(navigation image)
Home American Libraries | Canadian Libraries | Universal Library | Community Texts | Project Gutenberg | Biodiversity Heritage Library | Children's Library | Additional Collections
Search: Advanced Search
Anonymous User (login or join us)
Upload
See other formats

Full text of "Chemistry For Dental Students Vol II"

00 ir 

64875 



OSMANIA UNIVERSITY LIBRARY 

Call No, < l^O I ^ 4 C Accession No, / 
Author 



Title 



^ / /' / ? /4O- 

Jfc*- &*'*' " :> ^f 
ed on or before the date last marked below, 



BY THE SAME AUTHORS 



CHEMISTRY FOR DENTAL STUDENTS, VOL. I. 

Including qualitative analysis with specially 
adapted blowpipe tests, metallurgy with particular 
emphasis on extraction of metals and dental uses 
of metallic oxides, and the chemistry of dental 
alloys, amalgams, cements, etc. Fourth Edition, 
Revised and Enlarged. 

v + 180 pages. 6 by 9. 20 figures. 2 plates. Cloth, 
$2.50 net. 

PUBLISHED BY 

JOHN WILEY & SONS, INC. 



CHEMISTRY 



FOR 



DENTAL STUDENTS 

VOLUME II 

ORGANIC AND PHYSIOLOGICAL 
DENTAL CHEMISTRY 



BY 

H. CARLTON SMITH, Pn.G 

Assistant Professor of Dental Chemistry at Harvard University 
Dental School 

AND 

RACHEL M. SMITH, A.B. 

Research Assistant in the Laboratory of the Harriet Newell Lowell 

Society for Dental Research, Harvard 

University Dental School 



FOURTH EDITION, REVISED AND ENLARGED 



NEW YORK 

JOHN WILEY & SONS, INC. 

LONDON: CHAPMAN & HALL, LIMITED 
1923 



COPYRIGHT, 1906, 1912, 1917 

BY 
H. CARLTON SMITH 



COPYRIGHT, 1923 

BY 
H. CARLTON SMITH 

AND 

RACHEL M. SMITH 



Stanbopc prees 

TECHNICAL COMPOSITION COMPANY 

. H. GILSON COMPANY 

BOSTON, U. S. A. 



PREFACE TO VOL. II, FOURTH EDITION. 



The increasing tendency to place Dentistry on higher pro- 
fessional planes necessitates a deeper study of the causes of 
observed conditions, or, in other words, of the relationships 
between systemic and oral disease. In a consideration of 
systemic conditions in relation to oral disease, the majority of 
acute diseases which constitute so large a part of the physician's 
study may be disregarded. On the other hand, many of the 
habits of life, including diet, which lead to chronic conditions, 
healthful or otherwise, form an important part of Physiological 
Chemistry from the dental standpoint. All of this means that 
the student of Dentistry must make a more comprehensive 
study of Chemistry. 

It is the desire of the authors in this Second Volume to give 
to the student of Dentistry and to the dental practitioner the 
elements of Organic and Physiological Chemistry which are 
recognized to-day as essential to the scientific dentist, and 
which enable him at least to consider the many systemic fac- 
tors which may influence oral conditions. The authors hope 
that this volume will not only succeed in answering questions, 
but will also raise many questions in the minds of its readers 
and suggest new possibilities of dental research. 

The text contains frequent references to some of the more 
complete works listed below. The student is expected to have 
easy access to these, as well as to current dental literature, 
particularly to Folin's newer micro-methods for blood and urine 
analysis (Journal of Biological Chemistry). The student is 
urged to make use of these references in order that he may learn 
how to study a more important object in any course than the 
mere familiarity with present-day facts and theories. 

iii 



IV PREFACE 

Organic Chemistry Norris 

Organic Chemistry Holleman-Walker 

Physiological Chemistry . .Hawk, Eighth Edition 

Physiological Chemistry Mathews 

Metabolism Tibbies or Taylor 

H. C. S. 
R. M. S. 



TABLE OF CONTENTS 

PART I. 

ORGANIC CHEMISTRY 
CHAP. 

I. HYDROCARBONS AND SUBSTITUTION PRODUCTS i 

II. ALCOHOLS 15 

III. ETHERS 26 

IV. ORGANIC ACIDS AND ESTERS 29 

V. CYANOGEN COMPOUNDS 45 

VI. AMINES OR SUBSTITUTED AMMONIAS 50 

VII. UREA AND URIC ACID 54 

VIII. CLOSED-CHAIN HYDROCARBONS 60 

PART II. 
MICROCHEMICAL ANALYSIS 

IX. METHODS 74 

X. DENTAL PREPARATIONS 78 

PART III. 
PHYSIOLOGICAL CHEMISTRY 

XI. ENZYMES 93 

XII. CARBOHYDRATES 96 

XIII. FATS AND OILS 105 

XIV. PROTEINS no 

XV. BLOOD AND MUSCLE 129 

XVI. TEETH AND TARTAR 142 

XVII. DIGESTION 148 

XVIII. SALIVA, PROPERTIES, CONSTITUENTS AND ANALYSIS 156 

XIX. URINE 187 

XX. METABOLISM AND FOODS 219 

XXI. THEORIES OF TOOTH DECAY AND RELATION OF SYSTEMIC TO ORAL 

CONDITIONS 239 

EXPERIMENTS 248 

APPENDIX 294 

INDEX 307 

v 



DENTAL CHEMISTRY 



PART I. 
ORGANIC CHEMISTRY. 



CHAPTER I. 

THE HYDROCARBONS AND SUBSTITUTION PRODUCTS. 

In the present volume we are to consider briefly the organic 
compounds which will serve as a basis for the intelligent study of 
physiological chemistry, and also some which are of peculiar 
interest in dentistry. 

We shall touch but lightly on some of the subdivisions of the 
subject and take up a little organic chemistry proper, a little 
physiological chemistry, a little pathological chemistry, and 
from it all pick out such facts as may help us to a better under- 
standing of the problems of dentistry. 

As in many other departments of science, absolute rules for 
classification are impracticable; yet we may consider in a 
general way that the organic compounds are those containing 
carbon as a molecular constituent. The old conception that the 
organic compound must have been produced by a vital process 
of some sort (animal or vegetable) is of little value unless we con- 
fine our thought to substances found in nature only. 

The compounds of carbon are practically innumerable and 
very widely distributed, constituting the great bulk (aside from 
water) of all vegetable or animal substances. 

The carbon compounds contain the elements carbon and 
hydrogen, and when these two only are present the compounds 
are hydrocarbons. Compounds containing carbon, hydrogen, 
and oxygen, are of more frequent occurrence, and when the 



2 ORGANIC CHEMISTRY 

hydrogen and oxygen are present in the proportions in which 
they occur in water, the compound is a carbohydrate (with ex- 
ceptions). 

In the chemistry of the animal body the majority of sub- 
stances which we meet contain carbon, hydrogen, oxygen, and 
nitrogen, and often in addition sulphur or phosphorus. Many 
other elements, notably the halogens, and often the metals, may 
be found in organic compounds. 

In consideration of an organic substance, therefore, the ques- 
tion of its composition is the first one presenting itself. 

The analysis of organic bodies may be made from two distinct 
standpoints: first, to determine the various substances which 
may be separated from a given organized body, as from some 
part of a plant; second, to determine the constituent elements 
of one of the substances so separated. 

As an example of the first sort of analysis, we may find in a 
potato a certain basic principle (alkaloid), more or less water, 
and considerable starch. These may be called proximate prin- 
ciples, and the separation of them would be proximate analysis, 
while the second sort of analysis determines the composition of 
the starch molecule and is known as ultimate analysis. 

QUALITATIVE TESTS. 

Carbon. The presence of this element may be shown by 
the " carbonization/' or blackening, of the compound when 
heat is applied. 

Carbon may also be demonstrated by oxidizing the compound 
and producing carbon dioxide, as follows : 

Mix thoroughly a few grains of benzoic acid with about four 
times as much powdered copper oxide. Put the mixture into a 
glass tube about 6 inches in length and closed at one end, and 
shake well into the closed end of the tube. Then introduce three 
or four times as much coarsely powdered copper oxide and place 
the tube in a nearly horizontal position, tapping it to distribute 
powder through about half its length. Now, by means of a 
short rubber tube, connect a short delivery tube of glass which 



THE HYDROCARBONS AND SUBSTITUTION PRODUCTS 3 

dips into about 5 c.c. of lime-water contained in a test-tube. 
With the Bunsen burner in the hand, heat first the unmixed 
copper oxide, then gradually work toward the closed end of the 
tube until the whole is hot and the gas given off has produced a 
precipitate in the lime-water. What reactions have taken place? 

Hydrogen shows itself by the production of moisture in these 
same tests. 

Nitrogen may or may not be indicated by a preliminary 
test. It may be detected with certainty by either of the fol- 
lowing methods: 

(a) Conversion into a cyanogen compound. 

By means of a burette clamp, support a test-tube in a vertical 
position; drop into it a piece of clean, dry sodium, and with 
burner in the hand melt the sodium and continue heating until 
vapors begin to rise. Add a very small quantity of powdered 
albumin and continue to heat for a minute or two. Break lower 
portion of tube into mortar containing 2 c.c. of alcohol. Watch 
for the evolution of gas. When no more gas is given off, add a 
little sodium hydrate solution, and filter. To the clear filtrate 
add 2 or 3 c.c. of yellow ammonium sulphide, and evaporate the 
whole to dry ness over a water-bath. Dissolve residue in water 
which has been made slightly acid with hydrochloric acid, filter, 
and to the clear filtrate add a drop of ferric chloride. 

Explain steps in conversion of nitrogen into ferric thio- 
cyanate. 

(b) Conversion into free ammonia. 

Almost any nitrogenous substance may be made to evolve 
ammonia-gas by simply heating in a test-tube with several times 
its bulk of soda-lime. Test for ammonia by moistened red litmus 
paper or by odor. (This test is known as that of Wohler, also 
of Will and Varrentrap.) 

The Kjeldahl, or moist combustion, process is much employed 
as a quantitative method but may be used qualitatively as 
follows: The substance is heated in an ignition-tube with con- 
centrated sulphuric acid till a clear (not necessarily color- 
less) solution is obtained. The mixture is cooled, diluted with 
water, an excess of caustic soda added, and heat applied. Am- 



4 ORGANIC CHEMISTRY 

monia is then evolved, and may be detected by litmus paper or 
by odor. 

Sulphur and phosphorus are first completely oxidized, either 
by fusion of the substance with alkali nitrate and carbonate, 
or by treatment in the wet way with fuming nitric acid or mix- 
ture of potassium chlorate and hydrochloric acid. The result- 
ing sulphate or phosphate is detected by the usual qualitative 
methods.* 

A sulphur test may also be made by heating the substance 
with a little concentrated sodium hydroxide in the test-tube. 
A little sodium sulphide, which may be detected by dropping 
upon a bright silver coin or by testing with lead acetate solution, 
will thus be formed. 

Halogens. Chlorine, bromine, and iodine cannot be de- 
tected in organic combinations by the ordinary qualitative test 
with silver nitrate and dilute nitric acid, but must first be con- 
verted into corresponding inorganic haloid salts. This may be 
done by heating the organic substance strongly with pure lime, 
and thus forming calcium chloride, bromide, etc., which may be 
dissolved in water and tested in the usual way. (See Vol. I.) 

A test for chlorine or iodine may also be made by heating 
with copper oxide on a platinum wire in the Bunsen flame, chlo- 
rine giving first a blue and then a green color to the flame. 
Iodine gives green only (Beilstein). 

Test for presence of C, H, N and S in dried albumin. 

Test for P in casein precipitated from milk. 

Test a few drops of chloroform for the presence of chlorine. 

THE HYDROCARBONS. 

The hydrocarbons are organic compounds of carbon and 
hydrogen only. The simplest of these is marsh gas, or methane 
(CH4). The molecule of this substance consists of a single 

* Qualitative test for sulphates is the production of a white precipitate, BaSO 4 , 
insoluble in hydrochloric acid, upon the addition of BaCU to the unknown. 

Qualitative test for phosphates is the formation of the yellow ammonium phospho- 
molybdate when ammonium molybdate in nitric acid is added to the unknown. 



THE HYDROCARBONS AND SUBSTITUTION PRODUCTS 5 

carbon atom with each of its four points of atomic attraction 
(valence) satisfied by an atom of hydrogen. 

H \ / H 

r 



If one of these four atoms of hydrogen is replaced by a chlo- 
rine atom, for instance, we have a substitution product. Its for- 
mula will be CH 3 C1, its name monochlor-methane or methyl 
chloride. If two molecules of methyl chloride are brought to- 
gether and the chlorine removed by metallic sodium, the residual 
molecules (methyl radicals) will unite, forming a new hydrocar- 
bon, as follows: 

2 CH 3 C1 + Na 2 = 2 NaCl + C 2 H 6 (ethane). 

By a similar reaction we may form the third member of 
the series, propane, C 3 H 8 , from ethyl chloride, C2H 5 C1, and 
sodium; the fourth member, butane, C^io, from propyl chloride, 
etc. A tabulated list of the first five compounds of this series 
will plainly show their chemical relationship. 

CH 4 , methane or methyl hydride (CH 3 H). 
C2H 6 , ethane or ethyl hydride (C 2 H 5 H). 
C 3 H 8 , propane or propyl hydride (C 3 H 7 H). 
C 4 Hio, butane or butyl hydride (C 4 H 9 H). 
C 5 Hi2, pentane or amyl hydride (C 5 HnH). 

Note that the various members of this series differ from one 
another by CH 2 ; that is, each higher compound contains one 
carbon atom and two hydrogen atoms more than its predecessor. 
This holds true through the series, and the compounds of this 
or any such series are termed homologues and the series ho- 
mologous series. Note further that any member of this series 
(which is known as the paraffin series) may be represented by 
the general formula C^H 2n -f2. This likewise holds true through- 
out the series, and a compound having sixty carbon atoms will 
have a formula of C 6 oHi 22 . The first four hydrocarbons of this 
series are gaseous at ordinary temperatures; from C 5 Hi2 to 



6 ORGANIC CHEMISTRY 

about CieH34, the hydrocarbons arc liquid; from CioH^ (melt- 
ing at about 18) up, they are solids. 

The hydrocarbons of the paraffin series are known as straight- 
chain or aliphatic hydrocarbons, their graphic formulae consist- 

I I I 1 
ing of " chains " of carbon atoms, as butane, C C C C , 

till 

as distinguished from the closed-chain or cyclic compounds as 
represented by the "benzene-ring " (page 60), a carbon nucleus 
with the carbon atoms united in a continuous closed chain or 
" cycle." 

The paraffins are called saturated hydrocarbons because they 
are incapable of forming addition products by absorption of 
chlorine, for instance, without first giving off an equivalent 
number of atoms of hydrogen. This is because of the complete 
" saturation " or union of every carbon " bond " with some 
other atom.* Paraffin wax and mineral oil are mixtures of 
saturated hydrocarbons and resist chemical action even of strong 
nitric acid or sulphuric acid. 

The name paraffin is derived from the two Latin words parvus, 
little, and ajfinitas, affinity. 

The natural sources of hydrocarbons of the paraffin series are 
natural gas and crude petroleum, or rock oil. Many of these 
hydrocarbons exist as such in the petroleum, and some undoubt- 
edly are produced by the heat used to effect a separation of the 
various compounds. This separation may be effected by dis- 
tilling the oil in an apparatus similar to that pictured in Fig. i, 
and is known as fractional distillation, the different hydrocarbons 
passing over at different temperatures. Separation by this 
method, however, is by no means complete, and the resulting 
products are themselves mixtures of hydrocarbons, and are 
distinguished by physical properties rather than by chemical 
composition. 

When crude petroleum is thus distilled, the following products 
are obtained: first, rhigoline, which comes over at a temperature 

* Notice that while addition products of saturated hydrocarbons cannot be 
formed, substitution products are easily possible. See page 5. 



THE HYDROCARBONS AND SUBSTITUTION PRODUCTS 



of 20 to 22 C.; then petroleum ether, or benzine, at from 50 
to 60 C. ; then gasoline, or naphtha, 
at about 75 C.; then one or two un- 
important commercial products; and 
then kerosene, or burning oil which is 
obtained at 150 to 250 C. Above 
this, we may obtain paraffin oil or 
light lubricating oils; then the heavy 
lubricating or cylinder oils; and from 
the residue we obtain the solid sub- 
stances known as vaseline, or petro- 
leum jelly, and paraffin of various 
degrees of hardness. 

The first five hydrocarbons of this 
series we shall consider somewhat in 
detail, not only because they are 
important and comparatively com- 
mon, but also because they serve as 
types of all other compounds of the 
series, and reactions which we study 
with these compounds are, as a rule, 
general typical reactions which may 
be produced with other members of 
the series. 

Methane, CH4, occurs as marsh 
gas in stagnant ponds or pools and 
is a constituent of "fire damp" in 
coal mines. It is a colorless gas, 
odorless when pure, and very slightly 
soluble in water. Methane burns in 
the air with the production of carbon 
dioxide and water 

CH 4 + 2 2 = C0 2 + 2 H 2 0. 

It may be prepared synthetically 
by the direct combination of hydrogen FlG * 

and carbon. This reaction is usually brought about by allow- 




8 ORGANIC CHEMISTRY 

ing hydrogen to pass over heated nickel mixed with finely divided 
carbon. The nickel acts as a catalyzer; without it the com- 
bination of hydrogen and carbon takes place with difficulty and 
only at a temperature exceeding 1100. 
Other methods of preparation are: 

(a) Anhydrous sodium acetate and sodium hydroxide (marsh 
gas mixture*) heated together will react according to the following 
equation: 

NaC 2 H 3 2 + NaOH -> CH 4 + Na 2 CO 3 

This method is based on the fact that carbon dioxide can be 
liberated from acetic acid by means of a strong base, and it is 
the method recommended for general laboratory use. 

(b) Zinc methyl, a very loose chemical compound of two 
methyl groups and one atom of zinc, may be decomposed by the 
action of water, giving methane and zinc hydroxide. 

Zn (CH 3 ) 2 + H 2 O -> CH 4 + Zn(OH) 2 

This method and the previous one are of particular importance, 
as they are general methods adapted for the preparation of any 
hydrocarbon of the series. 

(c) Aluminium carbide treated with water reacts to yield 
methane and aluminium hydroxide. 

A1 4 C 3 + 12 H 2 O - 4 A1(OH), + 3 CH 4 

(d) Methyl chloride, a substitution product of methane, may 
be reduced with nascent hydrogen. 

CH 3 C1 + H 2 -> CH 4 + HC1. 

Ethane, C^He, the second member of the series, occurs natu- 
rally in a solution in crude petroleum, and can be artificially pre- 
pared by the electrolytic decomposition of a saturated solution 
of potassium acetate, as follows: 

2 CH 3 COOK C 2 He + 2 C0 2 + K 2 . 

* Marsh gas mixture also contains considerable calcium oxide to prevent too 
rapid action. See Appendix. 



THE HYDROCARBONS AND SUBSTITUTION PRODUCTS Q 

The free potassium, of course, decomposes water, liberating 
hydrogen gas which collects at the negative pole, and, if the 
solution contains sufficient potassium hydroxide, the carbon 
dioxide will be dissolved, allowing ethane to collect at the posi- 
tive pole. 

Ethane may also be made from a haloid derivative of marsh 
gas by the action of metallic sodium; that is, in methane we may 
replace one of the hydrogen atoms with iodine, forming CH 3 I, 
methyl iodide; then, by treatment with metallic sodium, the 
following reaction will take place : 

2 CH 3 I + 2 Na = C 2 H 6 + 2 Nal. 

This is the general method employed for building up all the 
higher hydrocarbons from the lower members of the series. 
By selecting the correct alkyl* halide, we may make any hydro- 
carbon from any other hydrocarbon that is a lower member of 
the series, by treatment with metallic sodium. 

Ethane, as indicated under preparation of methane, may also 
be made by either method (a) substituting sodium propionate 
for sodium acetate or (b) substituting zinc ethyl for zinc methyl. 

Ethane is slightly more soluble in water than methane. It 
may be condensed to a liquid at a pressure of forty-six atmos- 
pheres. 

Propane, C 3 H 8 , also occurs in petroleum, and can be made by 
treating a mixture of ethyl iodide and methyl iodide with metallic 
sodium : 

C 2 H 6 I + CH 3 I + 2 Na = C 3 H 8 + 2 Nal 

or by any of the synthetic methods before mentioned. 

Butane, C 4 H 10 , is the first of the series capable of existing in 
two forms, isomers. The structural formulae of these two com- 
pounds are shown in the illustration of the term isomer on page 
10. This compound and many of its higher homologues are 
of importance only in relation to some of their derivatives which 
will be subsequently studied. 

* Alkyl a term used to denote any hydrocarbon radical as CHa-, C2H 6 -, 
C 3 H 7 -, etc. 



10 ORGANIC CHEMISTRY 

Isomers. When two or more compounds are of exactly the 
same molecular composition, or when two compounds have the 
same percentage composition, the one being a multiple of the 
other, the compounds are said to be isomers or isomeric com- 
pounds. 

The isomerism of the first class is said to be metameric. It 
exists when the atoms of the several compounds are not only the 
same in kind, but also the same in the number of each kind. For 
example, C^H^On is the formula for cane sugar; C^H^On is also 
the formula for milk-sugar, and these two compounds have 
decidedly different properties, the difference being dependent 
upon the arrangement or relationship of the atoms in the mole- 
cule. Another example illustrating this difference may be 
found in the graphic formula for normal and isobutane, given 
below. 



H 



H 



H 



H H H H \ 

I I I I H /C 

H-C-C-C-C-H I 

I I I I TT C* C* 

III I AJL Vv \^ 

H H H H II 

H H C-H 

N H 

Note that each molecule has an empirical formula of C 4 Hi ; 
the normal compound may be represented as CH 3 .(CH2) 2 .CHs, 
the iso-compound as CH 3 .CH.(CH 3 ) 2 . These will be found to 
have quite different physical and chemical properties. 

The isomerism of the second class is called polymeric; one 
substance is the polymer of another when the molecules are of 
the same percentage composition but of different molecular 
weights. For example, CH 2 O is gaseous formaldehyde; (CH 2 O) 3 
is its polymer or polymeric form, known as paraform, a white 
crystalline solid. 



THE HYDROCARBONS AND SUBSTITUTION PRODUCTS II 

UNSATURATED HYDROCARBONS. 
DOUBLE-BONDED HYDROCARBONS. 

When a mixture of alcohol and strong sulphuric acid is heated, 
with the acid in considerable excess, water is withdrawn from 
the molecule of alcohol, and a gas, found to have the formula 
C2H 4 , is produced. (See Exp, 7.) The name of this gas is 
ethylene; it occurs in coal gas and in traces in solution in crude 
petroleum. It is the first of a series of hydrocarbons which 
contain double-bonded carbon atoms. The double bond is 
assumed because it is found to be impossible to produce a lower 
compound of this series, such as CH 2 , which might be called 
methylene, but which would necessitate a bivalent carbon atom; 
also because the hydrocarbons of this series are capable of 
formation of addition products as well as of substitution products. 

Note that the formula of ethylene does not conform to the 
general formula of the paraffins (C n H 2w +2), but is the first member 
of the new series of " unsaturated " hydrocarbons; the olefin or 
ethylene series with a general formula of C n H 2 . 

Addition Products. The hydrocarbons of this series are 
known as unsaturated hydrocarbons, because of the fact that 
by treatment with certain elements or compounds the double 
bond existing between two of the carbon atoms may easily be 
broken, thus giving an additional free bond to two carbon atoms. 

For example: when ethylene is treated with nascent chlorine, 
the double bond is broken and two chlorine atoms add themselves 
to the molecule, forming a saturated compound, ethylene chloride. 

H H H H 

II II 

H-C = C-H+C1 2 -> H-C-C-H 

I I 
Cl Cl 

Such a product is called an addition product, and the power 
of forming addition products is characteristic of all unsaturated 
compounds. Some of the addition reactions of ethylene, given 
below, may be considered as typical of the way in which addition 
products are formed. 



12 ORGANIC CHEMISTRY 

Ethylene can be converted into ethane by treatment with 
nascent hydrogen. Two atoms of hydrogen add themselves to 
the molecule, forming a saturated compound. Ethylene with 
any of the halogens produces a reaction similar to the one given 
above for chlorine. With the halogen acids, except hydrochloric, 
the acid molecule splits into the positive and negative ions, the 
positive hydrogen attaching itself to one carbon atom and the 
negative halogen ion going to the other carbon. The compound 
produced is CH 3 .CH 2 Br, or ethyl bromide. Sulphuric acid acts 
similarly, the acid ionizing into H+ and HS04~ and forming^ 
with ethylene, ethyl-hydrogen-sulphate, CH 3 CH 2 HS04. 

The hydrocarbons of this series take their names from corre- 
sponding members of the paraffin series, with " ene " as a dis- 
tinguishing termination ethylene, C 2 H4, propylene, C 3 He, 
butylene, C 4 H 8 , etc. They are unimportant in dental and physio- 
logical chemistry. Some of the higher oxygenated compounds 
of this class are, however, of great importance, as olein, which 
is a constituent of vegetable and animal fats and oils. 

TRIPLE-BONDED HYDROCARBONS. 

A third series of the straight-chain hydrocarbons is the ace- 
tylene series; these are triple bonded, and of course unsaturated, 
with a general formula of C n H 2 -2. 

The only members of this series of special interest are, first, 
acetylene, H C^C H, (C 2 H 2 ), made from calcium carbide 
and water (see Exp. 10, page 249). It is poisonous, combining 
directly with the hemoglobin of the blood, has a disagreeable 
odor, and is inflammable. Second, allylene, CsH 4 , derivatives of 
which occur in onions, garlic, mustard-oil, etc. 

HALOID DERIVATIVES OF THE PARAFFINS. 

Methane furnishes three chlorine substitution products which 
are more or less in common use: first, the monochlor-methane, or 
methyl chloride; second, the trichlor-methane, CHCla, or chloro- 
form; and third, the tetrachloride of carbon, CCU. 

Methyl chloride, CH 3 C1, may be made from methyl alcohol, 
zinc chloride, and hydrochloric acid. It is a colorless gas, con- 



THE HYDROCARBONS AND SUBSTITUTION PRODUCTS 13 

densing to a liquid at 23 C. ; it is used as a spray in producing 
local anesthesia (page 86), also as a constituent of anesthetics, 
such as anesthol, somnoform, etc. 

Dichlor-methane, CH 2 C1 2 , also known as methylene chloride, 
has been used as a general anesthetic, usually mixed in more or 
less chloroform and alcohol. Its use in this way is open to 
criticism because of its poisonous action upon the heart. 

Chloroform, CHC1 3 , trichlof-methane, is a general anesthetic 
prepared by distilling a mixture of chlorinated lime and acetone. 
Alcohol and water were formerly used in place of acetone (see 
Exp. 13, page 250). While it is not regarded as inflammable, 
its heated vapor can be made to burn with a greenish flame. 
The reaction with alcohol is probably as follows: 4 C 2 H 6 OH 
+ 8 Ca(C10) 2 = 2 CHC1 3 + 3 Ca(CH0 2 ) 2 + 5 CaQ 2 + 8 H 2 O. 

A delicate test for chloroform is made by treating a few drops 
with a mixture of aniline and alcoholic potassium hydroxide. 
Isobenzonitril, or phenyl-carbylamine, which has a very char- 
acteristic and intensely obnoxious odor, is thus produced. 

CHC1 3 + 3 KOH + C 6 H 6 NH 2 -> C 6 H 6 NC + 3 KC1 + 3 H 2 O 

Methyl chloroform, CH 3 CC1 3 , formed by replacing the hydro- 
gen atom of chloroform by a methyl group, CH 3 , has been used 
as an anesthetic. 

Tetrachloride of carbon is a colorless liquid used quite largely 
as a solvent. It also has anesthetic properties but, like dichlor- 
methane, is dangerous because of its action on the heart. 

Methyl bromide, or monobrom-methane, is used to some ex- 
tent as a constituent of anesthetics. 

Bromoform, CHBr 3 , tribrom-methane, is prepared from 
bromine and a solution of alcoholic potash. Its properties are 
similar to those of chloroform, but it is more poisonous. 

Methyl iodide, CH 3 I, is a heavy liquid, with pleasant odor, 
boiling-point 43 C.; it has been used somewhat as a vesicant. 

lodoform, CHI 3 , tri-iodomethane, is a much-used and very 
valuable antiseptic. It is a light yellow, crystalline powder 
with characteristic persistent odor (Plate III, Fig. 6, page n). 

lodoform may be made by heating in a retort two parts of 



14 ORGANIC CHEMISTRY 

potassium carbonate, two of iodine, one of strong alcohol, and 
five of water, till the mixture is colorless, 

QHfiOH + 4 I 2 + 3 K 2 CO 3 - CHI 3 + KCHOa + 5 KI + 2 H 2 O 

+ 3 C0 2 . 

lodoform is also produced from action of the above reagents 
with acetone in place of alcohol. This test is a very delicate 
one and advantage is taken of it in testing for acetone in saliva, 
see page 182. 

Cacodyl is an example of the arsenic derivatives of the hydro- 
carbons. It is one of several products which result from the 
distillation of a mixture of potassium acetate and white arsenic. 
Its composition is that of dimethyl arsine, (CH 3 ) 2 As. 

Ethyl chloride, C^H^Cl, chlorethyl, may be made by dis- 
tillation of a mixture of alcohol and hydrochloric acid and 
purification of the distillate. It is extremely inflammable, boils 
at 12 C., and is used as a local anesthetic in a similar manner to 
methyl chloride. Its higher boiling-point makes it the more 
convenient of the two preparations (see page 82). 

Ethyl bromide, QHsBr, is prepared from alcohol, sulphuric 
acid, and potassium bromide. It is a heavy, colorless liquid, 
does not burn, and has been used to a considerable extent as a 
general anesthetic. 



CHAPTER II. 
ALCOHOLS. 

If we substitute, for one of the hydrogen atoms of methane, 
a hydroxyl group (OH), we shall produce the first of a series of 
alcohols, several of which will claim our attention. 

The alcohols may be considered as hydroxides of alkyl radicals, 
CH 3 OH being methyl alcohol, C 2 H 5 OH being ethyl or ordinary 
alcohol; C 3 H 7 OH being propyl alcohol, and C 6 H n OH, amyl 
alcohol or fusel oil. 

The alcohols as a class may be prepared by the action of 
moist silver oxide on the corresponding halogen compounds; e.g., 

CH 3 Br + AgOH = CH 3 OH + AgBr. 

In many instances, the alkaline hydroxides will act in the 
same way. 

CH 3 Br + KOH = CH 3 OH + KBr. 

Alcohols treated with metallic sodium or potassium liberate 
hydrogen gas, forming compounds known as alcoholates; e.g., 

CH 3 OH + K = CH 3 OK + H; 
or C 2 H 5 OH + K = C 2 H 5 OK + H. 

While these compounds are, as just stated, called alcoholates, 
they may be distinguished, one from another, by using the name 
of the alkyl radical involved; CH 3 OK will be potassium methylate, 
while C 2 H 6 OK will be potassium ethylate. 

Alcohols may contain more than one hydroxyl group, and, 
according to the number of the OH groups, are termed mono-, di-, 
tri-atomic, etc. Thus, ordinary alcohol, C 2 H 5 OH, is mon- 
atomic; glycol, C 2 H 4 (OH) 2 , is diatomic; glycerol, C 3 H 5 (OH) 3 , 
is triatomic, while mannite, C 6 H 8 (OH) 6 , is a hexatomic alcohol. 

Alcohols may also be classified according to the relative po- 
sition of the hydroxyl group. By this classification, we may have 

15 



16 ORGANIC CHEMISTRY 

primary alcohols with OH replacing a hydrogen of the CH 3 
group; secondary alcohols with OH replacing the hydrogen of a 
CH 2 group; and tertiary alcohols with OH replacing the 
hydrogen of a CH group. This may be illustrated by the 
formula of an alcohol of each class. CH 3 CH 2 CH 3 being 
the hydrocarbon, a primary alcohol will have the formula CH 3 . 
CH 2 .CH 2 OH, and CH 2 OH may be considered the distinctive 
grouping of the primary alcohols. Again, from the same hydro- 
carbon, if OH is substituted for an H of CH 2 , then the secondary 
alcohol will be CH 3 ~CHOH-CH 3 and -CHOH may be 
regarded as a distinctive group of this class. 

The tertiary alcohols, however, must be produced from com- 
pounds having at least four carbon atoms, as a CH group is 
only possible when there are sufficient carbon atoms to produce 
a forked chain; that is, in a compound with three carbon atoms, 
one must of necessity be placed between the other two, while 
with four carbon atoms, the carbons may be attached in a 
straight chain, such as C C C C,or they may be arranged as 

/C 

a forked chain C C , and by supplying the hydrogen atoms 

\C 

necessary to satisfy the valence of each carbon, in this latter 
chain we find a CH group. OH introduced in place of the 
hydrogen of this group gives us the tertiary alcohol, 

/CH 3 
CH 3 -COH 

X CH 3 

Methyl alcohol, CH 3 OH, (H-CH 2 OH),* wood spirit, car- 
binol, is a product of the destructive distillation of wood, and can 
be made synthetically from methane. It is a colorless, inflam- 
mable liquid, with a gravity of 0.802 at 15 C., with solvent 
properties similar to ordinary alcohol. It boils at 66. 

It is customary to test for methyl alcohol by oxidizing it to 
formaldehyde and then obtaining a color reaction due to the 
formation of furfural. The details of the test are given in Exp. 
* Note that CEtOH is the "alcohol group" peculiar to this class of alcohols. 



ALCOHOLS 17 

24, page 253, a positive test being the production of a rose- 
colored ring.* In a mixture of alcohols the test should be 
applied to the first portion of distillate, as the boiling point of 
methyl alcohol is lower than that of any of the other common 
alcohols. 

Ethyl alcohol, C 2 H 5 OH, (CH 3 -CH 2 OH), methyl carbinol, 
grain alcohol, or ordinary alcohol, may be made by the action of 
silver hydratef on ethyl iodide or bromide, as suggested on page 
15. It is made commercially by fermentation of various car- 
bohydrates, and purified by distillation. Carbon dioxide is 
evolved as follows : 



= 2 QHaOH + 2 CO 2 . 



Ninety-five per cent alcohol has a specific gravity of 0.8164, 
boils at about 78 C., dissolves many inorganic salts, vegetable 
Waxes, resins (not gums), oils, etc., and is miscible with water, 
ether, or chloroform. 

Propyl alcohol, normal, CH 3 .CH 2 .CH20H, occurs with amyl 
alcohol as a constituent of fusel oil, or may be prepared by 
general method with moist silver oxide. It is a colorless liquid, 
and boils at 97 C. The iso-compound,ff CH 3 .CHOH.CH 3 , may 
be made by reducing acetone with nascent hydrogen; nascent 
hydrogen may be produced by sodium amalgam. 

Butyl alcohol, C 4 H 9 OH, occurs in four isomeric forms. The 
normal alcohol is CH 3 .(CH 2 )2.CH 2 OH. It h produced by the 
fermentation of glycerol. It boils at 117 C. The isobutyl al- 
cohol, (CH 3 ) 2 CH.CH 2 OH, obtained from fusel oil, boils at 107 C. 

Amyl alcohol, C 5 HnOH, (C 4 H 9 -CH 2 OH), consists of about 
87 per cent of isobutyl carbinol and about 13 per cent of an isomer 
known as active amyl alcohol. It is a colorless, oily liquid with a 
specific gravity of 0.818. It boils at about 130 C., and burns 
with a bluish flame. 

Fusel oil, or potato spirit, consists of amyl alcohol carrying 
traces of various other alcohols as impurities. 

* All the higher alcohols give a brown ring in this test. 
t Moist silver oxide. 
ft See note on page 22. 



l8 ORGANIC CHEMISTRY 

Amyl alcohol is a valuable solvent and is largely used in the 
manufacture of artificial fruit flavors, banana essence, and the 
like. 

Oxidation of the Alcohols. 

ALDEHYDES. 

The first step in the oxidation of a primary alcohol consists in 
the withdrawal of hydrogen; thus the oxidation of methyl 
alcohol produces formaldehyde (CH 2 O) and water, 
CH 3 OH + O = CH 2 O + H 2 O, 

and the oxidation of ethyl alcohol produces acetaldehyde and 
water. 

C 2 H 6 OH + = CHsCHO + H 2 O. 

The removal of the two hydrogen atoms may be considered 
as a result of the addition of oxygen to the molecule, as this 
gives a condition in which two hydroxyl groups are attached to 
one carbon atom. H This condition is an impossible 

I 
H - C - OH 

I 

one, and water OH immediately breaks off, remov- 

ing two hydrogen atoms and one oxygen atom, and leaving the 

H 

characteristic aldehyde grouping C 

With the exception of formaldehyde, all aldehydes may be 
considered compounds containing an alkyl radical and this dis- 
tinctive aldehyde group. Acetaldehyde, produced by the first 
oxidation of ethyl alcohol, as shown above, consists of the 
methyl group and the aldehyde group, CH 3 CHO; propionic 
aldehyde, of the ethyl group and the aldehyde group, C 2 H5CHO. 
In the case of formaldehyde, as with methyl alcohol, a hydrogen 
atom takes the place of the alkyl radical. 

Aldehyde reactions. The aldehyde structure and the con- 
sequent reactions to which aldehydes respond are of fundamental 
importance in the chemistry of carbohydrates. These reactions 
may be grouped as 



ALCOHOLS 19 

(1) Reduction of copper solutions and ammoniacal silver 
solutions. 

The products of the reduction of the copper solutions depend 
on the preparation of the reagent; but, in general, if much re- 
ducing agent is present, cuprous oxide is precipitated. The 
reduction of various copper solutions by carbohydrates has been 
used in part as a proof of the presence of an aldehyde structure 
in some carbohydrate substances and is used extensively as a 
test for carbohydrates (Fehling's Test, Benedict's Test, etc., 
page 271.) 

The reduction of an ammoniacal silver solution is one of the 
best known tests for aldehydes, Tollen's Test, Exp. 30, page 254, 
and is used in Howe's method for the sterilization of root canals, 
page 84. The aldehyde group is oxidized to form an acid, and 
metallic silver is precipitated. 

(2) Addition reactions. Aldehydes combine directly with 
ammonia, forming aldehyde ammonias white, crystalline 
compounds. 

CH 3 



CH 3 CHO + NH 3 = C 

jj / \ JNH2 

Hydrocyanic acid forms, with aldehydes, compounds known 
as oxynitrils or cyanides of oxy-organic acids. 

CH 3 

CH 3 CHO + HCN = C / OH 

H / \ CN 

It will be noted, in each case, that the molecule which adds itself 
to the aldehyde splits; the hydrogen goes to the oxygen of the 
aldehyde group, making a hydroxyl group and leaving a free 
bond which holds the rest of the new molecule. 

Aldehydes react similarly with phenyl-hydrazine, an aromatic 
compound, C6H 5 NH.NH 2 , forming phenyl-hydrozones or osa- 
zones, which are important in the detection of various sugars. 
(See page 99.) 



20 ORGANIC CHEMISTRY 

(3) Condensation of aldehydes. Two or more molecules of an 
aldehyde possess the property of uniting and thus forming new 
compounds. This union may take place either between the 
oxygen atoms, forming polymers, or between the carbon atoms, 
forming condensation products. Parafonn, a polymer of formal- 
dehyde, illustrates the way in which the molecules combine 
through oxygen, while aldol is an example of a condensation 
product. 

H H 

/ \ H 

C O C I 

/ \ / \ H-C-OH 

H O O H I 

\/ C-H 

C % 

/\ O 
H H 

Paraform Aldol 

It is of importance to note that when polymerization takes 
place the characteristic aldehyde grouping is lost and the new 
molecule does not possess aldehydic properties. When con- 
densation takes place, however, the aldehyde grouping is always 
left intact, allowing condensation products to react similarly to 
aldehydes. 

Formaldehyde, H.CHO, is a gas with a decidedly irritating 
odor, freely soluble in water. The commercial preparation, 
usually called formalin, is a 40 per cent solution. Formaldehyde 
is prepared by the oxidation of methyl alcohol. It is readily 
oxidized to formic acid and carbon dioxide, and possesses the 
reducing properties common to all the aldehydes. With am- 
monia its action is unique (page 194). 

Formaldehyde forms polymers, of which paraform, a combina- 
tion of three formaldehyde molecules, is the most important. 
This compound is known as tri-oxymethylene. Formose, a 
substance allied to glucose, has been considered as a higher 
polymer, but in a stricter sense it is a condensation product, 
as the union of the molecules is probably effected through the 
carbon atoms. 



ALCOHOLS 21 

Because formaldehyde coagulates albumin, hardens gelatin 
and renders proteins in general tougher and less digestible, it is 
utilized to a large extent in the preparation and preservation of 
anatomical specimens. It has been used quite generally as a 
food and milk preservative and is a valuable disinfectant. In 
dentistry its use is in connection with formo-cresol and will be 
studied under dental preparations (page 83). 

Acetaldehyde, aldehyde, CH 3 CHO or C 2 H 4 O, the aldehyde 
from ethyl alcohol, may be made by addition of H 2 S0 4 to a 
mixture of alcohol and bichromate of potassium. It is a color- 
less, inflammable liquid with pungent ethereal odor, and boils 
at 22 C. It responds to the general aldehyde reactions. 

Paraldehyde, (C2H 4 O) 3 , a polymer of acetaldehyde, is a "color- 
less liquid with a strong pungent odor, soluble in 8.5 parts of 
water at 15 C., miscible in all proportions with alcohol, ether, 
and fixed or volatile oils." (U.S.P.) It is a valuable hypnotic. 

Chloral, CC1 3 CHO, trichlor-aldehyde, is an oily liquid formed 
by action of dry chlorine gas on pure alcohol; it is soluble in 
ether and chloroform, boiling at from 94 C. to 98 C., and 
forming, with a molecule of water, chloral hydrate, CC1 3 CHO.H 2 O, 
a crystalline solid, the chloralum hydratum of the pharmacopoeia 
(see page 81). 

Chloral hydrate is decomposed by sodium or potassium hy- 
drate with liberation of chloroform (see Exp. 32, page 254): 
CC1 3 ~CHO + KOH = CHC1 3 + KCOOH (potassium formate). 

When a drop or two of aniline oil is warmed in an excess of 
alcoholic potash, chloral hydrate forms, first, chloroform, then 
phenyl-isocyanide, C 6 H 5 NC, the persistent disagreeable odor of 
which furnishes a delicate test for chloroform or chloral (see 
Exp. 33, page 255). If CHC1 3 is used as the reagent in place of 
the aniline, the same reaction becomes a test for aniline or 
organic compounds, from which aniline may be produced by 
heating with alcoholic potash as acetanilide. 

Other aldehydes from hexatomic alcohols are dextrose (glucose) 
and galactose. They are represented by the formula CH 2 OH 
(CHOH) 4 CHO, and will be considered more fully in a sub- 
sequent chapter. 



22 ORGANIC CHEMISTRY 

KETONES. 

The oxidation of secondary alcohols (page 16) will not yield 
aldehydes, but a class of substances known as ketones: 

CHs-CHOH-CHa + = CH 3 -CO-CH 3 + H 2 0. 

Secondary propyl alcohol. Dimethyl ketone. 

The converse of this reaction is possible, and, by reduction 
of a ketone with nascent hydrogen (sodium amalgam), the 
secondary alcohol will be formed: 

CH 3 -CO-CH 3 + H 2 = CH 3 -CHOH- CHs. 

Acetone. Isopropyl alcohol.* 

Likewise primary alcohols may be produced by the reduction of 
aldehydes: 

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

Acetaldehyde. Ethyl alcohol. 

It will be noted that the characteristic grouping of the ketone 

I 
is the C = O or carbonyl group. Ketones may be remembered 

I 

as alkyl radicals joined by the carbonyl group. Thus, acetone, 
the first ketone, formed as is shown above by the oxidation of 
secondary propyl alcohol, is dimethyl ketone, CH 3 .CO.CH 3 . 
Another example is methyl ethyl ketone, CH 3 .CO.C 2 H 5 . Note 
that whereas the aldehyde group is necessarily at the end of the 
molecule, the ketone grouping always falls between two carbon 
atoms. 

Although the two possess some properties in common, ketones 
differ from aldehydes in their inability to polymerize, their 
greater resistance to oxidation, and their lessened reducing 
properties. Ketones will not respond to Tollen's Test for alde- 
hydes, and in general they do not reduce ammoniacal silver 
solutions. When, adjoining the carbonyl group of a ketone, 
there is either a primary or a secondary alcohol group, the 
reducing properties of the ketone seem to be increased; a ketone 

* While the prefix "iso" generally means a forked chain compound, its use here 
indicates a secondary alcohol. 



ALCOHOLS 23 

with this structure reacts positively in the reduction of copper 
solutions. Fructose, a keto-sugar, is an example of this type of 
ketone and will be studied later in connection with sugars. 

When oxidation of a ketone is brought about, the molecule 
splits at the carbonyl group, forming two acids, the identification 
of which may sometimes serve as a means of determining the 
structure of the ketone. If ethyl methyl ketone is oxidized we 
may think of the melocule as probably splitting as indicated 
below 

C 2 H 5 /CO.CH 3 

(the smaller radical going with the carbonyl group), oxidation 
then taking place by the addition of a hydroxyl group to each 
half of the molecule. Acetic acid and ethyl alcohol, which 
readily oxidizes to acetic acid, are then formed. 

Acetone, or dimethyl ketone, CH 3 CO CH 3 , a colorless 
liquid of peculiar odor, boils at 56 C. and is made commercially 
by the dry distillation of acetate of lime. 

CH 3 OX) 

; Ca -> CaC0 3 + CH 3 COCH 3 
CH 3 COO 

Ketones in general may be prepared by heating the calcium 
salts of the organic acids containing the required alkyl groups. 
For example, if we wished to make propyl ethyl ketone we 
should distil dry a mixture of calcium propionate and calcium 
butyrate 

c* TT c*r\c\ r^ TJ c*f~\r\ 

l^JLlsLxL/vJ v^ 3 Jrl7v^ULJ 

) Ca+ ) Ca -> 2CaCO 3 + 2C 2 H 5 .CO.C 3 H 7 

C 2 H 5 COO C 3 H 7 COO 

Acetone may be a toxic substance when produced in the body 
under pathological conditions, and is therefore of clinical im- 
portance. From this standpoint it will be discussed in 
Chapter XX. 

OXIDATION OF TERTIARY ALCOHOLS. 

While the oxidation of a primary alcohol will produce an 
aldehyde and the oxidation of a secondary alcohol will produce 



24 ORGANIC CHEMISTRY 

a ketone, the tertiary alcohol, by action of a strong oxidizing 
agent, forms no new class of substances but is split into several 
simpler compounds. 

If we take as an example of a tertiary alcohol (CH 3 ) 2 COH. 
QHs, dimethyl ethyl tertiary alcohol, the oxidation may be 
illustrated as follows: one bond is broken first and then oxidation 
takes place, as with the ketone, by the addition of a hydroxyl 
group to each part of the molecule. Thinking of the mole- 

CH 3 \ 

cule as CH 3 COH, if the uppermost bond breaks the products 
C 2 H 5 / 

will be CH 3 OH and a hypothetical compound with the 

CH 3 \ 

structure C 2 H 5 COH. The methyl alcohol will oxidize in the 
OH/ 

usual way to formaldehyde and then to formic acid. The 
addition of a hydroxyl group to the other part of the molecule 
causes, as is shown above, the impossible condition in which 
two hydroxyl groups are attached to the same carbon atom. 
Water breaks off, and there remains a ketone which oxidizes to 
two acids, as has already been shown. 

Polyatomic Alcohols. 

Polyatomic alcohols are those alcohols containing more than 
one hydroxyl group. 

The term glycol is frequently applied to all those that are 
diatomic, although the name is specifically given to ethylene 
glycol, the first in the series and referable to ethylene. 

Glycol, ethylene glycol, C2H 4 (OH) 2 , may be regarded as a 
substitution product of ethane or as a hydroxy addition product 
of ethylene. It can be prepared from ethylene bromide by 
heating with water at a high temperature out of contact with the 
air. Its behavior is like that of a monatomic alcohol, in that it 
forms alcoholates with metals and replaces the hydroxyl groups 
with negative ions when acted upon by strong acids. 

Upon oxidation, glycol yields a series of products, as each 
primary alcohol group is oxidized separately until both have been 



ALCOHOLS 25 

converted into the carboxyl group, giving oxalic acid as a final 
product of oxidation. 



CH 2 OH 
1 
CH 2 OH 


CH 2 OH 
1 
CHO 


CH 2 OH 
1 
COOH 


CHO 

1 
COOH 


COOH 
1 
COOH 


glycol 


glycollic 
aldehyde 


glycollic 
acid 


glyoxylic 
acid 


oxalic 
acid 



Glycerol, or ordinary glycerine, is the first of the trihydric 
alcohols and the only one which we shall consider. It is a 
substitution product of propane and is assigned the structural 
formula CH 2 OH.CHOH. CH 2 OH, or C 3 H 5 (OH) 3 . It is the base 
of all common animal fat and vegetable oils and is produced 
when fat is saponified (page 107). Its dental use and detection 
are discussed in Chapter X. 

The sweet taste noticeable in glycerine seems to increase upon 
the addition of hydroxyl groups, the hexatomic alcohols those 
containing six hydroxyl groups being directly related to the 
sugars (see Chapter XII). 



CHAPTER III. 
ETHERS. 

Ethers may be regarded as oxides of the hydrocarbon radi- 

C 2 H 6 \ 
cals, as O, or as anhydrides of the monatomic alcohols, 



water having been removed from two molecules of the alcohol: 
2 QHsOH - H 2 O = (C2H 5 ) 2 O. 

Ethers may be simple or mixed. The simple ether is illustrated 
above by the formula for ordinary or ethyl ether, where two 
radicals of the same kind are united by an atom of oxygen. 

In a mixed ether, these radicals will be of different kinds; 
as, for example, CH 3 O C 2 H 5? methyl-ethyl ether. 

A general method for the preparation of simple and mixed 
ethers is that of distillation of the corresponding alcohols with 
sulphuric acid, as illustrated by Experiment No. 39, page 255. 
They may also be produced by the action of silver oxide on the 
corresponding alkyl iodides: 

2 C 2 H 5 I + A g2 - (C 2 H 6 ) 2 + 2 Agl, 

also, by treating the sodium alcoholate with an alkyl iodide, 
C 2 H 6 ONa + C 2 H 6 I = (C2H 6 ) 2 O + Nal 



or CH 3 ONa + CgHJ - ; O + Nal. 

CJSi 

Methyl Ether. Methyl oxide, (CH 3 ) 2 O, also known as 
formic ether, is isomeric with ordinary alcohol, and may be made 
in a manner similar to that used in the production of ethyl ether 
(q.v.). At ordinary temperature it is a gas, but it liquefies at 
20 C. (Bernthsen). It has been used as a general anesthetic, 
and the resulting anesthesia is said to be profound and quickly 

produced (U. S. D. from A. J. P., Sept., 1870). 

26 



ETHERS 27 

Methyl-ethyl Ether. This name, besides indicating a 
definite compound, as referred to in the preceding paragraph, 
has been applied to a mixture of methyl ether and ethyl ether, 
used for purposes of general anesthesia. 

Methylene Ether. A name applied to a mixture of methyl- 
ene dichloride and ethyl ether. It has been used as an anesthetic, 
but has been found unsafe (U. S. D.). 

Ethyl Ether. Ethyl oxide, (C 2 H 5 ) 2 O. The ether used for 
general anesthesia should contain not less than 95^ per cent or 
more than 97! per cent of ethyl oxide, the remainder consisting 
of alcohol with a little water (U. S. P.). It is a light, colorless 
liquid with a specific gravity of 0.715 at 25 C. ? and a boiling- 
point of about 35 C. It may be made by the action of sulphuric 
acid on ethyl alcohol, and from this fact has been known as 
sulphuric ether, but this name is, of course, incorrectly used. 

In the preparation of ether, sulphuric acid may be mixed with 
rather more than its own bulk of alcohol, the mixture heated to 
a temperature of from 130 to 138 C. in a suitable retort or 
still, the distillate (ether) being collected in a cold receiver. 

The reaction takes place in two steps, as follows: one mole- 
cule of acid and one of alcohol react to form ethyl sulphuric 
acid (ethyl acid sulphate) and H 2 O, H 2 SO 4 + C 2 H 5 OH = 
C 2 H 5 HSO 4 + H 2 0. Then the ethyl sulphuric acid reacts with 
a second molecule of alcohol to form ether and sulphuric acid, 
C 2 H 6 HSO 4 + C 2 H 5 OH - (C 2 H 5 ) 2 + H 2 SO 4 . Thus the sul- 
phuric acid, from two molecules of alcohol, has produced one 
molecule of ether and is in condition to repeat the process, hav- 
ing been changed only to the extent of adulteration with one 
molecule of water. In accordance with this theoretic forma- 
tion of ether by simple dehydration of alcohol by sulphuric acid, 
provision is made for a continuous process, by the introduction of 
a constant supply of fresh alcohol into the retort during the dis- 
tillation, and so regulated that the total bulk of liquid is neither 
increased nor diminished. The product is then purified, and 
freed from water and traces of acid by redistillation over a mix- 
ture of lime and calcium chloride. 

Ether, according to the U. S. P. requirements, is " a trans- 



28 ORGANIC CHEMISTRY 

parent, colorless, mobile liquid with characteristic odor and a 
burning and sweetish taste/' 

It is soluble in about twelve times its volume of water and 
in all proportions in alcohol, chloroform, petroleum ether, ben- 
zene, and oils. It is readily inflammable, and this fact, together 
with its easy volatility, makes it necessary to use considerable 
care when handling it. 

The action of sulphuric acid upon alcohol needs careful regu- 
lation; because there may be produced three other products in 
addition to the ethyl oxide already considered. These are, 
first, ethyl sulphuric acid, C 2 H 5 HSO4; second, ethyl sulphate 
(2115)2804, these being respectively the acid and neutral ethyl 
esters of H^SC^; third, the hydrocarbon ethylene, C2H 4 . This 
latter compound is the first of the ethylene series of hydro- 
carbons with the general formula C n H 2 and containing " double- 

H \ / H 

bonded " carbon atoms, ,C = C. or CH.CH 3 . 

IT N H 

These are unsaturated hydrocarbons (see page n). Ethylene 
is produced by the action of an excess of concentrated sulphuric 
acid, which abstracts water from each molecule of alcohol 
(C 2 H 5 OH H 2 O = C 2 H 4 ), whereas in the preparation of ether 
the more dilute acid abstracts water from two C2 



CHAPTER IV. 
ORGANIC ACIDS AND ESTERS. 

If the oxidation of an alcohol is carried beyond the formation 
of aldehyde or ketone, i.e., if the aldehyde or ketone be oxidized, 
an organic acid results. The first atom of oxygen involved in 
this process does not become a constituent part of the new 
molecule, but simply withdraws hydrogen from the old (the 
alcohol), as shown in the formation of aldehydes on page 18. 
The second atom of oxygen, however, attaches itself to the 
molecule and does become a part of the new substance (the acid) : 



CHs CHs CHs 

I + O = I + H 2 O I + O = I 

CH 2 OH CHO CHO COOH 

Alcohol. Aldehyde. Aldehyde. Acid. 

The group COOH is known as carboxyl and is the char- 
acteristic group of the acids. The hydrogen of the carboxyl 
differs from the other atoms of hydrogen in the molecule in that 
it is united to oxygen rather than to carbon, and constitutes the 
basic or replaceable hydrogen of the acid; hence, acetic acid is 
monobasic, and the only possible salt of potassium, for instance, 
is CH 3 -COOK. 

The basicity of the acid depends on the number of carboxyl 
groups it contains. 

Among the monobasic acids of the fatty or paraffin series 
which we shall study are the following: 

Representative Fatty Acids. 

H.COOH = formic acid or hydrogen formate; 
CH 3 .COOH = acetic acid or hydrogen acetate; 
QHs.COOH = propionic acid or hydrogen propionate; 
CsH 7 COOH butyric acid or hydrogen butyrate; 
valeric acid or hydrogen valerate; 
29 



30 ORGANIC CHEMISTRY 



= palmitic acid or hydrogen palmitate; 
Ci7H 35 COOH = stearic acid or hydrogen stearate. 

The acids of these series are represented by the general formula 
C n H 2n 62. They all are monobasic; i.e., they contain only one 
atom of replaceable hydrogen. 

All of the organic acids of this series may be prepared either 
by oxidation of the alcohol as suggested above or by treatment 
of the nitril with water, nitril being the term applied to an 
organic cyanide. 

CH 3 CN + H 2 -> 



More frequently potassium hydroxide is used in place of the 
water, the potassium salt of the acid then being formed. 

Formic Acid, (H.COOH), originally distilled from the bodies 
of ants (the name being derived from the Latin, formica, an ant,) 
is a colorless, easily volatilized liquid. It may be prepared in 
the laboratory by heating oxalic acid with glycerol, the oxalic 
acid breaking up into formic acid and C0 2 . 

C 2 H 2 4 = C0 2 + HCOOH. 

Carbon monoxide, passed over hot potassium hydroxide, 
results in the formation of potassium formate, 

CO + KOH = HCOOK. 

Also, by treatment of ammonium carbonate with nascent hydro- 
gen (sodium amalgam), 

(NH 4 ) 2 C0 3 + 2 H = HCOO(NH 4 ) + H 2 + NH 3 

and 

HCOO(NH 4 ) + NaOH = HCOONa + NH 3 + H 2 0. 

Formic acid, according to the above reaction, is apparently 
carbonic acid less one atom of oxygen, and the fact that formic 
acid acts easily as a reducing agent, taking away oxygen from 
other bodies and becoming H 2 CO 3 , is further proof of this re- 
lationship. 

Acetic acid, CH 3 COOH, is obtained commercially by the 
oxidation of ethyl alcohol. It is the acid of vinegar, which, 



ORGANIC ACIDS AND ESTERS 31 

according to Massachusetts law, should contain 4 per cent of 
acid. Glacial acetic acid is a commercial name of the acid con- 
taining i per cent or less of water; it is a colorless solid at a 
temperature below 15 C. The U. S. P. acetic contains only 
36 per cent (by weight) of the pure acid. 

Either one, two, or all three of the hydrogen atoms of the 
CH 3 group may be replaced by chlorine, forming respectively 
the mono-, di-, and tri-chloracetic acids, the tri-chloracetic acid 
being used to a considerable extent in dentistry (page 91). 

Acetic acid, by the abstraction of water, forms an anhydride, 
C 4 H 6 3 : 

2 HC 2 H 2 2 = (C 2 H 3 O) 2 O + H 2 O. 

This substance is of considerable importance in organic reac- 
tions. It is a colorless liquid with a boiling-point of 138 C., 
and, with the halogens, forms compounds such as acetyl chloride, 
C 2 H 3 OC1, the radical C 2 H 3 O being known as the acetyl radical. 

Propionic acid, CH 3 .CH 2 .COOH, is a colorless liquid, boiling 
at 140 C. According to Witthaus, it is best prepared by 
heating ethyl cyanide with caustic potash until the odor of the 
ester has disappeared: 

C 2 H 6 CN + KOH + H 2 O = C 2 H 5 COOK + NH 3 . 



Then, by treatment with H 2 SO 4 , the propionic acid is liberated, 
and may be separated by distillation. 

Butyric acid, C 3 H 7 COOH, occurs as a product of fermentation 
of butter, or other animal fat containing butyrin; also from the 
decomposition of lactic acid, two molecules of lactic acid fur- 
nishing one of butyric acid, two of carbon dioxide and two of 
hydrogen (H 2 ). It is an occasional constituent of the gastric 
contents, and may be detected by formation of the ethyl ester 
(page 43). The pure acid is a heavy, colorless liquid with 
characteristic odor, soluble in water in any proportion. See 
page 43 for the glyceryl ester of butyric acid (butyrin); also 
for stearic and palmitic acids. 

Valeric acid, C 4 H 9 COOH, may be made by the oxidation of 
amyl alcohol (C 6 H n OH). It is an oily liquid, boiling at 174 C. 



32 ORGANIC CHEMISTRY 

It occurs as a constituent of valerian, and in consequence has 
been called valeric acid. Its salts are used in medicine as seda- 
tives. 

The valeriate of amyl has an odor resembling that of apples, 
and is used in alcoholic solutions as apple essence. 

Palmitic acid, CuHaiCOOH, a solid " fat acid," occurs as a 
glyceryl ester in butter (to a very slight extent), in olive oil, 
palm oil, and bayberry wax. Combined with certain alcohols 
it occurs in white and yellow wax; also in spermaceti. 

Palmitin, C 3 H 5 (Ci6H3iO 2 )3, occurs in all animal fat and in 
large quantities in human fat. 

Stearic acid, Ci 7 H 3 5COOH, as glyceryl stearate or stearin, 
occurs in vegetable and animal fats, particularly in tallow. 
Stearic acid is only slightly soluble in alcohol or in ether. Its 
melting-point is 69.3 C. 

Acrylic Acid Series. 

If a hydrogen atom of a double-bonded hydrocarbon be re- 
placed by hydroxyl, a double-bonded alcohol is produced, 
which, like the saturated alcohol, may be oxidized to form first 
an aldehyde and then an acid. 

From propylene, the second double-bonded hydrocarbon, 
CH 2 CHCH 3 , we obtain acrylic aldehyde, CH 2 :CHCHO. This 
compound, known as acrolein, is a colorless liquid, boiling at 
52C. Its vapor has an irritating, pungent odor, sufficiently 
characteristic to be used as a qualitative test for glycerol, from 
which it is obtained by heating with KHSCX. 

By oxidation acrylic aldehyde gives acrylic acid, which is a 
type of the double-bonded acids the acrylic acid series. It 
is a liquid with boiling-point at 1 40 C . Nascent hydrogen breaks 
the double bond, forming propionic acid, CH 3 .CH 2 .COOH. 
Hydriodic acid will also break the double bond by direct union 
of its constituents, forming CH 2 I CH 2 COOH, /3-iodo 
propionic acid. 

The only other acid of particular interest in this series is oleic 
acid, CnHsaCOOH. It is an important constituent of oils, 
both animal and vegetable. 



ORGANIC ACIDS AND ESTERS 33 



Its glyceryl ester, C^(CnH^CO^)^ forms a large part of 
lard oil, cotton-seed oil, or any oil (of glyceryl base) obtained by 
cold expression. 

Because of its double bond, it has the property of forming 
addition products with the halogens. The iodine and bromine 
addition products are used in the identification of oils, as the 
proportion of olein, and hence the amount of iodine or bromine 
capable of being absorbed, varies in the different oils. 

Dibasic Acids. 

By the oxidation of glycol, shown on page 25, oxalic acid, the 
simplest acid containing two carboxyl groups, is produced. 
The presence of two carboxyl groups gives this acid the power to 
yield, upon ionization, two positive hydrogen ions; hence it is 
the first acid in the dibasic series. This series, like the other 
homologous series studied is built up by the addition of CH2 
groups. 



COOH 


COOH 


COOH 


1 


1 


1 


COOH 


CH 2 


CH 2 


Oxalic acid. 


1 


1 




COOH 


CH 2 




Malonic acid. 


1 






COOH 



Succinic acid. 

Each of these dibasic acids is referable to a diatomic alcohol 
and frequently may be formed from it, as glycol by oxidation 
may yield oxalic acid. The general method for the synthetic 
preparation of all organic acids, by treatment of a nitril with 
water, applies to the dibasic acids if a cyan-substituted acid is 
used in place of the alkyl cyanide. For example: 

CH 2 CN COOH 

I 
+ 2 H 2 = CH 2 + NH 3 

I 
COOH COOH 



34 ORGANIC CHEMISTRY 

Oxalic acid, which may be considered as a type of the di- 
basic acids, occurs as small, colorless crystals (four- or six- 
sided prisms) containing two molecules of water of crystallization 
(H 2 C 2 O 4 .2 H 2 O); it is but slightly efflorescent, and, if carefully 
crystallized, is suitable for the preparation of standard acid 
solution. Salts of oxalic acid occur in many plants; the acid 
potassium oxalate, " salt of sorrel/' is found in common red 
sorrel (Rumex acetora) and in wood sorrel (Oxalis acetocella). 
Oxalic acid in various combinations, often with lime, is widely 
distributed in articles of vegetable diet, particularly rhubarb, 
spinach, and asparagus; grapes, apples, tomatoes, and cabbages 
also carry oxalates but in smaller amounts. 

The source of oxalates in the system is twofold, the in- 
gested oxalates and those produced by oxidation, incident to 
metabolism, the exact nature of which has not been clearly 
demonstrated (see Calcium and Sodium Oxalates, under Urine 
and Saliva). 

Oxalic acid was previously made commercially by the action 
of strong nitric acid on starch or sugar; it is now prepared by 
heating cellulose (in form of sawdust) with a mixture of po- 
tassium hydroxide and sodium hydroxide, precipitating the acid 
as CaC 2 O 4 , and decomposing the salt by sulphuric acid. The 
acid is then purified by repeated crystallization. 

Malonic acid, COOH CH 2 COOH, is an oxidation product 
of malic acid (from apples), and is comparatively unimportant. 

Succinic acid, COOH(CH 2 ) 2 -COOH, occurs in amber, from 
which it takes its name (Latin, succinum, amber). It has been 
detected in the urine after asparagus and some fruits have been 
eaten. It occurs as colorless crystals, soluble in water, and ony 
slightly soluble in ether. Succinic acid may be obtained by the 
saponification of ethylene cyanide, C 2 H 4 (CN) 2 , and is a dibasic 
acid containing four carbon atoms. It is a constituent of some 
transudates and cyst fluids. It occurs in the spleen and thyroid 
gland, and has been found in sweat and in the urine (Ham- 
mars ten). 

Pyro-tartaric acid, glutaric acid, formed by the distillation of 
ordinary tartaric acid, is one of four isomers of formula 



ORGANIC ACIDS AND ESTERS 35 

and is of interest only in its relation to some of the amino acids 
which result from protein digestion. Formula for pyro-tartaric 
acid is CH 3 -CHCOOH-CH 2 ~COOH. 

/OH 

Carbonic acid, O = C , is dibasic in that it contains two 

\OH 

atoms of replaceable hydrogen, though not two carboxyl groups. 
It is claimed that a molecule of this sort cannot exist because 
a single carbon atom cannot hold more than one hydroxyl group 
in combination. This acid has never been isolated, all attempts 
to separate it in the pure form resulting in the formation of 
carbonic acid gas and water. Its compounds (carbonates) are 
very common and very important, both in organic and inorganic 
chemistry. Organic salts of carbonic acid may be made by 
treating silver carbonate with alkyl iodide. 

/OAg /OC 2 H 5 

CO + 2 C 2 H 5 I = CO + 2 Agl. 

N OAg X OC 2 H 6 

Oxy acids. 

Hydroxy-acids, or alcohol acids, contain hydroxyl in place 
of one or more hydrogen atoms of the fatty acids. Thus we 
may consider 

Carbonic acid as hydroxy-formic acid, HO COOH; 

CH 2 OH 

Glycollic acid as hydroxy-acetic acid, I ; 

COOH 

C 2 H 4 OH 

Lactic acid as hydroxy-propionic acid, I ; 

COOH 

CHOH-COOH 

Malic acid (from apples) as hydroxy- I 
succinic acid, CH 2 - COOH 

CHOH-COOH 

Tartaric acid as di-hydroxy-succinic I 
acid. CHOH-COOH 



36 ORGANIC CHEMISTRY 

Citric acid, from lemons, limes, etc., is in a class by itself. 
It is a tribasic acid (has three carboxyl groups and one hydroxyl) ; 
the formula is C 3 H 4 OH-(COOH)3. 

Glycollic acid occurs in nature in unripe grapes, and possibly 
as antecedent to oxalates in the system (Dakin, Journal of Biol. 
Chem., 3.57). Glycollic acid is formed from glycol by oxidation, 
and from glycocoll, by action of nitrous acid. 

Nitric acid will oxidize glycollic acid to oxalic acid. 

Lactic Acid. a-hydroxy propionic acid, or i*-ethylidene 
lactic acid, CH 3 CHOH COOH, is ordinary lactic acid 
produced by fermentation of milk-sugar, etc. It occurs in the 
gastric juice and in contents of the intestine, " particularly 
during a diet rich in carbohydrates," possibly in muscle (Chapter 
XV) and brain tissue (Foster). It is not volatilized at temper- 
atures below 1 60 C. 

The optical inactivity of the lactic acid obtained from these 
sources is due to the fact that it is a racemic acid, that is, it 
consists equally of d-rotary and 1-rotaryf compounds, which 
have a neutralizing effect on each other. 

Sarcolactic Acid. This is the lactic acid more commonly met 
with, in physiological chemistry, perhaps, and it may be con- 
sidered as the dextro-rotary portion of the racemic acid. It 
occurs in meat extract, and its presence causes the acid reaction 
of dead muscle, possibly of contracted muscle. It occurs in the 
blood and at times in the urine, and it is probable that it is this 
modification that may be found as lactates and acid lactates in 
the saliva and urine, the crystalline forms of which have been 
identified by Dr. E. C. Kirk of Philadelphia, by the use of the 
inicropolariscopic method of Dr. Joseph P. Michaels of Paris. 
See Chapters XVIII and XXI. 

The optical activity of the lactic acids depends upon the 
presence of an asymmetric carbon atom. This asymmetric 
carbon, as the name implies, is one holding four different groups 
or atoms, as illustrated by the following compounds. 

* Optically inactive. 

t dextro-rotary, levo-rotary. 



ORGANIC ACIDS AND ESTERS 37 



CH 3 OH (QjHsOs) OH H CH 2 .COOH 

\ / \ / \ / 

c c c 

/ \ / \ / \ 

H COOH H COOH HO COOH 

Lactic Acid. Tartaric Acid. Malic Acid. 

The truth of the above statement regarding the optical activity 
of these substances may be demonstrated quite readily by the 
reduction of the hydroxyl group in sarcolactic acid, as the in- 
active propionic acid results. 

CH 3 OH CH 3 H 

\ / \ / 

C C 

/ \ / \ 

H COOH H COOH 

Active. Inactive. 

The optical activity consists in the power of the substance 
to turn the ray of polarized light to the right or to the left. 

Both of these acids form characteristic crystalline salts of 
zinc and of calcium. In cold water the zinc sarcolactate is 
more soluble than zinc lactate; on the other hand, the calcium 
sarcolactate is rather less soluble than calcium lactate. 

/3-Oxybutyric acid, CH 3 -CHOH-CH 2 --COOH. If there 
is introduced into butyric acid, CH 3 CH 2 CH 2 COOH, an 
OH group, an oxybutyric acid results. If this alcohol group 
(OH) occupies the secondary or ]8 position (i.e., attached to 
the carbon atom twice removed from the carboxyl), the acid is 
the /J-oxybutyric as above. 

By oxidation of the compound, the alcohol group is broken 
up and hydrogen withdrawn to form water, leaving a keto acid, 
CH 3 -CO-CH 2 -COOH, known as diacetic acid. This in 
turn may give off carbon dioxide and become dimethyl ketone, 
or acetone, CH 3 CO CH 3 . These three substances, |8-oxy- 
butyric acid, diacetic acid, and acetone, are classed in von 
Noorden's " Autointoxication/' and in the works of other recent 
writers, as " the acetone bodies," and by this convenient term 



38 ORGANIC CHEMISTRY 

we may refer to them collectively. They occur in diabetic urine 
and, according to von Noorden, in other cases of perverted oxi- 
dation (not sufficient oxidation). 

Tartaric acid is a dihydroxy-succinic acid, COOH-(CHOH) 2 
COOH, obtained from grape juice. 

We see by an examination of the graphic formula of this 
acid that it contains two asymmetric carbon atoms. 

POOH ^ placing the hydrogen or the hydroxyl 

I on similar or opposite sides of the chain, we 

JI c H see how it might be possible to obtain a 

I new form of isomerism depending on the 

OH C OH relative position of the atoms in space and 

' not at all upon their attachment to other 

atoms of the molecule. This is found to 

be the fact and this sort of isomerism, resulting only in differing 

physical properties, such as optical activity, has been called 

physical isomerism or stereo-isomerism. 

A mixture of equal weights of these two kinds of tartaric 
acid crystallized together give an example of what is known as 
(//-forms or racemic compounds (optically inactive). 

The double tartrate of sodium and potassium (Rochelle salt), 
KNaC 4 H 4 Oe, is much used in medicine. 

Tartaric acid combines with potassium and antimony to form 
tartar emetic (KSbOC 4 H 4 6 )2 H 2 0. 

The " scale salts of iron" " ferri et ammonii tartras " and 
" ferri et potassii tartras," are prepared by dissolving freshly 
precipitated ferric hydroxide in the acid tartrate of ammonia or 
potash, and, after evaporation to thick syrup, solidifying in 
thin layers on glass plates. 

Potassium bitartrate, or acid tartrate, KHC 4 H 4 O 6 , is cream 
of tartar, and one of the few salts of potassium only sparingly 
soluble in water. Its commercial source is the wine vat. 

Ammo Acids. 

Amino acids are characterized by an NH 2 group in place of 
one hydrogen of the alkyl radical of the acid. Thus we have: 



ORGANIC ACIDS AND ESTERS 39 

Amino formic, NH 2 COOH, carbamic acid 
Amino acetic, CH 2 NH 2 COOH, glycocoll or glycine 
Amino propionic, CH 3 CHNH 2 COOH, alanine 

In the digestion and metabolism of protein substances amino 
acids are of prime importance. By hydrolytic enzyme action 
in the digestive tract, proteins are broken up eventually into 
amino acids which are capable of being absorbed into the blood 
stream. Here de-aminization takes place and urea is sub- 
sequently formed. (See Chapter XX.) 

Of the amino acids which we are considering, all are derived 
from protein except the amino formic;* all except the first two, 
amino formic and amino acetic, contain one or more asymmetric 
carbon atoms. While, as just stated, these acids are derived 
from protein, a number of them do not occur or have not as yet 
been found in animal muscle, which is our common source of 
protein substance. For example, neither glycine nor its methyl 
derivative, sarcosine, occur in meat as such. 

The amino acids are white, crystalline compounds, easily 
soluble in water, their solutions usually being sweet and in most 
cases giving a neutral reaction toward indicators. This char- 
acteristic is due to the fact that the amino group neutralizes the 
carboxyl group. The acid and basic properties are shown in the 
ability of an amino acid to react either with acids, forming such 
salts as glycine hydrochloride, or with metals, forming metallic 
salts such as copper glycine. 

Although amino acids occur naturally as products of the 
cleavage of the protein molecule, it is exceedingly difficult to 
separate them from this source. They may be prepared in 
general, however, by the action of a substituted halogen acid 

NH 2 

* Amino formic, or carbamic acid, I , is a hypothetical acid consisting 

COOH 

simply of an amino group, NH 2 , united to a carboxyl group, COOH. By the union 
of ammonia and carbon dioxide the ammonium salt of this acid is formed, 

NH 2 
2 NH 3 + CO2 = I 

COONH 4 

Ammonium carbamate is a constituent of commercial ammonium carbonate and 
an antecedent of ammonium carbonate in the hydrolysis of urea. 



40 ORGANIC CHEMISTRY 

with alcoholic ammonia. The ammonium salt of the acid is 
formed. 

