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
Calibration curve data:
1. According to the calculations required in the prelab question #1, prepare your
glucose standards starting form the stock solution 0.200 M. Prepare your dilutions
in distilled water, using appropriate glassware.
2. Set the LKB spectrometer wavelength to 505 nm and adjust the transmittance to
100% using a cuvette containing phosphate buffer only.
3. Rapidly mix 1 mL of standard with 5 mL of enzyme working solution. Transfer
the mix immediately to a clean cuvette and start spectrometer readings. Record
the initial absorbance reading and 6 subsequent readings at 1 min intervals.
4. Repeat step 3 TWO times with each sample.
Beverages data:
1. Choose 1 soda, its diet version and 1 fruit juice among the beverages available on
your bench.
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C2507 Intensive General chemistry – Spring 02 – E8: Kinetic Determination of Glucose in Beverages
2. 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 dioxide.
3. After stirring, dilute 5 mL of the beverage to 100 mL with phosphate buffer.
4. Set the LKB spectrometer wavelength to 505 nm and adjust the transmittance to
100% using a cuvette containing distilled phosphate buffer only.
5. Rapidly mix 1 mL of your diluted beverage with 5 mL of enzyme working
solution. Transfer the mix immediately to a clean cuvette and start spectrometer
readings. Record the initial absorbance reading and 6 subsequent readings at 1
min intervals.
6. Repeat step 5. TWO times with each sample.
Visible spectrum of oxidized sample:
1. Choose one of your most dilute oxidized glucose standards.
2. Obtain the visible spectrum of this sample by varying the wavelength on the LKB
spectrophotometer and measuring the absorbance of your sample versus phospate
buffer. Everytime you change the wavelength you will need to calibrate the
spectrophotometer to 100% transmittance using a cuvette containing phosphate
buffer only. Take a reading every 20 nm, except in the 450-550 nm region where
you need to take a reading every 5 nm.
CALCULATIONS
1. Use Excel to plot the Absorbance readings versus time.
2. Perform regression analysis to obtain the reaction rates in units of mA/min
(milliAbsorbance unit per min).
3. Construct the calibration curve by plotting the reaction rates of each glucose
standard versus its concentration.
4. Perform regression analysis to obtain the equation of the calibration curve. Use
this equation to determine the glucose concentration of the 3 beverages.
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C2507 Intensive General chemistry – Spring 02 – E8: Kinetic Determination of Glucose in Beverages
DISCUSSION
Include the answers to the following questions in your discussion section.
1. Compare your glucose concentrations with the values available on the labels or
literature values and determine the % difference. You might want to consult other
sources (for example the websites of the beverages companies). Comment on your
results and on the method. Discuss sources of error.
2. Which one of the 2 enzyme-catalyzed reactions is the rate-limiting step? Explain
your reasoning.
3. What makes the reacted solution pink? Comment on the choice of the LKB
wavelength for the kinetic determination.
4. What is the reason for not using a plot of Absorbance versus Concentration as a
calibration curve?
5. Provide 1 or 2 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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