 
peculiar and penetrating odour and turns brown in the air. It is 
soluble in water and is a strong base forming salts with acids. 

The natural substance is optically active and dextrorotatory. 

Constitution. 

Coniine has been shown to be a-propylpiperidine and has been 
synthesised as follows : 

0' 

CH 3 

W N N 

Pyridine. /\ a-picoline. 







THE ALKALOIDS 353 



OHC.CH 3 = \y'CH=:CH.CH, 

Acetaldehyde. N 

a-allyl-pyridine. 

CM 2 
H.jCr/N, CH, 

^^L/rl . L/rTg -f- 3*~^2 == *^2 

N 
o-propylpiperidine. 

The inactive coniine was separated into d- and /-coniine by the 
fractional crystallisation of its tartrate. 

Nicotine. C 10 H 14 N 2 . 

Nicotine occurs to the extent of '6-8 per cent, in tobacco leaves 
in combination with organic acids (malic or citric). 

Preparation. 

Tobacco leaves are boiled out with water. The aqueous solution is con- 
centrated, made alkaline with lime and distilled. The distillate is acidified 
with oxalic acid and evaporated. The concentrated solution is rendered 
alkaline with soda and extracted with ether. The ethereal extract on distilla- 
tion leaves the nicotine, which is purified by distillation in a current of 
hydrogen. 

Properties. 

Nicotine is a colourless oily liquid which boils at 241. It possesses 
an unpleasant smell and a burning taste. It is intensely poisonous. 
In air it undergoes oxidation, turning brown. 

It is a ditertiary base and forms salts with acids, which are dextro- 
rotatory, and combines with two molecules of methyl iodide. 

Constitution. 

On oxidation with chromic acid it yields nicotinic acid showing 
that it possesses a substituting group in 
the /3-position of the pyridine ring. This 
substituting group has been found to be 

N-methyl-pyrrolidine, the methyl group be- 

111 T HC* 1 

ing attached to the nitrogen atom. It is 

-N-methyl-pyrrolidine-pyridine. 

Hygrine. 

Hygrine has been shown to be /3-acetyl-N-methyl pyrrolidine. 




H 2 C 
H C 



CH-COCH, 



\y CU 2 

f 
CH 3 



354 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



Atropine or dl-Hyoscyamine. C 17 H 23 O 3 N. 

Atropine (or daturine) is found in the deadly nightshade, Atropa 
belladonna, in henbane, Hyoscyamus niger, and together with /-hyo- 
scyamine in Datura stramonium. 

The juice (i litre) obtained 4>y pressing the plant is made alkaline with 
potash (4 gm.) and extracted with chloroform (25 c.c.). The chloroform extract 
is evaporated and the residue is treated with dilute sulphuric acid which dis- 
solves the base. On adding potassium carbonate to the acid solution the 
atropine is precipitated and is crystallised from alcohol. 

Atropine is a white crystalline solid separating in prisms from 
dilute alcohol. It melts at 115, is almost insoluble in water but 
is easily soluble in alcohol, ether and chloroform. It is extremely 
poisonous, from 'O5- - 2 gm. being a lethal dose. It is a strong base 
and forms salts with acids which are easily soluble in water. The 
sulphate is most commonly used in medicine for dilating the pupils 
and other purposes. 

Atropine may be tested for as follows : on evaporating a small 
quantity with a drop of fuming nitric acid a yellow residue is left. 
This turns violet, changing to red, on adding a drop of alcoholic potash. 

Constitution. 

On hydrolysis by boiling with baryta, atropine is converted into 
tropine and tropic acid. 

Tropic acid is a-phenyl-y3-hydroxypropionic acid. 

Tropine has been shown to be the N-methyl derivative of a 
7-hydroxy-piperidine nucleus containing two extra 
methylene groups, or as a hydroxy derivative of 
a combined piperidine and pyrrolidine nucleus to 
which a methyl group is attached at the nitrogen 
atom. 

Atropine is the tropic acid ester of tropine : 




HC 



HC 



CH-0-OOCH-CH 2 OH 




and it has been synthesised from its constituents. 



THE ALKALOIDS 355 

Cocaine. C 17 H 21 O 4 N. 

Cocaine is found in the leaves of coca, Erythroxylon Coca, from 
which it is prepared as follows : 

The leaves are treated with hot water at a temperature of about 80. The 
filtered solution is precipitated with lead acetate to remove tannins, proteins, 
etc. The excess of lead is removed from the filtrate by adding sodium 
sulphate, and the solution, after again filtering, is made alkaline with soda 
and extracted with ether. The residue obtained on evaporation of the ether 
is crystallised from alcohol. 

Properties. 

Cocaine separates out in colourless prisms which melt at 98. It is 
not easily soluble in water, but is soluble in organic solvents. It is a 
strong base forming salts, the hydrochloride being the salt most fre- 
quently used in medicine. Its use in medicine depends upon its being 
a local anaesthetic. 

Constitution. 

On hydrolysis by acids cocaine is converted into methyl alcohol, 
benzoic acid and ecgonine. 

Ecgonine has been found to be closely related to tropine and is a 
carboxylic acid of tropine : 

CHOH 

iCH-COOH 




Cocaine is the double ester of ecgonine with benzoic. acid and 
methyl alcohol and has the following- constitution : 



CH.OOOC,H 5 



- COOCHo 



This formula has been proved by the synthesis of cocaine from 
these constituents. 

Other esters of ecgonine have been prepared using different acids in 
the place of benzoic acid and different alcohols in the place of methyl 
alcohol. 



356 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Cinchonine. C 19 H., 2 ON 2 . 

Cinchonine is present together with quinine in the varieties of cin- 
chona bark, the grey bark containing as much as 2-5 per cent. 

It is prepared from the solutions remaining from the preparation of quinine ; 
they are made alkaline with caustic soda and the precipitate so formed is 
dissolved in a small quantity of boiling alcohol ; on cooling, cinchonine is 
deposited. It is purified by converting it into its sulphate and crystallising 
from water. 

Cinchonine separates in colourless prisms which melt at 255, is soluble 
with difficulty in water, but more easily soluble in alcohol, ether, etc. It is a 
weak base and it forms salts with acids. It is a tertiary base, combining with 
two molecules of methyl iodide. 

Constitution. 

Cinchonine is an unsaturated compound and combines with two atoms of 
bromine, or with one molecule of hydrochloric acid. 

On oxidation it yields cinchonic acid or quinoline-y-carboxylic acid and 
another product which has been shown to be a piperidine derivative. 

Cinchonine is thus a derivative of quinoline and contains a piperidine 
ring. Its constitution is the following : 

CH 




Quinine. C 20 H 24 N 2 O 2 . 

Quinine, together with cinchonine, is contained in cinchona bark 
up to about 3 per cent. ; the yellow bark of Calisaya contains up to 
1 2 per cent. 

Preparation. 

The bark is powdered and treated with dilute sulphuric acid ; from the acid 
solution the bases are precipitated by adding soda. The mixture of bases is 
dissolved in alcohol and the solution is neutralised witli sulphuric acid. The 
sulphates, which are so obtained, are recrystallised from water. Quinine sul- 
phate is the most insoluble and separates out first. It is converted into 
quinine by precipitation with ammonia. 

Properties. 

Quinine crystallises from water with three molecules of water of crystallisa- 
tion. The anhydrous substance melts at 173. It is very slightly soluble in 
water, but is soluble in alcohol and ether. It has a bitter taste and is a feeble 
base. It forms salts with acids. The sulphate and hydrochloride are used in 
medicine. Like cinchonine it is a ditertiary base and combines with two mole- 
cules of methyl iodide. 

Quinine may be tested for by the following reaction : 

A solution of a quinine salt on the addition of chlorine water or bromine 
water followed by ammonia gives a green precipitate. This dissolves in ex- 
cess of ammonia giving an emerald-green solution. 



THE ALKALOIDS 357 

Quinine is very similar in constitution to cinchonine and is methoxy- 
cinchonine : 

CH 



CHOH 




Strychnine. C 21 H 22 O 2 N 2 . 

Strychnine is the poisonous alkaloid of the seeds of Strychnos nux 
vomica, in which it is present together with brucine. 

The powdered seed is extracted with hot dilute alcohol. The alcoholic 
solution is evaporated and the aqueous remainder is treated with lead acetate 
which precipitates tannin, proteins, etc. The excess of lead is removed by 
treatment with hydrogen sulphide and the filtrate from the lead 1 sulphide is 
freed from hydrogen sulphide. Magnesia is added to precipitate the alkaloids 
which are filtered off after standing. , The mixture of brucine and strychnine 
is separated by alcohol which dissolves the brucine. The strychnine is puri- 
fied by crystallisation from alcohol. 

Strychnine crystallises in colourless prisms which melt at 284. It is very 
slightly soluble in water and its solutions have a very bitter taste. It is a 
weak base and forms salts with acids. 

Though it contains 2 nitrogen atoms it is only a monacid base and com- 
bines with only i molecule of methyl iodide. 

Strychnine gives the following reaction by which it may be detected : 

On treating a small quantity of strychnine or a strychnine salt with con- 
centrated sulphuric acid in a porcelain basin and on adding a small amount 
of powdered potassium bichromate, a violet solution is produced which be- 
comes red and then yellow. 

The constitution of strychnine has not yet been definitely determined, 
but it is a derivative of quinoline. 

Brucine. C 21 H 20 (OCH 3 ) 2 O 2 N 2 . 

Brucine is present with strychnine in the seeds of nux vomica. 

Preparation. 

The alcoholic solution, in which the brucine has dissolved in the separa- 
tion from strychnine, is evaporated to dryness. The residue is dissolved in 
dilute acetic acid and this solution is evaporated so as to remove the strych- 
nine which is also contained in it. The strychnine separates out on eva- 
poration and is filtered off, the strychnine acetate being an unstable salt. 
The brucine acetate is dissolved in water and the brucine is precipitated by 
adding caustic soda. It is crystallised from alcohol. 



358 PRACTICAL ORGANIC AX!) BIO-CHKMISTRY 

Properties. 

Brucine crystallises from water in colourless prisms with 4 molecules 
of water of crystallisation. The anhydrous substance melts at 178. It is. 
slightly soluble in water and alcohol and is very similar to strychnine, but is 
not so poisonous. 

It gives the following reaction : 

A deep brown-red colour is formed on adding nitric acid to a brucine 
salt ; the colour changes to yellow on warming. The colour becomes violet 
on adding stannous chloride. 

Constitution. 

Brucine seems to be a dimethoxy derivative of strychnine and is a deri- 
vative of quinoline. 

Morphine. C 17 H 19 NO 3 . 

Morphine is the chief alkaloid contained in the heads of poppies, 
Papaver somniferum. The alkaloids are present as sulphates and 
meconates. 

Preparation. 

Incisions are made in poppy heads and the juice which exudes is col- 
lected and dried. This dry mass is termed opium. 

The opium is treated with boiling water and the solution containing 
the salts of the bases is made alkaline with milk of lime. Calcium meconate 
and the alkaloids are precipitated. The alkaline solution containing the mor- 
phine is concentrated and warmed with ammonium chloride, so as to form 
calcium chloride, as long as ammonia is evolved. On standing morphine 
separates out and is crystallised from alcohol. 

Properties. 

Morphine crystallises from alcohol in small colourless prisms with one 
molecule of water. It is slightly soluble in water and its solution has a bitter 
taste. It is soluble in alcohol. An alcoholic solution of opium is termed 
laudanum. 

It is a base which forms salts with acids, the hydrochloride being used in 
medicine ; it combines with i molecule of methyl iodide. 

Morphine may be detected by the following reactions : 

(1) A deep blue coloration is formed on adding ferric chloride to a solu- 
tion of a morphine salt. 

(2) On adding a little morphine solution to iodic acid solution, iodine is 
liberated and forms a brownish-red precipitate which reacts with starch. 

(3) On dissolving morphine in concentrated sulphuric acid and adding 
concentrated nitric acid, after about 15 hours a deep blue-violet colour, 
which changes to red, is produced. 

It is converted into apomorphine by loss of i molecule of water on heat- 
ing with concentrated hydrochloric acid. 



THE ALKALOIDS ' 



359 



Constitution. 

Morphine contains one hydroxyl group and one alcoholic group and on 
distillation yields pyridine and quinoline. Its constitution has not yet been 
definitely determined, but is probably 

CHCH 2 




CH 3 

It thus contains an isoquinoline nucleus and a phenanthrene nucleus. 

Codeine. C 17 H 17 NO(OCH 3 )OH. 

Codeine is also contained in opium. It can be obtained by methylating 
opium and is thus a methyl derivative of morphine. Its constitution is still 
not definitely known. 

Papaverine, Laudanosine, Narcotine, Narceine. 

These alkaloids are present in small quantities in opium with morphine. 
They are isoquinoline derivatives : ' 

OCH, 




CH.; 



Papaverine 

or 
etramethoxybenzylisoquinoline. 



OCH 




Laudanosine 
or 



N-methyl-tetrahydropapaverine. 



CH 3 CH 



CH 



xO 



H C 







OC- 

CH 2 CH 3 Ol 
CH 2 OCH, 

Narcotine. 




360 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



Narcotine yields cotarnine and meconin on hydrolysis : 
CH a O CHOH 




Cotarnine. 




BUG 




-CO 



NiCH 3 ) 3 HOOCr 

'H 2 HOOcl 

CH 2 OCH. 

Narceine. 




Berberine. C 20 H ]9 O 5 N. 

Berberine is an alkajoid found in Berberis vulgaris and in other' plants. 
It has a still more complicated structure : 




CH, 



The details of the determination of the structure of the alkaloids can be found 
in the special books dealing with alkaloids. 



THE PROTEINS. 



Proteins make up the greater part of the solid matter of all 
animal cells and tissues and are present in various parts of plants. 
Meat and eggs consist mainly of protein ; milk, seeds and some fruits 
contain a large proportion of it. Protein is thus an essential in- 
gredient of our food. 

Composition of Proteins. 

Proteins are composed of amino acids, which may be regarded as 
the units of the protein molecule just in the same way as a poly- 
saccharide is composed of monosaccharide units and a fat of glycerol 
and various fatty acid units. The amino acids are obtained by the 
hydrolysis of proteins with acids, alkalies or enzymes. Up to the 
present time 18 amino acids have been found to occur in the 
protein molecule hence its complexity but, though 18 units may 
be present in some proteins, others contain fewer, and in some 
cases a protein has been found to be composed of only 2 or 3 units. 
The percentage amounts of the various amino acids, which have 
been obtained by the hydrolysis of some of the proteins, are given in 
the following table : 





Salmine. 


Globin 
of Hamo- 
globin 
(Horse). 


Serum- 
albumin. 


Serum- 
globulin. 


Excelsin 
from 
Brazil Nut. 


Caseinogen. 


Gelatin. 


Silk- 
fibroin. 


Glycine - 






O 


3'5 


0-6 




16-5 


36'O 


Alanine- 




4-2 


27 


2'2 


2-3 


0-9 


0-8 


21 '0 


Valine - 
Leucine 
Isoleucine 


4 '3 


V 29-0 


V 20-0 


JI87 


I'5 

87 


I'O 

} 10-5 


I'O 

2'I : I'5 


Phenylalanine 




4-2 


3'i 


3-8 


3'5 


3-2 


0- 4 


i '5 


Tyrosine 


I'3 


2'I 


2'5 


3'i 


4 '5 


,9_ | 10-5 


Serine - 


7-8 


0-6 


0-6 






O'2 


o'4 ' i '6 


Cystine - 




0'3 


2-5 


0-7 




o-i 




Proline - 


ir-o 2-3 i-o 


2-8 


3-6 


3'i 


5'2 + 


Oxyproline 


I'D 








0-3 


3-0 , 


Aspartic acid 


4 '4 


3-1 


2'5 


3-8 


I '2 


0-6 + 


Glutamic acid 


1-7 


77 


8-5 


12-9 


II'O 


0-9 o 


Arginine 


8? '4 5 '4 






16-1 


4-8 


7'6 : i'o 


Lysine - 


o 4'3 






1-6 


5-8 


2-8' ' + 


Histidine 


II'O 






i"5 


2-6 


0-4 + 


Tryptophan - 


+ 


+ 


+ 


+ 


i '5 


o 



We may notice in particular that salmine contains 87 per cent, of 
arginine, that haemoglobin contains 1 1 per cent, of histidine, that 
silk-fibroin is composed chiefly of glycine, alanine and tyrosine. 
Some vegetable proteins, those of the cereals, contain about 40 per 
cent, of glutamic acid. Numerous other differences can be noted, e.g. 
that glycine is present in serum globulin, but absent in serum albumin. 
But no stress must be laid upon other small differences since the 
method of analysis is not a quantitative one. The amounts actually 

361 



362 PRACTICAL ORGANIC AND .BIO-CHEMISTRY 

present are in most cases greater than is given. The total sum of the 
isolated amino acids is generally about 50 per cent. ; it is 70 per cent, 
in the case of haemoglobin and nearly 90 per cent, has been obtained 
from a vegetable protein. 

The analytical data of all the proteins which have been analysed 
will be found in the "Chemical Constitution of the Proteins," Part I. 
In this book is also given a full account of the methods of analysis 
of the proteins. Here it is not possible to give more than a very 
brief outline of the method of separating the amino acids. 

Hydrolysis of Proteins and Separation of the Amino Acids. 

The hydrolysis of proteins is generally effected according to the par- 
ticular amino acid or group of amino acids required. The preparation of 
cystine, tyrosine and tryptophan is given on pp. 143, 267, 348. 

The other amino acids are divided into two groups monoamino acids 
and diamino acids. The latter is separated from the former by precipitation 
with phosphotungstic acid. 

If the diamino acids are required, the protein is hydrolysed by boiling for 
12-24 hours with 6 times its weight of 33 per cent, sulphuric acid. 

If the monoamino acids are required, the protein is hydrolysed by boil- 
ing for 6-12 hours with 3 times its weight of concentrated hydrochloric acid. 

If both groups are required, the protein is hydrolysed with sulphuric acid, 
the diamino acids are removed with phosphotungstic acid, the filtrate is freed 
from sulphuric acid and treated according to the procedure for monoamino 
acids. 

If all amino acids are required, tryptophan is first separated ; the solution 
after removing mercury is hydrolysed with sulphuric acid, the diamino acids 
are precipitated with phosphotungstic acid and the filtrate treated for mono- 
amino acids as mentioned below. 

The separation of the diamino acids depends upon the following re- 
actions : 

The phosphotungstate precipitate is decomposed with baryta ; the solu- 
tion is treated with silver sulphate and saturated with baryta. Arginine and 
histidine are precipitated. The lysine in the filtrate is finally precipitated 
by picric acid. Arginine and histidine are separated by a second precipita- 
tion with silver nitrate in neutral solution ; the histidine is thrown down ; the 
arginine is thrown down by saturating the filtrate with baryta. The diamino 
acids are then recovered from the precipitates. 

The separation of the monoamino acids is effected thus : 

The hydrolysed solution is evaporated and saturated with dry hydrogen 
chloride ; glutamic acid hydrochloride is precipitated. 

The filtrate is esterified by mixing with 3 volumes of absolute alcohol and 
saturating it with dry hydrogen chloride; glycine ester hydrochloride is pre- 
cipitated. 

The esters of the other amino acids are separated by extracting with ether 
after liberating them from their hydrochlorides with caustic soda and saturat- 
ing the solution with potassium carbonate. The esters, after distilling off the 
ether which has been dried with sodium sulphate, are separated by fractional 
distillation in vacua. 

The fractions of esters are hydrolysed by water, or by baryta, and the 

. mixture of two or three amino acids separated by fractional crystallisation, or 

by the fractional crystallisation of their copper and other salts. Proline is 

contained in the lower boiling fractions. It is soluble in alcohol and is thus 

separated from the amino acids which are insoluble. 



THE PROTEINS 363 

Constitution of the Proteins. 

The work of Emil Fischer and his pupils has shown that the 
amino acid units are combined together in the form of acid amides, i.e. 
the carboxyl group of one amino acid is combined with the amino 
group of another amino acid, e.g. : 

Glycyl-glycine - CH,(NH 2 ) . CO NH . CH 2 . COOH 
Alanyl-leucine - CH~ 3 . CH(NH 2 ) . CO NH . CH(C 4 H 9 ) . COOH 
Leucyl-alanine - C 4 H 9 . CH(NH 2 ) . CO NH . CH(CH 3 ) . COOH 

in which the units may either be the same or different, and combined 
in any possible order. 

These combinations of amino acids have been termed by Emil 
Fischer the polypeptides. The above compounds are dipeptides. In 
the same' way we may have : 

Tripeptides, e.g. 

Diglycyl-glycine or glycyl-glycyl-glycine, 

CH 2 (NH 2 ) . CO NH . CH 2 . CO NH . CH 2 . COOH. 

Glycyl-alanyl-tyrosine, 

CH 2 (NH 2 )CO NH . (CHCH 3 )CO NH . CH(CH 2 . C 6 H 4 OH) . COOH. 

Alanyl-glycyl-tyrosine, 

CH 3 . CH(NH 2 ) . CO NH . CH 2 . CO NH . CH(CH 2 . C 6 H 4 OH) . COOH. 

Tetrapeptides, e.g. 

Glycyl-alanyl-glycyl-tyrosine, 
' CH 2 (NH 2 ) . CO NH . CH(CH 3 )CO NH CH 2 . CO NH . CH(CH 2 . C 6 H 4 O'H) . COOH. 

Pentapeptides, hexapeptides, etc. 

The most complex polypeptide known is an octadecapeptide, 
which is composed of eighteen units made up of three leucine and 
fifteen glycine units. This compound, if it had been found in nature, 
would undoubtedly have been regarded as a true protein. 

The synthesis of these polypeptides has been effected in three 
ways. The simplest of the methods of combining two or more amino 
acids together is by the action of the acid chloride derivative of one 
unit upon the other unit, e.g. alanyl-leucine is formed by the action of 
alanyl-chloride upon leucine : 

CH 3 . CH(NH 2 ) . CO . Cl + H 2 N . CH(C 4 H 9 ) . COOH = 
Alanyl chloride. Leucine. 

HC1 + CH 3 . CH(NH 2 ) . CO NH . CH(C 4 H 9 ) . COOH. 
Alanyl-leucine. 

By the action of another amino acid chloride upon this compound 
a tripeptide will be formed and the continuation of the process will 
lead eventually to the most complex polypeptides. The new com- 
pound (di- or tripeptide) is again an amino acid and can be con- 
verted into its acid chloride. This acid chloride will react with 
another amino acid, a dipeptide, tripeptide, etc., yielding a tetra-, a 
penta-, a hexapeptide. 



364 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

The amino acids can be combined together in any order, e.g. a-b- 
c-d, b-c-a-d, etc. Consequently an enormous number of isomers 
is possible. 

The chief evidence which we possess in support of this polypeptide 
constitution of the proteins is (i) the hydrolysis of these polypeptides 
by trypsin and other proteoclastic enzymes into their constituent 
units in the same way as the natural proteins are hydrolysed, (2) the 
isolation of polypeptides from the natural proteins, e.g. glycyl-tyrosine. 

Classification. 

The known proteins are classified according to their origin, solu- 
bility, coagulability on heating and other physical properties without 
reference to their chemical composition. This classification is, how- 
ever, borne out by their actual chemical composition as far as it is 
known. 

The following is the classification adopted by the Chemical and 
Physiological Societies in 1 907 : 

1. Protamines. 

2. Histones. 

3. Albumins^., 

. , .. fCoagulable Proteins. 

4. GlobulmsJ 

5. Glutelins. 

6. Gliadins. 1 

7. Scleroproteins. 

8. Phosphoproteins. 

9. Conjugated Proteins. 

(a) Nucleoproteins. 
() Glucoproteins. 
(c) Chromoproteins. 
i o. Derivatives of Proteins. 
(a) Metaproteins^ 
() Proteoses. 
' (c) Peptones. 
(cT) Polypeptides. 

1 Prolamins is the American terminology of this group. 



THE PROTEINS 365 



THE GENERAL REACTIONS OF PROTEINS. 

Though proteins are classified into so many groups, they give a 
series of reactions which are very characteristic and which serve for 
their identification. 

The exact group to which a protein belongs is more difficult to 
determine, but it can be ascertained by reference to the physical pro- 
perties of the members of the different groups. 

The general reactions depend upon : 

(1) The constituent units (the colour reactions). 

(2) The basic character of the units, especially the diamino acid 
units (the precipitation by alkaloidal reagents). 

(3) Their colloidal nature and high molecular weight (coagulation 
reactions). 

All proteins do not give all the reactions ; if a particular unit is 
missing the colour reaction for that unit will be absent ; some of the 
coagulation reactions are negative with some of the groups. Con- 
sequently it is necessary to perform several of the reactions before the 
presence of protein is verified. 

The general reactions of a protein are given by a solution of egg- 
white, which is very conveniently prepared as follows : 

Egg-white is beaten to break up the membranes, filtered through 
calico and diluted with nineteen times its volume of water. A pre- 
cipitate of ovomucin (formerly regarded as globulin) separates out, 
but it passes into solution on adding a little salt solution (NaCl, 
Am 2 SO 4 ). 

Undiluted egg-white has a faintly alkaline reaction and contains 
about 10 or 12 percent, of protein. The solution may show very 
faint alkalinity to litmus and contains o - 5 to I per cent, of protein. 

A. COLOUR REACTIONS. 

(i) Biuret. 

To some of the protein solution is added caustic soda and then, 
drop by drop, dilute copper sulphate solution (i per cent), mixing 
after each addition : a violet colour appears. Excess of copper sulphate 
must be avoided as its blue colour masks the reaction. 

This reaction is due to the presence of at least two CO NH 
groups. (See under urea.) 



366 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

(2) Xanthoproteic. 

On heating a portion of the protein solution with concentrated 
nitric acid, a yellow colour is formed ; the colour changes to orange 
on adding ammonia or soda in excess to the cooled solution. 

This reaction is most probably due to the formation of nitro 
compounds with the aromatic units contained in the protein, namely, 
tyrosine, phenylalanine. 

(3) Millon's. 

On adding Millon's reagent to some of the protein solution, a 
white precipitate is formed ; it becomes red on heating. 

This reaction is due to the presence of tyrosine in the protein. 
(Compare hydroxy derivatives of benzene.) 

(4) Sulphur. 

A drop of lead acetate solution is added to some of the protein solu- 
tion and sufficient caustic soda to redissolve the precipitate which is 
first formed. A brown coloration, sometimes black, occurs on boiling. 

This reaction is due to the separation of hydrogen sulphide from 
the cystine unit, which gives lead sulphide with the lead acetate. 

(5) Adamkiewicz' or Glyoxylic Acid. 

