• Benedict’s solution?can be used to carry out a?semi-quantitative?test on a?reducing sugar solution?to determine the?concentration?of reducing sugar present in the sample
  • 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

   

• 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?green (low concentration)?to?brick-red (high concentration of reducing sugar present)

   

• A semi-quantitative test can be carried out by setting up?standard solutions?with?known concentrations?of a reducing sugar (such as glucose)
• These solutions should be set up using a?serial dilution?of an existing?stock solution
  • 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)

   

• 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
  • 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?standard 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

Colorimeters

• 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 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 colorimeter is used to obtain quantitative data that can be plotted to create a calibration curve to be used to find unknown concentrations