Cambridge International AS & A Level Biology 9700: Testing for biological molecules – Revision Notes

For full explanations and worked examples, use the testing for biological molecules study guide. These notes condense sub-topic 2.1, Testing for biological molecules, of the Cambridge International AS & A Level Biology 9700 syllabus for examination in 2025, 2026 and 2027. It is AS Level content: tested on Paper 1 and Paper 2, assumed in Paper 4, and used as a practical skill in Paper 3 (identifying unknown solutions, estimating concentrations) and Paper 5 (planning dilutions and controls).

Practise with the testing for biological molecules practice questions. For the rest of topic 2, see the biological molecules revision notes. The 9700 course hub, the printable checklist and the free 9700 AS diagnostic show where this fits.

The three outcomes

Outcome	In one line
2.1.1	Describe and carry out Benedict’s, iodine, emulsion and biuret tests
2.1.2	Standardise a Benedict’s test and estimate a reducing sugar concentration from time to first colour change or colour standards
2.1.3	Test for a non-reducing sugar with acid hydrolysis, neutralisation, then Benedict’s

2.1.1 Test summary table

Test	Detects	Reagent and method	Heat?	Positive	Negative
Benedict’s	Reducing sugars (glucose, fructose, maltose)	Equal volume of Benedict’s solution	Yes, water bath near boiling	Green → yellow → orange → brick-red precipitate	Stays blue
Iodine	Starch	Few drops of iodine in potassium iodide solution	No	Blue-black	Stays orange-brown
Emulsion	Lipids	Shake with ethanol, then pour into water	No	Cloudy white emulsion	Stays clear
Biuret	Proteins (peptide bonds)	Equal volume of biuret reagent, or potassium hydroxide then dilute copper sulfate	No	Purple (lilac)	Stays blue

Must-know reasons

• Benedict’s: reducing sugars reduce blue copper(II) ions, forming a coloured precipitate. More sugar, more precipitate, colour further along the series.
• Emulsion: lipid dissolves in ethanol; water is added; lipid is insoluble in water, so it comes out as tiny droplets, which look milky. Ethanol must come first.
• Biuret: detects peptide bonds, so it shows polypeptides and proteins.
• Controls: distilled water in place of sample (negative control) shows the reagent’s own colour; a known positive shows the reagent works.

Safety (from the syllabus materials list)

• Benedict’s solution: moderate hazard. Biuret reagent and potassium hydroxide: corrosive. Ethanol: flammable.
• Eye protection; water bath instead of a naked flame near ethanol; point heated tubes away from people.

2.1.2 Semi-quantitative Benedict’s test

Method in steps

1. Make a range of known concentrations of a reducing sugar (at least five) by serial or proportional dilution.
2. Use the same volume of each sample and the same volume of Benedict’s solution (in excess).
3. Heat all tubes in one thermostatically controlled water bath at the same temperature.
4. Either heat for a fixed time and keep the tubes as colour standards, or time how long each takes to show the first colour change from blue.
5. Repeat each concentration (at least three replicates) and calculate a mean time.
6. Test the unknown in exactly the same way. Compare with the standards, or read its concentration from a graph of time against concentration.

Variables to standardise

Variable	How
Volume of sample	Same syringe or graduated pipette volume each time
Volume of Benedict’s	Same volume, enough to be in excess
Temperature	Thermostatically controlled water bath, checked with a thermometer
Heating time (colour standards)	Timer; all tubes in and out together
Colour judgement	Same observer, white tile behind tubes, same lighting

Dilution formulas

Type	How it works	Example
Proportional	Volume of stock = (wanted ÷ stock concentration) × final volume, rest water	0.50 % from 2.0 % stock, 10 cm³ final: 2.5 cm³ stock + 7.5 cm³ water
Serial	Each solution diluted by the same factor to make the next	Tenfold from 1.0 %: 1.0, 0.1, 0.01, 0.001 %

Units

• 1 % (w/v) = 1 g of solute in 100 cm³ of solution.
• mol dm⁻³ → g dm⁻³: multiply by relative molecular mass (glucose = 180). Then g dm⁻³ ÷ 10 = % (w/v).
• Rate from time: rate = 1/t (units s⁻¹). Shorter time, higher rate, higher concentration.

Worked reminders

Converting units. 0.30 mol dm⁻³ glucose → 0.30 × 180 = 54 g dm⁻³ → 54 ÷ 10 = 5.4 % (w/v).

Reading an unknown from times. Standards: 0.25 % took 130 s, 0.50 % took 75 s. An unknown took 100 s. It changed colour faster than 0.25 % but slower than 0.50 %, so its concentration is between 0.25 % and 0.50 %. For a single value, plot mean time against concentration, draw a smooth curve through the points and read across from 100 s.