All of these acids obtained from protein are a-amino acids; 
that is, the NH 2 group is joined to the carbon atom next to the 
carboxyl group, and may be grouped as : 

(1) Glycocoll, amino-acetic, CH 2 NH 2 COOH 

(2) Amino-propionic acid derivatives 

Alanine, a-amino-propionic, CH 3 CHNH 2 COOH 
Phenyl alanine, a-amino-/3 phenyl propionic 
CH 2 C 6 H 5 .CHNH 2 .COOH 

Serine, a-amino-/J-hydroxy-propionic CH 2 OH.CHNH 2 COOH 
Tyrosine, a-amino-|8-para-hydroxy-phenyl-propionic 
CH 2 (C 6 H 4 OH).CHNH 2 COOH 

Tryptophane, a-amino-jS-indol-propionic 
CH 2 (C 8 H6N)CHNH 2 COOH 

Histidine, a-amino-/3-imidazol-propionic 
CH 2 (C 3 H 2 N 2 )CHNH 2 COOH 

(3) Di-amino acids 

Ornithine di-amino normal valeric 

CH 2 NH 2 CH 2 CH 2 CHNH 2 COOH 

Lysine a-e-di-amino-normal-caproic* 

CH 2 NH 2 (CH 2 ) 3 CHNH 2 COOH 

Cystine di-amino-dithio lactic 

CH 2 SCHNH 2 COOH 

I 
CH 2 SCHNH 2 COOH 

(4) Dibasic acids 

Aspartic, amino-succinic 

CHNH 2 COOH CH 2 COOH 

Glutaminic, amino-glutaric 

COOH.CH 2 CH 2 .CHNH 2 .COOH 

* It will be noted that caproic acid is the next higher homologue to valeric acid 
of the fatty acid series, and contains six carbon atoms. 



PLATE I. ORGANIC CHEMISTRY, 




FIG. i. 
Cystin. 




FIG. 3. 
Urea Nitrate. 





FIG. 2. 
Leucin. 




FIG. 4. 
Hippuric Acid. 




FIG. 5. 
Benzoic Acid (sublimed). 



FIG. 6. 
Tyrosin. 



ORGANIC ACIDS AND ESTERS 41 

In addition to the above acids three others may be included: 
Leucine, a-amino-iso-caproic (CHg^CHCHaCHNHaCOOH; 
Valine, a-amino-iso-valerianic (CH 3 ) 2 CH.CHNH 2 COOH; Argin- 
ine, 3-guanidino-a-amino normal valeric 

NH 2 . 

)C - NH(CH 2 )(CH 2 )(CHNH 2 )COOH 
NIT 

Proline, pyrrolidine carboxylic CH 2 : - CH 2 

I I 

CH 2 CHCOOH 



The properties of the individual amino acids will be considered 
in Chapter XIV. 

Esters. 

Ester is the term applied to compounds in which an alkyl group 
has taken the place of replaceable hydrogen of the acid.* They 
are produced by the action of the acid upon the alcohol, which is 
as nearly free from water as possible. 

Such action by the halogen acids would produce the alkyl 
haloids already considered; for example, CH 3 OH + HC1 = 
CH 3 C1 + H 2 O. As the water produces alcohol and hydro- 
chloric acid by action on CH 3 C1, it must be removed as the 
experiment proceeds. 

Other methods of preparation are (i) the action of an organic 
salt and the alkyl halides: 

CHsCOOK + C 2 H 6 I = CH 3 COOC 2 H 5 + KL 
(2) the action of the acid chloride on the alcohol: 

CH 3 COC1 + C 2 H 5 OH = CH 3 COOC 2 H 5 + HC1 

(acetyl chloride) 

The acetyl group, CHaCO, is the group remaining when the hydroxyl of the 
carboxyl group of acetic acid has been removed. Such a radical in general is 
called an acyl group, and the termination "yl" is commonly added to the name 
of the acid to differentiate between them, as acetyl, propionyl, etc. 

* The term ester is also applied to any organic salt in which either the positive 
or the negative ion is organic. 



42 ORGANIC CHEMISTRY 

(3) the action of the acid anhydride with an alcohol, producing 
the ester and an acid. 

CH 3 CO V 

;0 + C 2 H 6 OH = 
CH 3 CO ' 
CH 3 COOC 2 H 5 + CH 3 COOH. 

Anhydrides. The acid anhydride of a monobasic organic 
acid may be considered as an oxide in which two acyl groups are 
joined to the oxygen atom. Ethers, in contrast, are oxides having 
two alkyl groups combined with the oxygen. 

Acetic anhydride, to which reference has been made under 
acetic acid, is typical of organic acid anhydrides. It may be 
prepared from potassium acetate and acetyl chloride: 

CH 3 COOK + CHaCOCl = 

CH 3 CC> 

^0 + KC1. 
CH 3 CO / 

The acetyl chloride is often produced from phosphorus oxychlo- 
ride and the alkaline acetate, and a second molecule of the 
acetate is then allowed to react with the acetyl chloride, as given 
in the above reaction. 

Acetic anhydride with alcohol, as just shown, produces ethyl 
acetate; with ammonia it produces acetamide. 

These reactions are general for all organic acid anhydrides: 
with alcohols they form esters; with ammonia they yield amides; 
and with water, of course, the acid is produced. 

Various esters will be considered as occasion requires, through- 
out the course. A few are given below. 

Ethyl nitrite, C2HsN02, is a colorless liquid, boiling at 17 C. 
It is used in medicine as Sweet Spirits of Niter, which is an alco- 
holic solution containing traces of the ethyl nitrate, various 
oxidation products, and not less than 3.5 per cent nor more than 
4.5 per cent of the ethyl nitrite. It is prepared from alcohol, 
sulphuric acid and sodium nitrite, as given in Exp. 64. It is 



ORGANIC ACIDS AND ESTERS 43 

insoluble in water, but by action of boiling water or dilute 
alkalies becomes ethyl alcohol, C 2 H 6 NO 2 + KOH = C 2 H 5 OH 
+ KNO 2 . 

Ethyl acetate, CH 3 COO.C 2 H 5 , is formed by heating ethyl 
alcohol, sulphuric acid, and acetate of sodium. This reaction 
constitutes a qualitative test for acetic acid or acetates, the 
odor of the ester being sufficiently characteristic to furnish a 
delicate test. 

The acetic ether of the U. S. P. is " a liquid composed of 
about 98.5 per cent of ethyl acetate and 1.5 per cent alcohol. " 

Ethyl Butyrate, CH 3 -CH 2 -CH 2 -COOC 2 H 5 . This ester 
dissolved in 10 parts of alcohol forms pineapple essence. It 
may be made in a manner similar to the preparation of ethyl 
acetate, i.e., by heating together alcohol, butyric acid, and 
concentrated sulphuric acid. The production of the ester is 
likewise used as a qualitative test for the presence of the acid, 
and employed in the examination of gastric contents, as follows: 
" Heat 10 c.c. of contents with 5 c.c. of strong sulphuric acid 
and 4 c.c. of 95 per cent alcohol; odor of pineapple indicates 
butyric acid." (Hewes.) 

Amyl acetate and amyl butyrate may be obtained by heating 
the respective acids with amyl alcohol (C 5 H n OH) and strong 
sulphuric acid. These esters may also be used in detecting the 
presence of the acid, amyl alcohol being used in place of ordinary 
alcohol. Amyl acetate gives the odor of pears, amyl butyrate 
that of bananas. 

Amyl nitrite, CsHnNO 2 , is a compound used in medicine to a 
considerable extent, usually administered by inhalation. The 
U. S. P. preparation contains about 80 per cent of amyl nitrite. 
It is very soluble and inflammable. 

Fats are esters of glyceryl, C 3 H 5 , also called tritenyl, propenyl, 
etc. This radical forms with hydroxyl (OH) the propenyl 
alcohol, C 3 H 5 (OH) 3 , which is ordinary glycerin or glycerol. 

Glyceryl butyrate or butyrin (CH 3 - (CH 2 ) 2 - COO) 3 C 3 H 5 , 
constitutes (together with smaller quantities of the glyceryl 
esters of capric, caproic, and caprylic acids) about 7 per cent of 
butterfat. These esters are readily saponified by treatment with 



44 ORGANIC CHEMISTRY 

alcoholic potash; then, by decomposition of the potassium salts 
with [2804, the acids, being volatile, may be separated by dis- 
tillation. The amount of volatile fat acids thus obtained is a 
valuable test for the genuineness of the butter. 

For further consideration of fats see Chapter XIII. 



CHAPTER V. 
CYANOGEN COMPOUNDS. SULPHUR COMPOUNDS. 

Cyanogen, C 2 N 2 , is an intensely poisonous gas, colorless, 
heavy (specific gravity 1.81), and inflammable. It is very 
easily soluble in water or alcohol, forming unstable solutions, 
which, upon decomposition, give rise to various nitrogen com- 
pounds, among them ammonia, hydrocyanic acid, and urea. 

Cyanogen may be prepared by heating the cyanides of silver, 
mercury, or gold, or by the dry distillation of ammonium oxalate. 

Hydrocyanic acid, HCN, may be produced by the fermentation 
of the glucoside amygdalin from bitter almonds; also from the 
kernel of peach-stones, cherry-laurel leaves, etc. Hydrocyanic 
acid may be formed by direct synthesis of C 2 H 2 (acetylene) 
and nitrogen. The synthesis is induced by passing electric 
sparks through the mixed gases. It is conveniently prepared in 
the laboratory by distilling a mixture of dilute sulphuric acid 
with potassium ferrocyanide, K 4 Fe(CN) 6 + 5 H 2 SO 4 = 6 HCN 
+ FeSCX + 4 KHSO 4 . Hydrocyanic acid is a colorless, poi- 
sonous liquid, boiling at 26.5 C., with a characteristic odor often 
designated as a peach-stone odor. It is soluble in water, and a 
2 per cent aqueous solution constitutes the acidum hydrocyani- 
cum dilutum of the pharmacopoeia, also known as prussic acid. 

Potassium cyanide (KCN or KCy) occurs in trade as a white 
solid, sometimes granular, more often as a powder. It is intensely 
poisonous owing to the dissociation of the salt and the activity 
of the free cyanogen. 

Potassium cyanide is decomposed by carbonic acid of the air 
with liberation of hydrocyanic acid. The- aqueous solution 
of potassium cyanide hydrolyzes in two distinct ways: the most 
easily apparent at ordinary temperature is that resulting in 
the formation of hydrocyanic acid and potassium hydroxide, 
giving the solution an alkaline reaction: 

KCN + H 2 O = HCN + KOH. 

45 



46 ORGANIC CHEMISTRY 

Upon boiling a solution, the second hydrolysis may be dem- 
onstrated, whereby ammonia and potassium formate are pro- 
duced: 

KCN + 2 H 2 O = HCOOK + NH 3 (Exp. 70.) 

The organic cyanides are known as nitrils or isonitrils (car- 
bylamines) according as the hydrocarbon radical is attached 
directly to the carbon or to the nitrogen of the cyanogen group. 
That is, methyl cyanide would be represented by CH 3 CN, 
while the isocyanide would be CH 3 NC (methyl carbamine); 
the nitrogen atom being in the first place trivalent, in the second 
quinquivalent. 

Of these two classes of compounds, the isocyanides are of 
much greater interest to the student of dental medicine, owing 
to their relation to the isocyanates and to urea. 

Phenyl-isocyanide, C 6 H 5 NC, also known as isobenzonitril, 
is produced by warming aniline (C6H 5 NH 2 ) with alcoholic potash 
and chloroform. The intensely disagreeable odor of phenyl- 
isocyanide is utilized as a test for chloroform or chloral hydrate 
(page 81); or, with chloroform and potassium hydrate, the pro- 
duction of this isocyanide may become a test for aniline, 
acetanilide (antifebrin), and other derivatives of aniline. 

Potassium ferrocyanide, yellow prussiate of potassium, 
K 4 Fe(CN) 6 , is obtained by heating animal refuse with a little 
over one-third its weight of potassium carbonate and scrap 
iron. The mixture is covered so as to exclude the air, and after 
cooling the resulting mass is boiled with water and filtered. 
Upon evaporation of the filtrate, potassium ferrocyanide will 
separate as yellow, four-sided crystals with a formula K 4 Fe(CN) 6 . 
3 H 2 O. The complex acid ion (Fe(CN)e) is not regarded as 
poisonous but can be made to dissociate by the addition of acid. 
See Exp. 73. By the action of strong sulphuric acid the radical 
is broken up and carbon monoxide is evolved. Dilute sulphuric 
acid will yield hydrocyanic acid according to the reaction on 
page 45. 

Potassium f erricyanide, red prussiate of potassium, K 3 Fe(CN)6, 
contains iron in the ferric condition and may be made by oxidiz- 
ing the ferrocyanide by the action of chlorine gas. 



CYANOGEN COMPOUNDS. SULPHUR COMPOUNDS 47 

Cyanic acid, HCNO, may be made by distillation of its polymer, 
cyanuric acid (HCNO) 3. Cyanic acid cannot be made in the 
usual way by decomposition of its salts with mineral acids, since 
in the presence of water cyanic acid becomes ammonium car- 
bonate. 

Potassium cyanate may be prepared by direct oxidation of 
potassium cyanide with lead oxide. 

Ammonium cyanate passes, upon heating, directly into urea. 
See Exp. 79. 

Isocyanic acid, O = C = N H (carbimide) is supposed to be the 
acid of ordinary potassium and ammonium cyanates. 

Fulminic acid (C^N O H), isomeric with cyanic acid 
N^=C--O H and isocyanic acid (O = C = N H), is im- 
portant only because of its relation to the fulminates, which are 
explosive compounds of the acid, with some of the heavy metals, 
such as silver and mercury. 

Thiocyanic Acid or Sulphocyanic Acid. In this acid and 
its salts, the atom of sulphur replaces the oxygen of cyanic acid 
in the empirical symbol (HCNS); but, graphically, the sulphur 
is attached to the basic element (metal or hydrogen) rather than 
to carbon: thus, K S C=N, that is, the sulphocyanate is 
not an iso-compound. For occurrence and relations of HCNS in 
the human body, see Chapters XVIII and XXI. 

SULPHUR COMPOUNDS. 

Mercaptan, an organic sulph-hydrate. The name mercaptan 
comes from two Latin words signifying " taking mercury " 
(mercurium cap tans), because of compounds readily formed with 
mercuric oxide. Representatives of this class of compounds are 
found as derivatives of both the open and the closed-chain 
hydrocarbons. 

Ethyl mercaptan, thio-alcohol, C2H5SH, is a type of this class 
It is a colorless liquid, with bad odor, slightly soluble in water, 
boils at 37 C., and is used in the preparation of sulphonal. 

The mercaptans may be prepared by action of KHS on the 
alkyl haloids: 

C 2 H 5 I + KHS = C 2 H 5 SH + KL 



48 ORGANIC CHEMISTRY 

The thio-alcohols form potassium and sodium compounds 
similar to common alcohol, 

-f- K == C 2 H5oK -f- H. 



Mercaptol, a name which has been applied to the thioketones. 
The simple compounds of this class are not known, as they form 
polymers very readily. A dimethyl-diethyl compound is pro- 
duced in the process for preparation of sulphonal. 

Thio-ethers are organic sulphides prepared in a manner 
analogous to that employed in the preparation of the thio- 
alcohols, the inorganic sulphide being used in place of the sulph- 
hydrate; for example: 2 C 2 H 5 Br + K 2 S = (C 2 H 5 ) 2 S + 2 KBr. 

Sulphones are oxidation products of organic sulphides: as, 



for example, ethyl sulphone S 

CJEL, ^O 

Sulphonal is a complex derivative of mercaptan as previously 
stated. It may be prepared by the action of acetone and ethyl 
mercaptan with hydrochloric acid and subsequent oxidation of 
the resulting product. It possesses hypnotic properties. 

Sulphonic acids as a class may be obtained by the oxidation 
of an organic sulph-hydrate (mercaptan) . This oxidation may be 
produced by the action of nitric acid or potassium permanganate, 
and may be written as follows: 

C 2 H 5 SH + 30 = C 2 H 5 .S0 2 .HO. 

A simple method for remembering the structure of the sul- 
phones and sulphonic acids is to remember them in relation to 
sulphuric acid. If we consider the structure of sulphuric acid 

H-O O 
as S ' , then a sulphone is produced by the replace- 

H-0 X ^O 

ment of both hydroxyl groups with alkyl radicals, while a sul- 
phonic acid is produced by the replacement of one hydroxyl 
with an alkyl radical. 

Taurine is an important sulphonic acid of the paraffin series. 
Its graphic formula shows it to be an amino ethyl sulphonic acid, 



CYANOGEN COMPOUNDS. SULPHUR COMPOUNDS 49 

HS0 3 

v . Taurine is derived from taurocholic acid by 

NH 2 

hydrolysis. This acid is representative of one of the two prin- 
cipal acid groups occurring in the bile, the 4 salts of which may 
be found in pathologic conditions in the urine, or, according to 
Dr. J. P. Michaels and others, in the saliva. 



CHAPTER VI. 
AMINES OR SUBSTITUTED AMMONIAS. 

If one or more of the hydrogen atoms of ammonia, NH 3 , be 
replaced by a hydrocarbon group, the resulting compound is an 
amine; thus CH 3 NH 2 is methylamine, and (CH 3 ) 2 NH is di- 
methyl amine. Trimethyl amine, (CH 3 ) 3 N, has been found 
among the decomposition products of fresh brain, human liver, 
and spleen.* 

When one hydrogen atom only has been substituted in NHs 
the amine is known as a primary amine or amino compound 
(containing the NH 2 group). These may be prepared in a num- 
ber of ways, two of which we shall consider. 

If alkyl iodides or bromides are heated with alcoholic am- 
monia, compounds are produced analogous in composition to 
the ordinary ammonium salts: 

CH 3 I + NH 3 = NH 2 CH 3 .HL 

Upon distilling the methyl ammonium iodide (of this reaction) 
with caustic alkali, the amine results : 

NH 2 CH 3 HI + KOH = NH 2 CH 3 + KI + H 2 O. 

The second method is by the action of nascent hydrogen upon 
alcoholic solution of the nitrils: 

CH 3 CN + 2 H 2 = C 2 H 6 NH 2 . 

The disagreeable odor of carbylamine constitutes a char- 
acteristic test for the primary amines. This is known as Hof- 
mann's Carbylamine Reaction and may be easily brought about 
by warming the amine with a little chloroform and alcoholic 
potash. 

The reaction which takes place is analogous to that given on 

* Vaughn and Novy, Cellular Toxins. 
So 



AMINES OR SUBSTITUTED AMMONIAS 51 

page 13 as a test for chloroform, using aniline, which may be 
regarded as phenyl-amine. 

The secondary amines are those in which two hydrogen atoms 
of ammonia have been replaced, as in dimethyl amine (CH 3 ) 2 NH. 
These compounds have also been called imines (imides) or imino 
(imido) compounds because they contain the " imino " group 
(NH). 

Imides are formed with a number of the dibasic organic acids. 
The one of greatest interest is perhaps the imide of succinic acid 
which may be produced by the following reaction. Ammonium 
succinate subjected to heat splits off 2 H 2 O + NH 3 , becoming 
CH 2 .CO 

I NH. The hydrogen of the imide group may be 

CH 2 .CO X 

replaced by metals such as potassium, silver, or mercury. Suc- 
cinimide may also be produced by heating succinic acid, carbonic 
anhydride, and ammonia. This with mercuric oxide will give 
a white powder soluble in water, which is the mercuric suc- 
cinimide largely used for the treatment of pyorrhea. 

The secondary amines may be produced by further action of 
alkyl iodides and the primary amines. By action of sodium 
nitrite and hydrochloric acid upon fairly strong solution of a 
secondary amine, a nitrosamine is formed which, when mixed 
with phenol and strong sulphuric acid, gives a dark green solu- 
tion which becomes red upon dilution with water. This in turn 
becomes blue or green upon neutralization with a fixed alkali. 

Trimethyl amine, formed with the methyl and dimethyl 
amines, is a liquid with a not unpleasant odor. 

Diamines are derived from two molecules of ammonia, as 



ethylene diamine, C 2 H 4 v 

NH 2 

To this class of compounds belong many of the " ptomaines," 
produced by the putrefaction of organic matter, as putrescine 
(butylene diamine), CH 2 NH 2 (CH 2 ) 2 CH 2 NH 2 , and cadaver- 
ine (penta-methylene diamine), CH 2 NH 2 (CH 2 ) 3 ~CH 2 NH 2 . 

According to Matthews, the so-called carboxylase bacteria 
have the power of splitting off carbon dioxide from amino acids. 



52 ORGANIC CHEMISTRY 

If this bacterial change takes place before de-aminization of the 
amino acid, amines which are highly toxic are produced. Such 
amines are classed as ptomaines. 

AMIDES. 

If the hydrogen of ammonia be replaced by an acyl group, 
an amide results; thus NH 2 (C2H 3 O) is acetamide, or this com- 
pound may be regarded as acetic acid, CH 3 COOH, in which 
the OH has been replaced by NH 2 . 

It may be easier for the student to remember an amide as an 
organic acid with the OH of its carboxyl replaced by the " amido " 
or amino group NH 2 . 

Acetamide may be prepared by the action of strong ammonia 
upon ethyl acetate: 

CH 3 COOC 2 H 5 + NHa = CH 3 CONH 2 + C 2 H 5 OH. 

It forms colorless crystals soluble in both alcohol and water. 

Cyanamide (NH 2 in place of the hydroxyl of cyanic acid), 
NCNH 2 , is prepared by the action of ammonia on cyanogen 
chloride. The calcium compound is of commercial importance 
as a means of utilizing atmospheric nitrogen for agricultural 
purposes. CaC 2 heated with N 2 becomes NCNCa; this in a 
crude state is used as fertilizer. The calcium cyanamide by 
action of carbon dioxide, water, and soil bacteria becomes first 
urea, then ammonium carbonate. See page 54. 

Formamide, CHO.NH 2 , is a liquid miscible with both alcohol 
and water. It boils with partial decomposition at about 200 C. 
Upon heating quickly, it splits into carbon monoxide and am- 
monia. (Bernthsen.) 

Phenyl-formamide, CHO.NHC 6 H 5 , known as formanilide, 
occurs as yellow crystals soluble in water and in alcohol. 

Phenyl-acetamide, CH 3 CONHC 6 H5 (see acetanilide). 

HYDRAZINES. 

From diamide, NEk NH 2 , or hydrazine, may be derived 
such substitution products as methyl-hydrazine, CH 3 NH 
NH 2 ; ethyl-hydrazine, C 2 H 5 -NH-NH 2 ; and phenyl- 
hydrazine, C 6 H 5 NH-NH 2 . 



AMINES OR SUBSTITUTED AMMONIAS 53 

This last-named compound forms, with the monosaccharids and 
with many of the disaccharids, yellow crystalline compounds, 
known as osazones, which are precipitated in characteristic 
crystalline forms, recognizable upon microscopical examination 
and by their melting-points (see under Carbohydrates, page 99). 



CHAPTER VH. 
UREA AND URIC ACID. 

This substance forms about 50 per cent of the total solids and 
about 85 per cent of the nitrogenous matter contained in the 
urine. When we consider that only 5 per cent of the nitrogenous 
waste passes off in the feces and 95 per cent in the urine, the 
importance of urea as an index of the nitrogen excreted and of 
protein metabolism becomes apparent. 

Urea was the first organic substance synthesized from in- 
organic compounds. This was accomplished by producing a 
molecular rearrangement of ammonium isocyanate. The reaction 
is conveniently brought about by the double decomposition of 
potassium cyanate and ammonium sulphate and subsequent 
evaporation of the solution to dryness: 

2 CNOK + (NH 4 ) 2 SO 4 = 2 OCN.NH 4 + K 2 SO 4 . 
Then O=C=N NH 4 (ammonium isocyanate) + heat = 



HNrCT I (urea). 
X 

Urea was formerly considered as the amide of carbonic acid, 
CO(NH 2 ) 2 , and from this type has been explained the rapid 
transformation of urea into ammonium carbonate in stale 

X NH 2 

urine. O = C. with one molecule of H 2 O becomes 

NH 2 

/ ONH4 
O=C V or ammonium carbamate, and this, by addition 

N NH 2 

/ ONH 4 

of a second molecule of water, becomes O = C or am- 

ONH 4 

monium carbonate, (NH 4 ) 2 CO 3 . The last part of the reaction 
takes place whenever commercial " ammonium carbonate " 

54 



UREA AND URIC ACID 55 

[really a mixture of carbamate (NH 4 NH 2 CO 2 ) and acid 
carbonate (NH 4 HCO 3 )] is dissolved in water. 

Urea crystallizes in long needle-shaped crystals of the rhombic 
system. It is insoluble in water, somewhat soluble in alcohol, 
and nearly insoluble in ether. It fuses at 132, and at a somewhat 
higher temperature it gives off ammonia and ammonium car- 
bonate, and at 160 leaves a residue of ammelide, cyanuric acid, 
and biuret. Urea is decomposed by solutions of the alkaline 
hypochlorites or hypobromites, being broken up into N, C0 2 and 
H 2 O, as follows: 

CO(NH 2 ) 2 + 3 NaOBr = C0 2 + N 2 + 2 H 2 + 3 NaBr. 

The cyclic formula for urea was suggested by Werner in 1914, 
based on the action of urea and some closely allied compounds, 
In static condition and in neutral solution, according to Werner, 
the molecule of urea may be best represented by the formula: 

/NH 3 
HN:C I 
\0 

In the presence of strong acid, the equilibrium of the molecule 
is disturbed, and a hydrogen atom from the NH 3 group goes to 



the oxygen, forming a hydroxyl group: HN:C .Werner 

Isourea X OH 

also suggested that under some conditions, though he has not been 
able to determine them, the hydrogen atom may pass to the NH 
group, giving the carbamide formula to which we are accustomed. 

Cyanuric acid, N 3 C 3 03H 3 , is a polymer of cyanic acid (NCOH), 
which is, at first, formed in the above decomposition. 
X CO-NH 2 

Biuret, H N , may be obtained by heating 

X CO-NH 2 

urea. When pure, it occurs as white, needle-shaped crystals. 
With NaOH and i per cent CuSO 4 it gives the characteristic 
violet and rose-red shades obtained in the biuret reaction (Pio- 
trowski's protein test). Exp. 155, page 276. 

Urea nitrate may be precipitated from fairly concentrated 
urine by addition of HN0 3 . It separates in hexagonal crystals 



56 ORGANIC CHEMISTRY 

or plates, easily recognizable under the microscope (Plate I, 
Fig. 3, opposite page 40). 

Urea Oxalate. Upon addition of a solution of oxalic acid 
to concentrated urine, crystals of oxalate of urea are precipitated. 
They are rather more easily obtained in characteristic forms 
(Plate II, Fig. 5, opposite page 77) than are the crystals of 
nitrate, and, in consequence, treatment with oxalic acid con- 
stitutes a better method for the qualitative detection of urea in 
the body fluids than the nitric acid test formerly used. These 
crystals polarize light, and the use of the micropolariscope facili- 
tates their detection. 

Substituted Ureas. A hydrogen atom may be replaced by 
an alkyl radical in either one of the amino groups of urea, if 
we use the former carbamide formula, thus producing a methyl 



or ethyl urea (carbamide), O = C , or in the hydroxyl 

X NHCHa 

X OCH 3 

group of isourea as HN = C thus producing methyl 

NH 2 

isourea. Both methyl urea or methyl carbamide and methyl 
isourea have been known in the free state. 

Or by introducing an acid radical a ureide may be formed as 

,NH 2 
v 



acetyl urea represented formerly as O = C but 



now more correctly represented as HN= C 



NH(C 2 H 3 O) 
NHCOCH 3 

\ 



OH 

In the case of a dibasic acid, such as oxalic (COOH) 2 , entering 
into the reaction, one or both (OH) groups may be split off, form- 

NH 2 

X NH.CO.COOH 
NH.CO.COOH 



ing in the first instance a ureide acid, O = C ' 



More correctly represented perhaps as HN=C 



oxaluric acid 



UREA AND URIC ACID 57 

NH~C=O 

or, in the second case, a ureide, as O = C I parabanic 

X NH-C=O 
acid. 

If the residue of two molecules of urea enter into the composi- 
tion of the new molecule, the compound is a diureide. Of this 
class one of the most important is : 

Uric acid, trioxypurin, CsHUN^a- Its relation to urea may 

NH - CO 

I I 

be shown by the graphic formula O = C C = NH 

I II , C = O 

NH - C - NH / 

Uric acid is also referable to " purine," by the use of which the 
relationship of xanthin, hypoxanthin, and other " purine " or 
nuclein bases is easily demonstrated. 

These bases are of great physiological interest, in that they 
form an unquestioned link between the decomposition products 
of the proteins, nuclein, etc., on the one hand, and uric acid 
and the urates on the other. 

Uric acid normally occurs in the urine combined with alkaline 
bases, also with traces of calcium and magnesium. It is insoluble 
in alcohol, ether, or dilute acids; practically insoluble in water, 
but much more soluble in solutions of urea or of glycerin. A 
solution of uric acid does not redden blue litmus. 

Purine is a white, crystalline substance melting at 216 C., 
soluble in water and of weak basic character but not sufficiently 
basic to turn litmus blue (Holleman- Walker). Its formula is 

N=C-H 

I I 

C6H 5 N 4 , or graphically as H C C N H . If we now 

II )C-H 

N-C-N^ 
break all double bonds except those linking two carbon atoms 

i - N-C 6 

I I 
(4 and 5), we obtain a graphic nucleus, 2 = C C 5 N 7 

I II ) C = 8, 

3 - N-C 4 -N-Q 



58 ORGANIC CHEMISTRY 

by numbering the atoms of which we may easily designate any struc- 
tural formula of the group ; thus, 268, trioxypurin, is uric acid as 

H-N-C=O 

I I 

above, while xanthin is 2 6, dioxypurin, O = C C N H , 

I II >C-H 
H-N-C-N 

CH 3 -N-C = O 

I I 
and 13 7, trimethyl-xanthin, = C C N CH 3 , is caffein 

I II >C-H 

XT r* XT " 



and thein, alkaloids from coffee and tea. 

Traces of xanthin (2.6 dioxypurin), hypoxanthin (6 oxypurin), 
guanin (2 imino, 6 oxypurin), adenin (6 amino purin), and 
heteroxanthin (7 methyl xanthin) have been found in urine, 
and, in cases of leukemia, many of them in increased amounts, 
notably xanthin, hypoxanthin, and adenin (Witthaus). 

Upon heating uric acid, urea and cyanuric acid may be ob- 
tained; NHs and C0% are given off. We are not to infer from 
this decomposition that the uric acid is an antecedent of urea 
in the animal body; for such is not the case, except possibly 
to a limited extent. 

Uric acid produces, upon oxidation, a variety of compounds, 
according to the temperature and the oxidizing agent employed. 

Chlorine, hot, yields cyanuric acid, C 3 N 3 (OH) 3 . Chlorine or 

/ NHCO 
bromine, cold, forms oxalic acid, alloxan CO CO, 

NHCO ' 
/ NH - CO 
parabanic acid CO I and ammonium cyanate. 

^ NH- CO 

HNOs in the cold, forms alloxan, alloxan tin, and urea (Witthaus). 
Uric acid may be detected by the murexide* test. See Exp. 
84, page 263 , also by the more delicate phosphotungstic acid test, 
page 264. 

While uric acid is practically insoluble in H 2 O and the acid 

* Note. Murexide is a definite chemical compound (CgHs^Oe) and may be 
produced from alloxantin, an oxidation product noted above. 



UREA AND URIC ACID 59 

urates only sparingly soluble, the uric acid in the ^system is 
apparently held in solution as an acid urate (NaHU) by the 
presence of the sodium phosphates, NaH 2 PO 4 and Na^HPCX, 
possibly also aided by the presence of some unknown organic 
combination. 

NaHU + NaH 2 PO 4 forms, at 38 C., a solution with an acid 
reaction; if, however, the mixture is cooled to room tempera- 
ture, the reaction becomes alkaline from Na^HPO^ and uric 
acid is precipitated (Bunge) : 

NaHU + NaH 2 PO 4 = Na 2 HPO 4 + H 2 U. 

Na 2 HPO 4 is a normal constituent of the blood, and a tendency 
to precipitate uric acid may be met by the following reaction: 
Na 2 HPO 4 + H 2 U = NaH 2 PO 4 + NaHU. Because the acid 
urate of lithium is much more soluble in water than any of the 
other monometallic urates, lithium salts have long been used as 
uric acid solvents. But the fact that lithium solutions will 
precipitate from solutions of Na 2 HPO 4 crystals of Li 2 HPO 4 , has 
been made the basis for a claim that such use of lithium salts is 
without effect other than to decompose and render insoluble 
the alkaline phosphate, which has been acknowledged a valuable 
factor in keeping uric acid in solution. While the disodic phos- 
phate is regarded by many as superior to lithium salts as a uric 
acid solvent, the fact of comparative insolubility of Li 2 HPO 4 
can hardly be regarded as conclusive evidence that lithium com- 
pounds are not effective. 

The following in regard to our need for " sarsaparilla " in 
the spring is given by Dr. E. C. Hill, of the University of Den- 
ver, in his text-book of chemistry, page 370: " Reduced alkalinity 
of the blood, as in winter from eating meats freely, throws 
uric acid out of solution to collect in the more acid tissues (spleen, 
liver, and joints). With the vernal tide of alkalinity (due to 
freer sweating, with excretion of fatty acids) these deposits are 
swept out in the blood-current, irritating the nerves and giving 
rise to ' that tired feeling/ " 



CHAPTER VIII. 
CLOSED-CHAIN HYDROCARBONS. 

In illustrating the simpler relationships of organic compounds 
we have, as far as possible, carefully avoided reference to the 
closed-chain or aromatic compounds, as the characteristic group- 
ings are more easily seen by the use of simple formulae. The 
distinguishing feature of the aromatic (also called cyclic) com- 
pounds is a nucleus consisting of a closed chain of atoms; this 
chain may contain three, four, five, six, or seven members, but 
the six-carbon ring is by far the most important, and the only 
one that we are to consider. 

The hydrocarbons of the aromatic series have, for a general 
formula, C ff H 2w ~ 6 , the simplest being benzene or benzol, C 6 H 6 ; 
and we may consider that the aromatic compounds are derived 
from this. The structure of the benzene molecule is represented 
by Kekule's benzene ring. Note that there are three double 
bonds, which of course permit of addition products, as C 6 H 6 Cl2, 
benzene di-chloride, etc. The substitution products are, how- 
ever, of far greater importance. 

" The principal source of the hydrocar- 

P bons of the aromatic series is coal-tar. 

j, ^ When this substance is distilled, water, 

H C C H ammonium compounds and some of the 

I || lighter hydrocarbons are found in the 

H C C H first portions of distillate, coming over at 

^ ' a temperature below 105 C. The dis- 

I tillate obtained from 105 to 210 is 

jj known as " light oil," and in it may be 

found, among other substances, the first 

three members of the closed-chain hydrocarbons benzene, 
toluene, and xylene. 

Benzene, CeHe (benzol), is a colorless liquid from the " light 

oil " obtained by distillation of coal-tar. It boils at 80, has a 

60 



CLOSED-CHAIN HYDROCARBONS 6f 

gravity of 0.899, * s soluble in ether, alcohol, and chloroform, but 
insoluble in water. It may be made pure by distilling an inti- 
mate mixture of benzoic acid and quicklime, and at a temperature 
of about 5 C. may be obtained as a crystalline solid, C 6 H 5 COOH 
+ CaO = CaC0 3 + C 6 H 6 . (See Exp. 92, page 264.) 

Benzene may be considered as phenyl-hydride, C 6 H 5 H, and, 
as in the case of the straight-chain hydrocarbons, two of these 
phenyl groups may be made to combine, giving a hydrocarbon 
Ci 2 Hio, known as diphenyl. Reaction 2 C 6 HsBr + 2 Na = 
Ci 2 Hi + 2 NaBr. 

Toluene, (toluol). The next higher homologue of the series 
will be C 7 H 8 ; this is methyl benzene (C 6 H 5 CH 3 ) or toluene. 

The hydrocarbons of this series may be prepared in a manner 
similar to that used in the preparation of the hydrocarbons of 
the paraffin series. 

Toluene may be made by the action of metallic sodium upon 
a mixture of brombenzene and methyl iodide. 

C 6 H 5 Br + CH 3 I + Na 2 = C 6 H 5 CH 3 + NaBr + Nal. 

Toluene is a colorless liquid boiling at 110 C., and yielding 
upon oxidation a benzene derivative; i.e., the CH 3 , or so-called 
side chain, is the part of the compound changed by oxidizing 
agents, rather than the benzene ring, 

C 6 H 5 CH 3 +30 = C 6 H 5 CO 2 H + H 2 O. 

Xylene, C 8 Hio (xylol) or dimethyl benzene, the next hydro- 
carbon of this series, exists in coal-tar as a mixture of three 
isomeric compounds which may be graphically represented as 
follows: 



and 






These three possible positions of the second substitution are 
known as ortho-, meta-, and para-; thus, the first representation 



62 ORGANIC CHEMISTRY 

at the left will be ortho-xylene, or ortho-dimethyl benzene. The 
other two will be meta-xylene and para-xylene respectively. ' 

A tri-substituted benzene may be " adjacent/' if the sub- 
stituted element or group is attached to the carbon atoms 
i 2 3, or " unsymmetrical " i 2 4, or " symmetrical " 

1-3-5- 
A fourth isomer of dimethyl benzene would be an ethyl benzene, 

CeH^Hg. This, upon oxidation, yields benzoic acid, in a 
manner similar to toluene . (B ern thsen . ) 

Mesitylene, CgH^, is a trimethyl benzene. Only two isomers 
are possible. It can be prepared by dehydrating acetone by the 
use of sulphuric acid: 

3 C 3 H60 - 3 H 2 = C 9 Hi 2 . 

As the distillation of coal-tar proceeds to a temperature of 
210-240, the distillate is known as " carbolic oil " and contains 
some of the hydroxyl derivatives of benzene (phenols), and a 
hydrocarbon with a structural formula based upon the union of 
two benzene rings, naphthalene (CioHg). This is a white, crys- 
talline solid, which melts at 80 and boils at 218. From 240 
to 270, the so-called " creosote oils " are obtained; in these 
oils are found hydroxyl derivatives of toluene (cresol) and 
naphthalene (naphthol). 

Above 270 a third benzene ring links itself to the two of naph- 




thalene, giving us anthracene I I I J (CuHio), colorless 

plates melting at 213. The residue is pitch, used in preparation 
of roofing materials, etc. 

Of course, it is not to be understood that the various distillates 
contain only the substances indicated above. There are many 
others, and constituents of one fraction are pretty sure to be 
found in lesser quantities in the fractions above and below; e.g., 
the light oil contains some of the phenols, and some naphthalene 
comes over above 240. 



CLOSED-CHAIN HYDROCARBONS 63 

HYDROXY DERIVATIVES OF THE AROMATIC HYDROCARBONS. 

Phenol, carbolic acid, or oxybenzene, CeH^OH, is obtained 
from the distillation of coal-tar, and used as an antiseptic and 
disinfectant. For properties and test, see page 88. Phenol 
acts like an acid, in that it forms salts with the metallic bases, 
CeHsOK, potassium phenolate, but it does not have an acid 
reaction on litmus paper or other indicators, i.e., it does not 
have free hydrogen ions when in solution. It may be made in 
the laboratory from salicylic acid, Exp. 104. 

The three di-hydroxy-benzenes are all of interest and are 
graphically represented as follows: 

OH OH 

OH 0f/A0-dihydroxy / \ meta-dihydroxy 

benzene or I I benzene or 

pyrocatechol \ / OH resorcinol 




and 




para-dihydroxy 

benzene or 

hydroquinol 



OH 



The ortho-compound is pyrocatechol. Its ethereal sulphate 
(acid sulphate) is given by Hoppe-Seyler as a constituent of nor- 
mal urine, and its monomethyl ether, guaiacol, C 6 H 4 OH 
CHa, is obtained from beech-wood creosote, of which it constitutes 
the greater part (60 to 90 per cent U. S. D.). Guaiacol and 
various compounds produced from it have been widely recom- 
mended for tubercular diseases. 

Pyrocatechol has been found to be the most practical reagent 
for the detection of oxidizing enzymes in the saliva. 

Resorcinol is a white crystalline solid, becoming more or less 
colored upon exposure to the light. It melts at 118 C., and, 
in solution, gives a purple color with ferric chloride. Heated 
with sodium nitrate, it produces a substance known as " Lac- 
moid " which is used to a considerable extent as an indicator. 



64 ORGANIC CHEMISTRY 

The hydroquinol, or hydrochinon, is a white powder melt- 
ing at 169 C., and is largely used as a photographic developer. 

Pyrogallol,ortri-hydroxy-benzene,C6H3(OH) 3 (i 2 3), may 
be made by heating gallic acid, and because of this fact is usually 
called pyrogallic acid. It is a white, silky crystal which, like 
hydroquinol, is used as a photographic developer. Dissolved in 
a solution of caustic potash, it absorbs oxygen to a marked degree, 
and may be used as a reagent for the quantitative determination 
of oxygen in gas analysis. 

Phloroglucinol is another tri-hydroxy-benzene, isomeric with 
pyrogallol but with the hydroxyl groups occupying positions 
i 3 5 in the ring. The formula is C 6 H 3 (OH)3 (13 5). 

It crystallizes in rhombic prisms, soluble in water, alcohol 
and ether. This is used in physiological chemistry as a reagent 
with vanillin as a test for free hydrochloric acid. 

Thymol (3 methyl-6 isopropyl-phenol) , CeHsOH^CHa^CsHyce), 
is a solid of the nature of camphor, melting at 44 C., and is 
obtained from various volatile oils, particularly from the oil 
obtained from Thymus Vulgaris. It is very sparingly soluble in 
water. The addition of a little alcohol increases the solubility. 
It is largely used in the preparation of antiseptic dental prep- 
arations, mouth washes, etc. 

Eugenol is synthetic oil of cloves with a formula of CeHaOHd) 
-O~CH3( 2 )~CH 2 CH = CH 2 ( 4 ), largely used as an antiseptic in 
the preparation of dental " cements. " 

Naphthols. Important hydroxyl derivatives of naphthalene 
are the naphthols, d H 7 OH. Only two isomers are possible, 
/V\ /X/X-OH 



f the alpha-compound and [ the beta. Proper- 

\/\/ \A/ 

OH 

ties of the naphthols are not unlike those of the phenols. 

Alpha naphthol melts at 95 C., and is used in physiological 
chemistry as a test for the presence of carbohydrates. (Exp. 126.) 

Cresol, CcILiCHaOH, is a hydroxy-toluene. Three isomeric 
compounds of this formula are obtained from the distillation of 
coal-tar between 200 and 2ioC. The ortho- and para-cresols 



CLOSED-CHAIN HYDROCARBONS 65 

are solid at ordinary temperatures, the ortho compound melting 
at 31 C, the para at 36 C. Meta-cresol is a liquid which does 
not solidify except under extreme conditions of cold and pressure. 

The cresols are similar to phenol, not only in composition but 
also in physical and therapeutic properties; hence, cresol has been 
called cresylic acid, just as phenol has been called carbolic acid. 

A mixture of the cresols, said to be composed of meta-cresol 
40 per cent, ortho- 35 per cent, and para-cresol 25 per cent, con- 
stitutes the tri-cresol very largely used in dentistry as a germicide 
and antiseptic similar to carbolic acid. 

An emulsion of cresol, obtained by the solution of resin soap 
as an emulsifying agent, is known as creolin. Cresol is also a 
constituent of the disinfectant lysol. 

Tri-cresol is miscible with formalin in all proportions, and the 
mixture is recommended in the treatment of root canals. See 
Buckley's " Formo-cresol" page 83. 

AROMATIC ALCOHOLS, ALDEHYDES AND Acros. 

The various hydroxyl derivatives above considered are not 
true alcohols, in that the OH is attached directly to the car- 
bon nucleus, the characteristic alcohol groups, CH 2 OH and 
CHOH, are lacking, and these derivatives yield neither 
aldehydes nor ke tones upon oxidation. 

A type of the true aromatic alcohols is benzyl alcohol, C6H 5 CH 2 - 
OH. Benzyl alcohol is a liquid only slightly soluble in water. 
It gives none of the phenol reactions (Holleman), but may be 
oxidized to an aldehyde, like the primary straight-chain alcohols. 

Benzaldehyde (C 6 H 5 CHO), or " True oil of bitter almonds/' 
is the aldehyde constituting about 85 per cent of the natural oil. 
Benzaldehyde may be made by oxidation of toluene by methods 
which fall short of acid production. By action of KOH it is 
" saponified," producing the alcohol 2 C 6 H 5 CHO + KOH = 
C 6 H 5 CH 2 OH + C 6 H 5 COOK. Benzaldehyde is quite easily 
oxidized to benzoic acid, the simplest of the aromatic acids. 

Benzoic acid, C 6 H 6 COOH, was originally produced from gum 
benzoin, but may be made from hippuric acid (q.v.), which 



66 ORGANIC CHEMISTRY 

(from urine of horses) formerly constituted a commercial source. 
It is chiefly prepared, however, from toluene; (see Exp. No. 113) 
it crystallizes in colorless plates or long prismatic crystals (from 
solution). It is sparingly soluble in cold water, more soluble 
in hot water, easily soluble in alcohol. It sublimes and is in- 
flammable, burning without residue. (Plate I, Fig. 5.) 

Benzoates of sodium, ammonium, lithium, and lime are all 
used in medicine. Benzoated or benzoinated lard is prepared by 
digesting gum benzoin in hot lard. This is much used as a base 
for ointments, and keeps well. 

Salicylic acid, ortho-hydroxy-benzoic acid, C 6 H 4 OH.COOH, 
is a white, crystalline powder, odorless, irritating to mucous sur- 
faces, soluble in alcohol and ether, and in about 450 parts of 
water at 15 C. (U. S. D.). Salicylic acid may be made by 
action of carbon dioxide on sodium phenate and subsequent 
decomposition of the sodium salicylate. By heating rapidly the 
acid may be changed into phenol and carbon dioxide. 

Acetyl salicylic acid, C 6 H4.C 2 H3 Cfe.COOH, known in medicine 
as aspirin, may be obtained by heating salicylic acid with acetyl 
chloride. It occurs as white needles, slightly soluble in water, 
soluble in alcohol and ether. Aspirin is decomposed in the 
intestine, salicylic acid appearing in the urine twenty to thirty 
minutes after administration of aspirin. 

Salicylates have been used to considerable extent in various 
uric-acid diseases. Methyl salicylate constitutes 90 per cent of 
natural oil of wintergreen (Gaultheria). The alcoholic solution 
is essence of checkerberry. 

Salol is phenyl-salicylate, CeH^H.COCXCeHs), a white, crys- 
talline powder, practically insoluble in water and not decom- 
posed by the dilute acids of the stomach juices; in the intestine 
it becomes salicylic acid and phenol, as follows: 

C 6 H 4 .OH.COOC 6 H5 + H 2 = C 6 H 4 OH.COOH + C 6 H 6 OH. 

Gallic acid, a tri-hydroxy-benzoic acid, C6H 2 (OH) 3 COOH, 
(i : 2 :3:s), is prepared from tannic acid by action of dilute 
sulphuric acid, or by oxidation by exposure of powdered galls. 
It forms slightly brownish crystals; if pure, the crystals are 



CLOSED-CHAIN HYDROCARBONS 67 

colorless. At ordinary temperatures one part of acid is soluble 
in about one hundred parts of water, five parts of alcohol or 
twelve parts of glycerine. 

Tannic acid, or tannin, sometimes called di-gallic acid because 
its composition, C^HioOo, corresponds to two molecules of gallic 
acid less one molecule of water, occurs in galls, in many astringent 
drugs and bark from various trees, as hemlock and oak. Tannic 
acid causes dark-colored precipitate with ferric chloride, and 
precipitates gelatin, albumin and starch, differing in all of these 
particulars from gallic acid. (U. S. D.) 

Hippuric acid, benzoyl glycocoll, C 6 H 5 CO.NH.CH 2 -COOH, 
occurs in traces in human urine, to a considerable extent in the 
urine of the herbivora, but not at all in that of the carnivora. 
It crystallizes in prismatic needles (Plate I, Fig. 4, page 40), 
often resembling crystals of ammonium magnesium phosphate; 
but as these latter only occur in neutral or alkaline urine and 
hippuric acid usually in acid urine, there is little danger of con- 
founding the two substances. Hippuric acid is hydrolyzed by 
the urease of fermenting urine, forming benzoic acid and glycocoll 
(amino-acetic acid) : 
C 6 H 5 CO~NH-CH 2 -COOH + H 2 O 

- CeHsCOOH + CH 2 NH 2 COOH 

Tryosin, C 6 H 4 OH--CH 2 CH(NH 2 )~COOH, may be crystal- 
lized as fine, silky needles. It is an amino acid, formed from 
protein substances, particularly casein and fibrin, both by the 
action of proteolytic enzymes and by putrefactive processes. 
It rarely occurs in urinary sediment; when found it is in bundles 
or sheaves (Plate I, Fig. 6, page 40), and is usually indicative of 
acute liver disease, phosphorus poisoning, etc. 

X COOH 

Phthalic acid, CeHU , occurs in the form of rhombic 

COOH 

crystals. By heating phthalic acid, phthalic anhydride may be 
obtained. 

/ co \ 

Phthalic anhydride, CoH^ v . O, heated with phenol and 

^ CO / 



68 ORGANIC CHEMISTRY 

sulphuric acid, will give phenolphthalein, a valuable and familiar 
indicator in volumetric analysis. 

X HS0 3 

Sulphanilic acid, C 6 H 4 x , is made by treating aniline 

X NH 2 

with concentrated sulphuric acid. It is a strong acid, occurring 
as white crystals, is soluble in water, and is used in the manu- 
facture of aniline dyes and also with naphthylamine as a reagent 
for the detection of nitrites. 

Phenyl-sulphuric acid, C 6 H 5 HSO 4 , occurs only in combina- 
tion, the acid being unstable if attempt is made to isolate it. 
Its potassium salt is present in the urine as a product of in- 
testinal putrefaction. 

Phenyl-sulphonic acid may be made by action of oxygen upon 
the sulph-hydrate, as in the process described on page 48. 

C 6 H 5 SH + 30 = C 6 H 5 SO 2 HO. 

The potassium salt of this acid, heated with potassium hydroxide, 
is a commercial source of phenol. 

C 6 H 5 .SO 3 K + KOH = C 6 H 5 .OH + K 2 SO 3 . 

Phenol-sulphonic acid. When phenol is treated with several 
times its volume of cold, strong sulphuric acid, phenol sulphonic 
OH OH 

acid, I |HSO 3 or I ], results. If the mixture is heated for 

\/ \/ 

HS0 3 

some time over a water-bath, the di-sulphonic acid results. 
When this acid is wanned with a nitrate and the mixture treated 
with excess of ammonia, ammonium picrate is produced. This 
constitutes a delicate test for nitrates present in drinking water. 
Phenol-sulphonic acid has been used in dentistry as a thera- 
peutic agent (as antiseptic and otherwise). Such use is discussed 
in detail by Herman Prinz, M.D., D.D.S., in the Dental Cosmos 
for April, 1912, with the conclusion that the ortho compound is 
several times more active than either the meta or para compounds; 
that a one per cent solution is about equal in antiseptic strength 



CLOSED-CHAIN HYDROCARBONS 69 

to a one per cent phenol solution; but that in this strength it 
decalcifies the tooth structure, discolors the teeth, and should 
not be used in the mouth on account of its pronounced acid 
character. 

NITROGEN DERIVATIVES. 

Benzidine, a di-para-diamino derivative of diphenyl is made 
by the reduction of di-nitrophenyl; it is a solid substance melting 
at 122 C., and is used as a reagent in testing for blood. 

Nitrobenzene, C 6 H 5 NO 2 , may be produced by treating ben- 
zene with a mixture of nitric and sulphuric acid at reduced 
temperature. (Exp. 98, page 265.) It is a yellow, oily liquid, 
with the odor of bitter almonds, commercially known as oil of 
mirbane, and used in the manufacture of aniline. 

Aniline or Amino-benzene, C6H 5 NH 2 . By reaction of nitro- 
benzene with nascent hydrogen, the NO 2 group becomes an NH 2 
group and aminobenzene or aniline is produced. Aniline, a color- 
less liquid, also called aniline oil, is important from a commercial 
rather than from a medical standpoint, as it forms the basis of 
the aniline dyes. When pure it is a colorless liquid, but changes 
quite rapidly when exposed to the light. It is used in testing for 
chloral and chloroform. It is slightly soluble in water, and 
easily soluble in alcohol and ether. At 8 C. it becomes a crys- 
talline solid. 

Toluidine, amino toluene (C 6 H 4 .CH 3 .NH 2 ). 

By treatment of toluene with HN0 3 and H 2 S0 4 nitro deriv- 
atives, analogous to nitrobenzene are produced, and these 
by reduction will form the amino compounds. 

Ortho-toluidine is a liquid boiling at about 200. Para-to- 
luidine is a solid melting at 45. 

Diphenyl-amine, (C 6 H 5 ) 2 NH, is formed by the substitution of 
the phenyl group for one of the amino hydrogens of aniline. It 
crystallizes from petroleum ether in white crystals which melt 
at 54 C. 

Acetanilide, C 6 H 5 .NH.COCH 3 , also known as antifebrine, 
may be produced by heating aniline and glacial acetic acid; 
it crystallizes in colorless plates which melt at 115 C. Exp. 77. 



70 ORGANIC CHEMISTRY 

Amino-phenol may be formed by the reduction of nitro- 
phenol by the action of nascent hydrogen (tin and hydrogen 
chloride). The para compound forms an ethyl ester which by 
action of glacial acetic acid gives phenacetine or para-acet- 
phenetidine, 

/)C 2 H 6 . 
CeH4<f 

X NH.CO.CH 3 

Picric acid is tri-nitrophenol, C 6 H 2 .OH.(NO 2 )3. It may be 
formed by action of strong nitric acid, or mixture of sulphuric 
acid and nitric acid on phenol. It occurs as yellow plates slightly 
soluble in water, easily soluble in alcohol and ether, and is used 
in Esbach's reagent for the estimation of albumin in urine and 
as an alkaloidal precipitant. 

Salvarsan, (606), arsenobenzol, more accurately para-diamino- 
dioxy-arsenobenzene hydrochloride, is an arsenic derivative of 
benzene used in medical practice as a specific for syphilis. 

CH 

^ \ 

HC C CH 

Indol, C 8 H 7 N, I II II , is produced from protein 
HC C CH 
\ / \ / 
CH NH 

by the putrefaction occurring in the small intestine, also by 
action of the proteolytic enzyme of the pancreatic juice (trypsin). 
The indol, by oxidation (after absorption from the intestines) 
becomes indoxyl, CgHeNO, which, with potassium sulphate, forms 
indoxyl-potassium sulphate, CgHeNKSCX, and, as such, is elimi- 
nated (in part) by the kidneys. (See page 201.) This substance 
is a type of the so-called ethereal or conjugate sulphates, skatoxyl- 
potassium sulphate (skatol) and phenol-potassium sulphate being 
other compounds of this class. The ethereal sulphates are not 
precipitated by barium chloride in alkaline solutions, but may be 
decomposed by prolonged boiling with hydrochloric acid and then 
precipitated as usual. 



CLOSED-CHAIN HYDROCARBONS 71 

HETEROCYCLIC COMPOUNDS. 

Of the compounds containing elements other than carbon in 
the nucleus, we shall consider only a few. One of the simple 
compounds of this character Thiophene, C 4 H 4 S, is a liquid con- 
stituent of the benzene distillate from coal-tar, boiling at 84, 

- C- C- 

II II 

-C C- 
\ / 

s 

It forms nitro derivatives and reacts chemically with somewhat 
greater ease than does benzene. 

Furfuran, C 4 H 4 O C C , obtained from pine-tar, is a 

II II 
volatile liquid C C boiling at 32 and of much 

\ / 
less importance than O some of its derivatives, notably 

furfuraldehyde and pyromucic acid. 

Furfuraldehyde, C 4 H 3 O.CHO, is a liquid boiling at 162. 
It gives a red color with aniline and hydrochloric acid and is 
responsible for the color reactions of the aldehydes with milk and 
sulphuric acid, of carbohydrates and alpha naphthol, etc. Fur- 
furaldehyde, by action of alcoholic KOH or Ag 2 0, will yield the 
corresponding acid I |~COOH, known as pyromucid acid, 




also furfuryl alcohol, 1 CH 2 OH. 

\/ 

Pyromucic acid may be made by action of heat, dry distilla- 
tion, of mucic acid. It is white, crystalline, and will sublime 
unchanged. Upon distillation of the ammonium salt of pyromu- 
cic acid (or of mucic acid), pyrrol results. 

Pyrrol, C 4 H 4 NH, is of interest because of its relationship to 
the proteins. 

Prolin is pyrrolidine-carboxylic acid CH 2 CH 2 

II II 

CH 2 CH.COOH 

\ / 

NH 



72 ORGANIC CHEMISTRY 

found among the decomposition products of proteins generally 
(with exception of the protamines), particularly among those 
resulting from decomposition of casein and the vegetable pro- 
teins, hordenin and gliadin. 

Pyridine, C5H 5 N, obtained from the light oil of the coal dis- 
tillate and also from bone oil, is a colorless liquid, miscible with 
water. It has a boiling point of 115 C., a faintly alkaline reac- 
tion, and, like benzene, resists the action of chemical agents to a 

marked degree. 

H 
I 

C 

^ \ 
HC CH 

I II 

HC CH 

% / 

N 

Quinoline may be regarded as a condensation product of 
benzene and pyridine. It is a colorless liquid with a high boiling 

point, 236 C. Quinoline and its isomer, isoquinoline, 

' 'N 

Isoquinoline 

are of interest because of their relation to a number of im- 
portant alkaloids. 

H H 

I I 

C C 

^ \ ^ \ 

HC C CH 

I I II 
HC C CH 
^ / ^ / 
C N 
I 
H 

Quinoline 

The alkaloids are complex vegetable constituents frequently 
representing the active medicinal properties of the plant. They 
are basic in character and form salts with the common acids. 




CLOSED-CHAIN HYDROCARBONS 73 

Caffeine and theobromine, already considered in connection 
with uric acid, are purine derivatives obtained from coffee and 
cocoa respectively 

Nicotine is a liquid alkaloid found in tobacco leaves combined 
with malic and citric acids. Its empirical formula is Ci Hi 4 N2. 
It contains both the pyrol and pyridin groups. 

Morphine, Ci 7 Hi 9 NO 3 , and narcotine, C 22 H 23 NO7, are both 
derived from opium and contain the isoquinoline group. 

Heroin, an acetyl derivative of morphine, has been considered 
on page 84. 

Quinine, C2oH24N 2 2 , perhaps the alkaloid best known outside 
of the scientific world, represents the active principle of cinchona 
or Peruvian bark. There are more than twenty alkaloids that 
have been separated from cinchona, quinine being the most 
important and occurring in the largest quantity. Quinine is a 
quinoline derivative. 

Strychnine, C2iH 22 N 2 O 2 , a very powerful alkaloid, is associated 
with brucine in the seeds of Strychnos nux vomica, the so-called 
" Quaker button." Strychnine is quite easily detected by the 
" fading purple test " obtained by drawing a minute crystal of 
potassium dichromate through a drop of concentrated sulphuric 
acid and over the strychnine residue contained in a small porce- 
lain capsule obtained as follows: 

To an unknown aqueous solution or mixture, from which the 
fat has been removed, add acetic acid, producing a salt of the 
alkaloid. In this form the alkaloid is insoluble in chloroform. 
Agitate gently at intervals for several hours, with chloroform, in 
order to remove extraneous coloring matter which might interfere 
with the test. Remove this " acid " chloroform and change the 
reaction of the solution by adding a decided excess of ammonia. 
This precipitates the alkaloid as such. A repetition of the 
treatment with chloroform will now dissolve out the strychnine. 
By separation of the chloroform and evaporation over a water- 
bath, the strychnine residue referred to above is obtained. 



PART II. 
MICROCHEMICAL ANALYSIS. 



CHAPTER DC. 
METHODS. 

The advantages of microchemistry are many, as claimed by 
its enthusiastic advocates, and there are two particulars in which 
microchemical methods strongly recommend themselves to the 
dental practitioner: (i) Microchemical analysis deals with 
exceedingly minute portions of matter, making the examination 
of very small particles of substance easily possible. (2) Three 
or four one-ounce " drop-bottles " and a few two-drachm vials 
will contain all necessary reagents, and in consequence three feet 
of bench-room will furnish ample laboratory space. 

The principles of microchemical analysis are, of course, the 
same as those of any analysis, but the processes employed are 
quite different and need some explanation. In microchemical 
analysis the production of crystals of characteristic form furnishes 
perhaps the most rapid method of detection of an unknown sub- 
stance, and in this we are greatly aided by the use of polarized 
light, which not only helps in the differentiation of crystals but 
often makes it possible to see and distinguish small or trans- 
parent crystals which might otherwise escape notice altogether. 

Use of Microscope. For the examination of the crystals 
mentioned in this chapter, also for the work required on saliva 
or urine, lenses of comparatively low power are sufficient. For 
most of the microchemical tests, a No. 3 Leitz or a i6-mm. Bausch 
& Lomb objective will be found satisfactory. For a few micro- 
chemical tests and for urine, an 8-mm. Bausch & Lomb or a 
No. 5 Leitz objective will give better results in the hands of a 
beginner than one of higher power. 

74 



METHODS 75 

In using the microscope for microchemistry, the preparation 
should always be covered with a cover glass and the examination 
be made with the low-power lens if possible. The object in 
covering is to prevent any action by reagent upon the objective. 
As a further precaution, it is well to form the habit of first 
lowering the objective and then focusing by upward movement 
of the draw- tube. 

Formation of crystals may be brought about two ways : first, 
by precipitating insoluble crystalline salts by use of reagents, as 
in ordinary qualitative analysis; second, by allowing salts to 
crystallize by spontaneous evaporation of the solvent. 

If the first method is to be employed it is essential to have 
the dilution fairly constant in order to obtain crystals comparable 
with those obtained at other times or by other individuals. 
The tendency of strong solutions is to give amorphous precip- 
itates. Sometimes the precipitate will be amorphous when first 
thrown down, but upon standing will assume crystalline form. 
To secure the uniformity of results necessary to correct deduc- 
tions, the following method of procedure should be exactly fol- 
lowed every time. 

The reagent should be of uniform strength, usually one or 
two per cent. Place on a clean microscope-slide a small drop of 
the solution to be tested, and as close as possible without touching 
it, one of about equal size of the reagent to be used. Now bring 
the drops together by tapping the slide or with a small glass rod. 
If a precipitate forms immediately, cover with a cover-glass (this 
must always be done) and examine with the microscope. If the 
precipitate is crystalline, note the form, and in any case, whether 
crystalline or not, repeat the test after diluting the unknown 
solution one-half. If the second test gives an amorphous pre- 
cipitate, or crystals of different shape from the first, continue 
the dilution of the unknown till a point is reached when admixture 
with the drop of reagent gives no immediate precipitate, but one 
appearing in a few seconds' time (five to thirty). In this way 
we have produced the precipitate under standard conditions or 
as nearly so as is possible with unknown solutions. 

Until thoroughly familiar with the forms obtained by drying 



76 MICROCHEMICAL ANALYSIS 

the various reagents, it is well to evaporate a small drop of the 
reagent alone, on the same slide on which a test is made, for the 
sake of subsequent comparisons. 

Filtration in microchemical examinations, when perhaps only 
a few drops of solution are to be had, may be effected in a very 
satisfactory manner and without appreciable loss by absorption 
as follows: 

Cut a filter-paper about i cm. wide and 6 cm. long, double 
it and crease the middle so that it assumes the shape of an in- 
verted V. Put the solution to be filtered in a small watch-glass 
placed at a slight elevation above a microscope slide; now place 
one " leg " of the strip of filter-paper in the watch-glass, allowing 
the end of the other to touch the slide. By capillary attraction 
the clear solution will follow over the bend in the strip of paper 
and a drop or two of perfectly clear filtrate suitable for the test 
will be found upon the slide. 

Evaporation of a solution is best effected on a small watch- 
glass held in the fingers and moved back and forth over a low 
Bunsen flame, or else placed over a water-bath. 

The purpose of the microchemical tests here outlined is not 
so much a method of general qualitative analysis, to which they 
are not suited, as it is a specific application of well-known reac- 
tions to concrete examination of substances, the uses and prob- 
able composition of which are known. The details of the various 
tests will be given under classification furnished by the sub- 
stances investigated. 

Our study may include alloys and amalgams, teeth, tartar, 
dental anesthetics, cement, mouth-washes, antiseptics, disin- 
fectants, and sediments obtained from the saliva and from the 
urine. 

The following crystals are selected as among those most 
frequently met with in the analysis of the above substances, or 
best suited for the study of microchemical processes. The 
student should make each test here indicated and carefully draw 
the crystals produced. 

i. Calcium oxalate from 2 per cent H 2 C2O 4 and CaCl2 solu- 
tions (Plate II, Fig. i). 



PLATE II. MICROCHEMICAL ANALYSIS. 




FIG. i. 
Calcium Oxalate. 




FIG. 3. 
Strontium Oxalate. 





FIG. 2. 
Cadmium Oxalate. 




FIG. 4. 
Sodium Oxalate (P.L.). 




FIG. 5. 
Oxalate of Urea. 



FIG. 6. 
Zinc Oxalate. 



PLATE III. MICROCHEMICAL ANALYSIS. 




FIG. i. 
Ammonium Platinic Chloride. 




FIG. 2. 
Eucaine and Platinic Chloride. 




FIG. 3. 
Potassium Platinic Chloride. 





FIG. 4- 
Cocaine and Potassium Permanganate. 




FIG. 5. 
Tri-brom-phenol. 



FiG. 6. 
lodoform. 



PLATE IV MICROCHEMICAL ANALYSIS. 




;FIO. i. 

Morphine and Marine's Reagent. 




FIG. 3- 
Cocain with Tin Chloride. 





FIG. 2. 
Magnesium Ammonium Phosphate. 




FIG. 4. 
Morphine. 




FIG, S- 
Palmitic Acid. 



FIG. 6. 
Alypin and Potassium Iodide. 



METHODS 77 



2. Cadmium oxalate from 2 per cent H^dO* and CdS(>4 
solutions (Plate II, Fig. 2). 

3. Strontium oxalate from 2 per cent H 2 C 2 4 and Sr(N03)2 
solutions (Plate II, Fig. 3.) 

4. Sodium oxalate by evaporation of aqueous solution, also 
by evaporation of urine containing Na^C^C^ (polarized light) 
(Plate II, Fig. 4). 

5. Urea oxalate from 2 per cent H 2 C204 and urea solution 
(Plate II, Fig. 5). 

6. Ammonium-magnesium-phosphate from magnesium mix- 
ture* and sodium phosphate (Plate IV, Fig. 2). 

7. Ammonium platinic chloride (Plate III, Fig. i). For 
preparation of crystals see Vol. I. 

8. Potassium platinic chloride, K 2 PtCl 6 (Plate III, Fig. 3). 
For preparation of crystals see Vol. I. 

9. Sodium urate, from urine sediment (Plate IX, Fig. 3 
opp. page 214). 

10. Crystals formed from cocaine and potassium permanganate 
(Plate III, Fig. 4). 

11. Crystals formed from phenol and dilute bromine water 
(tri-brom-phenol) (Plate III, Fig. 5). 

12. Crystals formed from morphine solutions and ammonia 
(morphia) (Plate IV, Fig. 2). 

13. Crystals formed from morphine and Marine's reagent 
(Plate IV, Fig. i). 

14. Platinum chloride and /3-eucaine (Plate III, Fig. 2). 

15. Alypin and KI (Plate IV, Fig. 6). 

16. lodoform from acetone and iodine solution in potassium 
iodide (Plate III, Fig. 6). 

The list may be extended to include the crystals produced 
by various alkaloidal salts with the common reagents, also sub- 
stances usually employed in the manufacture of the various 
dental preparations. 

* Magnesium mixture as used in urine analysis to precipitate phosphates con- 
tains MgCl 2 (orMgSO 4 ), NH4C1, and NI^OH. 



CHAPTER X. 
DENTAL PREPARATIONS. 

Among dental preparations, anesthetics are perhaps the most 
important. In considering the chemistry of local anesthetics 
we may divide them into two classes as follows : 

First, those of definite or well-known compositions; and 

Second, preparations of a proprietary nature, the composition 
of which is always problematical. 

In the first class will be found cocaine, eucaine, tropacocaine, 
acoin, ethyl chloride, etc., which will be later alphabetically 
considered. The second class contains a large number of prep- 
arations of all degree of value, among them some of exceeding 
merit and largely used, others of doubtful worth, some worth- 
less if not dangerous. Many of the preparations of this class 
contain cocaine as the anesthetic, and frequently a little nitro- 
glycerin as a cardiac stimulant to counteract the depressant 
effect of the alkaloid. Carbolic acid and oil of cloves are also 
frequently used. 

Many of the constituents of this class of anesthetics may 
readily be identified by the processes of microchemical analysis 
to which previous reference has been made; others may be de- 
tected by special tests, some of which are given under the various 
substances in the following list. This list has been extended to 
include a considerable number of preparations of common 
occurrence. 

Acoin, a synthetic compound, chemically diparanisyl-mono. 

/ / (NC6H 4 OCH 3 )2 \ 
phenetyl-guanidine hydrochloride C HC1 

V \ (NC 6 H 4 OC 2 H 5 ) / 
soluble in both alcohol and water. It is strongly antiseptic and a 
valuable anesthetic, especially in conjunction with cocaine. 
Acoin should be used only in solution and this should be kept in 

a dark place. 

78 



DENTAL PREPARATIONS 79 

Adrenalin, a valuable hemostatic and frequently used in con- 
junction with dental anesthetics, is the active principle of the 
suprarenal gland or capsule. It occurs as very small white 
crystals which are not very stable and only slightly soluble 
in water; hence, the article is usually sold in solution with 
sodium chloride, according to the following formula taken from a 
commercial sample: 

Adrenalin chloride, i part; normal sodium chloride solution 
(with 0.5 per cent chloretone), 1000 parts. This solution is 
usually diluted with the normal (0.6 per cent) salt solution. 
According to the Druggists' Circular, preparations similar to the 
above are also marketed under the names of adrenol, adnephrin, 
hemostatin, suprarenalin (Armour & Co.), suprarenin, etc. 
(See Epinephrine.) 

Alypin. Benzoyl - dimethylamino - methyl -dimethylamino- 
butane hydrochloride, white crystalline, hygroscopic, melts at 
169 C. Soluble in water and alcohol. 

Alypin can be sterilized without decomposition, is not half 
so poisonous as cocaine and is cheaper. It is used in 2 per cent 
solution. The solution should be freshly made and prolonged 
boiling avoided. Alypin is sometimes used with adrenalin. 
(Cosmos, 1908, p. 889.) 

Alypin nitrate occurs as a white, crystalline powder melting 
at 159 C., readily soluble in ether. Mfrs.: Farbenfabriken of 
Elberfeld, Elberfeld (Germany) and New York. (Mod. Mat. 
Med., page 2.) 

Test. Alypin gives needle-shaped crystals with potassium 
iodide, easily produced. (Plate IV, Fig. 6.) 

Ammonium bifluoride is strongly recommended as a solvent 
for tartar by Dr. Joseph Head of Philadelphia. In Items of 
Interest, Vol. 31, page 174, Dr. Head gives the following method 
for its preparation. Hydrofluoric acid is neutralized with am- 
monium carbonate, the solution filtered and evaporated to half 
its bulk, the original volume restored by adding more hydro- 
fluoric acid, and then the resulting mixture is again concentrated 
to half its volume by evaporation. 



80 MICROCHEMICAL ANALYSIS 

Anesthol, or Anaesthol, is a mixture of ethyl chloride and 
methyl chloride, used as a local dental anesthetic. The name is 
also applied to a general anesthetic given by inhalation and con- 
sisting of a mixture of ethyl chloride 17 parts, chloroform 35.89 
parts, and ether 47.1 parts. 

Anaestheaine, a local anesthetic, contains 5 grains of stovaine 
to the fluid ounce. 

Argyrol, a protein compound of silver, occurs as dark brown 
crystals containing 30 per cent of silver. It is easily soluble in 
water. It does not precipitate chlorine nor coagulate albumin, 
and is recommended for use in place of ordinary silver nitrate. 

Aristol is given by the U. S. D. as a synonym for dithymol- 
diiodide which contains 45 per cent of iodine and is used as an 
antiseptic in the same way as iodoform. 

Atropine, an alkaloid obtained from belladonna, is usually used, 
combined with sulphuric acid, (CnH^NOa^EtSC^; the alkaloid 
is only sparingly soluble in water but the sulphate is easily sol- 
uble, dissolving in about one-half part of water at ordinary tem- 
perature. A one per cent solution is said to produce complete 
insensibility of the nerves in cases in which an artificial tooth is 
inserted in a living root. (U. S. D., page 249.) 

Tests. Atropine may be separated from a local anesthetic 
by first rendering the mixture alkaline with ammonia and shaking 
with chloroform. Upon evaporation of the chloroform solution 
on a watch-glass, the resulting residue may be tested by adding 
a drop or two of sulphuric acid and a trace of potassium bichro- 
mate and a little water. The odor of bitter almonds is produced. 
A more conclusive test is to convert the alkaloid, which has 
been dissolved by the chloroform, into a salt, by the addition of a 
few drops of acetic acid, evaporating to complete dryness, taking 
up in a few drops of distilled water and placing one or two drops 
of this solution in the eye of a cat. If atropine is present, a 
dilation of the pupil occurs in from fifteen minutes to an hour 
and a half, according to the amount present. 

Borax. Sodium tetraborate, Na^Oy, is used in antiseptic 
solutions and may be detected as follows: evaporate a little of 
the solution to dryness, add a little HC1, evaporate to dryness 



DENTAL PREPARATIONS 8 1 

a second time, then add a very dilute HC1 solution containing 
tincture tumeric. When this mixture is dried, a beautiful pink 
color appears. If much organic matter is present it may be 
burned off in the Bunsen flame before the addition of any acid. 

Carbolic Acid. See Phenol. 

Chloral hydrate, CC1 3 CHO.H 2 O, a crystalline solid composed 
of trichlor-aldehyde, or chloral, with one molecule of water 
(U. S. P.), easily soluble in water, may become with alcohol a 
chloral alcoholate comparatively insoluble in water. 

Tests. Chloral may be detected by adding to the suspected 
mixture a few cubic centimeters of fairly strong alcoholic solution 
of KOH or NaOH with one drop of aniline oil, and heating. 
Isobenzonitril, which has a peculiarly disagreeable and character- 
istic odor, is thus produced. This test is also given by chloroform, 
which is produced by heating chloral hydrate with caustic alkali. 
If more than traces of chloral are present this latter reaction may 
be a sufficient test. 

Chloretone, CC1 3 COH(CH3) 2 , is the commercial name of 
acetone-chloroform or tertiary trichlor-butyl alcohol. Made 
from chloroform, acetone, and an alkali, it occurs as small white 
crystals, with taste and odor like camphor. It is dissolved by 
alcohol and glycerol and to a slight extent by water. 

Chloroform, trichlor-me thane, CHCla, is prepared by action of 
chlorinated lime on acetone. Chloroform is a heavy colorless 
liquid with a specific gravity of 1.490 at 15 C. It is very volatile 
and used as a solvent for gutta-percha, caoutchouc, many 
vegetable balsams, camphor, iodine, bromine, and chlorine; 
it also dissolves sulphur and phosphorus to a limited extent. 

Tests. It may be detected by its odor, when heated, or by 
the isobenzonitril test, to which reference has been made under 
chloral hydrate. 

