The Benedict's Test (Cambridge International AS Biology)

The Benedict's Test for Reducing Sugars

Spec point — 2.1.2 · describe and carry out a semi-quantitative Benedict’s test on a reducing sugar solution by standardising the test and using the results (time to first colour change or comparison to colour standards) to estimate the concentration

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 colours of the standard solutions 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. In a serial dilution the concentration decreases by the same factor (e.g. halved in doubling dilutions) between each test tube. A range such as 0, 2, 4, 6, 8, 10 mmol dm^-3 is made by proportional (simple) dilution, not serial dilution

  • 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

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

Exam Hint For fair comparison in semi-quantitative tests, make sure volume of sample, volume of Benedict’s, temperature, and heating time are the same for every tube — exam questions may award marks for recognising these as control variables.