If excess of glacial acetic acid be added to the solution and con- 
centrated sulphuric acid be run underneath it, on standing, or on 
gently shaking, a reddish-violet colour appears at the junction of the 
fluids which gradually spreads throughout the solution. 

It has been shown by Hopkins and Cole that this reaction is due 
to the presence of glyoxylic acid in the glacial acetic acid ; it is there- 
fore better to use a solution of glyoxylic acid instead of glacial acetic 
which (if kept in the dark) may not contain this substance. 

A little glyoxylic acid solution is added to some of the protein solu- 
tion and concentrated sulphuric acid is run in as before to the bottom 
of the test tube. The reddish-violet ring as above described slowly 
forms. 

This reaction is due to the presence of tryptophan in the protein 
molecule. Some substance is formed from the glyoxylic acid which 
reacts with the tryptophan. 

A similar colour is produced on adding commercial sulphuric acid to 
a protein solution containing a minimal quantity of formaldehyde. This 
reaction is brought about by the presence of oxidising agents in the sulphuric 
acid which act upon the formaldehyde (Rosenheim). It is not due to the 
formation of glyoxylic acid by aldol condensation of formaldehyde and oxida- 
tion (Dakin). 

It should be noted that this reaction has been used for many years for 
detecting formaldehyde added to milk as a preservative. 



THE PROTEINS 367 



Proteins, precipitated by alcohol and washed with ether, give a blue colour 
when heated with concentrated hydrochloric acid. (Liebermann's reaction.) 
A reddish-violet colour, which ultimately becomes brown, is produced on 
heating proteins with concentrated hydrochloric acid. 

These reactions, according to Cole, are due to the presence of trypto- 
phan : in the first, glyoxylic acid is derived from the alcohol and ether : in 
the second, furfural is formed from carbohydrate in the protein and reacts 
with the tryptophan. 

The green to blue colour produced when proteins are heated with ben- 
zaldehyde, a drop of ferric chloride and concentrated hydrochloric acid 
(Reichl's reaction) is also due to the presence of tryptophan. 

(6) Molisch's. 

A few drops of a-naphthol solution are added to the protein solu- 
tion and mixed thoroughly. Concentrated sulphuric acid is run under 
the solution. At the junction of the two liquids a purple-red ring is 
formed. 

This reaction is due to the formation of furfural from the carbo- 
hydrate radicle in the protein and to its combination with a-naphthol. 






368 PRACTICAL ORGANIC AND BIO-CHEMISTRY 


B. COAGULATION REACTIONS. 

(1) Heat. 

On heating some of the solution an opalescence occurs, with 
perhaps a slight precipitate on the surface of the glass. But on faintly 
acidifying it, or another portion, with 1-2 drops of dilute acetic acid 
and again heating, a cloudiness and then a flocculent precipitate of 
coagulated protein is formed. This precipitate is not soluble in dilute 
acids and alkalies in the cold, but it gradually dissolves on heating 
with caustic soda. 

Coagulation did not occur at first as the reaction was alkaline ; it 
only occurs when the solution is faintly acid. 

(2) Alcohol. 

A precipitate is formed if excess of alcohol be added to some of the 
solution. This precipitate is at first capable of re-solution in water, 
but on prolonged contact with alcohol it is rendered insoluble, the 
protein being coagulated. 

(20) Ether. On adding about half a volume of ether to some of the solu- 
tion and mixing thoroughly by inverting the liquids, a gelatinous solution 
results, which contains coagulated protein. 

(3) Strong Mineral Acids. 

Hellers Test. Concentrated nitric acid is added to some of the 
solution by means of a pipette, or by gently pouring down the sides of 
the tube, so that the acid forms a distinct layer below the solution. 
At the junction of the two liquids a white ring of coagulated protein 
is formed. The precipitate does not dissolve in excess of the acid, 
if the liquids be mixed by shaking. 

C. PRECIPITATION REACTIONS. 

(i) Solutions of Heavy Metals. 

Mercuric Chloride. If 2 or 3 drops of mercuric chloride solution be 
added to some of the protein solution, a heavy white precipitate of the 
mercury compound is formed. This dissolves on adding some saturated 
sodium chloride solution. The mercury compound is reprecipitated 
from its solution in sodium chloride on adding a few drops of dilute 
hydrochloric acid. 

Copper sulphate, added drop by drop, forms a bluish-violet precipi- 
tate which dissolves in caustic soda, giving a violet solution (biuret 
reaction). 

Ferric chloride gives a precipitate soluble in excess. 

Lead acetate and basic lead acetate give white precipitates. 



THE PROTEINS 



369 



(2) Alkaloidal Reagents in Acid Solution. 

(a) Hydroferrocyanic acid. A few drops of glacial acetic acid are 
added to a little of the protein solution and then, drop by drop, potas- 
sium ferrocyanide solution. A voluminous precipitate is formed. 
This precipitation is less complete in the presence of neutral salts and 
does not occur in neutral solutions. 

(b} Picric acid. A yellowish precipitate is formed on adding picric 
acid to egg-white solution. 

(c) Potassio-mercuric iodide (Brilckes reagent}. A whitish pre- 
cipitate is formed when the protein solution is acidified with dilute 
hydrochloric acid and a few drops of potassio-mercuric iodide are added. 

(</) Trichloracetic acid. A white precipitate is formed on adding 
an equal volume of 10 per cent, trichloracetic acid. 

(e) Tannic acid. A brownish precipitate is formed. 

(f) Bromine water gives a white precipitate. 

(g) Phosphotungstic acid. A white precipitate is produced when 
phosphotungstic acid is added to the protein solution pre- 
viously acidified with hydrochloric or sulphuric acid. 

These reagents are the most commonly employed for 
removing proteins from solution, e.g. in the analysis of blood. 

The tannic acid compound is of commercial importance. 
Leather is made by tanning skins. 

The hydroferrocyanic acid reaction is often used clinically 
for detecting protein ("albumin") in the urine. 



ESTIMATION OF PROTEIN. 



s 

-* 



Picric acid is used in the estimation of protein by Es- 
bach's method. 

Some of the solution is poured into the Esbach tube (Fig. 
54) up to the mark U and then Esbach's reagent up to the 
mark R. The tube is corked, the contents are mixed by FIG. 54. 
inverting 2 or 3 times without shaking and allowed to stand for 24 
hours. The tube is graduated in amounts of protein in grams per 
litre ; the height of the deposit gives the amount. This method is 
often employed in estimating " albumin " in urine. 




24 



370 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

DERIVATIVES OF PROTEINS. 

Proteins are hydrolysed into their constituent amino acids by 
boiling with concentrated mineral acids or alkalies for 6-10 hours. 
This hydrolysis of the complex protein takes place in several stages, 
products intermediate between the large protein molecule and the 
amino acid molecule being formed. These are metaprotein, proteoses, 
peptones and polypeptides. In the formation of metaprotein only a 
comparatively small change in the protein molecule occurs. Proteoses 
and peptones are formed by the breaking up of the large molecule 
into several large complexes, each of which is gradually hydrolysed into 
smaller complexes, from which the amino acids are finally formed. 

The metaproteins, the proteoses and the peptones still possess 
some of the properties of a typical protein, but the amino acids do 
not. The polypeptides are intermediate between the peptones and 
amino acids ; some resemble the peptones, others the amino acids. 
In all probability peptone is a mixture of polypeptides. 

METAPROTEINS. 

The most typical metaproteins are formed from the mixture of 
albumin and globulin of egg-white or of blood serum, but they are 
also formed from the other proteins. 

Preparation. , 

Metaprotein is most easily prepared by the action of acid or 
alkali upon proteins. It is formed fairly rapidly at 60 and higher 
temperatures, more slowly at 37. 
(a) By Add. 

(i) Egg-white or serum is mixed with 10 times its volume of 0*4 
per cent, hydrochloric acid and kept in an incubator at 37 for at least 
24 hours. 

(ii) Egg-white or serum is mixed with one-third of its volume of 
glacial acetic acid and allowed to stand. The mixture sets to an 
opaque jelly. The opacity is due to the coagulation of the protein by 
the strong acid. On diluting with water the jelly dissolves leaving 
coagulated protein. 
(ff) By Alkali. 
(i) Egg-white or serum is mixed with 10 times its volume of OT 

per cent, sodium hydroxide and kept at 37 for about 18 hours. 

is (ii) Egg-white or serum is mixed with about one-third of its 

volume of 2N sodium hydroxide. On standing it sets to a transparent 

jelly (Lieberkiihn's jelly). The jelly dissolves on diluting with water. 

* Small quantities may be rapidly prepared by adding about one, 

quarter of the volume of dilute acid or alkali to 10 or 20 c.c. of an 

egg-white solution and keeping in water at 40-50 for 10-15 minutes. 



THE -PROTEINS 371 

Properties. 

The solutions prepared above are known as acid metaprotein or acid 
albumin and alkali metaprotein or alkali albumin. 

(1) No coagulation occurs on boiling a portion of the solution. 

(2) Metaprotein is insoluble in water. 

On carefully neutralising the solutions with 2N acid or alkali re- 
spectively, the metaprotein is precipitated when the solution is just 
acid to litmus ; it redissolves in an excess of either acid or alkali. 
The precipitate is allowed to settle, the bulk of the water decanted and 
the remainder filtered. The precipitate is washed with water and ex- 
amined as follows : 

(3) Metaprotein is soluble in dilute acid or alkali. 

A portion of the precipitate will dissolve in dilute acid or alkali, 
and will be precipitated on neutralising. 

(4) Metaprotein is coagulated by heating in neutral solution. 

A portion of the precipitate is suspended in water and heated. Coag- 
ulation occurs. This is verified by adding a drop of dilute acid to the 
cold solution when the precipitate is no longer found to be soluble. 

(5) Behaviour towards salt solutions. 

Acid metaprotein solutions are precipitated completely on saturat- 
ing the solution with (a) sodium chloride, (b] magnesium sulphate, or 
(c) by half saturation with ammonium sulphate. Alkali metaprotein 
solutions are not precipitated by saturation with sodium chloride, but 
are precipitated by saturation with magnesium sulphate or half-sat- 
uration with ammonium sulphate. 

(6) Both solutions give the colour reactions and some of the other 
general protein reactions. 

PROTEOSES AND PEPTONES. 

A mixture of these substances is formed by the hydrolysis of 
proteins. They are termed albumose, globulose, caseose, etc., fibrin- 
peptone, gelatin-peptone, histo-peptone, etc. , according to the name of 
the protein from which they arise. 

Preparation. 

The mixture of proteoses and peptones is most easily prepared by 
digesting a protein (egg-white, meat, etc. ) with 20 parts of '4 per cent, hy- 
drochloric acid and with about X)i gm. of pepsin at 37 for several days. 

Witte's peptone is a commercial peptone prepared in this way 
from fibrin. Similar commercial peptone preparations are made from 
other proteins. 

Properties. 

The commercial mixtures consist of amorphous powders, white 
or pale yellow in colour, easily and generally completely soluble in 
water, but sometimes a small residue remains undissolved. A solution 
of the mixture (5 per cent) shows the following reactions : 

24* 



372 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

A. Colour Reactions. 

The colour reactions for proteins are generally positive. 

B. Coagulation Reactions. 

(1) No coagulum is formed on boiling the solution after acidifying 
with a drop of acetic acid. 

(2) A white precipitate is formed on adding nitric acid, drop by 
drop, especially in presence of salts. This precipitate dissolves on 
heating, but reappears on cooling. 

(3) Alcohol precipitates the proteoses more or less entirely ; the 
precipitate is not coagulated by standing in contact with alcohol and 
redissolves on adding water. 

C. Precipitation Reactions. 
(a) Heavy Metals. 

The solution is precipitated by solutions of the heavy metals : 
copper sulphate, lead acetate, mercuric chloride. 
(6) Alkaloidal Reagents. 

(1) A white precipitate is formed on adding a drop of glacial 
acetic acid and -2 or 3 drops of potassium ferrocyanide. This pre- 
cipitate dissolves on heating but reappears on cooling. 

(2) Tannic acid gives a white precipitate. 

(3) Other alkaloidal reagents also produce precipitates. 

SEPARATION OF PROTEOSES AND PEPTONE. 

The mixture of derivatives obtained by the hydrolysis of a pro- 
tein is separated by salting out from the solution. Saturation with 
sodium chloride or magnesium sulphate produces the same effect as 
half saturation with ammonium sulphate or zinc sulphate. Ammon- 
ium sulphate is used almost exclusively. 

(i) Primary Proteoses. 

A white precipitate of primary proteoses is formed when an ex- 
actly equal volume of saturated ammonium sulphate solution is added 
to a solution of Witte's peptone (20 or 25 c.c. should be used in testing). 
If the mixture be well stirred with a glass rod covered at the end with a 
piece of rubber tubing, the precipitate may gather upon the end of the 
rod and can be almost completely removed in this way, otherwise it is 
separated by filtering. 

The mass thus collected may be dissolved in warm water. The 
cold solution will give the previous reactions for the mixture of pro- 
teoses and peptone. 

This precipitate according to Haslam cpntains three substances : 

a-protoproteose. 
/?-protoproteose. 
Heteroproteose. 



THE PROTEINS 373 

The a and y8 protoproteoses are very similar to one another and are easily 
soluble in water. Heteroproteose is very little soluble in water and can be 
separated from the others by dialysis ; it is precipitated from solution. 

Heteroproteose and the protoproteoses are more easily separated by 
means of alcohol. Heteroproteose is precipitated by 32 per cent, of alcohol ; 
protoproteose is soluble in alcohol up to 80 per cent. 

If an equal volume of alcohol be added to the above solution of primary 
proteoses, the heteroproteose will be precipitated. 

(2) Secondary or Deuteroproteoses. 

These proteoses are precipitated by complete saturation of the 
solution of proteoses and peptone with ammonium sulphate. The 
filtrate remaining after the precipitation of the primary proteoses is 
acidified with a drop of dilute sulphuric acid and saturated with finely 
powdered ammonium sulphate. 1 A flocculent precipitate comes down 
and is filtered off. 

If it be dissolved in water, it will be found not to give all the above 
reactions for proteoses, e.g. : 

The reactions with acetic acid and potassium ferrocyanide, con- 
centrated nitric acid, copper sulphate are negative. 

This precipitate also consists of a mixture of at least two deuteroproteoses, 
a and /?, and a third has been described. They differ in their behaviour 
towards ammonium sulphate. 

Both the primary and secondary proteoses are very indefinite substances 
and methods have still to be devised for a perfect separation. 

PEPTONE. 

Peptone is not precipitated by saturation with ammonium sulphate. 
It therefore remains in solution after the proteoses have been removed. 

Its chief characteristic is the biuret reaction : 

A portion of the filtrate is treated with excess of strong caustic 
soda solution (40 per cent, or solid substance) and a drop or two of 
I per cent, copper sulphate solution. A pink colour appears, which is 
characteristic of peptone. It is necessary to add a large excess of 
caustic soda if ammonium sulphate be present in the solution, in order 
to decompose it and in order that the alkalinity should be due to sodium 
hydroxide ; the alkalinity of ammonia does not produce the colour. 

Of the other colour reactions they are sometimes positive, some- 
times negative, depending on the peptone. 

Peptone is precipitated by some of the precipitating reagents, e.g. 
tannic acid, phosphotungstic acid, lead acetate, but not by others. 

Peptone again is a mixture which has not been perfectly separated ; 
at least two peptones are present. 

1 4 gm. to every 10 c.c. of half saturated solution. 



APPENDIX TO PROTEINS. 
COLLOIDS AND COLLOIDAL SOLUTIONS. 1 

The proteins, also the fats and soaps and the polysaccharides, the 
principal substances with which physiological chemistry has to deal, 
are colloids. Their properties depend so much upon this fact that 
it is necessary to examine the nature of colloids and colloidal solutions. 

Crystalloids and Colloids. 

Thomas Graham between 1861 and 1864, whilst studying the 
diffusion of dissolved substances through organic membranes, such as 
parchment paper, found that some substances dialysed, or passed freely 
through the membrane, but that other substances did not pass through 
or passed through very slowly. The substances belonging to the first 
class were salt, sugar, urea, etc., which crystallised well : the substances 
belonging to the second class like gelatin, albumin, gum, starch, did 
not crystallise. He distinguished the two classes as crystalloids and 
colloids. 

Natural and Artificial Colloids. 

The substances belonging to the group of colloids show amongst 
themselves many differences : 

Hot solutions of gelatin or agar on cooling form jellies which re- 
dissolve on warming. Solutions of albumin on heating coagulate, i.e. 
form an insoluble precipitate. Solutions of gum neither set to a jelly 
nor coagulate, but always form more or less viscous solutions. 

Graham found that substances like silicic acid, ferric hydroxide, 
etc., substances which are usually insoluble, could be made to form true 
solutions in their appearance to the eye, and that the solid matter in 
apparent solution did not diffuse through parchment membranes. 

These artificial solutions had one peculiar property : they under- 
went a marked and irreversible change on the addition of a small 
quantity of an electrolyte. The solid matter was either precipitated or 
the solution set to a jelly ; neither the precipitate nor the jelly could 
be redissolved to form a solution. 

1 An excellent description is given by Hatschek, " An Introduction to the Physics and 
Chemistry of Colloids," from which book most of these notes have been compiled. 

374 



COLLOIDS AND COLLOIDAL SOLUTIONS 375 

Variety of Solvent. 

It is now known that other solvents besides water can dissolve sub- 
stances forming colloidal solutions. 

Cellulose dissolved in Schweitzer's reagent or in zinc chloride forms 
a colloidal solution from which the substance is precipitated as a gelatin- 
ous mass. Nitrocellulose dissolved in acetic acid, acetone, or alcohol- 
ether, forms a colloidal solution. Sodium chloride can be made to form 
a colloidal solution in petroleum ether and the alkali metals in organic 
solvents. 

Sols and Gels. 

Graham called the apparent solutions of colloids colloidal solutions, 
or sols, and the precipitated or gelatinous substance, gels. We can 
further distinguish the solvent by prefixing its name, e.g. hydrosol, 
alcoholgel, etc. 

PREPARATION OF ARTIFICIAL COLLOIDAL 
SOLUTIONS. 1 

A. Colloidal Solutions of Metallic Sulphides. 

(a) Cadmium Sulphide. A fine suspension of cadmium sulphate, pre- 
viously washed with distilled water, is treated with hydrogen sulphide. The 
solution gradually becomes milky and finally has a yellow colour with a reddish 
surface. The excess of hydrogen sulphide is removed by a current of nitrogen 
or by boiling. 

(b] Arsenious Sulphide. About I gm. of arsenious acid is boiled 
for a few minutes with about 75 c.c. of distilled water ; the solution is 
filtered and allowed to cool. On passing hydrogen sulphide through 
the cold solution, it turns a yellow-orange colour with a greenish surface. 

B. Colloidal Solution of Ferric Hydroxide. 

I c.c. of a filtered 33 per cent, solution of ferric chloride is 
added to 100 c.c. of boiling distilled water. A reddish-brown solu- 
tion is obtained. 

A colloidal solution of ferric hydroxide may also be obtained by dialysing 
a solution of ferric chloride. 

C. Colloidal Solutions of Gold and Silver by Reduction. 

i c.c. of i per cent, gold chloride solution is diluted with 25 c.c. of 
distilled water. 2 gm. of tannic acid are dissolved in 100 c.c. of water. 

On mixing i volume of the gold chloride solution with 3 volumes of the 
tannic acid, a blue solution is formed. On mixing i volume of the gold 
chloride solution with i volume of the tannic acid, a red solution is formed. 

Similar solutions may be made by treating gold chloride solution with a 
solution of i gm. of hydroquinone or pyrogallol dissolved in 500 c.c. of water. 

Ammonia is added drop by drop to 10 c.c. of silver nitrate solution 
until the precipitate first formed just redissolves. The solution is 
diluted with 200 c.c. of water. On mixing equal volumes of this 
solution with the 2 per cent, tannic acid, a brown solution having 
a greenish colour in reflected light is formed. 

1 In preparing artificial colloidal solutions the glass vessels must be absolutely clean, 
preferably new, and washed with nitric or chromic acid. Freshly distilled water should 
also be used. 



376 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

D. Colloidal Solutions of Platinum and Silver by Disintegration. 

On forming an arc between two pieces of platinum or silver wire under 
distilled water, i.e. by separating the two poles when a suitable current is passed, 
a colloidal solution of the metal is formed. The larger particles which are 
formed settle and can be separated by decantation or nitration. 

The metal^ e.g. bismuth or chromium, is ground finely in a ball mill and 
treated for several days alternately with concentrated alkali and acid. On 
treating with water a colloidal solution results. 

Colloidal copper and silver solutions are obtained if distilled water be 
boiled in copper and silver vessels. 

Colloidal lead solutions are formed when water, free from oxygen and from 
which oxygen is excluded, is kept in contact with lead. 

A suspension of fine particles of kaolin is obtained by shaking 
some kaolin vigorously with water and pouring off from the larger 
particles which settle rapidly. 

E. Colloidal Solution of Lead Chromate Using Viscous 

Media. 

Sols of lead chromate and barium sulphate can be prepared if the 
reactions leading to their formation be carried out in a solution containing a 
colloid such as a solution of caseinogen. 

Colloidal solutions of some inorganic salts may be prepared by 
dissolving them in glycerol and pouring the solution into water (Craw). 

E.g. if some potassium chromate and lead nitrate be dissolved 
separately in glycerol, the solutions mixed and poured into water, a 
colloidal solution of lead chromate is formed. 

Detection of Colloidal Solutions. 

I. Dialysis. 

The simplest and most convenient way of showing the presence 
of a colloid in solution is that of dialysis as used by Graham. 

As dialyser Graham employed a piece of parchment paper fastened 
between two hoops forming a sort of tray which could be immersed in 
water or other liquids. As the object is to obtain as large a surface as 
possible the parchment paper is conveniently made in the form of a 
sausage skin. The colloidal solution is introduced into the sausage skin, 
the ends maybe tied tightly and the skin is immersed in water or 
other liquid, or it may be bent into U shape and suspended in a large 
tall vessel. 

Thimbles made of parchment paper are useful for small quantities 
of solution, and " soufflet " cases may also be used, especially for testing 
solutions. Fish-bladder is another material frequently employed. 

Collodion thimbles or tubes prepared by coating surfaces of test 
tubes, etc., with a solution of collodion in acetic acid, followed by im- 
mersion in water and removal of the tough membrane from the glass, 
form excellent dialysers. 



COLLOIDS AND COLLOIDAL SOLUTIONS 377 

In all cases a current of water is slowly circulated through the 
beaker or other vessel, or the dialyser may be put into several changes 
of .distilled water, e.g. : 

(a) Some litmus solution is placed with a drop or two of dilute 
hydrochloric acid in a parchment paper dish which is allowed to float in 
a beaker of distilled water. The litmus does not diffuse out, but the 
hydrochloric acid passes into the surrounding water. It may be tested 
for by silver nitrate in the presence of nitric acid. If the process of 
dialysis be continued sufficiently long (repeated changes of water), the 
red colour will disappear and the litmus will become blue. 

(b} The same experiment is repeated with a mixture of starch solu- 
tion and glucose; the former being a colloid does not diffuse out, but 
the latter, a crystalloid, diffuses out and can be tested for in the sur- 
rounding water by Trommer's or Fehling's test. 

(0 Egg-white solution treated in the same way does not diffuse 
out through a parchment paper membrane. The surrounding water, 
if tested for protein by the xanthoproteic, Millon's and the biuret 
reactions, will show that protein is absent. 

The globulin may be precipitated in the paper dish if the egg- 
white solution be dialysed long enough, as it is insoluble in distilled 
water ; it dissolves on adding a little salt. 

I 1. Tyndall Phenomenon. 

If a bright beam of light be passed through a colloidal solution 
contained in a vessel with parallel sides and the solution be viewed 
from the side, it will appear turbid, sometimes with a coloured sheen, 

III. Colloidal solutions are often opalescent, e.g. starch, glycogen. 
Some are coloured and show a pseudo-fluorescence : their colour in 
transmitted light is different to their colour in reflected light. 

IV. Colloidal solutions, especially those of natural substances, have 
a great tendency to froth if shaken. 

V. Colloidal solutions cannot generally be filtered through filter 
paper and behave like suspensions. 

E.g. a suspension of kaolin, prepared by shaking up kaolin with 
water, on filtration passes through, leaving only the large particles. 
Similarly, arsenious sulphide sol passes through filter paper. 



378 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

NATURE OF COLLOIDAL SOLUTIONS. 

Faraday, in 1857, who prepared a colloidal gold solution having a 
red colour by treating gold chloride with an ethereal solution of 
phosphorus, expressed the opinion that the gold was suspended in the 
liquid in an extremely fine state of division. 

Colloidal solutions have been shown by various methods to consist 
of suspensions of extremely fine particles. The colloidal condition is a 
state, not a form of matter. 

Suspensions and Emulsions. Suspensoids and Emulsoids. 

According as the suspension of fine particles may consist either of 
solid particles or of liquid particles, two classes are distinguished : 

(a) Suspensoid, in which the particles are solid, rigid and not 
deformable. 

() Emulsoid, in which the particles are liquid and deformable. 

Most of the natural colloidal solutions are emulsoids ; most of 
the artificial colloidal solutions are suspensoids. They are sometimes 
referred to as reversible and irreversible respectively, this terminology 
referring to their behaviour with electrolytes. 

i Continuous and Disperse Phases. 

It is usual to refer to the particles in suspension as the disperse 
phase and the medium in which they are suspended as the continuous 
phase. The continuous phase may be more concentrated in the form 
of a jelly or even a solid ; the disperse phase will then consist of drops 
of liquid or dilute solution in suspension. 

Filtration of the Particles Ultra-filtration. 

Though the minute particles in a sol cannot be filtered off through 
filter paper yet they are retained if they be filtered through paper im- 
pregnated with either gelatin hardened with formalin, or collodion 
(Bechhold), or if they be filtered through a clay filter impregnated with 
gelatin (Martin). The solution is forced through these filters by pres- 
sure and a clear solution free from particles results. 

Size of the Particles. 

(a) Knowing the strength of the gelatin or collodion filter, from 
which, the size of the pores can be determined, the size of colloidal 
particles can be estimated. The pores in a 2-5 and 5 per cent, collodion 
filter are from 21 //,//, to 930 /A/*. * Particles which are retained are 
probably larger than the size of the pores. 

(b} In the Tyndall phenomenon the particles in the solution which 
reflect the light must be smaller than the wave length of light, i.e. 
from 450 to 760 //./i for the visible spectrum. 

1 p. = -ooi mm. /J./JL = 'ooi /x = -000001 mm. 



COLLOIDS AND COLLOIDAL SOLUTIONS 379 

The particles may possibly be molecules with a high molecular 
weight, e.g. albumin, complex dye-stuffs. In the case 'of metallic and 
other inorganic sols the particles probably consist of aggregates of 
molecules. 

