Biochemical Tests

A-Level Biology requires you to know and apply a range of biochemical tests to identify the major classes of biological molecules: carbohydrates (reducing sugars, non-reducing sugars, starch), proteins, and lipids. You must also understand the difference between qualitative tests (which simply identify whether a substance is present) and quantitative tests (which measure the amount of substance present), as well as the use of colorimetry and calibration curves for quantitative analysis.

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Benedict's Test — Reducing Sugars

Principle

Reducing sugars (all monosaccharides and most disaccharides, except sucrose) have a free aldehyde or ketone group that can reduce Cu²⁺ ions in Benedict's reagent (an alkaline solution of copper(II) sulphate) to Cu⁺ ions, forming a coloured precipitate of copper(I) oxide (Cu₂O).

Method

1. Add approximately 2 cm³ of the sample solution to a test tube.
2. Add an equal volume of Benedict's reagent (a blue solution).
3. Heat the mixture in a water bath at approximately 80 °C for 5 minutes. (A water bath provides an even, controlled temperature; a Bunsen burner should not be used as the temperature would be uncontrolled.)
4. Observe the colour change.

Results

Colour	Interpretation
Blue (no change)	No reducing sugar present
Green	Trace amount of reducing sugar
Yellow	Small amount
Orange	Moderate amount
Brick-red precipitate	Large amount of reducing sugar

Exam Tip: The colour sequence is blue → green → yellow → orange → brick-red. The test is semi-quantitative in its basic form — the colour gives a rough indication of concentration. For a truly quantitative result, colorimetry is required (see below).

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Benedict's Test — Non-Reducing Sugars

Sucrose is the main non-reducing sugar you need to know. It does not react with Benedict's reagent because both anomeric carbons are involved in the glycosidic bond.

Method

1. First, test the sample with Benedict's reagent as above. If it remains blue, this suggests the sugar may be non-reducing.
2. Take a fresh sample. Add dilute hydrochloric acid (HCl) and heat in a water bath to hydrolyse any glycosidic bonds, releasing the component monosaccharides.
3. Neutralise the acid by adding sodium hydrogencarbonate (NaHCO₃) solution. (This is essential because Benedict's reagent is alkaline and will not work in acidic conditions.) Test with pH paper to confirm the solution is neutral or slightly alkaline.
4. Now repeat the Benedict's test (add Benedict's reagent and heat in a water bath).
5. If the solution changes colour (green → brick-red), a non-reducing sugar was present in the original sample.

Exam Tip: Always state that a control must be performed: test the original (unhydrolysed) sample with Benedict's reagent first to confirm it is negative. If the original sample gives a positive result, the sugar is a reducing sugar, not a non-reducing sugar. Also, you must state the neutralisation step — many students lose marks by omitting this.

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Iodine Test — Starch

Method

1. Add a few drops of iodine solution (iodine dissolved in potassium iodide, I₂/KI) to the sample.
2. Positive result: the solution turns blue-black (or dark blue). The iodine molecules become trapped inside the helical structure of amylose, producing the characteristic colour.
3. Negative result: the solution remains brown/yellow (the colour of iodine solution).

Exam Tip: The iodine test is specific for starch — it does not detect other polysaccharides such as glycogen (which gives a red-brown colour with iodine) or cellulose (no colour change). The test works because amylose forms a helix that traps iodine molecules.

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Biuret Test — Proteins

Principle

The Biuret test detects the presence of peptide bonds. Cu²⁺ ions form coordinate (dative) bonds with the nitrogen atoms in peptide bonds, producing a purple/violet colour.

Method

1. Add approximately 2 cm³ of the sample to a test tube.
2. Add an equal volume of sodium hydroxide (NaOH) solution to make the solution alkaline.
3. Add a few drops of dilute copper(II) sulphate (CuSO₄) solution. Mix gently by swirling (do not shake vigorously).
4. Positive result: the solution turns from blue to purple/violet.
5. Negative result: the solution remains blue.