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THE DETERMINATION OF SUCROSE IN PLANT
EXTRACTS. AN INVESTIGATION OF THE
VARIATION BETWEEN THE VALUES
OBTAINED BY THE POLARISCOPIC
AND CHEMICAL METITODS

BY

PAUL PETER SOMERS

THESIS

FOR THE

DEGREE OF BACHELOR OF SCIENCE

IN

CHEMISTRY

COLLEGE OF LIBERAL ARTS AND SCIENCES

UNIVERSITY OF ILLINOIS

1920

—— by the Internet Archive
in 2013

http://archive.org/details/determinationofsOOsome

UNIVERSITY OF ILLINOIS

THIS IS TO CERTIFY THAT THE THESIS PREPARED UNDER MY SUPERVISION BY

PAUT, PETER SOMERS

Methods.
IS APPROVED BY ME AS FULFILLING THIS PART OF THE REQUIREMENTS FOR THE

Instructor in Charge

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Acknowledgment

The author wishes here to

express his indebtedness to

Dr. Duane T. Englis upon whose
suggestion this investigation
was undertaken. A sincere
appreciation is teit for his
assistance in the organization
of the work and the writing of

this paper.

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Table of Contents.

Introduction - Nature and Object of the Investigation.

Experimental Part.

Hxperiment l. On the Possibility of the Hydrolysis of

Sucrose.by Fehling's Solution and Resulting Low Values for
Sucrose by Reduction Methods.

(a) Preliminary Discussion.

(bo) Methods, Materials and Procedure.

(c) Results.
Experiment 2. The Effect of Neutral Salts on the
Rotation of Invert Sugar.

(a) Preliminary Discussion.

(bo) Methods, Materials and Procedure.

(c) Results.
Experiment 3. The Possibility of Impurities Combining
with the Sucrose or Invert Sugar and Altering their
Rotation Values.

(a) Preliminary Discussion.

(bo) Methods, Materials and Procedure.

(c) Results.

III. Summary.

I¥. Bibliography.

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The Determination of Sucrose in Plant Extracte.

An Investigation of the Variation between the Values

Obtained by the Polariscopic and Chemical liethods.

Introduction.

The accurate determination of carbohydrates in plant tissues
is a problem of fundamental importance to any study of plant
metabolism. In most instances the experimental work which has
been carried on, was confined to the determination of certain
large groups, such as reducing sugars, and no effort has been
Made to estimate the quantity of each individual sugar.
Recently this problem has aroused considerable interest and
several investigators have made definite contributions towards
the solution.

It would seem at first glance that of all the sugars which
occur in plants in appreciable quantity, sucrose could be the
most easily and accurately determined. Since it is a non-
reducing sugar and on hydrolysis is converted into dextrose and
levulose, both of which are reducing sugars, the chemical method
involving a determination of the increase in reducing sugar
after hydrolysis can be used for its estimation. Furthermore
the fall in rotation accompanying the conversion of the cane
sugar to dextrose and levulose affords in the double polarization
method,a means of checking the value found by the first
mentioned method. Very few investigators in this field of
plant chemistry have attempted to check their sucrose values
by the two methods, but usually where such an attempt has been

made a considerable disparity is observed. Brown and Morris (1)

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who are perhaps among the earliest workers to make a
detailed study of the individual carbohydrates in plants call
attention to this disparity.

The magnitude of the difference between the two values

seems to vary with different plant materials and with different

portions of the same plant. With nasturtium (tropaeolum

majus), Brown and Morris report slightly higher values by the
polariscopic method. Parkins (2) working with the snow
drop (galanthus nivalis) states that the polarization value
is slightly higher, but usually the difference is negligible.
No experimental work is offered to justify his statement.
More recently Davis and co-workers (3) in extensive researches
on the carbohydrates of the mangold and potato find polarization
values which are sometimes as much as eighty per cent greater
than the reduction values, while sometimes they are as much as
ninety per cent lower. The variation is generally greatest
in the stem extracts. Muncie and Englis (4) find with leaf
extracts of nasturtium much the same results as Brown and liorris.

