Testing for Biological Molecules – Cambridge IGCSE/A‑Level (9700) 2.1
Learning‑outcome checklist (AO mapping)
| Skill / Knowledge | Assessment Objective(s) |
|---|
| List the four qualitative tests for the major biomolecule groups. | AO1 |
| Explain the chemical principle behind each test (redox, complex‑formation, etc.). | AO1 + AO2 |
| Carry out each test safely, accurately and with appropriate controls. | AO3 |
| Interpret the observed colour/physical changes and, where required, make a semi‑quantitative estimate. | AO2 |
| Describe how non‑reducing sugars are detected (acid hydrolysis + Benedict’s). | AO1 + AO2 |
General Practical Tips (AO3)
- Controls: Always prepare a blank tube containing only the reagent(s) and distilled water. This shows the colour of the reagent alone.
- Replication: Perform each test in duplicate (or triplicate) to check reproducibility.
- Record‑keeping: Use a table to note observations immediately. Include water‑bath temperature (≈ 100 °C for Benedict’s) and cooling time before observation.
- PPE: Gloves, goggles and a lab coat are mandatory. Follow the safety notes for each reagent.
1. Benedict’s Test – Reducing Sugars
Aim
Detect the presence of reducing sugars and, by comparison with a colour chart or standard, give a semi‑quantitative estimate of concentration.
Principle (Explain why the test works)
Reducing sugars contain a free aldehyde or ketone group that can reduce Cu²⁺ (blue) to Cu⁺**, precipitating red‑orange cuprous oxide (Cu₂O) under alkaline conditions. The overall redox reaction can be written as:
R‑CHO + 2 Cu²⁺ + 5 OH⁻ → R‑COOH + Cu₂O(s) + 3 H₂O
where R‑CHO represents the aldehydic form of the sugar. The amount of Cu₂O formed is proportional to the amount of reducing sugar present.
Materials
- Benedict’s solution (CuSO₄·5H₂O, Na₂CO₃, Na‑citrate)
- Test tubes (3 per sample: sample, blank, standard)
- Standard glucose solution (0.2 % w/v, or any known concentration)
- Distilled water
- Boiling water bath (≈ 100 °C)
Procedure (Qualitative & Semi‑quantitative)
- Label three tubes: sample, blank (water) and standard (glucose).
- Add 2 mL Benedict’s solution to each tube.
- Add 1 mL of the test solution to the sample tube, 1 mL distilled water to the blank, and 1 mL standard glucose** to the standard.
- Mix gently and place the tubes in a boiling water bath for 2–3 minutes.
- Remove, cool for ≈ 1 minute, then observe colour.
- Compare the colour of the sample tube with the colour chart supplied by the exam board (or with the standard tube) to obtain a semi‑quantitative estimate.
Observations & Interpretation
| Colour of precipitate | Relative amount of reducing sugar |
|---|
| Blue (no precipitate) | None |
| Green | Very low |
| Yellow‑orange | Low‑moderate |
| Brick‑red | High |
Semi‑quantitative use (AO2)
Match the observed colour with the official Benedict’s colour chart (5–6 bands). Each band corresponds to a concentration range (e.g., 0 %–0.2 % w/v, 0.2 %–0.5 % w/v, etc.). This satisfies the syllabus requirement that the test “can be made semi‑quantitative”.
Detection of Non‑reducing Sugars (e.g., sucrose)
- Place 1 mL of the sample in a test tube and add 1 mL of 1 M HCl.
- Heat in a boiling water bath for 5 minutes to hydrolyse the disaccharide into its constituent monosaccharides.
- Cool, then neutralise with an equal volume of 1 M NaOH (or add a few drops of NaOH).
- Carry out the Benedict’s test on the neutralised solution as described above.
A positive colour change after hydrolysis indicates the original presence of a non‑reducing sugar.
Possible Interferences (false results)
- Strongly coloured samples (fruit juices, tea) may mask the colour – dilute the sample.
- Ascorbic acid or other strong reducers give a false‑positive.
- Insufficient heating (< 90 °C) or a short heating time can produce a weak or false‑negative result.
Safety (AO3)
Benedict’s reagent contains copper(II) sulphate – avoid skin contact, wear gloves, and dispose of waste according to local regulations.
AO coverage for this test
- AO1 – list the test, write the redox equation.
- AO2 – interpret colour, discuss interferences, make semi‑quantitative estimate.
- AO3 – set up blanks, duplicate tubes, record temperature, observe safety.
2. Iodine Test – Starch
Aim
Detect the presence of starch (amylose/amylopectin) in a sample.
Principle (Explain why the test works)
Iodine (I₂) molecules fit inside the helical cavities of amylose, forming a charge‑transfer complex that absorbs light in the visible region, giving a blue‑black colour:
(I₂) + amylose → blue‑black I₂‑amylose complex
The intensity of the colour is roughly proportional to the amount of amylose present.
Materials
- Iodine solution (I₂ dissolved in KI)
- White tile or clear test tube
- Sample (≈ 0.5 mL solution or a pinch of solid)
Procedure
- Place the sample on a clean white tile (or in a test tube).
- Add 1–2 drops of freshly prepared iodine solution.
- Mix gently and observe within 30 seconds.
- Record the colour; compare with a colour chart if a semi‑quantitative estimate is required.
Observations
- Blue‑black – starch present (positive).
- Yellow‑brown** or no colour change – starch absent (negative).
Interferences & Notes
- Strongly coloured samples may obscure the colour change – dilute with water.
