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Food Technology and Nutrition Dept. 62-1
October 25, 1961
A Rapid Spectrophotometric Method of High Sensitivity for the
Determination of Sugar Constituents.

A. M. Furuholmen and R. A. Dennison

The procedure described in this report is based on the oxidation
of reducing sugars by highly-buffered alkaline ferricyanide. The

reduced form, ferrocyanide, reacts with arsenomolybdate forming a

blue-green complex which is measured spectrophotometrically at the

wavelength 745 mu and related through standard curves, to the

concentration of sugar present.

Glucose and fructose may be determined simultaneously through

differences in their oxidation rates at 100C and 55C under the

conditions used. Ten minutes at the higher temperature is sufficient

to oxidize both sugars completely. A 30-minute heating period at

the lower temperature is sufficient for complete oxidation of

fructose. During this time, however, only a small but reproducible

fraction of the glucose is oxidized. Thus the actual concentration

of each sugar can be calculated from the resulting absorptivity

coefficients and absorbance measured at the two temperatures. Total

sugars can be determined as the difference in reducing sugar content

before and after inversion.

The basic reactions were developed by S. V. Ting (1) into a
method for analysis of citrus juices. Ting's method involved the use

of a Lumetron colorimeter with a 515 mu filter. In our investigations

of the possible applications of this procedure, particularly to

potato extract, further modification was made and sensitivity -gatly

increased by measurement with a spectrophotometer at mu which.

the center of the absorption maximum of the complex. ;bLai; 'I

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determined, through comparative analyses, that the extract can be used

directly without clarification or removal of alcohol.

Procedure
Reagents

Reagents are prepared as recommended by S. V. Ting (1):

Alkaline Ferricyanide Solution. Dissolve 160 grams of anhydrous
sodium carbonate and 150 grams of disodium phosphate heptahydrate in
850 ml. of distilled water, add 4 grams of potassium ferricyanide, and

dilute to 1 liter.

Arsenomolybdate Solution. Dissolve 25 grams of ammonium molybdate

tetrahydrate in 450 ml. of distilled water. Add 21 ml. of concentrated

sulfuric acid, followed by 3 grams of disodium arsenate in 25 ml. of

distilled water. Heat at 55C for 30 minutes in a water bath with

constant stirring, or in an incubator maintained at 37C for 24 to 48
hours.

Sulfuric Acid Solution, 2N. Dilute 56 ml. of concentrated
sulfuric acid (specific gravity 1.84) to 1 liter.

Sodium hydroxide solutions, O1N and 1N.
Hydrochloric acid, 1 to 1 by volume.
Sample

The sample for analysis must contain from 0.01 to 0,05% reducing
sugar. Thus, in some cases, dilution will be necessary. A blank

determination is run with the samples, one at each temperature, and

used for setting 0.0 absorbance.

Total Reducing Sugars

Pipet 1 ml aliquot into 100 ml volumetric flask. Add, by pipet,

5 ml ferricyanide solution. Mix well and place in boiling water bath,
with solution below water level during entire heating period. Heat
for exactly 10 minutes, remove and cool quickly and completely by

immersing in cold water. When solution has cooled, add 10 ml 2N

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sulfuric acid and mix carefully until no more gas is evolved. Pipet

4 ml arsenomolybdate into flask, mix, and dilute to volume. Measure
absorbance, using Beckman DU (or equivalent spectrophotometer) set at

745 mu. Measurement should be made after one-half hour and before two
hours.

Fructose

The procedure is the same as for total reducing sugars except

that water bath is maintained at 55C and sample heated for 30 minutes.

Sugars by inversion

Pipet 50 ml aliquot into beaker. Add 10 ml 1:1 HCI and allow to
stand for 18 hours (may stand for 24 hours). Neutralize by adding 5

ml of 1ON NaOH and additional lN NaOH to bring sample to pH range of

5-7 (a pH meter is recommended). Quantitatively transfer to 100 ml
volumetric flask and dilute to volume. Procedure is then the same as

for total reducing sugar, again using a 1 ml aliquot. Other methods

of inversion should give equivalent results.