Some typical analyses are as follows:
Sucrose #1 #2 #3 #4
By Polarization Method 99.2 139.2 107.2 97.7
By Reduction Method 94.3 13223 101.9 92.8

In connection with the determination of Davis and co-workers
it may be well to call attention to the fact that they make the
common mistake of subtracting copper oxide values before
calculating their invert sugar equivalent. As pointed out
by Browne (20), low results are obtained. The following

recalculation from the data of Davis illustrates this error.

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0.3710 G. Cud (after inversion) -- 157.11 mg. invert sugar.

0.2210 G. CuO (before inversion)-- 90.05 mg. invert sugar.

Due to sucrose 67.06 mge invert sugar.

67.06 mg.

invert sugar -- 63.70 mg. sucrose.

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63.70 = 60.59 © 3.11 mg. sucrose error.

Calculation by Davis (.95) = 60.59 mg sucros¢

On this basis, all their reduction values are low and

recalculation of their data on the correct basis would tend

in cases where the polarization values are high, as they are

in most cases,

to bring the results by the two methods more

nearly together.

The theory advanced by Davis (7) to explain the difference
in the values found by the two methods is based on the assumption
that certain optically active nitrogenous impurities such as

glutamin and asparagin are present which cause a fluctuation

in the polarization value. The presence of such impurities
and their effect on the determination of sucrose in molasses

and cane products has been noted by Pellet (8), Saillard (9),

Ogilvie (10) and others. Since the rotation of most of these

impurities is altered by the presence of acids, Saillard proposes
the determination of sucrose by a double neutral polarization
and thus eliminate the disturbance due to the change in rotation

of the impurities caused by the addition of the acid to bring

about the hydrolysis of the cane sugar. The use of acid and
the introduction of neutral salts by neutralization of the acid

after hydrolysis can be avoided by carrying out the sucrose

inversion by means of the enzyme invertase. Assuming that

invertase is specific for the sucrose linkage, it would seem

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that if the neutrality of the solution is maintained
the rotation of all optically active substances other than
sucrose should be of a constant value before and after the
inversion and the change in rotation should be due to alteration
in the sucrose alone. Consequently the explanation of Davis
unless modified is untenable,

Other polysaccharides such as raffinose, gentianose and
stachyose contain the sucrose linkage and would be partially
hydrolyzed by invertase with an accompanying change in rotation
different from that corresponding to the change of sucrose to
invert sugar. Lowever, such polysaccharides should also give
an increase in reducing power on hydrolysis with acids. No
such increase has been noted where proper precautions were taken
to prevent enzymatic activity after the picking of the plant
material. There is practically no evidence for the occurrence
of these higher sugars in the plants which have been investigated
so they need not be considered.in this connection.

Object of the investigation.

This investigation is an endeavor to find a satisfactory
explanation for the difference in the amount of sucrose found by
polarization and reduction methods. During the progress of the
work several lines of attack have suggested themselves. The

experimental work and discussion in connection with each follows.

alt oy
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Experimental Part.

Experiment 1. On the Possibility of the Hydrolysis of
Sucrose by Fehling's Solution and Resulting Low Values for
Sucrose by the Reduction Method.

(a) Preliminary Discussion: Since in most instances
the polarization values were high, the first suggestion by
way of explanation of the problem, was that in the determination
of reducing sugars before inversion there might be a partial
hydrolysis of the sucrose by the Fehling's solution and
consequently a resulting low value for sucrose when calculated
from the increase in reduction after inversion. Browne (5)
has carried out a series of experiments in which he showed
that the reduction due to sucrose when accompanying reducing
sugars was quite appreciable. Allihn's (11) method, in which
the sugar is in contact with the Fehling's solution for two
minutes, was used by Browne and he states that the reduction
due to the sucrose would be greater in a longer boiling method.
Kjeldahl-Woy (12) have worked out a table for the determination
of reducing sugars in the presence of sucrose in varying
quantities. In this method the heating is continued for
twenty minutes but since the process of determination is
somewhat involved, it has not found general application.