The particles have been shown to behave like gases, filling the space 
in which they are contained and obeying definite laws. 

Visibility of the Particles. The Ultramicroscope. 

The particles in a colloidal solution are too small to be seen 
with an ordinary microscope, but in most cases the particles can be 
seen with the so-called ultramicroscope. With this instrument a strong 
beam of light is sent horizontally through the solution, which is viewed 
with a microscope. The particles reflect the light into the microscope 
and appear as bright specks. Instead of the ultramicroscope arrange- 
ment, many colloidal solutions will show particles by reflected light if a 
cardioid condenser be used with an ordinary microscope. 

Brownian Movement of Particles. 

The small particles visible in the ultramicroscope, like many larger 
particles under a microscope, show Brownian movement. 

Non-Settling of Particles due to Electric Charge. 

The mere smallness of the particles is not sufficient to account for 
the long time taken for a suspensoid to settle, nor is the fact that the 
particles are in Brownian movement. 

The non-settling of the particles is due mainly to the fact that they 
are electrically charged and are thus repelled from one another, 
preventing coalescence to form larger particles or aggregates. 

Almost any substance in contact with water assumes an electric 
charge ; most substances become negatively charged. The charge 
can be reduced to zero or even reversed in direction by the addition of 
a suitable electrolyte. The particles in a coarse suspension are also 
electrically charged. 

Determination of the Electric Charge of the Particles. 

The electric charge on the suspensoid particles may be determined 
by placing the sol in a U tube ; above the sol on each side is put a 
layer of distilled water. An electric current is passed through the 
contents of the U tube, the poles being in the water. The particles 
will travel to the positive or negative pole. 

This may also be done on a microscope slide furnished at each 
side with a platinum wire connected with an electric current. ' A drop of 
the sol is put on the slide and the particles, when the current is passed, 
will travel to one side or the other. 



380 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

PROPERTIES OF COLLOIDAL SOLUTIONS. 

A. Suspensions and Suspensoids. 

(1) Concentration. These colloidal solutions are generally very 
dilute and contain only a fraction of I per cent, of solid matter in 
suspension. 

(2) Osmotic Pressure. They have a low osmotic pressure. The 
freezing-point of the continuous phase is lowered very slightly and the 
boiling-point is raised very slightly. 

(3) Viscosity. The viscosity of a suspensoid sol is only slightly 
higher than that of water and is proportional to the amount of solid 
matter present. 

(4) Behaviour to Electrolytes : 

'(a) The suspended particles are precipitated immediately or in a 
short time by the addition of a small quantity of an electrolyte, e.g. : 

If a few drops of saturated sodium sulphate be added to colloidal 
ferric hydroxide solution, or of metallic silver, the solid matter is 
precipitated. 

(b) Salts containing divalent ions are more effective than salts with 
monovalent ions; salts with trivalent more than those with divalent. 
2 mol. NaCl = i mol. BaCl 2 ; i mol. A1CL, = 3 mol. NaCl. 

The particles are probably discharged by the oppositely charged 
ion so that they no longer repel one another, but coalesce to form larger 
aggregates. 

(5) Behaviour to other Suspensoids. 

Positively charged suspensoids will precipitate negatively charged 
suspensoids. Both suspensoids are precipitated together. If the two 
colloidal solutions contain an equal number of particles with a suitable 
number of electric charges both are completely precipitated, e.g. : 

Varying quantities (i, 2, 3 c.c.) of ferric hydroxide sol may be 
added to varying quantities of arsenious sulphide sol (3, 2, i c.c.). 
Precipitation will occur. The excess of either sol remains and the 
precipitate contains both substances, as can be seen from the colour of 
the precipitate and of the solution. 

(6) Influence of Emulsoids. 

Emulsoids protect suspensoids from precipitation by electrolytes. 
It seems that a layer of emulsoid particles is formed round the 
suspensoid and so alters its properties ; e.g. if ferric hydroxide sol or 
arsenious sulphide be diluted with (a) an equal volume of water, (^) an 
equal volume of albumin solution, and sodium chloride solution be 
carefully added to each, the amount required to precipitate in ($) will 
be considerably greater than in (a). 



COLLOIDS AND COLLOIDAL SOLUTIONS 381 

B. Emulsions. 

Emulsions are systems of two liquids insoluble in each other ; they 
consist of comparatively coarse liquid particles of one liquid in another 
with which it does not mix. 

Emulsions are of two kinds : (a) a small quantity of a liquid in 
suspension in a large amount of another liquid ; (b} a large amount of 
one liquid suspended in another liquid ; in this class the continuous 
phase must consist of a solution of a colloid such as soap, protein, or 
saponin. 

Formation of Emulsions. 

(a) An emulsion of oil in water is obtained if a fine stream of oil 
be injected into water. 

* An emulsion is formed if an alcoholic solution of oil be poured into 
water. 

* (&} Permanent emulsions are formed when colloids are present in a 
solution and the solution is shaken up with another liquid. The most 
typical permanent emulsions are observed with fats and oils. 

The fats are neutral substances, but generally they contain a little 
fatty acid, which gives them an acid reaction and causes the formation 
of an emulsion when they are shaken up with alkali : 

In five^-test tubes are placed : 

(i) (2) (3) (4) (5) 

10 c.c. H 2 O 10 c.c. H 2 O 10 c.c. H 2 O 10 c.c. H 2 O 10 c.c. H 2 O 

2 c.c. neutral l i drop 8/ NaOH 2 drops oleic acid i drop 8/ NaOH i drop 8/ NaOH 

olive oil. 2 c.c. neutral 2 c.c. neutral 2 drops oleic acid 2 c.c. ordinary 

olive oil. olive oil. 2 c.c. neutral olive oil. 

olive oil. 

Each is shaken thoroughly. Only in (4) and (5) is a permanent 
emulsion formed, separation occurring in (i), (2) and (3) after a short 
time. (5) shows that ordinary fat contains free fatty acid. 

The same result can also be seen by dropping a little neutral olive 
oil and a little ordinary olive oil on to the surface of some dilute sodium 
carbonate solution in watch glasses. The neutral oil drop remains 
clear, whilst the ordinary oil drop spreads out and gives a milky 
emulsion. 

The formation of emulsions is due to the fact that a layer of soap, 
formed by the combination of the free acid with the alkali, is made 
round the fat particle. 

1 Neutral olive oil is prepared by dissolving it in ether, shaking up with dilute sodium 
carbonate solution, washing free from alkali and finally distilling off the ether. 



382 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

In the same way an emulsion is obtained when oil or petroleum is 
shaken up with egg-albumin. In both cases a layer of coagulated egg- 
albumin is formed round each particle. 

To prove this : A little egg-albumin solution is shaken up in a test 
tube ; a fine layer of mechanically coagulated egg-albumin will be seen 
to be formed and it rises to the surface on standing. 

Protein solutions have free-surface coatings ; by mechanically 
shaking, these are heaped up to form solid masses of protein. The 
following simple experiment demonstrates the surface coating of a 
protein solution : 

Two beakers are taken ; in the first is placed clean water, in the 
second egg-white solution. On to the surface of each is floated a mag- 
netised needle and a magnet is brought near. In the first beaker, 
the needle spins round ; in the second, only a slight attraction or 
repulsion is seen. If the beaker be suspended by a wire, in the 
latter case the whole beaker would swing round, whereas in the former 
only the needle would rotate (Ramsden). 

Milk and rubber latex are examples of naturally occurring per- 
manent emulsions. Milk contains fat globules in a solution of the 
protein caseinogen ; rubber latex contains drops in a solution of vege- 
table protein. 

An extreme case is an emulsion of 99 per cent, of oil and I per 
cent, of soap solution which is of such a consistency that it can be cut 
into cubes. 



COLLOIDS AND COLLOIDAL SOLUTIONS 383 

Properties of Emulsions. 

(a) The properties of the first kind of emulsions in which a small 
quantity of liquid is present in another liquid I part in 10,000 are 
almost the same as those of suspensoids. The globules show Brownian 
movement, they are precipitated or coagulated by electrolytes and can 
be retained by ultra-filters. The particles are comparatively rigid and 
are separated from one another by thick films or layers of the continu- 
ous phase. 

() The properties of the second kind of emulsions in which the 
quantity of disperse phase is large are very different. 

(1) Viscosity. They are very viscous, an extreme case being the 
soap and oil emulsion mentioned above, which is almost a solid. 

(2) Closeness together of the Particles. If particles of a solid or 
rigid sphere be put together so that they touch, they will occupy 74 per 
cent, of the volume. Such a condition gives a thick paste, which is a 
solid. If particles of a liquid or a deformable sphere be put together 
so that they touch, the particles will become flattened and their face 
will have the shape of a dodecahedron. The whole system remains a 
viscous liquid. There is no limit to the ratio of the disperse phase to 
the total volume. 

(3) Surface Tension of the Continuous Phase. In these emulsions 
the continuous phase must be a solution of an emulsoid colloid. Such 
solutions froth when they are shaken. Frothing is due to a lowering 
of the surface tension of the solvent by the substance in solution. This 
lowering of the surface tension takes place at the points of contact 
between the two phases, i.e. the interfacial tension is lowered, which pre- 
vents tearing of the films of continuous phase between the partigles. 

(4) Structure of an Emulsion. The globules are flattened and form 
polyhedra, and they are separated by thin films of continuous phase. 
The whole system will be represented by a honeycomb structure filled 
with globules. On shearing, the whole surface of an emulsion becomes 
enlarged, the polyhedra moving over one another. Surface energy in 
spite of the lowered surface tension will be developed and it appears 
as viscosity. 



384 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

C. Emulsoids. 

Silicic acid sol is one of the few examples of an inorganic emulsoid. 
The organic emulsoids are very various. The types are represented 
by gelatin and agar, albumin, gum-arabic, cellulose and nitrocellulose 
solutions. 

Preparation of Emulsoid Sols. 

(a) Silicic Acid. A solution of sodium silicate is treated with excess 
of hydrochloric acid and dialysed. A clear solution remains in the 
dialyser. 

(b} Gelatin and Agar. These substances dissolve in hot water. 

(c) Albumin dissolves in cold water. 

(d) Cellulose dissolves in Schweitzer's reagent or zinc chloride solu- 
tion. 

(e) Nitrocellulose * dissolves in alcohol-ether, acetone, acetic acid, etc. 
General Properties of Emulsoid Sols. 

(1) Concentration. These colloidal solutions can be prepared of 
various strengths and are not necessarily dilute as suspensoid sols. 

(2) Osmotic Pressure. They have a low osmotic pressure. 

(3) Viscosity. They have a high viscosity. 

(4) Behaviour to Electrolytes. Silicic acid resembles suspensoid sols 
by being precipitated as a gel with a small quantity of electrolyte. 

The organic sols require larger amounts of electrolytes to precipitate 
them from solution, thus : 

(a) Sodium chloride is added to soap solution in small quantities 
at a time and occasionally shaken. The soap is precipitated after a 
large amount has been added. 

() On adding ammonium sulphate to starch solution precipitation 
of the starch occurs after a considerable quantity has been added if the 
solution be occasionally shaken so as to dissolve the salt. 

(c] If some egg-white solution be saturated with (i) sodium chloride, 
(2) magnesium sulphate, by grinding it in a mortar with the salt, a 
small quantity of globulin is precipitated. 

The same result is obtained by half-saturating the egg-white solu- 
tion with ammonium sulphate, i.e. by adding an equal volume of 
saturated ammonium sulphate solution. On saturating the filtrate with 
finely powdered ammonium sulphate crystals, the egg albumin is pre- 
cipitated. This method is employed for separating globulins, which 
are less soluble, from albumins, which are more soluble and are only 
precipitated from solution by completely saturating with ammonium 
sulphate (see under proteins). 

1 Cellulose nitrates. 



COLLOIDS AND COLLOIDAL SOLUTIONS 385 

(5) Behaviour towards Suspensoids, Suspensoids and emulsoids, if 
they have opposite electrical charges, mutually precipitate one another. 
This property is made use of in precipitating proteins from solution 
(many, if not all, of the alkaloidal reagents act in this way) : 

If to some egg-albumin solution or dilute serum an equal volume 
or more of colloidal ferric hydroxide be added, and then about .'5 to 
I gm. of sodium sulphate and the mixture be well shaken, a brownish 
mass containing the protein and excess of ferric hydroxide is pre- 
cipitated. The filtered solution will not contain protein as shown by 
the biuret reaction, Millon's reaction, etc. 

(6) Electrical Charge. The electrical charges on the particles of 
an emulsoid sol are chiefly due to the reaction of the medium, e.g. 
albumin in neutral solution is not charged and does not travel in an 
electric field. Albumin in faintly acid solution has a positive charge 
and travels to the negative pole. Albumin in faintly alkaline solution 
has a negative charge and travels to the positive pole. 

(7) Adsorption. If an emulsoid sol be precipitated by electrolytes 
or by suspensoids, dissolved substances are taken out of solution in the 
same way as with suspensoids. 

Special Properties of Emulsoids. 

The properties of emulsoids show many differences among them- 
selves and many differences from the properties of suspensoids and 
emulsions. 

(1) Silicic Acid. 

Silicic acid sol on treatment with an electrolyte behaves like a 
suspensoid ; a small quantity of electrolyte causes gel formation. The 
gel takes the form of a jelly which gradually becomes more viscous 
and sets to a hard mass with no separation of water. The change of 
state is continuous, proceeding until the mass sets. 

Thus, if excess of sodium silicate solution of sp. gr. IT 6 be added 
to 2N hydrochloric acid, an opaque gel containing the salt is. formed 
which gradually becomes more viscous and sets. The rigid gel cannot 
be redissolved. The colloidal solution thus resembles a suspensoid 
sol in that the transformation is irreversible. 

(2) Gelatin and Agar. 

Both gelatin and agar dissolve in hot water. The solution on 
cooling sets to a jelly. A quite stiff gel is formed by 2 per cent 
agar solution. These non-rigid or elastic gels can be dissolved again 
on warming. The transformation is reversible ; they show the pheno- 
menon of hysteresis. The setting-point is influenced . by the presence 
of salts : citrates raise the setting-point, thiocyanates lower it or may 
prevent setting. 

25 



386 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Albumin. 

Solutions of albumin coagulate on heating at temperatures varying 
from 50 to 70. The exact temperature depends upon the amount 
and kind of salt present ; in the presence of thiocyanates heat coagu- 
lation does not occur even at the boiling-point. The transformation 
is irreversible. 

Solutions of albumin are precipitated by high concentrations of 
electrolytes (Na 2 SO 4 , (NH 4 ) 2 SO 4 , MgSO 4 ). These precipitates redis- 
solve in water ; the transformation is reversible. 

The precipitates formed by CaCl 2 , SrCl 2 , BaCl 2 become insoluble 
on standing, whilst the precipitates formed by solutions of heavy 
metals are insoluble. 

Caseinogen, Gum- A rabic. 

These sols do not coagulate on heating and do not form gels. 
The solutions are simply more or less viscous at different temperatures. 

Cellulose, Nitrocellulose. 

These sols form coherent gels when the solvent is removed by 
evaporation or by washing out with water. 

Transition of Emulsoids to true Solutions. 

Some substances form emulsoid sols in one solvent, but true 
solutions in another solvent. 

E.g. soap in water is an emulsoid sol, in alcohol a true solution ; 
tannin in glacial acetic acid is a true solution, in water an emulsoid sol. 

There are many differences amongst the dye-stuffs ; eosin resembles 
a true solution ; fuchsin forms an emulsoid sol. 

Nature of Emulsoids. 

Emulsoids possess many of the properties of emulsions, especially 
high viscosity, and they show many differences from the suspensoids. 
Their behaviour can only be explained on the assumption that they 
are systems of two liquid phases, i.e. as systems consisting of dilute 
solutions of a colloid containing droplets or globules of more concen- 
trated solution. 

They differ from emulsions in the ease in which the solvent may 
pass from one phase into the other. Gelatin sol is a continuous liquid 
phase containing droplets of higher concentration : gelatin jelly is a 
continuous solid phase containing droplets of dilute liquid. In dis- 
solving gelatin, sol formation takes place by imbibition of water and 
swelling ; there is disintegration of the original system. 

The effect of salts upon the coagulation of albumin and upon jelly 
formation is to affect the distribution of the solvent between the two 
phases. They act by altering the compressibility of water. Solution 
of an emulsoid generally occurs with contraction. 



COLLOIDS AND COLLOIDAL SOLUTIONS 387 

Properties of Gels. 

The gels formed by silicic acid, gelatin, agar, etc., which may contain 
as much as 90 per cent, of water, possess some of the properties of solids. 
They may be put into two groups the rigid or nearly non-elastic 
gel like silicic acid and the elastic gel like gelatin, collodion, etc. 

(1) Behaviour to Water. 

(a) The rigid gel of silicic acid is translucent ; on exposure to the 
air it loses water, becoming opaque, and with loss of more water it 
again becomes clear. The amount of water present in the solid 
material corresponds with the tension of aqueous vapour, the ratio 
of the constituents, silicic acid and water, changing continuously. 
No definite hydrate is formed such as occurs with crystals containing 
water of crystallisation. The formation of many siliceous minerals 
may be accounted for in this way. The elastic gel, like gelatin, which 
is reversible, also behaves in a similar manner to water ; the amount 
of water present in it depends on the tension of aqueous vapour. 

() Gelatin behaves differently when immersed in water ; it swells 
and much more water is taken up ; it is given off again on exposure to 
the air. This absorption of water is of great physiological importance. 

(c) Though an absorption of water and swelling take place when 
an elastic gel is put into water, the actual volume of the gel and water 
is less than the total volume of the two substances. There is compres- 
sion of the water. 

The decrease in volume is demonstrated by Hatschek by placing 
a known weight of gelatin in a pycnometer, filling it with water, and 
immersing the vessel in water. When the gelatin has swollen, the 
vessel is taken out of the water, dried and weighed. There is an in- 
crease in weight which shows that water has entered the vessel. To 
compress water to an extent corresponding to 2 per cent, of the original 
volume requires 400 atmospheres. 

(d) Heat is liberated during the swelling of the gels. It has been 
measured and found to vary from 5 to 10 gm. calories per gm. of gel. 

(e) The total volume decreases, but the gel in water swells. 
This increase in volume has been measured and it has been found that 
against a pressure of 42 atmospheres a gel will swell by 16 per cent, 
of its volume ; against a pressure of I atmosphere the increase in 
volume is 330 per cent. 

From this it can be calculated that I gm. of gel on swelling will 
lift I kilo, to a height of 3 -3 cm. 

(2) The elasticity, the optical constants and thermal expansion of 
gels differentiate them from both liquids and solids. 

25* 



388 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

When not strained they resemble liquids. If stretched, (i) they 
contract on warming and rapid cooling produces expansion, (2) they 
become doubly refracting. 

They are deformed without change of volume (cross-section x 
length) ; on stretching a cylinder of gelatin its cross-section diminishes 
as the length increases. 

(3) Nature of Gels. In composition gels resemble the organic 
material of plants ; in these there is a cell structure. Liquid is enclosed 
in cells formed by a solid phase. Gels consist of a solid x continuous 
phase enclosing a liquid phase. 

The reversibility is accompanied by a distribution of water between 
the phases and is affected by the presence of salts. 

(4) Diffusion of Substances and Reactions in Gels. In dilute gels 
diffusion takes place as in water. The rate is slower with strong gels. 
The rate of diffusion is affected by various substances: urea, iodides, 
chlorides accelerate diffusion ; sodium sulphate, glucose, alcohol, 
glycerol retard diffusion. 

These substances affect the distribution of water between the two 
phases and probably also the relative volumes of the gel wall and the 
free liquid. Diffusion takes place chiefly in the liquid. The reactipn 
does not proceed continuously, but the product if insoluble is deposited 
in strata. Many substances can thus be obtained in the form of large 
crystals and often spherolites are formed. 

Some organic compounds on separation from hot solvents on cool- 
ing first form transient gels which gradually crystallise. Crystalline 
minerals may be formed from gels in a similar way. 

(5) Structure of Gels.- Gels resemble the organic matter of plants 
and animals in composition that is solid matter containing 80-90 per 
cent of water. Cell structure in animals and plants is visible with a 
microscope. Though apparent structure can be seen in gels with a 
microscope it is not real, but the presence of a structure in gels is 
indicated by the diffusion and reaction of substances in gels. 

The Phenomenon of Adsorption by Colloids. 

The phenomenon of adsorption by colloids is due partly to the 
large boundary surface between the particles and partly to the electrical 
charge upon the particles. In some cases the first cause may pre- 
dominate, in other cases the second cause. 

1 In this connection a solid is a substance less deformable than a liquid, but not non- 
deformable. 



COLLOIDS AND COLLOIDAL SOLUTIONS 389 

(1) The Large Boundary Surface. 

The surface of a liquid against its vapour or another liquid is in 
tension, known as surface tension or interfacial tension. Such a tension 
also exists between a gas and a solid, and a liquid and a solid. 

Work is required to produce or to enlarge a surface. A surface is 
a seat of energy and on this account a surface tends to become a 
minimum. The surface energy .is measured by the product of the 
surface and surface tension per unit length. 

The surface tension tends to reduce the surface and establish 
equilibrium with other forces acting in the body of a liquid. 

Gases*in contact with a surface produce a lowering of the surface 
tension, the amount of lowering being characteristic for each gas. With 
rising gas pressure or concentration there is a lowered surface tension. 
It is accompanied by condensation of the gas on the surface. The 
same occurs at the boundary of a solid and a gas. 

In a fr$th which has a large surface there is a higher concentration 
than in the liquid, and with froth formation there is lowered surface 
tension. There is thus an increase in concentration in the surface or 
adsorption with a lowered surface tension. If a dissolved substance in 
increasing concentration increases surface tension, it is less concentrated 
in the surface than in the liquid. If a dissolved substance in increas- 
ing concentration lowers surface tension, it accumulates on the surface. 
A small amount of a substance in solution can increase the surface 
tension only slightly, but a small amount can lower the surface tension 
greatly. 

The amount of adsorption is proportional to the active surface. It 
proceeds to a definite end point or equilibrium. 

This is expressed mathematically by 

y 

= ac" 
m 

where m amount of adsorbent, y = quantity adsorbed and c is the 
end or equilibrium concentration in the liquid after adsorption, a and 
n are constants depending on the solution and adsorbent. This equa- 
tion shows the peculiarities of adsorption. 

(2) The Electric Charge. 

In precipitating colloids by electrolytes the charge of opposite sign 
is the effective ion : equi-valent amounts of the ions produce the same 
effect. In the mutual precipitation of colloids the precipitation occurs 
with colloids of opposite charge. In the first case the ion is carried 
down with the colloid ; in the second case both colloids are pre- 
cipitated. The large surface of the colloid further influences the electric 
charge and in the case of emulsoids the viscosity also. 



390 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Peculiarities of Adsorption. 

1 i ) A mount of A dsorption. 

The amount adsorbed from a solution does not increase in direct 
proportion to the increase in concentration. It thus differs from 
chemical reactions, e.g. 10 times the concentration produces only 4 
times the adsorption. Relatively more substance is adsorbed from 
dilute solution than from a concentrated one. 

(2) Different Adsorption from Different Solvents. 

Usually the adsorption of substances from water is greater than 
from organic solvents. This peculiarity may be of practical use. Dyes 
can be removed from aqueous solution completely by chaVcoal, the 
particles being concentrated on the surface. On putting the charcoal 
into alcohol, the dye passes into the alcohol. This'is due to the surface 
concentration of the dye on the- charcoal being in excess ofcthat neces- 
sary to produce equilibrium between the phases. 

(3) Selective Adsorption. ^ 
Substances are not adsorbed to the same extent ; benzoic acid or 

salicylic acid are more adsorbed than acetic acid by charcoal. This 
selective adsorption has been put to practical use in capillary analysis, 
e.g. : 

Strips of filter paper are partly suspended in different solutions ; the 
liquid rises into the paper ; above a certain height there is only water ; 
the 'height to which the dissolved substance rises is different ; the 
more adsorbed substance does not rise so high as the less adsorbed. 

Lead salts on filtering through paper are retained by the surface of 
the paper and account for loss in the concentration of the solutions. 

(4) Adsorption by Different Adsorbents. 

Though the adsorbents may differ in active surface they adsorb the 
same relative amount of substance : thus if A adsorbs more X than 
Y, B also adsorbs more X than Y. 

(5) Reactions accompanying Adsorption. 

Chemical reactions may occur at the same time as adsorption, e.g. : - 

Alumina adsorbs the acid of congo red at the ordinary temperature 

without chemical reaction as seen by the colour, which is blue ; on 

warming, chemical reaction takes place ; the alumina becomes red in 

colour, the colour of the salts of congo red. 

(6) Effect of Adsorption on Extraction by Solvents. 

If there is adsorption of one substance by another, repeated extrac- 
tions must be made to separate them. 

(7) Filtration of Particles through Sand, etc. 

The sand particles having a negative charge will retain a definite 
quantity of positively charged colloids, such as colloidal ferric hydroxide 
and some dye-stuffs. This is apparently due only to the discharge of 
the electric charges on the particles. 



ENZYMES. FERMENTATION. 

In the previous sections an outline has been given of the organic 
compounds which are found in nature, both in plants and in animals. 
Their variety is very great, and they include not only simple compounds 
such as alcohol, glycerol, fatty acids, lactic acid, urea, amino acids, and 
many others, but also the more complex, such as chlorophyll, haemo- 
globin, and the three large groups, carbohydrates, fats and proteins. 
These three groups make up the main portion of the solid matter of 
plants and animals, and are concerned intimately with the functional 
activity of the organism. 

We have to investigate how \the change from the complex com- 
pound to the simple, such as starch and glucose to alcohol and carbon -^ 
dioxide, protein to amino acids, and vice versa from the simple to the 
complex, is effected in nature. \ 

The (hydrolysis and decomposition of the-complex compounds is ^ 
effected by the reagents grouped together under the term enzymes.) 

J * c> o i o _ - *^ t i 

^fhe formation of the complex compound from the simple ones is 
effected by the same reagents^ The decomposition is most easily 
ascertained antt followed, but the formation is only followed with 
difficulty and it has been actually observed only in a few instances. 
Nevertheless it is believed that the synthesis of all the complex com-X 
pounds is effected by enzymes. 

Historical. 