Cocaine is the alkaloid obtained from erythroxylon coca. 
The hydrochlorate, CnHoiNOdHCl, is the salt most usually 
employed. This is easily soluble in water and very largely 
used as a dental anesthetic in a T or 2 per cent solution. 

Tests. Cocaine solutions respond to the usual alkaloidal 
reagents. With i per cent solution potassium permanganate 



82 MICROCHEMICAL ANALYSIS 

gives pink plates resembling chloesterol (Plate III, Fig. 4) in 
form but not in color. 

Dilute cocaine solution with picric acid gives a yellow pre- 
cipitate which becomes crystalline on standing. Quite char- 
acteristic crystals may also be obtained from dilute cocaine 
solutions and stannous chloride in the presence of free HC1. 

Creosote. This is a mixture of phenols derived from the 
destructive distillation of wood tar. It is a heavy oily liquid, 
acting when pure as an escharotic. It is analogous in many 
respects to carbolic acid and may be used for similar purposes. 
To distinguish between creosote and carbolic acid, boil with 
nitric acid until red fumes are no longer given off. Carbolic acid 
will give yellow, crystalline deposit; creosote will not. An 
alcoholic solution of creosote is colored emerald green by an 
alcoholic solution of ferric chloride. Phenol is colored blue. 

Cresol is the next higher homologue to phenol, having a 
formula CoKUCHaOH, boiling at 198 C. It is largely used, 
usually together with allied compounds from coal-tar, as anti- 
septic and disinfectant solutions. 

Ektogan is peroxide of zinc, ZnCX designed for external use. 

Epinephrine. This is the active principle in the suprarenal 
glands. Chemically it is an 0-dihydroxyphenyl-ethanolmethyl- 
amine, C 6 H 3 (OH) 2 .CHOH.CH 2 NHCH3. It is a weak base 
which combines with hydrochloric acid to form the hydrochlo- 
ride, in which form it is usually used in dilutions of one part to a 
thousand. It acts as a cardiac stimulant, causing rise in blood 
pressure with slower heart action, acting somewhat in the same 
way as digitalis. 

Ethyl chloride, monochlor-e thane, C 2 H 5 C1, is a gaseous sub- 
stance at ordinary temperature, but when used as a dental 
anesthetic it is compressed to a colorless liquid which has a 
specific gravity of 0.918 at 8 C., is highly inflammable and usually 
sold in sealed glass tubes of 10 to 30 grams each. 

0-Eucaine is the hydrochlorate of benzoylvinyl-diacetone- 
alkamine, and occurs as a white, neutral powder, soluble in about 
thirty parts of cold water. It is used like cocaine as a local 
anesthetic, and is claimed to be less toxic, and sterilizable by 



DENTAL PREPARATIONS 83 

boiling without danger of decomposition. It is usually applied 
in i to 5 per cent solutions, which are conveniently prepared in 
a test-tube with boiling water. It is also marketed in the form 
of 1 1 and 5-grain tablets. (Druggists' Circular.} 

Test. /3-Eucaine gives characteristic crystals with platinic 
chloride. (Plate III, Fig. 2.) 

Eucain Lactate. "Eucain lactate is used in 2 to 5 per cent 
solution as a local anesthetic in ophthalmic and dental practice, 
and in 10 to 15 per cent solution when used in the nose or ear." 
(Review of American Chemical Research, page 97, 1905.) 

Eudrenin is a local anesthetic marketed in capsules of 0.5 c.c. 
containing 1/12 grain of eucain and 1/4000 grain of adrenalin 
hydrochloride. It is used as a local anesthetic, chiefly in den- 
tistry. The contents of one or two capsules, according to the 
number of teeth to be extracted, is injected into the gums ten 
minutes before extraction. Mfrs.: Parke, Davis & Co., Detroit, 
Mich. (Mod. Mat. Med., page 147.) 

Eugenol, Ci Hi 2 O 2 , synthetic oil of cloves, is miscible with 
alcohol in all proportions. Exposure to air thickens and darkens 
it. It should be kept in well-stoppered amber-colored bottles 
(U.S.D.). 

Europhen is recommended by Dr. J. P. Buckley as a sub- 
stitute for iodoform (Dental Review, Vol. 21, page 1284). 

Di-iso-butyl-cresol is described as a bulky, yellow powder of 
faint saffron odor and containing 28 per cent of iodine. (Mod. 
Mat. Med., page 152.) 

Formaline, formol, formine, etc., are commercial names for a 
40 per cent aqueous solution of formaldehyde, HCHO, prepared 
by the partial oxidation of methyl alcohol. Formaline is a 
powerful disinfectant, very generally used. (For test see page 
254, Exp. 28). 

Formo-Cresol. Buckley's Materia Medica combines cresol 
with formaline in proportion of three to one, under the name of 
formo-cresol. 

The putrescent pulp contains bacteria which produce hydrogen 
sulphide, ammonia, and certain amino acids, all of which will 
combine chemically with formaline. The cresol somewhat 



84 MICROCHEMICAL ANALYSIS 

retards the action, tends to make it more complete, and aids 
materially in the study of sterilization of the pulp chamber. 

Glycerol is a triatomic alcohol, C3H 6 (OH) 3 , a colorless liquid 
of syrupy consistency and sweetish taste, specific gravity 1.250 
at 15 C. It is easily soluble in either water or alcohol. 

Tests. Upon heating with acid potassium sulphate (solid) 
it is decomposed, giving off odor of acrolein, which is usually 
sufficient for its identification. A further test may be made by 
moistening a borax bead on a platinum wire with the suspected 
solution (after concentration) and holding in a non-luminous 
flame, to which it will give a deep green color which does not 
persist. Glycerol when present is apt to interfere with charac- 
teristic crystallization of many precipitates. 

Gram's solution, Kuhne's modification, contains 2 grams of 
iodine, and 4 grams of potassium iodide in 100 c.c. of water. 

Gutta-percha. The name signifies scraps of gum. It is ob- 
tained as a milky exudate from a number of tropical trees. It 
is soluble in ether, chloroform, carbon disulphide, toluene, and 
petroleum ether. It may be freed from impurities by shaking 
the solution with calcium sulphate, which will mechanically 
carry coloring matter and other impurities with it as it slowly 
settles out from the mixture. It is not soluble in alcohol or 
in water. 

Heroin is a diacetic ester of morphine. It is usually obtained 
as the hydrochloride and occurs as a white powder, soluble in 
two parts of water. Its action is similar to that of morphine; 
it answers to the usual color tests for morphine, but may be 
distinguished from it by the fact that it will yield acetic ether 
upon heating with alcohol and sulphuric acid. 

Howe's Silver Nitrate Solution. The silver nitrate solution 
used in Dr. Howe's method for sterilization of root canals is a 
nearly saturated ammoniacal solution. It is made by taking 
3 grams silver nitrate, i c.c. water and 3 c.c. strong ammonia. 
The ammonia should be poured into the silver nitrate slowly, and 
the solution shaken constantly. 

The formaline solution, applied a few minutes after the silver 
solution in the treatment, is made by taking 40 per cent U. S. 



DENTAL PREPARATIONS 85 

formalin solution i part, and 3 parts water. At present eugenol 
is used by a great many dentists in place of the formalin. 

Hopogan (also known as biogen) is a peroxide of magnesium, 
MgO2, recommended as a non-poisonous and non-astringent 
intestinal germicide. 

Hydrogen Peroxide, or dioxide, H 2 2 , is, when pure, a syrupy 
liquid without odor or color. It is sold under various trade 
names in aqueous solution containing about 3 per cent and 
yielding upon decomposition about 10 volumes of oxygen gas. 
It is used also as an escharotic in ethereal solutions containing 
25 to 50 per cent H 2 2 . Peroxide solutions may be concen- 
trated by heat without decomposition if kept perfectly free from 
dirt or traces of organic matter. Hydrogen peroxide is readily 
prepared by treatment of metallic peroxides, as Ba0 2 with dilute 
acids. 

Ba0 2 + H 2 S0 4 = BaS0 4 + H 2 2 
or BaO 2 + H 2 O + CO 2 - BaCO 3 + H 2 O 2 . 

This latter reaction has the advantage of producing an insolu- 
ble barium compound and at the same time introducing no 
objectionable acid. The peroxides of sodium, calcium, magne- 
sium, and zinc may also be used; ZnO 2 , however, is comparatively 
expensive and is used in powder form as an antiseptic dressing 
rather than as a source of H 2 O 2 . Na 2 O 2 is valuable as a bleaching 
agent, because for this purpose an alkaline solution is required 
and the solution of Na 2 O 2 in water produces both alkali and H 2 2 
according to the following reaction: 

Na 2 2 + 2 H 2 O = 2 NaOH + H 2 2 . 

Sodium perborate (page 89), also sold as euzone, is a powder 
which will produce H 2 2 in water. Commercial H 2 O 2 solutions 
are usually acid in reaction, as such solutions are more stable 
than if neutral or alkaline. 

Test. Add to a solution of H 2 O 2 a few drops of bichromate 
of potassium solution and a little dilute H 2 SO4. Shake cold with 
a little ether in a test-tube. The ether should be colored blue. 
(For further tests see experiments.) 



86 MICROCHEMICAL ANALYSIS 

Lugol's caustic iodine is made of iodine and potassium iodide, 
i part of each dissolved in 2 parts of water. 

Lugol's Iodine Solution. See appendix under Iodine Solu- 
tion. 

Menthol is the stearopten obtained from the oil of pepper- 
mint. It is a volatile, crystalline substance having a formula 
CeHQOHCHsCsHy. Menthol is but slightly soluble in water 
but freely soluble in alcohol, ether, chloroform, or glacial acetic 
acid. The presence of menthol may usually be detected by its 
odor. If the odor should be suggestive but not distinctive it 
is well to place a little of the substance on a filter-paper, rub it 
between the thumb and finger, thereby obtaining a " fractional 
evaporation," when the more easily volatile substance will pass 
off first, thus producing a partial separation of substances. 

Mercuric chloride, corrosive sublimate, HgCl 2 , is soluble in 
about 1 6 parts of water and 3 parts of alcohol. It is a powerful 
antiseptic, in aqueous solution i/iooo to 1/5000, but should 
never be used in mouth-washes. 

Tests. A drop of the suspected solution with a trace of 
potassium iodide will give a red precipitate of mercuric iodide 
soluble in excess of either reagent. With lime-water or fixed 
alkaline hydroxides a black precipitate is produced. A drop of 
mercurial solution placed on a bright copper plate will leave 
a tarnished spot due to the reduction of the mercuric salt and 
subsequent amalgamation of the metal. 

MethethyL Ethyl chloride mixed with a little methyl 
chloride and chloroform is said to be the composition of a local 
anesthetic sold under the name of methethyl (U. S. D.). 

Methyl chloride, CH 3 C1, is a colorless gas which condenses to 
a liquid at 23 C. Methyl chloride is easily soluble in alcohol, 
somewhat in water, and is used in a similar manner to ethyl 
chloride. 

Morphine, CnHigNOs, alkaloid from opium. Solutions for 
use are made from the sulphate, hydrochlorate, or acetate. The 
alkaloid itself is insoluble in water; its salts are easily soluble. 

Morphine may be separated from solutions containing it by 
making the solution alkaline with ammonia, and shaking out 



DENTAL PREPARATIONS 87 

the precipitated alkaloid with warm ethyl acetate. Upon 
evaporation of the solvent the residue may be tested with 
Frohde's reagent (sodium molybdate, i per cent, in strong sul- 
phuric acid). The color obtained should be a violet, changing 
usually to brown ; a pure blue color is not distinctive for morphine. 
If the morphine solution is of sufficient strength the addition of 
ammonia will produce minute crystals of the alkaloid, as shown on 
Plate IV, Fig. 4. Dental anesthetics containing morphine will 
give precipitates with the usual alkaloidal reagents. Marine's 
reagent (CdI 2 ) gives crystals represented on Plate IV, Fig. i. 

Nirvanin, hydrochloride of diethyl-glycocoll-^-amino-0-oxy- 
benzoic-methylester, has the formula 

(CH 2 N) = (C 2 H5) 2 HC1 
I 
CO.NH.C 6 H 3 (OH)COOCH 3 . 

White prisms, soluble in water and in alcohol, melt at 185 C., 
and give violet reaction with ferric chloride. 

Nitroglycerin, CsH^NOs^, is used as a cardiac stimulant in 
alcoholic solution, the U. S. P. Spiritus Glonoini, containing 
i per cent by weight of the substance. 

Test. Extract the dry substance, or the evaporated residue, 
with alcohol. Filter and evaporate to dryness. Add i c.c. of 
sulphuric acid and i c.c. of phenol-disulphonic acid. Heat over 
a water-bath for five minutes; add water and excess of ammonia. 
A deep yellow color of ammonium picrate indicates nitrates in 
the original substance. Exp. No. no, p. 266. 

Novocaine, discovered by Uhlfelder and Einhorn, is a hydro- 
chloride ^-aminobenzoyl-diethylamino-ethanol. It occurs as 
thin, colorless needles; melts at 156 C., soluble in i part water 
and 30 parts alcohol. It is seven times less toxic than cocaine, 
and three times less toxic than stovaine. It can be sterilized by 
boiling, and is used in 1/2 to 2 per cent solution, often with ad- 
renalin i/iooo. (Mod. Mat. Med., page 275.) 

Novocaine, if intended to represent a solution which is iso- 
tonic with the blood corpuscles, must be dissolved in a 0.92 
per cent sodium chloride solution. (Dental Cosmos, 1910, page 



88 MICROCHEMICAL ANALYSIS 

Oil of cloves, oil of Gaultheria, and other essential oils may be 
detected by the same process of fractional evaporation as sug- 
gested for menthol. In testing for the presence of any substance 
by its odor, it is usually necessary to make a comparative test on 
known samples, using the same methods. 

Orthoform, C 6 H 3 OH(NH 2 )COOCH3, methylpara-amino-meta- 
oxybenzoate, used as an anesthetic and antiseptic, is without 
odor, color, or taste, is slightly soluble in water, and easily soluble 
in alcohol or ether. 

Phenol. Carbolic acid, CeHsOH, is obtained from the de- 
structive distillation of coal-tar. It is a light, oily liquid of 
specific gravity 0.94-0.99. Carbolic acid is usually obtained as a 
white, crystalline mass, soluble in 20 parts of water. The pure 
acid turns pink with age, but does not suffer deterioration on 
account of this change of color. The addition of 5 to 8 per cent 
of water will result in liquefaction of the crystals and cause the 
preparation to become permanently liquid. It is easily soluble 
in glycerol, and strong solutions may thus be prepared. Car- 
bolic acid is sometimes added to local anesthetics with the in- 
tent of rendering the solution sterile, but, as shown by Di. 
Endelman (Dental Cosmos, Vol. 45, page 44), it would be neces- 
sary, in order to prevent the development of micro-organisms, to 
add the acid in proportion that would render the solution unfit 
for hypodermic purposes. 

Tests. Phenol may be detected in the majority of prepara- 
tions by the addition of bromine-water, which gives white crys- 
tals of tri-bromphenol (see Plate III, Fig. 5). See also Exp. 107. 

Phenol Compound. The following is Dr. Buckley's formula 
for treatment of root canals: menthol 1.3 grams, thymol 2.6 
grams, and phenol 12 c.c. 

Potassium hydroxide, KOH, gives an alkaline reaction to 
litmus paper and may be detected by the ordinary methods of 
inorganic analysis. 

Rhigolene is a light, inflammable liquid, obtained from petro- 
leum, boiling at about 18 C., used as a spray for the production 
of low temperature, in the same manner as methyl or ethyl 
chloride. It is readily inflammable, and the vapor, mixed with 



DENTAL PREPARATIONS 89 

certain proportions of air, is explosive. It should be kept in a 
cool place. 

Ringer's solution, which is used as a solvent for novocaine 
and other anesthetics, has the formula: 

Sodium Chloride 0.50 

Calcium Chloride 0.04 

Potassium Chloride 0.02 

Distilled water 100.00 

Saccharin. Saccharin is official in the ninth revision of the 
Pharmacopoeia as benzosulphinidum. It is a derivative of 
toluene, having a formula of C 6 H 4 COSO 2 NH, being benzoyl- 
sulphonimide. It is a white, crystalline powder, melting at 219 
to 222 C. 

It is said to be at least three hundred times sweeter than cane 
sugar and is used in mouth-washes, tooth-paste, etc., as a flavor 
and an antiseptic. 

Test. Add a few drops of potassium hydroxide solution to 
a little saccharin; heat for a few minutes. Acidify with hydro- 
chloric acid; add a few drops of ferric chloride; a reddish brown 
or purplish color is thus produced. 

Silver nitrate, AgNOs, crystallizes in colorless plates without 
water of crystallization; it is used as an antiseptic, disinfectant, 
or escharotic. It is freely soluble in water and may be detected 
by the ordinary methods of qualitative analysis. 

Sodium chloride, NaCl, is a constituent of many preparations 
designed to be used hypodermically. Experience has proved 
the value of such addition; a possible reason for its desirability 
is given by Dr. G. Mahe, of Paris, in the Dental Cosmos for Sep- 
tember, 1903, in the statement that sodium chloride added in 
excess to a toxic substance diminishes its toxicity by one-half, 
and this has been demonstrated particularly with cocaine. 

Sodium perborate is a powder having the composition NaBO 3 . 
4 H 2 O, which will furnish 10 per cent of available oxygen and 
produce H 2 O 2 with water; it is very stable and is recommended 
as a bleach-powder. 

Sodium perborate may be made by thoroughly mixing sodium 



QO MICROCHEMICAL ANALYSIS 

peroxide (Na 2 O2) with crystallized boric acid, and stirring the 
mixture gradually into cold water. The proportions recom- 
mended by V. E. Miegeville in the Dental Cosmos for 1905, 
page 1381, are 78 grams of the sodium peroxide, 248 grams of 
the boric acid, and 2 liters of water. The sodium perborate is 
formed spontaneously and separates from the solution as a 
white, crystalline powder. Its solubility is increased by addition 
of weak organic acids, citric or tartaric. 

Sodium peroxide, Na 2 O 2 , is a white powder, easily soluble 
in water, usually with evolution of more or less oxygen and forma- 
tion of hydrogen dioxide. 

Somnoform is a general anesthetic, administered in a manner 
similar to chloroform; it was introduced by Dr. Rolland, of 
Bordeaux, and consists of 60 per cent ethyl chloride, 35 per cent 
ethyl bromide, and 5 per cent methyl bromide. (Dental Cosmos, 
Vol. XLVII, page 236.) 

Stovaine. Benzoyl -ethyl -dimethyl-amino- propanol-hydro- 
chloride, Ci 4 H 2 iO 2 N.HCl, closely related to alypin, occurs in 
small shining scales, freely soluble in alcohol or water. It is 
incompatible with alkalies and all alkaloiclal reagents. It can 
be sterilized by boiling. (Mod. Mat. Med., 2nd edition.) 

It melts at 175 C., is very soluble in water, and gives reaction 
similar to cocaine, which is also a benzoyl derivative. (U. S. D., 
page 1 66 1.) 

It is less powerful than cocaine and physiologically incom- 
patible with adrenalin. (Dental Cosmos, 1905, page 146.) 

Test. Stovaine gives rather irregular but characteristic 
crystals with platinic chloride. 

Suprarenal Glands. The official preparation consists of 
dried glands obtained only from animals used for food by man. 
The glands must contain not less than 0.4 per cent nor more than 
0.6 per cent of epinephrine. 

Tannic acid, or tannin, sometimes called gallotannic acid, 
is an astringent organic acid obtained from nut-galls. It may 
be obtained as crystals carrying two molecules of water, 
HCi 4 H 9 O9.2 H 2 0. Tannic acid is a white or slightly yellowish 
powder, soluble in about i part water or 0.6 part alcohol. It is 



DENTAL PREPARATIONS 91 

used as an alkaloidal precipitate, also in astringent washes. 
It may be detected by the addition of ferric solutions, which 
form with it a black tannate of iron of the nature of ink. 

Thymol, C 6 H3(CH3)(OH)(C 3 H 7 ) 1:3:4. This is a phenol 
which occurs in volatile oils of thymus vulgaris (Linne). It 
melts at 44 C. ; is sparingly soluble in water, easily in alcohol and 
ether. 

Tests. It may usually be detected by its odor or by dis- 
solving a small crystal in i c.c. of glacial acetic acid; whereupon, 
if 6 drops of sulphuric acid and i drop of nitric acid be added, 
the liquid will assume a deep bluish-green color. (U. S. D.) 

Thymol iodide, di-iodo-dithymol, (C 6 H 2 .CH3.C 3 H7OI)2, is a 
valuable antiseptic containing 43 per cent of iodine. It is a 
brown powder, insoluble in water, slightly soluble in alcohol, 
easily soluble in chloroform or ether. 

Thymophen is a mixture of equal parts of thymol and phenol. 

Thyroids. The dried, powdered, thyroid glands of animals 
used for food by man, freed from connective tissue and fat, 
containing not less than 0.17 per cent or more than 0.23 per cent 
of iodine, constitute the official preparation used as a remedy in 
myxedema and other cases of perverted metabolism. 

Trichlor-acetic acid occurs as deliquescent crystals, readily 
soluble in water. It distils at 195 C. and is a powerful caustic. 
Dilute solutions are recommended for treatment of pyorrhea. 

Tropa-cocaine is an alkaloid, originally isolated by Giesel from 
the leaves of the small-leaved coca-plant of Java and introduced 
by Arthur P. Chadbourne, Harvard Medical School. It is used 
hypodermically in normal salt solution. It is probably superior 
to cocaine, but rather more expensive. It is obtained as an oil 
which, when quite dry, solidifies in radiating crystals, melting at 
49 C. It is easily soluble in alcohol. 

A number of commercial mouth-washes and local anesthetics 
will be given to the class for identification, the object being to 
familiarize the student with the more easily made tests for the 
principal ingredients of these preparations. Complete analysis 
will rarely be attempted. The following table, taken from the 
Druggist's Circular of June, 1910, may be helpful. 



MICROCHEMICAL ANALYSIS 



DIFFERENTIATION OF COCAINE AND ITS SUBSTITUTES. 





Iodine potassium 
iodide. 


Bromine water. 


Sodium hydroxide. 


Potassium per- 
manganate. 


Eucaine o. 
Eucaine 6. 

Cocaine 


Yellow-maroon 
precipitate, 
soluble on 
boiling. 
Deep-red pre- 
cipitate, solu- 
ble on boiling. 

Yel low-m aroon 


Yellow precipitate, 
soluble on heat- 
ing. 

Yellow precipitate, 
slightly soluble 
on heating, re- 
precipitated 
white on boiling. 
Yellow precipitate, 


White precipitate, 
insoluble in ex- 
cess and on boil- 
ing. 
White precipitate, 
insoluble in ex- 
cess and on 
boiling. 

White precipitate, 


Violet precipitate, 
blackening 
quickly. 

No precipitate 
immediately; 
color persists 
for a day. 

Violet precipitate, 


Novocaine . . . 


precipitate, 
soluble on 
boiling. 

Deep-red pre- 


soluble on heat- 
ing. 

Yellow precipitate, 


insoluble in ex- 
cess and on 
boiling. 

White precipitate, 


color persists 
for one hour, 
then deposits 
Mn0 2 . 
Violet precipitate, 


Stovaine 


cipitate, solu- 
ble on boiling. 

Deep-red pre- 


soluble on heat- 
ing. 

Yellow precipitate, 


insoluble in ex- 
cess and on boil- 
ing. 
White precipitate, 


blackening 
quickly. 

Violet precipitate 


Nirvanin 


cipitate, solu- 
ble on boiling. 

Deei>-red pre- 


soluble on heat- 
ing. 

Yellow precipitate, 


insoluble in ex- 
cess; aromatic 
odor on boiling. 
Precipitate, very 


blackening al- 
most immedi- 
ately. 
Precipitate, first 


Alypin 


cipitate, solu- 
ble on boiling. 

Yellow-maroon 
precipitate, in- 
soluble on 
boiling; orange- 
red deposit. 


soluble on heat- 
ing, but the 
liquid becomes 
red and gives an 
agreeable fruity 
odor. 
Yellow precipitate, 
soluble on gentle 
heating. 


soluble in excess 
of the reagent. 

White precipitate, 
insoluble in ex- 
cess and on boil- 
ing. 


maroon, then 
brown. 

Bluish-violet pre- 
cipitate, slowly 
blackening. 



PART III. 
PHYSIOLOGICAL CHEMISTRY. 



CHAPTER XI. 
FERMENTS OR ENZYMES. 

Physiological chemistry treats of the substances that go to 
make up the animal body, the changes which these substances 
undergo in the process of digestion and assimilation, and the 
final products of metabolism. 

This subject, like others, will receive our attention in outline, 
simply with a view to enabling the student to understand the 
conditions which at present seem to have the most direct bearing 
on dental science. The changes produced by the class of bodies 
known as ferments are of great importance and will be the first 
to be considered. 

If yeast is allowed to grow in a sugar solution of moderate 
strength, the sugar molecule is split into carbonic-acid gas and 
alcohol. The process is one of fermentation; the yeast is the 
ferment. There are various substances which cause similar 
vsplitting of complex molecules into simpler compounds.* 

The distinction between the organized and the unorganized 
ferments is no longer recognized, as it has been proved that the 
activity of an organized ferment is due to the presence of the 
unorganized ferment or enzyme. We shall, by preference, refer 
to these substances as enzymes. 

The enzymes, as a class, possess certain general properties 
which should be remembered: 

First. Their action is limited to a very few substances; 
i.e., the enzyme from yeast, referred to above, will convert a 

* Occasionally fermentation may produce a synthesis (putting together) rather 
than an analysis (pulling apart). 

93 



94 PHYSIOLOGICAL CHEMISTRY 

few sugars only, as indicated. It will not act in any other way 
nor upon other substances. 

Second. The enzymes act only at ordinary temperatures, 
usually showing the greatest activity at about the temperature 
of the animal body, 37 to 40 C. 

Third. Enzymes act only within very narrow limits as re- 
gards the chemical reaction (acid or alkaline) of the media. 

Fourth. Enzymes are destroyed (killed) by the heat of boil- 
ing water. 

Fifth. In regard to the nature of their composition, many of 
the enzymes are closely allied to the proteins. 

Hydrolytic Enzymes. An enzyme may be classified accord- 
ing to the sort of work it does. Many of the chemical changes 
involved in the utilization of food consist of breaking up a 
complex molecule and, by the use of a molecule of water, form- 
ing new and simpler compounds. This sort of change is called 
hydrolysis and an enzyme that will produce it is a hydrolytic 
enzyme. By hydrolysis or hydrolytic cleavage, the molecule 
of cane-sugar, C J2 H 22 On, becomes two molecules of a simpler 
sugar, such as glucose, C 6 Hi 2 O 6 . Ci 2 H 22 Oii + H 2 O = 2 C 6 Hi 2 O 6 . 

Hydrolysis is not dependent upon enzyme action, as the same 
change is produced by prolonged boiling with very dilute mineral 
acids. 

De-aminizing Enzymes. These are characterized by their 
ability to split off the amino group. They arc found in the blood 
and tissue cells, and their particular function is the de-aminization 
of the amino acids which are absorbed. 

Oxidizing Enzymes. Another large and very important class 
of enzymes consists of those which produce oxidative changes. 
They may be divided into the oxidases, which produce direct 
oxidation, and the peroxidases, which produce oxidation only 
in the presence, or by the aid, of peroxide. 

Oxidases have been found to exist in saliva, milk, blood, nasal 
mucus, tears, and semen, in many of the organs, and also in 
the muscular tissue. They exist, moreover, in the vegetable 
kingdom, in which the subject of oxidizing enzymes was first 



FERMENTS OR ENZYMES 95 

studied by Bertrand and Bourquelot* It is said that the urine, 
bile, and intestinal secretions do not contain any ferment of this 
kind. 

Besides the classification of enzymes by the character of the 
work they do, the name of the substance acted upon may also 
be used to designate an enzyme: thus, a proteolytic enzyme 
produces a cleavage of protein substances, a lipolytic enzyme 
(lipase) splits the fat molecule, etc. 

Several of the digestive enzymes, notably the proteolytic or 
flesh-digesting enzymes, such as pepsin, trypsin, etc., exist in 
the animal cell, not as active agents, but as inactive parent 
enzymes which are called pro-enzymes or zymogens. Enzymes 
of this class are set to work (liberated from the parent sub- 
stance) by a class of substances known as " activators " (illus- 
trated by the enterokinase of the intestine, page 151). 

Neither the zymogen nor the activator has of itself any diges- 
tive action whatever; a provision which results in the prevention 
of autodigestion (autolysis) of the cells containing them. 

In addition to the exocellular enzymes occurring in the digestive 
tract, there is a large class of so-called endocellular enzymes, 
occurring throughout the body cells. This class, of which the 
de-aminizing enzymes are an example, is chiefly responsible for 
the metabolic processes taking place in the animal body. 

Catalase is a term which has been applied to enzymes similar 
in action to the peroxidases, i.e., enzymes that destroy a peroxide 
with the formation of molecular oxygen; although, according to 
Hammarsten, they differ from both the oxidases and peroxidases 
in giving no reaction whatever with guaiac. 

The name of a specific enzyme usually ends in " -ase ": as 
zymase, the enzyme contained in yeast; lipase, a fat-splitting 
enzyme; urease, the urine ferment. 

* "Enzymes and their Application, " Effront: Prescott's translation. This 
work is also authority for statement immediately preceding regarding the source 
of oxidizing enzymes. 



CHAPTER XII. 
CARBOHYDRATES. 

The carbohydrates predominate in the vegetable kingdom. 
As the name signifies, they are compounds of carbon, hydrogen 
and oxygen, in which the oxygen and hydrogen usually occur in 
the same proportion as in water. There are a few carbohydrates 
in which this proportion does not hold; and there are a number 
of substances acetic acid, for example in which the pro- 
portion holds true though the substance is manifestly not a 
carbohydrate. 

As a rule, the carbohydrates are white, solid substances, some 
of them crystalline, many of them sweet, and some of them 
tasteless, as starch and glycogen. 

The carbohydrates are synthesized almost exlusively by the 
vegetable body. The exact manner of this synthesis is an open 
question, various theories having been suggested to explain it. 
Baeyer has suggested that formaldehyde, formed from carbon 
dioxide and water by the action of sunlight on the chlorophyll of 
the plant,* will condense and eventually produce CeH^Oe- 

CO 2 + H 2 O -> CH 2 O + O 2 
(CH 2 0) 3 -> C 3 H 6 3 



It is known that formaldehyde in a slightly alkaline solution 
will, by condensation of the molecules, become formose, a mixture 
of sugars. The plant, although practically neutral in reaction, 
seems to possess a catalytic agent capable of bringing about this 
condensation very rapidly. Different plants produce different 
carbohydrates; and it is therefore reasonable to assume that the 

* The exact action of the chlorophyll is not clear, but Priestly and Usher 
have demonstrated that extracted chlorophyll, with sunlight, CQa and water, can 
produce formaldehyde. 



CARBOHYDRATES 



97 



condensation taking place in the individual plant is regulated and 
controlled by an inherent property of the plant. 

By the action of electricity, formaldehyde has been converted 
into glycol aldehyde; and from this, sugar has been produced 
in the laboratory. 

However, although the exact nature of the processes which 
take place in the plant is at present unknown, it is established 
beyond question that the vegetable organism absorbs carbon 
dioxide, gives off oxygen and produces carbohydrates. 



Classification of Carbohydrates. 

Arabinose 
Xylose 



Sugars 



Pentoses. 



Starch 



Gum 
Cellulose 



Dextrose 
Levulose 
Galactose 

Saccharose 
Maltose 
, Lactose 
Starch 
Glycogen 

Dextrin 



Monosaccharides or monoses. 



Disaccharides or dioses. 



Polysaccharides or polyoses. 



Characteristics. The monosaccharides are reducing bodies 
of either the aldehyde or the ketone type. The termination 
" ose" is applied to all sugars, and may also be used in designating 
the type; thus dextrose is an " aldose," while levulose is a 
" ketose;" i.e., dextrose is an aldehyde, containing the char- 
acteristic CHO group, while levulose is a ketone containing 

I 
the C = O group. 

I 

The pentoses (C 5 Hi 5 ) are represented by two important 
compounds, arabinose and xylose. The first of these occurs 
occasionally in the urine (pentosuria), and can be prepared by 
boiling gum arabic with dilute mineral acids. The second, 



98 PHYSIOLOGICAL CHEMISTRY 

xylose, has been obtained from the pancreas, but may be pre- 
pared more easily from bran or straw by boiling with dilute 
hydrochloric acid (Exp. 124, page 270). 

The pentoses, as a class, boiled with dilute mineral acid 
(hydrochloric or sulphuric), yield furfuraldehyde by splitting off 
the elements of three molecules of water: 

C 6 H 10 6 - 3 H 2 - C 6 H 4 O 2 . 

The formation of furfuraldehyde can be easily demonstrated 
by various color reactions as given in experiment 124, page 270. 

The hexoses, CeH^Oe, also called monoses, occur quite gen- 
erally in nature (not true of the pentoses). They constitute the 
various fruit sugars, and may be obtained by hydrolysis of the 
dioses and polyoses. 

They all reduce Fehling's copper solution (galactose less 
easily than the others), and they are all fermented by yeast 
(galactose more slowly than the others.) 

Dextrose or glucose, CeH^Oe, also known as grape-sugar 
and as diabetic sugar, occurs in grapes, honey, etc. It is formed 
by the action of diastatic ferments on the disaccharides; also 
from many of the polysaccharides. Glucose thus occurs in the 
processes of digestion and constitutes the sugar of diabetic 
urine. It may be obtained commercially as a white solid, and 
also as a thick, heavy syrup, known as confectioners' glucose. 
The commercial glucose is prepared by the action of dilute acids 
on starch, hydrolysis taking place, as follows: 

C 6 HioO 5 + H 2 O - C 6 Hi 2 6 . 

Glucose is an aldose and may be represented graphically: 
CH 2 OH.CHOH.CHOH.CHOH.CHOH.CHO. The presence of 
the aldehyde group is responsible for many of its characteristic 
reactions. 

Dextrose can be oxidized first to gluconic acid (CH 2 OH.- 
(CHOH)4.COOH), and by further oxidation to dibasic saccharic 
acid: 

COOH.(CHOH) 4 .COOH. 







" FIO/I.- 
Glucosazone* 




FIG, 3. ' 





. .FIG* 2. 




FIG, 4* 
Wheat; 




_ 

B, 



' -Fie. 6, ' . ;' ' 

) B t 



CARBOHYDRATES 99 

This oxidation can be effected by the use of nitric acid. Sac- 
charic acid forms a definite soluble salt with calcium. Whether 
the fact has any bearing whatever on the relation between poor 
teeth and excessive use of candy has not been demonstrated. 

Tests. Glucose boiled with Fehling's solution precipitates 
the red suboxide of copper (CugO). 

Benedict's reagent with glucose gives a turbidity which is 
claimed by many to be more delicate than Fehling's test and in 
some instances more characceristic. 

Glucose responds to Molisch's test for carbohydrates, which is 
made with an alcoholic solution of a-naphthol and concen- 
trated sulphuric acid (Exp. 126). The monosaccharides, of 
which glucose is a convenient representative, may be distin- 
guished from the other carbohydrates by heating with Barfoed's 
solution (copper acetate in dilute acetic acid), which is reduced 
with precipitation of cuprous oxide. 

Heated with phenyl-hydrazine solution nearly to the boiling- 
point of water, glucose forms phenyl-glucosazone, which crystal- 
lizes, as the mixture cools, in characteristic yellow needles 
usually arranged in bundles or sheaves. (Plate V, Fig. i.) 

Osazones are the various compounds formed by the different 
sugars and phenyl-hydrazine, when treated as above. They 
crystallize in fairly distinctive forms and furnish valuable tests 
for the sugars. The phenyl-hydrazine test is considered at least 
ten times more delicate than Fehling's test. 

The formation of the osazones with glucose may be expressed 
by the following reactions: 

CH 2 OH CH 2 OH 

I I 

(CHOH) 3 (CHOH) 3 

I + C 6 H 5 NH.NH 2 -> I + C 6 H 5 NH.NH 2 -> 

CHOH CHOH 

I ,O Phenyl-hydrazine. I , N.NHC 6 H 5 + H 2 

cf cf 

X H X H 

Glucose. Phenyl-hydrazone. 



100 PHYSIOLOGICAL CHEMISTRY 

CH 2 OH CH 2 OH 

I I 

(CHOH) 3 (CHOH) 3 

I + C 6 H 8 NH.NH 2 - I 
C = O C = N.NHCeHs + H 2 O 

I . NNHC 6 H 6 + C 6 H 6 NH 2 + NH 3 I .N.NHC 6 H B 
/^ " r^ 

\ \ 

H Aniline. H 

Glucosazone. 

Glucose readily undergoes alcoholic fermentation, yielding 
C2H 5 OH and CCV (See Exp. 134, page 271.) 

Levulose, CeH^Oe, or fruit-sugar, turns the ray of polarized 
light to the left, and to a greater degree than glucose turns it to 
the right. It occurs in honey and in many fruits, and is produced 
with glucose by hydrolysis of cane-sugar. Levulose forms an 
osazone not to be distinguished from glucosazone. It reduces 
copper solutions in a manner similar to glucose, and, like it, is 
easily fermented by yeast. 

Levulose differs from glucose in that it is a ketose rather than 
an aldose. The keto group is joined to one of the CH 2 OH groups, 
and it may be represented graphically thus : 

CH 2 OH.CHOH.CHOH.CHOH.CO.CH 2 OH. 

The position of the keto group, between a primary and a 
secondary alcohol group, accounts for the fact that levulose 
possesses reducing properties, page 22. 

Galactose is the product of the hydrolysis of lactose, or milk- 
sugar, and some other carbohydrates. It is a crystalline sub- 
stance which reduces Fehling's solution and ferments slowly with 
yeast. 

DlSACCHARIDES OR DlOSES. 

Disaccharides have the general formula C^H^On. They are 
converted into the monosaccharides by hydrolysis, brought about 
either by action of enzymes or by boiling with mineral acid. 

Cane-sugar, C^H^On, sucrose or saccharose, is obtained from 
the sugar-cane (various varieties of sorghum), also from the 
sugar-beet (Beta wlgaris) and the sugar-maple (Acer sacchar 



CARBOHYDRATES 



IOI 



rinum). Cane-sugar is a white, crystalline solid, soluble in about 
1/2 part of water and in 175 parts of alcohol (U. S. P.). It does 
not reduce copper solutions, nor does it form an osazone with 
phenyl-hydrazine; but it is easily hydrolyzed with the formation 
of dextrose and levulose, and then, of course, the reactions 
peculiar to these substances may be obtained. It does not fer- 
ment directly, but, by the action of invertin contained in yeast, 
it takes up water, becoming glucose and levulose as above, these 
latter sugars being easily fermentable. 

The explanation of its failure to respond to the usual sugar 
tests is shown by its graphic formula, which is probably : 

CH 2 OH CH 2 OH 




CH 2 OH 



Note the lactone structure, i.e., the joining of the a and 5 
carbon atoms through oxygen. Note also the absence of either 
aldehyde or ketone grouping. 

Maltose, C^H^Ou, or malt-sugar, is an intermediate product 
in the hydrolysis of starch, and by further hydration becomes 
two molecules of dextrose: C^H^On + H 2 = 2 C 6 Hi 2 06. It 
is formed in the fermentation of barley by diastase (the ferment 
of malt), and with phenyl-hydrazine it produces an osazone 
distinguished from glucosazone and lactosazone by its micro- 
scopical appearance (Plate V, Fig. 2) and its melting-point. 

Lactose, Ci2H 22 On, obtained from milk, is a disaccharide with 
far less sweetening power than sucrose. It forms an osazone 
which crystallizes in small burr-shaped forms (Plate V, Fig. 3), 
often covered with long hair-like appendages. 



102 PHYSIOLOGICAL CHEMISTRY 

It reduces Fehling's solution, but does not reduce Barfoed's 
solution. It resists fermentation in a marked degree. Upon 
hydration it is converted into dextrose and galactose. 

POLYOSES POLYS ACCHARIDES. 

Starch. This well-known and widely distributed plant-con- 
stituent, is a carbohydrate represented by C 6 Hio0 5 , the actual 
molecule, however, being many times this simple formula. The 
microscopical appearance of the starch granule is quite charac- 
teristic, and recognition of the more common starches by this 
method is not at all difficult (see Plate V, page 99). 

Starch is not soluble in cold water; but in hot water, or in 
solutions containing " amylolytic " enzymes, or in solutions 
containing certain chemical substances, as chloride of zinc or of 
magnesium, dilute hydrochloric or sulphuric acid, capable of 
forming hydrolytic products, the starch granules swell up, and 
ultimately dissolve, being converted into dextrose. The con- 
version, however, takes place in several well-defined steps, as 
follows: Soluble starch is first formed, answering the same chem- 
ical test with iodine (Exp. 213, page 286); next, erythrodextrin, 
which gives a red color with iodine solution; then a, /?, and y 
achroodextrins (maltodextrin), which give no color with iodine, 
but react slightly with Fehling's copper solution; then maltose, 
also negative with iodine, but reacting strongly with Fehling's 
solution; and finally dextrose. 

Dextrin (CeHioOs) is a yellowish powder, also known as 
British gum; it is formed from starch, as indicated above, and 
constitutes to a considerable extent the " crust " of bread. It is 
soluble in water, the solution giving a red color with iodine, and is 
also distinguished from starch by its failure to give a precipitate 
with solution of tannic acid. 

Glycogen, or animal starch, is a carbohydrate, with the gen- 
eral formula CeHioOs, occurring principally in the liver, and to 
a lesser extent in nearly all parts of the animal body. Freshly 
opened oysters are a convenient source of the substance for 



CARBOHYDRATES 103 

laboratory demonstration. It occurs in horse-flesh in consider- 
ably larger proportions than in human flesh. 

Properties. Glycogen is a white powder without odor or 
taste. It dissolves in water, producing an opalescent solution. 
It is closely allied to the starches of vegetable origin in that the 
products of its hydrolysis are dextrin and ultimately dextrose. 
It differs in its ready solubility in water, and in the fact that it is 
precipitated by 66 per cent alcohol, also in its power of rotation, 
which is much stronger than that of starch. 

Physiology. Glycogen is formed by the liver, and stored by 
this same organ for future use. It is derived principally from 
carbohydrates, but may also be derived from proteins. It dis- 
appears during starvation. In dead liver or muscle it rapidly 
undergoes hydrolytic change with the production of a reducing 
sugar. 

Cellulose, CeHioOs, is a carbohydrate which occurs as a prin- 
cipal constituent of woody fiber, and which may be found in the 
laboratory in a nearly pure state, as absorbent cotton or Swedish 
filter-paper. It is insoluble in water, alcohol, or dilute acids; 
it may be dissolved, however, by an ammoniacal copper solution, 
also by Schweitzer's* reagent, and by a concentrated solution of 
antimony chloride or tin chloride. It is converted into mono- 
saccharides by acids, only after first treating with concentrated 
sulphuric acid, which partially dissolves it. Cellulose aids 
digestion in a purely mechanical way by separating the digestible 
matter and allowing easier access of digestive ferments. The 
celluloses may be divided into three classes : first, those resisting 
hydrolysis and consequently lacking nutritive value, such as 
flax, cotton fibers, and hemp; second, those which hydrolyze 
slightly, which include the ligno-celluloses and may be utilized 
as food by herbiverous animals; and third, the pseudo celluloses, 
or hemicelluloses, which hydrolyze, but instead of forming sugars 
give intermediate products such as the pentosans or hexosans, 
which, of course, will yield respectively pentose or hexose. 

The galactans, another class of these intermediate substances, 
are widely distributed in nature, and when pure will yield galao 
* A hvdrated solution of cupric oxide in ammonia. 



104 PHYSIOLOGICAL CHEMISTRY 

tose upon hydrolysis. An important example of this class is 
agar-agar. As a constituent of diet it absorbs moisture and 
prevents drying of the residual fecal matter found in the in- 
testine. In consequence of this property it tends to prevent 
constipation. 



CHAPTER XIII. 
FATS AND OILS. 

Natural fats and oils of animal or vegetable origin are mix- 
tures of several glyceryl esters (see page 43); they may be 
separated, by cold and pressure, into two portions, one solid 
with comparatively high melting-point, and the other liquid at 
ordinary temperatures. The solid portion is known as the ste- 
aropten, and the liquid, as the eleopten, of the fat. Thus, from 
beef-fat, we may express a fluid eleopten consisting largely of 
olein and obtain as a residue a stearopten, stearin. The stearop- 
tens of the volatile or essential oils are classed as camphors, on 
account of their resemblance to ordinary camphor. Menthol, 
from oil of peppermint, and thymol, from oil of thyme, are 
examples of this class of compounds, both of which are largely 
used in dental practice. 

Properties. Fats are insoluble in water, easily dissolved by 
ether, chloroform, and carbon disulphide, less easily by alcohol, 
crystallizing on evaporation of the solvent. (Plate VI, Fig. 3, 
page 132.) They are emulsified by mechanical subdivision of 
the fat globules, in the presence of some agent which prevents 
their reuniting. The vegetable mucilages, soap, jelly, etc., are 
such emulsifying agents. On exposure to the air, fats and oils 
are more or less easily oxidized, which causes a separation of the 
fat acid. This produces an unpleasant odor or taste, and the 
fat is said to become rancid. 

Chemistry. The principal organic acids entering into the 
composition of fat are stearic acid, HCisHs^, solid, white, 
without]odor or taste, melting at 70 C. ; palmitic acid, HCieHs^, 
resembling stearic acid in its physical properties but melting at 
62 C.; oleic acid, HCi 8 H3 3 O2, containing two CH = groups with 
double-bonded carbons in the middle of the chain. This last 
acid is fluid at ordinary temperatures and predominates in the 

105 



106 PHYSIOLOGICAL CHEMISTRY 

softer animal fat. Its glyceryl ester, olein, constitutes 70 to 85 
per cent of human fat (percentage said to increase with age) and 
36 per cent of butter. 

Physiology. Fats are not digested to any appreciable extent 
until they reach the intestine; here they are broken up by a 
fat-splitting enzyme, emulsified, and to a slight extent saponified, 
after which they may be absorbed by the system (see Pancreatic 
Digestion). 

Glyceryl palmitate, C 3 H5(Ci 6 H 3 iO 2 ) 3? tripalmitin; glyceryl 
stearate, C 3 H 5 (Ci8H 3 5O 2 ) 3 , tristearin, and glyceryl oleate, 
C 3 H 5 (Ci 8 H 33 2 ) 3 , triolein; these in varying proportions make up 
the greater part of animal and vegetable fats and oils. 

The prefix "tri" is used because the "mono" and "di" 
compounds, as monopalmitin, C 3 H 5 (OH) 2 Ci 6 H 3 iO 2 , etc., are 
possible and may be prepared by synthesis. Triolein is liquid 
at ordinary temperature, solidifies at 6 C., and is a " double- 
bonded " compound; hence it forms addition products with the 
halogens as stearin and palmitin cannot do, since they are 
" saturated hydrocarbons." 

The amount of chlorine, bromine, or iodine which a fat or oil can 
thus absorb is an index of the proportion of unsaturated fatty acids 
contained in it, and hence becomes a valuable method of identi- 
fication. For example, the iodine-absorption number of butter 
is 33-3, while for lard it is 55 and for cottonseed oil 109.5. For 
detail of determination see Exp. 149. Olive oil and lard oil 
contain large amounts of olein. 

Tripalmitin melts at 66 C., and is usually obtained from palm 
oil. Tristearin melts at 72 C., and occurs with palmitin and 
olein in beef-fat, mutton- tallow, etc., the consistency of the 
fat being dependent upon the proportions of the constituent 
esters. 

The fats, stearin for example, may be split into glycerol and 
fatty acid by steam under pressure, as follows: 

C 3 H 5 (C 18 H350 2 )3 + 3 H 2 = C 3 H 6 (OH) 3 + 3 



A partial result of this sort is brought about by the fat-splitting 
enzyme (lipase) of the pancreatic juice (see Steapsin). 



FATS AND OILS 107 

Saponification is the term applied when a glyceryl ester is 
acted upon by caustic alkali. When the glyceryl ester is an 
ester of palmitic, stearic or oleic acid, or a mixture of these esters, 
soaps similar to ordinary soap are produced. 

Saponification of the fats by caustic alkali takes place as fol- 
lows: 

C 3 H5(C 18 H 3 50 2 )3 + 3 KOH = C 3 H 6 (OH) 3 + 3 KC 18 H 36 O 2 . 

The potassium salts of the fatty acids constitute the soft 
soaps, while the sodium salts are in general the hard soaps. 
The " salting-out " process in soap manufacture brings about a 
double decomposition, resulting in the production of ordinary 
soap. 

When any determination of the fatty acids present in a fat 
is desired, the fat is usually saponified, and the fatty acids are 
thus fixed as soaps. A procedure frequently employed for 
determining the purity of butter-fat is the Reichert-Meissel 
determination of the volatile fatty acids. The fatty acids are 
first fixed as soaps; sulphuric acid is then added; and then, by 
distillation, the volatile acids are separated. See Exp. 148. 
Butter contains a much higher percentage of volatile fatty acids 
than animal fats, giving a Reichert-Meissel number of approx- 
imately 28. 

Emulsification consists in producing a more or less permanent 
intimate mixture of liquids not otherwise miscible e.g. oil 
and water. The emulsification is effected by means of emulsi- 
fying agents which mechanically hold apart the very finely 
divided particles of oil and prevent their reuniting. Gum 
arabic solution and albumin are examples of emulsifying agents. 
Milk is a natural emulsion. 

Volatile oils contain, instead of the glyceryl base, a group of 
hydrocarbons known as the " terpenes." The formula is 
(C 5 H 8 )2i and the mostimportant of the group is Ci Hi 6 from oil of 
turpentine and many of the essential oils. 

The odor of the volatile oils seems to be dependent upon the 
presence of water and air; for example, oil of clove distilled over 
lime and in atmosphere free from oxygen has little odor. The 



108 PHYSIOLOGICAL CHEMISTRY 

presence of oxygen and moisture restores the characteristic 
odor. 

Lanolin or wool fat, obtained from sheep's wool, constitutes 
about 45 per cent of the crude substance. It is a mixture of 
cholesterin esters. The fat may be partially saponified, and from 
the alkaline solution three alcohols have been separated, all of 
which are white, crystalline powders. One of these, with 
formula C^HsaOH, is known as lanolin alcohol and by oxidation 
with CrO 3 may be converted into lanolinic acid, C^IfeOs. 
(U. S. D.) 

Lecithin has been classified as a phosphorized fat; it occurs 
in nervous tissue, and in the bile, and is obtained in considerable 
quantity from the yolk of eggs. It contains two fat acid radicals 
combined with glycerol, phosphoric acid and choline. Lecithin 
is soluble in chloroform, alcohol, ether and benzene, and may be 
obtained in crystalline form from the alcoholic solution. The 
fatty acid radicals are not always the same or necessarily alike. 
Lecithin may be represented by the following formula: 



CH - C 17 H 33 CO 2 

I 
CH 2 

I 

O = P - OH 
I 
O - C 2 H 4 

I 
(CH 3 ) 3 N - OH 

and its decomposition by the following reaction: 

C 4 4H 9 oNP0 9 + 3 H 2 O = 2 C 18 H 36 2 + C 3 H 9 PO G + C 5 H 15 NO 2 

Lecithin Stearic Glycero- Choline 

acid phosphoric 

acid 

Cholesterol, as its name implies, is an alcohol containing one 
hydroxyl group and one pair of double-bonded carbon atoms. 
It is an important member of a group of sterols or " solid al- 



FATS AND OILS IOQ 

cohols." It is insoluble in water or dilute acids or alkalies, but 
is soluble in bile or solutions of bile salts. 

Cholesterol occurs in brain and nerve tissue, in bile, yolks of 
eggs and various glandular tissues. It is soluble in ether, chlo- 
roform, and hot alcohol, from which it may be recrystallized in 
characteristic plates with one corner more or less imperfectly 
formed. Exp. No. 239. 

Cholesterol responds to a number of color tests, of which Exp. 
151, and the following will furnish sufficient illustration: 

L. Kaplenberg, in the Journal of Biological Chemistry, May, 
1922, page 225, says, " In arsenic chloride, brain cholesterol or 
gall stone dissolves yielding a pink solution which gradually turns 
to a cherry red on standing, more rapidly on heating. Color is 
discharged by addition of benzene, toluene or chloroform." 



CHAPTER XIV. 
PROTEINS. 

Protein is a general term used to designate the nitrogenized 
bodies which constitute the greater proportion of animal tissue. 

While meat and " protein " are usually associated, it must 
not be forgotten that meat is not the exclusive source of protein, 
for we usually find protein in vegetable substances, often to a 
considerable extent. 

Unlike that of the other two great divisions of food substances 
(carbohydrates and fats), the structure of the protein molecule is 
so complex that, with a few exceptions of the simplest kind, its 
representation has not been attempted. 

The protein molecule contains nitrogen (often as the ammo 
group NH 2 ) in addition to the carbon, hydrogen, and oxygen of 
the carbohydrates and fats. It frequently contains sulphur, 
often phosphorus, and occasionally the metallic elements, par- 
ticularly iron. 

As examples of the complexity of protein molecules, the fol- 
lowing proposed formulae are given in Hawk's "Physiological 
Chemistry." 

Serum albumin C 4 5oH72oNii6S 6 Oi4o. 

Oxyhemoglobin, CessHnsi^oT^FeC^io. 

The protein molecule has been likened to a bundle of rods more 
or less loosely bound together, the several rods representing the 
several amino acids of which the protein molecule is largely com- 
posed. When the binders are broken, by process of digestion, 
the rods or amino acids fall more or less completely apart and 
are ready for absorption. There are about eighteen of these 
amino acids entering into the composition of the protein molecule 
which seem to be important, but not all of these acids are present 
in all proteins. For example, the protein zein, found in maize, or 
Indian corn, is lacking in the amino acid lysin; gelatin is lacking 

no 



PROTEINS III 

in tyrosine, cystine and tryptophane. It has been shown that 
animals forced to depend on such deficient proteins do not thrive; 
hence, the character of the protein contained in the food must be 
considered. 

Of great interest in this connection is the following paragraph, 
taken from page 65 of " The Newer Knowledge of Nutrition/' 
Second Edition, by Dr. E. V. McCollum. 

" It has been abundantly demonstrated by several workers, 
that the simplest of the amino acids, glycocoll, is readily syn- 
thesized by the body tissues. Casein contains no glycocoll, yet 
it is a complete protein, and can meet all the requirements of an 
animal for nitrogen in the form of amino acids. From these 
results it is safe to conclude that the tissues of the higher animals 
are capable of synthesizing certain amino acids, and that these are 
made use of in some way to conserve the body proteins, even 
though the list which can be so synthesized is incomplete. It 
appears certain that tyrosine, tryptophane, and probably all 
the other cyclic amino acids and cystine cannot be synthesized 
by the mammal.' 5 

We shall not take time to study the structure of all of these 
necessary amino acids; but a few, because of their importance 
as factors of nutrition, as above suggested, or because of impor- 
tance in certain laboratory tests, will be considered. 

For general properties see page 39. 

Glycocoll, amino acetic acid, CH 2 NH 2 .COOH, is the simplest 
obtained from protein, and may, as above stated, be synthesized 
within the body. (For relation to hippuric acid see page 67.) 

Alanine, a-amino propionic acid, CH 3 CHNH 2 .COOH, occurs 
in protein in comparatively small amounts, but related to it 
perhaps derived from it are serine, phenylalanine, tyrosine, 
and cystine. 

Phenylalanine is a-amino-/3-phenyl propionic acid, CH 2 C 6 H5.- 
CHNH 2 .COOH. 

Serine is c*-amino-/3-hydroxy propionic acid, CH 2 OH.CHNH 2 .- 
COOH. 

Tryptophane is a-amino-j3-indol propionic acid, CH^CsHeN).- 
CHNH 2 .COOH. The putrefaction of proteins containing tryp- 



112 PHYSIOLOGICAL CHEMISTRY 

tophane gives rise to indican in the urine. (See page 201.) 
This acid has been shown to be a growth-promoter in small 
animals. It is lacking in zein and gelatin. 

Tyrosine is a-amino-/3-parahydroxy-phenyl propionic acid, 
CH2(C 6 H 4 OH)-CHNH2.COOH. Tyrosine may be obtained 
from old cheese; it crystallizes in tufts of needle-shaped crystals 
and occurs rarely in urinary sediments, in cases of phosphorus 
poisoning and some other acute conditions. 

Lysine is a-ediamino-normal caproic acid, CH 2 NH2(CH 2 ). 
CHNH 2 .COOH. Lysine is one of the amino acids that are 
indispensable for growth, but is lacking in both zein, from maize, 
and gelatine. 

CLASSIFICATION OF PROTEINS. 

A chemical basis for the nomenclature of proteins seems at pre- 
sent impossible, but the following suggestions and groupings, 
based on the properties of the protein substances, are generally 
accepted. 

The word protein designates that class of substances which 
consist, so far as is known at present, essentially of a-amino acids 
and their derivatives: e.g., a-amino acetic acid, or glycocoll; 
a-amino propionic acid, oralanin; /3 phenyl-a-amino propionic 
acid, or phenylalanin; guamdine-ammo valerianic acid, or 
arginine, etc. Proteins are, therefore, essentially polypeptides. 

I. SIMPLE PROTEINS.* 

Protein substances which yield, on hydrolysis, only a-amino 
acids or their derivatives. 

Although no means are at present available whereby the 
chemical individuality of any protein can be established, a 
number of simple proteins have been isolated from animal and 
vegetable tissues, and have been so well characterized by con- 
stancy of ultimate composition and uniformity of physical 
properties that they may be treated as chemical individuals 
until further knowledge makes it possible to characterize them 
more definitely. 

* The classification and definitions herewith given are taken from the recom- 
mendations of the Committees of the American Physiological and Biochemical 
Societies as printed in Science, Vol. 27, No. 692, page 554. 



PROTEINS 113 

The various groups of simple proteins may be designated as 
follows : 

(a) Albumins. Simple proteins soluble in pure water and 
coagulable by heat; e.g., ovalbumin, serum albumin, lactalbumin, 
vegetable albumins. 

(6) Globulins. Simple proteins insoluble in pure water, but 
soluble in neutral solutions of salts of strong bases with strong 
acids;* e.g. serum globulin, ovoglobulin, edestin, and other 
vegetable globulins. 

(c) Glutelins. Simple proteins insoluble in all neutral 
solvents but readily soluble in very dilute acids and alkalies ;f 
e.g., glutenin. 

(d) Alcohol-soluble Proteins (Prolamines) . Simple proteins 
soluble in relatively strong alcohol (70 to 80 per cent), but in- 
soluble in water, absolute alcohol, and other neutral solvents ;{ 
e.g., zein, gliadin, and hordein. 

(e) Sclero Proteins, Albuminoids. Simple proteins which 
possess essentially the same chemical structure as the other pro- 
teins, but are characterized by great insolubility in all neutral sol- 
vents ; e.g., elastin, collagen, keratin, and reticulin. 

(f) Histones. Soluble in water and insoluble in very dilute 
ammonia and, in the absence of ammonium salts, insoluble even 
in an excess of ammonia; yield precipitates with solutions of 
other proteins and, on heating, a coagulum which is easily soluble 
in very dilute acids. On hydrolysis they yield a large number 
of amino acids, among which the basic ones predominate; e.g., 
globin, thymus histone, scombrone. 

(g) Protamines. Simpler polypeptides than the proteins in- 
cluded in the preceding groups. They are soluble in water, un- 

* The precipitation limits with ammonium sulphate should not be made a 
basis for distinguishing the albumins from the globulins. 

t Such substances occur in abundance in the seeds of cereals and doubtless 
represent a well-defined natural group of simple proteins. 

J The sub-classes defined (a, 6, c, d) are exemplified by proteins obtained from 
both plants and animals. The use of appropriate prefixes will suffice to indicate 
the origin of the compounds, e.g., ovoglobulin, myoalbumin, etc. 

These form the principal organic constituents of the skeletal structure of 
animals and also their external covering and its appendages. This definition does 
not provide for gelatin, which is, however, an artificial derivative of collagen. 



114 PHYSIOLOGICAL CHEMISTRY 

coagulable by heat, have the property of precipitating aqueous 
solutions of other proteins, possess strong basic properties and 
form stable salts with strong mineral acids. They yield com- 
paratively few amino acids, among which the basic amino acids 
greatly predominate; e.g., salmine, sturine, clupeine, scombrine. 

II. CONJUGATED PROTEINS. 

Substances which contain the protein molecule united to 
some other molecule or molecules, otherwise than as a salt. 

(a) Nucleo-proteins. Compounds of one or more protein 
molecules with nucleic acid; e.g., nucleo-histone. 

(b) Gly co-proteins. Compounds of the protein molecule 
with a substance or substances containing a carbohydrate group 
other than a nucleic acid; e.g., mucins and mucoids (osseomu- 
coid, tendomucoid.) 

(c) Phospho-proteins. Compounds of the protein molecule 
with some, as yet undefined, phosphorus-containing substance 
other than a nucleic acid or lecithins; e.g., casein, vitellin. 

(d) Hemoglobins. Compounds of the protein molecule with 
hematin or some similar substance; e.g., hemoglobin, hemo- 
cyanin. 

(e) Lecitho-proteins. Compounds of the protein molecule 
with lecithins; e.g., lecithans, phosphatides. 

III. DERIVED PROTEINS. 

i. Primary Protein Derivatives. Derivatives of the protein 
molecule apparently formed through hydrolytic changes which 
involve only slight alterations of the protein molecule. 

(a) Proteans. Insoluble products which apparently result 
from the incipient action of water, very dilute acids or enzymes; 
e.g., myosan, edestan. 

(b) M eta-proteins. Products of the further action of acids 
and alkalies whereby the molecule is so far altered as to form 
products soluble in very weak acids and alkalies, but insoluble 
in neutral fluids. 

This group will thus include the familiar " acid proteins " and 



PROTEINS 115 

" alkali proteins," not the salts of proteins with acids; e.g., acid 
meta-proteins (acid albuminate), alkali meta-protein (alkali 
albuminate). 

(c) Coagulated Proteins. Insoluble products which result 
from (i) the action of heat on their solutions, or (2) the action 
of alcohols on the protein. 

2. Secondary Protein Derivatives* Products of the further 
hydrolytic cleavage of the protein molecule. 

(a) Proteoses. Soluble in water, uncoagulated by heat, and 
precipitated by saturating their solutions with ammonium sul- 
phate or zinc sulphate ;f e.g., protoproteose, deuteroproteose. 

(6) Peptones. Soluble in water, uncoagulated by heat, but 
not precipitated by saturating their solutions with ammonium 
sulphate;J e.g., antipeptone, amphopeptone. 

(c) Peptides. Definitely characterized combinations of two 
or more amino acids, the carboxyl group of one being united 
with the amino group of the other, with the elimination of a 
molecule of water ; e.g., dipep tides, tripep tides, tetrapep tides, 
pentapeptides. 

GENERAL COLOR REACTIONS OF PROTEINS. 

Although there is wide variation in the structure of the protein 
molecule, proteins in general will respond to several color re- 
actions based on the presence of certain amino acid groupings in 
the protein molecule. 

Biuret Reaction, Piotrowski* s Test. 

This reaction is brought about by making the protein solution 
alkaline with sodium hydroxide and then adding a drop or two of 

* The term secondary hydrolytic derivatives is used because the formation of the 
primary derivatives usually precedes the formation of these secondary derivatives. 

t As thus defined, this term does not strictly cover all the protein derivatives 
commonly called proteoses; e.g., heteroproteose and dysproteose. 

J In this group the kyrins may be included. For the present we believe that 
it will be helpful to retain this term as defined, reserving the expression peptide 
for the simpler compounds of definite structure, such as dipeptides, etc. 

The peptones are undoubtedly peptides or mixtures of peptides, the latter 
being at present used to designate those of definite structure. 



Il6 PHYSIOLOGICAL CHEMISTRY 

very dilute copper sulphate. Upon standing for a few minutes 
a violet coloration is observed if protein is present. 

The shade of color produced varies from a blue violet in cases 
of simple proteins to a rose violet when proteoses or peptones are 
present. 

This reaction, although typical, is not distinctive for proteins, 
several other substances responding positively to it. The sub- 
stances that do give this reaction have been observed to be those 
containing at least one CONH^ group in which the amino group 
is free and one or more substituted amide groups. Also, accord- 
ing to Schiff, any amide of a dibasic acid in which the amide 
groups are attached to different carbon atoms will react positively, 
oxamide being an example, NH 2 CO CO NH 2 . Only 
one free amino group seems to be distinctly necessary, however. 

Xanthoproteic Test. 

If a solution of protein is made acid with concentrated nitric 
acid and heated, a yellow color is produced which changes to 
deep orange upon the addition of alkali. The alkali, generally 
ammonium hydroxide, should not be added until the solution 
is cool. This reaction is dependent upon the benzene ring, present 
in phenylalanine, tyrosine and tryptophane. 

A positive xanthoproteic test will show, therefore, the pres- 
ence of those amino acid groupings in the protein. The yellow 
color produced by the addition of nitric acid is indicative of the 
formation of a nitrobenzene product. This, upon the addition 
of ammonia, is converted into the salt formation which gives the 
orange coloration. 

Millon's Reaction. 

Millon's reagent, as given in the Appendix, consists of a mix- 
ture of mercuric nitrite and nitrate. When put in contact with 
protein and tested, it causes the protein to be precipitated; if 
allowed to stand, the precipitate will turn red. 

Millon's reaction seems to be dependent on the presence of a 
monohydroxy phenyl group and is consequently a distinguishing 
test for tyrosine. 



PROTEINS 117 

Hopkins-Cole Reaction. 

This reaction is effected by treating the protein solution with 
glyoxylic acid and underlaying the solution with concentrated 
sulphuric acid. In the presence of the indol grouping, trypto- 
phane, a condensation takes place producing a purple color. 
The exact chemistry of this reaction is not wholly known. 

Lieberman's Reaction. 

This reaction may be used as a test for a protein molecule 
containing both tryptophane and a carbohydrate grouping. It 
is made by treating the protein solution with strong HCL 

The action of the acid on the carbohydrate forms aldehydes 
which, we may assume, react with the tryptophane grouping to 
give a violet color. 

The carbohydrate grouping may also be detected by Molisch's 
a-naphthol test, as given under carbohydrates. 

PROTEIN PRECIPITANTS. 

The substances generally classed as protein precipitants may 
be grouped conveniently as acids, metals, and salts. Among 
the acids that produce a more or less insoluble compound with 
proteins are phospho-tungstic, phospho-molybdic, tannic, picric, 
chromic, and bichromic. Their precipitating action is now 
explained on the assumption that proteins contain several free 
amino groups, making them possess basic properties, and that 
the precipitate produced by action of an acid is an insoluble salt 
formation of that acid, as protein tannate, protein picrate, etc. 

Similarly, when any of the precipitating metals, as mercury, 
lead, copper, platinum, manganese, iron, or aluminium, reacts 
with a protein, an insoluble metal proteinate is produced. The 
acid properties of tiie protein, which account for its behavior 
with metals and other basic substances, are presumably due to 
the fact that proteins contain, in addition to the free amino 
groups, free carboxyl groups. They are consequently amphoteric 
substances. 

Hardy has shown that proteins in acid solution become electro- 



Il8 PHYSIOLOGICAL CHEMISTRY 

positively charged, while proteins in alkaline solution are nega- 
tively charged. From this it follows that if the precipitating ion 
is the negative part of the molecule, or the anion, as is the case 
with the acid precipitants, the protein solution must be electro- 
positively charged, or acid in reaction, to bring about complete 
precipitation. Likewise, if the precipitating ion is positive, 
cation, as with the metal precipitants, in order to secure pre- 
cipitation of the protein the solution must be negatively charged, 
or alkaline. 

Mathews gives some exceptions to this rule, which may be 
noted. Some of the most strongly acid proteins,* as casein, are 
not affected by slight amounts of alkali, and hence will sometimes 
react electro-positively in an alkaline solution. The reverse 
holds true for those proteins strongly basic in reaction. 

If we consider proteins as colloidal substances, we see that these 
statements are in direct accord with what was said in the first 
chapter in Vol. I, in regard to the manner of precipitating colloids; 
i.e., a negatively charged colloid is precipitated by the positive 
ion of the precipitating reagent, and vice versa. 

As the third class of protein precipitants, we include neutral 
salts. The most important salt of this group, and the one gen- 
erally used in laboratory work, is ammonium sulphate. All 
proteins, with the exception of the peptones and peptides, may be 
precipitated by completely saturating the solution with this salt. 
The form in which the protein is precipitated makes it easily 
soluble, and hence this precipitating reagent is well adapted for 
use in studying the properties of the protein. Magnesium 
sulphate and sodium chloride are also used as protein precipitants, 
but their action is not general. 

When acted upon by salts, proteins behave like any colloid, 
as suggested above; the precipitating ion of the salt being 
dependent on the electrical charge of the protein solution. 

The changes occurring during precipitation and solution of 
proteins are now regarded from a purely chemical viewpoint. 
Loeb and others have demonstrated the true ionization of pro- 
teins and have shown their characteristic reactions to be de- 

* Those which contain several comparatively strong acids in the molecule. 



PROTEINS 119 

pendent on their ionization, just as is the case with inorganic 
substances. The ionization of the protein is also stated to be 
responsible for its viscosity, and if the ionization of the protein 
is at a minimum the tendency will be for the viscosity to be at a 
minimum too. 

Another protein precipitant of practical importance is alcohol, 
95 per cent. Proteins, including peptones, are insoluble in 
strongly alcoholic solutions. The action of alcohol in bringing 
about protein precipitation may be considered as a process of 
dehydration. In this process, however, the protein is very apt 
to become denatured, so that for practical work on proteins it is 
inadvisable to make the precipitation with alcohol. The pre- 
cipitation is increased by the addition of a few drops of an elec- 
trolyte, as 10 per cent acetic acid. 

ISOELECTRIC PROTEIN. 

The term isoelectric protein means a protein in a perfectly 
pure state, in which it is chemically inactive; that is, a state in 
which it will combine neither with the anion nor the cation of a 
substance, and if placed in an electric field its particles will not 
be attracted to either pole. The point at which protein acts in 
this way is usually at a very definite hydrogen-ion concentration, 
designated as the isoelectric point of the protein. If a given 
protein is then in a solution with a hydrogen-ion concentration 
greater than its isoelectric point, it is capable of combining only 
with the cation of any solution which may be added. On the 
other hand, a protein in a solution having a hydrogen-ion con- 
centration less than its isoelectric point can combine only with 
the anion of any compound. This accounts for the fact that 
protein salts are formed in some cases, and in other cases metal 
proteinates. 

For application to formation of tartar, see page 143. 

ALBUMINS. 

The albumins are conveniently represented by egg-albumin 
and serum-albumin. They are soluble in water, respond to the 
general protein reactions (Exp. 152, page 276, etc.), and may be 



120 PHYSIOLOGICAL CHEMISTRY 

completely precipitated by saturation of the solution by am- 
monium sulphate. Albuminis coagulated by heat (75 to 80 C.). 

Egg-albumin differs from serum-albumin in that it is not 
absorbed when injected into the circulation, but appears un- 
changed in the urine. Egg-albumin is readily precipitated from 
aqueous solution by alcohol, while serum-albumin is precipi- 
tated only with difficulty. Albumins in general form, with 
acids or with alkalies, derived proteins known as acid or alkali 
albumins or albuminates (acid or alkali metaproteins). An acid 
albumin derived from myosin is known as syntonin. It differs 
but slightly from other acid albumins. The acid and alkali 
albumins are both precipitated by neutralization, but neither of 
them are coagulated by heat. 

Albumin normally occurs in all the body fluids except the 
urine. The amount in milk is extremely slight; the amount in 
saliva seems to vary in inverse proportion to mucin. Albumin 
occurring in urine in appreciable quantity is always abnormal, 
although in many cases it has no serious significance unless 
persistently present in more than the slightest possible trace. 

GLOBULINS. 

Globulins occurs in both plants and animals, and crushed 
hemp seed may be used as a convenient source for laboratory 
experiment. It is also associated with albumin in blood-plasma, 
and may be separated from it by half saturation with ammonium 
sulphate, which precipitates the globulin only; but it is not to 
be distinguished by the ordinary protein tests and reactions. 
The albumin of albuminous urine always consists of a mixture 
of these two proteins, globulin and albumin, not, however, al- 
ways in the same proportion. The globulins are not soluble in 
distilled water as the albumins are, but a very small quantity of 
neutral salt, such as sodium chloride, will serve to effect the solu- 
tion. Globulin is thrown out of solution by action of carbon 
dioxide as a white flocculent precipitate. By dialysis the in- 
organic salts necessary for its solution will be removed and the 
protein will be precipitated. It is also thrown out by saturation 
of sodium chloride or magnesium sulphate. Globulin is coagu- 



PROTEINS 121 

lated by heat at practically the same temperature as serum- 
albumin; i.e., 75 C. 

SCLERO-PROTEINS. 

Albuminoids or sclero-proteins are the simple proteins char- 
acterized by pronounced insolubility in all neutral solvents, 
and the common examples are keratin, from nails and hoofs, 
etc.; collagen, from bone and connective tissue; and elastin, 
from tendons and ligaments. 

The differences in these substances are slight, the keratin being 
less soluble and less easily acted upon by digestive ferments than 
either of the other two. Keratin also contains more sulphur. 
It is the principal constituent of horn, nails, hair, feathers, 
egg membrane, and some shells, such as turtle and tortoise. 
The sulphur content of these various sources differs considerably, 
ranging from about 5 per cent in hair, about 3 per cent in nail 
and horn, to 1.4 per cent in egg membrane. 

The keratins are characterized by the fact that the sulphur 
which they contain is loosely combined, i.e., easily separated by 
the formation of hydrogen sulphide and other sulphur com- 
pounds, as proved by Exp. 207. The keratins are insoluble in 
dilute acids and unaffected by any of the digestive ferments; 
they do, however, dissolve in the caustic alkali solutions, and 
may be used as the source of leucin, tyrosine, cystin, and other 
well-known products of protein digestion. 

Keratins heated with water, under pressure, to 150 C. will 
decompose with the formation of mercaptan, hydrogen sulphide, 
and a substance resembling the proteoses. 

Collagen, upon hydrolization with boiling water, produces 
gelatin, which is a characteristic property of this class of proteins. 
It may be dissolved by both the gastric and pancreatic juices, 
especially if previously treated with warm acidulated water. 
Collagen contains less sulphur than keratin and is obtained 
particularly from the tendo A chillis which contains about 32 
per cent of this albuminoid and 63 per cent of water. Collagen 
responds to the general color tests for the proteins. 

Elastin contains the least sulphur of any of the three sub- 



122 PHYSIOLOGICAL CHEMISTRY 

stances which we have considered. It may be obtained from 
the ligamentum nuchce of an ox, which contains about 31^ per 
cent of elastin and 58 per cent of water, by chopping the ligament 
finely and extracting for two or three days with /^//-saturated 
solution of calcium hydroxide. Like collagen, it is dissolved 
upon prolonged treatment with proteolytic ferments. 

Reticulin occurs as a fibrous part of lymph glands. It is 
insoluble in water and is not digested by pepsin or trypsin. It 
does not respond to Millon's test for proteins, indicating the 
absence of the hydroxy-phenyl group of ty rosin. 

