The Benedict's Test (Cambridge (CIE) A Level Biology): Revision Note

Exam code: 9700

Cara Head

Written by: Cara Head

Reviewed by: Alistair Marjot

Updated on

The Benedict's test for reducing sugars

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

Diagram explaining serial solution preparation with pipettes, stock solutions, and test tubes. Includes dilution calculations, concentration table, and doubling method.
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

Diagram of a colourimeter showing a display reading "1.08 Abs", with labelled buttons for calibration, measurement, on/off, and a slot for a cuvette.
Graph showing absorbance vs. glucose concentration with a linear trend. An orange dashed line helps determine an unknown sample's glucose level.
A colourimeter is used to obtain quantitative data that can be plotted to create a calibration curve

Examiner Tips and Tricks

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.

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Cara Head

Author: Cara Head

Expertise: Biology & Psychology Content Creator

Cara graduated from the University of Exeter in 2005 with a degree in Biological Sciences. She has fifteen years of experience teaching the Sciences at KS3 to KS5, and Psychology at A-Level. Cara has taught in a range of secondary schools across the South West of England before joining the team at SME. Cara is passionate about Biology and creating resources that bring the subject alive and deepen students' understanding

Alistair Marjot

Reviewer: Alistair Marjot

Expertise: Environmental Systems and Societies & Biology Content Creator

Alistair graduated from Oxford University with a degree in Biological Sciences. He has taught GCSE/IGCSE Biology, as well as Biology and Environmental Systems & Societies for the International Baccalaureate Diploma Programme. While teaching in Oxford, Alistair completed his MA Education as Head of Department for Environmental Systems & Societies. Alistair has continued to pursue his interests in ecology and environmental science, recently gaining an MSc in Wildlife Biology & Conservation with Edinburgh Napier University.