Testing for Biological Molecules
Four simple chemical tests identify the main classes of biological molecule: Benedict's test for reducing sugars, the iodine test for starch, the emulsion test for lipids and the biuret test for proteins. A fifth procedure, acid hydrolysis followed by Benedict's test, detects non-reducing sugars such as sucrose, and the Benedict's test can be made semi-quantitative to estimate concentration. These tests turn up constantly in Paper 3, where you may be given unknown solutions to identify, and in Papers 1 and 2 as "describe how you would test for" questions. Marks come from precise reagents, conditions and colours.
Summary of the tests
| Molecule | Test | Method in brief | Positive result | Negative result |
|---|---|---|---|---|
| Reducing sugar | Benedict's | equal volume of Benedict's solution; heat in a water bath at about 80–95 °C for a few minutes | brick-red precipitate (via green, yellow, orange) | remains blue |
| Non-reducing sugar | acid hydrolysis, then Benedict's | negative Benedict's first; boil fresh sample with dilute HCl; neutralise with sodium hydrogencarbonate; Benedict's test | brick-red precipitate after hydrolysis | remains blue |
| Starch | iodine | add a few drops of iodine in potassium iodide solution | blue-black | remains orange-brown (yellow-brown) |
| Lipid | emulsion | shake sample with ethanol; pour into water (or add water) | milky white emulsion | remains clear |
| Protein | biuret | equal volume of biuret reagent (or sodium/potassium hydroxide then dilute copper sulfate); no heating | lilac / purple / violet | remains blue |
Benedict's test for reducing sugars
Reducing sugars are sugars that can donate electrons to (reduce) another substance. All monosaccharides (glucose, fructose, galactose) and some disaccharides (maltose, lactose) are reducing sugars. Sucrose is a non-reducing sugar.
Benedict's solution is blue because it contains copper(II) ions, . When heated with a reducing sugar, the sugar reduces the copper(II) ions to copper(I), which forms a brick-red precipitate of copper(I) oxide:
- Place a known volume of the sample solution in a test-tube, for example . Solid samples are first ground with a little water.
- Add an equal volume of Benedict's solution.
- Heat in a water bath at about 80–95 °C (a beaker of near-boiling water) for a set time, for example 5 minutes. Do not heat the tube directly in a flame.
- Observe the colour.
The colour depends on how much copper(I) oxide forms, which depends on the concentration of reducing sugar:
| Reducing sugar concentration | Colour seen |
|---|---|
| none | blue (unchanged) |
| very low | green |
| low | yellow |
| moderate | orange |
| high | brick-red (with a lot of precipitate) |
The green and yellow colours occur because a small amount of red-orange precipitate is mixed with the remaining blue solution.
Write "brick-red precipitate" for a positive result, not "turns red". The red colour is a solid, which settles out. And remember that Benedict's solution must be heated: an unheated tube stays blue even with glucose present.
Semi-quantitative Benedict's test
The Benedict's test can estimate the concentration of a reducing sugar, not just its presence, as long as every other variable is standardised. This is called semi-quantitative because it gives an estimate within a range rather than an exact value.
- Make a series of glucose solutions of known concentration, for example 0, 0.2, 0.4, 0.6, 0.8 and 1.0%, by serial or proportional dilution of a stock solution (see Planning investigations).
- Standardise: the volume of each sugar solution, the volume and concentration of Benedict's solution, the temperature of the water bath, and the heating time (or record time).
- Test each known solution and the unknown in the same way, at the same time if possible.
- Then use one of two approaches:
- colour standards: after a fixed heating time, compare the colour of the unknown with the series of known standards and estimate that its concentration lies between the two closest standards;
- time to first colour change: start a timer when the tube goes into the water bath and record the time until the first change from blue is seen. The higher the concentration of reducing sugar, the shorter the time. Plot a calibration graph of time against concentration for the known solutions and read off the unknown.
- For a more objective result, filter off the precipitate and measure the absorbance (or transmission) of the remaining solution with a colorimeter (the less blue it is, the more sugar there was), or dry and weigh the precipitate.