Gelatin is made by hydrolysis of ossein or collagen brought 
about by prolonged boiling with dilute mineral acids. Gelatin, 
if first treated with cold water till soft, may be dissolved in hot 
water. The solution is precipitated by mercuric chloride, 
alcohol, tannic, and picric acids. It responds but feebly to the 
general protein reactions, but, by digestion with either pepsin or 
trypsin, compounds are obtained analogous to those resulting 
from similar protein digestion. 

Gelatin solutions respond to the biuret test, not to Millon's 
nor to the Hopkins-Cole test. This fact shows the absence of 
any tyrosine or tryptophane grouping in the molecule of 
gelatin. 

DERIVED PROTEINS. 

Meta-proteins Acid Meta-protein. The digestive action 
of the gastric juice on protein substances is the formation of an 
acid meta-protein, formerly called acid albuminate. The meta- 
proteins are characterized by the fact that they are precipitated 
on neutralization and are not coagulated by heat. They may 
also be precipitated by saturation with common salt. 

The Alkali Meta-protein or alkali albuminate is the stronger 
of these two classes of compounds when considered from a chem- 
ical standpoint; that is, the reactions are more marked, and some 
compounds will be formed with the alkali albuminate which are 
not produced when the acid albuminate is treated in a similar 
way. The acid meta-protein from the digestion of meat is known 
as syntonin. 



PROTEINS 123 

The Proteoses (albumoses) may be considered as the next 
well-defined product of protein digestion following the albu- 
minate. That is, leaving out the many intermediate prod- 
ucts between which sharp lines of demarcation cannot be drawn, 
the decomposition of albumin brought about by enzymes or 
digestive ferments gives, first, acid albumin; second, albumose; 
and third, peptone. Albumose may be taken as a type of this 
second class of digestive products. Other proteoses, such as 
globulose, etc., are the substances derived from other proteins 
at a corresponding point of decomposition or peptic digestion. 
Albumose may be coagulated by heat at a temperature ranging 
upwards from 56 C., but, as the temperature approaches the 
boiling-point, certain varieties of albumose go again into solution, 
and at a boiling temperature may be separated from albumin 
by filtration. In these cases, as the filtrate cools, the albumose 
will again precipitate. Albumose is also precipitated by nitric 
acid, by ferrocyanide of potassium and acetic acid (the precipitate 
in both cases being dissolved by heat), and the other general 
protein precipitants. The biuret test gives a distinctive color 
with proteoses and peptones, it being, a marked reddish shade 
rather than the violet or blue obtained with other proteins. 

Peptones are the final products of peptic digestion of the 
proteins. They are soluble substances which give the biuret 
test similarly to the proteoses, but are not precipitated by heat, 
by nitric acid, by potassium ferrocyanide and acetic acid, nor by 
saturation with ammonium sulphate. Peptones may be pre- 
cipitated by phospho-tungstic acid, phospho-molybdic acid, 
absolute alcohol and tannic acid. An excess of the reagent may 
dissolve the precipitate. 

PEPTIDES. 

The peptides differ from the peptones in that the peptide does 
not possess protein characteristics to any marked extent. They 
are hydrolytic products of the peptone and seem to be merely 
bunches of amino acids. Upon decomposition or hydrolytic 
splitting of peptides, the simpler amino acids without any protein 
characteristics result. 



124 PHYSIOLOGICAL CHEMISTRY 

CONJUGATED PROTEINS. 

These are substances which contain the protein molecule 
united to some other molecule or molecules, otherwise than as a 
salt. The conjugated proteins which we shall study are mucin, 
a type of glyco-protein, yielding upon decomposition a substance 
containing a carbohydrate group; casein (from milk), a phos- 
phorus-containing substance; and hemoglobin (from blood). 

The glyco-protein, mucin, a selected type of this class of 
protein substance, occurs in various forms in saliva, in urine, bile, 
and other body fluids. 

True mucins have been separated and examined from the 
secretion of the submaxillary glands, from snails, from mucous 
membranes of the. air passages, from synovial fluid, and from the 
navel cord. 

Mucin is quite readily converted to meta-protein by boiling 
with dilute acid, and, by action of strong acid, will yield a number 
of the simpler amino acids. Mucin itself is acid in reaction, but 
is, like all proteins, an amphoteric substance producing metal 
mucinates in solutions with hydrogen-ion concentrations above 
its isoelectric point, and mucin salts in solutions with acid 
concentrations less than its isoelectric point. 

The mucins are insoluble in pure water, but dissolve upon the 
addition of traces of alkali. The solution thus obtained will 
give the usual color reactions for the proteins. It is in reality a 
solution of the alkali mucinate which has been formed. From 
such a solution mucin may be precipitated by acetic acid. If 
pure isoelectric mucin is desired, a very dilute hydrochloric acid 
is preferable as a precipitating agent. 

Mucin in the saliva and its possible relations to bacterial growth, 
placque formation, and tartar deposit are discussed on pages 143. 

Closely allied to the true mucins are the so-called mucin 
substances. These are mucilaginous substances occurring in 
some of the lower forms of animal life, but they differ in their 
chemical reactions from the true mucins. They are not precip- 
itated from alkaline solutions with acetic acid. 

Casein, the second conjugated protein which we shall consider, 



PROTEINS 125 

is the principal nitrogenous constituent of milk and will be 
studied as such. 

MILK. 

Milk is the characteristic secretion of mammals and contains 
the three great classes of food material, viz.: proteins, carbo- 
hydrates, and fats. The fat is held as a permanent emulsion 
in so-called milk plasma. 

The plasma consists of water holding in solution casein, 
albumin with a trace of globulin, milk-sugar (lactose), and 
mineral salts. 

Specific Gravity. Milk contains two different sorts of sub- 
stances influencing the gravity : first, the fat, being lighter than 
the water, tends to decrease the gravity; second, the solids-not- 
fat, which are heavier than water, tend to increase the gravity of 
the milk. Consequently, it may happen that a very poor milk 
and a very rich milk will have the same specific gravity; e.g., the 
normal gravity of whole milk is about 1.031, while the gravity 
of skim milk will be about 1.035 or 1-036, and that in which cream 
occurs in large amount may be as low as 1.015 or 1-020. It can 
be easily seen that, starting with whole milk, the addition of 
cream or the addition of water will both alike reduce the gravity. 
Hence, taken alone, the gravity tells little or nothing as regards 
the quality of milk; but, if the gravity is taken together with 
the fat content, the two factors oftentimes give sufficient infor- 
mation. 

The relation between the gravity of the fat and the total 
solids is approximately constant, and the following formula 
will give the amount of total solids usually within o.io or 0.15 
of i per cent. 

^ . . r , Fat X 6 . Sp. gr. , 

Total solids = h + 0.46. 

5 4 

Reaction. The reaction of cow's milk, when perfectly fresh, 
is amphoteric to litmus; i.e., it will both redden blue litmus 
paper and turn red litmus blue, at the same time. This double 



126 PHYSIOLOGICAL CHEMISTRY 

reaction is due to the presence of various salts, probably the 
acid and alkaline phosphates. 

Cow's milk is acid to phenolphthalein, and this acidity is 
naturally increased by the multiplication of various acid-forming 
bacteria, which produce lactic acid by hydrolysis of the milk 
sugar. When the acid strength has increased sufficiently, the 
casein (English caseinogen) is decomposed, and paracasein 
(English casein) is produced and precipitated. 

This paracasein constitutes the curd, and the process is the 
ordinary souring of milk. 

Lactic acid is not the only acid produced in the spontaneous 
fermentation of milk, as traces of formic, acetic, butyric, and 
succinic acids have been demonstrated by different investigators. 

The degree of acidity of milk is conveniently determined as 
suggested by W. Thorner (Chem. Zeit., 1891, page 1108, abst. 
analyst XVI, 200) : 10 c.c. of milk with an equal volume of water 
and a few drops of phenolphthalein as indicator, are titrated with 
N/io alkali and every tenth of a degree of alkali used is con- 
sidered as representing one " degree " of acidity. 

By experimenting on samples kept under various conditions, 
Thorner found that milk coagulates on boiling when the acidity 
reaches 23. Adopting 20 as the permissible limit of acidity, 
he proposes the following test: 10 c.c. of milk, 20 c.c. of water, a 
few drops of indicator, and 2 c.c. of decinormal alkali are thor- 
oughly mixed; if any red color, however weak, results, the milk 
will not coagulate upon boiling.* 

This method is given partly for its own sake and partly be- 
cause exactly the same method is used by Dr. Eugene S. Talbot 
of Chicago and many others for the determination of acidity of 
urine. By slight modification it may be used for saliva. The 
record of slight amounts of acidity made in degrees in this way 
has several practical points in its favor. 

Casein is the principal protein found in milk. It exists in 
combination with calcium salts. This combination is broken 
up and paracasein precipitated by the action of rennin and other 
enzymes, by acids, and by certain inorganic salts. 

* From Allen's 'Commercial Organic Analysis/ Vol. 4. 



PROTEINS 



127 



Casein is classified as a pseudo-nucleo-albumin. The nucleo- 
proteins, so named because true nuclein may be obtained from 
them, are constituents of the cell nuclei, and differ in composi- 
tion from ordinary proteins by containing from 0.5 to 1.6 per 
cent of phosphorus. Casein from cow's milk contains, according 
to Hammarsten, 0.85 per cent of phosphorus. It has been 
classified as a pseudo-imcleo-albumm because, upon digestion 
with pepsin, pseudo-nuclein rather than true nuclein is obtained. 

Casein is practically insoluble in water, but dissolves readily 
in dilute alkaline solutions. Its precipitation as curd is de- 
pendent upon the presence of calcium salts. 

Lactalbumin is the only other protein substance worthy of 
note in milk. This may be found in the filtrate after separat- 
ing the casein. The total proteins contained in human milk 
average from 1.5 to 2.5 per cent while in cow's milk the proteins 
are 3.0 to 4.5 per cent. This difference, together with the vari- 
ation of reaction and sugar-content, makes it necessary to 
" modify " cow's milk when it is used as an infant food. 

The modification usually consists in the addition of lime- 
water (to change the reaction), of water (to reduce percentage of 
proteins), and of cream and milk-sugar (to increase fat and 
lactose). 

The following table shows comparative composition: 





Reaction. 


Total 
solids. 


Proteins. 


Sugar. 


Fat. 


Ash. 


Human milk . . 
Cow's milk. . . 


Alkaline 
Acid 


13.00% 
14.00% 


2.70% 
4-15% 


6.10% 
4.90% 


4-00% 

4-25% 


0.20% 

0.70% 



Fat. The fat of milk exists as microscopic globules appar- 
ently inclosed in a protein-like membrane separating substance^ 
the presence of which seems a necessary theory to account for 
the behavior of milk-fat toward various solvents, such as ether. 
The milk-fat, or butter-fat, consists largely of olein and palmitin 
with a slight amount of butyrin and traces of several other fatty 
acids. 

Milk, as has already been stated, undergoes lactic acid fermen- 



128 



PHYSIOLOGICAL CHEMISTRY 



tation readily and this may be induced by a considerable number 
of microorganisms. It is not, however, liable to alcoholic fermen- 
tation except under peculiar circumstances. Alcoholic fermen- 
tation may be induced by certain 
ferments, such as the Kephir 
grain used quite largely in the 
East, the product being known as 
Kumiss or milk wine. Kumiss 
originally was produced from 
mare's milk, but the name has 
also been applied to any milk 
which has undergone alcoholic 
fermentation. 

Colostrum is a peculiar sub- 
stance occurring at the very 
earliest stages of lactation. Its specific gravity is considerably 
higher than that of milk, being 1.040 to 1.060. It contains 
much more protein substance and is characterized by the presence 
of granular corpuscles known as colostrum corpuscles. (Fig. 2.) 




FIG. 2. Milk and Colostrum. 



CHAPTER XV. 
BLOOD AND MUSCLE. 

BLOOD. 

The blood, carrying oxygen and other forms of nutrition to 
all parts of the body, and returning carbon dioxide and the waste 
products of cellular activity, is an exceedingly complex substance. 
Its complexity is not surprising when we consider that it is the 
channel through which all internal exchanges are made. 

As very well expressed by Mathews* the function of the blood 
is fourfold. 

" i . It carries food from the intestine to the tissues ; and gaseous 
food, or oxygen, from the lungs to the tissues. 2. It removes 
waste products from the tissues and carries them to the kidneys, 
lungs, intestine, and skin, the excretory organs of the body. 
3. It provides for the metabolic co-ordination of the body, in 
that it distributes the internal secretions from each organ to 
other organs which utilize them. It thus keeps tissues, which 
may be far separated, in metabolic co-ordination or exchange 
with each other; it is the internal medium of exchange. 4. It 
plays a very important part in the defense of the organism against 
the invasion of parasites. " 

Blood normally is slightly alkaline, PH 7-35, and is remarkable 
for the constancy of its alkalinity, the P H being only slightly 
lowered in severe cases of acidosis. Emphasis should be laid 
on the fact that true acidity of the blood never occurs, the term 
signifying in this case merely a lessened alkalinity. It has been 
stated by Hawk that a change in the P H of the blood as great as 
that occurring between the PH of distilled water and the PH of 
tap water would be fatal to the individual. 

* Mathews' " Physiological Chemistry/' 3rd Edition, page 459. 

129 



130 PHYSIOLOGICAL CHEMISTRY 

COMPOSITION OF THE BLOOD. 

The composition of the blood itself, however, may be roughly 
described as a fluid (plasma) carrying in suspension the cellular 
constituents, red and white corpuscles. The plasma contains 
solid matter to the extent of about 8.9 per cent. This is largely 
protein, consisting of serum globulin, serum albumin, a slight 
amount of nucleo-protein, and fibrinogen; also a fibrin ferment, 
known as thrombase or thrombin. 

The last two constituents play an important part in the for- 
mation of the blood clot. Many theories* have been suggested 
in explaining this property of the blood, and although at present 
no theory has been technically proven the following seems to be 
well grounded and is quite extensively accepted. 

By action of a zymogen known as prothrombin with the calcium 
salts present in the blood, the fibrin ferment or thrombin is 
produced. The thrombin thus formed acts with the fibrinogen 
and produces the fibrin or blood clot. An interesting addition 
has been made to this theory by Howell,f who suggests that the 
prothrombin is held in combination by another substance called 
antithrombin. It is liberated only by the action of thrombo- 
plastin, which comes from the disruption of the blood platelets, on 
the antithrombin. The " clot " mechanically carries down the 
corpuscles, and as the clot contracts, the " serum " separates 
as a clear, amber-colored liquid, consisting of serum globulin 
(paraglobulin), serum albumin, and the fibrin ferment. 

Although their exact function may not be clearly understood 
at present, the blood platelets seem to be essential in the forma- 
tion of the blood clot. They may be considered as oval colorless 
disks about one-third the size of the red corpuscles. 

The clotting of blood is obviously brought about by contact 
with any foreign body and may be hastened by increasing the 
calcium content; the tendency to clot is decreased by the pres- 
ence of oxalates, citrates and a few other inorganic salts. 

* For a detailed discussion the student is referred to Mathews' 'Physiological 
Chemistry/ 3rd Edition, pages 514-538. 
t Howell, Am. Jour, of Physiology, 29, 187, igxz. 



BLOOD AND MUSCLE 131 

Fibrin. The fibrin may be obtained free from corpuscles 
by whipping fresh blood. Under this treatment the fibrin 
separates as shreds, while the remaining fluid constitutes " de- 
fibrinated blood. " 

Fibrin, as usually obtained, is in the form of brown, stringy, 
and " fibrinous " masses, which are kept under glycerin for labor- 
atory use. It is insoluble in water or alcohol. In dilute acid, 
(HC1), or alkali solutions, it swells and ultimately dissolves, 
although it may be several days before solution is effected. The 
fibrins from the blood of different animals differ in composition, 
as indicated by marked differences in solubility. 

In addition to the protein content of the plasma, just described, 
the blood plasma contains various other substances, the most 
important of which are glucose, urea, uric acid, creatinine, fat, 
amino acids, enzymes, lecithin and cholesterol, dissolved gases 
and mineral salts. Acetone is normally present in very slight 
traces, and its increase, as is true of many of the constituents, is 
indicative of a pathological condition. 

The chemistry of the red and white corpuscles is more complex 
and not so well known as the chemistry of the plasma, which 
we have considered. The red corpuscles consist of a frame of 
protoplasm, also called stroma, which contains lecithin, choles- 
trin, nucleo-albumin, and a globulin. (Hammarsten.) Upon 
and all through the stroma is the hemoglobin, which, together 
with its oxygen compound, oxy hemoglobin, is responsible for 
the color of the blood. Oxyhemoglobin may be obtained as 
silky, transparent crystals of blood-red color. 

Hemoglobin. Hemoglobin may be separated from blood 
by shaking with a little ether and water and allowing to stand 
twelve hours on ice; or sometimes crystals may be obtained by 
simply allowing a drop of defibrinated blood to partially dry on 
a microscope slide. The hemoglobin from different animals 
crystallizes in more or less distinctive forms: for example, from 
human blood the crystals will be diamond-shaped or rectangular; 
those from the blood of guinea pigs will be tetrahedrons or 
octahedrons resembling the crystals of white arsenic; and those 
from the blood of squirrels, six-sided plates. 



13* PHYSIOLOGICAL CHEMISTRY 

The composition of hemoglobin has been given as 96 per cent 
globin (a histone), and the remainder hemochromogen. 

Hemoglobin forms compounds with various gaseous sub- 
stances and furnishes a good example for the study of the law 
of mass action. In the lungs, excess of oxygen slowly drives 
other gases, particularly carbon dioxide, out of combination, 
and forms oxyhemoglobin, while in the capillaries excess of 
carbon dioxide in venous blood replaces the oxygen. Hydrogen 
sulphide, nitric oxide, nitrous oxide, and carbon monoxide all 
form compounds with hemoglobin of various degrees of stability, 
the most stable being formed by carbon monoxide which acts as 
a poison by preventing the formation of oxyhemoglobin. Blood 
containing carbon monoxide hemoglobin is of a bright red 
color, which darkens in the air much more slowly than ordinary 
blood. 

Hemoglobin is a conjugated protein, acid in reaction, de- 
composing into globin and hemochromogen^ which is the iron- 
containing radical of the hemoglobin. 

Oxyhemoglobin is similar in its properties, breaking into 
globin and hematin, the hematin being an oxidation product of 
hemochromogen and, like it, containing iron. Hematin, to 
which the formula CsJH&N^Fe has been assigned, is a brown, 
amorphous substance, acid in reaction, and forms with hydro- 
chloric acid hematin hydrochloride, or hemin, which crystallizes 
in characteristic form. See Plate VI, Fig. 2. 

If hematin is heated with strong hydrochloric acid, the iron 
is removed in the form of ferrous chloride, and iron-free hematin, 
or hematoporphyrin, is produced. 

Hematoporphyrin is a dark purplish powder, with the prop- 
erties of an acid and a base and is isomeric with bilirubin. It 
may be considered as the source of the bile pigments. 

These relationships may be expressed thus: 

Hemoglobin + Oxygen = Oxy hemoglobin 



Globin Hemochrbmog 



.vi. 




FIG. i. 

Edestlnu 




3. Fat Crystals, 
A, U, 




. . ' ' ' FIG; 5, : ' -. 
4, 
' - ' 




FIG. 2 e : _ : 




: . 4* 

t Fat 5 




" . ' - 6,..' ' 

'A 9 B> 

' " /" : ' 



BLOOD AND MUSCLE 133 

The iron from the blood may, by decomposition of the pigment 
and subsequent combination with sulphur (FeS), cause dis- 
coloration of teeth. This is the theory of Dr. E. C. Kirk, and 
in the author's opinion is perfectly sound, and far more probable 
than other explanations which have been offered, but which do 
not recognize the formation of a sulphur compound. 

The form of the red corpuscle is that of a biconcave disk 
without nucleus; by action of water it becomes swollen, and the 
hemoglobin may be washed away, leaving the " stroma." This 
is what takes place when blood is " laked, " i.e., when hemolysis 
takes place. The diameter of the red corpuscles of human blood 
is about 1/3200 of an inch. Of the domestic animals, the cor- 
puscles of the dog approach most nearly to the measurement of 
the human. The sheep, horse, and ox have smaller corpuscles 
than man, while those of birds, cold-blooded animals, and reptiles 
are larger (see Plate VI, Figs. 5 and 6). 

The white corpuscles are rather larger than the red, and occur 
in much smaller numbers, a cubic millimeter containing about 
5,000,000 red to 7500 white. The white corpuscles present a 
much greater diversity of character than do the red. They 
contain one to four nuclei, and are capable of amoeboid move- 
ments. The white corpuscles are also called leucocytes, and 
aggregations of them constitute pus. The leucocytes are di- 
vided histologically into various classes, lymphocyte, neutro- 
philes, eosinophiles, etc., according as they are acted upon by 
different staining-fluids or fulfill some particular office; but 
these classes are not to be distinguished chemically. 

BLOOD TESTS. 
Teichmari's Hemin Test. 

If to a drop of fresh blood on a microscope slide 2 drops of 
glacial acetic acid are added and the mixture is covered with a 
cover glass and gently warmed, upon cooling, dark brown, 
rhombic crystals of hematin hydrochloride, or hemin, are formed. 
These may be seen, frequently crossed, under the microscope. 
See Plate VI, Fig. 2. Hemin crystals can alsabe produced from 



134 PHYSIOLOGICAL CHEMISTRY 

dried blood, provided a small crystal of sodium chloride is added. 
These crystals are easily made from human blood and also from 
the blood of many other animals which fact tends perhaps to 
lessen the value of the test. 

Benzidene Reaction. 

This is considered one of the most delicate tests for blood. 
It consists in the development of a bluish-green color when a 
saturated benzidene solution and hydrogen peroxide are brought 
in contact with the sample. Equal volumes of the benzidene 
solution, prepared in glacial acetic acid or alcohol, according to 
the method given in the Appendix, and commercial hydrogen 
peroxide are used. Exp. 206. The hydrogen peroxide is de- 
composed by the hemoglobin of the blood, and the oxidation of 
the benzidene, due to the liberation of oxygen, gives the blue color. 

The blood of several different species of animals responds 
positively to this reaction, as to the hemin test. 

Guaiac Test. 

See Exp. 205. This test, according to Hawk, is of value if 
carefully performed, although it has lost favor with many 
because of its positive reaction with so many substances. 
The precautions necessary are that the unknown solution be 
boiled twenty seconds before the addition of the guaiacum and 
hydrogen peroxide solutions, and that the blue color does not 
develop until after the addition of the peroxide. Under these 
conditions it seems to be a fairly distinctive test for blood. 

Bordet Reaction. 

This test is regarded as the most satisfactory distinctive test 
for human blood. An antiserum, which will produce a precip- 
itate with human blood and with the blood of no other species, 
is prepared from rabbits who have previously been injected with 
human defibrinated blood or blood serum. 



BLOOD AND MUSCLE 135 

ANALYSIS OF BLOOD adapted from Folin. 

For a complete technique of blood analysis the student is 
referred to Folin's ' Laboratory Manual/ Only a small part 
of the procedure is outlined here, in the belief that it may be of 
value to the dentist. 

Precipitation of the Protein. 

The first step necessary in any blood analysis is the precipi- 
tation of the protein material and the subsequent formation of 
the clear, colorless filtrate, which is protein free. Folin ac- 
complishes this by means of tungstic acid, the procedure being as 
follows : 

The oxalated blood* is laked with seven volumes of water, and 
to this one volume of 10 per cent sodium tungstate (the tungstate 
must be pure) is added. Then one volume of two-thirds normal 
sulphuric acid is added slowly and with constant shaking. The 
flask which contains the solution is then stoppered and shaken 
thoroughly. After it has stood for five minutes, if the coagula- 
tion is complete, the color of the precipitate becomes dark brown. 
If the red color continues it usually indicates too much oxalate 
and it is necessary to add 10 per cent sulphuric acid, one drop at 
a time, with vigorous shaking, until the brown color is obtained 
and all foaming has ceased. The precipitate may then be fil- 
tered, and if the filtration is performed slowly and carefully the 
first filtrate will be clear and colorless. 

Determination of Non-Protein Nitrogen. 

This determination of the total nitrogen present, as urea, am- 
ino acid, ammonia, creatinine, uric acid, and other substances, 
has recently become one of the most valuable determinations 
made from a clinical standpoint. The nitrogen is determined 
by Folin's micro-Kjeldahl method, using the phosphoric sul- 
phuric acid mixturef to bring about digestion. 

Place 5 c.c. of the blood filtrate in a large dry pyrex test-tube 

* Blood which has been collected over potassium oxalate to prevent coagulation. 
t See Appendix. 



136 PHYSIOLOGICAL CHEMISTRY 

and add i c.c. of diluted acid mixture (i volume acid with i 
volume water). Boil vigorously from three to seven minutes, 
or until the tube is nearly filled with dense fumes; then quickly 
reduce the flame until boiling almost stops. Cover with a 
watch crystal and continue heating until oxidation is complete, 
i.e., until the solution is practically colorless. This usually 
happens in less than two minutes. Remove flame and let 
mixture cool for little more than a minute, then dilute with 15 
to 25 c.c. of water. The solution may then be nesslerized and 
the color obtained compared with a standard ammonium sul- 
phate solution. To obtain reliable results by this method the 
student needs practice in handling the colorimeter, and to give 
accurate results the colorimeter itself must be a fairly good one. 
The use of this method thus involves a considerable expense. 

Equally accurate results may be obtained quite as easily by 
the inexperienced, by distilling off the ammonia, which has been 
formed from the digestion, into standard acid and titrating the 
excess of acid. Because of the small quantities used the dis- 
tillation is not a long process. N/50 acid and alkali are recom- 
mended. 

The normal non-protein nitrogen is 25-30 mg. per 100 c.c. 
There is a marked increase in nephritis. 

Determination of Chlorine. 

Chlorides present in the blood filtrate may be determined 
directly by titration with potassium sulphocyanate by the same 
procedure as that given for chlorine in the saliva. Normal 
blood (whole blood) chlorine is 450-500 mg. per 100 c.c. about 
.45 per cent. The plasma content is slightly higher. 

Determination of Glucose. 
Reagents required: 
(i) Standard sugar solutions 

(a) i per cent glucose preserved with toluene, or made 
in a nearly saturated benzoic acid solution. 

(b) 5 c.c. of solution (a) diluted to 500 c.c. contains 
i mg. of glucose per 10 c.c. 



BLOOD AND MUSCLE 



137 



(c) 5 c.c. of solution (a) diluted to 250 c.c. contains 
2 mg. of glucose per 10 c.c. 

(2) Alkaline copper solution. 

In a 1000 c.c. flask dissolve 40 grams of anhydrous sodium car- 
bonate in 400 c.c. water. Add 7.5 grams of tartaric acid and, 
when dissolved, 4.5 grams of crystallized copper sulphate. 
Mix and fill to the mark with distilled water. 

(3) Molybdate phosphate solution. 

Place 5 grams of sodium tungstate and 35 grams molybdic 
acid in a large beaker (1000 c.c.). Add 200 c.c. 10 per cent 
NaOH and 'an equal volume of water and boil the solution 
vigorously for thirty or forty minutes. When cold, add water 
to make volume about 350 c.c., and then add 125 c.c. cone, 
phosphoric acid. Dilute to 500 c.c. 

PROCEDURE. 

For this determination a special tube (Fig. 3) is necessary. 
Place in the tube 2 c.c. of the blood filtrate and 
2 c.c. of the alkaline copper solution, making the 
volume of the solution such that it comes up to 
just the narrow part of the tube. In two other 
similar tubes place respectively 2 c.c. of standard 
sugar solutions (b) and (c) and to each add 2 c.c. of 
the alkaline copper solution. Heat the three tubes 
(for six minutes) in a boiling water-bath; then 
cool and add 2 c.c. of the molybdate phosphate 
solution to each tube. This will quickly dissolve 
the cuprous oxide formed. The blue solutions 
are then diluted to the 25 c.c. mark, and the flask 
stoppered and shaken. The color obtained in the 
unknown is compared colorimetrically with the 
standard which matches it most nearly. 




-8 nun. 

br{ 4 C.C. 



FIG. 3. 

Folin Sugar 
Tube. 



Reading of standard Mg. of glucose 



Calculations 



usually 20 



Reading of unknown 
mg. of glucose per 100 c.c. blood. 



X 



in standard 



138 PHYSIOLOGICAL CHEMISTRY 

Uric Acid Determination 

The Folin phospho-tungstic test for uric acid is given under 
saliva. By this test it has been found that the normal content of 
uric acid in the blood is 4-5 mg. per 100 c.c. These figures are 
somewhat higher than those usually given and are not correct 
if the older and less delicate methods are used. 

The value of blood analysis to physicians has long been known, 
but only recently has the importance to the dentist of a knowl- 
edge of systemic conditions been emphasized. That a relation- 
ship, and often a very close one, exists between oral disease and 
systemic conditions, is now a recognized fact; and in making 
this relationship clearer, blood analysis is essential. 

Dr. Toren* makes some interesting statements in regard to the 
value of the microscopical blood examination. 

" We wish to urge more thorough and painstaking work in 
the histological examination of blood. This work should not 
be delegated to an assistant or technician, but should be done by 
the physician or dentist himself." And further: 

" It was about ten years ago that we first noticed, in the course 
of the general diagnostic examination of patients, that a particular 
type of leucocyte was present in the blood of patients having 
infections around the teeth." 

MUSCLE. 

Muscle forms, in the adult, a little less than 45 per cent of the 
body weight; and of all the metabolic processes taking place 
in the animal body probably from 50 to 75 per cent take place 
in the muscle. These facts make the chemistry of muscle very 
essential and, at the same time, decidedly complex. 

The muscle changes rapidly upon the death of the animal, so 
much so that the liquid which may be expressed from living 
muscle (or from muscle frozen immediately upon the death of 
the animal) has been called muscle plasma, to distinguish it 

* Diagnosis of Oral Infection by Blood Examination, by Julius A, Toren, M.D.: 
Dental Cosmos, Sept. 1922, p. 917. 



BLOOD AND MUSCLE 139 

from muscle serum, which is obtained in the same manner from 
dead muscle. The chemical reactions and the composition of 
these solutions differ considerably. 

Muscle contains about 75 per cent water and about 25 per cent 
solid matter, the latter being chiefly made up of protein sub- 
stances. 

The two proteins of muscle plasma are given by Halliburton as 
paramyosinogen 25 per cent, and myosinogen 75 per cent. Of 
these the paramyosinogen seems to be a globulin, while the myo- 
sinogen, having many of the properties of a globulin, is soluble 
in pure water and is rather a mother protein from which the clot 
from muscle serum is produced. The protein of the muscle clot 
is known as myosin or myogen. Myosin may be precipitated 
from muscle serum by saturation with sodium chloride or mag- 
nesium sulphate. It has many of the properties of the globulins, 
but differs in the very important particular of not being pre- 
cipitated by dialyzation. 

In contrast with the soluble proteins of the muscle plasma, the 
proteins of the stroma, as the residue from which the plasma has 
been expressed is called, seem to be of an insoluble albuminoid 
character. 

According to Mathews, the stroma also contains nucleo- 
proteins, and phosphorus in the form of phospholipins, probably 
combined with a protein substance. 

MUSCLE EXTRACTIVES. 

When muscle is treated with boiling water several organic 
substances are dissolved out of the muscle substance. These 
so-called extractives are of importance, because from studying 
them we get some idea of the metabolism of the muscle cells, 
and we may believe that some of these extractives give rise to 
substances excreted in the urine. 

Nitrogenous Extractives. This group includes creatine and 
its anhydride, creatinine, as well as xanthine, hypoxanthine, 
uric acid, urea, guanine, methyl guanidine, taurine, and a 
few others. Most of these are white solids which crystallize 
more or less easily with characteristic forms. 



140 PHYSIOLOGICAL CHEMISTRY 

The exact source of creatine and creatinine has been the 
subject of much study, and it is still a disputed question. Crea- 
tine is methyl-quanidine acetic acid with the following formula : 

NH-C - N-CH 2 -COOH 

II I 
NH CH 3 

If creatine is dehydrated by means of boiling with dilute 
mineral acid, creatinine is produced. 

NH-C=O 

I 
NH = C 

I 
CH 3 -N - CH 2 

In the muscle, creatine seems to be a product of cell metabolism, 
diffused into the blood in small amounts. It has recently been 
suggested that it plays an important part in maintaining muscle 
tone. Creatinine, likewise a product of cell metabolism, may be 
present in living muscle in small amounts. It is found in the 
blood and urine, however, in much larger percentages than is 
creatine; the content seems to vary with the age, sex, and health 
of the individual, but is not appreciably altered by the protein 
intake. This fact is significant as regards its source, and points 
directly to it as a product of the vital processes of the muscle cell. 

The purine derivatives, pages 57 and 58, found among the ex- 
tractives of muscle are derived from the nuclei of the cells. 
Muscle tissue contains less nuclear material than most other 
body tissue, and therefore gives off a smaller amount of purines. 
These purine derivatives, according to Mathews, are diffused 
into the blood stream, particularly during muscular activity. 

Non-nitrogenous Extractives 

Under this group may be included glycogen, dextrin, lactic 
acid, sugars, inosite, and some fat. 

Glycogen exists in the muscle as stored carbohydrate, and is 
converted to glucose and oxidized as the muscle needs it. Lactic 
acid, commonly classed as a fatigue product of muscle, probably 
comes from the carbohydrate present. It is the a-hydroxy-lactic 



BLOOD AND MUSCLE 141 

acid, known as sarcolactic acid, that is responsible for the acid 
reaction of dead muscle. Inosite, C 6 Hi 2 O 6 + H 2 O, is a hexa- 
hydroxy-benzene, C 6 H 6 (OH) 6 . It has a sweet taste and was 
formerly erroneously classed with carbohydrates. Inosite may 
occur in the urine of diabetic individuals. 

The chief inorganic constituent derived from muscle is the 
acid phosphate of potassium. Like lactic acid and carbon 
dioxide, this may be considered as a fatigue product of the 
muscle. Chlorides of sodium, iron, magnesium and calcium are 
also found present. 

MUSCLE METABOLISM. 

In considering the metabolism of the muscle we must think of 
it in two distinct ways : the metabolism which is concerned with 
the maintenance of the muscle itself, or, as Mathews calls it, 
" formative metabolism "; and that which produces muscular 
activity, or " energy metabolism. " 

The former is similar to that taking place in all of the body 
tissues and is the process of building new protein material by 
re-synthesizing ammo acids. The exact nature of the synthesis 
is as yet merely conjecture. Unlike many other body tissues, 
muscle is capable of self-destruction; that is, it apparently 
contains proteolytic enzymes which must be constantly held in 
check. This checking is easily accomplished when there is no 
lack of food, but during starvation the muscle may be used as 
food for the more essential organs of the body. 

The so-called energy metabolism is unfortunately no more 
clearly known than the formative metabolism. The glycogen 
is converted to glucose by enzyme action, and energy and heat 
are produced by the oxidation of the glucose. Lactic acid, 
carbon dioxide, acetone, traces of alcohol, and traces of many 
other substances have been found in muscle, and different the- 
ories have been advanced as to their origin. However, no proven 
information is available at the present time. Further, it ha s 
been shown by Cannon that some secretions of the ductless 
glands, especially adrenalin, have a marked effect on the glycogen 
content of the muscle. 



CHAPTER XVI. 
TEETH AND TARTAR. 

The teeth consist of enamel, dentine, cement, and the pulp 
chamber, the first three of which may be considered chemically. 
The composition of the cement is practically that of true bone, 
the dentine and enamel differing principally in the proportion 
of organic matter which they contain. In all of these, the pres- 
ence of lime, phosphoric acid, carbonic acid, and traces of 
magnesium and calcium fluoride may be demonstrated. The 
tartar contains a greater proportion of carbonic acid, less calcium 
phosphate, and much less organic matter than the teeth, taken 
as a whole, or than dentine, but about the same as enamel. 
According to Berzelius, sodium chloride and sodium carbonate 
may also be found. 

The composition of the different parts of the tooth substance 
has been given as follows: 



Ash Ca ( p <)2. MgHP0 4 . CaC0 8 . 

Dentine ................... 23 . 2 76.8 70 . 3 4.3 2.2 

Cement ................... 32.9 67.1 60.7 1.2 2.9 

Enamel .................... 3.1 96 . 9 90 . 5 traces 2 . 2 

Also traces of magnesium carbonate, calcium sulphate, fluorides, 
and chlorides. An increase in the percentage of calcium phos- 
phate or fluoride increases the hardness of the tooth, while an 
increase of calcium carbonate decreases the hardness. 

Potassium sulphocyanate, ferric phosphate, sulphites, and 
uric acid have been found in tartar, as additional chemical 
constituents, while after the solution of the mineral matter 
the presence of epithelium cells, mucus, and the leptothrix may 
be demonstrated by the microscope. 

According to Vergness, Du tartre dentaire, quoted by Gamgee, 

the tartar from incisor teeth and that from molars show decided 

142 



TEETH AND TARTAR 143 

difference in their content of iron and calcium phosphates, the 
analysis being as follows: 

Tartar of Incisors. Tartar of Molars. 

Calcium phosphate 63 . 88-62 .56 55 . 11-62 .12 

Calcium carbonate 8.48- 8.12 7.36- 8.01 

Phosphate of iron 2.72- 0.82 12.74- 4.01 

Silica 0.21- 0.21 0.37- 0.38 

Alkaline salts 0.21- 0.14 0.37- 0.31 

Organic matter 24.99-27.98 24.40-24.01 

DEPOSITION OF TARTAR UNDER VARIOUS SYSTEMIC 

CONDITIONS. 

The presence of oxalates and urates has been reported in 
the black tartar from pyorrhea cases. The deficient oxidation 
and high acidity usually occurring in such cases is conducive to 
the production of large amounts of oxalic or uric acids in the sys- 
tem, not necessarily on the teeth, whether these substances have 
etiological relations to pyorrhea or not. 

The formation of ordinary hard tartar, consisting principally 
of phosphate and carbonate of calcium, is accounted for by 
Dr. Percy R. Howe* as follows: " An excess of calcium salts 
in the blood must be granted as one of the causes of calcification. 
These calcium salts are held in solution by two distinct factors: 
first, the excess of carbon dioxide; and second, by the presence 
of colloidal substances in suspension." 

The colloidal substances consist largely of mucin and it has 
been determined that the isoelectric point of mucin, or the PH at 
which mucin is most easily precipitated, is low, ranging with 
that of serum albumin, (PH 4-7)t an d serum globulin (P H 5.4), t 
thus putting the isoelectric point of the salivary proteins below 
the PH of any ordinary saliva. We know then why the calcium 
of the saliva may always be considered, as existing, in part at 
least, in the form of a calcium proteinate, and why we may dis- 
regard the occurrence of mucin as a protein salt (page 119). 
In the deposition of tartar, i.e., the precipitation of calcium 
phosphate and carbonate from saliva, the protein of the calcium 

* Dental Cosmos, 1915, p. 307. 

t Michaelis' 'Wasserstoffionenkonzentration,' Berlin, 1914. 



144 PHYSIOLOGICAL CHEMISTRY 

proteinate will be acted upon only by electro-positive ions 
(Vol. I) , furnished in the saliva by the alkaline salts. (Na 2 HPO 4 , 
NaHCO 3 ) which are always present. The calcium is thus left 
to combine with the anions of phosphoric and carbonic acids. 

We should then expect that the higher the P H of the saliva 
the more liable it would be to the formation of tartar. This has 
been shown to be not only possible but highly probable, by work 
done in the authors' laboratory. (Journal Dental Research, 
Vol. IV, No. i, March, 1922.) Modifying factors which must 
be taken into consideration are, of course, the relative quantities 
of calcium, phosphoric acid, and carbonic acid. The foregoing 
statements are in relation to the effect of PH only on tartar 
formation; large amounts of carbon dioxide would tend to keep 
calcium in solution and, of course, lower the PH accordingly. 

The important thing, however, is not exactly that the oral 
conditions which are most advantageous for the precipitation of 
tartar should be known, but rather that when this subject is 
under investigation all of the several factors influencing it should 
be considered. 

Barille holds that calcium phosphate occurs in the blood as an 
unstable carbonate-phosphate which tends to decompose into 
calcium acid phosphate and bicarbonate, and that in saliva we 
find both these salts held in solution by carbon dioxide as follows: 

Ca 3 (P0 4 ) 2 + 4 H 2 C0 3 = H 2 + P 2 O 8 Ca^H 2 .2 CO 3 (CO 3 H) 2 Ca. 

Upon the escape of the carbon dioxide, the calcium precipitates 
as the tri-metallic phosphate if the solution is alkaline, and as 
dicalcic phosphates if the solution is acid; and, of course, the 
loss of carbon dioxide will at the same time result in the pre- 
cipitation of the neutral carbonate (CaCO 3 ). 

That the general systemic condition is also a factor in the 
deposition of tartar is indicated by the experience of Dr. Wright 
of the Harvard Dental School, who has watched for a succession 
of years the fairly uniform increase in tartar deposits from Oc- 
tober to June, and has found the vacation period marked by 
smaller amounts of deposit. 



TEETH AND TARTAR 145 

Lactic and other organic acids have been found in minute 
quantities in tartar, but these, as well as the qualitative tests for 
urates, will be considered more in detail under the Chemistry 
of Saliva. 

ANALYSIS OF TEETH AND TARTAR. 

The substance for analysis should be reduced to a moderately 
fine powder by crushing in a mortar, and a fair sample of the 
whole taken for each test. 

Moisture may be detected by the closed-tube test (Vol. I.) 
and may be determined by accurately weighing out one gram 
of the substance in a counterpoised platinum dish or crucible 
and drying at 100 C. to constant weight. 

Inorganic matter may be determined by careful ignition of 
dried substance; raise the temperature slowly till full red heat 
is reached; cool in a desiccator and weigh. 

Organic matter may be ascertained by difference. 

Lactates and other organic acids may be detected by careful 
crystallization and examination with the micropolariscope. 

The several inorganic constituents may be demonstrated as 
follows : 

Phosphoric Acid. Dissolve a little of the powdered sub- 
stance in dilute nitric acid; then to a few drops of the clear 
solution add an excess of ammonium molybdate in nitric acid. 
A yellow crystalline precipitate of ammonium phospho-molyb- 
date will separate. Avoid heating above 60 C., as the ammo- 
nium molybdate may decompose and precipitate a yellow oxide 
of molybdenum. 

Carbonic acid may be detected by liberation of carbon dioxide 
and passing the gas into lime-water, as described in Vol. I, 
or with closed tube and drop of baryta-water. 

Chlorine may be detected in the dilute nitric acid solution by 
the usual silver nitrate test. 

Calcium and magnesium may be separated and identified by 
the usual methods of analysis in the presence of phosphates. 

Test for calcium and magnesium as follows: Add to the 
hydrochloric acid solution an excess of ammonia; calcium phos- 



146 PHYSIOLOGICAL CHEMISTRY 

phate and magnesium phosphate are precipitated, white. Filter 
and to the filtrate add ammonium oxalate; a white precipitate 
shows lime, not as phosphate. Wash the precipitate produced 
by ammonium hydroxide, dissolve in dilute hydrochloric acid, 
and add ferric chloride carefully till a drop of the solution gives, 
when mixed with a drop of ammonium hydroxide, a yellowish 
precipitate. Nearly neutralize with sodium carbonate and add 
barium carbonate, which precipitates ferric phosphate. Filter, 
heat the filtrate, precipitate the barium with dilute sulphuric 
acid, and filter again. From the filtrate calcium is precipitated 
as white calcium oxalate, by making it alkaline with ammonium 
hydroxide and adding ammonium oxalate as long as a precipitate 
is formed. Filter and add to the filtrate sodium phosphate, 
which precipitates magnesium as ammonio-magnesium phos- 
phate, white. 

LABORATORY EXERCISES may consist of the examination by 
microchemical methods of one or more samples of tartar. 

BONE. 

If all organic matter is burned off from bone, there remains 
the bone-earth, so-called, made up of the phosphates and car- 
bonates of lime and magnesia, with slight amounts of chlorine, 
fluorine, and of sulphates, the proportion being practically the 
same as given for dentine, under Teeth, on page 142. Because 
in some diseases, in which the bones are softened or decalcified 
(as osteomalacia), the relation of the calcium oxide and phos- 
phorous pentoxide remains unchanged, it has been claimed that 
these substances exist in the bone in the form of a definite phos- 
phate-carbonate containing three molecules of the tribasic 
phosphate to one of carbonate: 3 Ca 3 (PO 4 )2.CaCO 3 . 

If, by treatment with dilute hydrochloric acid, the mineral 
constituents are entirely dissolved out of bone, there remains 
a substance from which glue (gelatin) is derived, of similar 
composition to the collagen from connective tissue, and known as 
ossein. Neither of these (ossein or collagen) is soluble in water 
or in dilute acids. 



TEETH AND TARTAR 147 

Bone marrow is of two sorts, red and yellow. The red marrow 
contains erythrocytes, fat, lecithin, protein substance consisting 
of a globulin, a nucleo-protein, fibrinogen, traces of albumin and 
proteose. 

The yellow marrow is similar in composition, except that it 
contains fewer erythrocytes, more fat and more olein in the fat. 



CHAPTER XVII. 
DIGESTION. 

SALIVARY DIGESTION. 

Digestion begins with the action of the saliva upon the carbo- 
hydrates, and if mastication is sufficiently prolonged, the ptyalin 
may convert an appreciable quantity of starchy food into a more 
soluble form before it reaches the stomach. In the stomach 
the amylolitic action of the saliva is stopped by the contact 
with the gastric juice. A certain amount, however, of salivary 
digestion takes place within the stomach, due to the fact that 
considerable time necessarily elapses before the acid of the gastric 
juice has been secreted in sufficient quantity to completely 
permeate and acidify the mass of food received from the esopha- 
gus. As has been previously shown, a very feeble degree of 
acidity is conducive to the activity of the amylolytic ferment. 
The average alkalinity of the saliva, calculated as Na^CO 3 , is 
about 0.15 of one per cent. 

Saliva carries the digestion of starches to the maltose stage 
through action of the enzyme ptyalin. The first substance 
formed is soluble starch, which splits into erythrodextrin and 
maltose. The erythrodextrin hydrolyzes to give a-achroodextrin 
and maltose and the a-achroodextrin hydrolyzes in turn to /3- 
and 7-achroodextrin, some maltose also being produced each time. 
The further cleavage of the y- achroo-dextrin yields only maltose. 
Diagrammatically these changes may be represented as shown on 
the following page. 

These digestive changes may be indicated by the action of 
saliva on starch, using iodine as an indicator (see page 286); 
the color of the iodine test changes from a decided blue with the 
starch to purple and red with dextrine, finally becoming negative 

with maltose. 

148 



DIGESTION 149 

Starch 

I 

Soluble Starch 

/ \ 
Maltose Erythrodextrine 

/ \ 
Maltose a-Achroodextrine 

i/ \ 
Maltose /3-Achroodextrine 

i/ \ 
Maltose 7-Achroodextrine 

/ \ 
Maltose Maltose 

The amylolytic action of the saliva is best adapted to a neutral 
or slightly alkaline solution. It will act, however, in an acid 
solution, provided the acid is in combined form, i.e., as in the form 
of a protein salt; in the presence of free acid the action is pre- 
vented. 

In addition to the ptyalin of the saliva there is present also a 
small amount of maltase which hydrolyzes the maltose to glucose. 
By far the greater part of this hydrolysis, however, does not take 
place until the food comes in contact with the maltase of the 
intestinal juice. 

According to some authorities, the saliva also contains a trace 
of proteolytic enzyme; but in the author's opinion there has 
been no substantial experimental evidence differentiating be- 
tween the action of such a proteolytic enzyme and bacteria. 

GASTRIC DIGESTION. 

The first step in the gastric digestion is probably the union 
of the stomach hydrochloric acid with the proteins, forming 
meta-proteins or allied bodies which are changed by pepsin, 
which is the active digestive ferment of the stomach, into the 
proteoses, and slight amounts of the various peptones, following 
practically the changes produced experimentally on page 288. 

Pepsin is an active proteolytic enzyme occurring in the cells 
of the stomach- wall, probably as pepsinogen; this latter is 



150 PHYSIOLOGICAL CHEMISTRY 

decomposed by the hydrochloric acid, with the formation of free 
pepsin. Pepsin works only in faintly acid solutions, and in the 
stomach carries the digestion of proteins but little beyond the 
stage of the proteoses. 

The activity of pepsin is dependent on the hydrochloric acid, 
and for this reason the hydrochloric acid of the gastric juice is 
classed with those substances known as activators. They are 
not enzymes but are essential to the action of the enzyme. 

Hydrochloric acid is obtained from the fundus glands by an 
interchange of radicals between alkaline chlorides and the car- 
bonates of the blood. The quantity present varies from nothing 
to 0.3 per cent, the degree of acidity most favorable for peptic 
activity being about 0.18 per cent, PH 1.5-2.0. 

Aside from HC1, various organic acids may be present in the 
stomach contents; lactic acid, butyric acid, and acetic acid are 
the most important of this class, tests for which are referred to 
under analysis of gastric contents, page 288. 

Hydrochloric acid combines with protein substances of the 
food, forming a rather unstable compound, in which condition 
the acid is known as combined hydrochloric acid in distinction 
from the free hydrochloric acid which the gastric juice may also 
contain. The combined acid possesses only in modified form 
the properties of the free acid, and hence is less likely to stop the 
digestive action of ptyalin from the saliva. 

Rennin is a second enzyme found in the stomach. - This, like 
pepsin, also exists as a zymogen, and is liberated or developed 
by the presence of acid. Its action is particularly the curdling 
of milk, i.e., the decomposition of casein (Exp. 182), and con- 
sequent coagulation of the paracasein, the curd. 

This process involves a splitting of the casein into a slight 
amount of a peptone-like body, and soluble casein. From this 
latter substance the insoluble curd is produced by the action of 
the calcium salts contained in the milk. 

The activity of rennin is greatest in a slightly acid medium, 
PH 5.0, although it will act in a neutral or slightly alkaline 
solution. If the alkalinity becomes equal to that of the blood 
the enzyme is destroyed. 



DIGESTION 151 

It is interesting to note the difference in the optimum P H of 
pepsin and rennin, the two proteolytic enzymes of the stomach. 
In adult life the normal gastric acidity is very close to the PH 
best adapted for pepsin activity; while in the child the acidity 
is much less, and in very early life we find the pepsin virtually 
inactive and the condition to be that most favorable for the 
action of rennin. 

Gastric lipase, or stomach steapsin, a fat-splitting enzyme, also 
exists in the stomach, in very small quantities. Its action is 
comparatively weak and of but slight importance. 

It is to be noted that the digestive action of the stomach is 
only partial, the proteins being split into proteoses and to some 
extent into peptones, while further action is left for the more 
active ferments of the pancreatic and intestinal juices. 

PANCREATIC DIGESTION. 

It may be an aid, in remembering the various digestive fer- 
ments, to note that in the saliva we have one principal ferment, 
ptyalin; in the stomach we have two, pepsin and rennin; in 
the pancreatic juice, three, trypsin, amylopsin, and steapsin. 
In addition to these, the pancreatic juice contains a ferment 
similar to rennin, known as chymosin. 

Trypsin is the proteolytic enzyme of the pancreatic juice. 
It is a much more energetic digestive agent than pepsin, con- 
verting the proteoses into peptones, tyrosin, leucin, and other 
ammo acids. It also differs from pepsin in that it acts in an 
alkaline medium rather than an acid. Trypsin exists, like other 
proteolytic enzymes, as a parent enzyme, trypsinogen, which in 
itself is not a digestive ferment, but which is rendered active 
(activated) by another substance known as enterokinase. 

The enterokinase, another one of the activators, occurs in the 
intestinal juice, and seems to be secreted only as it is needed for 
the activation of the trypsinogen. Enterokinase does not in 
itself possess digestive power, but its action is destroyed by heat 
and in this it resembles the enzymes. 



152 PHYSIOLOGICAL CHEMISTRY 

Amylopsin, or pancreatic amylase, is the starch-digesting 
enzyme of the pancreatic juice. Here, again, we have an 
enzyme much more energetic in its action upon carbohydrates 
than the ptyalin of the saliva. It converts starch into maltose 
and to some extent to dextrin. The amylopsin is active in 
faintly alkaline or very faintly acid solution; more acid, however, 
retards its action. 

The starch-splitting enzyme of the pancreas is dependent 
upon the presence of electrolytes; if these are removed by 
dialysis a juice results which is devoid of starch-splitting power. 
A halogen ion, chlorine or bromine, is apparently essential to 
the activity of this enzyme.* 

Steapsin, lipase, is the fat-splitting enzyme of the pancreatic 
juice, inactive until it comes in contact with constituents of 
the bile. It splits the fat, as indicated on page 106, into glycerol 
and fatty acids, and also acts as an emulsifying agent. The 
free fatty acids thus formed unite with the alkaline bases found 
in the intestines, to form soaps, which are also active emulsifying 
agents. 

Chymosin, or pancreatic rennin, has practically the same 
action upon casein as the gastric rennin. 

The pancreatic juice and the bile enter the duodenum in very 
close proximity, and the digestive action of each is dependent, 
to a considerable extent, upon the presence of the other. 

The secretion of the pancreatic juice is brought about by a 
substance called secretin, secreted by the mucous membrane of 
the intestine. Secretin, according to some authorities, exists as 
prosecretin and is converted into secretin by the acidity produced 
in the intestine with the passage into the duodenum of the acid 
stomach contents. It belongs to that class of substances known 
as hormones, and differs from the activators in that it starts 
specific chemical action. Very little is known about the action 
of hormones, but that they are essential to the function of va- 
rious glands is a recognized fact. Hormones are secreted into 

* Journal of the American Chemical Society, Vol. 32, p. 1087, Kendall and 
Sherman. 



DIGESTION 153 

the blood, and the presence in the blood of these so-called " chem- 
ical messengers " seems to act as a co-ordinating agent between 
the various glands. 

The intestinal juice contains a number of substances playing 
an important part in the preparation of food material for assim- 
ilation. Among them is erepsin (erepase). This is a protein- 
splitting enzyme acting upon the products of tryptic digestion. 
It has little power upon the simple proteins, but will split the 
peptones into amino acids. There are also in the intestinal 
juice certain amylolytic enzymes, sucrase, lactase, and maltase, 
which continue the digestive action started by amylopsin or by 
ptyalin of the saliva. Their action is, respectively, the conver- 
sion of sucrose to glucose and levulose; of lactose to glucose and 
galactose; and of maltose to two molecules of glucose. 

Bile. This is a secretion produced by the liver and stored in 
the gall-bladder, from which it is delivered to the intestines, where 
it aids materially in emulsification and absorption of the fats. 

Composition of Bile. The composition of bile is very com- 
plex, as it contains a portion of the waste products of metabo- 
lism as well as substances playing an important part in digestion 
and designed to be re-absorbed into the circulation. 

Among the first class are the two principal bile pigments: 
the bilirubin (bile red) and its oxidation product, biliverdin, 
(bile green). The bile-pigments are derived from the coloring 
matter of the blood. The appearance of either of these or of 
their derivatives, in either urine or saliva, is indicative of patho- 
logical conditions either of the liver- or bile-ducts, causing 
obstructions to the outflow of the bile or a destruction of the 
red-blood corpuscles, f The blood pigments, according to 
Michaels, are easily demonstrable in the desiccated saliva by 
means of polarized light. 

Cholesterol (C 2 7H 4 50H?) may also be considered a waste 
product of the bile. It is excreted with the feces; when re- 
tained it is likely to produce " gall stones," which are often 
found to consist of fairly pure cholesterol with a little coloring 
matter. 

t Ogden. 



154 



PHYSIOLOGICAL CHEMISTRY 



< 
w 



o 


p 



8 






8 






,9 


M 


8 




8 11 




. 


J 


C-E 






























o 




O OO 








^ 












3 


TJ+ 5 


iT^ 
















11 




*73 o 






1 


&<> 


so 




v2 *1 






4J 




Cv / 










O) 


Action. 


Amylolytic 


Proteolytic 

Proteopyknotic 1 
Lipolytic 4 


Proteolytic 
Amylolytic 

Lipolytic 
Proteopyknotic 


Lipolytic 


.H 




3 


tfl 

4) 
So 

1 

U 


i| 


fProteoses (albu- 1 
1 moses) 1 
I Peptones, leucine [ 
ITyrosine, etc. J 
Casein (Neucleo- 
protein) 
Emulsion 


fProteoses \ 
\Peptones.Peptidesj 
Maltose, Dextrin 

Emulsion 

Casein (proteose- 
like body) 


Emulsion (glycerol 
and fatty acid) 


C * a> rt i i-i C 

^COi-jOOOf-^W 


V 

O 


Acts upon 


Carbohydrates 
(starch) 


Proteins by HCI 
into Metaproteins 

Casein (phos- 
phoprotein) 
Fat 


i|j 


-4-* 


Tryptic digestion 
Sucrose 

Lactose 

Maltose 
Trysinogen 
Fat 


?* 

99 to 
<V J> 

"C 
> ^ 

8 HH 

cfl m 


Ferment. 


1 


Pepsin 

Rennin 
Gastric Lipase 


Trypsin 
Amylopsin 2 

Steapsin 8 
Pancreatic Rennin 


Alkali activators 
(emulsifying 
agent) 


Erepsin (erapase) 
^ ^ Sucrase 

Qj 0) 

c J Lactase 

*"" Maltase 
Enterokinase 6 
Lipase 


nzyme. 2 Pancreat 
i. 7 Sugar-splitting. 


! 


> 


1 


! 
1 





ill 


nilk-curdling e 
itor for trypsir 


1 

3 


Salivary 


^ 

| 


i 


j 


. 


o -^ 

bfl -C 
.S 


Location. 


i 


CO 


1 




j 


.S ^ 

|j 



DIGESTION 155 

Two important acids of the bile are taurocholic and glyco- 
cholic, existing principally as sodium or potassium salts. Gly- 
cocholic acid upon hydrolysis splits into a simpler acid (cholic) 
and glycocoll, glycocoll being an amino-acetic acid (page in), 
which is undoubtedly an antecedent of urea. 

Taurocholic acid, on the other hand, splits into cholic acid 
and taurine, taurine being an amino-ethyl sulphonic acid (page 

49)- 



CHAPTER XVIII. 

SALIVA PROPERTIES, CONSTITUENTS, 
AND ANALYSIS. 

The saliva is a mixed secretion from the parotid, submaxil 
lary, and sublingual glands, together with a slight amounl 
obtained from the smaller buccal glands. The chemical com- 
position of the secretion from these various sources differs con- 
siderably, but from a dental standpoint we are much more 
interested in the mixed saliva and its constituents than the 
differences in the products of the various glands. The notable 
differences are that the mucin is practically wanting in the 
parotid saliva. The alkaline salts seem to be in smaller pro- 
portion in the parotid saliva than in the other two. Potassium 
sulphocyanate is a constituent of all varieties of saliva, although 
more constantly present in the submaxillary and in the sublingual 
than in the parotid. The parotid, on the other hand, contains 
a larger proportion of dissolved gases. The data on the com- 
position of these varieties differ to a considerable extent and 
comparisons are not wholly satisfactory. 

The mixed saliva contains, according to Professor Michaels, 
all the salts of the blood which are dialyzable through the salivary 
glands, and hence furnishes a reliable index of metabolic pro- 
cesses which are being carried on within the system. In order 
that this fact may be of practical value, two things are obviously 
of prime importance: First, methods of analysis which are not 
too complicated and which are at the same time conclusive; 
second, a knowledge regarding the source of the various con- 
stituents found, which will enable us to make a rational inter- 
pretation of the results obtained. In both of these fundamentals 
we are very much hampered by lack of knowledge; as yet there 
is much to be desired in the way of practical clinical tests for 
the various salivary constituents, and very much to be learned 

156 



SALIVA PROPERTIES, CONSTITUENTS, AND ANALYSIS 157 

as to their meanings, in order to make deductions which shall 
be conclusive. We are led to believe, from the work of an 
increasing number of specialists, that this subject of salivary 
analysis promises much and is worthy of careful investigation. 

The quantity of saliva secreted in twenty-four hours is vari- 
ously estimated from a few hundred c.c. to 1500; 1200-1500 c.c. 
is the more probable amount. As the measurement of the 
twenty-four hour quantity is practically impossible, Ferris has 
used the quantity secreted in twenty minutes as a basis for 
quantitative estimation. The quantity is diminished in fevers, 
severe diarrhea, diabetes, and nephritis, by fear and anxiety, 
and by the use of atropine. It is increased by smoking, by 
mastication, by the use of mercury, potassium iodide, or pilo- 
carpin. The flow of saliva is also increased by the action of the 
sympathetic nervous system, during pregnancy, and by local 
inflammatory processes. 

The methods outlined here for the analysis of saliva have been 
selected with two objects in view; first, to furnish some tests 
sufficiently simple to make them of use to the dental practitioner; 
and second, to give some determinations, usually of greater 
accuracy, suitable for the demands of investigators with the 
facilities of large chemical laboratories at their command. In 
some instances several methods will be given. 

Physical Properties. The physical properties of saliva in- 
clude its appearance, specific gravity, color, odor, and viscosity. 

Appearance. The appearance is clear, opalescent, frothy, 
or cloudy; normal saliva is usually opalescent. It may become 
turbid by precipitation of lime-salts caused by the escape of 
carbon dioxide. 

Specific Gravity. Specific gravity ranges from 1.002 to 1.009, 
the total solids being only from 0.6 to 2.5 per cent. 

Color. Saliva is usually colorless when fresh, but upon 
standing for twenty-four hours may assume various tints, 
which are developed from constituents derived from bile. (Pro- 
fessor Michaels.) Saliva may be colored red or brown by the 
presence of blood or blood pigments, but in such cases the 
source of the color is usually local and easily discovered. 



158 PHYSIOLOGICAL CHEMISTRY 

Odor. Normal saliva is practically odorless. In cases of 
pyorrhea there is usually a peculiar fetid odor, easily recognized. 
In other pathogenic conditions the odor may be slightly am- 
moniacal, or may occasionally resemble the odor of acetone or 
garlic. 

In any analysis physical properties of the saliva should first be 
noted. The color and appearance of the perfectly fresh sample is 
to be carefully compared with the appearance and color after 
standing for forty-eight hours in a small, tightly covered vial. 
The color may be yellowish, greenish, or brown, according to the 
variety of the derivative of biliverdin from which the color is 
obtained.* The general appearance may also change, inde- 
pendently of any color. A saliva that is hypoacid in character, 
when fresh, is usually markedly opalescent and of offensive 
odor after forty-eight hours, while a hyperacid saliva may have 
become clear or cloudy but without odor. 

We may add to this examination a viscosity test which will 
be of value as indicating the amount of mucin, as the mucin 
content affects the viscosity more than any other one constituent. 

The viscosity may be determined by use of the apparatus 
pictured in Fig. 4. 

The essential features of the viscosimeter are a straight 
graduated tube with the constriction (C) jacketed so that the 
conditions under which a given sample will pass through the 
opening will always be under absolute control. 

The apparatus is standardized by partly filling with distilled 
water in which the bulb of a thermometer is immerse^!. 

The temperature of the distilled water is brought to 25 C. 
The thermometer is removed to facilitate reading, and from 5 
to 10 c.c. of the liquid is allowed to run out, the time consumed 
being accurately determined by a stop watch. 

The viscosity of saliva is determined in the same way, care 
being taken that only a perfectly clear solution is used, as fine 
particles will clog the opening at C. The use of the stop cork 
as pictured in Fig. 4 is undesirable; in fact, it has been found 

* Dr. Joseph P. Michaels. S. S. White's reprint of paper read before Inter- 
national Dental Congress, Paris, 1900. 



SALIVA PROPERTIES, CONSTITUENTS, AND ANALYSIS 159 




FIG. 4. 



i6o 



PHYSIOLOGICAL CHEMISTRY 




FIG. 5. 



that straining the saliva, filtering through paper, or even cen- 
trifugalizing in order to separate the solid portions, will occasion 
a variation in the results obtained. The first determination 
should be carefully made and used, as repeated determinations 

result in a regular diminution of the 
viscosity figure, due to mechanical 
changes brought about by passing the 
saliva through the very small opening 
atC. 

If the constriction of the graduated 
tube is sufficiently great, i.e., if the 
opening is sufficiently small, compari- 
son may be made by counting drops delivered in a given time. 
This is not advised, as there is much greater difficulty in ob- 
taining the saliva so free from suspended particles as not to clog 
the tube. 

The inner tube should always be filled to the same mark in 
the determination as that used in the 
standardization of the instrument. 

Specific gravity may be taken by an 
ordinary urinometer or a specific gravity 
bulb if the quantity is sufficient, the read- 
ing to be made from beneath the surface of 
the liquid. If the quantity of the saliva is 
small, it may be diluted with an equal 
volume of water, and the last two figures 
multiplied by two will give the gravity of 
the undiluted sample, or the gravity may 
be taken by the pyknometer, in which the 
bulb of the instrument is filled with saliva 
accurately to the mark M (Fig. 6). The 
reading on this instrument, of course, will 
be from the bottom up, and the lower 
the bulb sinks the greater will be the gravity of the sample. 
This method, devised by S. A. De Santos Saxe, M. D., for use in 
examination of urine, has been suggested by Dr. Ferris and 
adopted by the National Dental Association as an official method. 




FIG. 6. Pyknometer. 



SALIVA PROPERTIES, CONSTITUENTS, AND ANALYSIS l6l 

For very accurate work the use of specific gravity bottles is 
recommended. These may be obtained holding i, 2, and 5 
cubic centimeters (Fig. 5), and with an accurate balance the 
gravity can be accurately obtained. 

Chemical Properties 

The first chemical property to be noted is the reaction. Saliva 
is normally alkaline to litmus paper or to lacmoid. Normal 
saliva, however, fails to give an alkaline reaction with phenol- 
phthalein owing to the presence of free carbon dioxide which may 
be present to the extent of nineteen parts in a hundred by volume. 

Acidity of Saliva. 

Perhaps the one determination which is of the most clinical 
importance to the dentist is the determination of the acidity of 
the saliva. The value of this determination lies in its simplicity 
and in its significance in regard to the general systemic condition 
of the patient. 

In discussing acidities in Vol. I, Chapter I, it will be remem- 
bered that they were divided into two kinds actual acidity 
and titratable acidity. A complete analysis of saliva includes 
the determination of both. 

Hydrogen-ion Concentration. 

The expression of the actual acidity, hydrogen-ion con- 
centration, or P H , is very clearly explained by Leon S. Medalia 
in his article on " Color Standards for the Colorimetric Measure- 
ment of H-Ion Concentration, PR 1.2 to P H 9.8."* He says, 
" The accumulation of free hydrogen-ions present in a given 
solution, i.e., the H. I. C. of that solution, can be measured to the 
minutest amount and has been expressed in terms of " normal 
solutions." The amounts are so minute that they run up to the 
billionth or trillionth normal, since the acid strength or the hydro- 

* " Color Standards " for the Colorimetric measurement of Hydrogen-ion Con- 
centration PH 1.2 to PH 9-8, by Leon S. Medalia: Jour, of Bacteriology, Vol. V f 
No. 5, Sept. 1920. 



162 PHYSIOLOGICAL CHEMISTRY 

gen-ion content of neutral or even alkaline solutions is measur- 
able. In order to overcome the unwieldiness of the figures 
necessary to express the H. I. C., Sorensen suggested the symbol 
PH to express one-tenth normal beginning on the acid side and 
going up in negative multiples of one-tenth towards alkalinity. 
Thus PH i equals N/io acid; PH 2 equals N/io X N/io = 
N/ioo; PH 3 equals N/ioo X N/io = i/iooo normal, etc. 
The lower the PH of a given solution, therefore, the more acid, 
or the higher its H. I. C., and the higher the PH the less acid, or 
the lower is its H. I. C." 

The hydrogen-ion concentration may be found either electro- 
lytically or colorimetrically. The latter method, though not 
quite as accurate as the former, is sufficiently accurate for all 
general purposes and is by far the simpler and more convenient. 

Principle of Method. 

In 1917, Clark and Lubs developed a series of indicators giving 
a range of PH from 1.2-9.6, each indicator being extremely 
sensitive to the hydrogen-ion concentration of solutions coming 
within its field. The range of P H and the color changes of these 
indicators are given by Clark and Lubs as: 

1. Thymol blue, acid range Red Yellow PH 1.2-2.8 

2. Brom-phenol blue Yellow Blue PH 3 . 0-4 . 6 

3. Methyl red Red Yellow PH 4 . 4-6 . o 

4. Brom-cresol purple Yellow Purple PH 5 . 2-6 . 8 

5. Brom-thymol blue Yellow Blue PH 6 . 2-7 . 8 

6. Phenol red Yellow Red PH 6.8-8.4 

7. Cresol red Yellow Red PH 7.2-8.8 

8. Thymol blue, alkaline range Yellow Blue PH 8.0-9 . 6 

Medalia noted the fact that each indicator covered a range of 
a PH of 1.6, so that by dividing this color range into eight equal 
parts he obtained a series of color changes at intervals of .2 P H . 
This was accomplished by taking a series of sixteen tubes, eight 
of which contained 10 c.c. acid and eight 10 c.c. alkali. Then, 
starting with .8 c.c. of the indicator in the acid tubes, he decreased 
the amount of indicator in each tube by .1 c.c., while in the tubes 
containing the alkali he started with .1 c.c. of indicator and 



SALIVA PROPERTIES, CONSTITUENTS, AND ANALYSIS 163 

increased to .8 c.c. Then by placing the acid tubes behind the 
alkaline, arranging them as shown (Fig. 7), so that each pair 
contained .8 c.c. of indicator, he succeeded in forming a series 
of tubes that gave the range of P H for any specific indicator at 
intervals of .2 PH- By matching the color obtained with an 
unknown with one of these pairs, one may determine the P H of 
the unknown. 

Procedure for PH Determination of Saliva. 

The most convenient apparatus is the " Comparator Black/ ' 
illustrated in the accompanying diagram. Shell tubes of equal 




lilM" 1 !! 1 ^*^ 



riG. 7. 

A contains the i c.c. sample, 4 c.c. distilled water and .4 c.c. indicator. 
B contains the i c.c. sample and 4 c.c. distilled water. 
C contains 5 c.c. distilled water. 

Each pair of tubes represents a definite hydrogen-ion concentration and the 
observer matches the color of the unknown with that of the known by looking 
through the holes in the front of the box. B and C are used so that the depth of 
liquid is the same in each case. 

diameter are used, and it has been found simpler in saliva work to 
use just half of the quantities given above; that is, to let each 
acid tube contain 5 c.c. HC1 (.1 per cent, made from .1 c.c. con- 
centrated HC1 and 100 c.c. distilled water) and each alkali tube 
5 c.c. N/20 NaOH. The quantity of indicator is also halved; 
that is, .4 c.c. is used and the amount is decreased or increased 
by .05 c.c. each time. Each pair of tubes, as shown in the 
diagram, will then contain .4 c.c. indicator divided between them 
differently, and the color change in the series will follow the table 



164 PHYSIOLOGICAL CHEMISTRY 

given on page 162, depending on the indicator used. Brcm- 
cresol purple and brom-thymol blue will cover the range of PH of 
practically all salivas, and these standard series will keep three 
months if carefully made. 

After the standards are prepared, the actual determination of 
the PH of an unknown solution is extremely simple. In each of 
two shell tubes similar to those used in the standard sets, 4 c.c. 
of distilled water and i c.c. of the centrifugalized saliva are 
placed. Then to one of these tubes .4 c.c. of the indicator is 
added, usually brom-thymol blue for saliva. The tubes are 
gently rotated and the one containing the indicator placed beside 
the tube in the standard set which matches its color best. In 
order to have the same depth and density of liquid to look 
through, two tubes containing 5 c.c. of distilled water are placed 
behind the unknown and, to insure an equal coloration in each 
if the unknown is slightly colored, the tube containing 4 c.c. 
distilled water and i c.c. of unknown is placed behind the acid 
and alkali pair with which the PH of the unknown is being 
compared. 

The hydrogen-ion concentration of resting saliva gives a PH 
Df 6.5-7.0, while activated saliva is normally 7.0-7.3, 

Titratable Acidity. 

The total titratable acidity of saliva is determined in a similar 
manner to that described under urine, page 194. 

Five c.c. of the centrifugalized sample is titrated with N/40 
NTaOH, using phenolphthalein as an indicator. Calculating in 
degrees as explained on page 126 (i being equivalent to each 
tenth of N/io NaOH used when 10 c.c. of unknown is used) 
the normal acidity for activated saliva is 2~4. The resting 
saliva will usually give a slightly higher degree of acidity and a 
dightly lower P H . 

The increased alkalinity of the activated saliva is due to the 
ncreased alkaline phosphate present. The acid phosphate of 
the blood is constantly reacting with the acid carbonate 

NaH 2 PO 4 +NaHC0 3 = Na 2 HPO 4 +H 2 O+CO 2 



SALIVA PROPERTIES, CONSTITUENTS, AND ANALYSIS 165 

yielding the alkaline phosphate and carbonic acid . It is reason- 
able to suppose that an increased activity of the glands increases 
the quantity of this salt which is dialyzed into the saliva. 

The acidity of saliva, referred to its behavior to phenol- 
phthalein, is in large part due to the presence of free carbon dioxide. 

The sources of carbon dioxide in saliva are probably three: 
carbon dioxide dialyzed through the salivary glands, traces 
from carbohydrate fermentation, and more or less absorbed 
from contact with expired air. 

The saliva obtained by chewing paraffin (a process calculated 
to furnish the maximum amount from the last two sources), 
may yield several times the amount of free carbon dioxide that 
another sample taken from the same patient by a saliva ejector 
will give. 

Acidity of saliva may be temporary, in which case it may be 
entirely removed by drawing air through the heated (not boiled) 
sample. The permanent acidity may be determined by titration 
of the sample after removal of carbon dioxide. 

Determination of Carbon Dioxide. 

Method I. The apparatus pictured in Fig. 8 has been used 
by the author for this acidity determination. 

The air is drawn from left to right, first through a potash 
bulb (A) to absorb atmospheric carbon dioxide, next through 
10 c.c. of saliva diluted with 20 c.c. of water contained in a small 
Soxhlet flask (B) whereby the carbon dioxide from the saliva 
is carried through the " test-tube " condenser and collected in 
baryta-water in the Erlenmeyer flask (C) at the left. This in 
turn is connected with a suction pump or aspirator. The 
" drip cup " (D) has been found necessary when working with 
very viscid samples. The thistle tube (E) holds water for 
maintaining the volume in (B) if the condenser is not used. 

The amount of free carbon dioxide may be determined by 
adding a standard carbonate solution (N/ioo Na^COs) to a 
Volume of baryta-water equal to that used in the Erlenmeyer 
flask and then comparing the degree of turbidity obtained. 
This may be done by viewing through flat-bottom tubes (shell 



i66 



PHYSIOLOGICAL CHEMISTRY 



tubes) of about 20 c.c. capacity, or, in many cases, better, by 
use of the Duboscq colorimeter used in other determinations 
(Fig. 9, page 176), or better still by the use of the nephelometer 
made with the Duboscq colorimeter after the method of Dr. 




FIG. 8. 

Bloor. (Journal of Biological Chemistry, Vol. 22, p. 145, 1915.) 
This apparatus may also be used to advantage in the determina- 
tion of calcium in saliva, or acetone bodies in urine. The 
nephelometer differs from the Duboscq colorimeter in that it 
makes use of reflected rather than transmitted light. 

