AQA A Level Biology

Revision Notes

6.4.8 Calculating the Concentration of Glucose in Urine

Required Practical: Producing a Dilution Series and Calibration Curve

  • 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

2.1 The Benedict’s test - Serial dilutions, downloadable AS & A Level Biology revision notes

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

2.1 The Benedict’s test - Colorimeter and Calibration Curve (1), downloadable AS & A Level Biology revision notes

Calibration curve percentage transmission, downloadable AS & A Level Biology revision notes

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

Calibration curve percentage transmission results, downloadable AS & A Level Biology revision notes

The calibration curve to be used to find unknown concentrations

Author: Lára

Lára graduated from Oxford University in Biological Sciences and has now been a science tutor working in the UK for several years. Lára has a particular interest in the area of infectious disease and epidemiology, and enjoys creating original educational materials that develop confidence and facilitate learning.
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