Choosing a dilution type. Use proportional dilution for evenly spaced concentrations (0.2, 0.4, 0.6 …). Use serial dilution for a wide range that spans several orders of magnitude (1, 0.1, 0.01 …). In serial dilution, an error at one step carries into every later solution.

Must-know distinctions

• Time to first colour change vs colour standards. Timing gives continuous data you can graph. Colour standards give ordinal data, so the answer is a range (“between 0.4 and 0.6 %”).
• Semi-quantitative vs quantitative. You estimate by comparison with known standards; you do not measure a concentration directly.
• Interpolation only. An unknown outside the range of the standards cannot be estimated from them.

Typical sources of error

• Subjective judgement of colour or of the “first” change.
• Tubes entering the bath at different times; the bath cooling when tubes go in.
• Benedict’s not in excess, so high concentrations all look brick-red.
• Unequal volumes from inaccurate measuring.

2.1.3 Non-reducing sugar test

Method in steps

1. Benedict’s test on the sample first. Non-reducing sugar alone: stays blue.
2. Fresh sample + dilute hydrochloric acid; heat in a water bath near boiling. Acid hydrolysis breaks glycosidic bonds.
3. Cool. Neutralise with sodium hydrogencarbonate until fizzing stops; check with pH paper or red litmus.
4. Benedict’s test again. Colour change (green to brick-red) = non-reducing sugar present.

Why each step

• Sucrose is non-reducing; hydrolysis releases glucose and fructose, both reducing sugars.
• Benedict’s needs alkaline conditions; leftover acid would stop a positive result.

Reading the two results

Before hydrolysis	After hydrolysis	Conclusion
Blue	Blue	No sugar of either type
Blue	Orange	Non-reducing sugar only
Green	Green (same)	Reducing sugar only
Green	Brick-red	Reducing sugar and non-reducing sugar

The last row only works if volumes and heating are the same in both tests.

Quick self-test

1. Name the reagent for the starch test and give its colour before and after a positive result.
2. Which three sugars named in the 9700 syllabus are reducing sugars?
3. Why must the sample be shaken with ethanol before water is added in the emulsion test?
4. State the negative result of the biuret test.
5. What is the purpose of a tube containing distilled water and Benedict’s solution?
6. Calculate the concentration made by adding 2.5 cm³ of 2.0 % glucose to 7.5 cm³ of water.
7. Express 1.0 % (w/v) glucose in mol dm⁻³ (Mr = 180), to 3 significant figures.
8. A tube shows its first colour change after 40 s. Calculate the rate in s⁻¹.
9. Why is sodium hydrogencarbonate added after acid hydrolysis?
10. A solution stays blue with Benedict’s before and after acid hydrolysis. What does this show?
11. Give two variables you must standardise when making Benedict’s colour standards.

Answers

1. Iodine in potassium iodide solution; orange-brown to blue-black.
2. Glucose, fructose and maltose.
3. Lipids dissolve in ethanol but not in water; adding water then makes the dissolved lipid form a cloudy emulsion. Water first, and the lipid never dissolves.
4. The solution stays blue.
5. A negative control: it shows the colour of the reagent with no reducing sugar, for comparison.
6. (2.0 × 2.5) / 10 = 0.50 %.
7. 1.0 g per 100 cm³ = 10 g dm⁻³; 10 / 180 = 0.0556 mol dm⁻³.
8. 1/40 = 0.025 s⁻¹.
9. To neutralise the acid, because Benedict’s test only works in alkaline conditions.
10. No reducing sugar and no non-reducing sugar is present.
11. Any two: volume of sugar solution; volume of Benedict’s solution; water bath temperature; heating time.

Where marks are usually lost

• Not stating that Benedict’s must be heated, or giving no temperature (water bath near boiling).
• Writing “goes red” or “changes colour” instead of naming the start and end colours.
• Calling the emulsion “a white precipitate”, or adding water before ethanol.
• Writing “biuret test turns blue-black” (the iodine result).
• Missing the neutralisation step, or neutralising with an acid.
• Saying sucrose gives a brick-red result with Benedict’s without hydrolysis.
• Forgetting that a shorter time to colour change means a higher concentration.
• Giving an exact value from colour standards instead of a range.
• Listing “amount of Benedict’s” without saying it must be the same volume and in excess.
• Doing fewer than five concentrations, or no replicates, in a planning answer.

Official syllabus

Cambridge International AS & A Level Biology 9700 syllabus for 2025, 2026 and 2027 (Version 1), Cambridge International (Cambridge University Press & Assessment). Topic 2, Biological molecules, sub-topic 2.1 Testing for biological molecules.