Limitations of the semi-quantitative test
- Judging colour by eye is subjective; colours such as yellow and orange are hard to distinguish, and different people judge the "first colour change" differently. Improvements: view against a white tile, use the same person, or use a colorimeter.
- The precipitate settles, so tubes should be shaken gently before comparing.
- The temperature of a water bath heated by Bunsen may vary; a thermostatically controlled water bath is better.
- The result is only an estimate between two standards. Using smaller intervals between standard concentrations narrows the range.
- Benedict's solution must be in excess: if all the copper(II) ions are reduced, a higher sugar concentration cannot give any more colour.
A student recorded the time for the first colour change with glucose standards and an unknown:
| Glucose concentration / % | 0.2 | 0.4 | 0.6 | 0.8 | 1.0 | unknown |
|---|---|---|---|---|---|---|
| Time to first colour change / s | 95 | 62 | 44 | 33 | 27 | 51 |
Estimate the glucose concentration of the unknown, and state two variables that must be standardised.
Solution
The time decreases as concentration increases. The unknown (51 s) lies between 0.4% (62 s) and 0.6% (44 s), so its concentration is between 0.4% and 0.6%. Reading from a calibration curve drawn through the points gives a value of about 0.5%.
Variables to standardise (any two): volume of glucose solution; volume and concentration of Benedict's solution; temperature of the water bath; the person judging the colour change (or the background used, e.g. a white tile); the size of test-tube.
A ruled straight line between 0.4% and 0.6% would give , but because the relationship is a curve, "about 0.5%" or "between 0.4% and 0.6%" is the expected answer.
Testing for non-reducing sugars
Sucrose (glucose + fructose) is the main non-reducing sugar. In sucrose the glycosidic bond links the parts of both monosaccharides that would otherwise act as reducing groups, so it cannot reduce Benedict's solution. If you hydrolyse the glycosidic bond, the free glucose and fructose are released, and both are reducing sugars.
- Carry out a Benedict's test on one sample. It must be negative (stays blue), showing no reducing sugar is present. (If it is positive, see below.)
- Take a fresh sample of the same solution. Add a few drops of dilute hydrochloric acid and heat in a boiling water bath for a few minutes. This hydrolyses the glycosidic bonds.
- Cool, then neutralise by adding sodium hydrogencarbonate (a little at a time, until fizzing stops; check with pH paper that the solution is neutral or slightly alkaline).
- Carry out a Benedict's test on the neutralised solution.
- A brick-red precipitate now shows that a non-reducing sugar was present.
Neutralisation is needed because Benedict's test works only in alkaline conditions: the acid would prevent the copper(II) ions from being reduced.
If the original sample already contained some reducing sugar, the first test is positive. A non-reducing sugar can still be detected by comparing the two tests: if there is more precipitate (or a colour further towards red) after hydrolysis than before, then a non-reducing sugar was also present.
A solution gave a green colour with Benedict's test. After boiling with acid and neutralising, a fresh sample gave a brick-red precipitate. What can you conclude?
Solution
The green colour shows that a small amount of reducing sugar is present. After hydrolysis there is much more reducing sugar (brick-red precipitate), so additional reducing sugars were released by hydrolysis. Therefore the solution also contains a non-reducing sugar (such as sucrose), which was hydrolysed into reducing monosaccharides.
A polysaccharide such as starch would also be hydrolysed by acid, so strictly the result shows a non-reducing carbohydrate. An iodine test on the original solution would rule out starch.
Iodine test for starch
Starch is a mixture of amylose and amylopectin. Amylose forms a helix, and iodine (as triiodide ions from iodine in potassium iodide solution) fits inside the helix, forming a complex that is blue-black.
- Add a few drops of iodine in potassium iodide solution to the sample (solution or solid, such as a slice of potato).
- A colour change from orange-brown to blue-black shows starch is present.
The test works at room temperature. Glycogen gives a red-brown colour rather than blue-black, because its highly branched chains form only short helices. In Paper 3, the iodine test is often used on a spotting tile to follow the disappearance of starch when amylase digests it; when samples no longer turn blue-black, all the starch has been hydrolysed.
