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Chapter i

Determination of Sucrose
(by polarimetry)

INTRODUCTION

For impure products containing other polarizing substances such as


raffinose, glucose, fructose, amino-acids etc., the determination of sucrose,
by use only of a polarimeter, requires methods of multiple polarization
of which the Clerget Method is a particular example.
If the product does not contain raffinose, two polarizations are made:
one direct and one after hydrolysis. The sucrose content is deduced from
the change in polarization on hydrolysis, it being assumed that the
polarizing impurities are not affected by the hydrolytic reagents, and
behave identically in both measurements.
If the product contains raffinose, this trisaccharide is partially hydrolysed
to fructose and melibiose under the conditions required to hydrolyse
sucrose, and a third polarization is required, either after complete
hydrolysis of the raffinose (and sucrose), or under some other pH con-
ditions so as to produce a predictable change in the optical activity of
the sugars. The sucrose can be deduced from two simultaneous equations
involving the three polarizations. Again it is assumed that the remaining
polarizing impurities are otherwise unchanged by the hydrolytic reagents.
The fate of these impurities, in either case, may not correspond to the
hypothesis for various reasons. Other optically-active compounds may
be hydrolysed or their rotations may be changed by the hydrolytic
reagents; the results obtained thus necessarily have an uncertain character.
It is essential to take into account such factors as the nature of the def ecant,
the content of dry solids of the solution to be polarized and the tempera-
ture and conditions of hydrolysis. Since many optically-active compounds
contribute to polarization, temperature corrections are necessarily un-
certain and it is recommended that, if possible, all polarizations should be
carried out at 20 C to avoid any temperature correction.
The simultaneous equations for three polarizations can be solved to
8 DETERMINATION OF S U C R O S E

deduce raffinose concentration, and formulae have also been proposed


for deducing raffinose from two polarizations if the material is free from
reducing sugars, or if the rotational contribution of the reducing sugars
can be estimated independently. Since the raffinose content is very much
less than the sucrose content, the uncertainties have a relatively greater
effect on the raffinose value and none of these procedures, except the
double-enzyme method of Paine and Balch, is recommended for esti-
mation of raffinose.

OFFICIAL RECOMMENDATIONS

(1) Determinations by multiple polarization are to employ preferably


hydrolysis by enzymes, owing to their specific action (invertase methods
for products derived from cane, the Paine and Balch method for products
derived from beet). In default, acid hydrolysis may be used (the Jackson
and Gillis method IV for products derived from cane, the Osborn and
Zisch method for products derived from beet).
(2) In the Jackson and Gillis method IV at 60 C, the time of heating after
3 min agitation is to be extended to 10 min, total time of heating.
(3) To avoid any temperature correction, all polarizations are to be
carried out at 20 C, if possible.
(4) The concentration correction in the Clerget divisor shall be based
on the total solids concentration, not on the sucrose concentration.
(5) In the determination of sucrose in cane products, the solution for
the invert polarization shall be of the same normality as that used for
direct polarization.
(6) Home's dry subacetate of lead, or its equivalent shall be used for
clarification of cane molasses. For deleading, dry ammonium dihydrogen
phosphate is recommended for use with the invertase method, and
anhydrous potassium oxalate with the Jackson and Gillis method IV.

DIVISORS

ICUMSA sub-committees have investigated values for the divisors,


concentration coefficients and temperature coefficients and the following
modifications to the originally published values have been officially
adopted:
Invertase method, divisor:
132.1 - 0.0833 (13-g) - 0.53 (7-20)
FOR CANE PRODUCTS 9

Jackson and Gillis method IV divisor:


(a) Inversion at 60 C:
1 3 2 . 5 6 - 0 . 0 7 9 4 (13-g) 0.53 (/-20)
(/?) Inversion at normal temperature:
1 3 2 . 6 6 - 0 . 0 7 9 4 (13-) 0.53 (V-20)
Paine and Balch double-enzyme method:
factor for (A) 2.201, factor for (B) 1.201
divisor: 132.22 + 0.0833(^-13)

In the above formulae, g is gram solids from original sample in 100 ml


of the solution read in the saccharimeter, / is the temperature at which the
readings are made and (A) and (B) are the two invert polarizations of the
Paine and Balch method.

For Cane Products

a. Invertase Method (official)

(i) Direct reading, - Dissolve the double-normal weight of the substance


in water in a 200 ml volumetric flask. (Depending on the colour multiples
or fractions of this weight may be used and the results calculated to a
basis of 26 g per 100 ml.) Add dry lead to clarify avoiding any excess,
shake, dilute to the mark with water, mix well and filter, keeping the
funnel covered with a watch glass. Reject the first 25 ml of filtrate. When
sufficient filtrate has collected, remove the lead from the solution by
adding ammonium dihydrogen phosphate in as small excess as po sible. c