Parkin (2), Davis,Daish and Sawyer (3), and Muncie and Englis (4)
have used the method of Brown, Morris and Iiillar (13) which

is one of the longer boiling methods. Parkin (2) claims to
have tested various mixtures of hexoses and sucrose and states
that the difference due to the sucrose is very small unless

present in preponderating proportions. No data is given as

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to concentration so it seemed advisable to confirm this

statement.

(b) Methods, Materials and Procedure.

Purification of Sucrose; Pure sucrose was prepared
by taking a boiling, saturated solution of saccharose and
precipitating the sucrose in absolute alcohol which had been
refluxed and distilled over lime. The liquid was decanted off
and the sucrose precipitate dried at a low temperature in an
electric oven. The dextrose used in the experiments was the
commercially pure product.

Determination of Reducing Sugars: The Brown-liorris-
Millar method was followed in the determination of the reducing
sugars. Exactly fifty cubic centimeters of mixed Fehling's
solution was pipetted into two hundred fifty cubic centimeters
beakers. These beakers were placed in a boiling water bath
at two minute intervals. By a series of experiments, it was
found that after five minutes in the bath, the beaker and contents
would be at a constant temperature. Then fifty cubic centimeters
of the sugar was added from a rapid delivering pipette. A
rapid delivering pipette was necessary in order to have a
homogeneous solution in the minimum time as otherwise layers
would be formed and the reduction would not be uniform. Twelve
Minutes after adding the sugar solution, the beakers were
removed and the contents filtered into suction flasks.

It was found that if the filtrate was then taken and
the copper determined by the Peter's (14) modification, the
process was much more rapid and a greater accuracy was obtained

than could be secured by dissolving the cuprous oxide and

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determining by Low's method. The filtrates were made up

to two hundred cubic centimeters after the addition of five

cubic centimeters of concentrated sulphuric acid. A portion

was then taken, potassium iodide added and the iodine titrated

with sodium thiosulphate, using starch as an indicator. A

pure sucrose blank was run with each set of reductions.

Plan of Experiment: Since the sucrose content of most of
the plants which have been investigated is seldom more than
three times the content of reducing sugars and is usually
nearly equal to it, the relative concentrations maintained in
this experiment were taken within this range. It seemed
advisable, also, to determine whether the quantity of unreduced
copper would have any influence in the results.

The experiment was carried out as follows: Two solutions
were prepared, the first containing seven grams of dextrose in
five hundred cubic centimeters of water and the second containing
ten and a half grams of sucrose in two hundred fifty cubic
centimeters of water. Portion of each solution were measured
carefully from a burette in amounts indicated below and the

mixtures made up to one hundred cubic centimeters with water.

Dextrose Sucrose Ratio S/D

50.00 00.00
50.00 16.66
50.00 50.00
28.55 00.00
28.55 9.51

28.55 28.55

14.27 00.00

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(c) Results.

Sample C.C. Na,5203 CuO equivalent Dextrose equivalent

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2. 5.94 » 3708 151.53
3. 5.92 ~ 5714 151.78
4. 10.86 »22352 87.90
5. 10.70 v 2280 89.15
6. 10.87 02257 S7«ihl
7. 14.97 ~0999 38.75
8. 14.57 ~1119 43.40
9. 14.97 -0999 38.75
Blank 18.30

From the results it can be seen that with the amounts of
Sucrose present there is no apparent increase in the reducing
Sugars and that the quantity of unreduced copper left in the
solution is also without influence. Repetition of the

experiment gave similar results.

