Biological Molecules: Question 7

Worked solution

Part (a): Testing for a reducing sugar with Benedict’s test

Add an equal volume of blue Benedict’s reagent to the food sample and heat the mixture in a boiling water bath for a few minutes. If a reducing sugar is present, the mixture changes colour, passing through green, yellow and orange, to form a brick-red precipitate (insoluble copper(I) oxide). If no reducing sugar is present, the mixture remains blue. The amount of precipitate formed gives a rough indication of the concentration of reducing sugar present. The test can therefore be used semi-quantitatively.

Part (b): Why sucrose does not reduce Benedict’s reagent

A reducing sugar reduces the blue Cu2+ ions in Benedict’s reagent to insoluble, brick-red Cu+ (as copper(I) oxide) because it has a free, exposed aldehyde or ketone group available to react. In sucrose, the glycosidic bond forms between the anomeric carbon of glucose and the anomeric carbon of fructose. Precisely the two carbon atoms that would otherwise carry the reactive aldehyde and ketone groups. With both of these carbons tied up in the glycosidic bond, sucrose has no free aldehyde or ketone group exposed, so it cannot reduce Cu2+ ions, and Benedict’s test gives a negative (blue) result.

Part (c): Modifying the test for a non-reducing sugar

Because sucrose gives a negative result even though it is a sugar, a further step is needed:

1. Take a fresh sample of the solution and add dilute hydrochloric acid.
2. Heat the acidified sample in a boiling water bath. This hydrolyses the glycosidic bond, breaking sucrose down into its two monosaccharides, glucose and fructose, each of which now has a free reducing group.
3. Neutralise the acid, typically by adding solid sodium hydrogencarbonate (or another alkali). Benedict’s reagent only works correctly under alkaline/neutral conditions.
4. Add Benedict’s reagent and heat again exactly as in part (a).

A brick-red precipitate at this stage confirms that a non-reducing sugar (such as sucrose) was present in the original sample, since it has now been hydrolysed into reducing monosaccharides.

Final answers

• (a) Heat with Benedict’s reagent in a boiling water bath; positive result is a colour change to a brick-red precipitate.
• (b) Both anomeric carbons are tied up in sucrose’s glycosidic bond, so no free aldehyde/ketone group is left to reduce Cu2+ ions.
• (c) Hydrolyse with dilute hydrochloric acid and heat, neutralise with an alkali, then repeat Benedict’s test; a brick-red precipitate indicates a non-reducing sugar was present.