Biochemical Tests: Reducing Sugars & Starch (OCR AS Biology)

• There are a number of tests that can be carried out quickly and easily in a lab to determine if a sample contains a certain type of sugar
• The following tests are qualitative - they do not give a quantitative value as to how much of each type of molecule may be present in a sample
• Sugars can be classified as reducing or non-reducing; this classification is dependent on their ability to donate electrons (a reducing sugar that is able to donate electrons is itself oxidised)
  • OILRIG in Chemistry

The Benedict’s test for reducing sugars

• Benedict’s reagent is a blue solution that contains copper (II) sulfate ions (CuSO₄); in the presence of a reducing sugar copper (I) oxide forms
  • Copper (I) oxide is not soluble in water, so it forms a precipitate

Method

• Add Benedict's reagent (which is blue as it contains copper (II) sulfate ions) to a sample solution in a test tube
• 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
• 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
• A positive test result is a colour change somewhere along a colour scale from blue (no reducing sugar), through green, yellow and orange (low to medium concentration of 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 approximate indication of how much reducing sugar is present without knowing the exact quantity

Benedict's test for reducing sugars produces a colour change from blue towards red if a reducing sugar is present

Reducing & Non-reducing Sugars Table

The test for non-reducing sugars

• Add dilute hydrochloric acid to the sample and heat in a water bath that has been brought to the boil
• Neutralise the solution with sodium hydrogencarbonate
  • Use a suitable indicator (such as red litmus paper) to identify when the solution has been neutralised, and then add a little more sodium hydrogencarbonate as the conditions need to be slightly alkaline for the Benedict’s test to work

• Then carry out Benedict’s test as normal
  • Add Benedict’s reagent to the sample and heat in a water bath that has been boiled – if a colour change occurs, a non-reducing sugar is present

Explanation

• The addition of acid will hydrolyse any glycosidic bonds present in any carbohydrate molecules
• The resulting monosaccharides left will have an aldehyde or ketone functional group that can donate electrons to copper (II) sulfate (reducing the copper), allowing a precipitate to form

The iodine test for starch

• To test for the presence of starch in a sample, add a few drops of orange/brown iodine in potassium iodide solution to the sample
  • The iodine is in potassium iodide solution as iodine is insoluble in water

• If starch is present, iodide ions in the solution interact with the centre of starch molecules, producing a complex with a distinctive blue-black colour
• This test is useful in experiments for showing that starch in a sample has been digested by enzymes

Iodine will change colour in the presence of starch