(The formation of alcohol and carbon dioxide from sugar was known to 
the ancients/iand bn account of the effervescence, or apparent ebullition of the 
liquid, during the aecomposition of the sugar, the process was called fermenta- 
tion, from/eryere, to boiU In the middle ages the decomposition of proteins 
was recognised as an analogous process to that of fermentation and the terms 
putrefaction and fermentation were frequently used to denote any process of 
decomposition. (Not until the beginning of the nineteenth century, mainly be- 
tween 1830 and 1840, was it recognised that fermentation was due to the 
presence of living cells) (Schwann, La Tour, Kiitzing, Pasteur). At about 
the same time it was discovered that extracts ofplants barley and almonds, 
and a little later that extracts of animal organs of the stomach and pancreas, 
frere able to effect theclecbmpdsiticm of the complex compounds, starch, 
amygdalin and proteins into simpler ones7\ The effective substance in barley 
extract was called diastase, that in almorids, emulsin, that in the stomach, 
pepsin, that in the pancreas, trypsin. There were thus two varieties of active 
agents the one living (yeast) the other not living (diastase, etc.), and they 
were called respectively organised ferments and soluble or unorganised fer- 



392 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Pasteur, who posited the necessity of life for fermentation, was op- 
posed by Liebig who, without clearly stating his ideas, was of the opinion that 
there was something in the yeast cell which actually produced the fermenta- 
tion. Traube, in 1858, clearly stated the position that the yeast cell contained 
a soluble ferment to which the decomposition was due. On account of the 
confusion of the terms it was suggested by Kiihne in 1878 that the soluble 
ferment should be termed enzyme (from tv vp.r), in yeast), which signifies 
that something in yeast which causes the fermentation of sugar. 

/Not until 1897 was it definitely shown by Buchner that yeast did contain 
an enzyme which fermented sugar in the absence of the living cells, and later 
it was shown that other cells and bacteria also contained soluble ferments. 
The term enzyme is now used for the active agent in all cases, and the term 
fermentation for the process of decomposition^ In France the term 'dias- 
tases ' is used as a general word for enzymes. 

Occurrence and Preparation. 

v Enzymes are present in all living_ce_ys. They are either excreted 
in the juices by definite cells or glands of the organism, e.g. by the 
salivary glands, the pancreas, etc. : that is, they act normally outside 
the cells which produce them (ectoenzymes) , or they are not excreted : 
that is, they act inside the cell envelope (endoenzymes). 

For purposes of investigation in the former case the juices of the 
glands, such as saliva and pancreatic juice, are collected. In the latter 
case the enzymes are extracted from the cells in which they are 
present ; the cells require to be ruptured so as to obtain their contents, 
tthe glands producing the secretion may also be extracted to obtain 
the enzyme\ 

The cells are ruptured by the following methods : 

(1) By drying the cells at a low temperature at 20 to 36 and 
sometimes subsequently warming the dried mass to 50 or 60. 

(2) By drying the cells by stirring up the tissue with alcohol or 
acetone and pouring off the liquid after a short time of contact. 

The dried material is treated with water ; the aqueous solution is 
filtered and precipitated with alcohol. 

(3) By autolysis in the presence of toluene or other antiseptics ; 
the cells are either mixed with antiseptic, or the tissue is minced and 
suspended in water containing toluene, etc. The enzymes in the cell 
dissolve the cell membrane and pass into solution. 

(4) By mechanical disintegration ; the cells are ground in a 
mortar with sand. The ground-up mass may be diluted with water, 
or the liquid contents may be separated from the cell walls by hyd- 
raulic pressure. 

This method was used by Buchner to show the presence of the en- 
zyme in yeast which ferments sugar. The ground-up yeast cells which 
formed a liquid mass, were mixed with siliceous earth to form a thick 
paste. The thick paste was pressed in a powerful hydraulic press. 



ENZYMES. FERMENTATION 393 

The liquid, which oozed out, was filtered (i) through paper and (2) 
through a clay candle to remove unbroken cells. 

The dried material is treated with water or with glycerin. The 
solution is filtered and precipitated with alcohol. 

The autolytic extracts, or the liquids produced by mechanical dis- 
integration, may be mixed with water or glycerol and precipitated with 
alcohol. 

The alcohol precipitate is dissolved in water, reprecipitated with 
alcohol, filtered off, washed with alcohol and ether and dried in vacua 
over sulphuric acid. 

Too frequent solution and precipitation by alcohol is avoided as 
much enzyme is lost in the process. 

Aqueous or glycerin extracts of the dried material or the fresh 
gland also contain the enzymes and may be used directly, as is usually 
the case when enzymes are to be detected in tissues. 

Chemical Nature. 

The chemical constitution of enzymes is still quite unknown ; they 
have been supposed to be proteins, nucleoproteins and carbohydrates 
from the fact that the enzyme~~solution gave the reactions of these 
classes of compounds. The purest preparations of invertase and amylase 
that have been prepared have contained carbohydrate ; the purest ^"~" 
preparation of pepsin has not contained nucleoprotein. 

(Though the chemical nature of enzymes is unknown they belong 
to the group of coljoidal substances ; thus, they do not diffuse through 
parchment paper and other membranes.) 

Properties of Enzymes. 

(1) Enzymes can only be recognised by their activity. u^ 

(2) Enzymes are specific in their action^ ArTenzyme acts only 
upon one compound, or a group of compounds, such as the fats and 
proteins The most striking instance of their specificity is observed 
intEe a- and /3- glucosides. The enzyme maltase acts only upon 
a-glucosides : the enzyme emulsin only upon /3-glucosides. 

(3) Enzymes act by combination, or by adsorption, with the com- ^" 
pound upon which they__act. From the combination of an enzyme 
with the substance upon which it acts and its specific property, 
arose the image of Emil Fischer, that the enzyme was to the sub- 
stance as a key is to a lock. Only the proper key will open the 
lock. In illustration of these properties Armstrong likened the speci- 
ficity and combination to the fitting of a glove upon the hand. Only 

the right-hand glove will fit the right hand. There may be combina- 
tion, but unless it is with every digit there is no enzyme action. 

(4) Enzymes act as catalysts, i.e. they increase the rate of a s 
reaction which is normally proceeding at so slow a rate that it cannot 

be detected. 



394 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

(5) Enzymes, like catalysts, act more rapidly at high temperatures, 
but there is a limit to the increase in the rate produced by enzymes. 

They are unstable catalysts ; they are usually destroyed at a 
temperature of 56 to 60 or 65. At O their action is nil, or consider- 
ably less than at room temperature ; at body temperature and up 
to 45 their catalytic action is at the optimum ; at higher temperatures 
it is more rapid, but the enzyme rapidly undergoes destruction so that 
the result of the action is generally less than at 37 to 45. 

(6) Enzymes are very sensitive to the presence of salts, acids and 
alkalies. Many enzymes will not act unless salt is present. Some, 
like pepsin, act only in the presence of very dilute acid ('iN) ; others, 

/ like trypsin, act best in the presence of dilute atlfali ( - iN). Most en- 
zymes act best in a very faintly alkaline medium. The action of all 
enzymes is stopped by acid or alkali exceeding 'iN. 

(7) Some enzymes require the presence of particular salts or other 
substances for their action, i.e. require a co-enzyme. E.g. phosphates 

V are essential in the fermentation of sugar to alcohol and carbon dioxide, 
the fat-hydrolysing enzymes require the presence of bile salts, oxidis- 
ing enzymes require the presence of iron or manganese salts. 

(8) Some enzymes in their action are inhibited by other enzymes or 
anti-enzymes. 

(9) Many enzymes require liberation from a precursor before they 
act proenzymes, e.g. trypsin and its precursor trypsinogen. 

(A full account of the action of enzymes is given by Prof. Bayliss 
in the "Nature of Enzyme Action". Only the general principles of 
the action of enzymes can be mentioned here.) 

Nomenclature. 

Enzymes are designated by the suffix -ase, the first part of the word 
being that of the name of the substance upon which the enzyme acts. 
The substance upon which the enzyme acts is known as the sjubstrate 
or hydrolyte. Most enzymes act by hydrolysis and are hydrolytic. 
Those which act upon the carbohydrates are sometimes termed sucro- 
clastic (sugar-splitting), upon fats, lipolytic or lipoclastic, upon pro- 
teins, proteolytic or proteoclastic. Other enzymes act by oxidation of 
the substrate and are termed oxidases. Another group of enzymes 
acts upon amino groupings forming hydroxy or keto groups and en- 
zymes can also remove carbon dioxide from carboxylic acid groups. 
They may therefore be classified as follows : 



ENZYMES. FERMENTATION 



395 



A. Ilydrolytic. 

I. Sucrodastic. 
ENZYME. 

Diastase 

or 
Amylase. 

Inulase. 

Invertase^ 

or 
Sucrase. J 

Lactase. 

Maltase 

or 
o-Glucase. 



Emulsin 

or 
3-Glucase. 

Zymase. 



J 
,} 



SUBSTRATE. 


PRODUCT. 




Starch or Amylum. 


Dextrin + Maltose. 




Glycogen. 







Inulin. 


Fructose. 




Cane Sugar. 


Glucose + Fructose. 




Raffinose. 
Lactose. 


Fructose + Melibiose 
Glucose + Galactose. 


/ Glucose. 
( Galactose. 


Maltose. 


Glucose + Glucose. 




o-Glucosides. 


a-Glucose. 




/3-Glucosides. 


^-Glucose. 




Amygdalin. 


Benzaldehyde, HCN, 


2 mols. Glucose. 


Glucose, 
x Fructose. 
Mannose. 
Galactose. 


CO 2 + Alcohol. 





IJ. Lipoclastic. 
Lipase. Fats. 

III. Proteoclastic. 

Pepsin. Proteins. 

Trypsin. 

Erepsin. 

Papain. 



Proteoses. 
Proteins. 



B. Oxidases. 



Catalase. 
Peroxidase. 



Hydrogen Peroxide. 
Peroxides : 
Hydrogen peroxides 

or 
Organic peroxides. J 



C. Deaminases. 



Guanase. 
Adenase. 
/Amino acid- 
^Deaminase. 

D. Carboxylases. 

Carboxylase. 



Guanine. 
Adenine. 

Amino acids. 



Keto acids. 
Amino acids. 



Glycerol + Fatty Acid. 



Proteoses + Peptone. 
Amino acids. 



Oxygen. 

" Active" Oxygen. 



Xanthine. 
Hypoxanthine. 

Hydroxy or Keto- 
acids. 



CO 2 + aldehyde. 
CO + amines. 



This list does not include all the known enzymes ; there are many 
more amongst the carbohydrate splitting enzymes. The lipoclastic 
enzymes can be subdivided into butyrinase, lecithinase. Xanthine 
is oxidised to uric acid by uricase and a whole series of enzymes are 
concerned in the hydrolysis of nucleic acid each acting upon a particular 
substrate. They are grouped together as nucleases and would include 
guanase and adenase as well as nucleotidases, etc. 



396 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

LOCALISATION OF THE ENZYMES AND THE 
CHEMICAL CHANGES IN THE ORGANISM. 

In the unicellular plants and animals the various chemical changes 
in the organic compounds must occur in the single cell. The colloidal 
substances are ingested and broken down by enzymes into crystalline 
and diffusible substances. These diffuse into the cell and are built up 
into colloids, which again are broken down and converted into other 
crystalline substances capable of diffusing out of the cell. In the 
process of evolution of the higher plants and animals a differentiation 
of groups of cells into special tissues or organs has occurred. Like 
the single cell, the cells of each organ must originally have been able 
to effect all the chemical changes, but during evolution the power 
of effecting some of them has become lost, whilst the power of effecting 
others has become increased. Individual organs can effect most 
changes, but not all ; some are effected by only one organ. Thus, 
for example, in animals, the organs of the alimentary canal are chiefly 
concerned in the hydrolysis of the proteins, carbohydrates and fats ; 
the liver is concerned mainly in the metabolism of fats and carbo- 
hydrates, the conversion of ammonia into urea and the destruction and 
removal of blood pigment ; the kidney is a filter which removes 
waste products; the ductless glands produce special substances to 
maintain the general equilibrium of the organism. The sequence of 
chemical processes taking place in the alimentary canal of animals is 
termed digestion. 

In plants, the differentiation is' much less than in animals : the 
growing points and the cambium layer in the stems are the active 
tissues, also the cells in the flower, or tuber, which produce the seed and 
embryo. Specialised cells are found in the insectivorous plants in Ne- 
penthes in the lining membrane of the pitchers, in Drosera in special 
tentacles. Certain cells in the surface of the scutellum of Zea Mais 
have a glandular appearance, but these glands have no lumen. 

Very frequently in plants the enzyme may be present in one cell 
and its substrate in an adjoining cell. They come into contact when 
the cell walls are broken, artificially by crushing the tissue or by the 
action of anaesthetics. 

Sucroclastic enzymes are present in greater variety and more 
abundantly in plants ; proteoclastic enzymes in animals. This accords 
with the general composition of the organic matter of plants and 
animals. 



ENZYMES. FERMENTATION 397 

Digestion in Animals. 

The three classes of organic compounds, the fats, the car- 
bohydrates and the proteins, are taken in as food and are hydrolysed 
into their constituents before they can pass through the wall of the 
alimentary canal and can be assimilated. 

I. Saliva. 

The first digestion of food occurs in the mouth by the action of 
the saliva, the secretion of the salivary glands. The saliva con- 
tains the enzyme diastase, or arhyla.se, which hydrolyses starch (and 
glycogen) converting it into dextrin and__mallQS. In the mouth, 
however, very little enzyme action takes place, the food being only 
moistened by the saliva and swallowed. The action occurs in the 
stomach, where the food is in the form of a mass in the fundus ; here 
only the exterior of the mass is in contact with the hydrochloric acid 
of the gastric juice, which Inhibits the action of the diastase ; the starch 
in the interior of the mass is slowly digested. 

I 1 . Gastric Juice. 

The first hydrolysis of proteins occurs in the stomach by the 
enzyme pgpsin. Pepsin is secreted by certain cells of the gastric 
mucous membrane. It acts only in the presence of hydrochloric 
acid, which is secreted by other cells. In disease, lactic acid is 
sometimes found in the contents of the stomach. Besides pejpsin, 
the enzyme, rejnniiT, is present in the cells of the mucous membrane, 
but it is very probable that pepsin and rennin are identical. Rennin 
acts upon caseinogen, the protein of milk, converting it into qgggiri 
(see under milk, p. 461). 

III. Pancreatic Juice. 

From the stomach the food passes into the intestine. The acid 
contents of the stomach, when they pass into the duodenum and 
come in contact with its mucous membrane, induce the secretion of 
secretin. The secretin passes into the blood and is carried to the 
pancreas, where it excites a flow of pancreatic juice. This juice has 
very little action upon proteins, but it contains lipase and diasta.se 
which hydrolyse fats and starch respectively. As soon as the pancre- 
atic juice, which contains trypsinogen, comes into the intestine, it 
becomes activated by the enzyme enterokinase and converted into the 
powerful proteoclastic enzyme, trypsin. Enterokinase is secreted by 
the glands of the duodenum. Trypsin acts upon unchanged proteins, 
proteoses, etc., from the stomach and converts them almost entirely 
into amino acids. A complex polypeptide is also formed which is not 
acted upon by trypsin. 



398 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

I V. Intestinal Juice. 

The cells of the small intestine also produce a secretion the 
succus entericus which contains erepsin, a peculiar proteoclastic 
enzyme discovered by Cohnheim ; it acts only upon proteoses and 
peptones, converting them into amino acids. This juice also contains 
invertase and lactase. These enzymes act not only in the secreted 
juice but also inside the cells of the mucous membrane. Any un- 
hydrolysed protein or polysaccharide is hydrolysed into it's constituents. 

V. Autolysis. 

Proteoclastic and other enzymes are present in all tissues ; they 
are concerned in the breaking down of the constituents of the tissue 
and in their synthesis. 

They are the cause of the auto- or self-digestion of the tissues 
after death ; in starvation, food is supplied by the breaking down 
of some organs at the expense of other organs by the autolytic 
enzymes. 

VI. Putrefaction. 

In the large intestine food remains are acted upon by bacteria. 
Amino acids are formed from proteins and they are further broken down 
into amines, carbon dioxide, indole, scatole, hydrogen sulphide, fatty 
acids. Unabsorbed carbohydrates and fats are also hydrolysed and 
decomposed. 

VII. The Liver. 

The liver is concerned most intimately with the products formed 
during digestion in the intestine, especially the monosaccharides and 
the fats, and with the regulation of their amount in the blood. 
Monosaccharides, chiefly glucose, are converted into glycogen and 
retained as reserve food-stuff to be broken down again when the 
amount of glucose in the blood sinks below its normal limit. I to 2 
per cent, of glycogen is present in the livers of well-nourished animals, 
but generally it varies from -I to -5 per cent. According to Kiilz 
the greatest amount is present in the liver 14 to 16 hours after a meal. 
The fats undergo changes in the liver cells the saturated become 
unsaturated and they are oxidised to simpler fatty acids and hydroxy 
acids which circulate in the blood. The amino acids undergo deaminisa- 
tion in the liver as well as in the other tissues. Liver cells contain 
arginase, which hydrolyses arginine to urea and ornithine. The liver is 
additionally concerned in the formation of urea from ammonia and car- 
bon dioxide ; in birds with that of uric acid as well. It also breaks down 
the haemoglobin of the blood and excretes the products, bilirubin 
and biliverdin, into the intestine through the bile duct 



ENZYMES. FERMENTATION 399 

DEMONSTRATION OF THE ACTION OF ENZYMES. 

Since enzymes can only be recognised by their action, their demon- 
stration necessitates the knowledge of the chemical and physical pro- 
perties of the compounds upon which they act ; e.g. starch and its 
products maltose and dextrin, fats and their products glycerol and 
fatty acids, proteins and their products the proteoses, peptones and 
amino acids. Either the disappearance of substrate, or the appear- 
ance of the products, or both, may be demonstrated. 

Frequently the amount of enzyme in a solution or in a preparation 
is very small and a considerable time must be allowed before its action 
can be demonstrated, e.g. from I day to 3 or 4 days. Under these con- 
ditions an antiseptic, preferably I per cent, of toluene or chloroform, 
is added to prevent the action of bacteria. The antiseptics destroy 
the bacteria or inhibit their growth ; they have no action on the 
enzyme. 

I. Diastase or Amylase. ' 

(1) Malt Diastase. 

The chief source of diastase is malt. Malt is prepared by steeping barley 
or other seeds of cereals in water and allowing them to germinate in a warm 
place until the plumules have reached a length of about - inch. The sprouted 
grain is dried and cured in a kiln. The composition of the grain alters under 
these conditions : the amount of starch decreases, the amount of reducing 
carbohydrates increases. Its colour . is light to dark yellow. The seeds 
should break easily, should have a white interior and a sweet flavour. Malt 
should be free from broken and damaged seeds and the dried rootlets. 

Malt extract is prepared from the dried material by treatment with water ; 
the aqueous solution may be evaporated to dryness. Its chief use is in 
brewing, but it is used in medicine as a food for its high content in maltose 
and for its diastatic action ; some varieties of malt extract contain no diastase 
as they have been boiled. 

A i to 2 per cent, filtered extract serves for the demonstration of diastase. 

An active diastase is prepared by treating malt, or ground barley, with 
2 to 4 parts of 20 per cent, alcohol, for 24 hours. The extract is precipitated 
by adding not more than 2^ volumes of alcohol. The precipitate is rapidly 
treated with absolute alcohol and ether and dried in vacua. (Lintner.) 

(2) Pancreatic Diastase. 

A solution containing diastase can be prepared from the pancreas of 
animals by allowing a fresh pancreas (free from fat), which has been finely 
minced, to stand with twice its weight of glycerin, for 12 to 24 hours and 
straining through muslin. Before use the solution may be diluted with i to 2 
volumes of water or preferably i to 2 drops of the concentrated extract may 
be used. Diastase is present in pancreatic juice and may be detected in 'i to 
i c.c. as described below. 

(3) Salivary Diastase. 

A solution of this diastase is prepared by rinsing out the mouth 
two or three times with 20 c.c. of distilled water, warmed to 40, for 
i to 2 minutes. The water is collected in a beaker and filtered. 

A i to 2 per cent, solution of soluble starch or starch paste 
solution is prepared as substrate. The presence of diastase is shown 
by the disappearance of starch and the appearance of erythrodextrin-, 
achrpodextrin and maltose ; thus : 



400 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

A series of drops of iodine solution are placed upon a porcelain 
plate, or upon a glass plate on white paper. 5 c.c. of starch solution 
are placed in each of two test tubes, 5 c.c. of diastase solution 
is placed in the one and 5 c.c. of boiled diastase solution in the other. 

The second tube acts as a control. The hydrolysis proceeds more 
rapidly at 40 so that the test tubes are placed in a bath at this tem- 
perature. 

A drop is removed from each solution immediately after the mix- 
tures have been made and placed against an iodine drop. 

The mixture in the first tube soon becomes less opaque, if starch 
paste solution has been used. 

At intervals of half a minute, or a minute, drops are removed from 
each test tube and placed against another iodine drop. The blue colour 
given by the control tube is given each time the test is made, but the 
colour becomes reddish-brown in the case of the other tube and finally 
no colour at all is given. Achroodextrin and maltose have been 
formed. Each test tube, after about 5 minutes, is tested with Feh- 
ling's solution. The control tube shows no reduction, but the pres- 
ence of maltose in the other tube is shown by a marked reduction. 

Effect of Temperature, o, 45, 100, upon Diastase. 

The same experiment is performed with two test tubes con- 
taining 5 c.c. of starch solution and 5 c.c. of diastase solution, but 
one of them is placed in cold water, or better, ice, and the second 
in a water- bath at 45. The time when the various colours are given 
and when no colour is given with the iodine drops is noted. 

The hydrolysis takes place more slowly in the tube kept at the 
lower temperature. A third test tube is prepared containing 5 c.c. 
of starch solution, but to it is added 5 c.c. of boiled and cooled diastase 
solution. It is placed in the bath at 45. A drop taken from this 
tube will give a blue colour with iodine. The enzyme has been 
destroyed by boiling. 

In experiments with enzymes a control experiment is carried out 
with boiled enzyme solution instead of water. The enzyme solution 
usually contains other substances besides the enzyme and the same 
amount of these is added to the substrate in each experiment. It is 
extremely important to carry out such a control experiment, especi- 
ally in cases where a rough measurement has to be made to demonstrate 
enzyme action, e.g. in the cases of lactase and maltase. 

Effect of Acid, Alkali and Salt upon Diastase. 

5 c.c. of starch solution, I c.c. of water and 5 c.c. of saliva solution 
are placed in one test tube. 

,5 c.c. of starch solution, I c.c. of dilute hydrochloric acid (-iN or 
0-4 per cent.) and 5 c.c. of saliva solution are placed in a second test 
tube. 



ENZYMES. FERMENTATION 401 

5 c.c. of starch solution, I c.c. of dilute acetic acid (0-5 per cent.) 
and 5 c.c. of saliva solution are placed in a third test tube. 

5 c.c. of starch solution, I c.c. of dilute alkali ('iN or 0*4 per 
cent.) and 5 c.c. of saliva solution are placed in a fourth test tube. 

5 c.c. of starch solution, I c.c. of sodium chloride solution (i per 
cent.) and 5 c.c. of saliva solution are placed in a fifth test tube. 

The five tubes are placed in the water-bath at 40 and at inter- 
vals drops from each are tested with iodine solution. 

Hydrochloric acid completely stops the action of diastase ; acetic 
acid hinders the action, i.e. the conversion of starch into achroodextrin 
and maltose takes longer ; alkali may hasten, but if strong will stop 
the action. A small concentration of sodium chloride hastens the 
action ; sodium chloride of a concentration of 5 per cent, will hinder 
the action. 

II. Invertase. 

The best source of invertase is yeast from which it may be prepared by 
several methods. Method of Autolysis. It is most conveniently prepared by 
grinding 500 gm. yeast with 30 gm. of calcium carbonate into a thick paste and 
placing the paste in a wide-mouthed bottle. 25 c.c. of chloroform are added 
and it is kept for 3-4 days in a warm room. The solution is filtered from 
the insoluble matter and treated with an equal volume of alcohol. The pre- 
cipitate is washed with alcohol and ether and dried in vacua over sulphuric acid. 

A 'i -i per cent, solution of the preparation is used to demon- 
strate the action of invertase. The autolysed yeast, if diluted about 100 
times with water, also serves for showing the presence of invertase. 

A cane-sugar solution of i per cent, is prepared as substrate ; 5 c - c - 
of the invertase solution are added to 5 or 10 c.c. of the cane sugar 
solution ;^t the same time 5 c.c. of boiled invertase solution are 
added to another 5 c.c. of cane sugar solution to act as a control. 

The solutions may be kept for about 5 minutes at room tempera- 
ture or at 40. They are tested with Fehling's solution. Reduction 
only occurs in the first tube due to the formation of glucose and 
fructose. 

Demonstration of Invertase in the Succus Entericiis or Intestinal 
Mucous Membrane. 

The succus entericus, or an extract of the cells of the mucous 
membrane of the small intestine, prepared by grinding the material 
with sand, allowing it to stand with water for 12 hours in the presence 
of toluene and straining through muslin, is divided into two equal 
portions. One portion is boiled and cooled. To each portion is 
added an equal volume of I per cent, cane sugar solution. I per 
cent, of toluene is added to each and the two mixtures are kept at 37 
for 12-24 hours. Each solution is tested with Fehling's solution. 
Reduction only occurs where the unboiled extract is present showing 
the presence of invertase. 

26 



402 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

III. Emulsin 

Emulsin is prepared most easily from almonds. The almonds are ground 
and the oil is pressed out. The residual cake is treated with water at room 
temperature. The filtered solution is acidified with acetic acid (2 drops per 
100 c.c.) to precipitate proteins. The filtrate is treated with an equal volume 
of alcohol. The enzyme preparation is thrown down, washed with alcohol 
and ether and dried. The precipitate can be immediately redissolved in water 
and used in experiments with emulsin. 

A substrate of 2 per cent, salicin is conveniently used. 5 c.c. of 
salicin solution are placed in each of two test tubes. To the one are 
added 5 c.c. of emulsin solution ; to the other are added 5 c.c. of boiled 
emulsin solution. The two tubes are placed in a water-bath at 40 
for 1 5-30 minutes or longer ; in the latter case I per cent, of 
toluene should also be added. The solutions are tested with Fehling's 
solution. Reduction occurs in the first tube in which the salicin has 
been hydrolysed to saligenin and glucose. 

Amygdalin may also be used as substrate and the formation of 
hydrogen cyanide tested for with picric acid paper. 1 This method has 
been used by Armstrong for detecting emulsin in plants. The plant 
leaf, etc., is put into a small test tube containing a drop of chloroform, 
and a few drops of amygdalin solution ; the test tube is closed with a 
cork, to which is attached a piece of picric acid paper. If emulsin be 
present, the paper becomes brick-red in colour from the action of 
hydrogen cyanide. In a similar way the presence of amygdalin, of 
emulsin together with amygdalin, may be tested for. 

