Method:
1. Plotting a calibration curve for one of the pH values:
Mix equal volumes of the fructose and glucose solutions to make a 0.8 mol/ dm3
stock solution of reducing sugar.
Collect 7 test tubes in a test tube rack. Label the first test tube ‘Blank’. The remaining
test tubes are numbered with concentrations of 0, 20, 40, 60, 80, and 100% reducing
sugar solution concentration.
Set up the colourimeter and cuvettes.
Add 2 cm3 of one of the pH buffer solutions and 2 cm3 of enzyme to each test tube.
Add 6 cm3 distilled water to the ‘Blank’ test tube.
Add 6 cm3 of 0, 20, 40, 60, 80 and 100% dilutions of reducing sugar stock solutions
to the 6 remaining test tubes according to their labels.
Mix, add bungs to each test tube and place in a 37°C water bath to incubate for 4
minutes.
Take out of the water bath, add 3 cm3 of Benedict’s reagent to each test tube, add
bungs and heat in the 80°C water bath for 5 minutes.
Transfer some of the ‘Blank’ sample to a cuvette, add a lid, using the red filter,
calibrate the colourimeter, record the absorbance readings for each of the reducing
sugar concentrations in a table, and plot a calibration curve.
2. Using the sucrose solution and the enzyme in the presence of the pH buffer used to plot the
calibration curve:
Collect 7 test tubes, and label one of them ‘Control’
To the control, add 2 cm3 of water, 6 cm3 of sucrose solution, and 2 cm3 of distilled
water.
Add 2 cm3 of pH buffer, and 6 cm3 of sucrose solution to the next 3 test tubes. These
are test tubes 1, 2, and 3.
To the last three test tubes add 2 cm3 of the enzyme.
Place all these test tubes in the 37°C water bath for about 10 minutes.
Add Benedict’s solution to the control test tube, and incubate at 80°C for 5 minutes,
then record the control absorbance.
Add the enzyme from one of the enzyme test tubes to Test tube 1 containing sucrose
and pH buffer. Immediately start the timer for 4 minutes.
After 4 minutes, add the benedict’s solution to Test tube 1, and place in the 80°C
water bath for 5 minutes.
Use the calibrated colourimeter to measure the absorbance of this sample, and
record it in a suitable data table.
Do the same for Test tubes 2 and 3.
Calculate the mean absorbance for test tubes 1,2, and 3, and calculate the reaction
rate at that particular pH, by dividing the difference between the control and mean
absorbance values by 4 minutes.
To confirm the accuracy of the calculated rate of reaction at that pH, use the
calibration curve to interpolate the control and mean absorbance readings, and divide
the difference between the concentrations by 4 minutes, giving the value of the rate of
change of concentration over time. Compare this with the rate of reaction calculated
only using the absorbance readings.
1. Do this for all the different pH values available and finally plot the rate of reaction on the y-
axis against the pH on the x-axis. A separate calibration curve is to be drawn for each pH
value as Benidict’s solution may behave differently at different pH levels (3).
