RPQ 11 - Determining the concentration of a glucose solution

• If it is suspected that a person has diabetes, a doctor may request that a sample of their urine is tested for glucose
• normally the concentration of glucose in urine is very low - 0-0.8 mM

  higher concentrations than this may indicate diabetes (although a blood test would be needed to confirm)
• use a quantitative Benedict's test
• Quantitative Benedict's reagent is different to normal Benedict's reagent:

  • when heated with glucose the initial blue colour is lost but a brick-red ppt is not produced
  • can use a colorimeter to measure the light absorbance of the solution after the quantitative Benedict's test has been carried out
  • the higher the concentration of glucose, the more blue will be lost - the paler the solution will become - decreasing the absorbance of the solution
• Initially need to make up several glucose solutions of different, known concentrations - serial dilution technique
• 5 serial dilutions with a dilution factor of 2 starting with initial glucose conc. 4mM:

  1. line up 5 test tubes in a rack
  2. add 10cm3 of the initial 4mM glucose solution to the first test tube and add 5cm3 distilled water to the other 4
  3. then using a pipette draw 5cm3 of the solution from the first test tube, add it to the distilled water in the second and mix the solution thoroughly - now have 10cm3 of solution that is half as conc as the solution in 1st test tube (2mM)
  4. repeat process 3x more to create solutions of 1mM, 0.5mM and 0.25mM
• when testing for a low concentration of glucose in a solution - quantitative Benedict's can give a more accurate result than normal Benedict's
• Measuring the absorbance of glucose:

  • make sure you have equal volumes of each of the solutions. Should also set up a test tube containing only pure water - act as a negative control
  • add the same amount of quant Benedict's reagent to each test tube and stir gently to mix
  • heat the test tubes for 4-5 mins in water bath brought to the boil - make sure heat them all for same amount of time
  • carefully remove the test tubes from the water bath and leave to cool
  • next use a colorimeter to measure the absorbance of the solution in each test tube
• Using a colorimeter:

  1. a device that measures absorbance (the amount of light absorbed by a solution)
  2. need to set up the colorimeter with a red filter (wavelength 635nm)
  3. after turning on the machine and allowing it time to stabilise - calibrate to 0 using a cuvette of distilled water
  4. then use a pipette to transfer a sample of solution you would like to test in a clean cuvette, and measure absorbance of the solution
  5. for each solution use a clean pipette and cuvette
• zero the colorimeter between each reading
• no glucose in the negative control - nothing for the Benedict's reagent to react with - solution will remain blue - should give you the highest absorbance value in the experiment
• Once measured the absorbance of each glucose solution - can make a calibration curve
• plot a graph of your results showing absorbance (y-axis) against glucose concentration (x-axis) - draw smooth line/curve of best fit through data points to create a calibration curve
• Then you can test the unknown solution ('urine' sample) in same way as the known concentrations - i.e. use the same volume of solution and quantitative Benedict's reagent, and heath the solution for the same amount of time

  once measured the absorbance of the unknown solution can use the calibration curve to find its concentration