Mix well and filter, again rejecting the first 25 ml of filtrate. Pipette one
50-ml portion of the lead-free filtrate into a 100-ml flask, dilute with water
to the mark, mix well and polarize in a 200-mm tube. The result, multi-
plied by 2 is the direct reading, P, of the formula given below. (If a
400-mm tube is used the reading equals P.) If there is a possibility of
mutarotation, allow sufficient time for its completion.
(ii) Invert reading. - First determine the quantity of acetic acid necessary
to render 50 ml of the lead-free filtrate distinctly acid to methyl red
indicator. To another 50-ml portion in a 100-ml volumetric flask, add
the requisite quantity of acid and 5 ml of invertase solution, fill the flask
with water nearly to 100 ml. Stand overnight (preferably at a temperature
10 D E T E R M I N A T I O N OF S U C R O S E

not less than 20 C), dilute to volume at 20 C, mix well and polarize at
20 C in a 200-mm tube. Correct the polarization for the optical activity
of the invertase solution and multiply by 2 to obtain the invert reading
for a normal solution (I).
Determine total solids by refractometer and multiply this figure by
the density at 20 C to obtain total solids from original solution in 100 ml
of the invert solution (g).
If a rapid inversion is required, use 10 ml of invertase solution and
stand in a water bath at 55-60 C for 15 min with occasional shaking.
Cool, add sodium carbonate until distinctly alkaline to litmus paper,
adjust to volume and polarize as described above. Allow the solution
to remain in the tube and again determine the polarization. If there is
no change from the previous reading, the mutarotation is complete.
If it is necessary to work at a temperature other than 20 C, both
polarizations should be made at the same temperature, which must be
as close as possible to 20 C.
Calculate the percentage sucrose, S, in the original sample from

b. JACKSON AND GILLIS'S Method IV (official, in default of a)

SCOPE. The method is applicable in the presence of invert sugar, but


inapplicable in the presence of optically active non-sugars which change
rotation with acidity. In principle sodium chloride equalises the effect of
hydrochloric acid on invert sugar present as an impurity.

PROCEDURE. - Prepare a normal solution of the sample or a solution of


such fractional normality as the nature of the substance will permit.
Defecate, if necessary, with dry basic lead acetate in the usual manner,
making to volume at the temperature at which the observations are to
be made. Filter. Delead with anhydrous potassium oxalate.
Pipette two 70-ml (or 50 ml + 20 ml of water) portions of the clear
filtrate into two 100-ml flasks. If preferred, 75-ml portions may be taken.
To one portion add 2.315 g of sodium chloride or 7.145 ml of a
saturated sodium chloride solution or 10 ml of a solution containing
231.5 g per litre; make to volume at the temperature at which the
observations are to be made and polarize. The temperature should be as
FOR BEET P R O D U C T S 11

close to 20 C as possible. Correct the rotation to the normal weight of


sample, P.
The other portion is inverted either at 60 C or at room temperature.
Either:
(i) add 10 ml of hydrochloric acid (d % 1.1029 or 24.85Bx. at 2 0 Q .
2

Immerse in a water bath at 60 C. Agitate the solution continually for


about 3 min and allow it to remain a total time of 10 min. Cool quickly.
Or:
(ii) add 10 ml of hydrochloric acid (d % 1.1029 or 24.85Bx. at 20C
2

and allow to remain 30.8 h at 20C, 14.6 h at 25C or 7.1 h at 30C. Cool.
Make to volume and polarize at the same temperature as used for
direct polarization. Correct the rotation to the normal weight of sample (P ).
r

Calculate the percentage sucrose, S, in the original form:


(a) Inversion at 60 C:

For Beet Products

a. Double-enzyme Method
(see chapter 3. Determination of Raffinose)

b. OSBORN AND ZISCH'S Method (official, in default of a)

PRINCIPLE. - The method involves the assumption that the material


is free from reducing sugars (or that the contribution of these compounds
to the direct polarization is zero) and that the optically active non-sugars
consist mainly of amino-acids contributing a value N to the direct
polarization. The rotation of the non-sugars is considered to be zero in
the acid solution after inversion but to be restored to its original value
on neutralisation. The difference between the invert polarization in acid
and neutral solution becomes a measure of N and hence it is possible
to deduce sucrose (and raffinose) from the three polarizations.
12 D E T E R M I N A T I O N OF S U C R O S E

S C O P E . - O w i n g to variation in composition of the impurities from


factory to factory, the coefficients used in the formulae giving sucrose
(and particularly raffinose) cannot rigorously have a general value but
may be satisfactory to give comparative results in any one factory.

PROCEDURE. - Dissolve 130 g (5 normal weights) of the sample in water


in a 500-ml volumetric flask, defecate with basic lead acetate, dilute to mark,
filter and delead with ammonium dihydrogen phosphate as in invertase
method. Polarize in a 200-mm tube to obtain the direct polarization, (P).
Pipette 50 ml deleaded filtrate and 15 ml water into each of two 100-ml
volumetric flasks and heat to 68-69 C in a 70 C water bath. Remove
from bath and immediately add 10 ml of hydrochloric acid (d % 1.1029).
2

Allow to cool spontaneously for 2 h, and then cool to 20 C Dilute one


sample to 100 ml at 20 C, mix, filter if necessary, and polarize at 20 C in
a 400-mm tube to obtain the invert polarization (/). To the second sample
add 1-2 drops of 0.2% methyl red indicator and neutralise with 6.34 N
ammonium hydroxide, adding the ammonia very slowly while constantly
whirling the flask. Then add exactly 1 ml in excess, dilute to 100 ml at
20C, filter if necessary and polarise at 20C in a 400-mm tube to obtain
the neutralised invert polarization, (/').
Calculate the percentage sucrose, S, in the original sample from:

where 7 V = / ' I + K,

K = P (0.0047 + 0.00017 v),


v = no. of ml basic lead added per 100 ml.