6.4.8 Calculating the Concentration of Glucose in Urine

• An alternative version of Benedict’s solution can be used to carry out a quantitative test on an unknown urine sample to determine the concentration of reducing sugars (glucose) in the sample
  • The Benedict's solution used contains potassium thiocyanate. This means that it does not produce a red copper oxide when it comes into contact with glucose
  • Instead, the presence of glucose is measured by the loss of the blue colour produced by copper sulfate along with the formation of a white precipitate
  • The white precipitate can be removed by filtering
  • The colour intensity of the resulting filtrate is then analysed

• The intensity of any colour change seen relates to the concentration of reducing sugar present in the sample
  • A positive test is indicated along a spectrum of colour from blue (low concentration) to colourless (high concentration of reducing sugar present)

• A quantitative test can be carried out by setting up standard solutions with known concentrations of reducing sugar (such as glucose)
• These solutions should be set up using a serial dilution of an existing stock solution
• Each solution is then treated in the same way: add the same volume of Benedict’s solution to each sample and heat in a water bath that has been boiled (ideally at the same temperature each time) for a set time (5 minutes or so) to allow colour changes to occur then filter the solution to obtain the filtrate
  • It is important to ensure that an excess of Benedict’s solution is used

• Any colour change observed for each solution of a known concentration in that time can be attributed to the concentration of reducing sugar present in that solution
• The same procedure is carried out on a urine sample with an unknown concentration of reducing sugar which is then compared to the stock solution colours to estimate the concentration of reducing sugar present
• To avoid issues with human interpretation of colour, a colorimeter could be used to measure the absorbance or transmission of light through the sugar solutions of known concentration to establish a range of values that an unknown sample can be compared against a calibration curve

Serial dilutions

• Serial dilutions are created by taking a series of dilutions of a stock solution. The concentration decreases by the same quantity between each test tube
  • They can either be ‘doubling dilutions’ (where the concentration is halved between each test tube) or a desired range (e.g. 0, 2, 4, 6, 8, 10 mmol dm^-3)

• Serial dilutions are completed to create a standard to compare unknown concentrations against
  • The comparison can be:
    • Visual
    • Measured through a calibration/standard curve
    • Measured using a colorimeter

  • They can be used when:
    • Counting bacteria or yeast populations
    • Determining unknown glucose, starch, protein concentrations

Colorimeter

• A colorimeter is an instrument that beams a specific wavelength (colour) of light through a sample and measures how much of this light is absorbed (arbitrary units)
• They provide a quantitative measurement
• They contain different wavelengths or colour filters (depends on the model of colorimeter), so that a suitable colour can be shone through the sample and will not get absorbed. This colour will be the contrasting colour (eg. a red sample should have green light shone through)
  • Remember that a sample will look red as that wavelength of light is being reflected but the other wavelengths will be absorbed

• Colorimeters must be calibrated before taking measurements
  • This is completed by placing a blank into the colourimeter and taking a reference, it should read 0 (that is, no light is being absorbed)
  • This step should be repeated periodically whilst taking measurements to ensure that the absorbance is still 0

• The results can then be used to plot a calibration or standard curve
  • Absorbance or % transmission of light against the known concentrations can be used
  • Unknown concentrations can then be determined from this graph

Apparatus

• A stock solution of glucose
• Distilled water
• Pipettes
• Test tubes
• Water bath
• Test tube rack
• Colorimeter
• Cuvettes
• Urine sample
• Eye goggles
• Gloves
• Labels
• Pen
• Graph paper
• Pencil
• Ruler

Method

• 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
  • Make sure to label the test tubes

Making serial dilutions

• Add a fixed volume of Benedict's solution (with potassium thiocyanate) to each labelled test tube
• Place the test tubes in a boiling water bath for 5 minutes
• After the time has elapsed filter the contents of each test tube and add a fixed volume into labelled cuvettes
  • Filtering removes the white precipitate

• Set the colorimeter wavelength to red
  • This is done as red is the complementary colour to blue, so a blue solution will absorb red light the strongest

• Calibrate the colorimeter using a cuvette containing only distilled water
  • This is known as the "blank" where there is 100% transmission of light through the solution

• Place each labelled cuvette in the colorimeter and measure the % transmission
• From the results plot a graph of glucose concentration against % transmission of light through the solution (this can also be referred to as absorbance) - this is the calibration curve

A colorimeter is used to obtain quantitative data that can be plotted to create a calibration curve

• Treat the urine sample in the same way as the glucose solutions
  • Add Benedict's solution, heat, filter and add to a labelled cuvette

• Place the cuvette in the colorimeter
• Obtain the % transmission of light through the solution for the sample
• Use this result and the calibration curve to work out the glucose concentration of the urine sample

The calibration curve to be used to find unknown concentrations