Method II. Another method consists in passing carbon 
dioxide, as above, into a measured volume of standardized 



SALIVA PROPERTIES, CONSTITUENTS, AND ANALYSIS 167 

baryta- water (N/2o) and titrating excess of barium hydroxide 
with N/2O oxalic acid. The end-point is determined by " spot- 
ting " on to fresh tumeric paper. When the paper ceases to 
turn brown-red the end of the reaction has been reached. 

Permanent Acidity. 

If the acidity of saliva is due to the presence of carbon dioxide, 
the carbon dioxide may be driven off by boiling the sample for 
ten minutes, and the resulting saliva will then be alkaline to 
phenolphthalein. In the activated sample this so-called tem- 
porary acidity is normal. In nearly all cases of resting saliva 
(saliva collected upon rising in the morning) and in many other 
cases which the writer considers pathological, a permanent acidity 
has been observed. Certain types of erosion and pyorrhea will 
as a rule yield salivas with a permanent acidity. 

The cause of this acidity is at present rather hypothetical, 
probably differing more or less with various samples. In several 
instances it has been shown to be at least partly due to lactic 
acid, and a qualitative test for lactic acid is therefore of con- 
siderable importance in the analysis of all samples showing a 
permanent acidity. The determination is most easily carried 
out by evaporating a few cubic centimeters of the sample with 
dilute hydrochloric acid, extracting with ether, evaporating the 
ether solution to dryness, dissolving the residue in distilled water, 
and making a careful test for lactic acid according to Exp. 60. 
A control test should always be made with water and the same 
amount of ferric chloride. 

Some salivas upon boiling will show a constantly increasing 
degree of acidity, which has been explained by Gies as probably 
due to decomposition of unknown compounds of the soluble 
calcium acid phosphate, CaHXPO^, with organic matter from 
the saliva. 

Alkalinity. 

Saliva normally is alkaline to litmus owing to the presence of 
various alkaline salts which are not neutralized by the acid factors 
present. The value of determining the alkalinity of saliva lies 



168 PHYSIOLOGICAL CHEMISTRY 

perhaps in the relationship existing between it and the titratable 
acidity. 

In the Pacific Dental Gazette for 1915, pages 335-345, Professor 
John A. Marshall of California published his technique for the 
determination of a " Salivary Factor," representing the difference 
in neutralizing power of resting and activated saliva from the 
same patient as an indication of immunity from, or tendency 
toward, dental caries. The factor was determined by obtain- 
ing the alkalinity (A) as described just below, and the 
acidity (J3) as described on page 164, using N/ioo acid and 
alkali in each case; then by adding (B) to (A) the so-called 
neutralizing power of the saliva was determined. This process 
was repeated with both the resting and the activated saliva, 
and by means of the following ratio the factor was obtained. 

neutralizing power resting saliva X 100 



neutralizing power activated saliva 



= Salivary Factor 



Professor Marshall claimed that a low factor, below 80, meant 
immunity, while 80 or above indicated a carious condition. 

It has been shown from experiments with dialyzed saliva that 
the inorganic constituents of the saliva are chiefly responsible 
for its neutralizing power. A high neutralizing power does not 
of necessity mean a high salivary factor, but it is usually asso- 
ciated with high inorganic constituents and a low percentage of 
proteins. 

Too much emphasis has apparently been placed on the re- 
lation of the Salivary Factor to the development of caries. The 
neutralizing power of the saliva, determined in some such manner 
as that adopted by Professor Marshall, is not at all unlikely to 
become a useful method in a comprehensive study of the saliva, 
but perhaps with a rather different significance from that thus 
far suggested. 

The alkalinity of a given sample may be easily determined by 
titration. To 5 c.c. of centrifugalized saliva, 10 c.c. of N/4O 
HC1 and a few drops of paranitrophenol are added. The excess 
acid is then titrated with N/40 NaOH, and the difference in- 
dicates the number of cubic centimeters of the standard acid 



SALIVA PROPERTIES, CONSTITUENTS, AND ANALYSIS 169 

necessary to neutralize the alkalinity of the saliva. Calculated 
in degrees, the alkalinity of normal saliva is between 15 and 20. 

Constituents. We should here distinguish carefully be- 
tween saliva proper and sputum. The constituents of sputum 
are derived from the air-passages rather than from the salivary 
glands, and are not at present under consideration. Among 
the normal constituents of saliva are included mucin, albumin, 
ptyalin, also oxidizing enzymes, ammonium salts, nitrites, 
potassium sulphocyanate, alkaline phosphates, and chlorides, 
with traces of carbonates, urea, creatinine and in fact practically 
all normal constituents of the blood; and, in the sediment, 
epithelium cells, occasional leucocytes, and fat globules. The 
abnormal constituents will include glycogen, dextrin, rarely sugar, 
cholesterin, derivatives from bile, lecithin, xanthin bodies or 
alkaline urates, acetone, lactic acid, and crystalline elements 
resulting from insufficient oxidation or perverted glandular func- 
tion. These latter are recognizable by the micropolariscope. 
Mercury and lead may also be found in saliva in cases of poison- 
ing by salts of these metals. 

Considerable work has been done in comparing some of the 
salivary constituents with those of the blood of the same patient, 
and the author is convinced that there may be a significant 
similarity between the two analyses. Particularly has this been 
observed to be true in cases of nephritis, where almost invariably 
a rise of urea nitrogen or creatinine in the blood will mean a 
corresponding rise in the saliva. 

PROTEINS. 

Mucin. The secretion from the parotid gland contains 
practically no mucin, but the sublingual saliva contains large 
amounts. Mucin is, according to Simon, the most important 
constituent of the saliva, not excepting ptyalin. The various 
glands contributing salivary mucin do not in all probability 
furnish just the same kind of protein; moreover, the mucin 
from different individuals seems to vary in composition and 
properties, some yielding more abundant acid decomposition 



170 PHYSIOLOGICAL CHEMISTRY 

products than others (see article by W. D. Miller, in Dental 
Cosmos for November, 1905), while, according to Professor 
Michaels, the mucin varies greatly in the same individual in 
health and disease. The changes in the characteristics of 
salivary mucin have been studied but little, and the investigation 
of these changes, as indications of diathetic states, promises much. 

An excess of mucin in the saliva tends to an increase of bacterial 
growth, from the fact that it furnishes increased facilities for 
multiplication; it has been suggested that it may also give rise 
to mucic acid, and thereby be a possible factor in tooth erosion. 
(Dr. G. W. Cook, in Dental Review, May, 1906, page 461.) 

Mucin may be separated, after taking the gravity, by the 
addition of a little acetic acid. It should then be filtered off, but 
it will be necessary to dilute and agitate, in order that a fairly 
clear filtrate may be obtained. 

A quantitative result may be obtained by weighing the pre- 
cipitated mucin after drying it. This result will of necessity be 
somewhat inaccurate as the character of the mucin is altered by 
both the acid and any heat to which it may be subjected. 

Albumin. Albumin is present in very small quantities, 
increased during mercurial ptyalism, usually in cases of pyor- 
rhea, and, according to some authorities, in various albumi- 
nurias. It may be detected by the usual methods after the 
separation of mucin. 

" According to Vulpian, the quantity of albumin is increased 
in the saliva of albuminurics of Bright's disease. The saliva 
of a patient with parenchymatous nephritis had mucin 0.253 
and albumin 0.182 per cent. The saliva of another patient, 
with albuminuria of cardiac origin, contained mucin 0.45, 
albumin 0.145 per cent. In a healthy man there was found 
mucin 0.320, albumin 0.05 per cent. This fact has been con- 
firmed by Pouchet, who found these substances in greater 
quantities. "* 

Albumin may be demonstrated in the filtrate, from which 
mucin has been separated by underlaying with strong nitric 

* Dr. Joseph P. Michaels. S. S. White's reprint of paper read before Inter- 
national Dental Congress, Paris, 1900. 



SALIVA PROPERTIES, CONSTITUENTS, AND ANALYSIS 171 

acid. This is Heller's test for albumin in the urine, and is best 
performed in a small wine-glass with round bottom and plain 
sides. 

ENZYMES 

Ptyalin. Ptyalin is the principal ferment of the saliva; it 
converts starch, by hydrolysis through the various dextrins 
(page 149), to maltose. The maltose in turn is converted into 
glucose by a second ferment, known as maltase, which exists 
in saliva in very small quantities. 

The activity of ptyalin is greatest at a temperature of 40 C. 
Very faintly acid saliva is the best medium. Neutral and faintly 
alkaline salivas are next in order. 

The amylolytic power of a given sample of saliva may be 
determined by the action on dilute starch paste. In making 
comparative tests it is essential that the conditions under which 
the ptyalin is allowed to act should be exactly the same, es- 
pecially as regards the temperature and duration of the process. 
A slight variation in the strength of the starch solution is of no 
consequence, as starch is supposed to be in excess. (See Exp. 
213 on page 286, also method on page 172.) 

Proteolytic Enzymes. Upon incubation with certain prod- 
ucts of protein digestion (dipep tides), proteolytic action of 
saliva has been noted; whether this action is due to an enzyme 
or to bacteria is an open question. (See eighth edition of Hawk's 
" Physiological Chemistry/' pages 56 and 57.) 

Oxydases. As a result of the work of Dr. C. F. MacDonald 
in the author's laboratory, the following conclusions were 
reached regarding these enzymes: 

First. That human mixed saliva contains an oxidizing 
enzyme distinct from ptyalin. 

Second. That the enzyme exhibits the properties of both 
an oxydase and a peroxydase. 

Third. That it is a product of the body (probably glandular) 
metabolism and may be increased in quantity or activity by 
mastication. 



172 PHYSIOLOGICAL CHEMISTRY 

Fourth. That it is more resistant to heat than ptyalin, but 
more easily destroyed by acids. 

Fifth. That the color obtained with a freshly prepared i per 
cent solution of pyrocatechol is sufficient test for this enzyme in 
saliva. 

The test for oxidizing enzymes may be made with the pyro- 
catechol as given on page 173, also by the use or phenolphthalin 
(reduced phenolphthalein). This last reagent has recently been 
rendered available by the work of Dr. H. L. Amoss, Harvard 
Medical School, who has given us a concise and simple method 
for its preparation. (Jour. Biolog. Chem., 1912.) 

Determination of Enzymes. 

Amylolytic Enzymes.* Preparation of starch paste. Put 15 
c.c. of distilled water to boil. Meanwhile, weigh out 3 grams of 
sterile starch and mix with 6 c.c. of cold distilled water. Add 
drop by drop under constant stirring to the boiling water, then 
rinse out with 5 c.c. of distilled water any particles of starch 
adhering to the dish and add to the boiling starch solution. 
Boil one minute under constant stirring. Cool to blood tem- 
perature and add gradually 4 c.c. of N/ioo iodine solution. 

This makes 30 c.c. of a 10 per cent starch solution, which, when 
colored, is of a dark blue, and can be kept several days in the 
ice-box. 

Filling the Tubes. Suck up the paste into glass tubes of 
1.5 mm. diameter, and cool in the ice-box. Just before using, 
make a file mark i cm. from the end of the tube and break off 
the piece of tubing so that it is full of the blue starch paste. 
Be sure that this small tube is broken so as to leave each end 
square and full of paste. Examine under low-power microscope. 

Determination of Enzyme. Immediately after delivery of 
the specimen, measure 2 c.c. of saliva into a test-tube. Place 
it in the small tube of starch paste, and heat the whole in a 
thermostat at from 37 to 38 C. for half an hour. The enzyme 
of the saliva will dissolve the paste from the ends of the tube, 

* Note Method taken from Dr. Ferris' Saliva Analysis, Dental Cosmos, Nov., 
1911, page 1295. 



SALIVA PROPERTIES, CONSTITUENTS, AND ANALYSIS 173 

leaving a blue column of paste unchanged in the center of the 
glass tube. After half an hour, measure with a micrometer 
gauge the total length of the tube and the length of the blue 
starch paste column remaining undissolved. The difference 
between these two measurements represents the amount of 
starch digested by the enzyme. Since the quantity of ferment 
in any fluid varies with the square of the length of the column 
digested, the quantity of ferment in the saliva is found by 
squaring this difference. Multiply by 100 to give the enzymic 
index. 

Oxidizing Enzyme. (Oxydase.) Treating 5 c.c. of saliva, 
diluted with an equal volume of water, with about i c.c. of a 
i per cent solution of pyrocatechol. The color obtained is a 
characteristic brown, developing within thirty minutes. 

SALTS OF THE SALIVA. 

Phosphates and Carbonates. These salts are probably present 
in both acid and neutral forms; that is, the phosphate may 
exist as Na 2 HPO 4 , also as NaH 2 PO 4 , and at times both of these 
may be present at once. The acid carbonate, NaHCO 3? is an 
undoubted constituent, while the neutral carbonate is probably 
not present at all. Chittenden says that mixed human saliva 
contains normally no sodium carbonate whatever. 

As explained by Dr. Kirk, the normal reaction by which 
overacidity of the blood is taken care of by renal epithelium 
is H 2 CO3+Na 2 HPO4 = NaH 2 PO4+NaHCO 3? and when condi- 
tions are such as to produce larger quantities of carbonic acid 
than the kidneys can eliminate in accordance with the above 
reaction, there is an increased acidity of the saliva as well as of 
the urine.* In the hypoacid individual, the so-called alkaline 
sodium phosphate, Na 2 HPO 4 , is present in the greater quantity. 

Determination of Phosphates. Five c.c. of saliva are heated 
with a few drops of 10 per cent sodium acetate, and the hot solu- 
tion titrated with standard uranium solution (for preparation 
see page 197). The end-point is obtained by " spotting " with 
K 4 Fe(CN)e until a permanent brown color is obtained. Each 
* International Dental Journal, February, 1904. 



174 PHYSIOLOGICAL CHEMISTRY 

cubic centimeter of uranium solution used is equivalent to .005 
gram of phosphate. 

A less accurate end-point may be obtained by using cochineal 
as an indicator. The appearance of a permanent green color 
denotes the end-point. 

Potassium Thiocyanate represents the salts of HCNS found 
in saliva. It occurs only in very slight traces in other body 
fluids, and in saliva only to the extent of o.ooi to 1.02 per cent. 
Dr. Michaels considered the proportion of thiocyanates relative 
to the ammonia to be of importance, and states that in health the 
ammonium salts and the thio-cyanates are present in very 
slight amounts, and the color tests, with Nessler's solution* 
and with ferric chloride, respectively, are of about equal in- 
tensity. In the hyperacid state the sulphocyanates are in 
excess of ammonia, while in hypoacid conditions, the ammonia 
exists in the greater quantity. Sulphocyanate is detected by 
means of ferric chloride, and distinguished from meconates and 
acetates, as indicated by Exp. 215, page 287. 

As we shall see in a subsequent chapter the intensity of color 
produced by ferric chloride and thiocyanate is not necessarily 
an index of the quantity of HCNS present; hence the above 
conclusions are of questionable value. 

The sulphocyanates are normal constituents of saliva, and 
consequently always present. According to A. Mayer (Deutsch. 
arch.f. klin. med., Vol. 79, No. 394), the sulphocyanates, without 
doubt, result from the decomposition of proteins, and exist in 
the urine in quantities variously estimated from 20 to 80 milli- 
grams per liter, while in saliva they have been estimated at 60 
to 100 milligrams per liter. Professor Ludholz of the University 
of Pennsylvania says that the sulphocyanates are eliminated in 
increased amounts in conditions where there is a lack of oxygen 
in the system, thus corroborating statements of Professor 
Michaels (see Ammonia). Dr. Fenwick (Lancet, 1877, Vol. II, 
page 303) demonstrated that the quantity of KCNS was directly 
dependent upon the bile salts in the blood. He found an increase 

* See page 300. 



SALIVA PROPERTIES, CONSTITUENTS, AND ANALYSIS 175 

of the salt in liver disorders attended with increase of bile salts 
in the blood, and marked increase in jaundice. In gout, rheu- 
matism, and conditions producing pyorrhea, it is also claimed 
to be present in considerable quantity. 

The sulphocyanates are usually present in more than normal 
quantity in the saliva of people addicted to smoking tobacco.* 
The claim has been made for this salt that it exerts a specific 
antiseptic action toward bacteria. 

While the sulphocyanates, or, in fact, any salt in sufficient 
concentration, will have an inhibitory action on the growth of 
bacteria, it is rather doubtful if this is the particular office of 
KCNS in the saliva. 

Thiocyanate (Sulphocyanate) Tests. To a large drop of 
saliva on a white porcelain surface, add about half as much 5 
per cent ferric chloride, acidified with hydrochloric acid. A 
reddish coloration indicates the presence of thiocyanate. 

Quantitative Method (Ferris).^ 

Into a 10 c.c. cylinder measure 2 c.c. of saliva, 5 c.c. N/io HC1, 
and 5 c.c. of 5 per cent ferric chloride. Dilute to the mark and 
mix. Similarly, make up a standard solution, using in place 
of the saliva i c.c. of standard ammonium thiocyanate solution 
(.5 gm. C.P. NH 4 CNS dissolved in one liter of water). One c.c. 
of this solution contains .5 mg. NHUCNS. 

Compare colorimetrically by placing a few cubic centimeters 
of the standard solution in one cup of a colorimeter (Fig. 9), and 
a few cubic centimeters of saliva in the other. Set the standard 
at 20 and then adjust the other cup so that the disk is of uniform 
intensity of color when observed through the lens. 

Calculation: 

20 

= mg. of NH 4 CNS per 100 c.c. saliva. 



reading 

* See article by Dr. J. Morgan Howe in Jour, of the Allied Societies, Vol. 4, 
p. 183. 

t Jan. Journal of the American Dental Association, 1923. "Composition of 
Human Saliva," Ferris. 



176 



PHYSIOLOGICAL CHEMISTRY 



Nitrites. That nitrites exist in most salivas is without ques- 
tion. So far as we know at present, the nitrites are apparently 
incidental, and occur as intermediate products in the oxidation 








FIG. 9. Colorimeter. 

of ammonia to nitrates, just as they do otherwise in nature out- 
side of the animal body. 

It is not at all improbable that the proportion of nitrates is 
dependent upon activities of the oxidases. This has, in some 
cases at least, been proven to be the case, as the same sample 
of saliva has frequently given steadily diminishing quantities 
of nitrites until they have wholly disappeared in cases containing 
active oxidizing enzymes. 



SALIVA PROPERTIES, CONSTITUENTS, AND ANALYSIS 177 

Considerable work* has been done recently in an attempt to 
prove the above statements, but the results have not been very 
satisfactory. It was conclusively shown, however, that in fresh 
saliva nitrites and oxydases were present and that if nitrates were 
present at all they were not in sufficient quantity to be detected. 
In the course of two or three weeks' time a slight positive test 
for nitrates was obtained, while the nitrites were diminished. 
That the oxidation was brought about by the oxydase was dem- 
onstrated by the fact that no change seemed to occur in the 
quantity of nitrites and there was no test for nitrates in samples 
in which the enzyme had been destroyed by boiling. 

Nitrites may be detected by adding to a large drop of saliva 
on porcelain a few drops of freshly prepared reagent, made 
by dissolving a very little naphthylamine chloride and an 
equal amount of sulphanilic acid in distilled water strongly 
acidulated with acetic acid. A pink coloration is a test for 
nitrites. 

This method could be made quantitative in a manner similar 
to the colorimetric methods for ammonia, or thiocyanate of 
potassium; but, at the time of the present writing, there seems 
to be no particular reason for undertaking this amount of work. 

Chlorides. 

Chlorides are always present in the saliva, usually as alkaline 
salts, but in very variable quantities. As a rule, the chlorine 
content of the saliva is less than that of blood, there being from 
75 to 125 mg. per 100 c.c., as compared with about 500 mg. per 
100 c.c. of blood. This difference may be accounted for by the 
fact that during the process of dialysis from the bloood the salt 
content of the lymph is increased, thereby diminishing the 
quantity in the saliva. 

Chlorine may be easily and accurately determined by taking 
5 c.c. of centrifugalized saliva, adding 10 c.c. of N/40 AgN0 3 and 
titrating back with N/40 KCNS, using ferric alum as an indi- 
cator. If for any reason the sample is not clear, it may be ashed 

* Thesis work of G. H. Leatherman, Harvard Dental School, 1924. 



178 PHYSIOLOGICAL CHEMISTRY 

with a few grams of calcium oxide. This treatment serves to fix 
the chlorine as calcium chloride, which may be dissolved in water. 
The chlorine content may then be determined in the manner 
described above. 

Calcium may be determined by the following volumetric 
method, recommended by Dr. Percy R. Howe, Dental Cosmos, 
April, 1912. To 5 c.c. of saliva, add as much more distilled 
water and a slight excess of oxalic acid or ammonium oxalate 
(5 c.c. of normal solution will be sufficient). Add ammonium 
water to alkaline reaction, heat nearly to the boiling-point, 
and allow to stand for twenty to thirty minutes. Filter through 
a hardened filter paper into a small beaker which is allowed to 
stand on a piece of black glazed paper. Under these circum- 
stances, a slight rotary motion of the beaker will show if any of 
the white precipitate of calcium oxalate is passing through the 
paper. 

After filtration is complete, wash five times in hot distilled 
water; then place the precipitate, together with the paper, into 
a small beaker, add about 30 c.c. of dilute sulphuric acid, and 
heat nearly to the boiling-point; then titrate with N/2O per- 
manganate solution. 

Gravimetric Calcium Determination (McCrudderi). 

To 50 c.c. of saliva add 10 c.c. of N/2 HC1 and 10 c.c. of 2\ 
per cent oxalic acid. Boil for fifteen minutes, during which time 
most of the calcium is precipitated. Cool and add 10 c.c. of 
3 per cent ammonium oxalate and 6 c.c. of 20 per cent sodium 
acetate. This aids in decreasing the acidity and in precipitating 
the calcium oxalate in large crystals. 

Filter through ashless filter-paper, washing the precipitate 
with cold i per cent ammonium oxalate and then with small 
portions of distilled water. Dry the filter-paper containing the 
precipitate and ignite, using a platinum evaporating dish if 
possible. Weigh carefully and calculate result as calcium oxide. 

Normally the calcium content of saliva is 10-20 mg. per 100 c.c. 

This method is accurate and fully as easy of manipulation as 



SALIVA PROPERTIES, CONSTITUENTS, AND ANALYSIS 179 

the volumetric method. However, it is often impossible to obtain 
sufficient saliva to make a gravimetric determination feasible. 

DETERMINATION OF UREA NITROGEN PLUS AMMONIA. 

Measure 2 c.c. of centrifugalized saliva into a heavy-walled 
test-tube fitted with a two-hole rubber stopper, one hole of which 
is connected with an aerating tube while the other one holds a 
delivery tube going into an Erlenmeyer flask. Place in the flask 
10 c.c. of N/ioo HC1, colored pink with methyl red, and a few 
drops of petroleum to prevent bumping. Put with the saliva 
about 2 c.c. of freshly prepared 5 per cent urease solution (Arlco- 
Urease) and allow digestion without heat to take place for twenty 
minutes. Then add 10 c.c. of saturated potassium carbonate 
and a few c.c. of petroleum to the digestion mixture, and blow 
air through the apparatus for fifteen or twenty minutes. This 
serves to blow over the ammonia formed from the urea into the 
standard acid, which fixes it as ammonium chloride. The excess 
acid is then titrated with N/ioo alkali and from the difference 
the amount of urea nitrogen and ammonia nitrogen is calculated. 

. OOP 14 X (difference) X 100 __ grams of urea nitrogen + ammonia 
2 ~~ nitrogen per 100 c.c. saliva. 

Normally the total urea and ammonia nitrogen is 10-15 mg. 
per 100 c.c. 

AMMONIA. 

Qualitatively, the presence of ammonia may be shown by 
treating one drop of saliva with one drop of Nessler's reagent. 
The intensity of the yellow color obtained gives an approximation 
of the amount of ammonia present. According to Dr. Michaels, a 
high quantity of ammonia tends toward a carious condition of 
the teeth with a general hypoacid diathesis. Little value is 
now attached to his statement, however. 

DETERMINATION OF AMMONIA Quantitative. 

Follow the directions outlined for urea nitrogen plus ammonia, 
omitting the addition of urease. This eliminates the necessity 



l8o PHYSIOLOGICAL CHEMISTRY 

of digestion, and the ammonia may be blown over directly after 
the action of the potassium carbonate has taken place. The 
calculation is the same as that given for urea nitrogen plus 
ammonia nitrogen. 

The ammonia content varies, but, calculated as nitrogen, 
is usually from 5-7 mg. per 100 c.c. 

DETERMINATION OF CREATININE. 

Place 5 c.c. of centrifugalized saliva and 5 c.c. of distilled water 
in a small Erlenmeyer flask. In another similar flask place 
5 c.c. of diluted standard creatinine solution* and 15 c.c. of 
water. Make a fresh alkaline picrate solution by mixing to- 
gether saturated picric acid 5 parts and 10 per cent sodium 
hydroxide i part. Add to the flask containing the saliva 5 c.c. 
of the freshly prepared alkaline picrate solution, and to the flask 
containing the standard creatinine add 10 c.c. Allow to stand 
four minutes and compare colorimetrically, setting the stand- 
ard at 20 (Fig. 9). 

Calculation : 

20 

X.oo75X2oX2 = mg. of creatinine per 100 c.c. 



reading 

Creatinine is normally present in very small amounts in saliva 
(.3 to .7 mg. per 100 c.c.). It has been found to be increased in 
cases of nephritis, where the blood creatinine has increased, and 
it has also been demonstrated that smoking directly before or 
during collection of the sample will cause a tremendous apparent 
increase.f That this is due to some action of the tobacco smoke 
on the picrate solution has been recently shown by drawing 
smoke through distilled water and obtaining a seemingly high 
creatinine content. Experiments have also been conducted 
with pure nicotine and have given nearly negative results, in- 

* This standard consists of the diluted standard referred to in the appendix 
(page 296) i part and distilled water 3 parts. Five c.c. then contain .0075 mg. 
creatinine. 

t Thesis Work W. E. Crocker, H. J. Cox, J. M. Collins, Harvard Dental 
School, 1924. 



SALIVA PROPERTIES, CONSTITUENTS, AND ANALYSIS 181 

dicating that it is some constituent of tobacco smoke, rather 
than of nicotine, that causes the error in creatinine determina- 
tions. This error is likely to be very large, unless care is taken 
to prevent it. 

DETERMINATION OF URIC Aero. 

To 2 c.c. of saliva in a small beaker add i c.c. of diluted phos- 
pho-tungstic acid.* Filter carefully into a 25 c.c. graduate, 
washing the precipitate repeatedly. Add from a burette 2 c.c. of 
7! per cent sodium cyanide solution and allow to stand two 
minutes. Simultaneously prepare a standard by using 5 c.c. of 
standard uric acidf solution, i c.c. diluted phospho-tungstic 
acid and 2 c.c. sodium cyanide. After both solutions have 
stood two minutes at room temperature transfer them to two 
test-tubes and place in boiling water for ninety seconds. Then 
replace them in the original cylinders and dilute them to volume. 
Mix and compare colorimetrically, placing the standard as 
usual at 20. 

Calculation: 

20 

X.02X5o = mg. of uric acid per 100 c.c. 
^ 



. 
reading 

The normal content is from 1-1.5 m - P er IO c - c See also 
page 245. 

TOTAL SOLIDS AND ASH. 

(Method I.) These should be determined immediately 
upon the arrival of the specimen, to avoid error through evapor- 
ation of moisture. 

Use a platinum or fused silica dish of constant weight, which 
has been kept in a desiccator over sulphuric acid. Weigh the 
dish accurately and rapidly, then introduce 2\ c.c. of the well- 
mixed specimen and heat in a drying oven, not over 100 C., 
for two hours. Then place in the desiccator over sulphuric acid 
for twelve hours or longer, and weigh accurately and rapidly. 

* Prepared by using 2 parts phospho-tungstic acid, Appendix, page 301, and 
i part distilled water. 

t Dilute standard for use referred to in Appendix, page 302. 
% Weight of uric acid contained in 5 c.c. of standard used. 



182 PHYSIOLOGICAL CHEMISTRY 

The difference between these weights represents the weight 
of total solids. To calculate the percentage, divide by two and 
one-half times the specific gravity. 

Add to the dish two or three drops of fuming nitric acid, 
and heat over a flame, keeping the dish two inches above the 
top of the flame, until the black color has become white. Heat 
in the direct flame until glowing, place at once in desiccator to 
cool for one or more hours, and weigh. Calculate the percent- 
age of ash in same manner as that of total solids. 

(Method II.) Total solids and ash are best obtained as 
follows: evaporate over a water-bath 5 grams of the sample 
thoroughly mixed with a weighed amount (half a gram) of 
ignited magnesium oxide. The weight of residue (less the 
magnesia) obtained by drying at 100 C., gives the total solids. 
These may be ignited until white ash is obtained, and again 
weighed. The second weight (less magnesia) gives the ash. 

The use of the magnesium oxide serves to retain carbonates 
and chlorides in the total solids and the chlorides in the ash. 
It also obviates the necessity of oxidation with nitric acid, which 
would decompose many of the inorganic constituents of the ash. 

To determine weight of sediment, obtain total solids as above; 
then if a portion of the saliva is carefully filtered and the solids 
determined in the clear filtrate by the same method, the differ- 
ence between the two determinations of solids will be the weight 
of sediment, epithelium, leucocytes, etc. 

ABNORMAL CONSTITUENTS. 

Acetone is of quite frequent occurrence in the saliva. In 
diabetic patients this substance is often present in comparatively 
large amounts, sometimes sufficient for the detection of the 
acetone by its characteristic odor. Acetone may appear in 
the saliva when it is not present in the urine. In such cases it 
has usually resulted from disordered digestion and a consequent 
faulty metabolism. (For further consideration of acetone, see 
Urine.) 

Acetone is most satisfactorily detected by the Gunning's 
lodoform Test) which may be performed as follows: 



SALIVA PROPERTIES, CONSTITUENTS, AND ANALYSIS 183 

To about 2 c.c. of saliva add a few drops of Gram's reagent 
and a few drops of dilute ammonia. This forms a black precip- 
itate of nitrogen iodide, which reacts slowly with the acetone to 
form iodoform. The iodoform will be observed as a yellowish 
sediment which upon microscopic examination will show the 
characteristic hexagonal iodoform crystals (Plate III, Fig. 6, 
page 77). 

Sugar. In diabetic patients, sugar has very rarely been found in 
the saliva. One case coming under the observation of the author 
was that of a woman of middle age, with diabetes of long stand- 
ing, with 8 per cent of sugar in the urine; from this case there 
were obtained a very few osazone crystals by subjecting a consid- 
erable quantity of saliva, after concentration, to the phenyl- 
hydrazine test. 

Choleslerin and lecithin have been found by Professor Michaels 
in pathological saliva, and leucin has been found by him in a 
case of lupus. According to Novey, leucin has also been found 
in a case of hysteria. 

Of the crystalline salts which may be separated by evaporation 
of dialyzed saliva, the sodium oxalate and the lactates and acid 
lactates of lime and magnesia are of the most importance and 
have been the most thoroughly studied. As these salts may 
likewise be separated from urine, their significance will be studied 
under that head. 

Lactic Acid has been considered: butyric and acetic acids may 
each be tested for, qualitatively, by the methods given under 
gastric digestion. 

Mercury. A very delicate test may be made for this metal 
as follows: Collect as large a sample of saliva as possible, dilute 
with an equal volume of water, acidify with a few drops of 
hydrochloric acid, throw in a few very small pieces of copper- 
turnings which have been recently cleaned in dilute nitric acid, 
and boil for at least one-half hour, keeping up the volume by 
occasional additions of water. Remove the copper-filings, dry 
thoroughly on filter-paper, and place in a large-sized watch- 
glass (3 inches). In another watch-glass of similar size place 
one droD of solution of gold chloride, and quickly invert so that 



184 PHYSIOLOGICAL CHEMISTRY 

the drop remains hanging on the under side of the glass. Now 
place this watch-glass directly over the one containing the 
copper, so that the chloride of gold is suspended directly above 
the turnings and perhaps half an inch from them; then gently 
heat the lower watch-glass with a very small flame, when the 
slightest trace of mercury, which may have been deposited upon 
the copper, will be volatilized, reducing the chloride of gold, and 
causing a purplish ring to appear around the edge of the drop. 
If no reduction of the gold occurs, mercury is absent. 

Lead, which occasionally occurs in saliva, may be detected 
by the methods given under urine. 

Microscopical examination of the sediment should be made 
in every instance. Normal saliva will contain epithelium from 
various parts of the oral cavity, an occasional leucocyte, and 
occasional mold fungi, leptothrix, etc. Constituents which per- 
haps are not properly classed as normal, and at the same time 
are not pathological, are fat globules, a rare blood-corpuscle, 
sarcinae, extraneous material, as food particles, starch granules, 
muscle fibers, etc. An excessive amount of blood, fat, pus, or 
micro-organisms would, of course, indicate pathogenic con- 
ditions. The bacteriological investigation of samples of saliva 
is always of interest, and may be necessary, but the detailed 
methods of such investigation do not lie within the scope of this 
work. 

CRYSTALS FROM THE DIALYZED SALIVA. 

To obtain characteristic crystals, as has been explained in 
considering the subject of micro-chemistry, uniformity as to 
conditions under which the crystallization takes place is a neces- 
sity. In the case of saliva, however, we are not producing 
new compounds, but simply searching for compounds, already 
formed and existing in unknown proportions in the samples 
tested. It is therefore necessary to make several preparations 
of each sample, in order that we may obtain the widest range of 
possibility for characteristic crystallizations. The following 
method of procedure will usually give satisfactory results: 
For a dialyzer use a fairly wide glass tube, over one end of which 



PLATE VII. - ANALYSIS OF SALIVA. 




FIG. i 




.- ' . 

J, '(P.- I 

f| f (P. L,) 





FIG, a, 




- . FIG, 4* 
i f Add 

B, 




- : '^ -FIG. s*'" . - . 

i% 



- . ; - FIG, 6. 
|%. 



SALIVA PROPERTIES, CONSTITUENTS, AND ANALYSIS 185 



has been tightly tied a piece of parchment (Fig. 10), or, better, a 
small dialyzing tube made entirely of parchment. Place about 
15 c.c. of saliva in the dialyzing tube, and suspend it in a small 
beaker or wine-glass which contains an equal volume of distilled 
water. At the end of twenty-four hours the distilled water will 
contain the dialyzable salts in nearly the same concentration as 
existed in the original saliva. Take four previously prepared 
cell-slides (microscope slides on 
which a ring of BelPs or other 
microscopical cement has been 
placed) and fill each cell full of the 
dialyzed saliva. Put number one 
in a warm place that it may evap- 
orate rapidly, leave number two 
exposed to the air at the room 
temperature and it will dry in from 
half to three-quarters of an hour. 
Place number three under a large 
beaker, or small bell-jar, and cover 
number four with a cover-glass, 
and from time to time examine the 

crystals that may be formed. Numbers three and four will 
probably take several hours, perhaps several days, before 
crystallization is complete. When the crystals have ap- 
peared, the preparation may be preserved by mounting in xylol 
balsam. In attempting to obtain crystals from the saliva 
before dialyzation, results are usually unsatisfactory, owing to 
the presence of mucin and other organic substances which 
interfere with the crystallization. The crystals obtained by 
this method are principally chlorides of the alkali metals, par- 
ticularly ammonium, and frequently sodium oxalate, lactates, 
and acid lactates of lime and magnesia, and rarely urates of 
the alkalies. (For forms of these crystals see Plate VII, Figs. 
3 and 4, page 185 and Plate II, Fig. 4, page 77.) 

The following is the type of analysis blank used for record- 
ing results. 




FIG. 10. 



i86 



PHYSIOLOGICAL CHEMISTRY 



NO. 



Date 



NAME 



Saliva Analysis by 



Appearance 



Odor 



Sp. Gr. 



Acidity permanent 



temporary 



CO 2 





Resting 


Resting 




Acidity Total 


PH 

Activated 


Activated 


Alkalinity 







Mucin 



Albumin 



Chlorine 



Ammonia 



HCNS 



Urea N. 



Creatinine 



Nitrites 



Oxydase 



Acetone 



P 2 6 



Viscosity 



Lactic Acid 



Uric Acid 



Soluble Salts 
Sediment 



Remarks 



CHAPTER XIX. 
URINE. 

Urine is a solution of waste products from the blood. It 
contains, normally, certain coloring matter, urea, uric acid in 
combination with alkaline bases, various organic constituents 
in slight amounts, including, perhaps, albumin and sugar, 
chloride of sodium, sulphates and phosphates of the alkalies and 
the alkaline earths. Abnormally, the urine may contain albu- 
min, sugar, uric acid as such, bile, salts of the heavy metals, 
lead, mercury, and arsenic; also occasionally albumose, peptones, 
lactates, acid lactates, oxalates, carbonates, hippuric acid, also 
organic compounds, resulting from insufficient or imperfect 
oxidations, as amino acids, leucin, tyrosin, and acetone bodies. 

We are to study the urine, not primarily with a view to the 
diagnosis of renal disease, which is more particularly the prov- 
ince of the physician, but to detect irregularities or deficiencies 
in the body metabolism: and, as far as possible, we are to study 
the methods whereby we may correct and regulate the mal- 
nutrition which lies at the foundation of many diseases of the 
oral cavity. It has been well stated that, if there are diseases of 
the oral cavity which may have their etiology in some systemic 
derangement not easily apparent, and if such diseases are to 
receive the attention of the dentist, he should obtain all possible 
light on every case, and at present a quantitative analysis of the 
urine is of greater value than any other laboratory aid except 
the analysis of the blood. In examining a sample of urine to ob- 
tain information as above indicated, it is essential that the sam- 
ple be a portion of the mixed twenty-four-hour quantity, and that 
the total amount of the twenty-four-hour excretion be known. In 
collecting samples for such analysis a convenient method is to give 
the patient a one- or two-dram vial, nearly filled with water, and 
containing three or four drops of a commercial formaldehyde 
solution, with instructions to empty this into a bottle, or other 
receptacle, in which the twenty-four-hour sample is to be col- 

187 



l88 PHYSIOLOGICAL CHEMISTRY 

lected. Formaldehyde if used in this amount has no effect on 
the subsequent analysis and is a sufficient preservative. 

PHYSICAL PROPERTIES. 

Quantity. The quantity of urine passed in twenty-four 
hours is normally about 1200 to 1400 c.c. for an adult female 
and 100 to 200 c.c. more than this for the male. The amount 
is increased in Bright's disease, in diabetes, and various other 
pathological conditions, also in cold weather when less moisture 
is given off from the skin. Normally, the quantity passed during 
twelve day hours, as 8 A.M. to 8 P.M., will exceed the amount 
overnight from 8 P.M. to 8 A.M. In cases of chronic interstitial 
nephritis the twelve-hour night quantity exceeds the day, hence 
it is desirable in collecting a twenty-four-hour sample to divide 
the time as suggested, and measure the amounts separately, 
especially if there is any suspicion of chronic kidney disease. A 
diminished quantity of urine may indicate simply a diminished 
amount of water taken into the system. The urine is diminished 
pathologically in acute conditions, such as fevers, etc., but such 
samples rarely reach the dental practitioner. 

Color. The normal color of the urine is usually given as 
straw color or pale yellow. If lighter than this the color is 
regarded as pale; if darker than normal it is regarded as high. 
The urine may also be colored by various abnormal constitu- 
ents; it may be bright red from the presence of blood, or choc- 
olate colored with a so-called coffee-ground sediment from 
decomposed blood coloring matter. It may be brown to yellow, 
bright blue or green, as a result of the ingestion of various drugs. 
If bile is present in any quantity in the urine, it will have a 
dark or smoky appearance, and, upon shaking, the foam will 
have a distinctly yellowish or yellowish-green tint. 

Appearance. In addition to the colors mentioned above, 
urine may sometimes have a smoky appearance, due to the 
presence of hematoporphyrin or iron-free hematin, often found 
in cases of lead-poisoning. It may have a milky appearance, 
due to presence of finely divided fat globules, as in chylous 
urine, due to the presence of chyme. It may be cloudy from four 



URINE 



189 



principal causes: first, amorphous urates; second, amorphous 
phosphates; third, pus; and fourth, bacteria. These may 
easily be distinguished. The application of a slight degree of 
heat (insufficient to cause coagulation of albumin) will redissolve 
the urates, and clear a urine which is cloudy from this cause. A 
deposit of phosphates is increased by the application of heat, but 
clears easily upon the addition of a few drops of acetic acid. A 
urine cloudy from the presence of pus is not cleared by either of 
these methods, but the cloud settles with comparative rapidity 
and pus corpuscles are easily recognized by microscopical ex- 
amination of the sediment. If bacteria are present in sufficient 
quantity to cause cloudiness, the sample is apt 
to be alkaline in reaction and will not clear 
upon filtering. If it is necessary to obtain a 
clear solution, a little magnesium mixture may 
be added to the urine, then a little sodium 
phosphate; warm gently with agitation, when 
the precipitated ammonium magnesium phos- 
phate will mechanically carry down the bac- 
teria, and a filtrate may be obtained which, 
after acidifying with dilute acetic acid, will be 
suitable for an albumin test. 

Specific Gravity. The gravity is most con- 
veniently taken with a urinometer (Fig. u). Care should be 
taken in the selection of this instrument so that the scale gradu- 
ation may be accurate. The fact that the instrument will sink in 
distilled water at the proper temperature (usually 60 F., 15^ C.) 
to the zero mark, is not a sufficient proof of its accuracy, as many 
cheap instruments will do "this, and give erroneous readings at 
the higher markings of the scale. Distilled water is represented 
by 1000, and the relative increase in the comparative gravity of 
urines will be easily represented on the scale ranging from 1000 
to 1050. As the first two figures of the specific gravity are 
always the same (10) they are usually omitted from the scale 
which is made to read from o to 50 or 60. The reading should be 
made, if possible, from underneath the surface of the liquid, as 
the liquid is usually drawn around the stem by adhesion, so that 




FIG. ii. 



190 PHYSIOLOGICAL CHEMISTRY 

accurate readings from the surface are difficult. The specific 
gravity of normal urine is from 1018 to 1022; it decreases in 
cases where the quantity is much above the normal (polyurias), 
unless sugar is present. It is increased by the presence of sugar 
or by concentration, whereby the normal solids are relatively 
increased. In case the quantity of urine is too small for the 
determination of the gravity in the usual way, the urinopyknom- 
eter, devised and recommended by Dr. Saxe in his " Examina- 
tion of the Urine/' may be employed. (See page 160, Fig. 6.) 

Reaction. The reaction of urine is normally acid to litmus 
paper, because of the presence of acid sodium phosphate, and be- 
cause of organic acid combinations, the composition of which is 
unknown. The degree of acidity is roughly indicated by the in- 
tensity of color produced with the carefully prepared litmus paper. 
More accurate results may be obtained by a regular volumetric 
examination (with N/2O alkali), or by the test for urinary acidities 
given by Freund and Topf er, who suggest the following method : 

" To 10 c.c. of the urine add two to four drops of a i per cent 
solution of alizarin. If the resulting color is pure yellow, free 
acids are present; if deep violet, combined acid salts. If none 
of these colors appear, there are present acid salts of the type 
of disodic phosphate. The amount of one-tenth normal hydro- 
chloric acid standard solution required to produce a pure yellow 
color represents the alkaline salts, while the amount of one-tenth 
normal sodium hydrate required to cause a deep violet represents 
the acid salts. " 

The average PH of normal urine is about 6.0, and this deter- 
mination should be made whenever practical. Note that the 
greater the hydrogen-ion concentration the stronger the acidity, 
but the lower the PH figure. Hawk says that the hydrogen-ion 
concentration is increased in most pathological conditions. 
(Hawk's " Physiological Chemistry/' 6th Ed., p. 511.) 

NORMAL CONSTITUENTS OF URINE. 

The more important normal constituents of the urine are 
urea, uric acid (combined as urates), chlorides, phosphates, 
sulphates, indoxyl, coloring matters; traces of mucin, organic 



URINE 



191 



acids, carbonates, hippuric acid, creatine, and creatinine may also 
be present. The total normal solids are composed approxi- 
mately of 50 per cent urea, 25 per cent chloride of sodium; at 
least one-half of the remainder are phosphates and sulphates. 
We see that the constituent which most influences the specific 
gravity is the urea, and in normal samples the specific gravity is 
an index of the amount of urea present. The total solids may be 
calculated by multiplying the last two figures of the specific 
gravity, taken at 25 C. by 2.6,* which will give approximately 
the number of grams of solids in one liter of urine; from this the 
solids in the twenty-four-hour amount may be easily calculated. 

UREA. 

The chemistry of urea has already been considered (page 54). 

Detection. A qualitative test for this substance is obvi- 
ously superfluous, although such may be made by obtaining 
the crystals of urea nitrate or oxalate (page 
56). The quantity of urea is of great impor- 
tance, especially in cases where there is any 
question in regard to the body metabolism or 
the amount of nitrogen excreted. By far the 
greater proportion of all nitrogenous waste is 
eliminated by the kidney a in the form of urea, 
a comparatively slight amount as other nitro- 
genous constituents of the urine, a still smaller 
amount in the feces, and traces only by other 
avenues. The urea may be quantitatively de- 
termined by various methods, the hypobromite 
method being the most practical. See reaction on page 297. 

Quantitative Determination. There are various forms of 
apparatus used in connection with this process. 

The Doremus-Hinds apparatus shown in Fig. 12 gives a 
perfectly satisfactory method for the estimation of urea by the 
hypobromite method. The reagent, equal parts of bromine 
solution and 40 per cent NaOH (Appendix, page 297), is intro- 

* Long's Coefficient. 




FIG. 12, 



PHYSIOLOGICAL CHEMISTRY 

duced into R and the tube completely filled. The tube U is 
next filled exactly to the zero mark; then by means of the stop- 
cock S i c.c. of urine is allowed to enter T a few drops at a time 
and slowly enough to prevent any escape of gas through R. The 
gas rises in small bubbles through a comparatively long tube 
and remains in contact with the reagent, which insures perfect 
absorption of CO2, thus overcoming the greatest objection to the 
Sqaibb's apparatus. 

The tube T is graduated to read centigrams of urea in i c.c. of 
urine. 

A more accurate determination of urea depends upon the 
conversion of urea into ammonia by various methods which 
make quantitative application of the Kjeldahl determination 

For detail of this test see page 280. 

URIC ACID. 

Uric acid and its antecedents, the xanthin bases, are derived 
from the decomposition of nuclein and nucleo-protein. See 
pages 57 to 59 and page 226. The uric acid is increased by a 
highly nitrogenous diet and certain vegetable substances which 
contain purine (page 58) derivatives, such as coffee, tea, and 
cocoa. Meats in general, and particularly those rich in nuclein, 
such as tongue, liver, sweet breads, etc., are regarded as the most 
abundant source of uric acid and urates. As previously suggested, 
uric acid does not occur as such in normal urine, but is combined 
with the alkaline bases. 

Determination. It is unnecessary to make a qualitative test 
in urine, as urates are always present. If a qualitative test is 
desired, the murexide test, as given on page 263, is available. 
Uric acid and allied constituents of the urine are conveniently 
determined quantitatively by the centrifugal method, as devised 
by Dr. R. Harvey Cook.* The detail of this method is as 
follows: Measure 10 c.c. of urine into a graduated tube, used in 
the centrifugal machine, add a few grains of sodium carbonate, 
and about 3 c.c. of strong ammonium hydrate. Place in the 
centrifuge, and allow to run for one or two minutes, then carefully 
* Medical Record, Mar. 12, 1898, page 373. 



URINE 193 

decant the clear urine into another graduate tube, leaving the 
precipitate which consists of earthy phosphates. The bulk of 
this precipitate may be noticed and an idea obtained as to whether 
the earthy phosphates are present in normal quantities or not. 
To the clear urine add 2 or 3 c.c. of ammoniacal silver-nitrate 
solution (AgN0 3 , 5 grams; distilled water, 80 c.c.; strong 
ammonia, 20 c.c.), and run in the centrifuge till the precipitate 
of silver urate has reached its lowest obtainable reading. The 
ammonia will prevent the precipitation of chlorides and, unless 
iodides or bromides are present, the precipitate will be fairly 
pure silver urate, each tenth of a cubic centimeter of the pre- 
cipitate being equivalent to 0.001176 gram of uric acid in the 10 
c.c. of urine used, or 0.01176 per cent. 

The silver precipitate is by no means pure silver urate, many 
of the other nitrogenous bases in urine forming insoluble silver 
salts. These occur only in very slight traces; so, for clinical 
purposes, the method is available unless the sample contains 
bromides or iodides, when iodide or bromide of silver will be 
formed, insoluble in the amount of ammonia usually used. 
More accurate results may be obtained by either Hopkins' or 
Folin's method. These are somewhat similar and consist of 
precipitation of the uric acid as ammonium urate. One hundred 
to 200 c.c. of urine is used and the precipitation effected by a 
saturated solution of NH 4 C1 (Hopkins' method) or 10 grams 
ammonium sulphate (Folin's method). 

The precipitate is washed in the reagent and dissolved in 
boiling water and the amount of uric acid determined by titra- 
tion with N/20 permanganate of potassium. Each cubic centi- 
meter of KMn0 4 used is equal to 0.00375 gram of uric acid. 

AMMONIA DETERMINATION. 

The amount of ammonia normally present in urine is about 
o. 7 gram in the twenty-four-hour amount. Ammonia is increased 
in any systemic condition resulting in an increase of acidic 
elements (Acidosis), or upon ingestion of ammonium salts of 
inorganic acids, i.e., salts not easily changed to urea. 

Normally the quantity of NH 3 follows more or less closely 



IQ4 PHYSIOLOGICAL CHEMISTRY 

the urea and the protein metabolism, and amounts to about 
one- twentieth of one per cent (0.05 per cent) or about 0.7 gram 
in twenty-four hours. In acidosis, even of the mild type, the 
ammonia is increased at the expense of the urea; i.e., the pro- 
portion of ammonia to urea is greater than normal, and uric acid 
is often high while urea is low. 

Formaldehyde Method. Place 10 c.c. urine in a 250 c.c. 
Erlenmeyer flask, add 10 or 20 c.c. H 2 O, titrate with N/io NaOH 
with phenolphthalein as an indicator. The amount of NaOH 
used will represent total acidity of sample. 

After exact neutralization add 10 c.c. of previously neutralized 
commercial formaldehyde solution and titrate again with N/io 
NaOH. The second amount of alkali added represents am- 
monia as follows: 



4 NH 4 Cl+6 CH 2 O+4NaOH = N 4 (CH 2 ) 6 +ioH 2 O+4 NaCl. 

As the ammonium salts and the caustic soda react molecule 
for molecule, it is possible to make calculation for quantity of 
NH 3 by multiplying the N/io factor (0.0017) by the number of 
cubic centimeters of N/io NaOH used. 

In cases of diabetes, when the ammonia reaches a compara- 
tively large amount the figures obtained by this process will be 
found to be a little high, as amino acids are also acted upon by 
the NaOH, and will be calculated as ammonia, but for ordinary 
work of clinical comparisons this method is very simple and 
sufficiently accurate. 

This method is not affected by urea, uric acid, creatine, crea- 
tinine, purine bases, or hippuric acid.* 

Zeolite Method for Ammonia (Foliri). 

This method is based on the property of zeolite powder (com- 
mercial permutit, a synthetic aluminate silicate) to absorb 
ammonia. The powder may be used repeatedly if it is carefully 

* Dr. Hans Malfatti in Zeit. fUr Anal. Chemie, 47, page 273. 
Note. See also the Vacuum Distillation Method, giving very exact results 
when properly carried out: 
H. Bj6rn Andersen und Marius Lauritzen, Zeit. fiir Physiol. Chemie, 64, page 21. 



URINE 195 

washed first with water and then with 2 per cent acetic acid and 
finally with water. 

Procedure: In a 100 c.c. volumetric flask, place about 2 grams 
of zeolite. Add 5 c.c. water and 2 c.c. urine. Add a little more 
water, 1-5, in order to rinse down the urine, and then shake 
gentl for five minutes. Rinse the powder to the bottom of the 
flask with 25-40 c.c. water, and decant the supernatant liquid. 
Repeat. Then add a few c.c. of water to the powder, i c.c. 10 
per cent NaOH, shake gently and let stand for a few minutes. 

Prepare a standard solution as follows: In another 100 c.c. 
flask place 5 c.c. standard (NH^SCX* solution, i c.c. 10 per cent 
NaOH. Dilute to about 75 c.c. and mix thoroughly. Measure 
10 c.c. of Nessler's reagent into a cylinder and add all at once 
to the solution in the flask while the contents of the flask are in 
vigorous motion. A perfectly clear solution should be obtained. 
Then dilute the urine flask to about 75 c.c. and add 10 c.c. of 
Nessler's solution as directed above. Compare the colors 
obtained in a colorimeter, setting standard at 20, and calculate 
result as follows: 

20 

~ r. ; X S X so = ing. of ammonia nitrogen per 100 c.c. 

Reading of cup 

CHLORIDES. 

The chlorides are represented in the urine chiefly by sodium 
chloride. This is present to the extent of 12 to 20 grams in 
twenty-four hours. An increase above this quantity is unusual, 
although it simply indicates an increase in the ingested salt, and 
is without clinical significance. The chlorine is diminished in 
dropsy, acute stages of penumonia, and in fevers generally. 

Determination. The usual qualitative test with silver nitrate 
and nitric acid is employed for detection of chlorine in the urine. 
If one drop of a strong solution of silver nitrate (i to 8) is al- 
lowed to fall into the wine-glass in which the albumin test has 
been made (q.v.), the appearance of the resulting precipitate 
will give a rough idea of the quantity of chlorine present. If a 
solid ball of silver chloride is formed which does not become 
* For preparation see Appendix. 



196 PHYSIOLOGICAL CHEMISTRY 

diffused upon gently agitating the contents of the glass, the 
chlorine is normal or increased. If the precipitate falls as a 
cloud distributed throughout the liquid, the chlorine is dimin- 
ished. The chlorine may be determined by precipitation with 
silver nitrate in 10 c.c. of urine, and the precipitate settled in a 
centrifuge-tube to constant reading, but this method is not 
recommended, as the precipitate is a bulky one, and usually 
takes a long time for thorough settling. 
An accurate titration of chlorine may be made as follows : 
To 10 c.c. of urine contained in a porcelain dish add about 5 c.c. 
of saturated solution of ferric alum strongly acidified with nitric 
acid. Add 20 c.c. of an N/io silver nitrate solution. This 
precipitates silver chloride, but silver urate, silver phosphate 
and any possible organic silver compounds are held in solution 
by the nitric acid. Titrate excess of silver nitrate with N/io 
potassium thiocyanate, the ferric alum acting as an indicator. 

Calculation: Subtract number of c.c. of thiocyanate used 
from 20, multiply result by .0035 and then by 10 to obtain per 
cent. 

PHOSPHATES. 

The phosphates in the urine are of two kinds, the alkaline 
phosphates, Na^HPCX and NaH 2 PO 4 , etc., and the earthy phos- 
phates represented by the magnesium and the calcium phos- 
phates. The phosphates are normally present to the extent of 
2\ to 3 1 grams, calculated as P 2 5 (in twenty-four hours). 

The triple phosphates, ammonium magnesium phosphates 
(Plate IX, Fig. 6, page 214), are the forms in which phosphoric 
acid is usually found in urinary sediment. Crystals of acid 
calcium phosphate are occasionally found, and resemble the 
acid sodium urate in form (Plate IX, Fig. 3, page 214), except 
that they are usually a little broader and more often occur in fan- 
shaped clusters. They may be distinguished by treatment with 
acetic acid, which dissolves the calcium phosphate promptly, 
while the urate is slowly dissolved and crystals of uric acid 
appear after a little time. The phosphates are deposited from 
neutral or alkaline urines, and when this precipitation takes 



URINE 



197 



place within the body the crystals cause more or less irritation 
to the urinary tract and may form aggregations which result 
in calculi. Phosphates are supplied by either a cereal or meat 
diet. They may be much increased in diseases accompanied 
by nervous waste, or by softening and absorption of bone. 
Phosphates are diminished in gout, in chronic diseases of the 
kidney, and during pregnancy. 

Determination. A qualitative test for earthy phosphates 
(E.P.) may be made by taking a test-tube half full of urine, and 
making alkaline with ammonium hydrate. When the precipi- 
tate has thoroughly settled, if it is about f to \ inch in depth, 
it represents normal earthy phosphates. If this mixture is now 
filtered, the alkaline phosphates (A. P.) may be determined in 
the filtrate by the addition to the solution of one-third its volume 
of magnesium mixture.* The precipitate after settling will be 
\ to f of an inch in depth if normal. The total phosphates may 
be determined in the centrifugal machine by adding 5 c.c. of 
magnesium mixture to 10 c.c. of urine. Each tenth of a cubic 
centimeter of the centrifugalized sediment will be equivalent to 
0.00225 gram of PzOs in the 10 c.c. used. 

A more accurate determination of the total phosphoric acid 
may be made by the titration with uranium nitrate or acetate 
solution as follows : 

Reagents Required. First. A standard uranium solution 
may be prepared as follows: Dissolve 35.5 grams of pure ura- 
nium nitrate or acetate in about 800 c.c. of distilled water; 
add 3 or 4 c.c. of glacial acetic acid and heat it enough to complete 
solution. Allow to stand over night, filter carefully, and make 
up to 1000 c.c. Standardize this solution against crystallized 
microcosmic salt by dissolving 14.721 grams of the pure salt 
(NaNH 4 HPO4 . 4 H 2 O) in sufficient water to make 1000 c.c. 
Then. titrate 20 c.c. of this solution, to which has been added 30 
c.c. of water and 5 c.c. of sodium acetate solution, with the 
uranium solution (method of titration is given under process 
below). 

The uranium solution should then be adjusted (diluted) so 

* See Appendix. 



198 PHYSIOLOGICAL CHEMISTRY 

that it will take exactly 20 c.c. for this titration, when i c.c. of 
the uranium will be equivalent to 5 milligrams of PiO&. 

Second. A sodium acetate solution containing 100 c.c. of 
30 per cent acetic acid and 100 grams of sodium acetate in enough 
distilled water to make 1000 c.c. 

Third. An indicator consisting of a saturated solution of 
potassium ferrocyanide. 

Process. Place 50 c.c. of urine with 5 c.c. of sodium acetate 
solution above described in a small Erlenmeyer flask and heat 
nearly to the boiling-point. Titrate, while hot (80 or above), 
with the standard uranium solution till a drop of the mixture 
placed on a white porcelain tile with a drop of the indicator 
K^Fe(CN)6, gives a distinct brown color. This method of de- 
termining the end-point is known as " spotting " and with a 
little practice gives very accurate results. 

The number of cubic centimeters of uranium solution multiplied 
by o.oi will give the weight of P 2 O 5 in 100 c.c. of urine (i c.c. of 
reagent being equal to 0.005 gram P20 5 ). 

This same process may be used for saliva by diluting the 
reagent one part to five, and preparing the sample for titration 
as follows: Take from 2 to 5 c.c. saliva, add sufficient alcohol 
to make 10 c.c. of mixture, warm, and filter. This serves to 
separate the protein substance. Take 5 c.c. of the filtered 
solution and titrate with the diluted uranium solution as by 
the process given above for urine. In this case, of course, i c.c. 
of the standard uranium will represent i milligram of P 2 5 rather 
than 5. 

SULPHATES. 

The sulphates in the urine are present as alkaline sulphates, 
K.2SO4 and Na^SC^; also as ethereal sulphates, represented by 
such compounds as indoxyl potassium sulphate, page 201. 

Detection and Determination. The sulphates may be de- 
tected by precipitation with barium chloride in hydrochloric 
acid solution. If the precipitate is obtained from 10 c.c. of 
urine and centrifugalized to constant reading, the per cent of 
sulphuric acid by weight will be one-fourth of the volume per 



URINE 199 

cent of the precipitate. The sulphates follow the urea rather 
closely, and their determination is not of great importance. 
They are increased in acute fevers, diminished in chronic diseases 
generally, and markedly diminished in carbolic-acid poisoning. 
(Ogden.) 

Determination of Total Sulphur. (J. Benedict, Biol. Chem., 
6, 363; W. Denis, /. Biol. Chem., 8, 401.) To 25 c.c. of urine 
contained in a porcelain evaporating dish (10-12 cm. diameter) 
add exactly 5 c.c. of a solution containing 25 per cent copper 
nitrate, 25 per cent sodium chloride, and 10 per cent ammonium 
nitrate. Evaporate to dryness over a water-bath. Then heat 
over a flame, gradually increasing the heat until the dish is 
red hot, and continue heating for ten to fifteen minutes. Allow 
to cool. Add 20 c.c. dilute hydrochloric acid and warm gently. 
Rinse into a flask or beaker by means of about 100 c.c. hot 
water. Heat to boiling, and add drop by drop 25 c.c. of a 10 per 
cent barium chloride solution. Filter, wash, ignite, and weigh. 

COLORING MATTER. 

Urochrome is a pigment to which the yellow color of urine 
is chiefly due. Uroerythrin and urorosein are less important, 
existing only in very slight quantities, 'but they are responsible 
for colors of some sediments and of decomposition products 
which are noticed in analysis. In an article on urochrome 
(K. F. Pelkon, /. Biol. Chem., March, 1920) the statement is 
made that all evidence points to the fact that urochrome is 
derived from the protein in the diet. A low protein diet de- 
creased the urochrome up to 50 per cent while a high protein 
diet increased it in all the experiments which the above in- 
vestigator performed. 

Urobilin, a less important coloring matter of the urine, exists 
as a parent substance, or chromogen, to which has been given the 
name urobilinogen. This undergoes decomposition by action 
of light with liberation of urobilin. 

Urobilin is without doubt derived from the bilirubin of the 
bile, which, in turn, comes from the hemochromogen of the 



200 PHYSIOLOGICAL CHEMISTRY 

blood. Dr. J. B. Ogden is authority for the statement that " it is 
safe to infer that the amount of urobilin in the urine is a measure 
of the destruction of the hemoglobin or blood pigment." 

Another pigment closely resembling and possibly identical 
with urobilin is hydrobilirubin. It is considered as an oxidation 
product of bilirubin and is normally produced in the fecal matter 
during its passage through the large intestine. 

SOLUBLE SALTS. 

An examination of the soluble salts of the urine is easily and 
often profitably made by simply allowing a large drop to evapo- 
rate spontaneously and examining the residue with the micro- 
polariscope. The alkaline chlorides are often seen but they 
do not polarize light. Crystalline phosphates, sulphates, urates, 
and oxalates do polarize light and may frequently be detected 
by their characteristic forms. The value of determination of 
soluble oxalates in this way is suggested on page 246. 

INDOXYL. 

The test for indoxyl in the urine is of great importance as it 
indicates an excessive degree of nitrogenous putrefaction taking 
place in the small intestine. An increase of this nature may 
be induced by an acute inflammatory process of the peritoneal 
cavity, but in the majority of cases liable to come under exam- 
ination in dental practice, the excess of indoxyl will mean intes- 
tinal indigestion. Ordinary constipation does not of itself 
cause an increase in the indoxyl. 

Detection and Determination. Place 15 c.c. of strong HC1 
in a wine-glass, and add a single drop of concentrated nitric acid; 
then stir 30 drops of urine into the mixture. If indoxyl is present, 
an amethyst color develops in five to fifteen minutes. If the 
color is purple the indoxyl is increased. Variation of the amount 
of indoxyl within normal limits is rather wide, and the indoxyl 
may be reported as high or low, normal, increased, or diminished. 

The particular constituent of the protein molecule responsible 
for appearance of indican in the urine is tryptophane which, it 



URINE 201 

will be remembered, is an a-amino-jS-indol-propionic acid. The 
chemistry of the changes involved may be represented as follows : 

I I 

C CH 2 C 

# \ / S \ / 

-C C - C -C C - C 

I II || CHNH 2 I II II 

-C C C I -C C C 

\ / \ / \ COOH \ / \ / \ 

C N H C N 

I I II 

Tryptophane Indol 

Tryptophane by putrefactive action becomes Indol 

By oxidation the indol becomes Indoxyl C 8 H 7 NO or C 8 HeNOH 

I 
C 

<c \ 

-C C - C-O-H 

I II II 
-C C C 
\ / \ / \ 
C N 
I I 
Indoxyl 

The indoxyl forms an ethereal sulphate, indoxyl potassium 
sulphate, or indican, and as such is eliminated by the kidney; 
i.e., the indican (CgHeNSO-iK) appears in the urine and then by 
action of the strong HC1 and trace of HNO 3 of the test, again 
becomes indoxyl. 

O O I 

I \ S C 

C S // \ 

// \ /I -C C - C-O-H 

-C C - C - O O +HC1 - I II II 

I II II I -C C C- 

-CCC K % / \ / 

% / \ / \ C N 

C N I 

I I Indoxyl 

Indoxyl Potassium Sulphate 



202 PHYSIOLOGICAL CHEMISTRY 

Two molecules of indoxyl by action of the oxidizing agent 
become one molecule of indigo blue. 

C 16 H 10 N 2 2 . 

I I 

c c 

^ \ ^o / % 

-c c - or o=c - c c- 

i ii i i ii i 

-c c c -c c c- 

\ / \ / \/\^ 

C N N C 

II II 
Indigo blue. 

ABNORMAL CONSTITUENTS OF URINE 

The principal abnormal constituents are albumin, sugar, 
acetone, bile, and various crystalline salts, discoverable either 
by microscopical examination of the sediment, or by evaporation 
of a clear fluid, and examination with the micropolariscope. 

Metallic substances, arsenic, lead, and mercury, are occa- 
sionally present, and tests should be made for them when gen- 
eral symptoms or the conditions of the kidney indicate metallic 
poison. ALBUMIN is probably present in minute traces in the 
majority of urines. When in sufficient quantity to be detected 
by the usual laboratory methods, it is essential that we learn 
the source from which it has been derived, for the simple pres- 
ence of even a considerable trace of albumin may be of but 
slight clinical importance. Albumin may indicate either a 
pathological condition of the kidney, which allows the entrance 
into the renal tubules of serum-albumin from the blood, or it 
may indicate a change in the composition of the blood, whereby 
the albumin passes more easily through the renal membranes, 
or its presence may be due to irritations from various sources 
of the urinary tract; and, as regards the bearing of albuminurias 
on dental disease, it is sufficient simply to determine whether 
renal disturbance is primary or secondary to some other trouble, 
such as heart disease; or purely local, as when caused by irri- 
tation due to crystalline elements. 



URINE 203 

Detection. Albumin may be detected by either of two 
simple methods. It is often desirable to use both of these 
methods, thereby eliminating possible confusion from the 
presence of substances other than albumin, which may respond 
to one of the two tests, but not to both. 

The first consists simply in underlaying about 25 c.c. of filtered 
urine in a wine-glass with concentrated nitric acid. The wine- 
glass should be tipped as far as possible and the acid allowed to 
run very slowly down the side. This method is preferable to 
the use of the apparatus known as the albuminoscope or Horis- 
mascope (Fig. 13). As this latter method does not provide for 





FIG. 13. FIG. 14. 

sufficient mixing of nitric acid with the sample, the albumin is 
shown by a narrow white ring at the plane of contact of the two 
liquids. A white ring above the plane of contact is not albumin, 
but is composed of acid urates, indicating an excess of urates in 
the sample (Fig. 14). The albumin, in distinction from this 
band, occurs directly above the acid and is usually reported as 
the slightest possible trace when just discernible; as a slight trace, 
when well marked, but not dense enough to be seen by looking 
through the liquid from above; as a trace, when the white cloud 
may be seen by looking down into the glass from above and a 
large trace if plainly visible in this way. 

The acetic acid and heat method of testing for albumin is the 
other method referred to in the preceding paragraph. It is of 
about the same delicacy as the nitric acid test, and is less liable 
to respond to substances other than albumin. It is made as 
follows: 




204 PHYSIOLOGICAL CHEMISTRY 

A test-tube is filled two-thirds full of perfectly clear filtered 
urine, one drop of acetic acid added and the upper half of the 
sample boiled. The tube can easily be held in the hand by the 
lower end. After boiling, if the tube is examined before a black 
background, a slight cloudiness or turbidity resulting from 
coagulated albumin can be easily detected in the upper part of 
tube. Anything more than a trace should be determined in 
the centrifugal machine by mixing 10 c.c. of filtered urine with 
about 2 c.c. of acetic acid and 3 c.c. of potassium ferrocyanide 
solution. Each tenth of a cubic centimeter of the 
precipitated albumin, when settled to constant read- 
ing, indicates one-sixtieth of one per cent albumin by 
weight. This factor is fairly correct up to four- or 
five- tenths of a cubic centimeter of precipitate; be- 
yond this it is of little value, and the albumin is best 
determined quantitatively by measuring 50 or 100 c.c. 
of urine into a small beaker, adding a drop of acetic 
acid, and boiling, which will completely precipitate 
the albumin. It may then be filtered into a 
counterpoised filter, thoroughly washed, first in water, 
next in alcohol, and lastly in ether, dried at a tempera- 
ture a little below the boiling-point of water, and 
jT*^ weighed. Esbach's method may be of value in some 

instances, and is carried out as follows: 

Fill the albuminometer (Fig. 15) with urine to the line U, 
and then add the reagent* to the line R; close the tube, mix 
the contents thoroughly, and allow to stand in an upright 
position for twenty-four hours. At the end of that time the 
depth of precipitate may be read by the figures on the lower 
part of the tube, these figures representing tenths of one per cent 
of albumin, or grams of albumin in a liter of urine. If a sample of 
urine contains more albumin than is easily estimated by the 
centrifugal or Esbach's method, approximate results will be 
obtained by diluting with several volumes of distilled water, 
until the quantity of albumin precipitated is within the limit 

* Esbach's reagent consists of picric acid, 10 grams; citric acid, 20 grams, and 
distilled water sufficient to make one liter. 



URINE 205 

of the test. The proteoses occasionally occur in the urine, and 
are distinguished from albumin by the fact that they redissolve 
at a boiling temperature. If filtered while hot, albumin, which 
usually accompanies them, will remain on the paper, while 
albumose will separate from the clear filtrate as it cools. 

SUGAR. 

Sugar in urine represents a perverted process of oxidation, 
for which the pancreas is largely responsible. The liver also 
often plays an important part in cases of diabetes, but just how 
this is done is not clearly known. Sugar in the urine does not 
of necessity indicate diabetes any more than albumin in- 
dicates B right's disease. Many cases of glycosuria are of a 
temporary nature and respond readily to dietary treatment. 
Whenever sugar is found it is desirable to make tests upon both 
a fasting and an after-meal sample, such as might be obtained 
before breakfast and one hour after dinner. If the fasting sample 
is comparatively free from sugar, it indicates that the glycosuria 
is of a temporary nature and due to faulty metabolism, rather 
than to any organic disease of the liver or pancreas. 

Detection. Sugar in the urine may be detected by several 
general carbohydrate tests, as previously given. 

Fehling's test. This test is very generally employed (Exp. 
129, page 271). It is best, however, to modify it by bringing 
the Fehling's solution to active ebullition, adding from 5 to 
30 drops of the suspected sample and allowing to stand without 
further heating. This prevents possible reduction of the sugar 
by xanthine bases or other occasional constituents of the urine, 
which might give misleading results if the mixture were boiled 
after addition of the sample. There is less danger of trouble 
of this sort if the gravity of the urine is below normal. If it 
is necessary to make a rapid test, the mixture may be boiled 
after the urine is added, and in case the result is negative there 
is no need of further test; if, however, a slight reduction of the 
copper solution takes place, it will be necessary to repeat the 
test, using the precaution above given. Quantitatively, sugar 
may be determined by the use of Fehling's solution as follows: 



206 PHYSIOLOGICAL CHEMISTRY 

Fehling's Quantitative Method. 

If the urine contains more than a trace of albumin, this sub- 
stance should be removed by adding a drop of acetic acid and 
heating; after filtration the sample should be cooled and restored 
to original volume with distilled water. If specific gravity of the 
urine is more than 1025, it should be diluted to ten times its 
volume with distilled water (urine, i part; water, 9). If the 
gravity is less than 1025, dilute it to five times its volume, mix, 
and fill a 25 c.c. burette. In a 250 c.c. flask place 10 c.c. each of 
the alkaline tartrate and copper sulphate solutions (Fehling's 
solution), and add about 100 c.c. of distilled water. Place the 
flask over a Bunsen burner, and bring to a boil. If no change 
takes place after a minute or two of boiling, add the solution 
from the burette gradually, until the precipitate bceomes suffi- 
ciently dense to obscure the blue color of the solution. Continue 
to boil for one or two minutes, then remove from the flame and 
watch carefully the line directly beneath the surface of the liquid, 
which will appear blue until all of the copper has been reduced to 
the red suboxide. The solution should be kept at the boiling- 
point throughout the entire operation, except in making the 
examination of the meniscus between the additions of the diluted 
urine. These additions must be made very carefully, and as the 
process nears completion not more than one or two drops should 
be added at a time. When the blue color has entirely disappeared, 
and the line of meniscus has become colorless, note the number of 
cubic centimeters of dilute urine used, and calculate that in that 
quantity there is an equivalent of 0.05 gram of glucose; in other 
words, 0.05 gram of glucose will exactly reduce the amount of 
Fehling's solution used, and from this fact the amount of glucose 
in the entire twenty-four hour amount of urine is easily cal- 
culated. If the titration is carried beyond the proper " end- 
point " the meniscus will appear yellow instead of colorless. 

Benedict's Sugar Determination. 

Qualitative. The following application of Benedict's so- 
lution to the detection of sugar in urine is taken from a paper 



URINE 207 

by Stanley R. Benedict in the Journal of the American Medical 
Association, October 7, 1911. " For the detection of glucose 
in urine about 5 c.c. of the reagent are placed in a test-tube 
and eight to ten drops (not more) of the urine to be examined 
are added. The mixture is then heated to vigorous boiling, 
kept at this temperature for one or two minutes, and allowed 
to cool spontaneously. In the presence of glucose the entire 
body of the solution will be filled with a precipitate, which may be 
red, yellow or greenish in tinge. If the quantity of glucose be 
low (under 0.3 per cent) the precipitate forms only on cooling. 
If no sugar be present the solution either remains perfectly 
clear, or shows a faint turbidity that is blue in color, and con- 
sists of precipitated urates. The chief points to be remembered 
in the use of the reagent are (i) the addition of a small quantity 
of urine (8 to 10 drops) to 5 c.c. of the reagent, this being de- 
sirable not because larger amounts of normal urine would cause 
reduction of the reagent, but because more delicate results are 
obtained by this procedure, (2) vigorous boiling of the solution 
after addition of the urine, and then allowing the mixture to 
cool spontaneously, and (3) if sugar be present, the solution 
(either before or after cooling) will be filled from top to bottom 
with a precipitate, so that the mixture becomes opaque. Since 
bulk, and not color, of the precipitate is made the basis of a 
positive reaction, the test may be carried out as readily in 
artificial light as in daylight, even when examining for very 
small quantities of sugar." 

Benedict's Quantitative Sugar Titration. 

Make a i-io dilution of the urine and fill a 25 c.c. burette 
with the diluted sample. In a large porcelain dish place 25 c.c. 
of Benedict's Quantitative Sugar Reagent (see Appendix) and 
about 10 grams of sodium carbonate, and some pumice stone 
to prevent bumping. Heat to boiling and allow the urine to run 
in from the burette. A heavy, white precipitate is produced. 
Continue allowing the sugar solution to run into the copper, 
which is kept at a boiling temperature throughout the deter- 
mination, until the blue color has been entirely discharged. 



208 PHYSIOLOGICAL CHEMISTRY 

Then note the reading on the burette and make calculations as 
follows: 

The 25 c.c. of Benedict's solution is reduced by .05 gram of 
glucose. Therefore : 

Reading on burette __ 100 
^5 ~"*~ 

This method is preferable to the Fehling's determination 
because other substances do not react to it; a sharp end-point 
is attainable even by the inexperienced, and the titration is more 
quickly made. 

