Calculating the Concentration of Glucose in Urine (AQA A Level Biology)

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

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