Experiment 2. The Effect of Neutral Salts on the
Rotation of Invert Sugar. |

(a) Preliminary Discussion; The second suggestion was that
neutral salts such as sodium acetate, which might be left after
deleading following the use of basic lead acetate might affect
the rotation of sucrose and invert sugar so that the apparent

drop in rotation on inversion would be in error and of the nature

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Some information is available concerning the effect of
neutral salts on sugars. Washburn (15) determined the effect
of such salts as sodium chloride, potassium chloride and lithium
chloride on the specific rotation of sucrose. In all cases
he found that the specific rotation of the sucrose was very
slightly decreased by the addition of the salts. Saillard (9)
found that the dextro-rotation of the sucrose was decreased by
chlorides and hydrochloric acid and that the laevo-rotation
of invert sugar was greatly increased. Owing to the very
general use of lead acetate as a clearing agent, followed by
deleading with sodium carbonate, the principle neutral salt
which accumulates is sodium acetate. Saillard does not
give any data regarding the effect of neutral acetate salts
but merely states that they would act similarly to the chlorides.
In order to see if this statement was justifiable, an experiment
was planned to secure data concerning the effect of such salts.

(db) Methods, Materials and Procedure

Polarization Apparatus: A Schmidt and Haensch polariscope,
a half shaded instrument, calibrated in Ventzke degrees was
used. The sugar solutions were polarized in a four decimeter
water jacketed tube, maintained at a constant temperature of

twenty five degrees Centigrade by the circulation of water from

the thermostat. This apparatus is illustrated in the accompanying

diagram.

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11

Invertase and Invert Sugar; The cane sugar used in the
preparation of the invert sugar was a portion of the sample
purified for the prededing experiment and was hydrolyzed with
invertase. The invertase was prepared by Sherman's (16)
method which consists in taking three pounds of baker's
compressed yeast and mixing it thoroughly with fifteen hundred
cubic centimeters of tap water and one hundred cubic centimeters
of toluene. This mixture, after being allowed to autolyze for
five days, was cleaned with lead acetate and filtered, the
excess lead being removed by hydrogen sulphide. The solution
was then dialyzed through a parchment membrane.

Plan of Experiment: Ten grams of sucrose was placed ina
five hundred cubic centimeter volumetric flask. Four hundred
cubic centimeters of water and five cubic centimeters of
invertase was added. After standing over night, five cubic
centimeters of alumina cream was added and the volume made up
to five hundred cubic centimeters and then filtered. To
Portions of this solution different amounts of sodium acetate

were added and the mixtures polarized.

(c) Results The following table gives the complete data.

Invert sugar Sodium acetate Average reading.
50.00 cc. 0.5 g. -2.00
50.00 1.0 22.00
50.00 2.0 “2.00
50.00 3.0 -2.00
50.00 4.0 22.00

Portions of the solution saturated with sodium oxalate

gave an average reading of -2.00.

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It seems from the results thus obtained that the amount

of sodium acetate or sodium oxalate in the invert sugar solution

does not affect the polarization values.

Experiment 3. The Possibility of Impurities Combining with
the Sucrose or Invert Sugar and Altering their Rotation Values.

(a) Preliminary Discussion. The third suggestion that
impurities either optically or non-optically active, may be
responsible for the disparity is indicated in part in an
experiment by Davis (17) with mangold extract. To each of
two portions of the extract, he added five grams of pure sucrose.
After inversion by the invertase and citric acid methods, it
was found that only four and ninety eight hundredths grams of
the sucrose could be accounted for by the reduction method.

On the other hand, the direct polarization value was equal to
five and twenty two hundredths grams of sucrose. Davis was
working on the methods of extraction and did not try to find
the cause of the high values since he was satisfied that no
sucrose was lost and correct values could be obtained by the
reduction method.

