The Benedict's Test (CIE AS Biology)

Method

• Add Benedict's reagent (which is blue as it contains copper (II) sulfate ions) to a sample solution in a test tube
  • It is important that an excess of Benedict’s solution is used so that there is more than enough copper (II) sulfate present to react with any sugar present
• Heat the test tube in a water bath or beaker of water that has been brought to a boil for a few minutes
• If a reducing sugar is present, a coloured precipitate will form as copper (II) sulfate is reduced to copper (I) oxide which is insoluble in water
• A positive test result is, therefore, a colour change somewhere along a colour scale from blue (no reducing sugar) to brown/brick-red (a high concentration of reducing sugar)
  • This test is semi-quantitative as the degree of the colour change can give an indication of how much (the concentration of) reducing sugar present

Semi-quantitative Benedict's test

• A semi-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
  • 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
  • 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 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

Alterations

• It is also possible to standardise this test but instead of waiting a fixed amount of time for a range of colours to be observed, time how long it takes for the first colour change to occur (blue to green)
  • The higher the concentration of reducing sugar in a sample, the less time it would take for a colour change to be observed
• To avoid issues with human interpretation of colour, a colourimeter 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 colourimeter
  • They can be used when:
    • Counting bacteria or yeast populations
    • Determining unknown glucose, starch, protein concentrations

Making serial dilutions

Using a 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 by the sample
  • Colour filters are used to control the light wavelength emitted
  • The colour used will be in contrast to the colour of the solution, e.g. Benedict's solution turns orange in the presence of sugar, so the colorimeter will assess the intensity of the orange colour; in order to do this a blue light filter would be used to shine blue light through the sample
    • Blue light is absorbed by an orange solution as orange light is reflected to give the orange appearance
    • The extent to which the blue light is absorbed will differ depending on the intensity of the orange colour; a solution that is orange/green will absorb less blue light than a solution that is brick red
    • The absorbance value therefore provides a quantitative measure of the strength of the orange colour
• Colorimeters must be calibrated before taking measurements
  
  • This is completed by placing a blank into the colorimeter 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 against the known concentrations can be used
  • Unknown concentrations can then be determined from this graph

Colorimeter diagram

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