Testing for biological molecules

· Know how to describe and carry out tests for reducing sugars, starch, lipids and proteins.
· Know the reagent, method, positive result and negative result for each test.
· For practical questions, always include sample + reagent, heating if required, and the expected colour change.
· Use standardised conditions when comparing results: same sample volume, reagent volume, temperature, heating time and concentration range.
· This subtopic contains no Additional HL content.

Benedict’s test for reducing sugars

· Tests for reducing sugars, e.g. glucose, fructose and maltose.
· Add Benedict’s solution to the sample.
· Heat in a hot water bath.
· Negative result: solution remains blue.
· Positive result: colour changes from blue → green → yellow → orange → brick-red precipitate.
· The further the colour change towards brick-red, the higher the reducing sugar concentration.
· Exam wording: Benedict’s test detects reducing sugars because they can reduce copper(II) ions in Benedict’s solution.

Benedict’s test gives a colour scale that can be used to estimate reducing sugar concentration. A blue result is negative, while green, yellow, orange or brick-red indicates increasing amounts of reducing sugar. Source

Semi-quantitative Benedict’s test

· Used to estimate reducing sugar concentration, not just detect presence/absence.
· Standardise the test by keeping the same: sample volume, Benedict’s volume, water bath temperature, heating time and test tube size.
· Method 1: record the time taken to first colour change.
· Faster colour change = higher reducing sugar concentration.
· Method 2: compare the final colour with colour standards of known reducing sugar concentrations.
· Use the closest matching colour standard to estimate the unknown concentration.
· For best exam answers, mention using a calibration scale or known concentrations for comparison.

Iodine test for starch

· Tests for starch.
· Add iodine solution to the sample.
· No heating required.
· Negative result: iodine remains yellow-brown / orange-brown.
· Positive result: iodine turns blue-black.
· Exam wording: a blue-black colour shows starch is present.

The iodine test is a quick qualitative test for starch. A positive test is shown by the formation of a blue-black colour, while a negative result stays brown or yellow-brown. Source

Emulsion test for lipids

· Tests for lipids.
· Add the sample to ethanol and shake to dissolve any lipid.
· Add the ethanol mixture to water.
· Negative result: solution remains clear.
· Positive result: a white / cloudy emulsion forms.
· Lipids are insoluble in water, so they form tiny droplets that scatter light, producing the cloudy appearance.
· Safety: ethanol is flammable, so keep it away from naked flames.

Biuret test for proteins

· Tests for proteins.
· Add Biuret reagent to the sample.
· No heating required.
· Negative result: solution remains blue.
· Positive result: solution turns lilac / purple / violet.
· The Biuret test detects peptide bonds, so a positive result indicates protein is present.
· Exam wording: proteins contain many peptide bonds, which react with Biuret reagent to give a purple colour.

Test for non-reducing sugars

· Used when the sample gives a negative Benedict’s test but may still contain a non-reducing sugar, e.g. sucrose.
· First carry out Benedict’s test to confirm no reducing sugar is present initially.
· Add dilute hydrochloric acid and heat to hydrolyse the non-reducing sugar.
· This breaks glycosidic bonds by acid hydrolysis, producing reducing sugars.
· Neutralise the solution with sodium hydrogencarbonate.
· Then repeat Benedict’s test.
· Positive result after hydrolysis: Benedict’s changes from blue to green / yellow / orange / brick-red, showing a non-reducing sugar was present.
· Exam trap: Benedict’s solution works in alkaline conditions, so the acid must be neutralised before adding Benedict’s reagent.

Results summary table

· Reducing sugar: Benedict’s solution + heat → blue to green/yellow/orange/brick-red precipitate.
· Starch: iodine solution → yellow-brown to blue-black.
· Lipid: ethanol then water → white/cloudy emulsion.
· Protein: Biuret reagent → blue to lilac/purple/violet.
· Non-reducing sugar: acid hydrolysis + neutralisation + Benedict’s → positive Benedict’s result after hydrolysis.

Practical exam technique

· Always name the reagent and the positive colour change.
· State whether the test needs heating: Benedict’s tests require heating, iodine/Biuret do not.
· For semi-quantitative Benedict’s, mention standardised conditions and comparison with known standards.
· Include a negative control or starting colour where relevant to make interpretation clear.
· Use correct safety: hot water bath, eye protection, and keep ethanol away from flames.