IV. Lactase and Maltase. 

Since both lactose and maltose reduce Fehling's solution their 
hydrolysis by enzymes is difficult to demonstrate. It can only be 
satisfactorily demonstrated by the measurement of the reducing 
power. Lactose and maltose do not reduce Barfoed's reagent which 
is reduced by glucose and monosaccharides. Hence this reagent will 
serve to show the presence of these enzymes thus : 

Demonstration of Lactase in Intestinal Mucous Membrane. 

The mucous membrane is scraped off, ground up with sand to break 
the cells and kept in water with I per cent, of toluene for 1 2-24 hours. 
The solution is strained through muslin and divided into two nearly 
equal parts, say of 105 and 1 10 c.c. The larger part is boiled and cooled. 

loo c.c. of unboiled solution are added to 100 c.c. of 5 P er cent, 
lactose solution in a small flask (i). 100 c.c. of boiled solution are 
added to 100 c.c. of 5 per cent, lactose solution in a small flask (2). 
I c.c. of toluene is added to each ; the two flasks are corked and 
kept at 37'for 1-4 days. 

Flask (i) will show a reduction when tested with Barfoed's reagent. 

Flask (2) will not show a reduction ; if there is a slight reduction, 
this will be due to the presence of glucose in the extract. 

1 Filter paper moistened with a solution of i gm. picric acid + 10 gm. of Na 2 CO 3 in 
100 c.c. of water. 



ENZYMES. FERMENTATION 403 

V. Zymase (Yeast). 

^east contains a mixture of several enzymes. Its principal 
enzyme is zymase^ which acts upon the four natural hexoses. It 
contains also maltase and invertase, but it does not contain lactase. 
Lactase is only present in special yeasts, such as kefir. It is owing to 
the presence of maltase and invertase that yeast is able to ferment 
maltose and cane sugar and convert them into alcohol and carbon 
dioxide. Lactose is not fermented as it is not hydrolysed into its 
constituent monosaccharides. Before alcoholic fermentation can occur 
hydrolysis into monosaccharides must take place. 

The action of yeast upon the sugars is most conveniently demon- 
strated with a series of Einhorn fermentation tubes (p. 233). They are 
filled with I per cent solutions of glucose, fructose, galactose, maltose, 
cane sugar and lactose, and a small piece of yeast is added to each. 
Fermentation proceeds slowly, but in 12 hours it will be observed 
that all the sugars except lactose have been fermented and that galac- 
tose is fermented more slowly, as shown by the smaller volume of 
carbon dioxide evolved. 

The presence of zymase in the yeast can be shown either by preparing 
yeast juice by Buchner's method (p. 392) or by preparing maceration extract 
by Lebedeff's method. Fresh yeast is carefully dried. 100 gm. of the dried 
material are treated with 300 c.c. of water for 2 hours at 37. The mixture 
is filtered rapidly on a large folded filter paper and the filtrate is collected in 
a vessel in ice. 

Portions of 5 c.c. or 10 c.c. are added to i per cent, solutions of glucose, 
fructose and the other sugars in Einhorn fermentation tubes. After some 
hours the formation of carbon dioxide will become visible. 

Specificity of the Action of Enzymes. 

5 c.c. of diastase solution are added to 5 c.c. of cane sugar 
solution and kept at 40 for some , time. There is no conversion of 
cane sugar by diastase into glucose and fructose as shown by testing 
for reducing sugar with Fehling's solution. 

The same experiment is performed with 5 c.c. of salicin solution 
instead of cane sugar. Again there is no reduction of Fehling's 
solution. 

In the same way the action of 5 c.c. of the invertase solution is 
tested upon 5 c.c. of starch solution at 40. There is no hydrolysis 
of starch by invertase. If any action occurs it is due to impurity in 
the invertase solution, i.e. to its containing a little diastase. It is very 
difficult to obtain enzyme solutions which contain only one enzyme. 
Most cell contents contain a mixture of enzymes. 

5 C.C. of emulsin solution will not hydrolyse 5 c.c. of starch 
solution or 5 c.c. of cane sugar solution. 

In the experiments with yeast neither invertase, nor maltase, nor 

zymase acted upon lactose. 

26* 



404 1'KACTICAL ORGANIC AND BIO-CHEMISTRY 

VI. Lipase. 

Preparations of lipase are most conveniently obtained from castor-oil seeds 
and from pigs' pancreas : 

(a) Lipase from castor-oil seeds. 

The seeds are shelled, freed from oil by pressure or by treatment with 
ether or petroleum ether and finely ground up with -iN acetic acid. 

The lipase is liberated by the treatment with acid. The insoluble matter 
is filtered off and washed free from acid. A suspension of it is made in water. 

(<$) Lipase from pancreas. 

The pancreas is freed from fat, weighed, finely minced and ground up 
with sand. It is then extracted for 24 hours with a mixture consisting of 
90 parts of pure glycerol and i o parts of i per cent, sodium carbonate solu- 
tion, 10 c.c. of this mixture being used for every gram of pancreas. The 
fluid is strained through muslin and is kept at o. The lipase is destroyed as 
soon as the fluid becomes acid ; this happens generally in about three days. 

An active extract may also be prepared by treating the fresh and finely 
minced pancreas with twice its weight of -5 per cent, sodium carbonate solu- 
tion for 1 2 hours and straining through muslin. 

As substrate for the action of lipase neutral olive oil (see p. 381), an 
emulsion of egg-yolk in water, milk and esters, such as ethyl butyrate, may 
be used. Hydrolysis occurs with the formation of fatty acids which are 
recognised by the acidity of the solution. 

The activity of the enzyme is shown as follows : 

(1)5 c.c. of oil, or ester, are mixed with 5 c.c. of suspension, or 
extract 

(2) 5 c.c. of oil, or ester, are mixed with 5 c.c. of boiled sus- 
pension, or extract. 

The two mixtures are placed in a water-bath at 40 for at least 
half an hour, preferably 2 or 3 hours, and the contents are occasion- 
ally mixed by shaking. At the end of this time a few drops of phenol- 
phthalein are added to each and they are titrated with -iN alkali, 
^lore acid will be required to neutralise the contents of (i). 

The presence of lipase in castor-oil seeds may be demonstrated 
as follows : i gm. of seed, freed from shell, is ground up with 25 c.c. 
of water saturated with chloroform ; two equal parts of the suspension 
(10 c.c.) are placed in two test tubes and to each is added i c.c. of 
dilute acetic acid to liberate the enzyme. One portion is immediately 
boiled. The substrate is the oil of the seed. Both test tubes are kept 
at 40 for half to one hour. A few drops of phenolphthalein are 
added and they are titrated with 'iN alkali. More alkali will be re- 
quired to neutralise the acid in the tube containing unboiled enzyme 
showing that fatty acids have been formed. 



ENZYMES. FERMENTATION 405 

Demonstration of the Presence of Lipase in Pancreatic Juice. 

The lipase in pancreatic juice may be demonstrated by adding a 
few drops, or I c.c., to neutral olive oil (about 5 c.c.) containing a 
drop of phenolphthalein. The mixture is coloured red by running in 
iN alkali from a burette. On keeping warm at 40 and occasionally 
shaking, decolorisation takes place. More alkali is run in, drop by 
drop, until the red colour again appears. It will disappear again. 
This can be repeated several times. 

Effect of Bile Salts on Pancreatic Lipase. 

Bile salts increase the rate^f hydrolysis of fats by lipase and act 
as a co-enzyme. This can be demonstrated by the following three 
experiments : 

(1) 5 c.c. neutral oil + 5 c.c. pancreas extract + I c.c. of water. 

(2) 5 c.c. ,, + 5 c.c. ,, + i c.c. of i percent. 

bile salt solution. 

(3) 5 c.c. ,, ,, + 5 c.c. boiled ,, + i c.c. of i percent. 

bile salt solution. 

These three mixtures are kept at 40 for half an hour and titrated 
with *iN alkali using phenolphthalein as indicator. No. 3 requires 
least alkali, No. 2 requires most. More hydrolysis therefore occurs 
in the presence of bile salts. 

VII. Pepsin. 

A solution of pepsin is readily prepared by treating the mucous membrane 
of the stomach with glycerin for 12-24 hours and straining the solution 
through muslin. Before use the glycerin extract is diluted with 2 or 3 
volumes of water. 

Dry preparations of pepsin in the form of powder or scales are obtained 
by precipitating aqueous extracts with alcohol, or evaporating them to dry 
at a temperature below 40. They generally dissolve slowly in water or 
hydrochloric acid. A i per cent, solution is convenient for the demonstfl? 
tion of pepsin. 

As substrate for detecting the presence of pepsin, threads of fibrin 
or pieces of coagulated white of egg, are generally used. 

The action of pepsin is demonstrated with five test tubes contain- 
ing the following mixtures : 

(1) 5 c.c. of water + i c.c. of pepsin solution + a piece of fibrin. 

(2) 5 c.c. of -iN HC1 + i c.c. of water + a piece of fibrin. 

(3) 5 c.c. of -iN HC1 + i c.c. of pepsin solution + a piece of fibrin. 

(4) 5 c.c. of -iN HC1 + i c.c. of boiled pepsin solution + a piece 

of fibrin. 

(5) 5 c.c. of -iN Na 2 CO :} + i c.c. of pepsin solution + a 

fibrin. 



UJCU. 

' 



406 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

The test tubes are placed in a bath at 40. 

Pepsin will only act in the presence of acid - f consequently digestion 
or solution of the fibrin will only take place in the third test tube, 
where, in about half an hour, the fibrin will have disappeared. In 
order to show that it is not the acid which has this effect, the second 
tube, containing no pepsin but only acid, was used as a control. In 
this tube the fibrin will have become swollen, but not dissolved. The 
first, fourth and fifth tubes will be seen to be unaltered. They both 
contained pepsin and they show that pepsin will not act in a neutral 
or alkaline medium. Hence pepsin oWly acts in the presence of acid. 

Action of Alkali on Pepsin. 

Pepsin is destroyed by the action of dilute alkaline solutions, such 
as are found in the intestine, where the action of pepsin ceases for this 
reason ; thus 2 c.c. of dilute sodium carbonate solution are added to 
5 c.c. of pepsin solution and it is put .in a water- bath at 40 for at 
least half an hour. It is neutralised with o - 4 per cent, hydrochloric 
acid, an equal volume of 0*4 per cent, hydrochloric acid and a piece 
of fibrin are added and it is again kept at 40. Digestion will not 
occur. 

The Products of the Action of Pepsin. 

Proteins are hydrolysed by pepsin and converted into metaproteins, 
proteoses and peptones. In a very prolonged digestion ammo acids 
may be formed in small quantities : they are most probably formed by 
the action of other proteoclastic enzymes the autolytic enzymes 
which have been extracted from the cells of the mucous membrane 
together with pepsin. 

Several grams (2-5) of fibrin or egg-white are placed in *iN 

rochloric acid solution and 5-10 c.c. of pepsin solution are added, 
e fibrin dissolves in the course of half to one hour. 

The presence of 

(1) Metaprotein is shown by neutralising and filtering. 

(2) Proteoses by boiling and acidifying the filtrate and testing a 
portion with concentrated nitric acid. The precipitate which is 
formed dissolves on heating and reappears on cooling. They are re- 
moved by saturating the solution with ammonium sulphate. 

(3) Peptone by testing the filtrate from the ammonium sulphate 
precipitate by the biuret reaction in the presence of excess of caustic 
soda (p. 373). 




ENZYMES. FERMENTATION 407 

VIII. Trypsin. 

Trypsin is the activated proteoclastic enzyme of the pancreas.- It may be 
prepared by extracting the minced gland with glycerin for 12-24 hours 
and straining through muslin. The solution is diluted with 2-3 volumes 
of wate^hefore use. 

An active solution of trypsin may also be prepared by treating the minced 
pancreas'with 3 times its weight of distilled water and an equal weight of 
alcohol for 3 days at room temperature with occasional shaking. The solu- 
tion is strained through muslin and filtered. To the filtrate i c.c. of con- 
centrated hydrochloric acid per 1000 c.c. is added. A precipitate which forms 
is allowed to settle and is filtered off. 

In preparing trypsin from the pancreas it is advisable to add a small 
amount of the mucous membrane of the intestine so as to activate the enzyme 
in case this is not done by contact with the intestine on removing the pancreas. 

Dry preparations of trypsin can be obtained by mincing the pancreas and 
drying, or by precipitating with alcohol or evaporating the extracts. 

Numerous preparations of trypsin can be obtained commercially, e.g. 
Benger's liquor pancreaticus, holadin of Messrs. Fairchild Bros. & Foster. 
This latter preparation also contains lipase and diastase. . 

Solution of fibrin or coagulated egg-white by the enzyme, as in 
the case of pepsin, is the simplest means of investigating the presence 
and action of trypsin. 

Four test tubes are filled with the following mixtures : 
L^(i] 5 c.c. of trypsin + 5 c.c. of -5 per cent. Na 2 CO 3 + a piece of 
fibrin. 

(2) 5 c.c. of trypsin + 5 c.c. of water + a piece of fibrin. 
/ (3) 5 c.c. of trypsin + 5 c.c. of -i'N HC1 + a piece of fibrin. 
^(4) 5 c.c. of boiled trypsin + 5 c.c. of -5 per cent. Na 2 CO 3 + a 
piece of fibrin. 

The four tubes are placed in a water-bath at 40. Only in the 
first tube will any change be seen. In the tube containing hydro- 
chloric acid the fibrin swells without dissolving. In the aqueous solu- 
tion there is no visible change, nor in the tube containing boiled enzyme. 
Trypsin thus acts only in faintly alkaline solution of 0-2-0-5 per cent, 
concentration. 

As substrate polypeptides, such as glycyl-tyrosine, glycyl-tryptophan and a 
peptone from silk have been used to detect trypsin. The tyrosine separates 
out and the bromine water reaction for tryptophan becomes positive. 






4 o8 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

. The Products of the Action of Trypsin. 

Proteins are hydrolysed by trypsin and converted into amino acids 
(and a polypeptide). 

The demonstration of the amino acids can be shown in a digest of 
protein, prepared by treating about 100 gm. of caseinogen|Bfr other 
protein, dissolved in 2000 c.c. of -iN ammonia or sodium carronate at 
37 for several days with about I gm. of dried pancreas preparation in 
the presence of toluene or chloroform. 

(1) The solution will most probably contain a white precipitate, 
which consists mainly of tyrosine. This is proved by filtering it off, 
washing and dissolving it in dilute acetic acid and testing with Millon's 
reagent (p. 268). 

(2) The filtrate from the tyrosine can be shown to contain trypto- 
phan by acidifying a portion of about 5 c.c. with acetic acid and add- 
ing bromine .water, drop by drop, as described on p. 349. 

(3) On evaporating the filtered solution on a water-bath to a small volume 
and allowing to stand for about 24 hours, a crystalline crust forms. This con- 
sists mainly of tyrosine as can be shown by microscopic examination, especially 
after solution in a drop of ammonia. The crystals will also give Millon's 
reaction. 

(4) On further evaporation of the filtrate leucine and glutamic acid sep- 
arate out on standing. Microscopic examination will show that the crystals 
consist mainly of rounded cones with a radiating striation (leucine). If free 
from tyrosine they will not give Millon's reaction. They dissolve in hot water 
and form copper salts, as can be shown by adding a very little caustic soda and 
a few drops of copper sulphate. The precipitate of cupric hydroxide dissolves 
on warming, giving a blue solution. 

IX. Trypsinogen. 

Trypsinogen is present in the pancreas and is contained in pancreatic 
juice; it may be prepared from the pancreas by the method de- 
scribed by Mellanby and Woolley : 

The pancreas is removed without contact with the intestines. It 
is finely minced and treated with twice its weight of -5 per cent, 
hydrochloric acid at room temperature for 12 hours. The solution is 
strained through muslin and neutralised with sodium carbonate. The 
precipitate is filtered off and the solution is kept under toluene. 

Preparation of Pancreatic Juice. 

The mucous membrane of the small intestine is ground up with sand and 
boiled with dilute hydrochloric acid. The boiling solution is neutralised with 
dilute alkali. Coagulable proteins are thus precipitated and filtered off. The 
solution contains secretin. Secretin is not an enzyme as it can be boiled, but 
belongs to the class of substances termed Jwrmones by Professor Starling. 

A cannula is placed in the pancreatic duct and the solution of secretin is 
slowly injected into the jugular vein. Pancreatic juice flows from the cannula 
after each injection and is collected in a clean vessel. It is mixed with an 
equal volume of 2 per cent, sodium fluoride to preserve it. 



ENZYMES. FERMENTATION 409 

Activation by Enterokinase. Conversion of Trypsinogen into Trypsin. 
A solution of enterokinase is prepared by making an aqueous ex- 
tract of the mucous membrane of the upper part of the small intestine. 

(a) As substrate a capillary tube (Mett's tube, cf. p. 421) of 1-2 mm. bore 
about 2 cm. long and filled with coloured gelatin is generally used. These are 
prepared by drawing up hot 10 to 20 per cent, gelatin solution stained with 
methylene blue or gentian violet into the tube, placing the tube horizontally 
and allowing the gelatin to set. The tube is cut into pieces 1-2 cm. long. 
They can only be used for experiments at room temperature ; at 40 the gelatin 
melts and flows out of the tube. 

Two of these tubes are placed in each of three small conical flasks together 
with 5 c.c. of '5 per cent, sodium carbonate solution. 

In the first is placed 1-2 c.c. of pancreatic juice or trypsinogen solution. 

In the second is placed 1-2 c.c. of pancreatic juice or trypsinogen solution 
+ a few drops of enterokinase solution. 

In the third is placed 1-2 c.c. of boiled pancreatic juice or trypsinogen solu- 
tion and a few drops of enterokinase solution. 

The flasks are kept at room temperature for 8 to 10 hours. 

No solution or digestion of the gelatin occurs in No. i or No. 3 which 
contained the trypsinogen or the boiled trypsinogen, but in No. 2 the gelatin 
will have been dissolved at both ends of the capillary tube. 

(b] H. Bierry and V. Henri have shown that milk is a very sensitive 
substrate for observing the activation of pancreatic juice by entero- 
kinase. The rnik is centrifugalised and filtered from fatty particles 
through wet paper and is sterilised by heating. 

In four clean test tubes are placed : 

(1) 5 c.c. of milk + 5 drops of pancreatic juice. 

(2) 5 c.c. of milk + 5 drops of pancreatic juice + 2 drops of 
intestinal extract. 

(3) 5 c - c - f milk + 5 drops of boiled pancreatic juice + 2 drops 
of intestinal extract 

(4) 5 c.c. of milk + 2 drops of intestinal extract. 

They are put in a water-bath at 40 for 10 to 15 minutes. No 
change will be found to have occurred in tubes No. i, No. 3 and No. 
4, whereas in No. 2 there is an immediate clarification of the milk, 
which becomes transparent after the lapse of the above time. 

X. Erepsin. 

Erepsin is contained in the cells of the mucous membrane of the 
small intestine and a solution is prepared by grinding the membrane 
with sand and treating with water, to which i per cent, of toluene has 
been added, for 12 to 24 hours. The solution is strained from sand 
and connective tissue through muslin. Erepsin acts upon proteoses 
and peptones forming amino acids ; a 2 per cent, solution of Witte's 
peptone is therefore used as substrate. 



410 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Two portions of Witte's peptone solution (50x3 c.c.) are placed in 
bottles ; to one portion 100 c.c. of erepsin solution are added ; to the 
other portion 100 c.c. of boiled erepsin solution. To both are added 6 
c.c. of toluene and they are kept at 37 for I to 3 days. 

A portion of each is examined for proteoses and peptones by the 
biuret reaction by adding exactly the same amount of caustic soda 
solution (5 c.c.) and exactly the same amount of I per cent, copper 
sulphate solution. 

The solution which contained boiled enzyme will show the biuret 
reaction (the substrate is unchanged). 

The solution which contained enzyme will either not show the 
biuret reaction or it will be fainter than in the other solution (the 
substrate has been hydrolysed completely or not quite completely). 

XI. Papain and Vegetable Proteoclastic Enzymes. 

Papain is prepared from the juice of the papaw tree by evaporation or by 
precipitation with alcohol. 

Bromelin is present in the juice of the pine-apple ; the juice is neutralised 
before testing its action. 

These enzymes can be demonstrated in a similar way to pepsin and trypsin 
by using fibrin or coagulated egg-white as substrate : 

In. six test tubes are placed : 

(1) 5 c.c. of water + 5 c.c. of papain solution + a piece of fibrin ; 

(2) 5 c.c. of water + 5 c.c. of boiled papain solution + a piece of fibrin ; 

(3) 5 c - c - f ' J N HC1 + 5 c.c. of papain solution + a piece of fibrin ; 

(4) 5 c.c. of - iN HC1 + 5 c.c. of boiled papain solution + a piece of fibrin; 

(5) 5 c.c. of -iN Na 2 CO 3 + 5 c.c. of papain solution + a piece of fibrin ; 

(6) 5 c.c. of - iN Na 2 CO 3 + 5 c.c. of boiled papain solution + a piece of 
fibrin ; and they are put in a water-bath at 40. 

The fibrin dissolves in (3) and (5) fairly rapidly ; very slowly or not visibly 
in (i). There is no solution in (2), (4), (6). 

Papain thus acts in the presence of either acid or alkali. 

The Products of the Action of Vegetable Proteoclastic Enzymes. 

In most respects the vegetable proteoclastic enzymes resemble trypsin. 
They form amino acids from proteins. Their action is very slow and complete 
conversion of protein to amino acids takes several weeks. The experiment is 
performed as described under trypsin with 100 gm. of caseinogen. 

The Presence of two Vegetable Proteoclastic Enzymes. 

By extracting seeds of Cannabis sativa with 10 per cent, salt solution and 
acidifying the solution with acetic acid, Vines has separated the proteoclastic 
enzymes of the plants into two groups. The precipitate contains a pepsin, the 
filtrate an erepsin. Since other extracts can also be separated in a similar 
manner, Vines considers that plants contain two kinds of proteoclastic enzymes : 
(i) Peptic, producing proteoses, etc. ; (2) Ereptic, producing amino acids from 
proteins and proteoses, etc. 



ENZYMES. FERMENTATION 411 

XII. Oxidases. 

A. Catalase. 

A catalase is present in most animal and vegetable tissues. 
Solutions may be prepared by extracting the tissues with water ; the 
extracts are usually not very active and a piece of tissue is* used 
directly. 

Since catalase acts upon hydrogen peroxide with the formation of 
oxygen only hydrogen peroxide can be used as substrate. 

E.g. a piece of liver is placed in a test tube and covered with a dilute 
solution of hydrogen peroxide. An evolution of oxygen occurs. 

B. Peroxidase. x 

Peroxidases are very abundant in plant tissues. Active solutions 
are best prepared from horse-radish, potato, or fungi, by grinding up 
the material, treating with water and filtering from insoluble matter. 

A substrate is usually present in the plant tissue together with 
the enzyme. Qn bruising the tissue it becomes brown like the cut 
surfaces of apples and pears, or it may become blue or red as in some 
species of fungi. The substrate is adihydric or trihydric phenol such as 
hydroquinone or pyrogallol. In the tissue an organic peroxide, or oxy- 
genase, is also frequently present. On bruising the tissue, oxygen is 
taken up from the air and the peroxide is formed. The peroxidase acts 
upon the peroxide giving " active " or nascent oxygen, which oxidises 
the substrate. These oxidases are sometimes called direct oxidases. 

Sometimes the colour is only given after hydrogen peroxide or 
other peroxides (especially organic peroxides), such as are present in 
oil of turpentine which has been exposed to the air, are added. Such 
oxidases are called indirect oxidases. 

For purposes of demonstration a variety of phenolic substances are 
used as substrate. 

(1) Guaiacum. A freshly prepared I per cent, solution in alcohol 
(tincture of guaiacum). It changes to blue on oxidation. 

(2) Guaiaconic acid, the constituent of guaiacum. A '5 to I per 
cent, solution in alcohol. 



412 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

(3) a-naphthol. A I per cent, solution in equal parts of water and 
alcohol. When oxidised it becomes lavender in colour. This sub- 

' strate has been largely used in botanical work. 

(4) Guaiacol. A 2 per cent, solution in alcohol. It is oxidised to 
tetraguaiacoquinone, which is red. 

(5) Benzidine. A I per cent solution in 50 per cent, alcohol. It 
becomes blue on oxidation and a brown precipitate is formed. 

(6) /-phenylenediamine hydrochloride. A I per cent, solution in 
water. It becomes greenish in colour. 

(7) Indophenol. A I per cent, a-naphthol solution in 50 per cent, 
alcohol and a I percent, aqueous solution of/-phenylenediamine hydro- 
chloride are required. 2 or 3 drops of each of these are added to the 
enzyme solution which is made faintly alkaline with sodium carbonate. 
A purple solution results. 

A few drops of any of these reagents are added to about 5 c.c. of 
the oxidase solution to which a few c.c. of hydrogen peroxide have 
been added. The colour slowly forms. 

Peroxidases are present in milk and blood (pp. 458, 476). 

The direct oxidase may be observed in potato : a drop of guaiacum 
solution is placed upon the cut surface ; in a short time it becomes 
blue. 

The presence of an oxidase in minced tissues is readily detected by 
the indophenol reaction as shown by Vernon. 1 The reaction takes 
place according to the equation : 

/ NH 2 

C 6 H 4 (NH 2 ) 2 4- C 10 H 7 OH + 9 = C 6 H/ + 2 H 2 O 

\N C 10 H.Q 
p-phenylene- a-naphthol. 
diamine. 

The substrate consists of -144 per cent, solution of a-naphthol 
(-01 M) and -I I percent, paraphenylenediamine (-oiM) in 50 per cent 
alcohol. 5 c.c. of the solution together with about 5 c.c. of -I per 
cent, sodium carbonate solution are poured upon -5 to I gm. of minced 
tissue in a flat dish (a Petri dish 8-8 cm.) and well stirred with the 
tissue. The indophenol begins to form almost at once. 

1 J. Physiol., 42, 402. 



ENZYMES. FERMENTATION 



413 



THE CATALYTIC ACTION OF ENZYMES. 

The resemblance of the action of enzymes to that of inorganic catalysts 
was pointed out by Berzelius. The agent producing the chemical change 
apparently takes no part in the reaction and can at the end be recovered 
unchanged. Minute quantities are capable of effecting a large amount of change ; 
as an example may be quoted O'Sullivan and Tompson's statement that invertase 
can hydrolyse 200,000 times its weight of cane sugar. 

The resemblance is most marked if the velocity of the action of enzymes 
be compared with that of inorganic catalysts, as is shown in the curves in 
Fig- 55- 



100 



- 900 




23456789 

F IG . 55. From Bayliss's " Nature of Enzyme Action ". 



10 



Curve B is the velocity of the action of hydrochloric acid upon cane 
sugar. Curve A is that of invertase upon cane sugar. Curve C is that of 
trypsin upon caseinogen. This latter curve is the most typical of enzyme action, 
that of invertase being more exceptional. 

