E8
Kinetic Determination of Glucose in
Beverages
C2507 Intensive General chemistry – Spring 02 – E8: Kinetic Determination of Glucose in Beverages
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C2507 Intensive General chemistry – Spring 02 – E8: Kinetic Determination of Glucose in Beverages
E8 - Kinetic Determination of Glucose in Beverages
INTRODUCTION
Glucose is a major component of animal and plant carbohydrates.
Quantitative
determination of glucose is important in clinical chemistry, biochemistry, and food
analysis. In this experiment you will determine the glucose content in fruit drinks and
carbonated beverages using kinetics and visible spectroscopy.
THEORY
Glucose is oxidized to -D-gluconolactone in the presence of an enzyme called glucose
oxidase (GOX):
GOX
-D-glucose + O2 + H2O  -D-gluconolactone + H2O2
(1)
To measure the reaction rate, the production of H2O2 is coupled to another enzymecatalyzed reaction. In this case the enzyme is horseradish peroxidase (HRP):
HRP
2 H2O2 + phenol + 4-amino-antipyridine 
4-N-(p-benzoquinoneimine)-antipyridine + 4 H2O (2)
The reaction product exhibits an absorption maximum at 505 nm with a corresponding
molecular extinction coefficient of 1.27 x 104 M-1cm-1. The reaction rate is measured by
monitoring the increase of absorbance with time.
Interferences could occur. Hydrogen peroxide produced through reaction (1) oxidizes
ascorbic acid (vitamin C) present in beverages, producing dehydroascorbic acid:
H2O2 + C6H8O6  C6H6O6 + 2 H2O
(3)
Because this reaction is faster than the HRP-catalyzed reaction (2), ascorbic acid
interferes in end-point assays, resulting in negative errors. This interference can be
eliminated by using ascorbic acid oxidase in the enzyme working solution. In kinetic
assays, the initial quantity of H2O2 produced through reaction (1) is consumed for
ascorbic acid oxidation, see reaction (3). Thus, a lag phase in the reaction rate curve can
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C2507 Intensive General chemistry – Spring 02 – E8: Kinetic Determination of Glucose in Beverages
be observed (see ref.). When all ascorbic acid has been consumed, the reaction rate can
be measured. The lag time is proportional to ascorbic acid concentration and inversely
proportional to glucose concentration (i.e. proportional to the ascorbic acid/glucose ratio).
The concentration of ascorbic acid even in vitamin C – fortified drinks is less than 6 mM,
while the expected glucose concentration in fruit drinks and carbonated beverages is in
the range of 55-295 mM. Therefore the ascorbic acid/glucose ratio will always be less
than 1/50 and the lag time less than 140 s (see below the experimental results published
in the Georgiou paper). If samples are pre-incubated with the enzyme solution for 21/2
min, ascorbic acid interference is eliminated.
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C2507 Intensive General chemistry – Spring 02 – E8: Kinetic Determination of Glucose in Beverages
EQUIPMENT AND REAGENTS
LKB Spectrophotometer
Glucose Stock Solution 0.200 M
Phosphate Buffer 0.10 M, pH 7.5
Enzyme Working Solution: Phenol 7.50 mM; 4-Amino-antipyridine 2.50 mM; GOX 0.5
IU.mL-1; HRP 20 IU.mL-1 in 0.10 M Phosphate Buffer pH 7.5.
HAZARDS AND WASTE
Phenol and 4-Amino-antipyridine are harmful if swallowed and are irritating to the eyes,
respiratory tract and skin.
All aqueous solutions must be disposed in the designated aqueous waste jar. Unused
beverages can go into the sink.
PROCEDURE
In appendix A you will find tips on how to use the LKB spectrophotometer.
1. Transfer a 20 mL sample of the beverage to be tested into a 100 mL beaker and
stir for 10 min to remove the excess of carbon dioxyde.
2. After stirring, dilute 5 mL of the beverage to 100 mL with phosphate buffer.
3. Set the wavelength of the spectrophotometer at 505 nm and adjust the
transmittance to 100% using a cuvette containing phosphate buffer only.
4. Rapidly mix 1mL of standard or diluted sample and 5 mL of the enzyme working
solution. Transfer the mix immediately to the spectrophotometer.
5. To eliminate ascorbic acid interference, samples are pre-incubated for 21/2 min
with the enzyme working solution. After pre-incubation, record the initial
absorbance reading and 3 subsequent readings acquired at 2 min intervals.
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C2507 Intensive General chemistry – Spring 02 – E8: Kinetic Determination of Glucose in Beverages
CALCULATIONS AND DISCUSSION
1. Use Excel to plot readings versus time and calculate the reaction rates in units of
mA.min-1 (milliAbsorbance.min-1). Construct the calibration curve. Comment on
the validity of Beer-Lambert’s law in the suggested range of concentrations.
Calculate the glucose concentration of the different beverages. Perform error
analysis on your results.
2. Which one of the 2 enzyme-catalyzed reactions is the rate-limiting step? Explain
your reasoning.
3. Take a visible spectrum of one of your colored standard and comment on the
choice of the wavelength of the spectrophotometer at 505 nm.
4. Compare your results with the values available on the labels. You might want to
consult other sources (for example the websites of the beverages companies).
Comment on your results and on the method. Discuss the sources of error.
5. Can you think of other methods suitable for the determination of the glucose
content in beverages? Compare with the method used in this experiment.
REFERENCES
Vasilarou A-M.G., Georgiou C.A., J.Chem.Ed., 77, (2000), 1327.
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