The fermentation test (Exp. 134, page 271) may also be used 
to detect the presence of sugar and, approximately, the amount. 

The fermentation test for sugar is a convenient and easily 
made qualitative test, it being only necessary to fill a fermen- 
tation tube (Fig. 26, page 271) absolutely full of urine to which 
a small portion of yeast has been added, and to allow the tube 
to stand in a warm place for several hours. Any collection of 
gas in the top of the tube will indicate the presence of sugar. 
This method may also be used as a quantitative test for sugar 
by taking two portions of the same sample, adding yeast to 
one, and using the other as a control. At the end of twenty- 
four hours, CC>2 is removed from fermented sample, the specific 
gravity of both samples is carefully taken, and the loss of density 
in the fermented sample is calculated as sugar by multiplying 
the number of degrees lost in gravity by 0.23, water being 
considered as 1000. 

The phenyl-hydrazine test may be used as a confirmatory test 
or in cases where very minute quantities are suspected. This 
test is considered about ten times as delicate as the Fehling's 
test; consequently, it may show small amounts of sugar which 
are not detected by the more rapid process. 

The optical analysis for sugar may be made with a polariscope, 
preferably constructed for use on urine. This determination 
depends upon the ability of glucose to rotate the plane of polar- 
ized light towards the right, the degree of rotation indicating 
the amount of sugar in a pure solution. Of course, allowance 



URINE 209 

or correction must always be made for the presence of any sub- 
stances which will rotate the light in the opposite direction, such 
as albumin, levulose and /3-oxy butyric acid. 

For the detail of construction and use of the polariscope, 
the student is referred to the more complete works on urine 
analysis by Ogden, Holland, or Purdy. 

ACETONE. 

Acetone may occur in the urine as a result of various patho- 
logical conditions and, according to von Noorden, they are all 
due to some one-sided perversion of nutrition. The acetonurias 
attendant on diabetes, scarlet fever, pneumonia, small-pox, etc., 
are of less practical interest to the dental practitioner than those 
which are more often overlooked by the medical profession, and 
which indicate improper diet, possibly resulting in serious mal- 
nutrition. The following points may be noted: In advanced 
stages of diabetes, acetone appears in the urine accompanied by 
diacetic acid. An increased ingestion of proteins may result 
in the appearance of acetone, in which case the direct cause is 
an " insufficient utilization of carbohydrates "* rather than the 
increase of protein. 

It should be remembered that while acetone may appear 
because of insufficient utilization of carbohydrates, the acetone 
bodies are produced from neither protein nor carbohydrates but 
are derived from the perverted metabolism of fat, as suggested 
in the chapter on Metabolism^page 230. 

Detection. Acetone may be detected in the urine by the 
production of iodoform, as described under analysis of saliva 
on page 182, but it is not in this case nearly so delicate a test 
on account of the odor and acid character of the urine. A 
more useful test is known as Legal's test and is made as follows: 
To a third of a test-tubeful of urine add a few drops of a freshly 
prepared and fairly concentrated solution of sodium nitro- 
prusside; next add two or three drops of strong acetic acid, and 
then a considerable excess of ammonia. If the contents of the 
tube are mixed by a rather rapid rotary motion without inverting 

* von Noorden's Diseases of Metabolism and Nutrition. 



210 PHYSIOLOGICAL CHEMISTRY 

or violent shaking, the ammonia will not reach the bottom of the 
tube, and the presence of acetone will be indicated by a violet-red 
band above the layer of acid liquid. If much acetone is present 
a deep violet to purple color is obtained. 

Diacetic Acid occasionally occurs in urine as an abnormal 
constituent, most commonly in advanced stages of diabetes, 
usually accompanied by acetone and j8-oxybutyric acid. It 
may be detected by adding to the urine a little ferric chloride, 
when a dark wine-red color is produced. If a precipitate of 
ferric phosphate is obtained, filter the urine and examine the 
filtrate for color. This test may be made fairly distinctive for 
diacetic acid by boiling and cooling a second portion of the 
urine previous to making the test, when the result will be nega- 
tive if the color at first produced was due to diacetic acid. 

/3-oxybutyric Acid. This substance usually accompanies 
diacetic acid, as above stated. Determinations of the quantity 
present cannot be made by any simple method. Perhaps the 
most practical method is by Bloor's nephelometer, page 166. 
See also page 229. 

BILE. 

Bile may occur in the urine as such, and may be due to path- 
ologic conditions of the liver or bile-ducts, as stated on page 153. 
The coloring matters of the bile may also occur from causes aside 
from lesions of the liver. A urine containing bile or bile-pig- 
ments is always more or less highly colored, and upon shaking, 
the foam will be of a yellow or greenish-yellow color. Albumin 
and high indoxyl accompany the presence of bile and there is 
also usually considerable renal disturbance. It may be de- 
tected by carefully adding to one-half a wine-glass of the sus- 
pected sample a few cubic centimeters of the alcoholic solution 
of iodine (tincture of iodine). A green color will be observed 
just beneath the line of contact of the two liquids (page 293). 
The test may be conveniently made by placing the iodine first 
in the wine-glass and then with a pipette introducing the urine 
beneath the iodine solution. 



URINE 211 

METALLIC SUBSTANCES. 

Arsenic, mercury, and lead are the three metals which it 
may be necessary to look for in a sample of urine. The method 
for the detection of mercury, given on page 183, is applicable 
for this purpose. 

Arsenic may be detected by the Marsh-Berzelius test (as 
given in Vol. I), after oxidizing all organic matter. The process 
may be carried out as follows: Evaporate to dryness a liter of 
urine, to which 200 c.c. of strong nitric acid has been added; add 
to the residue, while still hot, from 15 to 20 c.c. of concentrated 
sulphuric acid. This must be done in a large porcelain evapo- 
rating-dish, or else the acid must be added very slowly to prevent 
frothing over and loss of a portion of the sample. After the 
action has quieted down the whole mixture may be transferred 
to a 590 c.c. Kjeldahl flask and heat applied, gradually at first, 
and then more strongly. It will be necessary to add from time 
to time small portions of nitric acid and possibly a little more 
sulphuric acid; as the oxidation progresses the liquid in the flask 
becomes lighter in color and at the completion of the process is 
water-white, even when the temperature is increased so that 
sulphuric-acid fumes are given off. After cooling, the strongly 
acid liquid is diluted with four or five times its volume of water, 
filtered, if necessary, to remove excessive amounts of earthy 
sulphates, and is then ready for the arsenic test. 

Lead. The sample of urine to be tested for lead should 
measure at least 1000 c.c., and should be tested for iodine to 
establish the presence of potassium iodide to dissolve lead 
salts; otherwise a negative result may be obtained when lead 
is actually present and is poisoning the system. Oxidize the 
sample in precisely the same manner as when making the arsenic 
test, up to the point of diluting the strong acid solution with water; 
then, in this case, use rather less water for the dilution, allow to 
cool, and neutralize with Squibb's ammonia, acidify quite strongly 
with acetic acid, and pass H 2 S gas into the solution. It is desir- 
able to leave the solution saturated with H 2 S for at least twelve 
hours. Then filter, and without washing dissolve the precipitate 



PHYSIOLOGICAL CHEMISTRY 

in warm dilute nitric acid, evaporate the HN0 3 solution to dry- 
ness, add 5 c.c. of water, make alkaline with a drop or two of am- 
monia, and again acidify with acetic acid and add a solution of 
bichromate of potash.* Allow to stand several hours, filter off 
the chromate of lead, wash several times with distilled water, and 
lastly with H 2 S water, whereupon the lead chromate will blacken 
from the formation of lead sulphide. This stain is a superficial 
one and disappears upon standing, but when the process is 
conducted in this way it constitutes a very delicate and satis- 
factory test for lead in either urine or saliva. 

URINARY SEDIMENTS. 

The sediment which settles from a sample of urine upon 
standing consists normally of a slight amount of mucin and 
epithelial cells. It may also contain bacteria and a considerable 
variety of extraneous matter, including starch grains, various 
vegetable spores, yeast cells, fibers from various fabrics, cotton, 
wool, flax from linen, etc., diatoms, scales from insects' wings, 
and other particles which may occur as dust (see Plate VIII, 
Fig. 6; also Plate IX, Fig. 4). Under abnormal conditions 
the sediment may contain crystalline elements, including uric 
acid and urates, phosphates, oxalates, cystin, tyrosin, leucin, 
etc., also organized elements such as epithelium, renal or other 
casts (Plate VIII, Fig. 4), blood globules, pus cells (Plate VIII, 
Fig. 3), spermatozoa (Plate VIII, Fig. 2), fat, mucin (Plate VIII, 
Fig. 5), etc. Urinary sediment may be thrown down from a fresh 
specimen by the use of a centrifuge, or the urine may be allowed 
to stand in a glass tube with rounded bottom for several hours, 
when the sediment settles to the bottom by gravity. If possible 
it is best to examine sediments settled in both of these ways, as 
the centrifuge will show elements, such as small casts, that would 
settle slowly, possibly not at all, by the gravity method. On 
the other hand, the sediment allowed to settle spontaneously 
will often give a more correct idea of comparative numbers of 
the various elements observed, than when settled in a centrifuge- 

* Natural chromate of potash will precipitate copper, the acid chromate pre- 
cipitates lead only of the second group metals. 







FIG. i. 

Acid 




FIG. 3.- Pus. 

A, o! Acid, 




Fis, 5. 
and 




FIG, 2. 




FIG. 4- 




FIG. 6. 

A, 5 S C, 

I?, JE, 



URINE 213 

tube. A drop or two of formalin may be used to preserve 
urinary sediment, as suggested on page 187, but if too much of 
this substance is used, especially in urines containing high per- 
centages of urea, a compound is liable to be formed which has 
been called formaldehydurea (Plate IX, Fig. 5), which settles 
with the sediment and seriously interferes with the microscopical 
examination. This compound may form sheaf -like crystals 
similar to tyrosin and may be mistaken for crystals of sodium 
oxalate, especially when examined with a low-power objective. 

Uric Acid, Uric acid is deposited from normal urine, upon 
standing, with an excess of free acid (HC1). Urines that have 
a high degree of acidity will also produce a like deposit, and the 
finding of uric-acid crystals does not necessarily signify that the 
crystallization took place within the body, unless special care 
has been taken that the sample examined was perfectly fresh, 
although the tendency to deposit uric acid is, of course, indicated. 
The urine from which uric acid separates, as such, is usually 
rather concentrated and of strong acid reaction. These crystals 
vary in appearance (Plate IX, Figs, i and 2), but are almost 
always colored yellow to red. Colorless crystals are sometimes 
observed. They are usually quite small, but of the peculiar 
whetstone shape in which this acid usually crystallizes. The 
presence of uric acid has practically no effect upon the acid- 
ity of the sample; for, if the acid separates in a crystalline 
form, it is insoluble, and if it does not separate it is in combination 
as urates, possibly, of course, as acid urates. Uric acid exists 
normally in proportion to urea as about i to 50, but there is no 
necessary relationship between the quantities of the two sub- 
stances, and the one may be diminished while the other is in- 
creased. 

Urates. Urates may occur as crystalline or amorphous pre- 
cipitates. The crystalline urates are urate of sodium rarely, 
acid urate of sodium (Plate IX, Fig. 3), and acid ammonium 
urate (Plate VIII, Fig. i, page 213). The amorphous urates 
are of the alkaline bases, usually sodium, and are frequently 
precipitated by lowering of the temperature after the sample has 
been passed; in such cases the urine assumes a cloudy appearance 



214 PHYSIOLOGICAL CHEMISTRY 

which is cleared up by the application of heat. A sediment 
consisting of urates is usually of a pinkish color. 

Phosphates. Phosphates in the urinary sediment may be 
amorphous or crystalline. They are of the alkaline earths 
rather than of the alkaline metals, as the latter are soluble in 
both the acid and neutral forms. The amorphous phosphates 
deposit with the change of reaction from acid to alkaline, and 
usually in the form of a so-called triple phosphate of ammonia 
and magnesia (Plate IX, Fig. 6, page 214), This salt crystallizes 
in two forms. The prismatic form is the ultimate form; that 
is, if the crystallization takes place very slowly, the prismatic 
form is the one in which the salt is thrown out. If it takes 
place rapidly it may be precipitated in the feathery form, but 
this slowly changes over to the prismatic form. The acid 
phosphates may be precipitated in a form closely resembling in 
appearance the acid urates (Plate IX, Fig. 3), but may be dis- 
tinguished from them by their ready solubility in acetic acid 
and failure to produce, after solution in acetic acid, any crys- 
tals of uric acid such as are obtained from the urates. 

Acid Lactates. These are soluble salts, and are found in 
urine only by evaporation of a drop of the clear fluid and an 
examination of the residue by polarized light. When found 
in the urine, they have not the same significance as when found 
in the saliva, as in the urine they may possibly be formed from 
lactates, which indicate a faulty action of the liver, and of course 
they have no connection with tooth erosion. The lactates 
furnish evidence of similar character. 

Oxalates. Oxalates, if found in the sediment, usually occur 
as calcium oxalates. These crystals assume a variety of forms, 
as shown in Plate II, Fig. i, page 77. Sodium oxalate (Plate II, 
Fig. 4) may occur in the urine (not, however, in the sediment), 
and is detected only by evaporating a drop of the clear liquid 
and examining with polarized light. Dr. Kirk claims that a 
tendency to oxaluria may be detected in this way for a con- 
siderable time before the appearance of the oxalate of lime crys- 
tals, and hence such examination becomes a valuable aid to 
diagnosis. 







FIG, i. 




FIG. 3 . 
B, 





WIG. 2. 




FIG. 4, 
and 




FIG, 5. 

(P. L.). 



Fto. 6. 



URINE 215 

Cystin. Cystin occurs as six-sided plates. It is a com- 
paratively rare crystal, and indicates insufficient oxidation, 
particularly of the organic sulphur compounds. 

Epithelium. Epithelium from any part of the urinary tract 
may occur in the urinary sediment. In the male urine it is 
much easier to determine the character of the epithelium than 
in the female, as in the latter the comparatively large amount 
of mucous surface, from which epithelium may be gathered, 
furnishes a great variety of forms which are, of course, without 
clinical significance. The epithelium from the vagina may be 
quite readily distinguished as very large cells with small nuclei, 
lying usually in masses overlapping one another but with com- 
paratively slight density. Renal epithelium may be found as 
small, round cells, differing but slightly in size from a leucocyte. 
They may be a little larger, a little smaller, or about the same 
size. They are round and more or less granular in appearance. 

Epithelium from the bladder varies considerably, but the 
majority of cells would properly come under the general head 
of squamous epithelium, rather large and flat with a distinct 
nucleus of medium size. Epithelial cells from the neck of the 
bladder in male urine are quite typical, being round and com- 
paratively dense with a prominent nucleus. They are four 
or five times the size of a leucocyte and, in case of irritation 
at the neck of the bladder, are usually present in considerable 
numbers and of quite uniform appearance. 

Renal casts consist of molds which are formed within the 
tubules of the kidneys and retain the form of the tubules after 
expulsion into the bladder. According to Ogden the most 
probable theory of their formation is " that they are composed of 
coagulable elements of blood that have transuded into the renal 
tubules, through pathologic lesions of the latter, and have there 
solidified to be later voided with the urine, as molds of the 
tubules." Casts are termed blood casts, pus casts, epithelial 
or fat casts, as any of these elements may adhere with more 
or less profusion to the cast itself. Pure hyaline casts are pale, 
perfectly transparent cylinders, with at least one rounded end 
which can be plainly seen, and may occur occasionally in urine 



2l6 PHYSIOLOGICAL CHEMISTRY 

from perfectly healthy individuals. Fibrinous casts are highly 
refractive and when seen by white light are of a yellowish color 
and indicate acute renal disturbance. Waxy casts resemble 
the fibrinous casts as regards density, but they have no color, 
and usually indicate advanced and serious stages of kidney 
disease, while the presence of fibrinous casts has no necessarily 
serious significance. 

Blood and Pus are readily recognized under the microscope 
after a very little practice. The blood disks are circular and 
show a characteristic biconcavity in the alternate shading of 
the edge and center by slight changes of focus. The red cor- 
puscles usually show a shade of color by white light. The pus 
corpuscles or leucocytes are larger than the red corpuscles, and 
are granular in appearance. Treatment with acetic acid de- 
stroys the granular matter and brings into prominence the 
cell nuclei, two or three in number. If the leucocytes are free 
and scattered they should not be regarded as pus but should be 
reported simply as an excess of leucocytes; if they are very 
numerous and occur in clumps they constitute pus. 

Spermatozoa. Occasional spermatozoa may be found in 
sediment from either male or female urine and are without 
clinical significance. If persistent and in considerable numbers, 
seminal weakness is indicated (Plate VIII, Fig. 2, page 213). 

Fat occurs in urinary sediment as small globules, highly 
refractive and varying greatly in size. They are frequently 
adherent to cells or to casts. Fatty casts indicate a fatty de- 
generation, which may or may not result from chronic disease. 
Fat may be demonstrated by staining with osmic acid, which 
is reduced by the double-bonded fatty constituent (olein), 
leaving a black deposit which stains the globule. 

Mucin appears in the sediment as long and more or less 
indistinct threads. An excessive amount usually indicates irri- 
tation of some mucous surface. The source would have to be 
determined by other more characteristic elements (Plate VIII, 

Fig- 5). 

The salts which may be obtained by evaporation of a drop of 
clear urine and detected by the micropolariscope are similar 



URINE 217 

to those occurring in the saliva; sodium oxalate is probably 
most frequently found. If the gravity is above normal the 
urea often crystallizes, making it somewhat difficult to pick 
out the abnormal crystalline constituents. Phosphates are 
also usually observed, but these crystals are large and as a rule 
prismatic, not easily mistaken for anything else. 

RECORDING RESULTS. 

As stated at the beginning of the chapter on urine, our object 
has been the study of this secretion from the standpoint of 
general metabolism, rather than with a view to differentiating 
various forms of renal disease; and while it is important that the 
presence of renal disease should be recognized, its further in- 
vestigation constitutes a proper study for the physician rather 
than for the dentist. When such conditions are found to exist 
a patient's physician should be apprised of the fact. 

Uniformity of method in making out report cards is desirable 
although not absolutely necessary for the best class work; 
hence a few suggestions as to the use of the following blank. 
If no test is made, make no entry whatever on the blank. This 
permits the use of a dash, " ," to indicate a diminished (less 
than normal) quantity. If a substance is present in normal 
quantity use a capital " N," if increased above normal amount 
use " + " If absent use abbreviation " abs.," never the dash 
or minus sign. Observance of this method greatly facilitates 
correction of the report slips. 



218 



PHYSIOLOGICAL CHEMISTRY 



NO. 


Urine Analysis. 

NAME 




DATE 


PHYS. 




24 hr. Amt. 


Bracketed numbers are 
Sp. Gr. % 


average normal 
Grams 


React 


Urea 


(2.0 


(30. 


Color 


E. Phos Uric Ac 


(.033 


(0.6 


Ind 


A. Phos Ammonia 


(.050 


(o.75 




Chlor 


(0.67 


(10. 


Albumin 


Phos. Ac. 


(0.18 


(2.7 


Diac. Ac. 


Acetone Sugar 






Uric Ac. 


to Urea- 1 to 



Sediment 



CHAPTER XX. 
METABOLISM. 

Metabolism is the building-up and tearing-down process tak- 
ing place constantly in the living organism. Food is ingested, 
digested, absorbed, and by various complicated means eventually 
transformed into the living tissue of the organism or burnt up as 
fuel by the system. The term anabolism is applied to the con- 
structive metabolic process, while the destructive process and 
formation of waste is termed catabolism. 

CARBOHYDRATE METABOLISM. 

We have seen that the digestion of carbohydrates is accom- 
plished for the most part by means of five enzymes: first, the 
ptyalin of the saliva, which reduces starch to maltose; then the 
amylase of the pancreatic juice, which continues the action 
started by the saliva; and finally maltase, sucrase, and lactase 
of the intestinal juice, which convert the disaccharides to the 
simple sugars. Carbohydrates, then, are absorbed usually in the 
small intestine as glucose, levulose and galactose.* The blood 
circulating through the tissues carries these simple sugars to the 
cells where they are needed. 

We may believe that part of the sugar absorbed is almost 
immediately oxidized, while the remainder is stored by the system 
until it is required to satisfy the energy demands of the body. 
The oxidation of the glucose takes place in the muscle cells where, 
as was indicated under muscle metabolism, page 141, various 
substances are produced which may be considered as related 
directly to the oxidation. Lactic acid and alcohol are un- 
doubtedly intermediate products finally resulting in the pro- 
duction of carbon dioxide and water. 

The excess of glucose is converted by an endocellular enzyme, 

* There is considerable evidence for the belief that maltose is capable of absorp- 
tion in slight amounts. 

219 



220 PHYSIOLOGICAL CHEMISTRY 

glycogenase, into glycogen, and stored by the system as such. 
The liver seems to have a capacity for forming glycogen and it 
has been believed that this change, from glucose to glycogen, 
takes place mainly in this organ. At present, however, some 
authorities are inclined to believe that the conversion of glucose 
to glycogen is not localized and that the liver may act merely 
mechanically in holding back the sugar, since other substances 
seem to be held back also. The size of the liver and the natural 
relationship of its blood supply to the blood loaded with carbo- 
hydrate material from the intestine may also be factors ac- 
counting for the large quantities of glycogen found in the liver. 

Although glycogen is the storage form for carbohydrates, it is 
important to remember that during transportation, that is, while 
the carbohydrate is in the blood, and when oxidation takes 
place, it is in the form of glucose. Glycogenases seem to be 
abundant, and the conversion of the carbohydrate from one 
form to the other apparently takes place with the greatest of ease. 

Directly after absorption, and perhaps under some other 
conditions, levulose and galactose may be present in the blood 
in detectable quantities, but the normal blood sugar is always 
glucose. It is present to the extent of 90-110 mg. per 100 c.c., 
and these figures are given as the normal blood sugar level. Folin 
has stated that if the sugar content of the blood rises to 160 mg. 
per 100 c.c. there is a similar rise in all the body tissues; if it 
rises to between 160 and 170 mg. per TOO c.c. glucose will appear 
in the urine. 

The amount of carbohydrate which can be ingested in twenty- 
four hours without an appearance of sugar in the urine varies 
considerably with the individual; that is, each person seems to 
have what is termed a carbohydrate tolerance. These facts have 
been represented diagrammatically in the accompanying illus- 
tration, Fig. 16. * 

If the individual ingests more carbohydrate material than can 
be stored as glycogen, the system will convert the excess into fat; 
if there is a further excess, that is, if the carbohydrate tolerance 
is exceeded, the sugar is excreted by the kidney and a condition 
of glycosuria results. 



METABOLISM 



221 



Glycosurias may be divided into two main classes:* namely, 
those caused by a diseased pancreas, diabetes mellitus; and those 
caused merely by an over-ingestion of carbohydrate material, 
producing a renal glycosuria or an alimentary glycosuria. 




FIG. 16. 

The tank represents the blood sugar level. Outlet B represents the minimum 
requirement; when the blood sugar rises to C it is stored by the system as glycogcn, 
when it rises to D it is stored as fat, while if it rises to outlet E the carbohydrate 
tolerance is exceeded and sugar will appear in the urine. 

Concerning the role of the pancreas in carbohydrate meta- 
bolism, Stiles says, " A function of this organ even more necessary 
than its digestive contribution is the delivery to the blood of the 
hormone which makes it possible for the muscles, including the 
heart, to oxidize sugar. Abundance of this hormone insures a 
high tolerance for sugar; want of it produces, according to the 
degree of the lack, a low tolerance or substantial inability to make 
use of carbohydrate." 

In cases of true diabetes mellitus and in cases of over-ingestion 

* Another relatively unimportant but interesting class is the so-called emotional 
glycosuria. A glycosuria is produced in nearly all cases of intense emotion, 
probably because of some relationship between the secretion of adrenalin and 
the utilization of carbohydrate in the system. 



222 PHYSIOLOGICAL CHEMISTRY 

of carbohydrate, the blood sugar and the urine have been carefully 
studied and the following facts have been observed. 

After a large intake of glucose the normal individual will show 
a ^decided rise of blood sugar, 140 mg. per 100 c.c. perhaps, a 
condition of hyperglycemia. Shortly, however, the blood sugar 
will drop to the normal level, then become subnormal, and 
usually two or three hours after ingestion will be perfectly normal 
again. The subnormal level, hypoglycemia, may be explained 
on the ground that after a large intake of glucose there is the 
least possible need for transportation of glucose in the tissue. 
In the diabetic individual, on the contrary, there will be a decided 
rise but the return to normal will be very, very slow. These 
results, plotted, give the following curves (Folin). 




Normal Level 



Normal Diabetic 

FIG. 17. 

Further, if the normal individual ingests starch instead of 
glucose, a slight increase only is observed in the blood-sugar 
level; while with the diabetic, starch absorption gives a similar 
rise to the sugar. 

The urine of a diabetic patient will contain sugar in appre- 
ciable amounts either before or after eating. In cases of renal 
glycosuria, sugar is always found after eating but just before 
meals it will frequently disappear entirely. Renal glycosuria 
may be said to exist in those individuals who naturally have a 
low renal threshold for glucose, the renal threshold being the 
ability of the kidney to retain sugar. It is obvious that the 
carbohydrate tolerance of the individual is more or less de- 
pendent on this so-called 'renal threshold. Whether or not 
prolonged renal or alimentary glycosuria, produced by ingesting 
more carbohydrate than the system can take care of, will result 
in pancreatic diabetes is an open question. From a conservative 



METABOLISM 22$ 

viewpoint, however, the author would advise a lessened intake 
of carbohydrate food for individuals who show a tendency to- 
ward any kind of glycosuria. 

When the system is unable to utilize carbohydrates properly 
a perverted fat metabolism is nearly always sure to follow, 
with the production of acetone bodies formed by the insufficient 
oxidation of butyric acid (page 230). This, of course, accounts 
for the frequent presence of these substances in diabetic urine. 

The treatment* for diabetes has been modified recently. At 
present the diabetic specialist tries to discover just how much 
carbohydrate the individual can properly assimilate (it has been 
found that even in the most diseased condition of the pancreas 
a small amount of carbohydrate can be utilized) and then sees 
that the diet of protein and fat is in the right proportion to that 
amount of carbohydrate. The individual will usually lose 
weight, but this method of treatment has been found more 
beneficial than the former increase of protein and fat and the 
withdrawal of practically all carbohydrate food. 

This diet modification has been the result of a study of the 
relationship found to exist between the amounts of nitrogen and 
the amounts of sugar excreted. There has been found to be a 
direct ratio between them, the amount of sugar increasing if the 
nitrogen is increased, even though no carbohydrate is being fed, 
It has been determined that if the proportion of sugar to nitrogen 
excreted bears the ratio 3.65-1, the maximum amount of sugar 
is being excreted by the kidney. If this proportion is obtained 
with a patient eating no carbohydrate food it is usually called 
the fatal ratio, for it shows that the system is capable of utilizing 
almost no carbohydrate and is forming sugar from protein to an 
extent of 58 per cent. 

PROTEIN METABOLISM. 

It will be remembered that in the study of acids, amino acids 
Were considered important because of their relation to proteins. 

* Very recently a new preparation "insulin," obtained from the islands of 
Langerhan, has been found to be of great value in the control of the blood 
sugar level and hence in the treatment of diabetic. 



224 



PHYSIOLOGICAL CHEMISTRY 



The conversion of the complex protein molecule to amino acids 
takes place during digestion and may be briefly represented as 
follows : 



Substance. 


Enzyme. 


Place of digestion. 


Products. 


NATIVE PROTEIN 

Milk 


Pepsin 
Rennin* 


Gastric juice 

n 


Metaproteins 
Proteoses 
Peptones 
Coagulated protein 


Products of peptic 
digestion 
Milk 


Trypsin 
Chymosin* 


Pancreatic juice 


Proteoses 
Peptones 
Coagulated protein 


Products of tryptic 
digestion 


Erepsin 


Intestinal juice 


Peptones 
Peptides 
AMINO ACIDS 



* Rennin and chymosin both act on milk by precipitating the casein. The precipitated casein 
is then split by the other proteolytic enzymes. 

These amino acids are absorbed directly into the blood stream, 
and as the blood circulates certain of them are taken up by the 
body cells and are resynthesized into the living protoplasm. 

How this resynthesis takes place is entirely unknown, but it 
has been shown that while some amino acids are absolutely 
essential to the anabolic process others act as foreign substances. 
The essential amino acids undoubtedly differ with different 
species of animals, and probably the demands of one kind of 
tissue vary with those of another in the same animal. As a rule, 
the amino acids derived from the protein obtained from animal 
sources are utilized to a larger extent by man than those derived 
from vegetable protein. This may be due to the fact that animal 
protein yields a much larger variety of amino acids than does 
vegetable protein. 

In contrast to carbohydrates, proteins cannot be stored as 
such by the system, unless possibly in very small quantities. 
They may be converted into carbohydrate and fat, but this proc- 
ess is a difficult one and, as a rule, under normal conditions that 
which is not needed for actual repair of tissue is immediately 
converted into substances which are excreted. It follows, then, 



METABOLISM 225 

that the protein intake should not be greatly in excess of that 
necessary for the formative metabolism of the body cells. 

The excess of amino acids is in large part de-aminized by 
means of de-aminizing enzymes, and is converted chiefly into 
ammonia and subsequently urea. 

This conversion into urea takes place largely in the liver, 
although probably the action is not localized. Various theories 
as to how the change takes place have been suggested, and a 
few of them will be mentioned. Urea is probably formed in a 
number of ways, more or less dependent on other conditions 
present. 

After de-aminization of the amino acids takes place, some of 
the ammonia produced may combine with carbon dioxide 



2NH 3 +CO 2 -->0==C 

NH 2 

Ammonium carbamate may be formed, and, by oxidation, 
followed by reduction, may yield urea (Dreschol) 

NH 2 COONH 4 +0 -> NH 2 ~0- 

I 
NH 2 

> NH 2 



;C 
NH 2 NH 2 

Cyanamide may be first produced, and by hydrolysis may yield 
urea (Salkowski) 

NIL 

NH 2 C^N+H 2 O -> ; C = O 

NHa 

Excess of ingested protein means an excess of amino acids, 
urea, ammonia, and other products of nitrogenous metabolism, 
which must be eliminated. Young people, or older ones in 
robust health, can usually take care of this excess easily, but too 
often the system becomes overtaxed with the excess and harmful 
results follow. If these end-products are not properly eliminated, 



226 PHYSIOLOGICAL CHEMISTRY 

more ammonia may combine to form ammonium salts, resulting 
in a rise of ammonia nitrogen in urine and saliva and a lessened 
amount of urea nitrogen. The carbon dioxide may then convert 
neutral salts to acid salts, increasing the acidity of both urine 
and saliva. The amino acids not properly absorbed or elimi- 
nated are liable to undergo putrefaction in the intestine, giving 
rise to various toxic substances which aggravate, if they do not 
cause, skin disease, nephritis, etc. As tryptophane is a consti- 
tuent of nearly all protein foods, the amount of indoxyl obtained 
from tryptophane in the urine is an indication of this intestinal 
putrefaction. See page 200. 

In addition to the protein metabolism already discussed, the 
conversion of the purine base derivatives into uric acid is also 
taking place. This type of metabolism occurs both in the meta- 
bolism of the cell itself (endogenous uric acid) and as a result of 
the ingestion of foods containing the purine nucleus (exogenous 
uric acid). The uric acid found in the system is chiefly derived 
from nucleic acid, and a few facts regarding the chemistry of 
nucleic acid may be worthy of mention. 

Nucleic acid, as the name implies, comes from the cell nuclei 
of plant and animal material. There is a slight difference in the 
composition of that obtained from plant tissue and that from 
animal sources, but in general they both contain a molecule of 
phosphoric acid, a carbohydrate group, purine bases, and pyra- 
midine bases. During metabolism the purine bases are con- 
verted into uric acid. 

Both plant and animal nucleic acids are insoluble in cold water 
and soluble in warm water, and with an acid mixture are precip- 
itated with proteins. A precipitate formed in this way may be 
a salt of the acid, but in general the term nucleo-proteins has 
been used to designate this class of substances. See page 114. 

These nucleo-proteins, which are present in many protein foods, 
particularly glandular meat, tongue, and beef, are hydrolyzed 
during the process of gastric digestion into protein and nuclein. 
The latter, which may be considered as a simpler nucleo-protein, 
splits again during pancreatic digestion into protein and nucleic 



METABOLISM 227 

acid. During intestinal digestion the nucleic acid is further 
broken up into its constituent parts referred to above. 

In discussing muscle metabolism, creatinine was referred to 
as a product of the endogenous metabolism of the living cell. 
Its production is similar to that of the endogenous uric acid, but 
in direct contrast to that of the exogenous uric acid, urea, am- 
monia and amino acids, which are products of food metabolism. 
This fact, demonstrated by Folin, accounts for the inverse ratios 
found to exist between the excreted creatinine nitrogen and 
other protein nitrogen that is, on a low protein diet Folin has 
shown that creatinine nitrogen increases in proportion to the 
total nitrogen eliminated, while other protein nitrogen decreases. 
In consequence, the creatinine nitrogen in the body fluids is 
remarkably constant for the individual in normal conditions and 
quite independent of protein intake. Although it has been 
shown that an increase of creatinine nitrogen in the urine does 
not follow from a high protein diet, an increase has been observed 
in various diseases, as typhoid fever, tetanus, pneumonia. In 
cases of anemia, advanced nephritis, and paralysis, the creatinine 
has been found to decrease. 

Protein metabolism, as we have considered it, has been wholly 
from the angle of nitrogen metabolism. A study of nitrogenous 
metabolism is the best index of protein metabolism, because 
proteins contain a fairly uniform amount of nitrogen while there 
is no uniformity in the amounts of other elements present, such 
as sulphur. Also, simpler and more accurate methods for nitro- 
gen determinations are available. However, since sulphur is a 
common constituent of a large group of proteins, we shall add a 
few facts regarding its metabolism. 

In general, the sulphur is split off from the protein and becomes 
hydrogen sulphide which is oxidized to sulphuric acid. The 
sulphuric acid eliminated may be divided into that which com- 
bines with inorganic elements present and is thrown off as in- 
organic sulphates, and that which unites with organic com- 
pounds as phenol and indol and is classed as ethereal sulphates. 
Then, as a third way in which sulphur is eliminated, may be 
added unoxidized or neutral sulphur, which corresponds closely 



228 PHYSIOLOGICAL CHEMISTRY 

to the creatinine of nitrogen metabolism. It seems to come from 
cell metabolism. 

The inorganic sulphates compose the largest part, while the 
ethereal sulphates are of particular interest as they are the prod- 
ucts of the excess amino acids and putrefying proteins in the 
intestine combined with sulphuric acid. (Page 201.) 

METABOLISM OF FATS. 

In the intestine, by action of the pancreatic lipase and the 
intestinal lipase, the fats are split into fatty acids and glycerol, 
and as such they are absorbed. The absorption is greatly helped 
by the presence of the bile salts. In part at least they are re- 
sorbed with the fatty acids. After the absorption, a resynthesis 
between the glycerol and fatty acid takes place in the cells of the 
intestinal wall, forming neutral fat. What happens to part of 
this neutral fat seems to be unknown, but about 60 per cent is 
carried, in a state of fine emulsion, in the lymphatics up to the 
jugular vein, where it enters the blood stream. According to 
Taylor, the neutral fat formed in this way is not the fat charac- 
teristic of the species, but is the same as that ingested. This 
statement he demonstrates by feeding a starving dog mutton 
fat, whereupon the dog lays on the fat peculiar to sheep, instead 
of making dog fat. As the neutral fat passes into the blood 
stream it apparently enters into a complex combination, in 
which state it is soluble and diffusible. In this condition it is 
carried to the tissues which need it, and any excess is stored in the 
fat reservoirs of the body. If it is stored directly, as in the case 
of the dog cited above, it will be of the same kind as that ingested; 
if further splitting takes place and the fat is resynthesized from 
its elemental constituents derived either from fat or from sugar, 
human fat or fat characteristic of the species is produced. This 
in turn may combine in various ways, forming some of the com- 
plex lipoids present in the body tissues. 

Fat is the fuel of the human machine, and by its oxidation 
heat is produced. The combustion of fat seems to take place 
first by hydrolysis of the fat into fatty acids and glycerol, and 



METABOLISM 



229 



then by oxidation of the fatty acids and conversion of the glycerol 
into glucose. 

In the process of oxidation the acid seems to split each time at 
the j8-carbon atom : 



CH 3 


CH 3 


CH 3 


1 


1 


1 


(CH 2 ) 14 


(CH 2 ) 14 


(CH 2 ) 14 


1 +0 


-> 1 +O - 


-> 1 


CH 2 


CHOH 


c=o 


1 


1 


1 


CH 2 


CH 2 


CH 2 


1 


1 


1 


COOH 


COOH 


COOH 


Stearic Acid 






CH 3 


CH 3 




1 


1 




(CH 2 ) ]4 


(CH 2 ) 14 





+H 2 



I +2O 2 - I +2C0 2 +H 2 O 

C = COOH 

I Palmitic Acid. 

CH 2 
I 
COOH 

The oxidation continues as shown above, yielding carbon dioxide 
and water as end-products until butyric acid is formed. This by 
oxidation gives diacetic (aceto-acetic) acid, which in turn is 
oxidized to formic acid and finally to carbon dioxide and water. 
At the same time a small part of the diacetic acid, through loss 
of carbon dioxide, is converted into acetone: 



CH 3 

I 

CHOH 
I +0 

CH 2 

I 
COOH 

0-oxy-butyric acid 



CH 3 

I 

c=o 

I +H 2 O -> 2 CH 3 COOH + 30-2 - 
CH> 

I 2HCOOH+ 2 CO2+2H 2 O 

COOH | 

Diacetic acid C0 2 +H2O 



230 PHYSIOLOGICAL CHEMISTRY 

Then to a limited extent 

CH 3 

I CH 3 

C = O I 
I -C0 2 -> C = O 

CH 2 I 

I CH 3 

COOH Acetone 

Under normal conditions by far the greater portion of the 
diacetic acid is oxidized, as shown by the first reaction. When 
there is insufficient oxidation in the system, or when the meta- 
bolism is perverted as in diabetes, a greater proportion of diacetic 
acid forms acetone, and acetone is excreted in the urine. The 
normal oxidation of diacetic acid may be considered as in some 
way interdependent upon the normal oxidation of glucose. 



FOODS. 

We have discussed in considerable detail the composition of 
body fluids and tissues, and have found them to contain protein, 
carbohydrate and fat, salts and water. The food must of 
necessity, then, supply at least these substances; and as a matter 
of fact we know that two additional food substances are essential 
to the proper utilization of those mentioned, the vitamines to 
produce growth and maintain health, and a certain amount of 
more or less indigestible material classed as bulk, which serves 
to prevent undue concentration of the nutritive portion. We 
may then classify food substances according to the purposes they 
serve as follows: 

Proteins Tissue Repair 

Carbohydrates Energy 

Fats Heat 

Water - Distribution during Metabolism 

Mineral Salts Regulation and Activation 

Vitamines Growth 

Bulk Distribution during Digestion. 

We have suggested the dual nature of metabolism, resulting 
in the maintenance of heat and the repair of tissue, but we have 
come to accept the measure of food value as expressed in terms 
of heat production alone. This method may not be ideal, but 
as yet we have no unit of value which will measure the usefulness 
of all kinds of food material. The unit generally used is the 
calorie, which may be defined as the degree of heat necessary to 
raise one kilo of water one degree centigrade, and is a thousand 
times as great as the small calorie (seldom used). 

Of the above-named groups the first three contain the foods 
which furnish calories. The caloric value of fats is higher than 
that of the other two, the combustion of i gram yielding a heat 
equivalent of 9.3 calories, while a gram of either pure protein or 
pure carbohydrate will furnish only 4.1 calories. These figures 
are not absolutely accurate, because of slight discrepancies be- 

231 



232 PHYSIOLOGICAL CHEMISTRY 

tween the combustion of metabolism and the combustion of the 
calorimeter, but they are accepted as the basis for computation. 

The amount of these foods required differs with the individual, 
depending primarily on the age, sex, weight and work of the 
person. An average adult male doing average work, neither 
wholly sedentary nor wholly muscular, will require perhaps 2500 
calories per day. This should be made up of a " balanced " 
diet consisting approximately of 80 grams of protein, 120 grams 
of fat, and 300 grams of carbohydrates. The digestibility and 
adaptability of food should also receive careful attention, but 
as this is largely a matter of individual peculiarities tables and 
rules are impractical. During the period of growth the body 
naturally demands a somewhat greater proportion of protein, 
as new tissue is being formed constantly, in addition to the 
waste which is being repaired. 

In considering the caloric value of food, an essential point, and 
one frequently overlooked, is that some foods furnishing an 
equal number of calories vary widely in their real nutritional 
value to the individual. This is particularly true of the protein 
content of certain foods. For example, 63 per cent of the protein 
of milk is available, while with cereal protein only 20 to 26 per 
cent is available. 

Although not yielding many actual calories, the other con- 
stituents of the diet as grouped under the remaining four headings 
are hardly less important than those having caloric value. 

As just suggested, among the sources of protein food, which is 
essential for tissue building, we may consider milk as one of the 
most important. During the first few months of life it is an 
amply sufficient source not only of protein but of all other foods. 
In adult life a milk diet is hardly sufficient for the normal indi- 
vidual, and proteins from other sources must be used. 

Food proteins may be classed as being derived from two 
sources, animal and vegetable. As already suggested above 
those derived from animal sources are much better adapted to 
human needs than those from vegetable sources, because of the 
incomplete character of the amino acid content of many of the 
vegetable proteins. To-day we believe that the human organism 



METABOLISM '233 

cannot synthesize many of the most important amino acids, 
(page m), and if the protein is lacking in these it follows that 
the system will suffer in consequence. Therefore, contrary to 
the vegetarian's ideas, a certain amount of animal protein is 
very desirable for the adult. However, an excess of protein 
is not desirable, and as has already been shown on page 226, 
when protein metabolism was discussed, such excess may give 
rise to conditions of high acidity and high uric acid and the pro- 
duction of toxins. 

Little need be said about the sources of our carbohydrate food. 
The cereal grains and vegetable tubers furnish the largest 
quantity; cow's milk contains about 4 per cent, human milk 
about 6 per cent of lactose. 

Fats are obtained from both animal and vegetable sources and, 
as in the case of proteins, the fat from milk, eggs, and other 
animal sources is rather more desirable for the organism. With 
fats, however, the advantages lie not in the actual composition 
of the fat but in the fact that those fats mentioned above contain 
the fat-soluble vitamines. 

Mineral Salts. A well-balanced diet will furnish the proper 
amounts of mineral solids (excepting perhaps sodium chloride); 
btlt all diets are not balanced and it is well to know what part the 
various salts have in maintaining the health of the individual. 

Sodium chloride is essential to digestion. It has been re- 
peatedly demonstrated that if sodium chloride is withheld 
hydrochloric acid will not enter the stomach. Excess of sodium 
chloride may cause irritation or place an undue strain upon weak 
or diseased kidneys and in such cases should be avoided; on the 
other hand, acidosis usually results from a salt-free diet. 

Potassium salts are said to keep the tissues soft and pliable, 
to prevent hardening of the arteries, etc., but potassium salts 
may cause a diminution of necessary sodium according to Bunge 
(Physiologic and Pathologic Chemistry, 2nd Edition), who says 
that potassium salts will react with sodium chloride in the system, 
forming potassium chloride and undesirable organic sodium 
salts, both of which are eliminated by the kidneys, and thus 
cause loss of sodium. 



234 PHYSIOLOGICAL CHEMISTRY 

Tibbies quotes Cahn in Zeit. f. Physiol. Chem., in practically 
the same statement. 

Calcium salts. See Vitamines. 

Magnesium occurs generally distributed in the system, the 
bones containing about one per cent. By increasing the amount 
of magnesium ingested, the percentage in the bone may be 
increased but it does not take the place of calcium. The com- 
pounds of magnesium are generally more soluble than those of 
calcium. Magnesium oxide, as milk of magnesia, is used exten- 
sively as an antacid. An excessive amount, however, may act 
in removing necessary calcium in just the same way that potas- 
sium acts in removing sodium, as indicated by the following from 
Pickerills' 'Prevention of Dental Caries and Oral Sepsis/ page 144. 

" Weiske's experiments also support these findings. For 
instance, of two rabbits, one received one gram CaCO 3 daily in 
addition to its food; the other one gram of MgCOs for three 
months. The rabbits were then killed, and it was found that, 
although they were of equal body-weight, the total weight of 
the bones (dried and fat-free) in the first rabbit exceeded that of 
the second rabbit (77.45 grams: 69.52 grams); and, further, that 
the amount of organic matter in the bones of the MgC0 3 rabbit 
was in excess of that in the CaCO 3 rabbit." 

Iron is an essential constituent of blood, derived from food, 
and perhaps more than in the case of any other mineral con- 
stituent, it is necessary for iron to be taken in natural organic 
combination. 

Phosphates are essential for the development of all cellular 
tissue. Phosphates are credited with preventing the deposition 
of uric acid by the reaction given on page 59, also with keeping 
calcium oxalate in solution. Phosphate acts beneficially in the 
bowels by slightly stimulating the peristaltic action. 

Iodine occurs in the ductless glands, and is apparently neces- 
sary for their best development, although this fact has been 
seriously questioned. 

It is impracticable to give tables of food composition, but the 
following may be noted : 

Strawberries, beans, and potatoes are rich in potassium 



METABOLISM 



235 



compounds; beets, spinach, turnips, and cherries are rich in 
sodium salts; milk, oranges, turnips, and parsnips are rich in 
calcium oxide; almonds and walnuts are rich in magnesiuni 
oxide; carrots and rice are rich in iron; meat, cheese, beans, 
eggs, and wheat are rich in phosphates; cocoa powders, rhubarb, 
and spinach, are rich in oxalates. 

Vitamines. There are recognized to-day four very important 
food constituents, which are called vitamines for the want of a 
better name. The substances are vital in their importance, but 
it is very doubtful if they contain nitrogen in any form to warrant 
the use of " amine " as part of the name. The term " food 
accessories " has been sometimes used. Three of these substances 
are. specifically known as fat-soluble A, water-soluble B, and 
water-soluble C, and a fourth vitamine has recently been dis- 
covered by E. V. McCollum. The fat-soluble A vitamine, first 
recognized by McCollum and 
Davis, occurs in cod-liver oil, 
yolk of egg, milk-fat (butter), 
to a less extent in other 
animal fats, and in slight 
amounts in vegetables and 
cereals. It is anti-rachitic 
and essentially growth-pro- 
moting in its action. 

The water-soluble B vita- 
mine is antineuritic and 
occurs in fresh green vege- 
tables, yeast, milk, whole 
cereal grains, and to some 
extent in lean meat. 

The water-soluble C is the 
antiscorbutic vitamine and 
occurs in the juice of citrus 
fruits, particularly oranges, 
lemons, and limes. It is also found in considerable quantity in 
fresh cabbage and tomatoes. 

Water-soluble C is much more easily destroyed by heat than 





Permission Dr. P. R. Howe. 

FIG. 18. 

A and B were twin-sister guinea-pigs. 
Water soluble C was withheld from the 
diet of A while B had an unrestricted 
diet. The illustration shows the stunted 
growth of A. 



236 PHYSIOLOGICAL CHEMISTRY 

either of the other two. Canned tomatoes, however, contain the 
vitamine in such proportion that they are usually recommended, 
in absence of fresh orange juice, for infant feeding. 

Dr. Percy R. Howe has shown the intimate relationship be- 
tween this antiscorbutic vitamine and the proper utilization 
of calcium in the formation of teeth and bone and its consequent 
importance in the prevention of caries. 

Dr. Howe found that he could produce, at will, decalcification 
or recalcification of the bones of guinea pigs, by simply with- 
drawing or restoring vitamines in the diet, calcium salts always 
being given in abundance. Fig. 19. 

Hart-Steenbock and Hoppert* have shown (/. Biol. Chem., 
September, 1921, page 33) that the antiscorbutic vitamine, as 
contained in orange juice, fresh green vegetables, etc., is without 
effect on calcium metabolism. We must therefore regard Dr. 
Howe's experiments as showing that lack of antiscorbutic vi- 




Permission Dr. P. R. Howe. 

FIG. 19. 

A indicates softened, decaying bone produced on a diet lacking in water soluble 
C. B indicates the new calcification formed on top of the softened bone when 
the water-soluble C vitamine is included in the diet. 

tamine produces non-calcification only as it allows the develop- 
ment of scurvy. 

Hart-Steenbock and Hoppert have also shown in the above 
article that fresh green oats, or oat-hay dried out of direct sun- 
light, and cod liver oil will increase calcium metabolism. 

* These experiments were performed with goats, animals naturally immune to 
scurvy, and the effect of the vitamine was thus studied independently of the 
disease. 



METABOLISM 237 

While the water-soluble C vitamine has a marked influence on 
calcium metabolism of animals (including human) subject to 
scurvy, it has also been shown (Bogart and Trail, /. BioL Chem. 
LIV, October, 1922, page 39) that yeast or pure butter-fat 
(water-soluble B and fat-soluble A) added to basal diet would 
decrease calcium excretion, i.e., aid in retaining calcium in the 
system. On page 386 of the above Journal, Bogart and Kirk- 
patrick conclude that calcium is more apt to be retained on a 
base-forming diet than on either a balanced or an acid-forming 
diet. 

In connection with calcium metabolism it is worthy of note 
that " children do not seem to utilize the calcium of vegetables as 
efficiently as they do that of milk. Calcium balances were more 
variable and always less favorable when vegetables replaced 
about half the milk as sources of calcium. " This paragraph is 
from " Calcium and Phosphorus Metabolism in Childhood/ ' by 
H. C. Sherman and E. Hawley (/. BioL Chem., August, 1922, 
page 398). 

Dr. E. V. McCollum has recently shown that the vitamine 
most essential to the proper utilization of calcium is neither A, B, 
nor C, but a fourth vitamine, D, occurring for the most part 
with the fat-soluble A but not identical with it. This, of course, 
raises a question as to whether the fat-soluble A vitamine pos- 
sesses antirachitic properties. Dr. McCollum found this fourth 
vitamine to be present in considerable quantities in cod-liver oil, 
in lesser quantities in butter-fat and in small quantities in cocoa- 
nut oil, differing in this last source from fat-soluble A, which does 
not occur in cocoanut oil. 

Bulk. Unless the diet includes considerable bulk un- 
digested cellulose the material in the intestine becomes too 
concentrated, peristaltic action is decreased, and the action of 
enzymes is retarded. This condition favors the production of 
toxic substances in the intestine and gives rise to all the evils 
resulting from constipation. 

To furnish bulk, fruit, vegetables as lettuce, spinach, carrots, 
celery, etc., are advised and such cereals as contain considerable 
bran. 



238 PHYSIOLOGICAL CHEMISTRY 

A well-balanced diet and sufficient out-door exercise are two 
of the greatest factors contributing to health. The diet correct 
for one individual may not be correct for another, but in general 
the diet most beneficial to the average person is one low in 
protein and rich in carbohydrate foods, with an abundance of 
vegetables, fruits and milk. 

ACID-FORMING AND BASE-FORMING FOODS. 

One of the reasons for the above recommendations is the 
desirability of producing a tendency toward alkalinity in the 
system rather than a condition favoring acidosis. Most vege- 
tables and fruits are alkaline-forming foods; that is, the final 
products of metabolism are alkaline. On the other hand, meats, 
bread and pastries are acid-forming. Prunes and cranberries 
are two fruits which do not follow the general rule but are acid- 
forming. This is because they contain benzoates which give 
rise in the system to hippuric acid. One of the most strongly 
alkaline-forming foods is potatoes. 



CHAPTER XXI. 

RELATION OF SYSTEMIC TO ORAL CONDITIONS AND 
THEORIES OF TOOTH DECAY. 

The establishment of relations between oral and systemic 
conditions is, of course, the goal toward which the dentist's study 
of physiological chemistry should be directed. 

It has been too much the practice to study a single relation 
and jump at conclusions without regard to co-relation of factors 
which may not appear to be closely allied but which nevertheless 
exert important influences. Witness the effort to establish the 
relationship of tartar deposition to calcium content of the saliva 
without considering the quantity of carbon dioxide present or 
the fact that certain colloidal substances (such as occur in saliva) 
may prevent precipitation of calcium salts. 

The relations of potassium sulphocyanate to dental caries, 
and other problems, have been studied in much the same way, 
and the object of this chapter is to emphasize the necessity of 
getting all possible viewpoints on a given question before attempt- 
ing to draw positive conclusions regarding it. 

History has repeatedly shown us that early, and frequently 
discarded, theories have ultimately been proven to be of more 
or less value; hence, a brief review of theories bearing on our 
subject will form a part of this chapter. The history of these 
theories is also valuable for its own sake. 

Dr. J. P. Michaels, in a review of this subject, tells us that 
" in 1835 Dr. A. Donne published in French a study on the 
chemical characteristics of saliva considered as a means of 
diagnosing certain diseases of the stomach. This was a very 
interesting special study, but chemistry was not sufficiently 
advanced to throw enough light on these questions." 

In 1884 Dr. Binet published in Paris a thesis on saliva, in 
which he noted " a correlation of biochemical principles between 

239 



240 PHYSIOLOGICAL CHEMISTRY 

the sweat and the saliva. " The importance of such " correla- 
tion" does not to-day appeal to us as great, compared with 
some other facts which Dr. Binet presented, as for example the 
determination of the density of saliva or an idea as to the twenty- 
four-hour amount. Dr. Binet also confirmed the previously 
demonstrated fact of the presence of urea as an organic salivary 
constituent. 

In 1900 Dr. J. P. Michaels presented to the Third Interna- 
tional Dental Congress in Paris a paper on the " Application 
of the Analysis of Saliva to the Diagnosis of Oral Disease," and 
gave us what is perhaps the first " relationship " worthy of 
serious consideration. Dr. Michaels classified people in general 
as (a) " Adiathetic " i.e., without indication as to future con- 
ditions of oral disease, (b) " Hypoacid " individuals who he 
said were liable to have trouble from carious teeth, and (c) 
" Hyperacid " individuals with a tendency to immunity from 
caries. His work led him to believe that the hyperacid condition 
was characterized by a high thiocyanate content of the saliva 
and a correspondingly low ammonia, while in cases with hypoacid 
tendencies the opposite condition prevailed. 

A considerable number of the leading dentists of this country 
took up this single test of high or low sulphocyanates, gave it a 
place of first importance in the prognosis of dental disease, made 
hundreds, probably thousands of tests and experiments, in- 
cluding the administration of sulpho-cyanate in tablet form to 
overcome the carious tendency, and apparently forgot all about the 
more important question of hypoacidity and hyperacidity, of 
which the thiocyanate was only supposed to be indicative. We 
know now that there are much better methods of determining 
the systemic acidities than a qualitative sulphocyanate test, but 
let us keep in sight the original divisions made by Dr. Michaels, 
because the markedly hypoacid and hyperacid salivas are with- 
out doubt indicative of systemic conditions. 

Whether or not hyperacidity is accompanied by immunity to 
dental decay has not been demonstrated, but the condition is 
consistent with pyorrhea and in many cases with erosion. As a 
rule, the more advanced the pyorrhea the more pronounced the 



THEORIES OF TOOTH DECAY 241 

hyperacidity. This does not argue for any etiological rela- 
tionship between the two conditions; in fact, it is very im- 
probable that hyperacidity ever caused pyorrhea and more 
improbable that pyorrhea ever caused hyperacidity; but the 
fact is that the hyperacid condition is the one favorable to the 
development of pyorrhea. Dr. Michaels described hyperacidity 
as a condition of insufficient oxidation, and therefore one that is 
often accompanied by the appearance of oxalates, acid lactates 
and urates (so called " acidic principles ") in the saliva. The 
discussion of soluble salts of the saliva is taken up later; and 
pyorrhea, as usually accompanying a condition of slight acidosis 
and consequent poor oxidation, has now been conceded by nearly 
everyone. These facts are of value to the dentist because from 
a few saliva and urine tests he can easily get some idea of the 
systemic condition of the patient. A patient of this type is very 
liable to show a considerable salivary acidity together with a 
high urinary acidity. Increased indoxyl, high ammonia and 
frequently high uric acid in both saliva and urine are usually 
found. 

As a rule, the insufficient oxidation indicated by such analyses 
is benefited not by more oxygen necessarily, but by modifications 
of diet and improved habits of living which will tend toward the 
correction of the systemic acidosis. This treatment combined 
with the local treatment is of far more permanent value than 
local treatment alone. 

Glycosuria and Pyorrhea. 

A condition of glycosuria is an indication of low oxidation; 
therefore we should expect, perhaps, to find conditions of this 
kind more or less associated with pyorrhea. Let us bear clearly 
in mind, first, that glycosuria and diabetes are not synonymous 
terms, and while diabetic pyorrhea has upon occasion been 
suggested as one of the types of the disease, it is doubtful if there 
is any direct relationship between the two disorders. 

From the examination of urine from more than sixty patients 
attending the pyorrhea clinic at the Harvard Dental School we 
obtained only one case of undoubted diabetes, while about 



242 PHYSIOLOGICAL CHEMISTRY 

fifteen of the patients had alimentary or renal glycosuria. That 
this proportion is larger than normal is shown by some figures 
obtained from the Life Extension Bureau of New York. These 
and also some Community Hygiene Experiments conducted at 
Framingham, Mass., by the National Anti tuberculosis Asso- 
ciation, showed less than 3 per cent of renal glycosuria among 
supposedly healthy individuals. We conclude, therefore, that 
in this 3 per cent the sugar in the urine is incidental while in the 
pyorrhea patients it is an indication of systemic acidosis. 

Pyorrhea is a disease of lowered resistance and reduced vitality, 
whatever the initial cause may be (malocclusion if you choose). 
The gums are " end organs/' physiologically considered, and as 
such are among the first to suffer from diminished blood supply. 
Dr. Walter Cannon has demonstrated that where there is dim- 
inished blood supply the fatigue products of metabolism are 
likely to be found. These, as we know from our study of physi- 
ological chemistry, include lactic acid and di-hydric-phosphate. 
This is only a statement of two and two; whether or not they 
make four is yet to be proven. 

SUGAR AND TOOTH DECAY. 

Caries is a disease of childhood, and there is an undoubted 
relation between the decaying teeth of children and excessive 
use of carbohydrates; but the relation is an indirect one and 
often resolves itself into a case of malnutrition, which might be 
produced by fine white bread as well as by sweets, provided the 
bread was taken in the same way i.e., between meals and when- 
ever opportunity offers. Any food taken in this way " spoils 
the appetite " for regular meals, with the result that less vege- 
tables are eaten. The consequences are disorders of digestion, 
intestinal stasis, lack of vitamines, and consequent lowered 
resistance to bacterial activity or other factors producing tooth 
decay. 

MILLER'S LACTIC ACID THEORY. 

Miller's theory that all tooth decay was a matter of decal- 
cification of tooth substance, caused by lactic acid produced 
from acid-forming bacteria growing on carbohydrate debris. 



THEORIES OF TOOTH DECAY 243 

furnishes the starting point for our present theories. Granting 
that decay is due to bacterial activity of some sort, we have to 
include a study of conditions favorable for bacterial growth and 
a study of conditions favorable to immunity, i.e., resistance. 

Among the conditions favorable for growth of acid-forming 
bacteria, the hydrogen-ion concentration is of prime importance, 
but must be considered in connection with the carbohydrate 
content and the presence of so-called " buffer substances," e.g., 
alkaline phosphates. 

Dr. Lawrence F. Foster has shown in the Journal of Bacteri- 
ology, Vol. 6, 1921, that for the growth of streptococcus hem- 
olyticus (which will produce lactic acid from glucose) a PH of 
7.6-8.1 is optimum concentration, and that a medium contain- 
ing i per cent glucose and i per cent potassium phosphate 
(K 2 HPO4) is best suited for acid production. Figures also 
quoted as optimum PH for streptococcus viridans, which has 
been repeatedly found in cavities of decayed teeth, are 7.6, 
7.8, and 6.8, and we also know that these acidities are within 
the range of ordinary salivas. 

It is safe to say that a number of acid-producing bacteria 
would be influenced in similar ways and by similar conditions. 
We know that in many mouths, and in restricted areas of many 
more, carbohydrates occur in various concentrations aside from 
the carbohydrate constituents of the saliva, glycogen (?). Also, 
we know, as Dr. Howe has shown in an article to which previous 
reference has been made, that phosphates increase the growth 
of bacteria in the mouth. Considering, then, the probable 
carbohydrate content of the saliva together with the fact that 
the presence of phosphates tends to increase the alkalinity of the 
saliva, thus making a more favorable medium for bacterial growth, 
we must at least admit a possible relationship between these 
factors and tooth decay. We know that in our own experience 
cases of marked decay have usually been accompanied by a PH 
higher than normal, while as a rule the lower PH has been found 
in cases of pyorrhea and erosion. 



244 PHYSIOLOGICAL CHEMISTRY 

DR. PERCY R. HOWE'S THEORY OF TOOTH DECAY. 

Dr. Howe, after years of experiments at the Forsy th Infirmary, 
concludes that the lactic acid theory of Dr. W. D. Miller is 
erroneous. He claims that sugar and local fermentations are of 
secondary importance and that the prime cause of tooth decay 
is malnutrition, and lack of proper mastication. He has shown 
that the lines of lowered resistance can be plainly traced in many 
cases and that they extend from the inside, the pulp chamber, 
outward toward the enamel. As the author understands Dr. 
Howe's theory (after intimate discussion with him on the subject) 
he does not deny the part played by bacteria in breaking down 
the tooth substance but does maintain that the initial cause is 
as above stated, and bacterial action is incidental. 

The author would differ with Dr. Howe in the use of terms, 
considering the lack of resistance a predisposing condition and 
the action of bacteria the immediate or exciting cause of dental 
caries. 

SALIVA AND BLOOD. 

That the constituents of saliva follow closely the constituents 
of the blood has long been a recognized fact, but that there is any 
relation existing between the quantities of the various constit- 
uents which are common to both has only recently been asserted. 
In the author's experience, however, there is a very direct re- 
lationship which may frequently prove of value to the dentist 
in detecting pathological conditions. The percentages of the 
constituents present, of course, vary, the blood containing a 
much higher concentration of most substances than the saliva, 
but the rise and fall of many substances in the blood is accom- 
panied by a similar change in the saliva. This has been found 
to be particularly true of urea nitrogen, creatinine, and uric acid. 
An analysis of nephritic saliva will invariably show these sub- 
stances high, following the same curve as the blood analysis. 

Aside from the point of view of nephritis, which may but 
seldom come to the dentist's attention, the uric acid content of 
the saliva, although of very recent investigation, seems now at 
least to be one of the most valuable from a diagnostic viewpoint. 



THEORIES OF TOOTH DECAY 245 

In every case of apical infection or pus absorption which has 
come under the author's observation recently (about fifty cases), 
the uric acid content of the saliva has been double or more than 
double what it seems to be in people with perfectly healthy 
mouths. The determination is so simple and the results so 
far have been so consistent that it would certainly seem to be one 
of the most promising new suggestions in regard to salivary anal- 
ysis. In an article by R. N. De Niord and B. J. Bixby on focal 
infection,* the following conclusions are drawn in regard to 
blood uric acid, and since the saliva curve seems to follow the 
blood curve as a rule, it is reasonable to suppose that these 
same conclusions may be applicable to saliva. 

" i. High uric acid value in the blood is indicative of nuclear 
degeneration which in turn may mean focal in- 
fection. 

" 2. Other factors productive of high uric acid, aside from 
nuclear degeneration, are comparatively easy to 
determine, i.e., leukemia, primary anemias, ca- 
chexiasf from whatever cause and massive doses of 
x-ray or radium. 

" 3. Elimination of all foci of infection invariably is followed 
by a return of the uric acid to normal, 

" 4. Failure to eliminate all foci will prevent the return to a 
normal uric acid, and this substance therefore fur- 
nishes a reliable index to the complete elimination 
of foci of infection." 

In another paragraph of this paper we read 

" The presence of a high blood uric acid does not of course 
point to any particular part of the body as its source. Hence the 
diagnosis of focal infection through this medium does not indicate 
that the teeth or tonsils or any other organ is at the bottom of the 

* Studies in Focal Infection. The Journal of Laboratory and Clinical Medicine, 
Vol. VII, No. 10, July, 1922. 

t Cachexia means any severe diseased condition nephritis and gout are 
perhaps the two most common ones to be eliminated. 



246 PHYSIOLOGICAL CHEMISTRY 

disturbance. But it does point to the necessity for a thorough 
search for all possible primary foci, and it gives definite authority 
for the removal of such foci when found." 

In the author's experience, cases of pyorrhea, where there is 
pus absorption, invariably show high salivary uric acid, and 
cases of abscessed teeth and sinus infection have been diagnosed 
and verified by the high uric acid content of the saliva. 

The value to the dentist of being able to determine from the 
saliva the approximate normality or abnormality of the blood, 
as regards these three constituents, and of thus being able to 
detect pathological systemic conditions, is self-evident. Further- 
more, the determination of the possible direct relationship be- 
tween high uric acid in the blood, as well as in the saliva, and pus 
absorption is of unquestionable value. 

POLARIZED LIGHT PICTURES AND ORAL CONDITIONS. 

Of the soluble salts which may be found by evaporation of a 
drop of clear urine or of dialyzed saliva, the majority have not 
been identified with sufficient certainty, or else their relation 
to definite conditions of any sort has not been sufficiently es- 
tablished to make the examination particularly valuable as yet. 
However, a few statements regarding our findings may help 
as a basis for further investigation. 

The test is very simple and in the author's experience it has 
seemed worth while to make it, and some interesting results have 
been obtained. The alkaline chlorides, sodium, potassium, and 
ammonium, are usually present in saliva; various forms of 
phosphates and frequently soluble oxalates are found in the 
urine. Crystals consistent with lactates are more rarely ob- 
served, and these have sometimes led to the detection of lactates 
by chemical means, Dr. E. C. Kirk suggested that the soluble 
oxalates in the urine may antedate the appearance of calcium 
oxalate and thus give warning of an undesirable metabolic 
process. 

The presence of oxalates, sometimes in the saliva and more 
frequently in the urine, has in our experience gone hand in hand 



THEORIES OF TOOTH DECAY 247 

with a condition of nervousness of the patient and of an acidosis 
more or less pronounced. 

Lactates in the urine are indicative of insufficient oxidation 
by endocellular enzymes and have occasionally given evidence 




FIG. 20. 

The long needle-shaped crystals resemble lactates and the round ones are 
consistent with sodium oxalates. 

of faulty liver action. The accompanying photomicrograph 
(Fig. 20), obtained from a drop of urine, indicated the presence 
of lactic acid, which was subsequently proven by careful chem- 
ical tests. 



EXPERIMENTS. 

ORGANIC CHEMISTRY. 
Experiments with Carbon and Hydrocarbons. 

Exp. i. Carbon as a decolorizing agent. To 25 or 30 c.c. 
of a dilute solution of aniline color, contained in a small beaker, 
add a teaspoonful of bone charcoal. Heat to the boiling- 
point, rotate or stir thoroughly for a few minutes, and filter. 

Exp. 2. Absorption of metallic salts. To 25 c.c. of solution 
of lead acetate of such strength that H 2 S water gives marked 
color but no precipitate, add a teaspoonful of bone charcoal and 
treat as in preceding experiment. Test the filtrate with H 2 S 
water and note whether lead has been removed. 

Exp. 3. Perform an experiment with a view to determining 
whether bone charcoal will absorb H 2 S from H 2 S water. 

Exp. 4. Repeat any one of the three preceding experiments, 
using wood charcoal in place of bone charcoal. Does the wood 
charcoal work as well as the bone charcoal in the absorption of 
color or other substances? How does bone charcoal differ in 
composition from wood charcoal? 

Exp. 5. Arrange apparatus as shown in Fig. 21. To the 
boiling flask (B) provided with a thermometer registering 200 C. 
connect a beaker condenser, C, immersed in ice water. In this 
apparatus distil slowly 25 c.c. of crude petroleum until at least 
four fractional products are obtained, with boiling points differing 
by at least 15. Compare the physical properties of the distil- 
lates thus obtained. 

Exp. 6. Charge an ignition- tube with dry " marsh-gas mix- 
ture," found on side shelf (consisting of NaC 2 H 3 O 2 , NaOH, 
and CaO 2 H 2 ). Fit with a delivery tube and collect two small 
bottles of the gas over water. 

248 



EXPERIMENTS 



NaC 2 H 3 2 +NaOH = CH 4 +Na 2 CO 3 . 



249 



Test the inflammability of this gas. Notice the odor, 
Exp. 7, Mix carefully in a test-tube 2 c.c. of alcohol and 
8 c.c. of strong sulphuric acid. Heat gently and notice odor of 
gas. Fit a bent glass tube to the test-tube and collect over 
water a test-tube full of the gas. To this apply a flame. Note 
the color of the burning gas. 

C 2 H 6 OH-H 2 = C 2 H 4 . 

Exp. 8. Collect a test-tube full of ethylene (Exp. 7), add 
a few c.c. of dilute permanganate solution and shake. Then 




FIG. 21. 

repeat, using marsh gas in place of the ethylene (test for un- 
saturated hydrocarbons). 

Exp. 9. Shake together, in separate test-tubes, small quan- 
tities of petroleum and sulphuric acid in one tube, and petroleum 
and nitric acid in the other. If no action results, mix contents 
of the two tubes and shake again. Explain any change or 
absence of change which may be apparent. 

Exp. 10. In a small generator (see model) place a few small 
pieces of calcium carbide (CaC 2 ), add strong alcohol through the 
funnel tube till the lower end of the tube is " sealed." Now 



250 EXPERIMENTS 

add very slowly a little water till a brisk evolution of gas is 
obtained. Collect over water two or three test-tubes full of the 
gas. (Acetylene.) 

Test with a lighted splinter. Note odor of gas cautiously, 
as it is poisonous when inhaled in quantity. 

CaC 2 +2 H 2 O = Ca(OH) 2 +C 2 H 2 . 

Exp. ii. Conduct a little of the acetylene gas into an am- 
moniacal cuprous chloride solution.* What is the red pre- 
cipitate? 

Exp. 12. If the evolution of gas (Exp. 11) has not been in- 
terrupted the delivery tube may be replaced by a short tube 
drawn out to a fine point, and the gas ignited. Note color of 
flame. If it smokes badly, explain the reason for it. 

Experiments with the Halogen Derivatives of the Hydrocarbons. 

Exp. 13. Place in a test-tube a little bleaching-powder, cover 
with strong alcohol and heat the mixture to boiling. Notice 
carefully the odor of the vapor produced and compare with a 
little chloroform (CHC1 3 ) from side shelf. 

4 C 2 H 5 OH+8 Ca(C10) 2 =2 CHCl 3 +3 Ca(CH0 2 ) 2 

(Formate of Ca) 

+5CaCl 2 +8H 2 O. 

Exp. 14. Heat i c.c. of chloroform with about 5 c.c. of one 
per cent NaOH. Test a portion of the resulting solution for 
inorganic chlorides. Distil the remainder of the solution and 
test the distillate, collected in a test-tube, with litmus paper. 

Exp. 15. Place in a test-tube about i gram of crystallized 
carbonate of sodium, about half as much iodine and i or 2 c.c. of 
alcohol. Now add 10 or 15 c.c. of H 2 and keep the mixture 
at moderate heat (not boiling) till the color of the iodine is dis- 
charged. Allow to cool; collect on a small filter paper some of 
the yellow crystals which have been formed and examine under 
the microscope. What are the crystals? (See Plate III, Fig. 6.) 
Explain their relation to marsh gas. 

* See Appendix for preparation of reagent. This test is characteristic of the 
triple-bonded hydrocarbons. 



EXPERIMENTS 251 

Exp. 1 6. Cover one or two small pieces of calcium carbide, in 
a small porcelain dish, with a mixture of three parts water and 
one part alcohol. While the gas is being evolved hold over the 
mixture a test-tube full of chlorine. 

Exp. 17. Oxidation of chloroform. If chloroform is oxidized 
by heating a few drops with a crystal of potassium dichromate 
and 2 or 3 c.c. of concentrated sulphuric acid, phosgene gas is 
produced, COC12. Note the odor very cautiously and write 
reaction. 

Exp. 18. Ethyl iodide from ethyl alcohol. Place 2 1/2 grams 
of red phosphorus, and 20 grams of absolute alcohol in a small 
flask of 100 c.c. capacity. To this mixture add gradually 25 
grams of finely powdered iodine, shaking the flask frequently 
and keeping it cool by allowing cold water to run over it. Con- 
nect the flask with an upright air-condenser and allow the 
reaction to continue for at least four hours. It may continue 
overnight without detracting from the value of the experiment. 
Then heat the flask over a water-bath for two hours, the air- 
condenser acting as a reflux condenser. After the reaction is 
completed, distil off the ethyl iodide. To facilitate distillation 
a rapidly boiling water-bath should be used, or a naked flame if 
used cautiously. 

To obtain the distillate free from alcohol and the iodine which 
gives it its brown color, wash several times with water and then 
with a dilute solution of sodium hydroxide. Separate the color- 
less oil in a separating funnel, dry with a small piece of granular 
calcium chloride and redistil. The boiling-point of ethyl iodide 
is 72 and the yield should be about 25 grams. 

Exp. 19. Prepare ethyl bromide from alcohol, potassium 
bromide and sulphuric acid as follows: Using the apparatus 
suggested for Experiment 62, place in the distilling flask about 
30 c.c. of 50 per cent alcohol. Add slowly with constant agi- 
tation 30 c.c. of strong sulphuric acid. Cool thoroughly, then 
add 30 grams of powdered potassium bromide. Distil carefully 
until condenser is nearly full of distillate. Pour about a quarter 
of the product into excess of water. Shake well to wash the ethyl 
bromide. Remove from the wash water by means of a pipette 



252 EXPERIMENTS 

and dissolve in a little alcohol. Test this alcoholic solution for 
bromine with alcoholic silver nitrate. 

To another portion of the ethyl bromide add 5 to 10 c.c. of 
alcoholic potassium hydroxide (5 per cent in absolute alcohol). 
Boil for a minute or two, dilute with water and make the usual 
qualitative test for bromides. 

Write reactions. 

Ethyl bromide may also be prepared by distilling a mixture of 
i part of alcohol and 5 parts of strong hydrobromic acid. 

Exp. 20. Pr eparation of dithymol-di-io dide (C6H 2 CH 3 C3H 7 OI)2. 
Dissolve 1.5 grams of thymol in 30 c.c. of 6 per cent NaOH. 
It will take considerable time to dissolve the thymol and it 
may be necessary to add some stronger alkali. In a separate 
beaker dissolve 6 grams of iodine in 30 c.c. of water, using 
sufficient potassium iodide to put the iodine into solution 
easily. Then slowly and with constant stirring add the iodine 
solution to the thymol solution. A brown precipitate of thymol 
iodide results; this should be washed until there is no trace of 
alkali in the wash water, and dried at a temperature not exceeding 
50 C. 

Experiments with Alcohols. 

Exp. 21. The detection of water in alcohol. Prepare a little 
anhydrous copper sulphate by heating a few crystals of CuSO 4 
on a crucible cover until the water is driven off and a nearly white 
powder results. If this white powder is added to half a test-tube 
full of alcohol, the absorption of water, if present, will result in 
the reforming of the crystallized salt and a consequent production 
of blue color. 