The curve B is a logarithmic curve. The enzyme curves deviate from 
this in two important particulars. They are linear at the commencement and 
at the end. The cause of the deviation of the enzymic curve from that of a 
proper catalytic curve has been found to be due to three causes : 

(1) Disappearance of the enzyme during the course of the action. Enzyme 
solutions, as previously stated, are never pure ; they contain other substances 
which act upon and remove or destroy the enzyme. 

(2) Effect of the products of the action ; they hinder the reaction. 

(3) Combination of the enzyme with the substrate which also takes an 
appreciable time. 

The linear part of the curve is the result when either substrate is in excess 
at the beginning, or enzyme in excess at the end. 



4H PRACTICAL ORGANIC AND BIO-CHEMISTRY- 

Demonstration of the Catalytic Action of Enzymes. 

The catalytic action of enzymes is readily followed by analysing the amount 
of decrease of the substrate or the amount of the products formed at intervals 
during the progress of the reaction. 

Physical methods of analysis are preferable to chemical methods as they are 
easier of manipulation, e.g. : 

(1) Optical activity. The rotation of the solution of enzyme and substrate 
is taken immediately the mixture is made and at intervals of i minute, i hour, 
i day afterwards. Throughout the experiment a constant temperature must 
be maintained. 

(2) Electrical conductivity. This method can be employed when electro- 
lytes are produced as end products, e.g. fatty acids from ethyl butyrate and amino 
acids by the action of trypsin upon proteins. 

(3) Viscosity. Protein solutions are viscous and the decrease in viscosity 
can be measured. The results by this method are not so accurate. 

When chemical methods are employed samples of the solution must be re- 
moved at the beginning and at intervals from a larger bulk of solution. The 
action of the enzyme must be stopped immediately after removing the solution. 
This cannot be effectively accomplished by boiling as it is impossible to 
raise the temperature of the samples to boiling at the same rate ; it can only 
be done by pouring the enzyme solution into boiling water. In this case 
dilution occurs and the manipulation of the solution is troublesome. 

The action of the enzyme is most effectively stopped by adding an excess 
of alkali or acid. In the case of carbohydrates the mutarotation l of the solu- 
tion takes place immediately on adding the alkali. Since enzymes are associated 
with proteins or complex carbohydrates, precipitation with heavy metals or 
tannic acid is very convenient. A definite volume of the solution is added to 
a definite volume of the reagent. A known volume of the filtrate is analysed 
after removing the heavy metal or other reagent. Each sample is analysed 
in exactly the same way. 

1 The sugar is split off in either the a or /3 forms ; the equilibrium mixture is thus 
obtained immediately ; by noticing the change in rotation on adding alkali it can be ascer- 
tained whether the a or form of the sugar is contained in the substrate. 



ENZYMES. FERMENTATION 415 

THE SYNTHETICAL ACTION OF ENZYMES. 

The majority of the chemical changes effected by enzymes are hydrolytic 
changes. The substrate consists of a compound which can be hydrolysed 
into two or more constituents and in most instances the organic compound 
has been synthesised from its constituents. The reactions are reversible. 
The typical example of a reversible reaction is the formation and hydrolysis 
of methyl acetate : 

CH 3 OH + CH 3 COOH <^ H 2 O + CH 3 . COOH 3 . 

These reactions have been measured and it has been found that an 
equilibrium is reached from whichever side the reaction is started when the 
composition of the mixture of the four substances is 

f mol. ester + 3 mol. water + mol. alcohol + ^ mol. acid. 

Other similar reactions have also been measured and their equilibrium 
positions have been determined. Reversible reactions proceed according to 
the Law of Mass Action. The effect of a catalyst upon reversible reactions, 
as it effects the reaction to the same degree from both sides, is not to alter 
the position of equilibrium of the reaction. The final position in the case of 
enzymes is usually reached when the products of hydrolysis make up over 90 
and sometimes nearly 100 per cent, of the mixture. This is on account of 
the large proportion of water present. Enzymes as catalysts should there- 
fore accelerate the reaction in both directions, i.e. be capable of synthesising 
the compounds which they hydrolyse. The synthetic power of enzymes has 
been demonstrated in only a few cases, e.g. that of maltase by Croft Hill, of 
lipase, of emulsin and of trypsin. The demonstration of the synthetical 
action of enzymes is difficult as the equilibrium point is generally so near the 
point of -complete hydrolysis. It can be.shown most easily in the cases of 
lipase and of emulsin. The following experiment devised by Bayliss shows 
the synthetical action of emulsin : 

18 parts of pure anhydrous glucose are dissolved in 12 parts of water and 
cooled. 40 parts of dry glycerol and 3 parts of emulsin are added. The 
rotation of the mixture (+ 2 '83) is taken immediately the mixture is made. 
The mixture is kept at 47 for seven days and the rotation again observed 
(+ ''80) ; in fifteen days the rotation is - -16, which corresponds to 75 per cent, 
of synthesis. A control experiment is made with emulsin alone omitting the 
glucose. In order to show that glucose has not disappeared by other re- 
actions, the mixture is diluted with 2 to 3 volumes of water and a fresh 
quantity ('5 gm.) of emulsin is added. The rotation of the solution is taken 
immediately and again after 2 or 3 days when hydrolysis is complete. It 
will be found to be the same as the original rotation of the mixture, allowing 
for the dilution. 

Croft Hill and Bayliss point out that though the synthetical reaction is so 
small it suffices for synthesis in nature ; the synthetical product is usually a 
colloid and insoluble ; it is removed from the reaction and synthesis will con- 
tinue. Bayliss considers that we have no cause for believing that there are 
enzymes which act specially as synthetical catalysts of the natural compounds. 



416 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

THE MEASUREMENT OF THE ACTIVITY OF 
ENZYMES. 

In order to determine the activity of an enzyme solution four 
factors must be taken into account. This was first clearly established 
by Kjeldahl in 1879 in the case of the diastatic enzyme of malt, 
namely : 

(1) The temperature at which the action takes place. 

(2) The time during which the enzyme acts. 

(3) The amount of enzyme solution. 

(4) The concentration of the substrate solution. 

The amount of enzyme solution must be small in comparison with 
the amount of substrate and the change must not exceed 30-40 per 
cent, of the total change. These statements correspond to the curve 
of the catalytic action of enzymes. The curve is linear at the com- 
mencement and again at the end. The measurement is proportional 
only during the linear part of the curve where the amount of enzyme 
is small and the substrate large in comparison. The linear portion of 
the curve corresponds to 30-40 per cent, of hydrolysis. 

The temperature and the concentration of the substrate are fixed, 
and by fixing either the time or the amount of enzyme solution .the 
fourth factor can be determined. As the basis of comparison it is 
best to determine the time taken to effect an equal change. This is 
most important where the reaction takes place in stages ; comparable 
values can be obtained only in this way. More frequently the 
amount of enzyme required to produce an equal change, or the 
amount of change produced by equal amounts of enzyme solution in 
a given time, is determined. 

The measurements are made either by chemical methods or by 
physical methods, depending upon the properties of the substrate and 
the products. 

I. Diastase. 

The measurement of the diastatic activity of malt is of practical importance 
in brewing and certain standard methods of making the extract and of de- 
termining the hydrolysis have been adopted. 

Preparation of the Extract. 

25 gm. of malt are treated with 500 c.c. of water for 3 hours at 21. 
The solution is filtered and the first 100 c.c. are rejected. The activity of 
the perfectly clear extract is determined by method (a) or (b}. 



ENZYMES. FERMENTATION 417 

(a) Lintners Method. 

In each of a series of ten clean test tubes is placed the same quantity of 
soluble starch solution (10 c.c. of 2 per cent.) and then a progressively in- 
creasing quantity of enzyme solution, thus cri c.c. in No. i, 0-2 c.c. in No. 
2, o'3 c.c. in No. 3, and so on. The contents are mixed and placed in a 
water-bath at 21 for exactly i hour. 5 c.c. of Fehling's solution are now 
placed in each tube, the tubes are heated in a boiling water-bath for 20 
minutes and then examined. Some of the tubes in the series will show no 
blue colour, or are faintly yellow, whilst others are still blue. The amount 
of enzyme in the first colourless tube is that amount which will just convert the 
fixed amount of starch into maltose in the given time. 

The diastatic power is based upono'i c.c. enzyme solution and called 100. 
.. if the result was between the sixth and seventh tubes the diastatic power, 
D, is 

100 

x o-i = i<5'55. 
0-65 

Generally 1*5 is deducted as due to reducing sugars in the extract. The 
extract is diluted with an equal volume of water, if D is very high. 

It is generally necessary to repeat the experiment once or twice, 
i 

() Lings Method. 

3 c.c. of the diastase extract are added to 100 c.c. of 2 per cent, soluble 
starch solution in a 200 c.c. measuring flask heated to 21. It is allowed to 
act for i hour at this temperature. 10 c.c. of - iN caustic alkali are added 
to stop the action; the solution is cooled to 15 -5, made up to 200 c.c. and 
well mixed. The amount of reducing sugar is estimated against 5 c.c. 
Fehling's solution heated to boiling over a naked flame, the solution being 
added slowly, 5 c.c. at a time, and kept boiling until the reduction is com- 
plete as ascertained by Ling's indicator. The result is calculated from 

p _ I00 

xy 

where D is the diastatic activity, x, = c.c. of malt extract in 100 c.c. of 
diluted solution, j> = c.c. of liquid required to reduce the Fehling's solution. 
It is not accurate for values of D above 50 so that less malt extract 
(z c.c. or i c.c.) must be taken and the measurement repeated. 

(c) Roberts' Method. 

Though not so accurate, this method is the most rapid to carry 
out. Here the time taken to effect the change of i per cent, starch 
solution into achtoodextrin is measured. The stage at which no 
colour is given by iodine solution, i.e. when the last traces of erythro- 
dextrin have been converted into achroodextrin, is known as the 
achromic point. The time taken to reach this point is termed the 
" chromic period ". The time taken to reach the achromic point must 
be between 2 and 10 minutes. ,The diastatic power D is the number 
of c.c. of starch solution which can be converted by i c.c. of enzyme 
solution in 5 minutes, or 

D = " x 5 

V t 

where n = number of c.c. starch solution taken, v = volume of enzyme 
solution (dilution must be known), t = time, 5 = 5 minutes. 

: 27 



4 i8 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

5 c.c. of i per cent, starch solution are warmed to 40 and 1-5 c.c. 
of diastase solution are added. The time is noted at which the mix- 
ture is made. At intervals of ^ to I minute a drop is removed and 
tested against a drop of iodine solution. The time is taken at which 
no colour is given, i.e. when the achromic point is reached. 

Vernon 1 states that this method is very exact if corrected by 
means of a table which he gives. 

(d) WohlgemutWs Method. 

This method is carried out in a similar way to Lintner's method with 
varying quantities of enzyme solution, but the disappearance of starch as 
shown by the iodine reaction is taken account of. 

5 c.c. of i per cent, starch solution are placed in each of a series of 10 
tubes and cooled to o, whilst an increasing quantity of enzyme solution is 
added to each tube in the series. They are transferred to a bath at 40 for 
30 or 60 minutes and after this time again cooled to o to stop the action. 
Each tube is filled with water and one drop of -iN iodine solution is added 
to each. The colours are blue, blue-violet, reddish and yellow. The limit 
is taken as that tube which still shows a violet colour. The activity of the 
solution is then calculated on the basis of the power of i c.c. enzyme solution. 
Thus if the tube in the series contain 0^3 c.c. enzyme solution, then the 

40 . 5 
diastatic power at 40 in 10 minutes or D -, is -- = 16-6. 

3 o'3 

It has been shown by Evans 2 that this method only gives certain values 
for D, that the values are only approximate and that it is not accurate when 
the volumes of saliva added are in geometrical progression. He finds that 
the achromic point method is more delicate. 

(e) Other Methods. 

The reducing sugar may be estimated gravimetrically or by any other 
method for estimating carbohydrates. Proteins, etc., should be removed before 
the estimation (see under lactase). 

Pancreatic Diastase. 

The diastase of the pancreas does not hydrolyse properly except in the 
presence of salts; '3 gm. of sodium chloride and 7 c.c. of '2N disodium 
phosphate should be added per 100 c.c. of reaction mixture. 3 

*J. Physiol., 27, 182. ' 2 Ibid., 44, 220. 

3 Sherman, Kendall and Clark, J. Amer. Chem. Soc., 1910,32, 1073. 



ENZYMES. FERMENTATION 419 

II. Invertase, Emulsin. 

In measuring the activity of these enzymes either the reduction is estimated, 
preferably after removing proteins as under lactase, or the change in optical 
activity is observed. 

A known weight of preparation is dissolved or extracted with water and 
made up to a definite volume. 

i to 5 c.c. or other suitable volume of the solution or extract, is added 
to 100 c.c. of 2-5 per cent, substrate solution. The solution is observed 
in the polarimeter at once and at definite intervals and the readings noted ; 
or after a fixed interval of time at room temperature, or at 37, the action is 
stopped with 10 c.c. of - iN alkali, the volume made up to 200 or 250 c.c., 
and the reduction measured (a) against 5 or 10 c.c. of Fehling's solution, or 
(b) gravimetrically. 

III. Lactase, Maltase. 

The measurement of the activity of lactase and maltase on account of 
the small differences in reduction, and rotation also in the case of lactase, is 
somewhat tedious. A control experiment must be performed. Lactase 
solutions generally contain a large amount of protein if prepared from the in- 
testine and this must be removed before an estimation can be carried out, 

Two portions of 100 c.c. of 5 per cent, lactose solution are placed in two 
250 c.c. flasks. To each 50 or 100 c.c. of lactase solution or intestinal ex- 
tract are added. To one of them (C), the control, 5 or 10 c.c. of neutral 
mercuric nitrate solution are added. 1 To both are added 5 c.c. of toluene. 
They are kept in corked flasks at 37 for 3-4 days. (Instead of adding 
mercuric nitrate to (C), 50 c.c. of boiled and cooled enzyme solution might 
have been added and mercuric nitrate added to both after 3-4 days.) The 
same volume of mercuric nitrate solution is added to the other flask contain- 
ing enzyme (E) and mixed. The two flasks are now in the same stage of 
operations and their contents are treated as follows : 

(1) Filtered through dry papers into dry flasks. 

(2) Equal volumes of filtrate (as much as possible) are taken and 
neutralised to litmus with sodium hydroxide from a burette (about 4-8 c.c.). 
The same quantity is added to both. 

(3) The precipitate is filtered off through a dry paper and the filtrate 
collected in a dry flask. 

(4) Equal volumes of filtrate (as much as possible) are taken and treated 
with hydrogen sulphide. The gas is passed on to the surface of the solution. 
Only a small quantity is required to remove the last traces of mercury. 

(5) Excess of hydrogen sulphide is removed by adding copper sulphate 
solution until the smell disappears and it has a faint blue colour. 

(6) The solution is filtered from the sulphides and the volume is made up 
to 250 or 500 c.c. 

The reducing sugar is estimated gravimetrically or by Bertrand's method. 

The percentage of hydrolysis is calculated as follows : 

The reducing power of the control multiplied by V- is the reducing 
power of lactose if completely hydrolysed. The difference between this 
figure and the original is that of total hydrolysis T. 

The difference between the control figure and that of the enzyme solution 
is the actual hydrolysis A. 

Hence percentage is A : T = x : TOO. 

1 220 gm. of mercuric oxide are suspended in about 200 c.c. of water and dissolved by 
adding concentrated nitric acid. The solution is treated with caustic soda until a per- 
manent precipitate of mercuric oxide is formed. The solution is filtered and made up to 
1000 c.c. 27 * 



420 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Zymase. 

The activity of zymase is measured by estimating the amount of carbon 
dioxide which is evolved. This is effected most simply by determining the 
loss in weight : 20 c.c. or a known volume of zymase solution are placed in 
an Erlenmeyer flask with a known amount of sugar solution and i per cent, 
of toluene is added. The flask is closed with a Meissl tube and the loss in 
weight determined. 

It may also be determined by mixing known amounts of the zymase 
solution with sugar solution and some toluene in a flask connected to 
another flask containing excess of N sodium hydroxide. The first flask is 
fitted with a valve and the second, containing the sodium hydroxide, is con- 
nected with a soda lime tube. Air is sucked through the flask at the end 
of the experiment through the sodium hydroxide, the enzyme solution being 
boiled to expel the last trace of carbon dioxide. The sodium hydroxide 
solution is titrated firstly against phenolphthalein and secondly against 
methyl orange. The difference in the values gives the amount of carbon 
dioxide. 

Harden, Thompson and Young l have described a very accurate method 
in which the estimation is effected volumetrically. 

Lipase. 

Lipase acts upon fats producing fatty acids. Its activity is measured 
by titrating the mixture of enzyme, fat and fatty acids. A known volume 
of enzyme solution is added to a known amount of ester (ethyl butyrate), 
of fat (neutral olive oil), or egg-yolk, suspended in water ; the mixture is kept 
at 37 and titrated after 2-24 hours. A control is made with boiled enzyme 
or a similar mixture titrated immediately after the addition of the enzyme 
solution. 

1 Biochem. J., 1910, 5, 230 ; see also Harden's monograph on " Alcoholic Fermentation ". 



ENZYMES. FERMENTATION 421 

Proteoclastic Enzymes. 

The numerous methods which have been employed for comparing 
the strengths of two proteoclastic enzymes may be divided into two 
main groups. 

I. Those in which the action of the enzyme is determined by ob- 
serving the rate of solution of an insoluble substrate. 

II. Those in which the rate of formation of the products of the 
action of the enzyme is observed. 

Of these the former is generally used for pepsin, the latter for trypsin. 

A. Pepsin. 

(a) Briickes Method. 

In this method the time taken to dissolve equal-sized threads of fibrin 
is noted. The more active enzyme produces solution in the shorter time. 

(b} Metfs Method. 

Numerous results, especially those in Pavloff s laboratory, upon digestion 
by enzymes have been obtained by this method. It consists in directly 
measuring the amount of protein digested in a given time, the protein being 
contained in narrow tubes open at both ends and known as Mett's tubes. 

Melt's tubes consist of a small length of coagulated egg-white or serum 
in a narrow glass tube of 2 mm. bore, and are made by drawing up egg-white 
into the glass tubing (no air bubbles must be present in the egg-white) and 
placing it in nearly boiling water for 2-3 minutes. This tubing is then 
cut up into lengths of about i cm. The coagulated egg-albumin must form 
a continuous layer free from air spaces. Two small pieces of tube are placed 
in the enzyme solution and after a definite lapse of time the pieces are laid 
upon a mm. scale and the amount dissolved from each end measured. The 
mean of these readings is taken. 

5 c.c. of o'4 per cent, hydrochloric acid, 5 c.c. of pepsin solution and 
i or 2 Mett's tubes are placed in a small conical flask. The flask is stoppered 
and kept at 37 for 8-10 hours. It is then removed and the amount of pro- 
tein digested from each end of the Mett's tubes is measured. The mean is 
taken. In this experiment where the action is for a long period of time, ac- 
cording to the Schutz law the squares of the lengths digested represent the 
activity of the enzyme more accurately than the direct ratio. 

(c) Grutzners Method. 

This method is the one which has been most frequently em- 
ployed on account of the rapidity with which the results are obtained. 
Fibrin, stained with carmine, is the substrate used ; a definite quantity 
is added to the enzyme solution and according to the rate of diges- 
tion of the fibrin more or less of the dye-stuff passes into solution. 
Comparison is made by observing the depth of the colour. 

Roaf has suggested the use of fibrin stained with congo red instead 
of carmine. This possesses the advantage that the stained fibrin can 
be used in both acid and alkaline media ; carmine-stained fibrin can 
only be used in acid media as carmine is dissolved out of the fibrin by 
alkalies. 



422 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

The fibrin is prepared in the following way : 

Fresh fibrin is minced, washed till free from blood and placed 
for 24 hours in a 0*5 per cent, solution of congo red in the propor- 
tions of 50 gm. of fibrin to 100 c.c. of congo-red solution. The mass 
is poured into a large volume of water heated to 80 to fix the dye 
and kept at this temperature for about 5 minutes. The fibrin is then 
placed in a cloth and washed in running water ; the excess of water is 
squeezed out and the fibrin is preserved in a mixture of equal parts of 
glycerol and water, a little toluene being added as a preservative. 

Another modification consists in the use of ^ stained cubes of 
coagulated egg-white. 

A known quantity of 0*5 gm. of congo-red fibrin, 5 c.c. of 0*4 per 
cent, hydrochloric acid and 5 c.c. of the pepsin solution are placed in 
a test tube and put in a water-bath at 40 for half an hour. After 
this time sufficient solid anhydrous sodium carbonate is added to 
change the blue colour of the congo red to red. This also stops the 
action of the enzyme. A measured volume is removed ; in order 
to compare two pepsin digests water is added from a burette to the 
deeper one till the tints of the two solutions are the same. The 
amount of water added is noted. The strengths of the enzymes are 
to one another as the amount of dilution : thus if an equal volume of 
water be added the strengths are as 2 : I. 

(d} Fuld's Method. 

By making use of edestin as substrate and its precipitability by 
salts from its solution in hydrochloric acid, Fuld has devised a very 
simple method for measuring the activity of pepsin solutions. Vary- 
ing amounts of the enzyme are added to definite volumes of the 
edestin solution in a series of tubes and after a prescribed lapse of 
time sodium chloride is added ; the first tube in each series in which 
a precipitate of edestin is no longer formed is noted, i.e. the tube con- 
taining least enzyme. Thus : 

5 c.c. of the 0*5 per cent, edestin solution in 0*4 per cent, hydro- 
chloric acid are measured out with a pipette into each of a series of 
five test tubes. To these tubes is added in order, O'2 c.c., o - 4 c.c., 
o - 6 c.c., O'8 c.c., 1*0 c.c. of the pepsin solution A from a burette 
(generally 2 drops = OT c.c.). The same operations are performed 
with pepsin solution B. 

The tubes are kept at the ordinary temperature for half an hour, or 
longer, but the same time for each series ; then to each tube is added 
I c.c. of saturated sodium chloride solution. The first tube in which 
a precipitate of edestin is no longer produced is noted. 



ENZYMES. FERMENTATION 423 

(<?) Hatas Method. 

It was shown by Hata in 1909 that a suspension of coagulated 
egg-white was a very delicate substrate for estimating the activity of 
pepsin solutions. Under the influence of the enzyme the clqudy 
solution becomes quite clear. This method also possesses the 
advantage that egg-white is obtainable everywhere and the suspension 
is readily prepared. The comparison is carried out by adding varying 
quantities of enzyme solution to the substrate and observing in which 
tube clarification is produced by the least amount of enzyme after a 
given time. 

The substrate is prepared by rubbing up egg-white in a basin 
until it is of a uniform consistency. It is then slowly mixed and 
rubbed up with water until it has been diluted five times. The solu- 
tion is strained through muslin and heated in a water-bath at 60 for 
20 minutes, after which it is once more strained through muslin. A 
homogeneous suspension is thus obtained. Before use it is diluted 
with 9 volumes of water. 

5 c.c. of the above substrate are measured out into each of a series 
of five test tubes. To each is added 5 c.c. of 0-4 per cent, hydro- 
chloric acid solution and then in order, o - 2, 0*4, 0*6, 0*8, and I c.c. of 
pepsin solution. The tubes are placed in a water-bath at 40 for 15 
or 30 minutes. It is noted in which tube in the series the smallest 
amount of enzyme first produces complete clarification. 

(f) Gross' Method. 

Gross has suggested a solution of caseinogen instead of edestin for 
estimating peptic activity. It is prepared by dissolving I gm. of pure 
caseinogen in 16 c.c. of 25 per cent, hydrochloric acid of sp. gr. 1*124 
in a 1000 c.c. flask on a water-bath and diluting to 1000 c.c. It is pre- 
cipitated by a 20 per cent, solution of sodium acetate. 

A series of tubes are filled with varying quantities of the pepsin 
solution from T to I c.c. To each 10 c.c. of the caseinogen solution 
warmed to 40 are added. The series is placed in a bath at 38-40 for 
1 5 minutes. A few drops of the sodium acetate solution are added 
to each tube ; undigested caseinogen is precipitated. The smallest 
quantity required to digest the 10 c.c. is the value noted. 

If I c.c. of pepsin solution be the basis of the calculation and -025 
c.c. were sufficient, the pepsin solution corresponds to .^^ or 40 units. 

Other methods for estimating pepsin are those of Hammerschlag, who 
uses Esbach's reagent to precipitate unchanged protein ; of Volhard, who 
digests caseinogen and titrates the amount of hydrochloric acid used up in 
combination with caseoses ; of Jacoby, who digests the protein ricin in sus- 
pension in dilute hydrochloric acid in a similar way to Hata. 



424 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

B. Trypsin. 

(a) Metis Method. 

The activity of a trypsin solution can be measured by Mett's method in 
the same way as described for pepsin. The tubes may contain either egg- 
white or gelatin (p. 409). 5 c.c. of '4 per cent, sodium carbonate solution 
and 5 c.c. of the trypsin are placed in a small conical flask together with i or 
2 Mett's tubes, i c.c. of toluene, or chloroform, is also put in the flask as 
antiseptic. After periods of 2, 4, 8-24 hours the lengths digested from each 
end are measured. 

The Squares of the lengths digested more nearly represent the activity 
than the actual lengths which are measured. 

(b] Roberts Method. 

The action of trypsin upon milk serves as a very convenient and 
simple property for determining the activity of the enzyme. Roberts 
in 1 88 1 discovered that trypsin acting upon milk produced a striking 
change in its properties at an early stage of its digestion, namely, 
coagulation on heating due to the formation of casein (metacasein as 
it was termed). This property disappears at the end of the digestion. 

30 c.c. of milk are diluted with 30 c.c. of water in a small flask 
and warmed to 40; 1-5 c.c. of the trypsin solution are added. The 
time is noticed and at the end of every minute a portion of 5 c.c. is 
withdrawn and heated to boiling. It is noticed at what time a 
distinct curdling or precipitation occurs. A sample on heating shows 
incipient curdling before the actual appearance of the curdling ; this 
indicates that the next sample will probably coagulate properly. 

The activities of trypsin solutions are proportional to the time 
taken to produce curdling. 

Vernon l considers this method very exact if the values are cor- 
rected by reference to a table. 

(<T) Fuld-Gross Method. 

Another convenient method of measuring the activity of trypsin solutions is 
that of Fuld-Gross. A casemogen solution is digested with trypsin ; acidulated 
alcohol is added to precipitate unchanged caseinogen after a certain time. 

