6-4-8-required-practical-determining-the-concentration-of-glucose-in-urine

Required practical: using colorimetry to determine the concentration of glucose in urine

• It is possible to use colorimetry to determine the concentration of glucose in a urine sample of unknown concentration

• This test uses quantitative Benedict’s reagent that contains potassium thiocyanate

  • This means that it does not produce a red precipitate when it comes into contact with glucose

  • The presence of glucose is instead measured by the loss of blue colour, along with the formation of a white precipitate

    • The white precipitate can be filtered out prior to analysis

• A positive test is indicated along a spectrum, from:

  • blue = low concentration, to

  • colourless = high concentration

• A quantitative test of this kind involves setting up a calibration curve, to which the unknown sample can be compared

Apparatus

• A stock solution of glucose

• Distilled water

• Pipettes

• Test tubes

• Water bath

• Test tube rack

• Colorimeter

• Cuvettes

• Urine sample, or equivalent solution of unknown glucose concentration

• Goggles

• Gloves

• Labels

• Pen

• Graph paper

• Pencil

• Ruler

Method: calibration curve

1. Prepare a dilution series of glucose solutions

   • Different volumes of stock solution and distilled water are added to each test tube using pipettes to produce glucose solutions of different concentrations

2. Add a fixed volume of quantitative Benedict’s solution to each labelled test tube

3. Place the test tubes in a water bath that has been heated to at least 70 °C for 5 minutes

4. Filter the contents of each test tube and add a fixed volume of into separate, labelled cuvettes

   • Filtering removes the white precipitate

5. Set the colorimeter wavelength to red

   • Red is the complementary colour to blue, so a blue solution will absorb red light and allow a reading to be taken

6. Calibrate the colorimeter using a cuvette containing only distilled water

   • This results in 100% transmission of light through the solution and gives a point of comparison for all other readings

7. Place each labelled cuvette in the colorimeter and measure the % transmission

8. Plot a graph of glucose concentration against % transmission

   • This is the calibration curve

Method: concentration of unknown sample

1. Treat the unknown sample in the same way as the glucose solutions

   • Add the same fixed volume of Benedict’s solution,

   • Heat in the same water bath for 5 minutes

   • Filter

   • Add to a labelled cuvette

2. Place the cuvette in the colorimeter

3. Obtain the % transmission of light through the solution for the sample

4. Use this result and the calibration curve to work out the glucose concentration of the urine sample