Standard Curve

Standard curves are run for glucose and fructose at 100C and

55C by regular procedure. The concentrations used are 0.01, 0.02,

0.03, 0.04 and 0.05 gm/100 ml.
Calculations

From valuesobtained by analysis of standard solutions, the

average K values are calculated, where K is the reciprocal of the

absorptivity coefficient, as, from Beer's Law: As = asbC

As = measured absorbance

b = cell width, cm

C = concentration

As K
C = A- =K As where 1 cm cell used,
asb b C = KAs
s


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1. total reducing sugar
Rt = KrAsD Rt = concentration total

reducing sugar (gm/100 ml)
Kr = K value at 1000C
D = dilution factor

2. fructose and glucose (550C)

apparent fructose = Fa = KfAsD Kf = K value of

fructose at 55C

glucose = G = (Rt-Fa) Q Q = KG/KF as obtained at 550C
Q-1
KG = K value of glucose at 55C

fructose = F = (Rt-G)

3. sugar after inversion
Calculated as the difference in total reducing sugar
before and after hydrolysis x 0.95.

Discussion

A study was made of the complex spectra, using a Beckman DK-2
recording spectrophotometer in the visible region (340-900 mu). The
spectra showed an absorption maximum in the region of 740-750 mu.
With the instrument set for the scale of 0-1.0 absorbance units, the

peak was on scale for sugar concentration of approximately 0.05% and
lower. High sensitivity is obtained by measurement at 745 mu as shown

by an absorbance value of 0.37 for 0.02% solution and 0.77 for 0.04%.
Spectra were run of the complex formed by standard glucose and fructose
solutions and by potato extract, at 100o and 550C. The extract
paralled the standard solutions in the region above 500 mu; however,
divergence occurred in the region toward the uv with increased
absorption by the extract. In considering the application of this


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procedure to other systems where there are natural components present

with strong absorption near 745 mu, a check of the complex spectra

should be made from 500-800 mu.

Several types of spectrophotometers were used to determine their

accuracy in measurement of absorbancy. To obtain reproducible results

in the range of 0.01 to 0.05%, it is recommended that an instrument

such as Beckman Model B or its equivalent be considered minimum

adequate specifications for selection and that a maximum cell size of

1 cm be used. A Beckman DU was selected for the current work with

potato analyses.

A study of the variation of absorbance with time was made. There

was no shift in the peak during the 24 hours of study, but absorbance

decreased after two hours. Based on data obtained, then, the extract

should be measured between one-half hour and two hours after reaction

with arsenomolybdate. It is to be expected that the stability of the

complex will vary according to the system; therefore a check should be

made to be assured of reproducible results in the two hour period.

Reproducibility by this analytical procedure is extremely good.

Six replicates had a standard deviation of 0.0015, which is the same

range of accuracy as the reading of the instrument.

Comparison with the Shaffer-Somogyi method showed good agreement

between procedures, and recovery determined by use of model systems

showed an average recovery of 98.3%, the major loss (2-3%) being at

the upper limit of concentration.

Several substances were investigated by Ting (1) to determine

their effect on the values obtained. He found that ascorbic acid will

interfere but only if present in a concentration of 100 mg/100 ml or

higher. Other substances checked but which did not develop color

include naringin, hesperidin, citric acid and several amino acids.


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Polyphenolic compounds were included in our investigation. It

was found, as expected, that they are oxidized by ferricyanide under

the conditions of the reaction and thus would constitute an interference

if present in sufficient quantity (approximately 50 mg/100 ml).

However, analysis of the extract by chromatography and by the

colorimetric method of Arnow (2) gave no indication of the presence

of these substances in the alcohol extract.

In summation, the procedure described will provide an accurate,

rapid method for determining quantitatively sugar constituents as

glucose, fructose, total reducing sugars, and hydrolyzable sugar.

No clarification was necessary and alcohol extracts may be used

directly. The concentration range is 10-50 mg/100 ml. The method

is applicable where the above mentioned sugars are the major reducing

sugar components and where dilution may be made to yield samples in

the desired range. Very small differences may be determined because

of the high sensitivity obtained.

References

1. Ting, S. V.; Rapid Colorimetric Methods for Simultaneous

Determination of Total Reducing Sugars and Fructose in

Citrus Juices; J. Agr. Food Chem. 4, 263 (1956).

2. Arnow, L. E.; Colorimetric Determination of the Components

of 3,4-Dihydroxyphenylalanine-Tyrosine Mixtures; J. Biol.
Chem. 118, 531 (1937).


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