The substrate is prepared by dissolving 'i gm. of pure caseinogen in 5 c.c. 
of *iN sodium hydroxide + 25 c.c. of water and heating to boiling. The 
solution is cooled ; about 4^5 c.c. of - iN HC1 are added to neutralise the excess 
of alkali and the volume is made up to 100 c.c. It keeps for 48 hours; 
it is not advisable to add toluene which tends to precipitate the caseinogen. 

The acid solution is prepared by mixing together i part of glacial acetic 
acid, 49 parts of water and 50 parts of 96 per cent, alcohol. 

Ten test tubes are filled in series with increasing amounts of trypsin solu- 
tion -oi to i c.c. Water is added to make the volumes, where necessary, 
equal to i c.c. 2 c.c. of the caseinogen solution are added to each tube and 
they are placed for i hour in a bath at 37. They are removed and 6 drops 
of the acid alcohol added to each. In those tubes containing undigested 
caseinogen there is a flocculent precipitate. The first tube containing no 
precipitate in which there is the smallest amount of enzyme is noticed. The 
result is calculated according to the number of c.c. of caseinogen solution 
which can be digested by i c.c. of enzyme solution : thus supposing it were '2. 
02 : 2 = i : x x = 100 

The trypsin solution is spoken of as containing 10 units. 

1 J. Physiol., 27, 182. 



ENZYMES. FERMENTATION 425 

(d) Sbrensens Method. 

The most accurate and simple method is that of Sorensen. In 
this method the rate of formation of the products of the action of the 
enzyme, namely, the amino acids, is measured. The amino acids 
contain both a carboxyl group and an amino group and consequently 
their reaction is neutral. By combining the amino group with formalde- 
hyde, its basic character is destroyed and the carboxyl group is free 
to exert its acid character. The reaction which takes place is : 

R. CH.NH 2 + OHCH R . CH . N : CH 2 + H 2 O 
COOH COOH. 

Samples of a trypsin digest of caseinogen, gelatin, etc., are treated 
at intervals with neutral formaldehyde. They show a gradual in- 
crease in acidity as the action of the enzyme proceeds ; the rate of the 
increase depends on the strength of the enzyme. 

60 c.c. of formalin are diluted with two volumes of water and 
neutralised by running in 'iN alkali from a burette until the colour is 
just red to phenolphthalein, which is added as indicator. 

100 c.c. of a 4 per cent, caseinogen solution in O'4 per cent, 
sodium carbonate solution are measured out into a smail flask, warmed 
to 40 and then 5 c.c. or more of the trypsin solution are added ; the 
mixture is kept at 40. Immediately after the addition a sample of 
25 c.c. v is removed with a pipette and 30 c.c. of the previously 
neutralised formaldehyde solution are added. At intervals of half an 
hour, one hour, one hour and a half, two hours, further samples of 25 
c.c. are removed and to them are added 30 c.c. of the formaldehyde 
solution and a few drops of phenolphthalein. Each sample as it is 
obtained is titrated with the - iN alkali in the burette until the solution 
has a distinctly red colour. The amount of alkali used for each 
sample is noted. 

(e) Method by Estimating the Nitrogen of the Amino Acids. 
Proteins are precipitated by the various alkaloidal reagents. These 

reagents are of use in measuring the action of trypsin. Most frequently 
tannic acid is used, but trichloracetic acid and phosphotungstic acid are also 
employed. 

100 c.c. of a solution of protein, as in Sorensen's method, are mixed 
with a known amount of trypsin solution and digested for 2-48 hours in the 
presence of toluene. Immediately after mixing and at intervals of 15, 30, 
or 60 minutes a saniple of 10 c.c. is removed and put into 10 c.c. of tannic 
acid or trichloracetic acid solution. When the precipitate has settled the 
solution is filtered through a dry paper into a dry vessel. A nitrogen 
estimation by Kjeldahl's method is made with 5 c.c. or any aliquot portion 
of the filtrate. 

(/) Physical Methods. 

Several physical methods can be used to estimate trypsin, but they are 
adapted mainly for the study of the catalytic action. The change in electri- 
cal conductivity, in viscosity, in rotation has been frequently observed in 
experiments with trypsin. 



426 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Oxidases. 

A. Catalase. 

The measurement of catalase is effected by estimating volumetrically the 
amount of oxygen produced. 

B. Peroxidases. 

The estimation of the activity of a peroxidase is most easily effected 
colorimetrically ; the colour produced may be estimated with a spectrophoto- 
meter. 

If an insoluble precipitate is produced it can be filtered off and weighed, 
or it can be centrifuged and its bulk measured in the centrifuge tube. 

The methods are so various and depend so much upon the particular 
reaction suitable to the peroxidase that they must be referred to in the larger 
text-books. 

The indophenol reaction is very convenient as shown by Vernon. 1 

The indophenol which is insoluble and is deposited on the tissue is 
dissolved by adding a known volume of alcohol (10 c.c.); after 25 minutes 
the alcohol solution is filtered and compared colorimetrically with a standard 
prepared by adding i part of the substrate to 200 parts of 50 per cent, 
alcohol and sufficient bleaching powder (i part of -5 per cent, solution) to 
give a maximum tint on standing (1-2 days). The colour of the standard 
keeps for some weeks and then fades. The standard is kept in a sealed 
tube, a known volume of the indophenol is put into a similar test tube and 
diluted till the colours match. The colour varies from purplish pink to 
violet, depending on the amount of alcohol present, and can be made to 
match by adding water or alcohol. The colour is white with alcohol and 
pink with water. 

1 J. Physiol., 42, 402. See p. 412. 



APPENDIX TO DIGESTION. 

I. THE ACIDS IN THE GASTRIC CONTENTS. 

Normally hydrochloric acid to the extent of about -4 per cent, is 
secreted by the gastric mucous membrane, but in . disease it may be 
absent and lactic acid may be found. 

Detection of the Acids. 

The presence of an acid in a solution is shown by the colour change 
it produces in an organic dye-stuff or indicator. In pure aqueous solu- 
tions mineral acids give a distinct colour change, organic acids give 
a less distinct colour change. The colour change is masked in the 
presence of proteoses and peptones which are present in the gastric 
contents, owing to the combination of the acid with the protein in the 
form of a salt, so that it is difficult to decide whether hydrochloric 
acid, free or combined, is present or absent. A decision may be 
arrived at by making use of a series of indicators : 

(a) methyl violet ; 

(&) methyl orange, or dimethylaminoazobenzene (Topfer's reagent) ; 

(c) Congo red ; 

(<a?) Uffelmann's reagent 2 per cent, phenol treated with dilute 
ferric chloride till of an amethyst-violet colour (it is used especially for 
lactic acid, p. 112); 

(Y) Gunzberg's reagent 2 gm. phloroglucinol, I grn. vanillin, 30 
gm. absolute alcohol (it is used especially for hydrochloric acid). 

The test with Gunzberg's reagent is carried out thus: 

About 10 drops of the solution are placed in a small basin, 2-3 
drops of freshly prepared Gunzberg's reagent are added, and they are 
evaporated very carefully over a small flame, oscillating the basin and 
blowing upon the mixture. Charring must be prevented. 

The differences in the colours of the indicators under the various 
conditions may be seen by carrying out the following six experiments 
with 1-2 drops of each of the indicators : 

(1)2 c.c. of 0-4 per cent, hydrochloric acid (the strength occur- 
ring in gastric juice). 

(2) 2 c.c. of dilute lactic acid (8 c.c. in IOOO c.c. water). 

(3) 2 c.c. of O'4 per cent, hydrochloric acid and 2 c.c. of dilute 
lactic acid. 

(4) 2 c.c. of 0-4 per cent, hydrochloric acid and 2 c.c. of 2 per 
cent. Witte's peptone solution in 5 per cent, sodium chloride solution. 
(This is to simulate the products of gastric digestion.) 

(5) 2 c.c. of dilute lactic acid and 2 c.c. of 2 per cent. Witte's 
peptone solution in 5 per cent, sodium -chloride solution. 

427 



428 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



(6) 2 c.c. of 0*4 per cent, hydrochloric acid, 2 c.c. of dilute lactic 
acid and 2 c.c. of Witte's peptone solution. 

A survey of the results will be given if the colour be written in the 
table : 





Methyl- 
violet. 


Methyl, 
orange. 


Congo red. 


Uffelmann's 
reagent. 


Gunzberg's 
reagent. 


HC1 - 












Lactic acid -"-'-- 












HC1 + lactic acid 












HC1 + peptone 












Lactic acid + peptone - 












HC1 + lactic acid + peptone 













In performing experiments with gastric contents it may be advis- 
able to reproduce some of these experiments and compare the colour 
with these. 

Estimation of the Acids. 

There is no very satisfactory way of estimating the amount of free 
hydrochloric acid, combined hydrochloric acid and organic acid in 
gastric contents. Topfer's method of titrating a known volume with 
(i) phenolphthalein, (2) dimethylaminoazobenzene to an orange yellow, 
not yellow tint, (3) alizarin red, gives the following information: 

(1) total acids, i.e. free mineral acid + combined mineral acid + 
organic acid. 

(2) free mineral acid. 

(3) free mineral acid + organic acid. 
(i) minus (3) combined mineral acid. 
(3) minus (2) organic acid. 

The data are not absolutely correct, but they serve well for com- 
parative determinations. If the above six mixtures of acids + pep- 
tone be titrated figures such as the following are obtained : 





HC1 

2 C.C. 


Lactic 
acid 

2 C.C. 


HC1 

2 C.C. + 

Lactic acid 

2 C.C. 


Peptone 

5c.c. 


Peptone 
5 c.c. + 

2 C.C. 

HC1. 


Peptone 
5 c.c. 
+ 2 c.c. 
Lactic acid. 


Peptone 
5C.C. 
+ 2 c.c. HC1 

+ 2 C.C, 

Lactic acid. 


Phth 


1-7 


17 


3H 


0'3 


2'O 


2-0 


3-8 


Topfer 


1-6 


I'5 


3'I 


o 


0-8 





2'2 


Alizarin red 


17 


17 


3 '4 


o 


i'3 


I'3 


2-8 



THE CONSTITUENTS OF BILE 429 

i 

II. THE CONSTITUENTS OF BILE. 

The constituents of the bile are : 

(1) The colouring matters, bilirubin and biliverdin, the latter formed 
by the oxidation of the former. 

(2) The bile salts, the sodium salts of glycocholic and taurocholic 
acids. 

(3) A small quantity of mucin or nucleoprotein (the more recent work 
insists on the presence of the latter, but both are probably present). 

(4) Cholesterol, which gives rise to gall-stones in certain conditions. 

Examination of Ox or Sheep Bile. 

(1) It has a faintly alkaline reaction to litmus ; the bitter taste 
and peculiar odour should be noticed. 

(2) It does not coagulate on heating. 

(3) On acidifying a small quantity with acetic acid a precipitate is 
formed which is insoluble in excess of acetic acid. As above stated, 
this precipitate was considered to be mucin owing to its insolubility in 
excess of acid, nucleoprotein being soluble ; in the presence of bile 
salts the precipitate of nucleoprotein is insoluble. 

(3*2) No pigment is extracted on shaking up a little bile with ether. 
If a few drops of dilute hydrochloric acid be added, both nucleoprotein 
and pigment are liberated as free acids from their sodium compounds. 
The nucleoprotein is precipitated, but the pigment passes into solution 
on shaking with the ether. 

(4) Gmelin's Test for Bile Pigments. A little bile is carefully 
placed on the surface of some fuming nitric acid in a test tube, either 
by pouring it carefully down the side of the tube or by means of a 
pipette. On shaking the tube very gently a play of colours will be 
seen as the bile becomes oxidised. Generally the colours are yellow, 
red, violet, blue, green ; 

or, 

A drop of fuming nitric acid is placed on a thin film of bile in a 
porcelain basin. Rings of the various colours will be seen ; 

or, 

A little bile is filtered several times through an ordinary filter paper 
and a drop of fuming nitric acid is placed on the paper. The colours will 
be seen. 

(5) Huppert's Test for Bile Pigments. This test is especially 
useful for detecting bile pigments in urine. 

5 c.c. of bile are diluted with 25-50 c.c. of water and 4 c.c. 
of sodium phosphate solution and 6 c.c. of calcium chloride solution 
are added. The precipitate is filtered off. It carries down the pigment 
mechanically or may contain an insoluble calcium compound of bili- 
rubin ; it is heated with 5 c.c. of alcohol and a few drops of concentrated 
hydrochloric acid. A fine green colour is formed. The formation of 
the green colour may require the addition of an oxidising agent such 
as a few drops of ferric chloride or potassium chlorate solution (Cole). 



430 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

(6) Pettenkofer's Test for Bile Salts. A fragment of cane sugar 
is dissolved in a little bile which has been diluted 10 times with 
water ; when it has dissolved, about 5 c.c. of concentrated sulphuric 
acid are run into the bottom of the test tube and shaken gently. A 
purple colour develops slowly. Furfural is formed by the action of 
the concentrated sulphuric acid on the sugar ; this reacts with the 
bile acids, giving the purple colour. Excess of sugar must be avoided 
as it may be charred by the strong acid and spoil the colour. The 
colour disappears on diluting with water and is only stable in the 
presence of strong sulphuric acid. If a portion of the purple liquid be 
diluted with 50 per cent, sulphuric acid and examined in the spectro- 
scope, two absorption bands, the one between C and D, nearer D, and 
the other in the green, can be observed. 

This test is sometimes performed by shaking up the bile with a 
little sugar solution so as to obtain a froth ; on pouring in the 
concentrated sulphuric acid the colour appears where it has come in 
contact with the froth. 

(7) Hay's Test for Bile Salts (Surface Tension Test). A little 
bile in a test tube is diluted with water and some flowers of sulphur 
are sprinkled on the surface. They sink. If the experiment be repeated 
with pure water, the particles of sulphur will float. 

On performing the same test with strong mineral acids, ammonia, 
brine, ammonium sulphate solution, etc., the sulphur floats. It sinks 
in alcohol, ether, chloroform, olive oil, etc., in fact in all liquids with a 
surface tension less than 60 dynes per sq. cm. 

This test depends upon the power of the bile salts to lower the 
surface tension of water. It is particularly valuable for detecting bile 
salts in urine where other coloured substances may interfere with 
Pettenkofer's test. Alcohol, which has a low surface tension, must, if 
present, be previously removed by evaporation. 

Grunbaum has described a method of estimating bile salts in urine which 
depends on this property of bile salts. The rate of escape of urine from 
standard capillary tubes is measured ; the higher the concentration the greater 
is the rate. 

Draughtsmen employ this property of bile salts in making tracings 
on oiled paper on which the ink collects in drops and does not spread. 
On treating the paper with ox bile and allowing it to dry the difficulty 
is overcome owing to the reduction in surface tension. This experi- 
ment with oiled paper treated with bile may be tried with advantage. 

In the same way oil will pass through a filter paper moistened with 
'dilute bile solution, whereas it will not pass through a paper moistened 
with water. This statement may easily be verified. 



THE CONSTITUENTS OF BILE 431 

(8) Solvent Action of Bile Salts on Fatty Acids containing 
Oleic Acid. If some fatty acids from mutton or beef fat be stirred with 
water, they do not dissolve ; on adding a little bile and stirring up again, 
fatty acids can be detected in the filtrate by evaporating to dryness, heating 
and noting the characteristic odour. This process no doubt occurs in the 
digestion and absorption of fats. 

(9) Oliver's Test for Bile Salts. This depends upon the power of 
the bile acids to precipitate peptone in acid solution and is useful for showing 
the presence of bile salts in the urine, e.g. 

About 20 c.c. of bile are evaporated to complete dryness on the water- 
bath. The residue is heated with 20 c.c. of alcohol on the water-bath stirring 
the mixture thoroughly with a glass rod. A little more alcohol is added and 
it is filtered. The filtrate is evaporated to dryness on the water- bath and the 
residue extracted with about 30 c.c. of hot water. A solution of the pigments 
and the salts of bile, free from proteins, is obtained on filtering. 

If a portion of this solution be acidified with glacial acetic acid, the bile 
acids are not thrown down, but on adding an equal quantity of i per cent. 
Witte's peptone solution a turbidity or a precipitate is obtained, insoluble in 
excess of acid. 

As applied to urine, it is only necessary to acidify with acetic acid, 
filter till quite clear and treat with an equal volume of i per cent. Witte's 
peptone solution. 

(10) Cholesterol may be detected as follows (Roaf) : 

10 c.c. of bile are evaporated to dryness on the water-bath. The 
residue is extracted several times with small quantities of ether, pour- 
ing each ether extract into another evaporating basin. The ether is 
allowed to evaporate and the residue is dissolved in about 2 c.c. of 
chloroform. It gives Salkowski's and Liebermann's reactions. 

HI. GALL STONES. 

Calculi of various sizes and shapes and of variable number occur in the 
gall bladder. Three kinds have been distinguished : 

(1) Pigmented Chalk Stones. 

In man these stones are small ; in the ox and pig stones as large as a 
walnut have been found. They are heavier than water. They consist almost 
entirely of the calcium salt of bilirubin and contain very little or no biliverdin. 
Sometimes black or greenish-black metallic-looking stones, which ' consist 
of bilifuscin and biliverdin, occur. Iron and copper are generally present. 

(2) Cholesterol Stones. 

The shape and size of the cholesterol stones are very variable ; they are 
generally lighter than water and are composed of concentric layers. Their 
surface if fractured appears crystalline ; if cut, waxy. If the fractured surface be 
rubbed with the nail it also looks waxy. By rubbing against one another in 
the gall bladder they are generally faceted. They are almost white and usually 
show pigmented edges (pigmented chalk). 

(3) Calcium Carbonate and Phosphate. 

These stones are very rare in man. 



THE INDIVIDUAL GROUPS OF PROTEINS. 

PROTAMINES. 

The protamines occur in ripe fish sperm in which they are present as 
salts of nucleic acid. Salmine, the first known member of the group, was 
discovered by Miescher in salmon sperm. The other members have been 
isolated by Kossel and his pupils from the sperm of other fishes. They are 
named according to the fish from which they are obtained, e.g. sturine from 
sturgeon, clupeine from herring, scombrine from mackerel, cyprinine from 
carp. Our knowledge of these proteins is almost entirely due to Kossel and his 
pupils, who have shown that they are composed principally of diamino acids, 
especially arginine, which in some cases makes up over 80 per cent, of the 
molecule. It seems that they are the diamino acid constituents of muscle 
protein since the testicles grow at the expense of the muscles in the spawning 
season, the fish taking no food and living upon the mono-amino acid 
portion. 

Preparation. 

The ripe or nearly ripe testicles are smashed up, shaken continuously 
with water and the liquid strained through a cloth. The milky fluid, which 
contains the spermatozoa, is acidified with a few drops of acetic acid which 
causes them to clot together. The clotted mass is filtered off, boiled 
several times with alcohol and then with ether to remove fats and dried in 
the air. The dry matter (about 100 gm. portions) is shaken for 15 minutes with 
5 times its volume of i per cent, sulphuric acid, filtered off and the extraction 
with acid repeated several times. The combined extracts are treated with 
3 volumes of alcohol. Protamine sulphate is precipitated, filtered off after 
12-24 hours, redissolved in water and precipitated with alcohol. It is dis- 
solved in about 1500 c.c. of hot water ; on cooling, protamine sulphate separates 
as a yellowish or brownish oil. The solution is concentrated and allowed to 
stand in a separating funnel. A further quantity of oil, but contaminated with 
nucleic acid, collects. It is dissolved in warm water and treated with sodium 
picrate. The precipitate of protamine picrate is filtered off, washed and re- 
converted into sulphate by extraction with ether in presence of an excess of 
sulphuric acid. The aqueous acid solution is precipitated with alcohol ; 
solution in water and precipitation with alcohol is repeated. Solution and 
precipitation must be repeated until the protamine sulphate forms a loose, white 
precipitate and is not sticky. 



43? 



THE INDIVIDUAL GROUPS OF PROTEINS 433 

Properties. 

The protamines in the free state have not been much investigated ; 
they are strong bases, blueing litmus, and absorb carbon dioxide from the 
air. They are easily soluble in water, but insoluble in alcohol and ether. 
They are not coagulated by heating, do not diffuse, give the biuret reaction, 
sometimes other colour reactions and are laevorotatory. 

They form salts with acids of which the sulphate is the principal. The 
chlorides are more easily soluble, as also the carbonates and nitrates. They 
form insoluble double salts with platinum chloride and mercuric chloride. 

They dissolve copper hydroxide giving solutions of a violet colour. 

They are precipitated by alkaloidal reagents in neutral or faintly alkaline 
solution and are precipitated from solution by salts. They give precipi- 
tates with other proteins, excepting the secondary proteoses, peptones and 
polypeptides. 

They are precipitated from solution by a solution of sodium nucleate as 
protamine nucleate. 

They contain 25-30 per cent, of nitrogen in their molecule, no sulphur 
and no phosphorus. 



HISTONES. 

The group of proteins termed histones was established by Kossel, who 
isolated a histone from the red blood corpuscles of the goose. Other members 
of the group have since been isolated from the unripe testicles of fish and 
from the thymus. During maturing of the sperm the histone in some cases 
remains unchanged, but in other cases changes into protamine. 

Preparation. 

(1) From Unripe Fish Sperm. 

The material is first treated as in the preparation of protamines, but it is 
extracted with dilute hydrochloric acid. The extract is saturated with sodium 
chloride, the precipitate is freed from salt by dialysis and the solution preci- 
pitated by ammonia. 

(2) From Red Blood Corpuscles. 

The paste of corpuscles is treated with water and ether ; the insoluble 
residue after washing with water is extracted with dilute hydrochloric acid. The 
solution is saturated with sodium chloride and the precipitate is freed from salt 
by dialysis. The histone is then precipitated by adding ammonia. 

(3) From Thymus Nucleohistone (p. 466). 

The nucleohistone is treated with 0-8 per cent, hydrochloric acid and the 
solution is precipitated with ammonia. 

Thymus histone is also formed when solutions of nucleohistone are saturated 
with salt. The histone is precipitated on adding ammonia. 



28 



434 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Properties. 

The various histones vary considerably in properties. They contain about 
18-19 P er cent, of nitrogen ; some contain sulphur, others do not. 

They are basic and are intermediate between albumins and globulins and 
the protamines, yielding on hydrolysis a larger proportion of arginine than 
albumins and globulins, but less than protamines. 

Their properties resemble in part the coagulable proteins, in part the pro- 
tamines and in part the proteoses. 

A neutral solution, free from salt, is precipitated by ammonia, but accord- 
ing to Bang the histone from red blood corpuscles is soluble in excess, but 
comes down on saturating the solution with ammonium sulphate. Histones are 
also precipitated by caustic alkalies and alkaline earths. Towards nitric acid 
they behave like the proteoses. They are precipitated by the alkaloidal re- 
agents also in neutral solution. Like the protamines they are precipitated 
by albumin and primary proteoses* They are not coagulated by heat, but 
coagulation occurs in the presence of salt. The coagulum dissolves in dilute 
hydrochloric acid. 

Globin. 

Globin, the protein moiety of the conjugated protein, haemoglobin (p. 
4.72), has been considered to be a histone, though in its properties it has many 
points of difference. 

Preparation. 

A solution of haemoglobin is dialysed to remove salts and treated with 
very dilute hydrochloric acid 20 c.c. of *iN HC1 to 200 c.c. of haemo- 
globin solution containing 1-84 gm. (Gamgee and Hill) until a flocculent 
brown precipitate which forms is just redissolved. One fifth of the volume 
of 80 per cent, alcohol is added and the solution shaken several times with 
half its volume of ether. The clear aqueous solution on neutralisation gives 
a flocculent precipitate. It is rapidly filtered off, washed with water, dis- 
solved in dilute acetic acid and dialysed. The globin is precipitated on 
adding alcohol. 

Properties. 

Globin dissolves in water and it differs from histones in that the neutral 
solution, in the absence of salts, gives a precipitate which is readily soluble in 
excess of ammonia, and that ammonium chloride only precipitates it when a 
large excess of ammonia is not present. 

Globin gives most of the colour reactions of proteins ; it is not precipitated 
by most of the heavy metals. 

It contains about 17 per cent, of nitrogen of which 29 per cent, is in the 
form of diamino acids. In this respect it resembles the histones, but the 
chief diamino acid is histidine, not arginine. 



THE INDIVIDUAL GROUPS OF PROTEINS 435 

COAGULABLE PROTEINS. ALBUMINS. GLOBULINS. 

These proteins generally occur together in most tissues and 
fluids of animals, e.g. in egg-white, blood and muscular tissue. They 
are also present in various parts of plants, especially in the fruits and 
seeds. 

The coagulable proteins are the most typical proteins and are often 
called native proteins. They have the common property of being 
changed into insoluble modifications when their solutions are heated to 
boiling in the presence of a little acetic acid. The insoluble form is 
also present in the various animal tissues such as muscle. 

The chief distinction between albumins and globulins is their 
behaviour towards water and solutions of salts. They can be separ- 
ated from solution in an unchanged condition in this way. A large 
number of salts have been used for this purpose, the principal ones 
being sodium chloride, magnesium sulphate and ammonium sulphate. 

Albumins are soluble in water and in dilute salt solutions. 
Albumins are not precipitated by saturating their aqueous solutions 
with sodium chloride or magnesium sulphate nor by half-saturation 
with ammonium sulphate (i.e. adding an equal volume of saturated 
ammonium sulphate), but they are precipitated by saturation of the 
solution with ammonium sulphate. 

Globulins are insoluble in water, but soluble in dilute salt solu- 
tions. Globulins are precipitated from dilute salt solutions by 
saturation with sodium chloride or magnesium sulphate, or by half- 
saturation with ammonium sulphate. 1 

There are, however, several globulins which behave slightly dif- 
ferently by being soluble in water, or by being precipitated with less 
salt than is required for complete saturation. These are atypical 
globulins. 

1 The amount of salt required to saturate an aqueous solution is 
3'6 gm. of sodium chloride lor every 10 c.c. 
10-2 gm. of cryst. magnesium sulphate for every 10 c.c. 
4'o gm. of ammonium sulphate for every 10 c.c of half-saturated solution. 
It is better to weigh out the requisite amount of salt than to add it until no more 
dissolves, as an excess is in this way avoided and does not interfere with further operations. 



28 



436 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

THE COAGULABLE PROTEINS OF EGG-WHITE. 

White of egg is made up of a pale yellow fluid contained in a net- 
work of a fibrinous material, which is broken up by beating the egg 
and the egg-white obtained by straining it through calico. 

Egg-white has a faintly alkaline reaction to litmus and a specific 
gravity of 1*045. It contains 13-3 per cent of solid matter, 85-88 
per cent, being water. Of the solid matter 12*2 per cent, is protein ; 
the remainder is glucose '5 per cent, ash '66 per cent, and traces of 
soaps, fat and cholesterol. Of the protein 67 per cent, is globulin, 
10 per cent is ovomucoid or ovomucin, a glycoprotein (p. 471). 

