Colorimetric determination of glucose using Benedict’s reagent
Background
A Benedict’s test is used to determine the presence of reducing sugars such as fructose,
glucose, maltose and lactose.
In a Benedict’s test, a blue chemical reagent known as a Benedict’s reagent or solution is
used.
A positive test with Benedict’s reagent is indicated by a change in colour from blue to
orange or brick-red.
When a reducing sugar such as glucose is heated in a water bath, benedicts solution will
change from a transparent blue colour to an orange or brick-red precipitate depending on
the level of glucose present (see picture below):
The benedicts test is a qualitative analysis – it is based on an observable feature (colour
change) that provides a rough indication of how much glucose is present.
A quantitative (numerical based) analysis can be carried out via colorimetric analysis.
In this experiment, you will prepare a selection of glucose solutions of differing, accurate
concentrations and use them to construct a calibration graph. This calibration graph will
then be used to determine the unknown concentration of a glucose solution labelled X.
Before proceeding with this experiment, you should re-familiarise yourself with the
colorimetry handout issued earlier in the course.
Apparatus required
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Colorimeter (set to red filter)
Glucose powder
Benedicts reagent
Centrifuge
Graduated plastic droppers
10ml & 25ml measuring cylinders
250ml beaker
Access to kettle
Ammonia solution (s.g. 0.88)
1 mol l-1 sulphuric acid
10vol hydrogen peroxide
Wash bottle
5 x 100ml volumetric flasks
Safety
Sulphuric acid is an irritant.
0.88 ammonia is corrosive and produces a toxic gas. Ammonia can cause asthma attacks in
susceptible people so work in a well ventilated room.
Ensure the centrifuge conforms to modern safety standards.
Method
Part 1: Preparing the glucose solutions
Part 2: Processing the samples
1. Using a plastic dropper, transfer 1ml of the 0.2% glucose solution to a centrifuge
tube.
2. Add 10ml of Benedicts solution and place the tube in a beaker of boiling water for 5
minutes.
3. Transfer the tube to a centrifuge and spin for 2 minutes until copper oxide is
precipited.
4. Discard the supernatant (the liquid around the precipitate) and wash the precipitate
by adding distilled water. Centrifuge again (2 minutes) then discard the supernatant.
5. Add 4ml of sulphuric acid followed by 2ml of hydrogen peroxide and warm gently
over a Bunsen flame to dissolve the precipitate.
6. Cool and add 4ml of ammonia solution. This mixture should then be transferred to a
100ml standard flask and filled to the graduation mark with water (using correct
technique). You may need to stopper and invert the flask several times to mix.
7. Calibrate the colorimeter using distilled water as a reference sample.
8. Transfer a small quantity of the solution into a cuvette and measure its absorbance
using a blue filter (note – run it with a green one as well).
9. Repeat this procedure for the other glucose samples and plot a calibration graph of
concentration (x-axis) against absorbance (y-axis).
Part 3: Identifying the unknown sample
1. Calibrate the colorimeter using distilled water as a reference sample.
2. Measure the absorbance of glucose sample X.
3. Use your calibration graph to work out the concentration of sample X.