Emulsion test for lipids
Lipids such as triglycerides are insoluble in water but soluble in ethanol.
- Add about of the sample to about of ethanol (absolute ethanol) in a dry test-tube. Shake well to dissolve any lipid.
- Pour this solution into a test-tube containing an equal volume of water (or add water to it).
- A milky white emulsion (cloudiness) shows that lipid is present.
The lipid dissolves in ethanol, but when water is added the lipid comes out of solution as tiny droplets dispersed through the water. These droplets scatter light, so the mixture looks milky. If no lipid is present, the solution stays clear.
The order matters. Lipid must be dissolved in ethanol first. Adding water first and then ethanol does not give a clear result. Ethanol is flammable: keep it away from Bunsen flames.
Biuret test for proteins
Biuret reagent contains copper(II) ions in alkaline solution (sodium or potassium hydroxide with dilute copper sulfate). The copper(II) ions form a purple complex with the nitrogen atoms in peptide bonds, so the test detects any molecule with peptide bonds (proteins and polypeptides, but not individual amino acids).
- Add an equal volume of biuret reagent to the sample. (Alternatively, add a few drops of sodium or potassium hydroxide solution, then a few drops of dilute copper sulfate solution.)
- Mix gently. Do not heat.
- A colour change from blue to lilac / purple / violet shows protein is present.
Choosing and interpreting tests
Four solutions, A to D, contain starch, glucose, sucrose and albumen (a protein), one in each. Results:
| Solution | Benedict's | Acid hydrolysis then Benedict's | Iodine | Biuret |
|---|---|---|---|---|
| A | blue | brick-red | orange-brown | blue |
| B | brick-red | brick-red | orange-brown | blue |
| C | blue | brick-red | blue-black | blue |
| D | blue | blue | orange-brown | purple |
Identify each solution.
Solution
- B is glucose: positive Benedict's without hydrolysis (reducing sugar).
- D is albumen: purple biuret; no sugar or starch.
- C is starch: blue-black with iodine. It is also positive after acid hydrolysis because acid hydrolyses starch to glucose.
- A is sucrose: negative Benedict's, positive only after hydrolysis, and no starch.
The key reasoning: both C and A are positive after hydrolysis, so the iodine test is needed to distinguish starch from sucrose.
A student tested a solution of sucrose. She boiled it with dilute hydrochloric acid, cooled it, added Benedict's solution and heated it. The solution stayed blue. Explain this result and describe how the procedure should be corrected.
Solution
- The acid hydrolysed the glycosidic bond in sucrose, producing glucose and fructose (reducing sugars).
- However, the student did not neutralise the acid before adding Benedict's solution.
- Benedict's test requires alkaline conditions; in acid, the copper(II) ions are not reduced, so no precipitate formed and the solution stayed blue.
- Correction: after cooling, add sodium hydrogencarbonate until effervescence stops (check with pH or universal indicator paper that the solution is neutral or slightly alkaline), then add an equal volume of Benedict's solution and heat in a water bath at 80–95 °C. A brick-red precipitate should form.
- Always name the reagent, the condition (heat in a water bath for Benedict's; no heating for biuret) and the colour change from what to what ("from blue to brick-red precipitate").
- "Iodine" in a question means iodine in potassium iodide solution; the colour change is orange-brown (or yellow-brown) to blue-black, never "turns black" alone.
- For the emulsion test, state "dissolve in ethanol, then add to water" and "milky white emulsion".
- For a non-reducing sugar test you need all of: negative first Benedict's, hydrolysis with acid and heat, neutralisation, repeat Benedict's, red precipitate.
- In semi-quantitative work, identify the standardised variables and the method of comparison, and describe the result as an estimate within a range.
Hazards and risk (Paper 3 may ask you to assess risk as low, medium or high):
- Ethanol is flammable (F) and harmful: no naked flames nearby. Medium risk if Bunsens are in use.
- Biuret reagent and sodium/potassium hydroxide are corrosive (C): wear eye protection; wash off skin immediately.
- Benedict's solution is a moderate hazard: eye protection; care with hot water baths (scalds).
