1-2-10-biochemical-tests-reducing-sugars-and-starch

Biochemical Tests for Reducing Sugars & Starch

• 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