Cambridge A-Level Biology 9700 – Testing for Non‑Reducing Sugars
Testing for Biological Molecules
Objective
Describe and carry out a test to identify the presence of non‑reducing sugars in a sample, using acid hydrolysis followed by Benedict’s solution.
Background Theory
Non‑reducing sugars (e.g., sucrose, trehalose) lack a free aldehyde or ketone group because the anomeric carbon is involved in a glycosidic bond. They do not react directly with Benedict’s reagent. Hydrolysis with a strong acid cleaves the glycosidic bond, converting the non‑reducing sugar into its constituent reducing monosaccharides, which then give a positive Benedict’s test.
Principle of the Test
Acid hydrolysis: \$\text{R–O–R'} + \text{H}_2\text{O} \xrightarrow{\text{H}^+} \text{R–OH} + \text{R'–OH}\$
Benedict’s reaction: Reducing sugars reduce Cu²⁺ (blue) to Cu⁺ (red precipitate of Cu₂O) in an alkaline medium.
Materials and Reagents
Item
Quantity / Concentration
Purpose
Test sample (unknown solution)
2 mL
Source of possible non‑reducing sugar
Hydrochloric acid (HCl)
1 M, 2 mL
Acid hydrolysis
Distilled water
10 mL
Dilution and washing
Sodium hydroxide (NaOH)
2 M, 2 mL
Neutralise excess acid & provide alkaline medium
Benedict’s solution (freshly prepared)
5 mL
Detect reducing sugars
Test tubes (minimum 4)
–
Reaction vessels
Safety Precautions
Handle HCl and NaOH with gloves and eye protection – both are corrosive.
Work in a well‑ventilated area or fume hood.
Dispose of copper‑containing waste according to local regulations.
Procedure
Label four test tubes as “A – Sample”, “B – Hydrolysis”, “C – Positive control (glucose)”, and “D – Negative control (water)”.
Place 2 mL of the unknown sample into tube A.
Add 2 mL of 1 M HCl to tube A, mix gently, and heat in a boiling water bath for 5 minutes to hydrolyse any non‑reducing sugars.
Allow tube A to cool, then add 2 mL of 2 M NaOH to neutralise the acid.
To tube A add 5 mL of Benedict’s solution, mix, and heat in a boiling water bath for 2 minutes.
Prepare controls:
Tube C: 2 mL of 0.1 M glucose solution + 2 mL NaOH + 5 mL Benedict’s solution (positive control).
Tube D: 2 mL distilled water + 2 mL NaOH + 5 mL Benedict’s solution (negative control).
Heat both control tubes as in step 5.
Observe the colour and precipitate formed in each tube and record the results.
Observations and Interpretation
Tube
Colour after heating
Precipitate
Interpretation
A – Sample (hydrolysed)
Brick‑red / orange‑red
Cu₂O precipitate
Positive – non‑reducing sugar present (hydrolysed to reducing sugars)
C – Positive control
Brick‑red
Cu₂O precipitate
Test working correctly
D – Negative control
Blue
None
No reducing sugar; confirms absence of contamination
Key Points to Remember
Non‑reducing sugars do not give a direct Benedict’s test.
Acid hydrolysis breaks the glycosidic bond, producing reducing monosaccharides.
Neutralisation with NaOH is essential before adding Benedict’s reagent to avoid acid‑catalysed side reactions.
Colour intensity of the precipitate correlates with the amount of reducing sugar present.
Common Errors and Troubleshooting
Insufficient heating: Hydrolysis may be incomplete – extend boiling time to 7–10 min.
Excess acid not neutralised: Leads to a false‑negative Benedict’s result – always add NaOH in excess.
Old Benedict’s solution: May give weak colour change – prepare fresh solution.
Suggested diagram: Flowchart of the test – sample → acid hydrolysis → neutralisation → Benedict’s test → observation.