- Hydrochloric acid (dilute) is an irritant: eye protection.
- Iodine solution stains skin and clothes and is hazardous to the aquatic environment: avoid contact; dispose of correctly.
- Benedict's (heat in water bath) detects reducing sugars: blue to green, yellow, orange, brick-red precipitate as concentration increases.
- Glucose, fructose and maltose are reducing sugars; sucrose is non-reducing.
- Non-reducing sugars: negative Benedict's; boil with dilute HCl to hydrolyse glycosidic bonds; neutralise with sodium hydrogencarbonate; Benedict's gives red precipitate.
- Semi-quantitative Benedict's: standardise volumes, concentration, temperature and time; compare with colour standards or time to first colour change.
- Iodine in potassium iodide: orange-brown to blue-black with starch.
- Emulsion test: dissolve in ethanol, add to water, milky white emulsion for lipid.
- Biuret: blue to lilac/purple with protein (peptide bonds), no heating.
Practice questions
- State the reagent and the positive result for the test for (a) starch, (b) protein, (c) lipid.
- Name two reducing sugars and one non-reducing sugar.
- Explain why the Benedict's test can give a range of colours.
- Describe how you would show that a solution contains sucrose. (4 marks)
- Explain why the solution must be neutralised before the second Benedict's test in the non-reducing sugar test.
- Explain why the emulsion test gives a milky appearance.
- A student wants to compare the glucose concentration in three fruit juices using Benedict's solution. State four variables she should standardise.
- A tube of Benedict's solution and a sugar solution were heated for 5 minutes and became yellow. A second tube of the same sugar solution, after boiling with acid and neutralising, gave an orange-red precipitate. Explain these results.
- Describe how you would use colour standards to estimate the concentration of glucose in a urine sample, and explain one limitation of this method. (5 marks)
- Suggest why the time to first colour change becomes a less reliable measure at very high glucose concentrations, and suggest an alternative way to measure the result more objectively.
Answers
- (a) Iodine in potassium iodide solution; orange-brown to blue-black. (b) Biuret reagent; blue to lilac/purple. (c) Ethanol then water; milky white emulsion.
- Reducing: any two of glucose, fructose, galactose, maltose, lactose. Non-reducing: sucrose.
- The amount of red copper(I) oxide precipitate depends on the concentration of reducing sugar; small amounts of red precipitate mixed with the blue solution give green, yellow or orange.
- Benedict's test on one sample: remains blue (no reducing sugar); boil a fresh sample with dilute hydrochloric acid (hydrolyses sucrose to glucose and fructose); cool and neutralise with sodium hydrogencarbonate; Benedict's test, heat in water bath; brick-red precipitate shows sucrose (a non-reducing sugar) was present.
- Benedict's test only works in alkaline conditions; the acid would prevent the reduction of copper(II) ions, giving a false negative.
- Lipid dissolves in ethanol; when water is added, the lipid is insoluble and forms tiny droplets (an emulsion) which scatter light, making it look milky white.
- Any four: volume of juice; volume of Benedict's solution; concentration of Benedict's solution; temperature of water bath; heating time; same person judging colour; same size of test-tube; juice diluted to the same extent.
- The first tube shows some reducing sugar (yellow: low concentration). After hydrolysis the colour is nearer red, so more reducing sugar is present: a non-reducing sugar (e.g. sucrose) was hydrolysed into reducing sugars. The solution contains both.
- Prepare glucose solutions of known concentrations (e.g. by serial dilution). Carry out Benedict's test on equal volumes of each and of the urine, with equal volumes of Benedict's, the same temperature and the same heating time. Compare the colour of the urine sample with the standards; its concentration lies between the two closest. Limitation: colour judgement is subjective / colours are hard to distinguish / only gives a range; improve with a colorimeter or more standards with smaller intervals.
- At high concentrations the colour change happens very quickly, so the times are short and the percentage error in timing (reaction time) is large; differences between concentrations are small. Alternatives: dilute the samples; use a colorimeter to measure absorbance after a fixed time (after removing the precipitate); or filter, dry and weigh the precipitate.