Cambridge A‑Level Biology 9700 – Testing for Non‑Reducing Sugars (Topic 2 Biological Molecules)
1. Objective
- Describe and carry out a test to identify the presence of non‑reducing sugars in an unknown sample by (i) acid hydrolysis and (ii) a Benedict’s test.
- Perform the semi‑quantitative version of the Benedict’s test.
- Recall the other routine tests for biological molecules (iodine, emulsion, biuret) and the chemical principles behind each.
2. Syllabus Context & Learning‑Outcome Checklist (AO1 & AO2)
These activities address the following Cambridge AS/A‑Level Biology requirements (Topic 2.1 – Testing for biological molecules):
| Learning outcome | Evidence in the notes |
|---|
| State the principle of the Benedict’s test. | Section 3.1 – Reducing‑sugar reduction of Cu²⁺. |
| Explain why non‑reducing sugars give a negative direct Benedict’s test. | Section 3.1 – No free aldehyde/ketone. |
| Describe the role of acid hydrolysis in converting non‑reducing sugars to reducing sugars. | Section 3.1 – Hydrolysis reaction. |
| Carry out the procedure safely and evaluate the reliability of the results. | Sections 5–7 (procedure, safety, evaluation). |
| Identify the reagents, observations and biomolecules detected by the iodine, emulsion and biuret tests. | Section 3.2 – Summary tables. |
| Link carbohydrate testing to structure‑function relationships (e.g., digestion, storage). | Box “Why it matters” (Section 3.1). |
3. Background Theory
3.1 Non‑Reducing Sugars and Acid Hydrolysis
- Non‑reducing sugars (e.g., sucrose, trehalose) have their anomeric carbon involved in a glycosidic bond, so no free aldehyde or ketone group is available to reduce Cu²⁺.
- Acid hydrolysis cleaves the glycosidic bond:
R–O–R′ + H₂O → R–OH + R′–OH (catalysed by H⁺)
The products are monosaccharides (glucose, fructose, etc.) that are reducing sugars.
- Why it matters: Distinguishing reducing from non‑reducing sugars helps explain why sucrose must be broken down by sucrase before it can enter glycolysis, whereas glucose can be used directly.
3.2 Benedict’s Test (Reducing Sugars)
- Reagent: alkaline copper(II) carbonate solution (CuSO₄ + Na₂CO₃ + NaOH).
- Reaction: reducing sugar reduces Cu²⁺ (blue) to Cu⁺, precipitating cuprous oxide (Cu₂O) which appears orange‑red to brick‑red.
- Colour intensity is proportional to the amount of reducing sugar present.
3.3 Other Routine Tests (Syllabus 2.1)
| Test | Reagent | Observed result | Biomolecule detected | Chemical basis |
|---|
| Iodine test | I₂/KI solution | Blue‑black colour | Starch (polysaccharide) | I₂ fits into the helical cavities of amylose, forming a charge‑transfer complex. |
| Emulsion test | Lauryl sulphate solution + ethanol | Milky emulsion | Lipids (fats, oils) | Lipid droplets are suspended in the aqueous medium by the surfactant, scattering light. |
| Biuret test | CuSO₄ + NaOH (Biuret reagent) | Violet colour | Proteins (peptide bonds) | Cu²⁺ complexes with peptide nitrogen atoms under alkaline conditions. |
4. Materials and Reagents
| Item | Quantity / Concentration | Purpose |
|---|
| Unknown aqueous sample | 2 mL | Source of possible non‑reducing sugar |
| Hydrochloric acid (HCl) | 1 M, 2 mL | Acid hydrolysis |
| Sodium hydroxide (NaOH) | 2 M, 2 mL (per tube) | Neutralise HCl and provide alkaline medium for Benedict’s |
| Benedict’s solution (fresh) | 5 mL per tube | Detect reducing sugars |
| Glucose standard solution | 0.1 M, 2 mL (per control tube) | Positive control & calibration |
| Distilled water | 10 mL (for dilutions & negative control) | Blank |
| Test tubes (minimum 6) | – | Reaction vessels |
| Boiling water bath (thermostatically controlled, ≈100 °C) | – | Heating steps |
| pH paper (optional) | – | Check neutralisation (target pH ≈ 7 ± 0.5) |
| Colour‑chart or spectrophotometer (620 nm) (optional) | – | Semi‑quantitative measurement |
5. Safety Precautions
- Wear lab coat, nitrile gloves and safety goggles – HCl and NaOH are corrosive.
- Carry out heating in a fume hood or well‑ventilated area.
- Cuprous oxide precipitate is toxic; avoid inhalation and dispose of copper‑containing waste according to local regulations.
- Handle hot test tubes with tongs or a tube holder.
- Do not taste or inhale any reagents.
6. Procedure
Part A – Qualitative Test (Hydrolysis + Benedict’s)
- Label six test tubes as follows:
- A – Sample (hydrolysed)
- B – Sample (no hydrolysis)
- C – Positive control (glucose)
- D – Negative control (water)
- E – Blank (reagents only)
- F – Spare (optional duplicate)
- Hydrolysis tube (A)
- Add 2 mL of the unknown sample.
- Add 2 mL of 1 M HCl, mix gently.
- Place in a boiling water bath for 7 min (ensure a rolling boil). Longer (10 min) if the sample is viscous.
- Cool to room temperature.