Globulin (Ovoglobulin). 

Preparation. 

The globulin is precipitated from egg-white or a solution of egg- 
white by saturation with sodium chloride or magnesium sulphate, or 
by half-saturation with ammonium sulphate. 

An equal volume of saturated ammonium sulphate solution is 
added slowly to egg-white, or a solution of egg-white in water, with 
constant stirring. After standing the precipitate is filtered off, dis- 
solved in water 1 and precipitated again with ammonium sulphate. 

This process is repeated several times. The final solution is dialysed to 
remove salt and the protein can be obtained by evaporation of the solution at 
a low temperature. 

In a coagulated state it may be separated by acidifying and boil- 
ing (p. 368), washing the coagulum with water, alcohol and ether, and 
drying in the air, or by precipitating with alcohol, drying with alcohol, 
washing with ether and exposing to the air. 

Properties. 

In the uncoagulated state, globulin is an amorphous yellow mass, 
insoluble in water but soluble in dilute salt solutions ; it is precipit- 
ated from solution by saturation with sodium chloride, magnesium 
sulphate, or by half-saturation with ammonium sulphate. 

The solution in salt solutions shows all the general reactions for 
proteins. , . 

In the coagulated state, globulin forms an amorphous white powder 
which is insoluble in water and dilute salt solutions. It dissolves 
slowly on warming in dilute acids and alkalies, undergoing hydrolysis 
into derivatives (metaprotein). 

A suspension in water will show most of the colour reactions for 
proteins. 

This substance has been termed ovomucin by Osborne and 
Campbell. It is not certain if it is a single protein. 

1 Sufficient salt is still present to make the solution a dilute salt solution. 



THE INDIVIDUAL GROUPS OF PROTEINS 437 

Albumin (Ovalbumin). 
Preparation. 

Ovalbumin remains in solution after the globulin has been precipi- 
tated by half-saturation with ammonium sulphate. 

The filtrate is saturated with finely powdered crystals of ammonium sul- 
phate. The precipitate is dissolved in water and again precipitated with 
ammonium sulphate and the process is repeated several times. The last pre- 
cipitate is dissolved in water and the solution is dialysed to remove the salt. 
The albumin is obtained on evaporation in vacua at 40. 

Coagulated albumin is obtained by acidifying the solution and boil- 
ing, washing the coagulum with water, alcohol and ether, and drying, 
or by pouring it into several volumes of alcohol. The coagulum is 
washed by decantation with alcohol and ether and dried. 

Preparation of Crystalline Ovalbumin (Hopkins}. 

Fresh egg-white is beaten into a froth with an exactly equal volume of 
saturated ammonium sulphate solution and the mixture is filtered after stand- 
ing for some hours. 10 per cent, acetic acid is added to the filtrate from a 
burette with constant stirring until it becomes distinctly turbid, i c.c. of 
acetic acid is then added for every 100 c.c. of filtrate. An amorphous pre- 
cipitate is first formed, but on standing it becomes crystalline and with frequent 
shaking the whole of the Ovalbumin crystallises in 5-6 hours. After 24 hours 
it is filtered off, washed with ammonium sulphate containing - i per cent, of 
acetic acid and dissolved in water (i part in 10). Saturated ammonium sul- 
phate solution is carefully added with gentle snaking until a permanent pre- 
cipitate results, and then for every litre 2 c.c. more of ammonium sulphate. 
The ovalbumin crystallises out and is washed as before. The crystalline mass 
contains ammonium sulphate. A solution free from ammonium sulphate is 
obtained by dialysis, or as above either in the uncoagulated or coagulated 
condition. The yield is 50 gm. from 1000 c.c. of egg-white. 

Preparation of Conalbumin. 

The whole of the ovalbumin can never be obtained in a crystalline 
condition. According to Osborne and Campbell only 50 per cent, of the 
albumin can be crystallised. The remainder is termed conalbumin. It is 
prepared from the filtrate by saturation with ammonium sulphate as described 
under albumin. 

Properties. 

Ovalbumin in an uncoagulated state is an amorphous mass of a 
yellowish colour, soluble in water and dilute salt solutions. It is not 
precipitated from solution by saturation with sodium chloride, magnes- 
ium sulphate or half-saturation with ammonium sulphate, but is pre- 
cipitated by complete saturation with ammonium sulphate. 

A 2*5 per cent, solution in water coagulates at 60-64 ; m IO P er 
cent, salt solution at 68-70. 

Conalbumin is very similar to ovalbumin, but it coagulates at a 
lower temperature and has a higher rotation. 

The solutions of ovalbumin, crystalline ovalbumin and conalbumin 
give all -the general reactions for proteins. 

The coagulated albumins are insoluble in water and salt solutions, 
but dissolve slowly in acid and alkali yielding solutions of derivatives 
(metaprotein). 



438 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

THE COAGULABLE PROTEINS OF BLOOD. 

Blood is a fluid which contains proteins, salts, glucose, amino acids 
and other simple compounds in solution, and red and white blood cor- 
puscles in suspension. The presence of blood platelets in living blood, 
i.e. in the blood vessels, is denied by some observers, but they are un- 
doubtedly present when the blood is shed, their formation being in- 
stantaneous. 

I. Clotting of Blood. 

Blood drawn from a blood vessel clots spontaneously into a 
solid mass or clot. On standing the clot slowly contracts, express- 
ing the almost colourless blood-serum. The clot consists of the insol- 
uble protein, fibrin, which has entangled the blood corpuscles. The 
fibrin, almost free from corpuscles, can be obtained by whipping the 
blood whilst it clots and washing the fibrin threads with water to 
remove the entangled corpuscles ; defibrinated blood remains, which 
contains the corpuscles. Thus 

, Fibrinogen + Corpuscles Whipped f Fibrin 

u. A I - Blood J 

j Defibrinated Blood 
^ Serum I (Serum + Corpuscles) 

This may be readily observed as follows : 

A little freshly drawn blood is collected in two watch glasses. The 
one is allowed to clot ; after a time the clot contracts, expressing the 
serum. The other is defibrinated by stirring it with a pin or needle. 
The fibrin adheres to the needle and defibrinated blood remains. The 
fibrin can be washed free from corpuscles by water and an almost 
colourless mass remains. 

II. Factors concerned in Clotting of Blood. 

In the process of clotting the insoluble protein fibrin is formed. 

It arises from fibrinogen, a soluble protein contained in the blood 
plasma, by the action of fibrin 'ferment or thrombin. 

Thrombin again does not exist .'normally in the blood, but is 
formed from precursors after the blood is shed in the presence of 
calcium salts. According to Morawitz the precursors are thrombogen, 
which exists as such in the plasma, and thrombokinase, which is pro- 
duced by the corpuscles or platelets, or may come from other tissues. 
The evidence for the presence of thrombokinase was obtained from 
experiments with birds' blood ; if it be carefully collected and the cor- 
puscles separated without damage, it does not clot, but it clots on 
adding damaged corpuscles or a scraping of muscular tissue. These 
substances were previously described by Wooldridge under the names 
B fibrinogen (= thrombogen), A fibrinogen (= thrombokinase), and 
C fibrinogen (= fibrinogen). The older observers, Alex. Schmidt, 
Hammarsten, Arthus, only recognised the stage prothrombin which 
was converted into thrombin by calcium salts. 



THE INDIVIDUAL GROUPS OF PROTEINS 439 

Thrombin has been considered to be a ferment or enzyme, but 
the observations of Howell and Rettger show that a solution of 
thrombin, if obtained almost free from protein, is stable to heat. 
Enzymes are generally recognised to be more or less easily destroyed 
by heating. 

The following scheme represents the processes which occur in the 
formation of fibrin : 

, . ( Thrombogen "| 

Prothrombm. , >, 

( Thrombokmase 1 

" I Fibrin + (Soluble globulin ?) 
Calcium salt ) 

FibrinogenJ 

For the elucidation of the above factors in the scheme of blood clot- 
ting it was necessary to prevent the blood from clotting, to prepare 
fibrinogen in a pure state from non-coagulated blood, to prepare a 
solution of fibrin ferment or thrombin and to determine the factors 
leading to the formation of thrombin. 

III. Prevention of Clotting of Blood. 

Blood may be prevented from clotting by collecting it in various 
salt solutions when it is drawn, e.g. sodium sulphate, magnesium 
sulphate, potassium oxalate, sodium fluoride, sodium citrate. Salt 
plasmas are thus obtained. 

Clotting may also be hindered or prevented by keeping the drawn 
blood at a low temperature (o), or by adding leech extract to it im- 
mediately after it is drawn. 

If peptone be injected into the circulation, or if leech extract or the 
active principle of leech extract, termed hirudin, be injected, the blood 
when drawn does not coagulate. 

Preparation of Salt Plasmas. 

(a) Sodium sulphate. i part of blood is collected in i part of saturated 
sodium sulphate solution (500 c.c. to 500 c.c.). 

(b) Magnesium sulphate. 3 parts of blood are collected in i part of 
saturated magnesium sulphate solution (750 c.c. to 250 c.c.). 

(c) Oxalate. 9 parts of blood are collected in i part of potassium 
oxalate solution (i per cent.) (900 c.c. to 100 c.c.). 

(d) Fluoride. 9 parts of blood are collected in i part of sodium 
fluoride solution (3 per cent.) (900 c.c. to 100 c.c.). 

During the mixing each is well shaken. The plasma is then separated 
from the corpuscles by centrifugalising. All these plasmas should be quite 
free from blood corpuscles, as the stromata of these may serve as the mother 
substance of the ferment. It is difficult, however, to obtain them quite free 
from haemoglobin, which gives them a reddish colour. 

Fibrinogen. 

Fibrinogen can be prepared from the salt plasmas by precipitation with 
sodium chloride or ammonium sulphate. 

(a) From sodium sulphate plasma. 

A precipitate, the plasmine of Denis, is formed when sodium sulphate 
plasma is saturated with sodium chloride. This precipitate consists of 
fibrinogen and serum globulin, 



440 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

Fibrinogen is precipitated by adding an equal volume of saturated sodium 
chloride solution. The precipitate is filtered off, washed with half-saturated 
sodium chloride solution, dissolved in water and reprecipitated. 

(ft) From magnesium sulphate plasma. 

Fibrinogen is precipitated by adding an equal volume of saturated 
sodium chloride solution to magnesium sulphate plasma. The precipitate 
is filtered off and washed with half-saturated sodium chloride solution. It is 
purified by dissolving in water and reprecipitating with sodium sulphate. 

(c) From oxalate plasma. 

(i) The fibrinogen is precipitated by adding an equal volume of saturated 
sodium chloride solution (Ca-free). It is filtered off arid washed with half- 
saturated sodium chloride solution, redissolved and reprecipitated. 

(ii) The fibrinogen is precipitated by quarter-saturation with am- 
monium sulphate. Every 12 c.c. of oxalate plasma are diluted with 
30 c.c. of water and 20 c.c. of saturated ammonium sulphate are added. 
The precipitate is filtered off, washed with quarter-saturated ammonium 
sulphate, dissolved in water and reprecipitated. 

Fibrinogen gives the colour reactions, coagulation reactions and 
precipitation reactions of proteins. 

It is a globulin, being soluble in dilute salt solutions but insoluble 
in water. It is precipitated from solution by salts, but less than com- 
plete saturation (half-saturation) with sodium chloride and less than 
half-saturation (quarter-saturation) with ammonium sulphate throws it 
out of solution. It is, therefore, an atypical globulin. 

Its temperature of heat coagulation in a dilute salt solution is 56. 

It is converted into insoluble fibrin by thrombin : 

(a) Fibrinogen from Sodium Sulphate Plasma. 

The fibrinogen on solution in water, 1 if it has been well washed, will not 
clot if kept at 40 for 10-15 minutes, but it is converted into fibrin if a 
little serum or fibrin ferment solution be added. 

(b} Fibrinogen from Magnesium. Sulphate Plasma. 
A solution of the well- washed fibrinogen in water 1 does not clot on warming 
to 40, but it gives fibrin if a drop of serum or thrombin solution be added. 

(c) Fibrinogen from Oxalate Plasma. 

A solution of fibrinogen in water 1 may clot at 40 on adding calcium 
chloride as it will be contaminated with thrombokinase and thrombogen. 

It is converted into fibrin if a drop of serum or thrombin solution 
be added. 

Fibrin. 

Fibrin is not usually prepared from fibrinogen, but directly from 
blood. The blood, when drawn, is immediately whipped with a 
bundle of twigs. Threads of fibrin collect on the twigs ; they are re- 
moved, placed in a muslin bag and washed with running water. 
1 It dissolves in water as sufficient salt is still present with it. 



THE INDIVIDUAL GROUPS OF PROTEINS 441 

The freshly prepared threads of fibrin are nearly colourless ; on 
drying by exposure to the air they form a brownish mass. The fresh 
threads are best preserved in glycerin, which is easily removed by 
washing. 

Fibrin is insoluble in water, salt solutions, cold dilute solutions of 
acids and alkalies. It dissolves on warming in dilute acid or alkali, 
but undergoes hydrolysis into its derivatives. 

A suspension of fibrin in water will give the colour reactions for 
proteins. The solution in acid or alkali will behave like metaprotein, 
or proteoses and peptone, depending on the length of time the solu- 
tion has been heated. It will be precipitated by heavy metals and 
alkaloidal reagents. 

Thrombin. 

Thrombin is not present in blood plasma, but is formed in the process of 
clotting : it will therefore be present in the serum and upon the fibrin. 

(1) Preparation from Serum or Defibrinated Blood. 

i volume of serum or defibrinated blood is mixed with 15-20 volumes 
of alcohol and the mixture is allowed to stand for some weeks. The pre- 
cipitate, which is formed, is filtered off, washed with alcohol and dried in 
a desiccator. It contains thrombin, which is extracted by water (Schmidt). 

(2) Preparation from Fibrin. 

It is best to use fibrin which has been obtained from blood diluted with 
10 volumes of water and which has been washed with water. This fibrin is 
preserved in weak alcohol. A solution of thrombin is obtained by extracting 
the fibrin with 8 per cent, sodium chloride solution (Gamgee). 

IV. The Formation of Fibrin by Interaction of Calcium Salts, 
Thrombogen and Thrombokinase. 

The formation of fibrin is easily observed by the following experi- 
ments with the salt plasmas : 

(a) Sodium Sulphate Plasma. 

Clotting occurs on diluting a small quantity with 5 volumes of 
water and keeping the solution warm at 40. The clotting is more 
rapid if a drop of serum or thrombin solution be added. 

This plasma cdntains fibrinogen arid also thrombin. They have 
been prevented from interacting by the presence of neutral salts, hence 
on dilution the plasma clots. 

(b) Magnesium Sulphate Plasma. 

This plasma on dilution with 9 volumes of water and warming 
to 40 does not c[ot. 

It clots on diluting as above and adding a drop of serum or throm- 
bin solution. 

This plasma contains fibrinogen, but no thrombin. The formation 
of thrombin has been prevented by the presence of magnesium sul- 
phate. 



442 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

(f) Oxalate Plasma. 

On diluting with 5 volumes of water, no clotting occurs at 40. 
Clotting occurs on diluting and adding a few drops of calcium chloride 
and warming to 40. 

On diluting and adding a drop of serum, freed from calcium salts 
by precipitation witjh oxalate, and warming to 40, clotting occurs. 

This plasma contains fibrinogen, but the formation of thrombin 
has been prevented by the absence of calcium salts. On their addi- 
tion thrombin is formed and clotting takes place. 

Serum contains thrombin, and hence, when added free from cal- 
cium salts to plasma free from calcium salts, clotting occurs. Calcium 
salts are therefore necessary for the formation of thrombin. 

(d) Fluoride Plasma. 

On diluting with 2 volumes of water, it does not clot on warm- 
ing to 40. 

No clotting occurs on diluting and adding a few drops of calcium 
chloride and warming to 40. 

Clotting occurs on diluting and adding a drop of serum or throm- 
bin solution. 

Clotting occurs on diluting and adding a scraping of muscle or 
other tissue. 

These results are only obtained if the plasma has been thoroughly 
centrifugalised ; if improperly centrifugalised, corpuscles will be present 
which yield thrombokinase. 

The plasma contains fibrinogen and thrombogen, but no throm- 
bokinase. Thrombin cannot be formed in the absence of throm- 
bokinase when calcium salts are added. 

Composition of Blood. 

Blood plasma consists mainly of water ; the solid matter amounts 
to less than I o per cent. The proportions of the constituents have 
been frequently determined and some of the analyses for 1000 c.c. are 
given in the following tables : 

Chicken. Frog. 

,- N Animals. 

Hoppe Seyler. Hammarsten. 
Water 
Solids .... 91-6 82*4 % 701097 54 



Fibrin . 
Globulin 
Albumin 



Horse. 


Man and other 
Animals. 


Hoppe Seyler. 


Hammarsten. 




908-4 


917-6 




91-6 


82-4 


' 70 to 97 


lOOO'O 


1000-0 




f} IO*I 


6-5 







38-4 







24-6 




7T6 


69 '5 


55 to 84 


: '-ol 


} 




f.. f *3j 


1 - 12-9 




.' i- 7 J 


j 





Total Proteins . . 77-6 69-5 55 to 84 39-5 25-4 

Fat 

Extractives 
Soluble Salts . 
Insoluble Salts 



THE INDIVIDUAL GROUPS OF PROTEINS 443 

Total Protein. Fibrinogen. Globulin. Albumins 



Man 

Dog 

Sheep 

Horse 

Pig 



I 



72-6 4-2 28-3 40-1 

60-3 6-0 22-6 317 \ by 

72*9 4'6 30*0 38*3 j Lewinsky. 

80-4 4-5 47'? 28-0 I 



80-5 6-5 29-8 44-2 

The ratio globulin to albumin in man is usually i : 1-15 but i : 1-39 to 2-13 has been 
found. In starvation the globulin increases. 

Man Other Animals 

(by Schmidt). (by Bunge and Abderhalden). 
0-387 to 0-40 0-225 to 0-270 



Na,0 
CaO 
MgO 
Cl . 
P.O. 



4*290 4*251 to 4*442 

0-155 0-119 to 0*131 

0*101 0*040 to 0*046 

3-565103-659 3-627104-170 

0-052 to 0-085 



Serum. 

Blood plasma contains four coagulable proteins, fibrinogen, serum 
globulin, pseudoglobulin and serum albumin ; also a nucleoprotein 
in small quantities. With the exception of fibrinogen the same 
proteins are contained in serum. Serum also contains the other 
.constituents of blood. 

Defibrinated blood contains the red and white corpuscles in addi- 
tion. These are removed by centrifuging and serum probably more or 
less pigmented by haemoglobin results. 

Serum, free from haemoglobin, is a faintly yellow fluid of sp. gr. 
1030 and alkaline to litmus. 

A dilute solution of serum (i-io of water) acidified with a drop of 
acetic acid coagulates on heating at 70-80, chiefly between 73-75 

It gives all the general reactions for proteins. 

The Coagulable Proteins. 

According -to Haslam l the three coagulable proteins of serum have 
the following properties by means of which they can be separated : 

/-i u r t u T i ui 1 precipitated by half- 

Globulin, or euglobulm insoluble in water 
u i i , . i- , ,, ^saturation with 

Pseudoglobulin soluble in water 

J ammonium sulphate. 

Albumin soluble in water, precipitated by complete saturation 
with ammonium -sulphate.- 

Globulin or Euglobulin. 

Preparation. 

(a) Globulin is prepared by dialysing serum ; the precipitate is redissolved 
in dilute salt solution and again dialysed. This process is repeated several 
times. 

(b) Globulin is precipitated from serum by acidifying it with acetic acid 
and passing carbon dioxide through it. The precipitate is dissolved in dilute 
salt solution and again thrown out, preferably by dialysis or by sodium 
chloride. 

1 Biochem. J., 1913, vol. vii. 



444 PRACTICAL ORGANIC AND BIO-CHEMISTRY 

(c) Serum is half- saturated with ammonium sulphate. The pre- 
cipitate is treated with saturated sodium chloride solution. The 
globulin is insoluble. It can be purified by dissolving in dilute salt 
solution and precipitating with sodium chloride. 

The globulin is obtained in a coagulated state by treating the 
precipitate with alcohol and ether, or it may be obtained by dissolving 
in dilute salt solution, acidifying and boiling. The coagulum is 
washed with boiling water and dried with alcohol and ether. 

Properties. 

The uncoagulated protein is a white amorphous substance, insoluble 
in water, but soluble in dilute salt solutions. It is precipitated by 
saturating its solution with sodium chloride or magnesium sulphate or 
by half-saturation with ammonium sulphate. 

It gives the general reactions of the proteins and is a typical 
globulin. The coagulated protein is insoluble, but dissolves on warm- 
ing in dilute acids and alkalies, undergoing conversion into derivatives. 

Pseudoglobulin. 

Preparation. 

Serum is half-saturated with ammonium sulphate solution. The pre- 
cipitate is- treated with saturated sodium chloride solution. The pseudo- 
globulin dissolves and is thrown out again by half-saturation with ammonium 
sulphate. It is dissolved in sodium chloride solution and the solution 
dialysed. 

In a coagulated state it is obtained by precipitating with alcohol and 
drying with alcohol and ether, or by acidifying the solution and boiling, drying 
the coagulum after washing with alcohol and ether. 

Properties. 

The uncoagulated protein, if its solution in water be evaporated in vacua, 
will form an amorphous glassy mass of a yellow to brown colour. The coagu- 
lated protein is an amorphous white or nearly white powder. 

Pseudoglobulin is not a typical globulin as it is soluble in water and in 
saturated sodium chloride solution. 

Its solution gives the general reactions for proteins. 

Serum Albumin. 

Preparation. 

Serum albumin remains in solution after the globulins have been 
precipitated by half-saturation with ammonium sulphate. It is pre- 
cipitated by complete saturation of the filtrate with ammonium sulphate. 

It is purified by dissolving in water, half-saturating with ammonium sul- 
phate, filtering and completely saturating with ammonium sulphate and re- 
peating the process several times. The final solution is dialysed to remove 
ammonium sulphate and the coagulated protein obtained either by acidifying 
and boiling, or by precipitation with alcohol, or it may be evaporated in vacuo. 



THE INDIVIDUAL GROUPS OF PROTEINS 445 

Preparation of Crystalline Serum Albumin. 

The globulins are removed by slowly adding an equal volume of saturated 
ammonium sulphate to serum and stirring thoroughly ; after 4 or 5 hours they 
are filtered off. The filtrate is treated with 'aN sulphuric acid until there 
is a permanent turbidity (10-14 c.c. per roo c.c.). Crystals separate out as 
the solution stands. They are filtered off, dissolved in water and recrystallised 
by adding acid and ammonium sulphate. This is repeated several times. 

Coagulated protein is obtained from it by pouring its solution in water into 
alcohol, washing the coagulum with water and drying with alcohol and ether. 

Properties. 

Uncoagulated serum albumin, obtained by evaporation of the 
dialysed solution, forms an amorphous yellowish mass. It is soluble 
in water and its solution is coagulated by heating when acidified with 
acetic acid. It shows all the general reactions of the proteins. 

Coagulated serum albumin forms a white amorphous powder 
which is insoluble in water and salt solutions. It is dissolved by dilute 
acids and alkalies on warming and is hydrolysed into derivatives. It 
is more resistant to acids than egg-albumin. 

Protein Content of Serum. 

The total amount of protein in blood serum varies considerably. 
Hartley 1 found that bovine serum contained from 6-9 per cent, and that the 
relative amounts of -the three proteins varied, but that the composition in 
protein of the serum of a healthy animal remained constant for periods of 
1 1 -2 1 days. In disease, the total amount was found to diminish, the 
amount of albumin varied little, but the amount of euglobulin diminished 
considerably. Albumin formed 37-50 per cent., euglobulin 21-31 per cent, 
of the total proteins in healthy animals ; in diseased animals the euglobulin 
diminished to 10 per cent, and less. 

Hartley refers to observations by other workers and gives the following 
method of estimation. 

Estimation of Coagulable Proteins in Blood or Serum. 

The blood is defibrinated and centrifugalised. 

Total protein is estimated by adding 10 c.c. of blood to 1.90 c.c. of distilled 
water, acidifying the solution and coagulating by heat. The precipitate is 
filtered off, washed with water and alcohol, dried at 100 and weighed. 

Albumin is estimated by adding to c.c. of blood to 90 c.c. of distilled water 
and half-saturating the solution with 100 c.c. of saturated ammonium sulphate. 
An aliquot portion of the filtrate is acidified and boiled. The coagulum is 
filtered off, washed with water, alcohol and ether, dried at 100 and weighed. 

Pseudoglobulin is estimated by adding 10 c.c. of serum to 20 c.c. of 
distilled water ; 10-5 gm. of sodium chloride are slowly added and the volume 
is made up to 100 c.c. with saturated sodium chloride solution. The pre- 
cipitate is filtered off after 4 hours. An aliquot part of the filtrate is acidified 
and boiled. The coagulum is filtered off, washed with alcohol and ether, 
dried at 1 00 and weighed. 

The globulin is estimated by calculating the difference. 

1 " Memoirs of the Department of Agriculture in India," 1914, Vol. I, No. iv. 



446 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



THE COAGULABLE PROTEINS OF MILK. 

In addition to the protein caseinogen (p. 460), milk contains small 
quantities of coagulable proteins, lactoglobulin and lactalbumin, which 
closely resemble those of blood. 

Lactoglobulin. 

Milk is saturated with finely powdered sodium chloride to remove the 
caseinogen. The filtrate is saturated with magnesium sulphate. The pre- 
cipitate is dissolved in water and again precipitated and the process is repeated. 
The precipitate is dissolved in water and. dialysed to remove salts, or coagu- 
lated by heat in acid solution. 

Lactoglobulin closely resembles serum globulin. Crowther and Raistrick's 
analysis of lactoglobulin points to its identity with serum globulin. 

Lactalbumin. 

Milk is saturated at 30 with magnesium sulphate which precipitates the 
caseinogen and lactoglobulin. The filtrate is acidified with acetic acid so 
that the content of acid is about i per cent. The precipitate is filtered off, 
pressed out and dissolved in water ; the solution is neutralised and dialysed ; 
the lactalbumin is obtained on evaporation in vacua, or as coagulated protein 
by heat coagulation or by precipitation with alcohol. 

Lactalbumin is very similar to serum albumin but differs in rotation and 
percentage composition. It has been obtained in a crystalline state in the 
same way as serum albumin. It behaves like serum albumin in other 
respects. 



THE INDIVIDUAL GROUPS OF PROTEINS 447 

THE COAGULABLE PROTEINS OF MUSCLE. 

The solid matter of muscle consists essentially of* proteins, the 
principal other constituents being fa