Exp. 22. Water may be separated from alcohol by saturat- 
ing with potassium carbonate. To demonstrate this, take a 
mixture of alcohol and water, containing 15 or 20 per cent of 
alcohol, and add solid potassium carbonate until the salt will no 
longer dissolve. Agitate and allow to stand. Two layers will 
form, one consisting of alcohol, the other of the water solution of 
K 2 CO 3 . 

Exp. 23. To about 75 c.c. of a 10 per cent glucose solution add 
a little yeast and allow to stand for twenty-four hours at a tern- 



EXPERIMENTS 253 

perature of about 37 C.; then distil by means of gentle heat, 
10 or 15 c.c., and test distillate for alcohol by iodoform test, as 
given on page 250, Exp. 15. The production of CO 2 may also 
be demonstrated if the gases evolved during the fermentation 
are passed into clear lime water: 

C 6 H 12 O 6 = 2 C 2 H 5 OH+2 CO 2 . 

Exp. 24. A test for methyl alcohol. This test is applicable 
only to slight traces of methyl alcohol and may be made with 
a i to 2 per cent solution or with the first cubic centimeter of 
distillate from the substance suspected of containing methyl 
alcohol. Place 2 or 3 c.c. of very dilute methyl alcohol in a 
test-tube, heat a spiral of copper wire to white heat in a Bunsen 
flame and plunge immediately into the solution to be tested. 
Cool the contents of the tube by immersion in freezing mixture 
or ice water, and repeat the treatment with the hot copper wire. 
Cool again, and a third time introduce the hot copper wire. 
The copper spiral can be made by winding copper wire around a 
lead pencil, and should be of such a length that it is not wholly 
covered by the liquid in the tube. 

This process serves to oxidize a portion of the alcohol to 
aldehyde. Now add to the solution which is being tested a few 
drops of a 1/2 per cent water solution of resorcinol and underlay 
the mixture with strong sulphuric acid. A pink ring will indi- 
cate the presence of methyl alcohol. The higher alcohols will 
give red or brown rings when similarly treated. 

Exp. 25. Repeat Experiment 24, using ethyl alcohol in place 
of methyl alcohol. 

Exp. 26. In 5 or 10 c.c. of absolute alcohol, dissolve 1/4 
to 1/2 gram of metallic sodium. Test the gas given off. 

Write reaction. Save the product. 

Experiments with Aldehydes and Ketones. 

Exp. 27. Mix about i c.c. of a very dilute solution of for- 
maldehyde with four or five times its volume of milk in a test- 
tube. Keep at a temperature of 40 to 50 C. for half an hour, 
then carefully underlay the mixture with commercial sulphuric 



254 EXPERIMENTS 

acid of a specific gravity of 1.80. At the point of contact of the 
two layers of liquid, a violet-colored ring indicates the presence 
of formaldehyde. It is necessary that time be allowed for the 
casein of the milk to unite with the formaldehyde, also that the 
sulphuric acid should contain a trace of iron; this the commercial 
acid usually does. It is undesirable that the acid should be 
stronger than of 1.80 specific gravity; for, if it is, a reddish-brown 
ring may be formed, due to partial carbonization of the casein. 

Exp. 28. To a very dilute solution of formaldehyde add a 
few drops of 1/2 per cent resorcinol solution and underlay the 
mixture with H 2 SO 4 as in Exp. 24. The appearance of a violet 
ring will constitute a test for formaldehyde. 

Exp. 29. To about 5 c.c. of a strong aqueous solution of 
potassium dichromate add a little sulphuric acid, then a few 
cubic centimeters of alcohol, and notice the odor of acetaldehyde. 
produced by oxidation of the alcohol. Note also the reduction 
of the dichromate to Cr 2 (SO 4 ) 3 , as follows: 

K 2 Cr 2 7 + 4 H 2 SO 4 +3 C 2 H 5 OH = 

K 2 S0 4 +Cr 2 (S0 4 ) 3 +3 C 2 H 4 O+ 7 H 2 O. 

Exp. 30. Test dilute solutions of acetone, formic and acetic 
aldehydes by Tollen's test for aldehyde as follows: Into a clean 
test-tube which has been rinsed with NaOH solution, place 5 c.c. 
of Tollen's reagent, add 10 c.c. of solution to be tested, shake; 
the silver is reduced, forming a metallic mirror on the inner sur- 
face of the tube. 

To make Tollen's reagent, dissolve 3 grams of silver nitrate 
in 30 c.c. of ammonia water and add 3 c.c. of solution of sodium 
hydroxide. 

Exp. 31. Prepare acrolein in each of the following ways: 

ist: From glycerol according to the test given on page 84. 

2nd : Oxidize one or two drops of allyl alcohol with potassium 
bichromate and H 2 S0 4 , as in the oxidation of ethyl alcohol 
in Exp. 29. 

Exp. 32. To about 5 c.c. of an aqueous solution of chloral 
hydrate add a few cubic centimeters of strong NaOH solution 
and boil. Note odor of chloroform. 



EXPERIMENTS 255 

Exp. 33. Isobenzonitril test for chloral or chloroform: Place a few 
drops of a dilute chloral hydrate solution (or a small drop of 
chloroform) in a test-tube, add 5 c.c. of an alcoholic solution 
of alkali hydrate* (NaOH or KOH) and one drop only of fresh 
aniline oil. Heat till the mixture just begins to boil and note 
the odor of the nitril. For reaction see test for chloroform, 
page 13. 

Exp. 34. Test 2 or 3 c.c. of an aqueous solution of aldehyde 
with an equal volume of Schiff 's reagent. 

Exp. 35. Preparation of Schiff 's Reagent. Into a dilute solu- 
tion of fuchsin, pass sulphur dioxide gas until color is entirely 
discharged. 

Experiments with Acetone. 

Exp. 36. Preparation of acetone. Heat a few grams of 
dried calcium acetate in an ignition-tube, collect the distillate, 
which consists of an impure acetone. If this is mixed with a 
little water and filtered, part of the impurities may be removed, 
and the filtrate tested for acetone by the following experiment. 

Exp. 37. Dilute the filtrate from the last experiment with 
distilled water; add a crystal of sodium nitroprusside. After 
the crystal is dissolved, add a few drops of acetic acid, and then 
an excess of ammonia water. A violet or purple color indicates 
the presence of acetone. Using a dilute solution of acetone in 
place of the alcohol in Experiment 15 on page 250, produce iodo- 
form crystals by similar reaction with iodine and sodium or po- 
tassium carbonate. 

Exp. 38. Acetone may be dissolved or mixed with water in 
all proportions; but, upon saturating the water with KOH, 
the acetone will form a separate layer which may be drawn off as 
in the separation of alcohol in Experiment 22, page 252. 

Experiments with Ethers. 

Exp. 39. Into a large test-tube put a little alcohol and about 
half its volume of strong H 2 SO 4 . Warm gently and notice the 
odor. 

* If alcoholic potash or soda is not at hand, the test may be performed with 
5 c.c. of alcohol and i or 2 c.c. of a 40 per cent aqueous solution of NaOH. 



256 EXPERIMENTS 

Ether is formed by two reactions. First, C 2 H 6 OH+H 2 SO 4 
= C 2 H 6 HS0 4 -}-H 2 O. Then the ethyl-hydrogen sulphate (C 2 H 5 - 
HS0 4 ) is acted upon by a second molecule of C 2 H 5 OH, as fol- 
lows: C 2 H 5 HS0 4 +C 2 H 6 OH- (C 2 H 6 ) 2 O+H 2 SO 4 . 

Exp. 40. Test the inflammability of ether by applying a 
lighted match to a few drops on a watch-crystal. 

Exp. 41. Determine the solubility of water in ether by shaking 
10 c.c. of ether with an equal quantity of water. Then turn the 
ether off into a dry test-tube, preferably allowing it to pass 
through a dry filter-paper first. The filtered ether may be 
tested for water by treating it with some anhydrous copper 
sulphate. 

Exp. 42. Into a 10 c.c. graduate introduce exactly 6 c.c. of 
distilled water and 4 c.c. of ether. Close tightly (thumb may 
be used) and shake gently for a minute. Without uncovering 
tube, allow liquids to separate and read volume of water. Con- 
clusion? 

Exp. 43. Try the solubility of ether in each of the following: 
alcohol, benzene, petroleum ether, dilute hydrochloric acid, 
dilute sodium hydroxide. 

Experiments with Organic Acids (C n /7 2n 2 ). 

Exp. 44. Introduce into a small flask (250 c.c. capacity) 
about 30 c.c. of anhydrous glycerin and an equal weight of 
oxalic acid crystals. Boil for several minutes; C0 2 is given 
off and a compound formed between the acid and glycerin; 
then, upon addition of more acid and continued heating, formic 
acid may be distilled. Collect about 10 c.c. of distillate; test 
reaction with litmus paper. Make silver-mirror test, described 
on page 254, Exp. 30. The silver solution will be reduced, but 
difficulty will be experienced in obtaining the mirror. 

Exp. 45. To 5 c.c. of formic acid solution add 2 or 3 c.c. of 
dilute H 2 SO 4 (1-5) and a little potassium permanganate solu- 
tion; heat the mixture and conduct the gas evolved into a tube 
containing lime-water. 

Exp. 46. From a mixture of formic acid, alcohol, and sul- 
phuric acid; ethyl formate may be evolved in a manner similar 



EXPERIMENTS 257 

to that recommended in the production of ethyl acetate or 
butyrate (page 259). Compare the odors of these two ethers. 

Exp. 47. To a dilute aqueous solution of acetone add po- 
tassium permanganate slowly until the mixture is permanently 
colored pink; filter, add dilute sulphuric acid and distil until 
i or 2 c.c. of distillate is obtained. This may be tested for acetic 
acid by litmus paper and ferric chloride. 

Exp. 48. To a dilute solution of ferric chloride add a little 
acetic acid; divide the solution into two parts; to one add mer- 
curic chloride and to the other HC1, and note results. 

Exp. 49. Repeat Exp. 48, using diacetic acid in place of 
acetic. 

Exp. 50. Repeat Exp. 48, using meconic acid* in place of 
acetic. 

Compare results of these three experiments and save record 
for future use in the study of saliva. 

Exp. 51. In a small flask saponify a little butter by heating 
with alcoholic potash over a steam-bath till mixture is dry. 
Dissolve in water, add dilute H 2 SO4, and distil off a portion of 
the butyric acid. Record whatever can be learned from this 
experiment regarding the physical properties of the butyric acid. 

Exp. 52. In separate test-tubes take about 5 c.c. of solu- 
tions of stearic and oleic acids in carbon tetrachloride. Add to 
each about i c.c. of a one-tenth per cent solution of iodine, also 
in carbon tetrachloride, allow to stand for some time, and ex- 
plain fully the difference in action exhibited by the two fatty 
acids. 

Experiments with Organic Acids not of the C w H 2fl O2 Series. 

Exp. 53. To a dilute solution of permanganate of potassium 
add a few drops of sulphuric acid and heat nearly to boiling. 
Note if any change takes place. Now add a few crystals of ox- 
alic acid and watch carefully. Explain the use of sulphuric acid. 

Exp. 54. In separate test-tubes, insoluble oxalates may be 
produced by adding a solution of ammonium oxalate to a solu- 

* Laudanum diluted with water till color is light brown may be used 



EXPERIMENTS 

tion of (a) calcium chloride, (6) silver nitrate, (c) zinc sulphate, 
(d) copper sulphate, (e) lead nitrate. 

Exp. 55. Place in an ignition-tube, fitted with delivery tube 
to collect evolved gas in test-tube, about 3 grams of dry calcium 
oxalate. Heat strongly and test gas evolved with lighted match 
or splinter. After ignition tube has become cold add dilute 
H 2 S04 and pass gas evolved into lime-water. 

Exp. 56. Dissolve about 3 grams of dry oxalic acid (100 C.) 
in a test-tube half full of methyl alcohol. It will probably be 
necessary to boil the mixture before solution is complete, and 
great care must be used to avoid burning of the alcohol. The 
use of a water-bath is recommended. As the hot mixture cools, 
dimethyl oxalate will crystallize out. 

Separate sufficient of the crystals to obtain melting-point, 
which should be about 54 C. 

Exp. 57. The ester prepared in above experiment may be 
dissolved in alcohol and upon addition of NH 4 OH will give a 
precipitate of oxamide. 

Exp. 58. Take a test-tube half full of calcium chloride (10 
per cent), make strongly alkaline with NH 4 OH and pass CO 2 
into the mixture for several minutes. A solution of calcium 
carbonate will result. 

Write reaction, CaCl 2 +2 CO 2 +4 NH 4 OH=?. Heat the solu- 
tion of calcium carbonate just produced till a precipitate of 
CaC0 3 is produced. 

Write reactions showing the formation of CaH 2 (C0 3 ) 2 and the 
precipitation of CaCOs from the acid salt. 

Exp. 59. To one-third of a test-tube of cider vinegar add a 
few cubic centimeters of basic acetate of lead solution; a bulky 
precipitate of lead malate separates. 

Exp. 60. Dilute a few drops of neutral ferric chloride solu- 
tion until no color is discernible, then to 10 c.c. of this dilution 
add 4 to 5 drops of 1/2 per cent solution of lactic acid. A 
greenish-yellow color constitutes a positive test. 

In practical application of this test, it needs further con- 
firmation by boiling the unknown solution with a drop or two 
of HG1 and then extracting with ether. Evaporate the ether, 



EXPERIMENTS 259 

take up the residue in 2 or 3 c.c. of water and repeat the test as 
given above. If the yellow color persists, it is due to lactic acid. 

Exp. 61. The production of esters may be demonstrated 
by the test for acetic acid, forming ethyl acetate, or by the 
following experiment used to detect butyric acid in gastric 
contents : 

Exp. 62. Mix in a test-tube 5 c.c. of a dilute 1/2 per cent 
solution of butyric acid with an equal volume of strong H 2 SO 4 
and as much strong alcohol. Heat gently and note the odor 
of ethyl butyrate (pineapples). 

Exp. 63. The action of fixed alkalies on esters is known 
as "saponification." It may be illustrated by heating 10 c.c. 
of ethyl acetate with 80 c.c. of a 10 per cent NaOH solution for 30 
to 40 minutes, when the odor of ethyl acetate should be destroyed. 
The flask should be connected with a reflux condenser and the 
heat applied by immersing the flask in boiling water. Write 
reaction. 

Exp. 64. Preparation of ethyl nitrite. Take 8 c.c. of strong 
H 2 SO 4 and add 25 c.c. of water. Cool the liquid and add 17 c.c. 
of alcohol previously diluted with an equal volume of water. 
Place the whole in a 200 c.c. flask surrounded with ice-water. 
Dissolve 20 grams of NaN0 2 in 56 c.c. of water. Filter, and 
from a separatory funnel allow the sodium nitrite solution to 
drop into the cold acid solution previously prepared. 

Allow the crystals which may have been formed to settle, and 
decant the liquid into a clean separatory funnel. The ethyl 
nitrite will rise to the top and the water may be drawn off. 
Wash the ethyl nitrite with 10 c.c. of ice- water to remove acid, and 
then with 10 c.c. of ice-water to which Na 2 CO3 has been added. 

Exp. 65. Test for ethyl nitrite. Fill the large tube of a 
Doremus Hind's ureometer with a mixture containing 20 c.c. of 
saturated NaCl, 5 c.c. of 20 per cent KI, 5 c.c. of 20 per cent 
H 2 SO 4 . In the small arm place 2 c.c. of a saturated solution of 
NaCl and 2 c.c. of the prepared ethyl nitrite. Add cautiously 
i c.c. of the mixture in the small arm. Nitric oxide is evolved 
according to the following equation: 

C 2 H 6 N0 2 +KH-H 2 SO4^C 2 H 6 OH+KHS04+I+NO. 



860 EXPERIMENTS 

Experiments with Cyanogen Compounds. 

Exp. 66. In a large test-tube dissolve 1/2 gram or less of 
potassium ferrocyanide in about 4 c.c. of water. Add a little 
HsSCX and boil, conducting the gas evolved into a beaker con- 
denser (Fig. 23) by means of a bent glass tube. Note the odor 
of this dilute solution. (Do not smell of the contents of generat- 
ing tube, as the strong acid is intensely poisonous.) Write 
reaction. 

Exp. 67. To one half of the dilute hydrocyanic acid prepared 
in the previous experiment add a drop or two of AgN0 3 solution 
with a little HN0 3 . After the precipitate has settled, decant the 
fluid, then add an excess of ammonia water. 

Exp. 68. To the other half of the HCN from Exp. 66 add 
a little solution of ferrous sulphate; also a few drops of ferric 
chloride solution; then a little KOH solution; mix thoroughly 
and acidify with HCL A blue precipitate, Fe 4 [Fe(CN) 6 ]3, is a test 
for HCN or any soluble cyanide. 

Exp. 69. To a few drops of KCN solution add a little yellow 
ammonium sulphide, (NH 4 ) 2 S, and evaporate to dryness. Dis- 
solve in water; acidify with HC1 and add Fe 2 Cl6 solution. 

Exp. 70. In a small flask boil a solution of KCN. While 
boiling, test the vapors for ammonia gas. Solution of potassium 
formate remains in the flask. 

Complete reaction, KCN +2 H 2 O= ? 

Exp. 71. To a little dilute (2 per cent) solution of K 4 Fe(CN) 6 
add a little bromine water and boil. Prove the formation of 
K 3 Fe(CN) 6 by use of FeCl 3 . 

Judging from this experiment, what is the relative valence of 
iron in the two compounds? Why? 

Exp. 72. To a fresh solution of K 3 Fe(CN) 6 add a little 10 
per cent KOH solution and some PbO, shake and filter. To the 
dear filtrate add FeCl 3 . 

Give reason for the statement that the PbO has acted as a 
reducing agent. 

Exp. 73. Dissolve a piece of potassium ferricyanide, as 
large as a pea, in 5 c.c. of water, add 2 c.c. of a solution of potas- 



EXPERIMENTS 



261 



sium ferrocyanide. Dilute to the capacity of a test-tube with 
distilled water and put equal amounts of this solution into 2 
shell tubes. Examine the color through the length of tube, then 
add to one tube 2 or 3 drops of strong HCL Examine again 
and notice that a trace of prussian blue has been produced. 
Explain. 

Experiments with Amines and Amides. 

Exp. 74. Distil 60 c.c. of ammonium acetate in a glass 
retort fitted with a thermometer, as in Fig. 22. Acetamide 




FIG. 22. 

should distil at about 222 C. and condense as a white solid in 
the receiver. 

Exp. 75. In a 500-c.c. flask place 10 grams of strong, fresh, 
bleaching powder; add 3 grams of acetamide dissolved in about 
10 c.c. of water. Mix as thoroughly as possible and add slowly 
25 c.c. of a 20 per cent solution of NaOH. Distil with steam, 
collecting distillate in 15 c.c. of cold water. 

Exp. 76. To a little of the water solution of methyl amine 
prepared in the last experiment, add 2 or 3 drops of chloroform 
and a little alcoholic potash. This mixture upon warming 
will give carbylamine. Note the odor. Warm a little of the 
solution with a little 5 per cent NaOH. Test the vapor given off 
with litmus paper and compare with ordinary qualitative test for 
ammonia. 



262 EXPERIMENTS 

Exp. 77. Prepare acetanilide (phenyl acet-amide) by mixing 
intimately 10 c.c. each of acetic anhydride and aniline oil. Cool 
and recrystallize from repeated portions of hot water. 

Exp. 78. Preparation of camphor sulphonic acid. In a small 
beaker place 10 to 12 c.c. of acetic anhydride. Place the beaker 
in an ice-salt bath and keep the temperature below 10 C. When 
the acetic anhydride is thoroughly cold, add slowly 3.5 c.c. of 
concentrated H 2 SO 4 . Then add slowly about 7 grams of cam- 
phor. Stir until all the camphor is dissolved. Let stand at 
room temperature for two days, when crystals of camphor 
sulphonic acid will appear. Wash the crystals with ether, and 
dry. During the addition of the camphor it is necessary to 
keep the temperature low or the sulphuric acid is apt to char it. 

Urea and Uric Acid. 

Exp. 79. Make separate solutions of 10 grams of potassium 
cyanate* and 8.25 grams of ammonium sulphate. Mix and 
evaporate on a water-bath in a shallow dish. Separate the 
potassium sulphate as the evaporation proceeds; finally, evapo- 
rate to dryness and extract with absolute alcohol. Evaporate 
alcohol and reserve the urea for subsequent experiments. (See 
Urea, page 54.) 

Exp. 80. Heat a few crystals of urea in a test-tube until they 
fuse and no more gas is given off; cool, and dissolve the fused 
mass in water; add i or 2 c.c. of strong NaOH solution, then 
not more than i or 2 drops of a i per cent CuSO 4 solution. 
Note the pink to violet color produced. This constitutes the 
biuret reaction used in physiological chemistry as a test for 
albumoses and peptones. Biuret is formed from urea as follows: 

NH 2 = C 

20 = C( = 

X NH 2 O = C 

X NH 2 

Exp. 81. Produce crystals of urea nitrate and oxalate (page 

* For method of making potassium cyanate, see Preparation of Reagents and 
Organic Compounds, in the Appendix. 



EXPERIMENTS 263 

77) and examine under the microscope. (Repeat with urea 
obtained from urine). 

This experiment may be performed by concentrating a little 
urine to about one-fifth of its bulk and using the concentrated 
solution as a solution of urea. 

Exp. 82. Treat 5 c.c. of urea solution (urine may be used) 
with a little sodium hypochlorite or hypobromite; note results 
and study reaction given on page 55. 

Exp. 83. Heat one- third of a test-tube of urine with barium 
hydroxide (baryta- water) ; test vapor with red litmus for NH 3 . 

Exp. 84. Murexide test for uric acid: Place a very small 
quantity of uric acid on a porcelain crucible cover, or in a small 
evaporating dish. Add 2 or 3 drops of strong nitric acid and 
evaporate to dry ness over a water-bath. A yellowish-red residue 
remains, which changes to a purplish red upon addition of a 
drop of strong NH 4 OH, and to purple-violet upon further 
addition of a drop of KOH solution, the color disappearing upon 
standing or upon the application of heat. (Difference from 
xanthine, which also gives a deeper red color.) 

Exp. 85. Repeat No. 84, using caff em in place of uric acid. 

Exp. 86. Heat a little sodium acid urate in a dilute solution 
of NaH 2 PO 4 . Allow to cool, and examine any deposit for uric 
acid crystals. Test reaction of solution both hot and cold 
(page 59). 

Exp. 87. Mix, and allow to stand for some time at reduced 
temperature, 30 c.c. of urine (a 2 per cent urea solution), 2 or 3 
c.c. of strong Na 2 C0 3 solution, and 5 c.c. of saturated NH 4 C1 
solution. 

A precipitate consists of ammonium urate. 

Examine under the microscope and make murexide test. 

Exp. 88. To a mixture of sodium urate, sodium chloride, and 
sodium phosphate, add a little magnesium mixture. Filter. 
Make filtrate quite strongly alkaline with NH 4 OH and add 
ammoniacal silver nitrate. Precipitate is AgMg urate. The 
three salts used in this experiment are all found in urine, and the 
uric acid is precipitated and may be roughly determined by use 



264 



EXPERIMENTS 




FIG. 23. 



of ammoniacal AgNO 3 (page 193). Use of mixture? Use of 
excess of ammonia? 

Exp. 89. Separate caffein from a spoonful of coffee as follows: 
Boil with about 200 c.c. of water for twenty minutes. Filter, 
remove coloring matter and tannic acid by careful addition of 
subacetate of lead solution. Filter and concentrate filtrate to 

100 c.c. or less and extract with 
30 c.c. of chloroform. Allow 
chloroform to evaporate without 
heat, and dissolve residue in 
about 5 c.c. of water. Test this 
solution according to Exp. 90. 

Exp. 90. To a few cubic 
centimeters of uric acid solution 
add i c.c. of phospho-tungstic 
acid and 2 c.c. of sodium cyanide 
(7 1 per cent). Compare the 
color obtained with a blank test 
in which distilled water has been used in place of the uric acid. 

Exp. 91. Schiffs test for uric acid. To a solution of sodium 
carbonate add a trace of uric acid. Pour the solution upon a 
piece of paper wet with silver nitrate, and note the reduction of 
the silver salt. 

Experiments with Aromatic Hydrocarbons. 

Exp. 92. Into a small and thoroughly dry flask (250 c.c.) 
introduce about 50 grams of a mixture consisting of i part of 
benzoic acid and 2 parts of quicklime; connect with a beaker 
condenser (Fig. 23) and heat. Benzene (benzol) distils over: 

CaO+C 6 H 5 COOH = CaCO 3 +C 6 H 6 . 

Exp. 93. Turn a little of the benzene prepared in the last 
experiment on to some water contained in a porcelain capsule. 
Set fire to it and note that it burns with a smoky flame. Cool 
a few cubic centimeters of pure benzene, contained in a narrow 
test-tube, by immersion in a freezing mixture of ice and salt. 

Exp. 94. Test benzene for double bond. Exp. 8. 



EXPERIMENTS 265 

Exp. 95. Test benzene samples A and B for CS 2 . Use 10 c.c. 
of sample and add to each 2 drops of phenylhydrazine. Presence 
of CS 2 will be indicated by formation of crystalline precipi- 
tate. (C 6 H 5 NH.NH 2 ) 2 CS 2 . 

Exp. 96. Test solubility of naphthalene in alcohol, water, 
and gasolene. 

Exp. 97. Determine melting-point of naphthalene. 

Exp. 98. In a wide test-tube mix 5 c.c. of concentrated 
H 2 SC>4 with about half its volume of strong HNO 3 ; cool in ice- 
water or cold running water, and add very slowly about 2 c.c. 
of benzene. Nitrobenzene is formed and may be separated as 
a heavy oily liquid by pouring the mixture into an excess of 
water. Notice the odor of oil of bitter almonds. 

Exp. 99. Observing the same precaution against overheating 
as given in Exp. 98 reduce nitrobenzene to amino-benzene as 
follows: In a large test-tube or small flask place i or 2 c.c. of 
nitrobenzene with three times its weight of tin powder. To 
this add 10 or 15 c.c. of strong HC1 in successive small portions, 
keeping cool as indicated. The odor of nitrobenzene should be 
replaced by that of aniline. 

Exp. 100. Heat a mixture of 2 c.c. of aniline, 5 c.c. of water 
and i c.c. of strong sulphuric acid to the boiling point; then set 
aside where it may cool slowly. Crystals of aniline sulphate 
will separate. 

Exp. 101. Repeat preceding experiment, using 5 c.c. of 
aniline, 5 c.c. of water and 10 c.c. of strong hydrochloric acid. 
When the mixture has become thoroughly cold, filter off the 
crystals of aniline hydrochloride and dry in a current of air. Test 
solubility in water, using only a very little of the crystallized salt. 

Exp. 102. Place 5 c.c. of an aqueous solution of aniline in 
each of three test-tubes. Add to the first a few drops of bromine 
water; to the second a few drops of dilute ferric chloride; and 
to the third a solution of hypochlorite of calcium or sodium. 

Exp. 103. Shake together in a test-tube i part of aniline oil 
and 5 parts of water. Is the oil soluble in water? 

Agitate with HC1 added in small portions till liquid becomes 
clear. Explain. 



266 EXPERIMENTS 

Exp. 104. Mix in a large test-tube or small flask a little dry 
slaked lime and salicylic acid, connect with a beaker condenser 
(see cut on page 264) and distil. Test distillate for phenol. 
Write reaction. 

Note. After the first heating, the tube containing the lime and acid may be 
inclined so that any moisture in distillate will run into collecting tube rather than 
back on to the mixture. 

Exp. 105. To a few cubic centimeters of a 3 per cent phenol 
solution add dilute bromine water. A yellowish-white, crystal- 
line precipitate of tribromphenol is produced (see page 77). 

Exp. 1 06. To an aqueous solution of phenol add a few drops 
of solution of ferric chloride. 

Exp. 107. To 5 c.c. of an aqueous solution of phenol add 
one quarter of its volume of ammonia water and then a few drops 
of sodium hypochlorite solution. Mix and warm. A blue-green 
color develops, turning red upon addition of hydrochloric acid 
to slight acid reaction. 

Exp. 108. Repeat Exps. 105 and 106, using an aqueous solu- 
tion of cresol in place of phenol. 

Exp. 109. To a test-tube one-third full of nitric acid (50 per 
cent absolute HNOs), add, i drop at a time, about i c.c. of phenol 
with constant agitation. When all the phenol has been added, 
heat carefully to boiling. Allow to cool slowly, when trinitro- 
phenol will be precipitated. 

Exp. no. Evaporate a few drops of a i per cent solution of 
potassium nitrate to dryness in a small porcelain capsule. Add 
2 c.c. of phenoldisulphonic acid;* stir thoroughly, and keep hot 
for three to five minutes; dilute with water, make strongly 
alkaline with ammonia, and note the intense yellow color of 
ammonium picrate. The reaction is used as a test for nitrates 
in drinking water. 

Exp. in. Determine melting-point of benzoic acid. 

Exp. 112. Arrange two watch glasses of equal size with the 
concave surfaces together and a piece of filter-paper stretched 
between them. The glasses may be held together with a small 
brass clamp. 

* For method of preparation see Appendix. 



EXPERIMENTS 267 

A little benzoic acid placed in the lower glass may be sub- 
limed by means of a gentle heat through the paper, and collected 
upon the upper glass. Examine the sublimate by polarized 
light. See Plate V, Fig. 5, opposite page 99. 

Exp. 113. Preparation of benzoic acid from toluene (oxidation 
of side chain). Place 5 c.c. of toluene in a 250 c.c. boiling-flask 
containing 125 c.c. of distilled water and 7 grams of KMn0 4 . 
Shake mixture well. Connect flask with a reflux condenser and 
boil for at least thirty minutes. If the permanganate color 
disappears during this time add 3 grams more of KMnC>4. 
Remove condenser and filter solution. The filtrate will contain 
excess permanganate and potassium benzoate. Let cool slightly 
and then add 20 per cent H 2 S0 4 . Heat to boiling and add 
sufficient oxalic acid to cause the solution to become colorless. 

When the solution has been cooled by immersion in ice-water, 
crystals of benzoic acid will separate out, and may easily be 
filtered off and dried. 

Exp. 114. With an aqueous solution of benzaldehycle, deter- 
mine whether Tollen's test for aldehydes (Exp. 30) is applicable 
to aromatic compounds. 

Exp. 115. Boil 10 c.c. of oil of wintergreen with a little 
20 per cent NaOH; keep the volume constant by frequent 
addition of water. When the oil has entirely disappeared, cool 
and add HC1 to acid reaction. Salicylic acid will separate, 
white and crystalline. 

Exp. 1 1 6. To a dilute solution of sodium salicylate, or satu- 
rated aqueous solution of salicylic acid, add a few drops of 
Fe2Cle. A slight amount of salicylates in the urine will produce 
this color when a test is being made for diacetic acid (q.v.). 

Exp. 117. Preparation of methyl salicylate (synthetic oil of 
wintergreen). Dissolve 15 grams of salicylic acid in 60 c.c. of 
absolute methyl alcohol. Very gradually add 30 c.c. strong 
H 2 SO 4 and let stand in a warm place for twenty-four hours, 
covered. Add half its volume of water and distil slowly. 



268 



EXPERIMENTS 



EXPERIMENTS FOR PHYSIOLOGICAL CHEMISTRY. 
Preparation of Oxidase. 

Exp. 1 1 8. Clean thoroughly a small potato and grate the 
skin into a small beaker; cover with water and allow to stand 
in a cool place for an hour. Filter through coarse paper. Turn 

B C 






FIG. 24. 

about 5 c.c. of the filtrate slowly into 25 c.c. of strong alcohol. 
The enzyme will be precipitated. Filter and test as follows: 

Exp. 119. Transfer the moist precipitate from the above 
experiment into a test-tube half-filled with distilled water. Shake 
frequently for about ten minutes and filter. The filtrate will 
contain oxidizing enzymes in solution. Divide the solution into 
two parts; to one add a few drops of tincture of guaiacum, and 
to the other a little of a i per cent solution of pyrocatechol. The 
guaiacum gives a blue color, and the pyrocatechol a red-brown 
color in the presence of oxidizing enzymes. 



EXPERIMENTS 



269 



Q-G 



Experiments with Enzymes. 

Hydrolytic enzymes produce cleavage of the molecule, 

Exp. 120. Take four test-tubes a-b-c-d. Make a thin paste 
by rubbing one-sixth of a yeast cake with water, and place a 
little in each of the four tubes; then fill a with a dilute glucose 
solution; b with a dilute solution 
of milk-sugar; c with dilute solu- 
tion of cane-sugar; prepare d in 
the same manner as c, but before 
adding the sugar solution, boil the 
enzyme (yeast) for at least one 
minute. Have each tube full and 
fit with a delivery tube (Fig. 24, 
page 268), making provision to 
collect any liquid which may be 
forced out of the tube. Allow the 
four tubes to stand overnight and 
then test the liquid which has 
been forced out of the tubes during 
the night for alcohol (Exp. 15), 
and the gas for C0 2 by means of 
forcing it into baryta-water. (This 
may be done by use of a special 
stopper fitted with thistle tube and 
delivery tube.) 

Exp. 121. Take four test-tubes, a-b-c-d, arrange as indicated 
in Fig. 25, and half fill each with some thin starch paste (see page 
301 of Appendix). Into a put a little of the yeast from last 
experiment; into b a little pepsin solution; into c a little saliva 
(the enzyme of the saliva is ptyalin) ; into d a little invertase, as 
used in preceding experiment. Warm all the tubes to about 37 
or 38 C., and allow to stand overnight; then test contents of 
each tube for a reducing sugar which may have been produced 
from the starch. (Use Exp. 129.) 

Exp. 122. The student may prepare a fat-splitting enzyme 
(lipase) from an animal source, pig's pancreas, or from a 




FIG. 25. 



270 EXPERIMENTS 

vegetable source, castor beans, according to direction in the 
Appendix, 

Fat Digestion with Lipase (Castor Bean). Grind with the 
powder,* in the order named, 5 c.c. N/io sulphuric acid, 5 c.c. of 
neutral cotton oil (sp. gr. 0.92) and 5 c.c. lukewarm water. The 
water should be added a little at a time and thoroughly worked 
into the mixture so that at the end of the operation a good 
emulsion is secured. Cover the evaporating dish and let 
stand in a warm place overnight. 

Add 50 c.c. of alcohol, 10 c.c.of ether, and a few drops of 
phenolphthalein, and titrate with N/i sodium hydrate. Calcu- 
late the amount of fatty acid and the per cent of fat digestion. 

Exp. 123. In a test-tube one- third full of milk, colored 
slightly blue with nearly neutral litmus solution, place half as 
much solution of lipase (fresh pancreatic extract) and keep at 
about 40 C. for twenty to thirty minutes. Sufficient fat acid 
should be separated to change the blue litmus to red. Write 
reaction. 

Experiments with Sugars. 

Exp. 124. Fill a test-tube about one-third full of dry straw. 
Cover with 10 per cent hydrochloric acid; boil, collecting the 
distillate in a dry tube. Divide the distillate into two parts, 
and make the following tests for furfuraldehyde which has been 
produced from the pentose contained in the straw. Treat the 
contents of one tube with a little aniline and hydrochloric acid. 
Red coloration indicates the presence of furfuraldehyde. To the 
contents of the other tube add a little solution of casein (skimmed 
milk) and underlay with strong sulphuric acid. Furfurol will 
give a blue or purple line at the point of contact of the two liquids. 

Monosaccharides. Exp. 125. Test for C and H, using 
cane-sugar. Make closed-tube test for H, which is given off as 
H 2 O, and for C, which remains as such in tube. (See Vol. I.) 
Write reactions. 

Exp. 126. Molisch's test for Carbohydrates. To a few cubic 
centimeters of a 3 per cent glucose solution add a few drops of 

* For preparation of powder, see page 299. 



EXPERIMENTS 



271 




an alcoholic solution of a-naphthol, and carefully underlay the 
mixture with strong H 2 SO 4 . 

Exp. 127. To a few cubic centimeters of CuSO 4 solution 
in a test-tube add a little NaOH. Boil and write reaction. 

Exp. 128. Repeat Exp. 127 with the addition of Rochelle 
salt; if solution remains clear on boiling, add a few drops of a 
glucose solution. 

Exp. 129. Fehling's Test for Sugars. Take about 5 c.c. of 
Fehling's solution, made by mixing equal parts of the CuS0 4 
solution and the alkaline tartrate on side shelf. 
Boil and add immediately a few drops of glu- 
cose solution. Set aside for a few minutes, 
watching the results. 

Exp. 130. Repeat Exp. 129, using diabetic 
urine instead of glucose. 

Exp. 131, Repeat Exp. 129 without heat 
and allow to stand for twenty-four hours. 

Exp. 132. To 5 c.c. of Benedict's solution 
(for preparation see Appendix) add 8 or 10 drops 
of a 2 per cent glucose solution. Heat the mix- 
ture to boiling; keep at this temperature for 
one or two minutes. 

Exp. 133. Barfoed's Test. To about 5 c.c. 
of Barfoed's reagent add a few drops of glucose 
solution; boil and set aside for a few minutes, 

riu. 2u. 

watching results. 

Exp. 134. Fermentation Test. Fill the " fermentation 
tube " (Fig. 26) found in the desk with glucose solution; add a 
little yeast; insert stopper, with long arm of tube extending into 
glucose mixture nearly to bottom of tube, and allow it to stand 
upright, in a warm place, overnight. On the next day, test the 
gas, with which the tube is filled, with lime-water. 

Exp. 135. Phenylhydrazine Test. Place about 5 c.c. of 
glucose solution in a test-tube; add an equal volume of phenyl- 
hydrazine solution; keep the tube in boiling water for thirty 
minutes. Allow to cool gradually. Examine the precipitate 
microscopically and sketch the crystals. 



272 EXPERIMENTS 

Disaccharides. Exp. 136. Use dilute solutions of cane- 
sugar, milk-sugar, and maltose, and make on each Fehling's 
test (Exp. 129), Barfoed's test (Exp. 133), and the phenylhy- 
drazine test (Exp. 135). Sketch the different osazone crystals 
obtained. 

Exp. 137. To a dilute solution of cane-sugar add a few 
drops of dilute H 2 SO 4 and boil for five minutes. Cool the 
mixture and make slightly alkaline with NaOH. With this 
solution perform Exps. 129, 133, and 135. Explain results. 
Compare with Exp. 136. 

Experiments with Starches and Cellulose. 

Polysaccharides. Exp. 138. Examine potato, corn, and 
wheat starch under the microscope, use a drop of water and a 
cover glass. Sketch the granules of each in notebook, and, 
while still on the slide, treat with a dilute iodine solution. Note 
changes in appearance of granules. 

Exp. 139. Preparation of starch. Grate a little raw potato. 
Mix thoroughly with water and strain through " bolting " cloth 
or stout, coarse muslin. After the liquid has run through, com- 
press the cloth by twisting till no more liquid can be squeezed 
out. The starch has passed through the cloth and may be 
washed by decantation, dried on filter paper, examined, and used 
for the following experiments: 

Exp. 140. Make some starch paste by rubbing i gram of 
starch to a smooth, thin paste with water; then slowly pour it 
into 100 c.c. of boiling water, stirring constantly. With this 
solution compare a i per cent solution of dextrine and a solution 
of glycogen* as follows: 

(a) Treat each by boiling with Fehling's solution. 

(b) Add to 5 c.c. of each a few drops of tannic-acid solution. 

(c) To each solution add a drop of iodine solution. Note 
color of mixture while cold. Heat nearly to boiling and allow 
to cool again, watching the color during process. 

(d) To 5 c.c. of each solution add twice its volume of 66 per 
cent alcohol. 

* For the isolation of glycogen, see Appendix. 



EXPERIMENTS 273 

(e) Tabulate results of the tests and formulate method of 
distinguishing these three substances from one another. 

Experiments with Fats and Oils. 

Exp. 141. Test solubility of olive oil in water, ether, chloro- 
form, and alcohol, carefully avoiding the vicinity of a flame. 

Exp. 142. Let one or two drops of an ether solution of the 
oil drop on a plain white paper, also an ether solution of a volatile 
oil found on side shelf. Watch behavior of the two oils, and 
report differences, if any. 

Exp. 143. Dissolve a little butter in warm alcohol, examine, 
with the microscope and micropolariscope, the crystals which 
separate on cooling. 

Note. If possible perform the next experiment in triplicate, i.e., carry three 
experiments along at the same time, using for " fat " the glyccryl ester of the three 
most common fat acids: olein (lard oil or olive oil), stearin (beef fat or tallow), 
palmatin (bayberry wax or tallow, which contains a large amount of free palmitic 
acid) . 

Exp. 144. Saponification. To about 2 grams of solid fat 
placed in a narrow beaker, or i5o-c.c. Erlenmeyer flask, add 10 
or 15 c.c. of alcoholic solution of potassium hydroxide. Allow 
the beaker to stand on the water-bath till the alcohol is entirely 
evaporated, then dissolve the resulting soap in water; filter, if 
necessary, to obtain a clear solution, and make the following tests: 

(a) Add to a portion of solution a saturated solution of so- 
dium chloride. What takes place? 

(6) To another portion add a few cubic centimeters of a so- 
lution of calcium or magnesium chloride. Explain the results. 

(c) Pour the remainder slowly, and with constant stirring, 
into warm dilute H 2 SO 4 , and heat on the water-bath. What is 
the result? Write the equation. Transfer the mixture to a 
filter-paper which has been moistened with hot water, and wash 
with hot water till all H 2 SO 4 is removed. Reserve the filtrates. 

Exp. 145. Fatty acids. 

(a) Dissolve a portion of the above precipitates (144 c) by 
warming with strong alcohol. Test the reaction of the solution. 
Examine the crystals, which separate upon standing, with micro- 
scope and micropolariscope. (Plate VII, Fig. 3, page 185.) 



274 EXPERIMENTS 

(6) Add to a portion a few cubic centimeters of a strong 
Na 2 CO 3 solution, and heat till the fatty acids dissolve. Cool. 
What takes place? Explain the reaction. Reserve the jelly. 

Exp. 146. Neutralize the filtrates of Exp. 144 c and evaporate 
almost to dryness on the water-bath. Extract with alcohol 
and evaporate. Note the taste. Heat another portion of the 
residue with a little powdered dry KHSO4 in a dry test-tube, 
and note the odor, which is due to acrolein, CH2 = CH CHO. 
Fuse some borax and glycerin on a platinum loop: green color. 

Exp. 147. Emulsification. (a) Put i to 2 c.c. of a solution 
of sodium carbonate (0.25 per cent on a watch glass, and place in 
the center a drop of rancid oil. The oil-drop soon shows a 
white rim, and a white milky opacity extends over the solution. 
Note with the microscope the active movements in the vicinity 
of the fat-drop, due to the separation of minute particles of oil 
(Gad's experiment). 

(6) Take six test-tubes and arrange as follows: 

1. 10 c.c. of a 0.2 per cent Na 2 CO 3 solution+2 drops of 

neutral oil. 

2. 10 c.c. of a 0.2 per cent Na 2 CO 3 solution+2 drops of 

rancid oil. 

3. 10 c.c. of soap-jelly, warm, + 2 drops of neutral oil. 

4. 10 c.c. of albumin solution+2 drops of neutral oil. 

5. 10 c.c. of gum-arabic solution+2 drops of neutral oil. 

6. 10 c.c. of water +2 drops of neutral oil. 

Shake all the mixtures thoroughly and note the results. 
What conclusions do you form relative to the influence of con- 
ditions upon emulsification? 

(c) Examine a drop of an emulsion under the microscope. 

Identification of Fats. 

Exp. 148. Determination of the Reichert-Meissel number for 
volatile fatty acids. In an Erlenmeyer flask of 250 c.c. capacity, 
saponify 5 grams of fat with N/2 alcoholic NaOH until solution 
is free from fat globules. Evaporate on a water-bath and 
dissolve the dry soap in 100 c.c. of warm water. Cool and add 



EXPERIMENTS 275 

a few cubic centimeters of 10 per cent H 2 SO 4 . Connect flask with 
a condenser, and distil. After the distillation has continued for 
thirty minutes, disconnect apparatus and titrate the entire 
distillate with N/io NaOH. 

The number of cubic centimeters of N/io alkali necessary to 
neutralize the volatile fatty acids obtained from 5 grams of fat is 
the Reichert-Meissel number of the fat. 

Exp. 149. Determination of the iodine absorption number for 
fat. Place i gram of fat or .2 to .4 gram of oil in a 250 c.c. 
flask. Dissolve in 10 c.c. of chloroform and then add 30 c.c. of 
prepared iodine solution (iodine and mercuric chloride). Shake 
and place flask in a dark place for three hours, shaking frequently. 
Add 20 c.c. of KI solution and 100 c.c. of water. Titrate excess 
iodine solution with standard thiosulphate solution, using starch 
paste as an indicator. 

Calculate the amount of iodine absorbed by i gram of fat. 

Lipoid Experiments. 

Exp. 150. Boil 75 grams of brain tissue with 150 c.c. strong 
alcohol for thirty or forty minutes, using a reflux condenser and 
a water-bath. Filter hot, and allow to cool overnight. A 
cloudy precipitate will consist of a mixture of the so-called 
lipins or lipoids (cerebrin, cholesterol, lecithin, cephalin.) If 
the precipitate is very slight, concentrate the solution by evapo- 
rating and cool again. Filter on black paper and make following 
tests. 

(1) A little of the precipitate mixed thoroughly with water 
and examined under the microscope will show myelin movements 
(due to cerebrin). 

(2) With another portion of the precipitate, make micro- 
chemical test for phosphorus (showing presence of phosphorized 
fat). 

(3) In a small test-tube, heat a portion with fixed alkali and 
if possible detect fishy odor of trimethylamine (chlolein). 

(4) Warm (on a microscope slide) a little precipitate with a 
few drops of alcohol. Look for crystals of cholesterol. See 
also Exp. 239. 



276 EXPERIMENTS 

(5) If sufficient precipitate remains, hydrolyze for one hour 
with dilute hydrochloric acid, neutralize and test for a reducing 
sugar (galactose). 

Exp. 151. In a thoroughly dry test-tube place 2 c.c. of chlo- 
roform, in which dissolve a little cholesterol. Add about 10 
drops of acetic anhydride and two drops of concentrated sul- 
phuric acid. The mixture produces a violet color quickly 
changing to blue-green. (Liebermann-Burchard acetic an- 
hydride test). 

General Protein Reactions. 

Exp. 152. Test dried egg-albumin for C, H, S, and N, ac- 
cording to the methods described on pages 2, 3 and 4. Test 
casein for phosphorus, and dried blood for iron. 

There are several reactions which are common to nearly all 
proteins. For the following tests use a solution of egg-albumin 
(1/50) in water, as a general type of a protein. 

i. Color Reactions. 

Exp. 153. Xanthoproteic test. To 10 c.c. of the albumin 
solution add one- third as much concentrated HN0 3 ; there may 
or may not be a white precipitate produced (according to the 
nature of the protein and the concentration). Boil; the pre- 
cipitate or liquid turns yellow. When the solution becomes 
cool add an excess of NH 4 OH, which gives an orange color. 
(This color constitutes the essential part of the test.) 

Exp. 154. Millon's test. Add a few drops of Millon's re- 
agent* to a part of the albumin solution. A precipitate, which 
becomes brick-red upon heating, forms. The liquid is colored 
red in the presence of non-coagulable protein or minute traces 
of albumin. 

Exp. 155. Piotrowski } s test. To a third portion add 2 
drops of a wry dilute solution of CuSO 4 , and then 5 to 10 c.c. 
of a 40 per cent solution of NaOH. The solution becomes blue or 
violet. Proteoses and peptones give a rose-red color (biuret 

* Mercuric nitrate in nitric acid. For the preparation of this and other re- 
agents, see Appendix. 



EXPERIMENTS 277 

reaction) if only a trace of copper sulphate is used; an excess 
of CuSO 4 gives a reddish-violet color, somewhat similar to that 
obtained in the presence of other proteins. All proteins respond 
to this test. 

Exp. 156. Hopkins-Cole reaction. Mix 2 or 3 c.c. of the 
unknown protein solution with 3 or 4 c.c. of the reagent (gly- 
oxylic acid). Then carefully superimpose upon 5 c.c. of strong 
sulphuric acid in another test-tube. 

The glyoxylic acid is made by the reduction of oxalic acid 
with nascent hydrogen produced by the action of sodium amal- 
gam and water. Formula is CHO.COOH. 

2. General Precipitanis. 

Proteins are precipitated from solution by the following re- 
agents (peptones are exceptions in some cases) : 

Exp. 157. Acetic acid and potassic ferrocyanide. Make 
part of the solution to be tested strongly acid with acetic acid, 
and add a few drops of potassic ferrocyanide solution. A white 
flocculent precipitate is formed (not with peptones). 

Exp. 158. Alcohol. To another part add one or two vol- 
umes of alcohol. 

Exp. 159. Tannic acid. Make the solution acid with 
acetic acid, and add a few drops of tannic-acid solution. 

Exp. 1 60. Potassio-mercuric iodide. Make acid another 
portion with HC1, and add a few drops of the reagent. 

Exp. 161. Neutral salts. Certain neutral salts precipitate 
most proteins. (NH^SO^ added to complete saturation to 
protein solutions, faintly acid with acetic acid, precipitates all 
proteins, with the exception of peptones. 

Experiments with Albumin and Globulin. 

The albumins and globulins respond to all the general protein 
reactions. Experiments 153 to 161. 

Exp. 162. A specimen of solid egg-albumin, prepared by 
evaporating a solution to dryness at 40 C., is provided. Test 
its solubility in water, alcohol, acetic acid, KOH solution, and 
concentrated HC1. Report results. 



278 EXPERIMENTS 

Perform the following additional experiments, using a dilute 
(1/50) solution of egg-albumin. 

Exp. 163. Nitric-acid test. Take 15 c.c. of the solution in 
a wine-glass, incline the glass, and allow 5 c.c. of concentrated 
HNOs to run slowly down the side to form an under layer. 
What other proteins respond to this test? 

Exp. 164. Picric-acid test. Take a portion of the albumin 
solution and add a few drops of a solution of picric acid acidified 
with citric acid (Esbach's reagent). What other proteins re- 
spond to this test? 

Exp. 165. Action of ( N H^SO*. To 10 c.c. of the albumin 
solution in a test-tube add some solid (NH 4 ) 2 SO4, shaking until 
solution is thoroughly saturated. Allow to stand a little while, 
shaking occasionally, then filter, saving the filtrate to test for 
albumin by the heat test. Report result. Test the solubility 
of the precipitate on the filter-paper. 

Exp. 166. Action of MgSO. Perform an experiment 
similar to Exp. 165 using solid MgSCX instead of (NH 4 ) 2 SO4. 
With what results? 

Exp. 167. Salts of the heavy metals. Note the action of 
the following: AgNO 3 , HgCl 2 , CuS0 4 , Pb(C 2 H 8 2 )2. Use solu- 
tions of the salts and of albumin. 

Why is white of egg an antidote in cases of metallic poisoning? 

The following tests serve to distinguish the globulins from 
other proteins. 

The tests may be made upon blood serum, or upon a globulin 
(edestin) which may be separated from hemp seed according to 
preparation in Appendix, page 306. 

Globulins. 

Exp. 168. Action of CO 2 . To 5 c.c. of blood serum add 
45 c.c. of ice-cold water. Place the mixture in a large test-tube 
or cylinder, surround it with ice-water, and pass through it a 
stream of C0 2 . A flocculent precipitate (paraglobulin) will be 
formed. 

Exp. 169. Precipitation by dialysis. Into a parchment 
dialyzing tube, previously soaked in distilled water, pour 20 c.c. 



EXPERIMENTS 279 

of serum; swing the tube, with its contents, into a large vessel 
of distilled water, which is to be changed at intervals. Let 
stand twenty-four hours, then examine the serum in the dialyz- 
ing tube; it will contain a flocculent precipitate of paraglobulin. 
Give explanation of cause of precipitation. 

Exp. 170. Pour a solution of globulin, drop by drop, into a 
large volume of distilled water (in a beaker). What takes 
place? Explain. 

Exp. 171. Precipitation by magnesium sulphate. Saturate 
about 5 c.c. of globulin solution with solid magnesium sulphate. 
A heavy precipitate will be formed. Compare this with the 
action of the same salt on the egg-albumin solution. Paraglob- 
ulin is so completely precipitated by this salt that the method 
is used for its quantitative estimation. 

Experiments with Keratin and Gelatin. 

Keratins are characterized by their insolubility, and by their 
high content of loosely combined sulphur. 

Exp. 172. Test solubility of keratin (nail or horn) in water, 
acids, alkalies, gastric and pancreatic juices. 

Exp. 173. Warm a bit of keratin with 5 c.c. strong NaOH 
solution for a few minutes, and add a few drops of a lead acetate 
solution. What is the result? 

Exp. 174. With a solution of gelatin make the usual tests 
for protein. 

Exp. 175. Precipitate gelatin from dilute solution with the 
following reagents: 

(a) Tannic acid. 

(6) Alcohol. 

(c) Acetic acid and potassium ferrocyanide. 

(d) Mercuric chloride. 

(e) Picric acid. 

Experiments with Milk. 

Exp. 176. Examine microscopically whole milk, skim-milk, 
and cream. Note the relative amounts of fat in the three 
varieties. 



2&0 EXPERIMENTS 

Exp. 177. Shake a little cream with chloroform in a test- 
tube; separate the chloroform, evaporate, and melt the fat 
residue obtained; allow it to cool slowly , when fat crystals will 
be obtained, which may be examined under the microscope and 
micropolariscope . 

Exp. 178. With a lactometer take the specific gravity of 
whole milk and skim-milk and explain the difference in results. 

Exp. 179. Test the reaction of milk with litmus. 

Exp. 1 80. Dilute some milk with six or seven times its 
volume of water, and add acetic acid, drop by drop, till the 
paracasein is precipitated. Filter and reserve the precipitate. 
Test the filtrate for proteins, if any remain; determine their 
character if possible. 

Exp. 181. Test another portion of the filtrate for carbohy- 
drates, determining the variety present. 

Exp. 182. To 50 c.c. of milk add a few drops of rennin solu- 
tion; keep at a temperature of 40 C. for a few minutes, and 
explain results. 

Exp. 183. Determination of total nitrogen- Kjedahl method. 
Take 5 c.c. of milk (dilute i-io) in a 300 c.c. long-neck Kjehdahl 
flask. Add 15 c.c. of strong H 2 S0 4 and .1 to .2 gram of CuS0 4 . 
Heat, cautiously at first, gradually raising the temperature until 
white fumes appear in the flask. Cover with a watch-crystal. 
Reduce the heat somewhat and digest until clear. It may be 
necessary to add more sulphuric acid during the process. Allow 
to cool, then dilute with 100 c.c. of ammonia-free water. 
Add a slight excess of strong NaOH solution and introduce into 
the flask a few glass beads or granular zinc to prevent bumping 
during distillation. Connect with a condenser and distil off 
about half of the solution, conducting the distillate into 30 c.c. of 
N/io HC1. 

Titrate the excess acid with N/io NaOH and calculate the 
number of cubic centimeters of N/io acid used in neutralizing 
the ammonia produced from the nitrogen of the protein. Cal- 
culate as nitrogen. 

Exp. 184. Take a portion of the precipitated paracasein from 
Exp. 180, digest at 40 C. with pepsin HC1 for twenty minutes 



EXPERIMENTS 281 

or half an hour. While digesting, test other portions of para- 
casein, for solubility in water, in dilute acid and dilute alkali. 
Also test a portion for phosphorus by boiling in a test-tube with 
dilute nitric acid, cooling to at least 50 C., and adding ammo- 
nium molybdate solution. 

Exp. 185. To a little skim-milk contained in a test-tube add 
a saturated solution of ammonium sulphate. 

Experiments with Mucin. 

Exp. 1 86. To a solution of mucin* found on the side shelf 
add acetic acid till precipitation takes place. Settle, filter, 
wash, and test solubility in water, dilute alkali solution and 
5 per cent HC1. 

Exp. 187. Make color-tests for proteins. 

Exp. 188. Boil a little mucin solution with dilute HC1 for 
several minutes. Cool, neutralize, and test for sugar. 

Experiments with Protein Derivatives. 

Exp. 189. Preparation of meta-protein. To a solution of 
egg-albumin add a few drops of a 0.5 per cent solution of NaOH, 
and warm gently for a few minutes. With the solution thus 
obtained make the following tests : 

Exp. 190. (a) Effect of heating. Boil some of the solution 
and report result. 

(b) Effect of neutralizing. Add a drop of litmus solution, 
and cautiously neutralize. 

Acid Meta-protein. 

Exp. 191. Add a small quantity of a 0.2 per cent HC1 solution 
to a solution of egg-albumin, and warm at 40 C. for one-half to 
one hour. Or cover with an excess of 0.2 per cent HC1 some meat 
cut into fine pieces, and expose for a while to a temperature of 
40 C. Filter. With either of the solutions thus obtained 
make same tests as on alkali meta-protein, and compare results. 
How distinguish between them? 

Exp. 192. Determine whether cheese forms a meta-protein, 

* For preparation of mucin solution from navel cord, see Appendix. 



282 EXPERIMENTS 

and if so whether the acid or alkali is most easily produced. 
Write detail of the experiment in laboratory notebook. 

Experiments with Proteases. 

Albumoses (hemialbumose) . This name includes four closely 
allied forms of albumose, namely: (i) protoalbumose, (2) 
deuteroalbumose; (3) heteroalbumose; (4) dysalbumose, an 
insoluble modification of heteroalbumose. Commercial peptone, 
which is substantially a mixture of albumoses and peptones, will 
be given out for use. 

Exp. 193. Make a solution of the peptone in water, filter 
if necessary, and saturate with solid (NH 4 ) 2 SO 4 . Filter. The 
precipitate contains the albumoses, the filtrate the peptones. 
Reserve the filtrate for subsequent tests for peptone. Wash the 
precipitate with a saturated solution of ammonium sulphate; 
dissolve in water, and, with the solution obtained, perform the 
following tests, noting especially the tendency of albumose pre- 
cipitates to dissolve upon the application of heat and to reappear 
upon cooling. 

Using this solution of albumose, repeat Exps. 153, 154, 155, 
163, 164. If no precipitate forms with HNO 3 in Exp. 153, add 
a drop or two of a saturated solution of common salt. (Deutero- 
albumose gives this reaction only in the presence of HCL) 

Exp. 194. Saturate some of the solution with (NH 4 ) 2 SO4. 
Report the result. 

Exp. 195. To some of the solution add 2 or 3 drops of acetic 
acid and then a saturated solution of NaCl. A precipitate 
forms, which dissolves on heating, and reappears on cooling. 

Experiments with Peptones. 

Exp. 196. Using the peptone solution prepared, in the manner 
above described, from commercial peptone, repeat the experi- 
ments indicated in Exp. 193. 

Exp. 197. Effect of heating. Boil some of the peptone 
solution. Report the result. 

Exp. 198. Power of dialyzing. Dialyze some of the peptone 
solution. Use 10 c.c. of the peptone solution, and in the outside 



EXPERIMENTS 283 

vessel about 100 c.c. of water, which in this case is not to be 
changed. After twenty-four hours test the outside water for 
peptone, employing the biuret test. 

Exp. 199. Action of ammonium sulphate. Saturate some 
of the peptone solution with solid (NH 4 ) 2 S0 4 . Report the result. 

A number of unknown solutions will be given out to be tested 
for carbohydrates and proteins. A report of the results, to- 
gether with the methods employed, is to be made. 

Experiments on Blood. 

Exp. 200. Test the reaction of blood with a piece of litmus 
paper which has been previously soaked in a concentrated NaCl 
solution. To what is reaction due? 

Exp. 201. Blood-corpuscles. (a) Examine a drop of blood 
under the microscope. Sketch the red and white corpuscles. 

(b) Note the difference between the corpuscles of mammals 
and those of birds and reptiles. 

(c) Note the effect upon the red corpuscles produced by the 
addition of (i) water, (2) a concentrated solution of salt. 

Exp. 202. Hemoglobin crystals. Place a drop of defibrinated 
rat's blood on a slide; add a drop or two of water; mix, and 
cover with a cover-glass. Sketch the crystals which separate 
after a few minutes. Or instead of above, add a few drops of 
ether to some blood in a test-tube; shake thoroughly until the 
blood becomes " laky," and then place the tube on ice till crystals 
appear. 

Exp. 203. A spectroscope will be found ready for use in the 
laboratory, and the absorption-bands given by oxyhemoglobin 
and hemoglobin will be demonstrated. The student may pre- 
pare solutions for examination as follows: 

(a) Oxyhemoglobin. Use dilute blood (i part of defibrinated 
blood in 50 parts of distilled water). 

(b) Hemoglobin (reduced hemoglobin). Add to blood a few 
drops of strong ammonium sulphide, or i or 2 drops of freshly 
prepared Stoke's reagent.* Note the change in color produced 

* Stoke's reagent consists of 2 parts of ferrous sulphate and 3 parts of tartaric 
acid dissolved in water, with ammonia added to distinct alkaline reaction. There 
should be no permanent precipitate. 



284 EXPERIMENTS 

by the addition of the reducing agent. Shake with air and note 
the rapid change to oxy hemoglobin. 

(c) Hemochromogen. To a little of the hemoglobin, re- 
duced with ammonium sulphide, add a few drops of concen- 
trated NaCl, and note the spectrum of reduced hematin or 
hemochromogen. 

(d) Carbon-monoxide hemoglobin. Pass a current of illumi- 
nating gas through a dilute oxyhemoglobin solution for a few 
minutes and filter. Note the change of color. Try the effect on 
the solution of (i) ammonium sulphide; (2) Stoke's reagent; 
(3) shaking with air. Note the stability of the compound. 

Exp. 204. Take the specific gravity of blood by filling a test- 
tube one-half full of benzene; add one drop of blood, and then 
add chloroform, a drop at a time, with careful but thorough mix- 
ing, until the drop of blood floats at about the middle of the 
mixture, indicating that the gravity of the mixture and of the 
blood are the same. The specific gravity of the benzene and 
chloroform may be taken in any convenient way. 

Exp, 205. Make the guaiacum test for blood on a sample 
of dried blood; also on potato scrapings. The method is as 
follows : 

Boil a little clear solution of blood for twenty seconds. Add 
one drop tincture of guaiacum and then a few drops of an ethereal 
solution of hydrogen peroxide; shake the mixture and note the 
blue color obtained. 

Try the same test with material obtained from potato scrapings 
with and without boiling. 

Exp. 206. The benzidine reaction consists in adding to a 
few cubic centimeters of a saturated benzidine solution in glacial 
acetic acid or alcohol acidified with acetic acid an equal volume of 
commercial H 2 O 2 and i c.c. of the suspected solution. If blood is 
present a green or blue color will develop. It is better to make a 
blank test to insure purity of reagents. 

Exp. 207. Hemin crystals (Teichmann's test). Place a 
bit of powdered dried blood on a glass slide; add a minute 
crystal of NaCl (fresh blood contains sufficient NaCl) and two 
drops of glacial acetic acid. Cover with a cover-glass and warm 



EXPERIMENTS 285 

gently over a flame until bubbles appear. On cooling, dark- 
brown rhombic crystals, often crossed, separate (chloride of 
hematin). Similar crystals can be obtained by using an alka- 
line iodide or bromide in place of NaCl. 

Exp. 208. Coagulation of blood. Observe the phenomenon 
of coagulation as it takes place (a) in a test-tube; (b) in a drop 
of blood examined under the microscope. Explain fully. 

Exp. 209. Proteins of blood-plasma. (a) Serum-albumin. 
(6) Serum-globulin. Using blood-serum, separate and identify 
these two proteins. 

(c) Fibrinogen. Fibrinogen is a globulin found in blood- 
plasma, lymph, etc., together with paraglobulin. Like para- 
globulin it responds to all the general precipitants and tests, and 
in addition gives the reactions with CO 2 , dialysis, and MgSO4. 
It is easily distinguished from paraglobulin by two reactions, viz., 
its power to coagulate, i.e., to form fibrin when acted on by fibrin 
ferment, and its temperature of heat coagulation, which will be 
found to be from 56 to 60 C. 

Exp. 210. Fibrin. (a) Note its physical properties. 

(6) Note action of 0.2 per cent pepsin hydrochloric acid. 

(c) Apply the protein color tests. 

Experiments with Muscle. 

Exp. 2ii, Place 25 grams of fresh, finely chopped muscle 
in a beaker with 75 c.c. of 5 per cent solution of common salt, and 
allow to stand for about one hour, with frequent stirring. (In 
the meanwhile perform Exp. 212.) Then filter off the liquid and 
make the following tests with the filtrate. 

(a) Test for proteins. 

(b) Having found proteins, pour a little of the solution into 
a beaker of water. Result. Inference (myosin). 

(c) Make a fractional heat coagulation in the following man- 
ner (upon the care with which the temperatures given are ad- 
hered to, depends the success of the separation) : Warm to from 
44 to 50 C., and keep at that temperature for a few minutes. 
The coagulum is myosin [synonyms: paramyosinogen (Halli- 
burton), musculin (older authors)]. In solutions the myosin, 



286 EXPERIMENTS 

which has the properties of a globulin, becomes insoluble after a 
time, because it changes to myosinfibrin. In heating the solu- 
tion as above, a slight cloud may appear at from 30 to 40 C. 
This is due to coagulation of soluble myogenfibrin. Now filter 
off the coagulated myosin. 

Heat filtrate to from 55 to 65 C. The coagulum is myogen 
(synonym: myosinogen). In spontaneous coagulation of its 
solutions it forms, first, soluble myogenfibrin, and, finally, in- 
soluble myogenfibrin. Filter. 

Heat to from 70 to 90 C. Coagulum is serum-albumin from 
the blood within the muscle, and is not a constituent of the 
muscle plasma. Filter. 

Test filtrate for proteins. If it shows a slight biuret test, 
this is due either to incomplete precipitation by coagulation 
or to the post-mortem formation of albumose or peptone by 
auto-digestion (autolysis). 

Exp. 212. Make an aqueous extract of muscle, and test for 
lactic acid by acidulating with H 2 SO 4 , extracting with ether, 
and testing the ethereal extract with very dilute ferric chloride 
solution. The presence of lactic acid is shown by a bright- 
yellow color. (See Exp. 60.) 

DIGESTION EXPERIMENTS. 
Experiments with Saliva. 

Exp. 213. Action of saliva upon starch. Take some fil- 
tered saliva in a test-tube and place in the water-bath at 40 C., 
for five or ten minutes. Put some starch paste into a second 
test-tube and place this also in the water-bath for a while, then 
mix the two (10 c.c. of starch paste to 3 c.c. of undiluted saliva) 
and return to the water-bath. The starch is changed first to 
soluble starch (if originally a thick paste, it becomes fluid and 
loses its opalescence), then to erythrodextrin, which gives a 
red color with iodine, and finally to achroodextrin, which gives 
no reaction with iodine, and to maltose. Prove these changes 
as follows: Every minute or two take out a drop of the mixture, 
place it on a porcelain plate, and add a drop of iodine solution. 



EXPERIMENTS 287 

This gives first a blue color, showing the presence of starch; later 
a violet color, due to the mixture of the blue of the starch reaction 
with the red caused by the dextrin; next a reddish-brown color, 
due to erythrodextrin alone (starch being absent), and finally no 
reaction at all with iodine, proving the absence of starch and 
erythrodextrin. The fluid now contains achroodextrin and 
maltose. Test for the latter with Fehling's solution and with 
Barfoed's reagent. 

Exp. 214. Influence of conditions on ptyalin and its amylolytic 
action. Report and explain the results of the following ex- 
periments : 

(a) Boil a few cubic centimeters of the saliva, then add some 
starch paste, and place in the water-bath at 40 C. After five 
minutes test for sugar. 

(6) Take two test-tubes: put some starch paste in one, and 
saliva in the other, and cool them to o C., in a freezing mixture. 
Mix the two solutions, and keep the mixture surrounded by 
ice for several minutes, then test a portion for sugar. Now 
place the remainder in the water-bath at 40 C., and after a 
time test for sugar. 

(c) Carefully neutralize 20 c.c. of saliva with very dilute 
HC1 (the 0.2 per cent diluted), and dilute the whole to 100 c.c. 
Test the action of this neutralized saliva on starch. 

(d) To 5 c.c. of starch paste add 10 c.c. of 0.2 per cent HC1 and 
5 c.c. of neutral saliva, and expose the mixture for a while at 
40 C., and test for sugar. 

(e) To 5 c.c. of starch paste add 10 c.c. of a 0.5 per cent solution 
of Na 2 CO 3 and 5 c.c. of neutral saliva, and expose the mixture 
for a while at 40 C., and test for sugar. 

(f) Carefully neutralize (d) and (e), and again test the action 
of the two on starch. 

(g) Mix a little uncooked starch with saliva, expose to a 
temperature of 40 C. for a while, and test for sugar. 

Exp. 215. In three separate test-tubes place a few cubic 
centimeters of dilute solutions of KCNS or NH 4 CNS, of meconic 
acid, and of acetic acid; add to each a few drops of ferric chloride, 
and notice that a similar color is obtained in each case. Divide 



288 EXPERIMENTS 

the contents of each tube into two portions, and to one set add 
HC1; to the other add mercuric-chloride solution. Formulate a 
method of distinguishing from the sulphocyanates, meconates, 
and acetates. 

Analysis of Gastric Contents and Experiments with Pepsin. 

The following solutions will be found in the laboratory: 

A. A 0.2 per cent Solution of HCl. This is prepared by 
diluting 6.5 c.c. of concentrated HCl (sp. gr. 1.19) with distilled 
water to i liter. 

B. A Solution of Pepsin. Prepared by dissolving 2 grams 
of pepsin in 1000 c.c. of water. 

C. A Pepsin-hydrochloric-acid Solution. Prepared by dis- 
solving 2 grams of pepsin in 1000 c.c. of solution A. 

Or, add to 150 c.c. of solution A about 10 c.c. of the glycerol 
extract of the mucous membrane of the stomach. 

Exp. 216. Take five test-tubes and label a, b, c, d, e. Fill 
as indicated below. Place in a water-bath at 40 C., and ex- 
amine an hour later, and again the next day. 

(a) 3 c.c. pepsin solution + 10 c.c. water + a few shreds of 
fibrin. 

(b) 10 c.c. 0.2 per cent HCl + a few shreds of fibrin. 

(c) 3 c.c. pepsin solution + 10 c.c. 0.2 per cent HCl, and a few 
shreds of fibrin. 

(d) 3 c.c. pepsin solution + 10 c.c. 0.2 per cent HCl, boil, and 
then add a few shreds of fibrin. 

(e) 3 c.c. pepsin solution + 10 c.c. 0.2 per cent HCl, and a few 
shreds of fibrin which have been tied firmly together into a ball 
with a thread. 

Make a note of all changes. 

Exp. 217. Filter c. Neutralize with dilute Na 2 CO3- Filter 
again. Why? Test the filtrate for the biuret reaction. 

Exp. 218. To 5 grams fibrin add 30 c.c. of the pepsin solution 
and 100 c.c. 0.2 per cent HCl. Set in the water-bath at 40 C., 
stirring frequently, and leave in the water-bath overnight. 
Observe the undigested residue, on the following day, and also 
a slight flocculent precipitate. What is this precipitate? 



EXPERIMENTS , 289 

Filter and carefully neutralize the filtrate. A precipitate 
varying with the progress of the digestion will form. What is it? 

Remove this by filtration, and saturate this filtrate with 
(NH 4 ) 2 SO 4 . Filter. Save precipitate and filtrate. Of what 
does each consist? 

Exp. 219. Dissolve the last precipitate of Exp. 218 in water, 
and try the following tests: 

(a) Biuret reaction. 

(6) Effect of boiling. 

(c) Test with HNO 3 , as in performing test for albumin in the 
urine, page 203. 

Exp. 220. To the last filtrate of Exp. 218 add an equal vol- 
ume of 95 per cent alcohol, and stir thoroughly. The peptones 
will collect in a gummy mass about the stirring-rod. 

(a) Determine the solubility of peptones in water. 

(6) What is the effect of heat when they are so dissolved? 

(c) Try the biuret reaction. 

Exp. 221. Demonstration of rennet enzyme. Place 10 c.c. 
of milk in each of three test-tubes. Label the test-tubes i, 2, 3. 

To i add a drop of neutralized glycerol extract of the mucous 
membrane of the stomach (made from the stomach of the calf). 

To 2 add a drop of neutralized glycerol extract, and boil 
at once. 

To 3 add a few cubic centimeters of (NH 4 )2C 2 4 solution, 
and then a drop of a glycerol extract. 

Place these tubes in the water-bath at 40 C., and examine 
after five to ten minutes. Explain results in each case. 

Continue heating tube 3 for half an hour, then add 2 or 3 
drops CaCl 2 solution. The liquid instantly solidifies. Why? 

Exp. 222. Digestion of paracasein. Determine the products 
of the digestion of the curd from the last experiment. 

Exp. 223. Tests for free hydrochloric acid. - Try each of the 
following tests with (a) HC1 (0.2 per cent, 0.05 per cent, and 
o.oi per cent successively); (6) lactic acid (i per cent); (c) 
mixtures containing equal volumes of (a) and (6). Tabulate the 
results. 

(a) Dimethylaminoazobenzene. Use i or 2 drops of a 0.5 



290 EXPERIMENTS 

per cent alcoholic solution. In the presence of free mineral acids 
a carmine-red color is obtained. 

(b) Gunzburg's reagent. Phloroglucin, 2 grams; vanillin, 
i gram; alcohol, 100 c.c. Place 2 or 3 drops of the solution to 
be tested in a porcelain dish, add i or 2 drops of the reagent, 
and evaporate on a water-bath. In the presence of free hydro- 
chloric acid a rose-red color develops. 

(c) Boas' reagent. This is prepared by dissolving 5 grams 
of resublimed resorcinol and a gram of cane-sugar in 100 grams 
of 94 per cent alcohol. Take three or four drops each of the 
reagent and the solution to be tested, and cautiously evaporate 
to dryness. In the presence of a free mineral acid a rose or 
vermilion red color is obtained. This gradually fades on cooling. 

(d) Trop&olin OO. Use i or 2 drops of a saturated alcoholic 
solution. 

(e) Congo-red. Use filter-paper which has been dipped into 
a solution of the reagent and then dried. 

Exp. 224. To 5 c.c. egg-albumin in solution add i c.c. of 
0.2 per cent HC1. Mix thoroughly, and test for the presence of 
free HC1. What is the result? How do you explain it? Repeat 
the test, using a solution of peptone in place of the egg-albumin. 

Exp. 225. Tests for lactic acid. Ujfelmanrfs reagent. Mix 
10 c.c. of a 4 per cent solution of carbolic acid with 20 c.c. of 
water, and add a drop or two of ferric chloride. 

To 5 c.c. of the reagent add a few drops of the lactic-acid 
solution. Note the canary-yellow color. 

Does the presence of free HC1 interfere with this reaction? 

A more delicate reagent is obtained by adding 3 or 4 drops of a 
10 per cent ferric-chloride solution to 50 c.c. of water. Such 
a solution has a very faint yellow color, which is distinctly inten- 
sified by lactic acid. (See Exp. 60.) 

Using 5 c.c. of this nearly colorless solution for each experi- 
ment, note the effect of (a) 0.2 per cent HC1; (6) acid phosphate 
of sodium; (c) alcohol; (d) glucose; (e) cane-sugar. What con- 
clusions do you reach concerning the value of this test, when 
applied directly to the gastric contents? 

