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 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.