Stanek (18) offers evidence that nitrogenous pigments are
formed by the condensation of amino acids with sugar. When
solutions of invert sugar or sucrose are heated in an autoclave
at one hundred five to one hundred thirty degrees Centigrade
with sodium glutamate or aspartate or with asparagin, carbon
dioxide is liberated, the solution becomes acidic, and dark
pigments are formed which are almost completely precipitated

with lead acetate. Asparagin and aspartic acid give rise to

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13
much more of this product than does glutamic acid.
Maillard (19) in an earlier work finds that when glycocoll
is warmed with four parts of glucose in three to four parts of
water, the liquid slowly assumes a characteristic yellow color,
changing to a dark brown and followed by foaming (carbon dioxide).
It has been shown that the carbon dioxide came from the carboxyl
group of the glycocoll. Then assuming that this loss of carbon
dioxide is accompanied by a union of the nitrogen with the
|} aldehyde carbon of the sugar, the glucose molecules forming part
}of the new compounds, must suffer dehydration resulting in the
}appearance of double bonds or possibly rings. it was also
demonstrated that other amino acids worked the same way on
| glucose and similarly, various sugars act this way on glycocoll.

Apparently the presence of amino compounds in the plant
extracts alter both the direct polarization value of the added
sucrose and also that of invert sugar. Since asparagin and
glutamin have been found in many plants and are suspected of
}veing the generally occuring nitrogenous impurities, it seemed
advisable to determine the effect of the addition of these
compounds to sugar solutions.

(bo) Methods, Materials and Procedure.

The invert sugar was made up as previously described.
The asparagin was from a laboratory stock solution and the
apparatus was the polariscopic apparatus previously described.
The asparagin and invert sugar solutions were mixed and polarized

in accordance with the following table.

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(c) Results.

invert Sugar

Polarization Reading - 320 -1.435 -1.060

The mixed asparagin and invert sugar solution give a
greater negative rotatory value than that obtained by adding the
rotation values for the constituents determined separately.
This seems to indicate that there is some sort of loose combination
between the sugar and amino groups. Under the conditions of
low concentration and low temperatures, no formations of pigments
Or insoluble compounds analogous to those described by Stanek and

Maillard was observed so it is hardly possible that the reaction

is of the same character. However, it seems plausible that it

may be responsible for at least part of the disparity between the
two methods for the estimation of sucrose.

An attempt was made to repeat the experiment with a neutral
solution of glutamic acid hydrochloride but there was such a
development of color that it was impossible to determine the

polarization value of the solution.

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i. There is a disparity between the polariscopic and
chemical methods for the determination of sucrose in plant

extracts.

2. In the concentration in which sucrose is present

in plant extracts, there is no hydrolysis due to the Fehling's

solution which would result in low values for sucrose by the
reduction method.

Se Neutral salts such as sodium acetate and sodium
oxalate, do no have any affect on the rotation of the invert
sugar.

A. As an impurity, Asparagin causes an increased value
for the sucrose by the polarization method and serves as

one explanation for the disparity between the two methods.

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Bibliography.
Brown and Morris J. Chem. Soc., 63, 664, (1893).
Parkin sBiochem. J., 6, 18, (1911-12).
Davis, Daish and Sawyer J. Agr. Sci., 7, 255, (1916-17).
Muncie and Inglis (Unpublished).
Browne J. Am. Chem. Soc., 28 451, (1906).
Davis J. Agr. Sci., 7, 316, (1916-17),
Davis J. Agr. Sci., (1916-17).
Pellet Seen muaer J., 17, 421, (1915).

Saillard Gomp. rehd., 165, 116, (1917).

Ogilvie Int. Sugar J., 14, 89, (1912).
Allihn J. prakt. Chem., (2), 22, 46.
Kjeldahl-Woy them. Centralblatt, 1897, (2),

Brown, Morris and Millar ds Ghem. Soe.,; 7h;

Peters Js Am. Chem. Soc., 34, 928, (1912).
Washburn Z. Ver. Zuckerind., 60, 381.

Sherman J. Am. Chem. Soc., L566, (1914).

36,
(2

Davis J. Agr. Sci., 7, 274,

916-17) e
Stanek Oe onem. SoOc., L12, I, 544.
Maillard Comp. rend., 154, 66.

Browne "Handbook of Sugar Analysis", 937.

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