The test is best applied to an aqueous solution of the ethereal 



EXPERIMENTS 2QI 

extract of the gastric contents. Add to the contents 2 drops 
of HC1, boil to a syrup, and extract with ether. Dissolve the 
residue obtained upon evaporation of the ether in a little water, 
and test for lactic acid. 

Exp. 226. Test for butyric acid; see ethyl butyrate, page 259. 

Exp. 227. Test for acetic acid similarlarly forming ethyl 
acetate. 

Exp. 228. The acidity of the gastric contents may be deter- 
mined as follows: To 5 c.c. of the filtered contents, diluted with 
2 S to 30 c.c. of water in an Erlenmeyer flask, add 2 or 3 drops 
of a solution of dimethylaminoazobenzene. Titrate with N/io 
alkali till the color changes to a yellow which fairly matches the 
indicator; this represents the free HC1. To this mixture add 
a few drops of phenolphthalein solution, and continue the 
titration until a permanent pink color is obtained. The N/io 
alkali used will represent the total acidity, combined HC1, and 
organic acids. The organic acids will not be present in gastric 
contents in the presence of any appreciable amount of free 
HC1, as they are derived almost entirely from fermentations 
which are inhibited by the hydrochloric acid. 

Experiments with Pancreatic Juice. 

Exp. 229. Proteolytic action. To 25 c.c. of a i per cent 
solution of Na 2 C0 3 add a few drops of the pancreatic extract. 
Place some pieces of fibrin in this liquid, and keep in the water- 
bath at 40 C. till the fibrin has disappeared (one or two hours 
probably). Observe the digestion from time to time. Note 
that the fibrin does not swell and dissolve as in gastric digestion, 
but that it is eaten away from the edges. 

Filter. What is the precipitate? Carefully neutralize the 
filtrate with 0.2 per cent HCL Another precipitate may appear. 
What is this? 

Again filter, if necessary, and test the filtrate for proteoses 
and peptones as directed under gastric digestion. 

Exp. 230. Amylolytic action. To some starch paste in a 
test-tube add a drop or two of the pancreatic extract and place 
in the water-bath at 40 C. After a few minutes test for sugar 
and report the result. 



2Q2 EXPERIMENTS 

Exp. 231. The Piolytic (fat-splitting) action. For the 
demonstration of this action use natural pancreatic juice, or 
finely divided fresh pancreas, or a recently prepared extract. 

To some perfectly neutral olive oil, colored faintly blue with 
litmus, add half its volume of the pancreatic extract, shake 
thoroughly, and keep at 40 C. for twenty minutes. Record 
the result. Reserve for next experiment. 

Exp. 232. Emulsifying Action. To 10 c.c. of a 0.2 per cent 
solution of Na 2 C0 3 add a few drops of the mixture used in Exp. 
231. Shake thoroughly, and report the result. Referring to the 
earlier experiments on emulsification (see .Fats), explain the 
efficacy of the pancreatic juice in emulsifying fats. 

Experiments with Bile. 

Exp. 233. Color. Note the difference in color between 
human bile and ox bile. Explain. 

Exp. 234. Reaction. Dilute some bile with 4 parts of 
water. Immerse a strip of red litmus paper, then remove and 
wash with water. Note the reaction. 

Exp. 235. Nucleo-albumin. Dilute bile with twice its 
volume of water, filter if necessary, and add acetic acid. What 
is the precipitate? How distinguished from mucin? 

Exp. 236. Filter 235 and test the filtrate for proteins. Report 
the result. 

Exp. 237. Separation of Bile Salts. Mix 20 c.c. of bile 
with animal charcoal to form a thick paste, and evaporate on the 
water-bath to complete dryness. Pulverize the residue in a 
mortar, transfer to a flask, add 25 c.c. of absolute alcohol, and 
heat on the water-bath for half an hour. Filter. To the fil- 
trate add ether till a permanent precipitate forms. Let the 
mixture stand for a day or two, and then filter off the crystalline 
deposit of bile salts. Save the filtrate which contains choles- 
terin. (Plate VI, Fig. 4, page 132.) 

Exp. 238. Bile-pigments. (a) Gmelin's test. Take some 
bile in a wine-glass and underlay with yellow HNO 3 , in the 
manner described in testing saliva for albumin. Notice the 
play of colors, beginning with green and passing through blue, 



EXPERIMENTS 293 

violet, and red, to yellow, at the junction of the two liquids. 
Explain. 

(6) Iodine test. Place 10 c.c. of dilute bile in a test-tube, 
and add slowly 2 or 3 cubic centimeters of dilute tincture of 
iodine, so that it forms an upper layer. A bright green ring 
forms at the line of contact. 

Exp. 239. Cholesterol. Examine under the microscope the 
crystals obtained by the cautious evaporation of the alcohol- 
ether filtrate of Exp. 237. 

Concentrated H 2 SO4, containing a little iodine, gives with 
cholesterol a series of colors passing from violet to blue, then to 
green, and finally red. 

Exp. 240. Action of Hie in digestion. (a) Take three 
test-tubes. In one mix 10 c.c. of bile and 2 c.c. of neutral olive 
oil; in the second, 10 c.c. of bile and 2 c.c. of rancid olive oil; 
in the third, 10 c.c. of water and 2 c.c. of neutral oil. Shake and 
place in a water-bath at 40 C. for some time. Note the extent 
and the permanency of the emulsion in each case. 

(6) Into each of two funnels fit a filter-paper. Moisten one 
with water and the other with bile, and into each pour an equal 
volume of olive oil. Set aside for twelve hours (with a beaker 
under each funnel). Do you notice any difference in the rate 
of filtration? 

(c) Add, drop by drop, a solution of bile salts to (a) a solution 
of egg-albumin; (b) a solution of acid-albumin; (c) a solution 
obtained by digesting a bit of fibrin in gastric juice and filtering. 
Explain the results. 



APPENDIX. 
REAGENTS. 

It is desirable that all reagents be made with reference to the 
molecular weights of the substances employed. These may be 
from one to ten times the molecular weight per liter, while the 
solutions for practice are from one- tenth to one-fourth the 
molecular weight per liter. Salt solutions used as reagents are 
conveniently from five to ten per cent; that is, a molar concen- 
tration bringing the strength within these limits is selected. 

In the following list a few exceptions will be noted. 

Ammoniacal Cuprous Chloride. This may be made by dis- 
solving copper oxide with metallic copper in dilute hydrochloric 
acid with the aid of heat. To the clear, cool, resulting solution 
add ammonia to marked alkaline reaction. 

Ammoniacal Silver Solution. Dissolve 10 grams of silver 
nitrate in 200 c.c. of water and add about 50 c.c. of strong 
ammonia, or an amount considerably in excess of that required 
to dissolve the precipitate first formed. 

Ammonium Molybdate Solution for Phosphates. This may 
be made by dissolving 20 grams of ammonium molybdate in 
a mixture of 250 c.c. NH 4 OH and 250 c.c. of water. Then this 
solution is added to 1000 c.c. of nitric acid, making 1500 c.c. of 
reagent. In using this solution as a test for phosphates it is 
necessary to heat the mixture to about 60 C. 

If the reagent is prepared in the following manner it reacts 
without heating, is more sensitive than that produced by the 
first formula and is recommended as the better of the two. Dis- 
solve 100 grams of molybdenum trioxide (molybdic acid) in 
400 c.c. of dilute NH 4 OH (loper cent). Allow to cool and add 
all at once 1000 c.c. of dilute HNO 3 (HNO 3 three parts, H 2 O two 

parts). The precipitate first formed is immediately redissolved 

294 



REAGENTS 295 

and the product should be a perfectly clear, nearly colorless 
solution. 

Ammonium Oxalate M/4, 35.52 grams per liter. 

Ammonium Sulphate. (Standard for ammonia determina- 
tion). Dissolve .4716 gram of the pure salt in a liter of water. 
Ten c.c. then contains i mg. of nitrogen. 

A solution of twice this strength may be used and seems 
preferable to the author. Ten c.c. then contains 2 mg. of nitrogen 
and 5 c.c. used in the determination will contain i mg. 

Ammonium Sulphate (for protein precipitation). A satu- 
rated solution is required that is, the solution of protein must 
be saturated with the salt. 

Note. In case a half-saturated solution is called for, take 
equal volumes of the unknown protein solution and saturated 
ammonium sulphate. 

Barfoed's Reagent. Dissolve i part of copper acetate 
in 15 parts of water; to each 200 c.c. of this solution add 5 c.c. 
of acetic acid containing 38 per cent of glacial acetic acid. 

Benedict's Qualitative Solution has the following composition: 

Grams or c c. 

Copper sulphate (pure crystallized) 17.3 

Sodium or potassium citrate 173 .o 

Sodium carbonate (crystallized) 200.0 

or one-half the weight of the anhydrous salt 
Distilled water to make 1000.0 

The citrate and carbonate are dissolved together (with the aid 
of heat) in about 700 c.c. of water. The mixture is then poured 
(through a filter if necessary) into a larger beaker or casserole. 
The copper sulphate (which should be dissolved separately in 
about 100 c.c. of water) is then poured slowly into the first 
solution with constant stirring. The mixture is then cooled and 
diluted to i liter.* 

Benedict's Quantitative Solution. 

CuSO 4 .5 H 2 O 1 8 grams 

KCNS 125 " 

Na 2 CO 3 200 " 

* Journal American Medical Association, Oct. 7, 1911, page 1193. 



296 APPENDIX 

Sodium or potassium citrate 200 grams 

K 4 Fe(CN)e 5 c.c. of a 5 per cent solution 

Distilled water to make a total volume of 1000 c.c. 

The citrate, cyanate, and carbonate are dissolved by aid of 
heat, and the solution filtered if necessary. The copper sulphate 
is dissolved in a separate portion of water and added to the hot 
filtered solution, the flask being shaken vigorously during the 
addition. 

Benzidine Solution. Saturated solution of benzidine in 
glacial acetic acid with an equal volume of peroxide of hydrogen 
solution. The two solutions are to be mixed when used as a test 
for blood. 

The following method of making the benzidine solution is 
suggested by Hawk's 'Physiological Chemistry': 4.33 c.c. of 
glacial acetic acid is warmed in a small Erlenmeyer flask to about 
50 C., a half gram of benzidine added, and the mixture heated 
eight or ten minutes at 50 C. and then the solution diluted with 
19 c.c. of distilled water. If kept in a dark place it is fairly per- 
manent. 

Brom-cresol Purple. See page 297. 

Brom-thymol Blue. See page 297. 

Cochineal. Extract cochineal with 30-50 per cent alcohol 
and filter. 

Congo Red. Two per cent aqueous solution. 

Copper Sulphate. One per cent solution for Biuret Test. 

Creatinine Solution. Dissolve 1.6106 grams of creatinine 
zinc chloride in i liter of N/io HC1. This solution contains i 
mg. of creatinine per c.c. 

Standard for Saliva and Blood. Take 6 c.c. of the above 
standard (i.e., 6 mg. creatinine), and 10 c.c. of N/i HC1 and 
make up to a liter with distilled water. Add a few drops of 
toluol and mix. Five c.c. of this solution contains .06 mg. 
creatinine. 

Dimethylaminoazobenzene. 0.5 per cent alcoholic solution. 

Esbach's Reagent. Dissolve 10 grams of picric acid, and 20 
grams of citric acid in sufficient water to make i liter of solution. 

Fehling's Solution. The Fehling's solution recommended 



REAGENTS 297 

for experiments in this book is one-half the strength frequently 
employed, and is prepared in separate solutions as follows: 
Dissolve 34.639 grams of pure crystallized copper sulphate in 
water, and make solution up to i liter. This constitutes the 
first part of the reagent. The second part may be made by 
dissolving 173 grams of Rochelle salts and 52.7 grams of caustic 
soda (NaOH) in water and making up to i liter. When prepared 
in this way 10 c.c. of each of these solutions mixed together 
will be reduced by 0.05 gram of glucose. 

Ferric Alum. For use in silver and chlorine determinations. 
A saturated aqueous solution to which sufficient strong HN0 3 
is added to make about a 10 per cent solution. 

Ferric Chloride. 2.5 per cent solution acidified with HCL 

Goulard's Extract. A solution of lead subacetate, q.v. 

Gram's Solution. See iodine solution. 

Gunzburg's Reagent. Phloroglucin, 2 grams; vanillin, i 
gram; alcohol, 100 c.c. 

Hopkins-Cole Reagent (glyoxylic acid, CHO.COOH.H 2 O). 
Prepared by saturating a liter of water with oxalic acid, adding 
60 grams of sodium amalgam and allowing to stand until re- 
duction is complete or until hydrogen ceases to be evolved. 
For use this solution should be filtered and diluted with two or 
three volumes of water. 

Hydrochloric Acid (dilute). Hydrochloric acid, strong, 
(sp. gr. i. 20) i part; distilled water, 2 parts. 

Hypobromite Solution for Urea. Consists of a mixture of 
equal parts of the following solutions kept separately and mixed 
for use. 

Bromine Solution for Urea. 125 grams KBr and 125 grams 
Br to one liter water. 

NaOH Solution for Urea. A 40 per cent solution, or a ten 
molar solution. 

Indicators for H-Ion Concentration Determination. 

Brom-thymol Blue. A .02 per cent watery solution. 
Brom-cresol Purple. A .02 per cent watery solution. 
Methyl Red. A .02 per cent watery solution. 



298 APPENDIX 

If a flocculent precipitate occurs in the case of the methyl red 
it may be cleared away by the addition of .5 c.c. of N/20 NaOH 
(sterile). This does not interfere with obtaining accurate results. 

Instead of being prepared directly, these indicators may be 
prepared from " stock alcoholic solutions " containing .1 gram 
of indicator dissolved in 50 c.c. of 95 per cent alcohol. 

To prepare the .02 per cent watery solution in this way, take 
45 c.c. of distilled water and 5 c.c. of the alcoholic solution. 
This makes 50 c.c. of the dilute watery solution. 

Note. All these solutions should be kept in amber colored 
bottles and should be prepared under as nearly sterile conditions 
as possible. 

Iodine Solution. For determination of iodine absorption 
number of fats. 

Solution I. 

26 grams iodine 

500 c.c. 95 per cent alcohol 

Solution II. 

30 grams mercuric chloride 
500 c.c. 95 per cent alcohol 

Mix the two solutions and allow to stand at least twelve hours 
before using. 

Iodine Tincture. See Tincture. 

Invertase. Mix 500 grams of " beer yeast/' 200 c.c. of water 
and 10 grams of sugar; allow to stand one hour. Add 50 c.c. of 
60 per cent alcohol and a little thymol. Filter, press or allow to 
dry, put the nearly dry mass in a flask, add 20 grams of sugar 
and shake till solution is effected. Keep in ice chest. 

If " beer yeast " is not available, a solution of invertase, rather 
less satisfactory than the above, can be made as follows: Take 
one dozen compressed yeast cakes, grind with sand and mix 
with 500 c.c. of water, and a little chloroform as preservative. 
Allow to stand twelve hours and filter. 

Iodine Solution. 

LugoVs solution. Iodine 5 grams, potassium iodide 10 
grams, and sufficient distilled water to make 100 grams. (U. S. P.) 



REAGENTS 299 

Gram's solution. Iodine i gram, potassium iodide 2 grams, 
and sufficient distilled water to make 200 grams. 

Lead Subacetate (basic acetate of lead). The U. S. P. 
method of preparation is as follows: lead acetate 180 grams, lead 
oxide no grams, distilled water to make 1000 grams. Rub lead 
oxide to a paste with 100 c.c. of water, dissolve lead acetate in 
700 c.c. of boiling distilled water; add slowly with constant 
stirring to lead oxide and boil the mixture for half an hour. 
Cool and filter and make up to 1000 c.c. with water free from 
carbon dioxide. 

Leucin. See under Cystin, page 303. 

Lipase. From castor bean (see page 270). Remove the 
shells from 10 grams of fresh beans, break them up as fine as 
possible and allow to stand overnight in a loosely stoppered test- 
tube full of alcohol ether mixture. Pour off; grind the beans to 
a powder in a small mortar, transfer to a test-tube and let stand 
under ether overnight. Filter with suction and wash two or 
three times with small amounts of the alcohol ether mixture. 

Lipase. From pancreas. Take a pig's pancreas, remove 
all fat, grind and allow to stand overnight. Then add four 
times its weight of 25 per cent alcohol and allow to stand three 
days. Syphon off clear fluid and neutralize with sodium car- 
bonate. The solution will contain a fat-splitting enzyme. 

Lithium Sulphate (for use in uric acid determination). 
Twenty per cent aqueous solution. 

LugoPs Solution. See Iodine. 

Magnesia Mixture. Dissolve 125 grams of ammonium 
chloride, and 125 grams of magnesium sulphate, in sufficient water 
to make i liter of solution; then add 125 c.c. of strong ammonia 
water. 

Marine's Reagent. Ten grams of potassium iodide, 5 grams 
of cadmium iodide, 100 c.c. of water. 

Mercuric Chloride Solution. Five per cent HgCl 2 in dis- 
tilled water. 

Millon's Reagent. To one part of mercury add two parts of 
nitric acid of specific gravity 1.4, and heat on the water-bath 



300 APPENDIX 

till the mercury is dissolved. Dilute with two volumes of water. 
Let the precipitate settle, and decant the clear fluid. 

Molisch's Reagent for Carbohydrates. Fifteen per cent 
solution of a-naphthol in alcohol. 

Nessler's Solution* An alkaline solution of potassio-mercuric 
iodide, made as follows: Dissolve 35 grams of potassium iodide in 
about 200 c.c. of water. Dissolve 17 grams of mercuric chloride 
in 300 c.c. of hot water. Add the potassium iodide to the mer- 
curic chloride, until the precipitate at first formed is nearly all 
redissolved. If the precipitate should entirely dissolve, add a few 
cubic centimeters of a saturated solution of mercuric chloride, 
until a slight permanent precipitate is obtained. After the 
mixture is cold, make up to i liter with a 20 per cent solution 
of caustic potash. Allow to settle and use the clear solution. 

Nitric Acid (dilute). Strong HNOa (sp. gr., 1.42) one part, 
and water three parts. 

Pancreatic Extract. Obtain a fresh pancreas and soak in 
four times its weight of 25 per cent alcohol for two or three days. 
Filter and make the solution neutral or very slightly alkaline 
with sodium carbonate. This solution will contain the fat- 
splitting enzyme. 

Phenoldisulphonic Acid. Phenoldisulphonic acid, for esti- 
mation of nitrates in water analysis, may be prepared by heat- 
ing on a water-bath for several hours a mixture of 555 grams of 
concentrated sulphuric acid and 45 grams of pure carbolic-acid 
crystals. 

Phenolphthalein. Make a i per cent alcoholic solution and 
then dilute with an equal volume of water. 

Phenyl-hydrazine Solution. Dissolve i gram of phenyl- 
hydrazine hydrochloride and 2 grams of sodium acetate in 10 c.c. 
of water. 

Phosphoric-Sulphuric Acid Digestion Mixture (Folin). 

50 c.c. 5 per cent CuSO 4 (Crystallized) 

300 c.c. 85 per cent H 3 PO 4 

100 c.c. cone. H 2 S0 4 (ammonia-free) 

Mix and prevent absorption of ammonia from air. This acid 



REAGENTS 301 

mixture acts on glass and consequently can only be used in 
microchemical analyses when the digestion takes place in a few 
minutes. 
Phospho-tungstic Acid (Folin-Denis uric acid reagent). 

750 c.c. distilled water 

100 grams sodium tungstate must be very pure 

80 c.c. 85 per cent H 3 P04 

Let boil slowly for two hours and then dilute to a liter and mix. 

Picric Acid (for creatinine determination). To make a 
saturated solution of picric acid, dissolve with heat (do not boil) 
an excess of picric acid in distilled water. Then cool the solution, 
and as crystallization takes place pour off the supernatant fluid. 

Picric-acid Solution (Esbach's Reagent). Dissolve 10 grams 
of picric acid and 20 grams of citric acid in sufficient water to 
make one liter. 

Potassium Ferrocyanide Solution. K 4 Fe(CN)6, one-fourth 
molar solution (9.2 per cent). 

SchifFs Reagent. Into 50 c.c. of a 2 per cent solution of 
fuchsine or rosaniline pass S0 2 gas until the solution is colorless. 
Then dilute with an equal volume of water and keep in small full 
bottles in a dark place. 

Silver-nitrate Solution. Drop solution, i : 8, used as a 
qualitative test for chlorine in urine. 

Silver-nitrate Solution (for sterilization of root canals). 
See Howe's silver-nitrate preparation, page 84. 

Sodium Cyanide (for use in uric acid determination). Make 
a 7.5 per cent aqueous solution and allow to stand at least one 
week before using. 

Starch Paste (thin). Rub about one-half gram of starch to 
a thin paste with cold water. Add sufficient boiling water to 
dissolve, then dilute to 100 or 150 c.c. 

Sulphuric Acid (dilute). Twenty per cent strong H 2 S0 4 in 
distilled water. 

Tincture Iodine for Bile Test. Dilute the U. S. P. tincture 
with alcohol until just transparent in test-tube. 

Tollen's Reagent. Make a 10 per cent solution of AgNOs in 



302 APPENDIX 

dilute ammonia, and just before using mix an equal volume of 
this solution with a 10 per cent solution of NaOH. 

Tropseolin oo. Saturated alcoholic solution. 

Tungstic Acid (for protein precipitation). 

10 per cent solution sodium tungstate (must be C.P,) 
2/3 N sulphuric acid 

Equal volumes of each are added for complete protein pre- 
cipitation. 

Urease Solution. Five per cent water solution of arlco 
urease. This urease dissolves easily, but the solution does not 
keep well. It should be made fresh each time. 

Uric acid Solution (Folin). Weigh 2 grams of uric acid on 
watch glass. On another watch glass weigh i gram lithium 
carbonate; dissolve the lithium salt in 300 c.c. of water and heat 
to 60. With the lithium solution rinse the uric acid through 
funnel into a 2-liter flask. Shake to solution and cool. Add 
50 c.c. of 40 per cent formalin and shake. Then add 10 c.c. of 
glacial acetic acid and shake until rid of C0 2 . Dilute to volume 
and mix. Put up in loo-c.c. bottles. This solution keeps in 
the dark indefinitely. 

For Use: 

Transfer i c.c. of the above standard to a 25<>c.c. volumetric 
flask. Half fill with distilled water and add i c.c. of formalin. 
Dilute to volume and mix. This solution keeps at least ten days. 

Uffelmann's Reagent. Mix 10 c.c. of a 4 per cent solution 
of carbolic acid with 20 c.c. of water, and add a drop or two of 
ferric chloride. 

Uranium Solution (standard for phosphate determination). 
See page 197. 

PREPARATIONS. 

Creatine may be most conveniently prepared from a strong 
solution of Liebig's extract. Dissolve the extract in 20 parts 
of water, add basic lead acetate drop by drop, to avoid more 
than a slight excess, then remove excess of lead; concentrate to 
a syrup over a water-bath and allow to stand in a cool place, 



REAGENTS 303 

whereupon creatin crystals will separate out. Two or three 
days' time may be required before the crystals are obtained. 
They may be washed with 88 per cent alcohol and purified by 
recrystallization from water. Hypoxanthin and sarcolactic acid 
may be obtained from the mother liquor.* 

Creatinine may be prepared from creatine by boiling for ten or 
fifteen minutes with very dilute sulphuric acid. Neutralize the 
acid with BaC0 3 , filter, evaporate to dryness on a water-bath, 
and extract the creatinine with alcohol. Upon evaporation, the 
creatinine is obtained in the form of crystals. 

Cystin. i. Clean 200 grams of hair by washing with dilute 
HC1 and then with ether. Boil the clean hair with 600 c.c. of con- 
centrated HC1 (specific gravity, 1.19) for four hours (in a 3- 
liter flask with condenser) on a sand-bath in hood. Then let cool. 

2. Add concentrated NaOH solution (750 c.c. H 2 O, 500 grams 
NaOH) till the reaction is only faintly acid. 

3. Add to the solution, which has begun to boil on neutral- 
ization, plenty of animal charcoal, and boil three-quarters of 
an hour. 

4. Filter hot, being careful to moisten filter and funnel with 
hot water to prevent funnel from cracking. 

5. The filtrate should be faintly yellow. On cooling, a 
crystalline precipitate forms, mainly cystin, with some tyrosin 
and leucin. If this is not the case, or if the precipitate is slight, 
the solution must be concentrated. Save the filtrate, which with 
the filtrate from 6 is to be worked up later for tyrosin and leucin. 

6. Filter off the precipitate after letting it stand overnight. 

7. Dissolve this precipitate in 350 c.c. of hot 10 per cent 
NH 4 OH (hood) and let cool. Then continue the cooling with 
finely chopped ice or with snow. Filter off any tyrosin that may 
have precipitated, and combine it with the filtrate of 6. 

8. Add glacial acetic acid, being careful not to acidify. The 
precipitate is a mixture of tyrosin and cystin. Filter. 

9. Make filtrate from 8 quite acid with glacial acetic acid. 
The precipitate is almost pure cystin. Let stand twenty-four 
hours. Then filter, and wash with H 2 O and alcohol. 

* Lea's 'Chemical Basis of the Animal Body/ 



304 APPENDIX 

10. Recrystallize by redissolving in the smallest quantity of 
hot 10 per cent ammonia that will effect solution, cooling and 
precipitating with glacial acetic acid. 

The preparations should be pure and should contain no 
tyrosin, for which test may be made with Millon's reagent. 

Reactions. Put a trace of cystin into a test-tube with some 
dilute NaOH and a little lead acetate. Boil. H 2 S is formed 
because S is split off. 

Tyrosin. i. Concentrate the neutralized filtrate of 6 of 
cystin preparation till, on cooling, tyrosin crystallizes out. 

2. Filter, and save filtrate for the preparation of leucin. 

3. Dissolve the tyrosin crystals in a very little hot water. 

4. Add amyl alcohol till a heavy precipitate forms. 

5. Filter precipitate. 

6. Redissolve in a very little hot water, and let crystallize out 
by cooling. 

Examine crystals under the microscope. 
Test with Millon's reagent. 

Leucin. i. Take the filtrate of 2 in the preparation of 
tyrosin, and evaporate to dryness on the water-bath. 

2. Extract with alcohol. 

3. On standing, the leucin crystallizes out of the alcoholic 
extract as it evaporates. 

4. Filter, and dry the crystals. 
Examine under the microscope. 

Gelatin. Take about 10 grams of bone, preferably small 
pieces of the shaft of a long bone, clean carefully, and allow to 
stand for a few days in 60 c.c. of dilute HC1 (1/20). The dilute 
acid dissolves the inorganic portion of the bone, leaving the 
collagen. Note the effervescence due to the presence of carbon- 
ates. The acid solution is poured off and kept for further 
investigation. The remains of the bone are allowed to stand 
overnight in a dilute solution (i/io) of Na 2 C0 3 , and then boiled 
in 100 c.c. of water for an hour or two. The collagen undergoes 
hydrolysis and is converted into gelatin, which dissolves. A 
core of bone untouched by the acid usually remains. Evaporate 
the solution to 25 c.c. bulk and allow to cool. A firm jelly is 



REAGENTS 305 

formed if the solution is sufficiently concentrated. If the solu- 
tion gelatinizes, add an equal bulk of water and heat anew. If 
the solution thus obtained is sufficient in quantity it may be 
used for Experiments 208 and 209. 

Gelatin may also be prepared from tendons, which consist 
almost wholly of white fibers. Collagen is the substance of 
which white fibers are made up. 

Glycogen (CeHio0 5 ) n . ~ Use a liver taken from an animal 
just killed, or, if the season permits, oysters just removed from 
the shell. Cut an oyster, as rapidly as possible, into small 
pieces, and throw it into four times its weight of boiling water, 
slightly acidulated with acetic acid. After boiling the first por- 
tion for a short time, remove the pieces, grind in a mortar with 
some sand, return to the water, and continue the boiling for 
several minutes. Filter while hot. The opalescent solution 
thus obtained is an aqueous solution of glycogen and other 
substances. 

If a purer solution is desired, continue as follows: Add to the 
filtrate alternately a few drops of hydrochloric acid and potassio- 
mercuric iodide, until a precipitate of protein ceases to form. 
This may be determined more conveniently by filtering off a 
small portion of the liquid from time to time, and adding to the 
clear filtrate the hydrochloric acid and potassio-mercuric iodide. 
When the precipitation of the proteins is complete, filter, and to 
the milky filtrate add double its volume of alcohol; the glycogen 
will precipitate as a white powder. Filter this off, wash with 
66 per cent alcohol (one part of water to two of alcohol), and 
dissolve in water. 

Mucin Solution. Cut a portion of a navel-cord into small 
pieces. Shake in a flask with water, changing the water several 
times. This removes salts and albumin. Extract for twenty- 
four hours with N/ioo NaOH in a corked flask. Add N/ioo 
acetic acid, which precipitates the mucin. Let settle, filter, and 
wash with water. 

Mucin may also be prepared from the saliva by precipitation 
with acetic acid. 

Potassium Cyanate (KCNO). Melt in an iron ladle, of at 



306 APPENDIX 

least 50 c.c. capacity 5 grams of commercial potassium cyanide, 
and stir in gradually 20 grams of litharge. When the entire 
amount has been added, pour the mass out upon an iron plate, 
and allow to cool. Separate as far as possible the reduced lead 
from the potassium cyanate that has been formed, powder the 
latter, and dissolve in 25 c.c. of cold water. Filter if necessary 
and purify by repeated crystallization. 

Tyrosin. See paragraph under Cystin, page 303. 

Urea, Synthesis of. Add to a filtered solution of KCNO 
a cold saturated solution of ammonium sulphate, containing 
at least 6 grams of (NH) 2 SO 4 . Heat the mixture slowly on a 
water-bath at a temperature of 60 C., and maintain at that 
point for one hour. By this process ammonium cyanate is 
formed and then changed to urea, which may be obtained in an 
impure state by evaporating the solution to dryness on a water 
bath, and extracting the residue with hot, strong alcohol. The 
urea will crystallize from the alcohol as it cools. 

Vegetable Globulin: e.g. Edestin. Extract about i ounce of 
crushed hemp seed with water containing about 5 per cent so- 
dium chloride. This extraction should take from one-half hour 
to one hour at a temperature of about 60 C. Filter while hot. 
Upon cooling, a portion of the globulin (edestin) will probably 
separate out. Use the clear separated fluid for the general 
protein reactions and precipitates. Boil the cloudy portion 
until the precipitated globulin has dissolved. Then set aside 
for twenty-four hours in order that the edestin may crystallize 
slowly, whereupon hexagonal plates should be obtained. Ex- 
amine by the microscope. (See Plate VI, Fig, i, page 



INDEX. 



Absorption 

of carbohydrates, 219 

of fat, 228 

of protein, 224 

of starch and sugar in diabetes, 222 
Acetaldehyde, 21 
Acetamide, 52 

preparation of (Exp. 74), 260 
Acetanilide, 69 

preparation of (Exp. 77), 262 

test for, 46 
Acetic acid, 30 

anhydride, 31, 42 

ether see Ethyl acetate 

test for (Exp. 48), 257 
Acetone, 23 

bodies, 37 

in relation to fat metabolism, 229 

chloroform, 81 

in saliva, 182 
test for, 182 

in urine, 209 

Legal's test for, 209 

preparation of, 23 (Exp. 36), 255 

test for (Exp. 37), 255 
Acetyl chloride, 41, 42 
Acetyl group denned, 31, 41 
Acetyl-salicylic acid, 66 
Acetyl-urea, 56 
Acetylene, 12 

reparation of (Exp. 10), 249 
roo-dextrine, 102, 148 
Acid: 

acetic, 30 
acrylic, 32 ^ 
amino-acetic, 40, in 
amino-formic, 39 
benzoic, 65 
butyric, 31 
carbonic, 35 
citric, 36 
formic, 30 
glacial acetic, 31 
glutaric, 34 
glycollic, 35, 36 
glyoxylic, 25 
hippuric, 67 



Acid: lactic, 35, 36 

malic, 35 

malonic, 33, 34 

mucic, 170 

oleic, 32 

oxalic, 34 

oxaluric, 56 

palmitic, 32 

propionic, 31 

pyrogallic, 64 

pyromucic, 71 

pyrotartaric,f34 

saccharic, 98 

stearic, 32 

succinic, 33, 34 

tartaric, 35, 38 

uric, 57 

valeric, 31 

Acid-albuminate, 122 
Acid ammonium urate, 213 
Acid-anhydride, 42 
Acid-forming foods, 238 
Acidity: 

of saliva, 161 

of urine, 190, 194 
Acid-carbonate of lime, preparation 

(Exp. 58), 258 
Acid-lactates, 214 
Acid metaprotein, 122 
Acidosis, 129 
Acid phosphates, 214 
Acid potassium oxalate, 34 
Acid urates, 213 
Acids: 

amino, 38 

organic, 29 

preparation of, 30 
Acoin, 78 
Acrolein, 32 

preparation of (Exp. 31), 254 
Acrylic acid, 32 

Acrylic aldehyde see acrolein 
Activators, 95, 150 
Acyl group, 41 
Addition products, n 
Adenine, 58 
Adnephrine, 79 
Adrenalin, 79 
Adrenol, 79 



307 



INDEX 



Agar-agar, 104 
Alanine, 40, in 
Albumin, 113, 119, 170 

in saliva, 170, in urine, 202 

exps. with albumin and globulin, 277 
Albuminates, 115, 122 
Albuminoids, 113, 121 
Albumose, 123 
Alcohol: 

amyl, 17 

butyl, 17 

ethyl, 17 

test for (Exps. 23, 25), 252, 253 

grain, 17 

methyl, 16 

test for (Exp. 24), 253 

propyl, 17 

protein precipitant, 119 

separation of water from (Exp. 22), 

252 

Alcoholates, 15 
Alcohols: 

classification of 15, 16 

oxidation of, 18 

polyatomic, 24 

solid, 108 

Aldehyde ammonia, 19 
Aldehydes, 18 

condensation of, 20 

reactions of, 18 

tests for (Exps. 29, 30, 34), 254, 255 
Aldol, 20 
Aldose, 97 

Alimentary glycosuria, 221, 222 
Aliphatic hydrocarbons, 6 
Alkali albuminate, 122 

metaprotein, 122 
Alkalinity of saliva, 167 
Alkyl (term defined), 9 
Alloxan, 58 
Allylene, 12 
Alpha-naphthol, 64 
Alypin, 79 

Alypin and potassium iodide, 79 
Amides, 52 
Amines, 50 
Amino acids, 38 

absorption of, 224 

classification of, 40 

in protein digestion, 224 
Amino-acetic acid, 39, in 

-benzene, 69 

preparation of (Exp. 99) 

-ethyl-sulphonic acid, 48 

-formic acid, 30 

-glutaric acid, 34 

-phenol, 70 

-propionic acid, 31 

-succinic acid, 33, 34 

-toluene, 69 



Amino-valeric acid, 31 
Ammelide, 55 
Ammonia : 

determination in saliva, 179 

determination in urine, 193 
formaldehyde method, 194 
zeolite method, 194 

metabolic origin of, 225 
Ammoniacal cuprous chloride, Appen- 
dix, 294 

Ammoniacal silver solution, Appen- 
dix, 294 

Ammonias substituted, 50 
Ammonium acid urate, 213 
Ammonium bifluoride, 79 

molybdate solution for phosphates, 
Appendix, 294 

oxalate solution, Appendix, 295 

piatinic chloride (PL 3, Fig. i), 77 

salts in saliva, 179 

sulphate as a protein precipitant, 295 
preparation of, Appendix, 295 

sulphate standard for ammonia, 

Appendix, 295 
Amyl acetate, 43 

alcohol, 17 

butyrate, 43 

nitrite, 43 

Amylolytic enzymes in saliva, 172 
Amylopsin, pancreatic, 152 
Anabolism, 219 
Anaestheaine, 80 
Anaesthetics, 78 
Anaesthol, 80 
Aniline, 69 
Anthracene, 62 
Antifebrin, 69 
Antithrombin, 130 
Apple essence, 32 
Arabinose, 97 
Arginine, 41 
Argyrol, 80 
Aristol, 80 

preparation of (Exp. 20) 
Aromatic alcohols, acids and aldehydes, 

6 .5 
Aromatic hydrocarbon, 60 

Arsenic in urine, 211 
Aspartic acid, 40 
Aspirin, 66 
Asymetric carbon, 36 
Atropine, 80 
test for, 80 
Autolysis, 95 



B 



Baeyer's Hypothesis, 96 
Barfoed's reagent: 
preparation of, Appendix, 295 



INDEX 



309 



Barfoed's reagent: test for sugar (Exp, 

133) 

Basic forming foods, 238 
Bayberry wax, 32 
Benedict's solution: 

preparation of, Appendix, 295 

test for sugar, 207 (Exp. 132) 
Benzaldehyde, 65 
Benzene, 60 

preparation of (Exp. 92), 264 
Benzidene, 69 

reaction, 134 

solution, Appendix, 296 

test for blood (Exp. 206) 
Benzine, 60 
Benzoated lard, 66 
Benzoic acid, 65 

preparation from toluene (Exp. 113) 
Benzol, see benzene 
Benzoyl glycocoll, 67 
Benzyl alcohol, 65 
/Seucaine, 82 

/Soxybutyric acid, 37; see also acetone 
bodies 

in urine, 37, 210 
/Snaphthol, 64 
Bile, 153 

experiments with, 233-240 

in relation to fat absorption, 228 

in urine, 210 

pigments, test for, 153 

salts, separation of (Exp. 237), 292 

salts in saliva, 153 
Bilirubin, 153 
Biliverdin, 153 
Binet, Dr., contribution toward saliva 

analysis, 239 
Biogen, 85 

Bitter almonds, oil of, 65 
Biuret, 55 

Biuret reaction, 115 
Blood, 129 

analysis, 135 

and saliva, 244 

benzidene reaction, 134 

Bordet reaction, 134 

chicken (PL VI, Fig. 6), 132 

chlorine in, 136 

clot, 130 
Howell's theory of, 130 

composition of, 130 

corpuscles, 131 
size of, 133 

dog (PL VI, Fig. 5), 132 

experiments with, 200-209 

fish (PL VI, Fig. 6), 132 

frog (PL VI, Fig. 6), 132 

glucose in, 136 

guaiacum test for, 134 

horse (PL VI Fig. 5), 132 



Blood: human (PL VI, Fig. 5), 132 

hydrogen-ion concentration of, 129 

non-protein nitrogen in, 135 

plasma, 130 

platelets, 130 

precipitation of protein in, 135 

serum, 130 

specific gravity of, Exp. 204 

spectroscopic examination of (Exp. 
203), 283 

stroma, 131 

sugar in, 220 

sugar level, 220 

Teichman's test for, 133 

tests, 133 

uric acid in, 138 

urinary sediment, 216 
Boas' reagent test (Exp. 223^), 290 
Bone, 146 
Borax, 80 

Bordet reaction, 134 
Brick dust deposit; see Uric acid 
British gum, 102 

Brom-cresol purple, Appendix, 297 
Brom-thymol blue, Appendix, 297 
Bromoform, 13 
Buckley's formo-cresol, 83 
Bulk in diet, 237 
Butane, 9 

Butter crystals (PL VI, Fig. 3), 132 
Butter fat, 43 
Butyric acid, 31 

preparation of (Exp. 51) 
Butyrin, 43 



Cacodyl, 14 
Cadaverin, 51 
Cadmium oxalate, 77 
Caffeine, 58, 73 

and murexide test (Exp. 85) 

preparation of (Exp. 89) 
Calcium : 

acid lactate (PL VII, Fig. 4), 185 

determination in saliva, 178 

lactate (PL VII, Fig. 3), 185 

in blood clotting, 130 

in milk digestion, 126 

in teeth and tartar, 142 

metabolism, 236 

oxalate, 214 

in urine, 214 

phosphate in bone, 146 
Calories, 231 
Camphor, 105 
Camphor-sulphonic acid, preparation 

of (Exp. 78) 
Cane sugar, 100 
Carbamic acid, 39 



; io 



INDEX 



Carbamide, see urea 
Carbimide, 47 

Carbinol, see methyl alcohol 
Carbohydrates, 96 

caloric value of, 231 

characteristics, 97 

classification, 97 

experiments with, 124-140 

metabolism of, 219 

Molisch's test for (Exp. 126), 270 

synthesis of, 96 

tolerance, 220 
Carbolic acid, see phenol 
Carbolic oil, 62 
Carbon, 2 

Carbon as a decolorizing agent (Exp. 
i), 248 

qualitative test for, 2 
Carbon dioxide in saliva, 165 

determination of, 165 
Carbon monoxide hemoglobin, 13 
Carbon tetrachloride, 13 
Carbonates in saliva, 173 
Carbonic acid, 35 
Carbonyl (term denned), 22 
Carboxyl (term defined), 29 
Carboxylase bacteria, 51 
Carbylamine reaction, Hoffman's, 50 
Carbylamines, 46 
Casein, 126 

digestion, 150 
Caseinogen, 126 
Casts in urine sediment, 215 
Catabolism, 219 
Catalase, 95 
Cellulose, 103 

Dement, composition of in tooth, 142 
Chloral, 21 

alcoholate, 81 

hydrate, 21, 8 1 

tests for (Exps. 32, 33) 
test for, 21 
Chlorethyl, 14 
Chloretone, 81 
Chlorine: 

determination of in saliva, 177 

determination of in urine, 195 

in blood, 136 
Chloroform, 13, 8 1 

preparation of (Exp. 13), 250 
Cholesterol, 108, 153 

in saliva, 183 

test for, 109 (Exp. 151), 276 
Chylous urine, 188 
Chymosin, 152 
Citric acid, 36 

Closed-chain hydrocarbons, 60 
Cloudy urine, causes of, 189 
Cloves, oil of, 88 
Coagulated proteins, 115 



Cocaine, 81 

differentiation from substitutes, 92 
with permanganate (PL 3, Fig. 4), 77 
with SnCl 2 (PL 4, Fig. 3), 77 
tests for, 8 1 

Cochineal, preparation of, Appendix, 
296 

Collagen, 121 

Collodion, 104 

Colloidal protein and tartar formation, 

143 . 

Color reactions for proteins, 115 
Coloring matter in urine, 199 
Colostrum, 128 
Condensation products, 20 
Congo red solution, Appendix, 296 
Conjugate proteins, 114, 124 
Cook, Dr. G. W., on mucin in saliva, 170 
Cook, Dr. R. H., on determination of 

uric acid, 192 

Copper sulphate solution, Appendix, 296 
Cork in urine sediment (PL VIII, Fig. 

6), 213 

Corn starch (PL V, Fig. 5), 99 
Cotton fibers (PL VIII, Fig. 6), 213 
Cotton seed oil, 33 
Cream of tartar, 38 
Creatine in muscle, 140 
Creatinine: 

in blood, 131, 135 

in muscle, 140 

in saliva, 180 

metabolism of, 227 

standard solution, Appendix, 296 
Creolin, 65 
Creosote, 82 
Cresol, 64, 82 
Cresylic acid, 65 
Crystals: 

method of forming, 75 

from saliva, 184 
Cyanamide, 52 
Cyanic acid, 47 
Cyanogen compounds, 45 
Cyanogen, 45 
Cyanuric acid. 55 
Cystin, formula for, 40 

in urine sediments, 215 
Cyclic formula for urea, 54, 55 

D 

Defibrinated blood, 131 
Dental preparations, 78 
Dentine, composition of, 142 
Derived albumins, 123 
Derived proteins, 114, 122 
Deuteroalbumose, 282 
Dextrine, 102 
Dextrose, see glucose 



INDEX 



Diabetes, causes of, 221 

treatment of, 223 
Diabetic sugar, 98 
Di acetic acid, 37, 210 

test for (Exp. 49) 
Dialysis of saliva, 185 
Diamines, 51 
Diamino acids, 40 
Diastase, 101 
Dibasic acids, 33 
Dichlormethane, 13 
Diet, 232-238 
Digestion, 148 

salivary, 148 

gastric, 149 

pancreatic, 151 

intestinal, 153 
Digestion chart, 154 
Dimethyl amine, 50 
Dimethyl-amino-azo-benzene, test for 
HC1, 289 (Exp. 2 23 A) 

preparation of, Appendix, 296 
Dimethyl arsine, 14 
Dimethyl benzene, 61 
Dimethyl ketone, 23 
Dimethyl oxalate: 

preparation of (Exp. 56) 
Dioses, 100 
Diphenyl, 61 
Diphenylamine, 69 
Disaccharides, 100 
Diureides, 57 

Doremus-Hinds urea apparatus, 191 
Double-bonded hydrocarbons, n 
Dysalbumose, 282 

E 

Earthy phosphates in urine, 196 
Edestin : 

preparation of. 306 

(PL VI, Fig. i), 132 
Egg albumin, 120 
Ektogan, 82 
Elastin, 121 
Eleopten, 105 

Emotional glycosuria, 221, footnote 
Emulsification, 107 
4 experiment on, 147 
Enamel, composition of, 142 
Enterokinase, 151 
Enzymes, 93 

deaminizing, 94 

endocellular, 95 

exocellular, 95 

hydrolytic, 94 

oxidizing, 94 

properties of, 93 
Epinephrin, 82 

Epithelium in urine sediment, 215 
Erepase, 153 



Erepsin, 153 

Erosion and hyperacidity, 240 

Erythrodextrine, 102, 148 

Esbach's method for albumin in urine, 

204 
Esbach's Reagent 

preparation of, Appendix, 296 
Essence of checkerberry, 66 
Esters, 41 
Ethane, 8 

Ethereal sulphates, 198, 201 
Ethers, 26 

ethyl ether, 27 

preparation of (Exp. 39) 
solubility of (Exp. 43) 

methyl ether, 26 

methylene ether, 27 

mixed ether, 26 
Ethyl acetate, 43 

alcohol, see alcohols 

benzene, 62 

bromide, 14 

preparation of (Exp. 19) 

butyrate, 43 

chloride, 14, 82 

hydrazine, 52 

iodide, preparation of (Exp. 18) 

mercaptan, 47 

nitrite, 42 

preparation of (Exp. 64) 
test for (Exp. 65) 

oxide, 27 

sulphonc, 48 

urea, 56 
Ethylene, n 

preparation of (Exp. 7) 
Ethylidene lactic acid, 36 
Eucaine, 82 
Eucaine and platinic chloride (PL III, 

m Fig. 2), 77 
Eucaine lactate, 83 
Eudrenin, 83 
Eugenol, 64, 83 
Europhen, 83 



False casts and mucin (PL VIII, Fig. 5), 

213 
Fat acid: 

crystals (PL VI, Fig. 4), 132 

in urine sediment, 216 

of milk, 127 
Fats, 43, 105 

caloric value of, 231 

experiments with, 273 

metabolism of, 228 

metabolism of in relation to carbo- 
hydrate metabolism, 230 

properties of, 105 

saponification of, 107 



312 



INDEX 



Fatal ratio, 223 
Fatty acids : 29 

preparation of (Exp. 145) 
Fatty casts, 216 
Fehling's solution: 

preparation of, Appendix, 296 

test for sugar (Exp. 129) 
Fehling's sugar determination, quanti- 
tative, 206 

Fen wick, Dr. S., on KCNS in saliva, 174 
Fermentation test for sugar, 208 (Exp. 

134) 

Ferments, 93 
Ferric alum solution, preparation of, 

Appendix, 297 
Ferric chloride solution, preparation of, 

Appendix, 297 

Ferris, Dr. H. C., quantitative deter- 
mination for KCNS in saliva, 175 
Fibrin, 130, 131 

properties of (Exp. 210) 
Filtration, microchemical, 76 
Firedamp, 7 
Folin : 

ammonia determination, 194 

blood analysis methods, 135 

sugar tube, 137 

uric acid solution, 302 

uric acid test, 181 
Folin-Denis, uric acid reagent, 301 
Food values, 231 
Formaldehyde, 20 
Formaldehyde urea in urine sediment 

(PL IX, Fig. 5), 214 
Formaline, 83, see formaldehyde 
Formamide, 52 
Formanilide, 52 
Formic acid, 30 

preparation of (Exp. 44) 

test for (Exp. 66) 
Formine, see formaline 
Formo-cresol, 83 
Formol, see formaline 
Formpse, 96 

Fractional distillation, 6 
Freund and Topfer, test for urinary 

acidity, 190 
Frohde's reagent, 87 
Fructose, see levulose 
Fruit-sugar, 100 
Fulminic acid, 47 
Furfuraldehyde, 71 
Furfuran (furfural), 71 
Furfuryl alcohol, 71 
Fusil oil, 17 



Gad's experiment (Exp. 147), 274 
Galactan, 103 



Galactose, 100 
Gallic acid, 66 
Gasoline, 7 
Gastric contents: 

acidity of (Exp. 228), 291 

analysis of, 288 
Gastric digestion, 149 
Gastric lipase, 151 
Gelatin, 122 

precipitation of (Exp. 175) 
Glacial acetic acid, 31 
Globin, 132 
Globulins, 113, 120 
Glonoin, 87 
Gluconic acid, 98 
Glucose, 98 

in Wood, 136 

in urine, 205 

properties of, 98 
Glucosozone, 100 
Glutaminic acid, 40 
Glutaric acid, 34 
Glutelins, 113 
Glycerol, 25, 84 

in relation to fat metabolism, 228 

test for, 84 
Glyceryl butyrate, 43 

oleate, 106 

palmitate, 106 

stearate, 106 
Glycine, see glycocoll 
GlycoJ, 24 
Glycocoll, in 
Glycogen, 102 

formation in metabolism, 220 

function in metabolism, 220 

in muscle, 140, 141 

occurrence of, 102 

test for (Exp. 140), 272 
Glycogenase, 220 
Gly collie acid, 25, 35, 36, 155 
Glycollic aldehyde, 25 
Glycoproteins, 114 
Glycosurias, 221 
Glycosuria and pyorrhea, 241 
Glyoxylic acid, 25 
Gmelin's test for bile (Exp. 238), 

292 

Goulard's extract, 297 
Grain alcohol, 17 
Gram's solution (iodine), 84 

preparation of, Appendix, 297 

strength of, 299 
Grape sugar, see glucose 
Guaiac blood test, 134, Exp. 205 
Guanine, 58 
Gunning's iodoform test for acetone, 

182 

Gunzberg's reagent, 297 
Gutta-percha, 84 



INDEX 



3*3 



H 

Halogens, qualitative tests for, 4 
Head, Dr. Joseph, bifluoride of am- 
monia, 79 
Hematin, 132 
Hematoporphyrin, 132 
Hemicellulose, 103 
Hemin, 132 
Hemochromogen, 132 
Hemoglobin, 114, 131 

preparation of crystals (Exp. 204) 
Heroin, 73, 84 
Heteroalbumose, 282 
Heterocyclic compounds, 71 
Heteroxanthine, 58 
Hexoses, 98 
Hippuric acid, 67 
Histpnes, 113 
Histidine, 40 

Hoffman's carbylamine reaction, 50 
Homologues, 5 

Hopkin's Cole reaction, 117 (Exp. 
156), 277 

preparation of reagent, Appendix, 

297 

Hopogan, 85 
Hormones, 152 
Howe, J. Morgan, Ref. 175 
Howe, Dr. Percy R., calcium determi- 
nation in saliva, 178 

silver nitrate treatment for sterili- 
zation of root canals, 84 

theory of tooth decay, 244 
Human fat: 

composition of, 106 

formation of, 228 
Hydrazines, 52 
Hydrobilirubin, 200 
Hydrocarbons, 4 

sources of, 6 

Hydrochloric acid, preparation of, Ap- 
pendix, 297 
Hydrochloric acid in stomach, 150 

test for in gastric contents (Exp. 223) 
Hydrocyanic acid, 45 

preparation of (Exp. 66) 

reactions of (Exp. 67-70) 
Hydrogen, qualitative test for, 3 
Hydrogen-ion concentration, 161 

determination of, 163 

favorable for gastric digestion, 151 

favorable for mouth bacteria, 243 

of saliva, 164 
and tartar formation, 143 

of serum albumin and globulin, 

143 

of urine, 190 
Hydrogen peroxide, 85 
Hydroquinol (hydroquinone), 64 



Hyperacidity, 240 

in relation to pyorrhea, 241 
Hypoacidity of oral fluids, 240 
Hypobromite solution, Appendix, 297 
Hypoxan thine, 58 



Imides, 51 
Imines, 51 
Imino group, 51 
Indican, 201 
Indigo blue, 202 
Indol, 70, 201 
Indoxyl: 

in urine, 201 

relation to tryptophane, 201 

test for, 200 

Indoxyl-potassium sulphate, 201 
Inosite, 141 
Insulin, 223 

Intestinal digestion, 153 
Invertase (Invertin), 101 
Iodine : 

absorption number of fat, 106 (Exp. 

149) 

Gram's solution, 84 

Lugol's solution, 86 

in diet, 234 

test for in bile pigments, 153 
lodoform, 13 

preparation of (Exp. 15) 

test for, 182 
Iron: 

in diet, 234 

scale salts of, 38 
Isobenzonitril, 46 

test for chloral, 21 
Isocyanic acid, 47 
Isoelectric point, 119 
Isoelectric protein, 119 
Isomers, 10 
Isomerism : 

metameric, 10 

polymeric 10 
Isoquinoline, 72 
Isonitrils, 46 

K 

Kekule's benzene ring, 60 
Kephir grain, 128 
Keratins, 121 

experiments with, 172, 173 
Kerosene, 7 
Ke tones, 22 
Ketose, 97 
Kjeldahl test for nitrogen, 3 

quantitative (Exp. 183), 280 
Kumiss, 128 



314 



INDEX 



Lacmoid, 63 

Lactalbumin, 127 

Lactase, 153 

Lactates, acid in urine sediment, 214 

Lactic acid, 35, 36 

and tooth decay, 242 

ethylidene lactic acid, 36 

in muscle, 140, 141 (Exp. 212) 

in saliva, 167 

optical activity of, 37 

paralactic acid, see sarcolactic acid 

sarcolactic acid, 36, 141 

test for (Exp. 60) 
Lactose, 100 

Lactosazone (PL V, Fig. 3), 99 
Lanoline, 108 
Lanolinic acid, 108 
Lard crystals (PL VI, Fig. 3), 132 
Lead in urine, 211 
Lecithin, 108 
Lecithoprotein, 114 
Leucine, 41 

in saliva, 183 

(PL I, Fig. 2), 40 

preparation of, 303 
Leucocytes, 133 
Levulose, 100 

formula for, 100 

properties of, 100 
Lieberman's reaction, 117 
Lignocellulose, 103 
Lipase, 95 

from castor bean, preparation of, Ap- 
pendix, 299 

in digestion, 151, 152 
Lipoids, experiments with, 150, 151 
Lithium phosphate (uric acid solvent), 

59 

Local anesthetics, 78 
LugoFs caustic iodine, 86 
LugoFs iodine solution, 86 
Lycppodium (PL VIII, Fig. 6;, 213 
Lysine, 40, 112 



M 



MacDonald, Dr. C. F., Oxidases in sal- 
iva, 171 
Magnesium: 

acid lactate (PL VII, Fig. 4), 185 

ammonium phosphate, 214 (PL IV) 

in diet, 234 

in teeth and tartar. 142 

lactate (PL VII, Fig. 3), 185 

mixture, Appendix, 299 
Malic acid, 35 

test for (Exp. 59) 



Malonic acid, 33, 34 
Maltase, 153 

in saliva, 149 
Maltose, 101 

absorption of (footnote), 219 
Maltosozone (PL V, Fig. 2), 99 
Mannite, 15 

Marine's reagent, Appendix, 299 
Marsh gas, see methane 
Marsh gas mixture, 8 
Marshall's salivary factor, 168 
McCrudden's calcium determination, 

178 

Meconic acid, test for (Exp. 50) 
Menthol, 86 
Mercaptan, 47 
Mercaptol, 48 
Mercury in saliva, 183 
Mercuric chloride, 86 

test for, 86 

Mercuric succincimide, 51 
Mesitylene, 62 
Metabolism, 219 

carbohydrate, 219 

fat, 228 

in diabetes, 222 

of carbohydrates in relation to fat, 
230 

of carbohydrates in relation to pro- 
teins, 223 

of pyorrhea patients, 241 

protein, 122, 223 

salts in, 233 

sulphur, 227 

Meta compounds, defined, 61 
Meta cresol, 65 
Metal proteinates, 117 * 
Metaproteins, 114 

experiments with, 189-191 
Methane, 7 

preparation of, Exp. 6 
Methethyl, 86 
Methyl amine, 50 
Methyl bromide, 13 
Methyl-alcohol, see alcohols 

chloride, 12, 86 

chloroform, 13 

ether, 26 

ethyl ether, 27 

iodide, 13 

salicylate, 66 

preparation of (Exp. 117) 
Methylene chloride, 13 

ether, 27 
Michaels, Dr. J. P., on saliva analysis, 

240 

Microchemical analysis, 74 
Microchemical filtration, 76 
Milk, 125 

acidity of, 125 



INDEX 



315 



Milk: alcoholic fermentation of, 128 

composition of, 127 

composition of human, 127 

experiments with, 176-185 

fat, 127 

modified, 127 

plasma, 125 

reaction of, 125 

specific gravity of, 125 

solids by calculation, 125 

sugar of , 127 

vitamines in, 235 

Miller's lactic acid theory of tooth de- 
cay, 242 
Millon's reaction (for proteins), 116 

Exp. 154 

reagent, Appendix, 299 
Mineral salts in diet, 233 
Mixed ether, 26 
Molisch's reagent: 

test for carbohydrates, 99 

(Exp. 126), 270 
Monobrom-methane, 13 
Monochlor-methane, see methyl chlo- 
ride 

Monosaccharides, 97 
Monoses, 97 
Morphine, 73, 86 

and Marm6's reagent (PI. IV, Fig. i), 

77 

microchemical test for, 87 

(PI. IV, Fig. 4), 77 
Moth scales (PI. VIII, Fig. 6), 213 
Mouth conditions and bacteria, 243 
Mucic acid, 170 
Mucin, 124 

experiments with, 186-188 

from navel cord, 305 

in saliva, 169 

in urine sediment, 216 

substances, 124 
Mucoids, 114 
Murexide, 58 

Murexide test for uric acid (Exp. 84) 
Muscle, 138 

experiments with, 211-212 

extractives, 139, 140 

metabolism, 141 

plasma, 138 

serum, 139 

stroma, 139 
Musculin, 285 
Myogen, 286 
Myosin, 139 
Myosinogen, 139 



N 



Naphtha, 7 
Naphthalene, 62 



Naphthol, 64 
Narcotine, 73 
Nephritic saliva, 244 
Nessler's reagent, 300 
Neutral fat, 228 
Nicotine, 73 
Nirvanin, 87 

Nitre, sweet spirits of, 42 
Nitrils, 30, 46 
Nitrites: 

test for in saliva, 176 
Nitrobenzene, 69 

preparation of (Exp. 98) 
Nitrocellulose, 104 
Nitrogen : 

qualitative test for, 3 

Kjeldahl determination of, 3 (Exp. 
183) 

metabolism, 223-227 
Nitroglycerine, 87 

test for, 87 

Non -protein nitrogen in blood, 135 
Novocaine, 87 
Nucleic acid, 226 
Nucleohis tones, 114 
Nucleoproteins, 114 

in metabolism, 226 

O 

Oil of bitter almonds, 65 

of cloves, 88 

gaultheria, 66 

gaultheria, test for, 88 

mirbane, 69 

wintergreen, 66 
Oils, 105 

Olefin series of hydrocarbons, n 
Oleic acid, 32 

Optical analysis, sugar solution, 208 
Organic acids, 29 

experiments with, 256 
Organic chemistry: definition, i 
Organic matter in teeth and tartar, 143 
Ornithin, 40 

Ortho-compounds, defined, 61 
Orthocresol, 64 
Orthoform, 88 
Osazones, 99 
Ovaglobulin, 113 

Oxalate, acid potassium, see salt of sor- 
rell 

calcium in urine sediment (PL II, 

Fig. i), 77 
Oxalates: 

insoluble (Exp. 54) 

in urine, 214 

occurrence in vegetables, 235 
Oxalic acid, 34 
Oxaluric acid, 56 



316 



INDEX 



Oxidases, 94 

preparation of from potato (Exp. 118) 
Oxidases in saliva, 171 
Oxidation of fat, 229 
Oxidation in muscle cell, 141 
Oxyacids, 35 
Oxybenzene, 63 
Oxybutyric acid, 37, 210 
Oxy hemoglobin, no, 132 
Oxypropionic acid, 36 



Palmitic acid, 32 (PL IV, Fig. 5), 77 
Palmitin, 32, note Exp. 143 
Pancreas, role in diabetes, 221 
Pancreatic digestion, 151 

extract, 300 

juice, digestion with (Exps. 229-232) 

rennin, 150 
Paracasein, 126 
Para-compounds, defined, 61 
Parabanic acid, 57 
Paraffin, 7 

Paraffin series of hydrocarbons, 5 
Paraform, 20 

Paraformaldehyde, see paraform 
Paraglobulin, 279 
Paralactic acid, see sarcolactic 
Paraldehyde, 21 
Paramyosinogen, 139 
Pentane, 5 
Pentpses, 97 
Pepsin, 149 

digestion with (Exps. 216-220) 
Pepsinogen, 149 
Pep tides, 115, 123 
Peptones, experiments with, 197-199 

preparation of (Exp. 196) 
Peroxidases, 94 
Peroxide of hydrogen, 85 
Petroleum ether, 7 
Petroleum jelly, 7 
Phenacetine, 70 
Phenol, 63, 88 

compounds, 88 

preparation of (Exp. 104) 

tests for (Exp. 105-107) 
Phenol-sulphonic acid, 68 

use as therapeutic agent, 68 
Phenyl-alanine, 40, in 
Phenyl-formamide, 52 
Phenyl-hydrazine, 52 

test for sugar (Exp. 135) 
Phenyl hydrozone, 99 
Phenyl isocyanides, 46 
Phenyl-sulphonic acid, 68 
Phenyl-sulphuric acid, 68 
Phloroglucmol, 64 
Phosgene, preparation of (Exp. 17) 



Phosphates and mouth bacteria, 243 

in saliva, 173 

determination of, 173 

in urine, 196, 214 

in urine sediment (PL IX, Fig. 6), 214 

metabolic function of, 234 

titration with uranium, 197 
Phospho-proteins, 114 
Phosphorus, qualitative test for, 4 
Phospho-tungstic acid test for uric acid 

(Exp. 90), see also uric acid 
Phthalic acid, 67 

anhydride, 67 
Picric acid, 70 
Pineapple essence, 43 
Piotrowski's test (for proteins), 115 

Exp. 155 
Pitch, 62 

Polymer, term defined, 10 
Polyoses, 102 
Polysaccharides, 102 
Potassium: 

acid phosphate 
in muscle, 141 

bitartrate, 38 

chloride (PL VII, Fig. 5), 185 

cyanate, 47 

cyanide, 45 

ethylate, 15 

ferricyanide, 46 
preparation from KtFe(CN)e (Exp. 

7i) 

ferrocyanide, 46 

preparation from K 3 Fe(CN)e (Exp. 
72) 

hydroxide (in dental preparations), 88 

iodo-hydragerate, see Nessler's solu- 
tion 

methylate, 15 

platinic chloride (PL III, Fig. 3), 77 

red prussiate of, 46 

salts, function of in metabolism, 233 

sulphocyanate, see thiocyanate 

thiocyanate, 174 

test for in saliva, 175 

yellow prussiate of, 46 
Potato spirit, 17 
Potato starch (PL V, Fig. 6), 99 
Primary alcohol, 16 

amine, 50 
Proenzyme, 95 
Prolamines, 113 
Proline, 41, 71 
Propane, 9 
Propionic acid, 31 
Propylene, 12 
Prosecretin, 152 
Protamines, 113 
Proteans, 114 
Proteins, no 



INDEX 



317 



Proteins: alcohol-soluble, 113 

caloric value of, 231 

classification of, 112 

coagulated, 115 

color reactions of, 115 

conjugated, 114, 124 

derived, 114, 122 

digestion of, 149, 151, 1 53 

electrical charge on, 118 

hydrogen-ion concentration of, 143 

ionization of, 118 

isoelectric, 119 

digestion of, 224 

metabolism of, 223 

precipitants of, 117 

experiments with, 157-161 

properties of, no 

secondary derivatives of, 115 

simple, 112 

synthesis of, 224 
Proteoses, 115, 123 

preparation of (Exp. 193) 
Prothrombin, 130 
Proximate analysis, 2 
Proximate principles, 2 
Prussic acid, 45 
Pseudocellulose, 103 
Ptomaines, 52 
Pytalin digestion, 148 
Ptyalin, enzyme of saliva, 148, 171 
Purine, 57 

Purine nucleus derivatives, 58 
Pus in urine sediment, 216 (PL VIII, 

Fig. 3), 213 
Putrescine, 51 
Pyorrhea and systemic conditions, 241 

and glycosuria, 241 

urine and saliva, 241 
Pyridine, 72 

Pyrocatechol (pyrocatechin), 63 
Pyrogallic acid, 64 
Pyrogallol, 64 
Pyromucic acid, 71 
Pyrotartaric acid, 34 
Pyrrol, 71 
Pyrollidine carboxylic acid, 71 



Quinine, 73 
Quinoline, 72 



R 



Racemic compounds, 38 
Red blood corpuscles, 131 
Red prussiate of potassium, 46 
Reichert-Meissel number of fats, 107 

(Exp. 148) : 
Renal casts, 215 



Renal glycosuria, 222 

Renal threshold for glucose, 222 

Rennin, 150 

experiment with, 221 
Resprcinol, 63 
Reticulin, 122 
Rhigoline, 6, 88 
Ringer's solution, 89 
Rochelle salts, 38 
Rock oil, 6 



Saccharic acid, 98 
Saccharin, 89 

test for, 89 
Saccharose, 100 
Salicylates, 66 
Salicylic acid, 66 

preparation of from oil of winter- 
green (Exp. 115) 
Saliva, 156 

acetone in, 182 

acidity of, 161 

permanent, 165, 167 
temporary, 165 
titratable, 164 

action on starch, 149 

albumin in, 170 

alkalinity of, 167 

ammonia in, 179 

analysis blank for, 186 

and blood, 244 

calcium in, 178 

constituents of, 169 

carbon dioxide in, 165 

methods for determining, 165 

carbonates in, 173 

chlorine in, 177 

creatinine in, 180 

dialysis of, 185 

enzymes of, 171 

experiments with, 213-215 

hydrogen-ion concentration of, 161 

lactic acid in, 167 
determination of, 167 

mercury in, 183 

mucin in, 169 

nitrates in, 177 

nitrites in, 176 

of pyorrhea patients, 241 

oxidase, test for, 173 

phosphates in, 173 

physical properties of, 157 

polarized light crystals from, 246 

potassium thiocyanate in, 174, 240 

proteins in, 169 

ptyalin, 171 

quantity of, 157 

salts of, 173, 246 



INDEX 



Saliva, sediment of, 184 

soluble salts of, 184, 246 

specific gravity of, 157 
determination of, 160 

sugar in, 183 

total solids and ash of, 181 

urea in, 179 

uric acid in, 181, 245 

variation of secretions, 156 
from different glands, 156 

viscosity of, 158 
Salivary digestion, 148 
Salivary factor, 168 
Salol, 66 

Salt of Sorrell, 34 
Salts in metabolism, 233 
Salvarsan "606", 70 
Saponification, 107 

Exp. 144 

Sarcolactic acid, 36 
Saturated hydrocarbons, 6 
Scale salts of iron, 38 
Schiff's reagent: 

preparation of (Exp. 35) 

test for in uric acid (Exp. 91) 
Schweitzer's reagent, 103 
Scleroproteins, 113, 121 
Scombrone, 113 
Secondary alcohol, 16 
Secondary amines, 51 
Secondary protein derivatives, 115 
Secretin, 152 
Sediment, salivary, 184 
Sediment, urinary, 212 
Serine, 40, in 
Serum albumin, no, 143 

blood, 130 

globulin, 143 
Silver nitrate, 89 
Silver nitrate, Howe's method for 

root canal treatment, 84 
Simple proteins, 112 
Sodium acid phosphate, 173, 214 
Sodium acid urate, 213 (PL IX, Fig. 3) 
Sodium chloride, 89 (PL VII, Fig. 2), 

,- 185 
in diet, 233 

perborate, 89 

peroxide, 89, 90 

oxalate, 214 (PL II, Fig. i), 77 

phosphate (uric acid solvent), 59 

urate (PL IX, Fig. 3), 214 
Soluble salts: 

in saliva, 184, 246 

in urine, 200 
Soluble starch, 148 
Somnoform, 90 
Specific gravity: 

of blood (Exp. 204), 284 

of milk, 125 



Specific gravity: of saliva, 157 

of urine, 189 
Spermatozoa in urine sediment, 216 

(PL VIII, Fig. 2), 213 
Spiritus Glonoini, 87 
Starch, 102 

experiments with, 272 

hydrolysis of, 149 (Exp. 140), 272 

paste, 301 

preparation of (Exp. 139) 

soluble, 102, 148 
Steapsin, 152 
Stearic acid, 32 
Stearin, 32 
Stereopten, 105 
Stereoisomerism, 38 
Sterols. 1 08 
Stomach steapsin, 151 
Streptococcus hemolyticus, 243 
Streptococcus viridans, 243 
Stovaine, 90 

Straight-chain hydrocarbons, 6 
Strontium oxalate (PL II, Fig. 3), 77 
Strychnine, 73 

test for, 73 
Sturine, 114 

Substitution products, 5 
Succinic acid, 33, 34 
Succinimide, 51 
Sucrase, 153 
Sucrose, 100 

digestion of, 153 

formula for, 101 

inversion of, 101 
Sugar in blood, 220 

in saliva, 183 

in urine, 205 

determination of, 205-208 

of milk, 101 

Sugar in relation to tooth decay, 241 
Sulphanilic acid, 68 
Sulphates in urine, 198 
Sulphocyanate in saliva, see saliva 
Sulphocyanic acid, 47 
Sulphonal, 48 
Sulphones, 48 
Sulphonic acids, 48 
Sulphur compounds, 47 
Sulphur elimination, 227 
Sulphur, qualitative test for, 4 
Sulphur, metabolism of, 227 
Sulphocyanate in saliva, 174, 240 
Suprarenal glands, 90 
Suprarenalin, 79 
Syntonin, 122 



Tannic acid, 67, QO 
Tannin, 90 



INDEX 



319 



Tartar: 

analysis of, 145 

composition of, 142 

formation of, 143 
Tartar emetic, 38 
Tartaric acid, 35, 38 
Taurine, 48, 155 

in muscle, 139 
Taurocholic acid, 155 
Teeth: 

analysis of, 145 

composition of, 142 
Teichman's hemin crystals 

test for blood, 133 (Exp. 207) 
Temporary acidity of saliva, 165 
Terpenes, 107 
Tertiary alcohols, 16, 23 
Tetrachloride of carbon, 13 
Thein, 58 ^ 
Theobromine, 73 
Thio-alcohols, 47 
Thiocyanate in saliva, 174, 240 
Thiocyanic acid, 47 
Thio-ether, 48 
Thio-ketones, 48 
Thiophene, 71 
Thrombase, 130 
Thrombin, 130 
Thromboplastin, 130 
Thorner on acidity of milk, 126 
Thymol, 64, 91 
Thymol iodide, 91 
Thymophene, 91 
Thyroids, 91 

Tollen's reagent, Appendix, 301 
Toluene (Toluol), 61 
Toluidene, 69 
Tooth decay: 

and sugar, 241 

Howe's theory of, 244 

Miller's theory of, 242 
Tribromphenol (PI. Ill, Fig. 5), 77 

Exp. 105 

Trichloracetic acid, 91 
Trichloraldehyde, 21 
Trichlor-me thane, 81 
Tricresol, 65 
Trimethylamine, 50 
Trimethylbenzene, 62 
Trinitrophenol, preparation of (Exp. 

109) 

Triolein, 106 
Tripalmitin, 106 
Triple-bonded hydrocarbons, 12 

test for (Exp. n) 
Triple phosphates, 214 
Tristearin, 106 
Tritenyl, 43 
Tropa cocaine, 91 
Tropeolin, Appendix, 302 



Trypsin, 151 
Trypsinogen, 151 
Tryptophane, in, 201 

formula, 40 

relation to protein metabolism, 226 

test for in proteins, 117 
Tyrosin, 67, 112 

formula, 40 

preparation, Appendix, 303 

test for in proteins, 116 



U 



Uffelman's reagent: 

test for lactic acid (Exp. 225) 
Ultimate analysis, 2 
Unsaturated hydrocarbons, n 

test for (Exp. 8) 

Unsymmetrical hydrocarbons, 62 
Uranium, standard solution for phos- 
phates, 197 
Urea, 54 

cyclic formula for, 55 
determination of in saliva, 179 
determination of in urine, 192, 213 
experiments with, 80-83 
formation of from protein, 225 
in muscle, 139 

nitrate, 56 (PL I, Fig. 3), 40 
oxalate, v 56 (PL II, Fig. 5), 77 
synthesis of (Exp. 79) 
Ureas, substituted, 56 
Urease, preparation of, 302 
Ureides, 56 
Uric acid, 57 

and lithium salts, 59 
and disodium phosphate, 59 
determination of in blood, 138 
in saliva, 181, 245 
in urine, 192, 213 
formula for, 57 
in muscle, 139 
in urine sediment (PL IX, Fig. i, 

2), 214 

metabolic origin of, 226 
murexid test for, 263 
phosphotungstic acid test for, 

Folin, 181 
Urine, 187 

abnormal constituents, 202 
acetone in, 209 
acidity of, 190, 195 
albumin in, 202 

alkaline phosphates in, 196, 214 
ammonia in, 193 
analysis blank and use, 218 
appearance of, 188 
bile in, 210 
causes of cloudy, 189 



320 



INDEX 



Urine: chlorine in, 195 

color of, 1 88 

coloring matter in; 199 

epithelium in, 215 

hydrogen ion concentration of, 190 

indoxyl in, 200 

method of collecting, 187 

normal constituents of, 190 

phosphates in, 196, 214 
estimation of, 197 

physical properties of, 188 

quantity of, 188 

reaction, 190 

soluble salts in, 200 

specific gravity, 189 

sugar in, 205 

sulphates in, 198 

uric acid in, 192, 213 
Urinary sediment, 212 
Urinometer, 189 
Urobilin, 199 
Urochroifte, 199 
Uroerythin, 199 
Urorosein, 199 



Vitellin, 114 
Volatile oils, 107 



W 



Water, detection of in alcohol, 252 
Wheat Starch (PL V, Fig. 4), 99 
White blood corpuscles, see leucocytes 
Will and Varrentrap's test for nitro- 
gen, 3 

Wohler's test for nitrogen, 3 
Wood spirit, see methyl alcohol 
Wood fibers (PL VIII, Fig. 6), 213 

X 

Xanthine, 58 

Xanthroproteic test for proteins, 116 

Exp. 153 
Xylene, 61 
Xylose, 97 



Yeast cells in urine sediment (PL IX, 

Fig. 4), 214 
Yellow prussiate of potassium, 46 



Valeric acid, 31 

Valine, 41 

Vaseline, 7 

Vegetable globulin, 306 

Vinegar, 30 

Viscosity of saliva, 158 

Vitamines, 235 



Zein, 113 

Zeolite method for ammonia determi- 
nation, 194 

Zinc oxalate (PL II, Fig. 6), 77 
Zymase, 95 
Zymogen, 95