- Very low starch concentrations give a faint colour; a magnifying lens or comparison with a standard improves detection.
Safety
Iodine stains skin and clothing; handle with gloves and avoid inhalation of vapour.
AO coverage
- AO1 – state the principle (charge‑transfer complex).
- AO2 – interpret colour, discuss interferences.
- AO3 – use a control (iodine alone) and record observations promptly.
3. Emulsion Test – Lipids
Aim
Detect the presence of lipids (fats, oils, waxes) in a sample.
Principle (Explain why the test works)
Lipids are non‑polar and insoluble in water but soluble in ethanol. When a lipid‑containing ethanol solution is mixed with water, the lipid forms a fine suspension of tiny droplets – a milky white emulsion – because water cannot dissolve the lipid.
Materials
- Ethanol (96 % or absolute)
- Distilled water
- Test tube with stoppered cap
- Sample (oil, butter extract, plant material dissolved in ethanol)
Procedure
- Add 2 mL ethanol to a clean test tube.
- Add 1 mL of the sample and swirl to dissolve any lipid.
- Add 2 mL distilled water and shake vigourously for 5–10 seconds.
- Observe the formation of a cloudy, milky layer.
Observations
- Milky white/cloudy emulsion – lipid present (positive).
- Clear, colourless solution – no detectable lipid (negative).
Interferences
- Proteins or polysaccharides soluble in ethanol can give a faint turbidity; confirm with a second lipid‑specific test (e.g., Sudan III) if required.
- Very small amounts of lipid may produce a faint emulsion – use a white background for better visibility.
Safety
Ethanol is highly flammable; keep away from open flames, work in a well‑ventilated area and wear gloves.
AO coverage
- AO1 – describe the principle (insolubility in water).
- AO2 – interpret the emulsion, note possible interferences.
- AO3 – follow the procedural steps, use a blank (ethanol + water only).
4. Biuret Test – Proteins
Aim
Detect the presence of proteins (or any compound containing peptide bonds) and give a semi‑quantitative estimate.
Principle (Explain why the test works)
In an alkaline medium, Cu²⁺ ions coordinate with the peptide nitrogen atoms of at least two peptide bonds, forming a violet‑coloured Cu‑protein chelate. The reaction can be represented as:
Cu²⁺ + 2 –NH‑C(=O)– → [Cu(NH‑C(=O)–)₂] (violet complex)
The intensity of the violet colour is roughly proportional to the number of peptide bonds (i.e., to protein concentration).
Materials
- Biuret reagent (CuSO₄·5H₂O dissolved in NaOH)
- Test tubes (sample, blank, standard 1 % w/v albumin)
- Distilled water
Procedure (Qualitative & Semi‑quantitative)
- Label three tubes: sample, blank (water) and standard (albumin).
- Add 2 mL Biuret reagent to each tube.
- Add 1 mL of the test solution to the sample tube, 1 mL distilled water to the blank, and 1 mL standard protein to the standard.
- Mix gently and allow to stand for 1–2 minutes at room temperature.
- Observe the colour change; compare with the standard or a colour chart for a semi‑quantitative estimate.
Observations
- Purple/violet – protein present (positive).
- Blue‑green** or no colour change – protein absent or very low concentration (negative).
Interferences
- Strongly coloured substances (e.g., phenols) may mask the violet colour – dilute the sample.
- Very high protein concentrations can give a dark brown colour; perform a dilution series.
Safety
The Biuret reagent is strongly alkaline (NaOH). Wear gloves and eye protection; avoid splashes on skin.
AO coverage
- AO1 – write the coordination reaction.
- AO2 – interpret the violet colour, discuss interferences, make semi‑quantitative estimate.
- AO3 – use blanks, duplicate tubes, record observations, follow safety rules.
Summary Table – Quick Reference
| Molecule | Reagent(s) | Positive result (colour/physical change) | Underlying chemical principle |
|---|
| Reducing sugars | Benedict’s solution (CuSO₄, Na₂CO₃, Na‑citrate) | Green → yellow‑orange → brick‑red Cu₂O precipitate | Free aldehyde/ketone reduces Cu²⁺ to Cu⁺ (redox) |
| Non‑reducing sugars (after hydrolysis) | 1 M HCl (hydrolysis) + Benedict’s solution | Same colour series as above after acid treatment | Acid hydrolysis yields reducing monosaccharides |
| Starch | Iodine solution (I₂/KI) | Blue‑black complex | I₂ fits into amylose helices → charge‑transfer complex |
| Lipids | Ethanol + water (emulsion test) | Milky white/cloudy emulsion | Lipids are insoluble in water; form dispersed droplets |
| Proteins | Biuret reagent (CuSO₄ in NaOH) | Purple/violet colour | Cu²⁺ chelates with peptide bonds under alkaline conditions |
Key Points for Examination (AO1 + AO2 + AO3)
- All four tests are qualitative; Benedict’s and Biuret can be made semi‑quantitative by comparison with a colour chart or a standard tube.
- Always include a blank control and record the temperature (≈ 100 °C for Benedict’s) and cooling time before observation.
- For non‑reducing sugars, the extra acid‑hydrolysis step must be performed before Benedict’s.
- Safety reminders:
- Copper salts (Benedict’s) – avoid skin contact.
- Iodine – stains, handle with gloves.
- Ethanol – flammable, keep away from flame.
- Biuret – strong alkali, wear eye protection.
- Potential interferences (coloured samples, other reducing agents, insufficient heating) should be mentioned in answers to demonstrate deeper understanding.