- Add 2 mL of 2 M NaOH to neutralise the acid. Check pH; it should be 6.5–7.5.
- Non‑hydrolysed tube (B)
- Add 2 mL of the unknown sample.
- Add 2 mL of 2 M NaOH (no HCl).
- Positive control (C)
- Add 2 mL of 0.1 M glucose solution.
- Add 2 mL of 2 M NaOH.
- Negative control (D)
- Add 2 mL distilled water.
- Add 2 mL of 2 M NaOH.
- Blank (E)
- Add 2 mL distilled water.
- Add 2 mL of 2 M NaOH.
- To each tube (A–E) add 5 mL of freshly prepared Benedict’s solution. Mix gently by tapping.
- Place all tubes in the boiling water bath for 2 min. Remove and observe the colour immediately.
Part B – Semi‑Quantitative Benedict’s Test (Optional)
- Prepare a series of glucose standards (0, 0.025, 0.050, 0.075, 0.100 M). In each standard tube add 2 mL of the standard solution, 2 mL of 2 M NaOH and 5 mL of Benedict’s solution.
- Heat as in step 8 and record the colour using the provided colour‑chart or measure absorbance at 620 nm.
- Plot a calibration curve (absorbance or colour grade vs. glucose concentration).
- Compare the colour/absorbance of the hydrolysed sample (tube A) with the curve to estimate the concentration of reducing sugar released.
7. Observations and Interpretation
| Tube | Colour after heating | Precipitate | Interpretation |
|---|
| A – Sample (hydrolysed) | Orange‑red to brick‑red (intensity varies) | Cu₂O (red‑brown) | Positive – non‑reducing sugar present; hydrolysis produced reducing monosaccharides. |
| B – Sample (no hydrolysis) | Blue (no change) | None | Confirms the original sugar was non‑reducing. |
| C – Positive control (glucose) | Brick‑red | Cu₂O | Test working correctly. |
| D – Negative control (water) | Blue | None | No reducing sugar; validates absence of contamination. |
| E – Blank (reagents only) | Blue | None | Reagents are uncontaminated. |
8. Data‑Analysis Worksheet (AO3 – Evaluation)
- Record raw observations – colour, precipitate amount, any cloudiness.
- Assign a colour grade (0 = blue, 1 = green, 2 = yellow, 3 = orange, 4 = brick‑red) or enter absorbance values if a spectrophotometer is used.
- Calculate the concentration of reducing sugar in tube A using the calibration curve:
Concentration (mol L⁻¹) = (Absorbance – Intercept) / Slope
- Optional – % hydrolysis (theoretical maximum assumes complete conversion of the original disaccharide to its two monosaccharide units):
% Hydrolysis = (Measured reducing‑sugar concentration ÷ Theoretical maximum) × 100
- Evaluation questions
- Were the controls as expected?
- Was the hydrolysis time sufficient? (Consider extending to 10 min if colour is weak.)
- Did the pH after neutralisation fall within 6.5–7.5?
- Identify systematic errors (e.g., incomplete hydrolysis, excess NaOH redissolving Cu₂O, colour‑blindness).
- Suggest improvements (duplicate trials, thermostatically controlled water bath, use of a standardised colour chart, precise pH measurement).
9. Key Points to Remember
- Non‑reducing sugars give a negative direct Benedict’s test because the anomeric carbon is locked.
- Acid hydrolysis breaks the glycosidic bond, liberating reducing monosaccharides that give a positive Benedict’s reaction.
- Neutralisation with excess NaOH (pH ≈ 7 ± 0.5) is essential; residual acid prevents Cu²⁺ reduction.
- Colour intensity (or absorbance at 620 nm) is proportional to the amount of reducing sugar present.
- Always run a positive (glucose) and a negative (water) control.
- Link the test to biological relevance: distinguishing sucrose from glucose helps explain carbohydrate digestion and energy metabolism.
10. Common Errors and Troubleshooting
| Error | Possible cause | Remedy |
|---|
| Weak or no colour change in tube A | Insufficient hydrolysis time; incomplete neutralisation | Extend boiling to 10 min; verify pH and add an extra 0.5 mL of 2 M NaOH if pH < 6.5. |
| Colour change in negative control (D) | Contamination of reagents or glassware | Use fresh glassware, rinse thoroughly with distilled water, prepare fresh Benedict’s solution. |
| Precipitate dissolves on standing | Excessive NaOH (pH > 12) redissolves Cu₂O | Use the calculated amount of NaOH (2 mL of 2 M for 2 mL of 1 M HCl) and check pH. |
| Variable colour intensity between replicates | Inconsistent heating or timing | Use a thermostatically controlled water bath; start timing when the water reaches a rolling boil. |
| Faint colour with fresh Benedict’s solution | Solution stored too long; Cu²⁺ complexes degrade | Prepare Benedict’s solution weekly, store in a dark bottle, keep at room temperature. |
11. Suggested Diagram (for classroom hand‑out)
Flowchart showing two parallel pathways:
- Sample → No hydrolysis → NaOH → Benedict’s → Observation (blue = non‑reducing)
- Sample → HCl (hydrolysis) → NaOH (neutralise) → Benedict’s → Observation (orange‑red = reducing sugars released)
Include an inset colour‑chart (blue → green → yellow → orange → brick‑red) for semi‑quantitative scoring.