Biochemical Tests for Biological Molecules

Biochemists and biologists use a standard set of qualitative and quantitative tests to identify biological molecules in samples. These tests form a core part of OCR A-Level Biology Practical Activity Group (PAG) requirements and are examinable both in theory papers and in written assessments of practical skills. This lesson covers OCR specification point 2.1.2 (f): the principles and procedures of tests for reducing sugars, non-reducing sugars, starch, lipids and proteins, including quantitative analysis.

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1. Overview of Biochemical Tests

Molecule	Test	Positive Result
Reducing sugars	Benedict's test	Blue → green → yellow → orange → brick-red precipitate
Non-reducing sugars	Benedict's after acid hydrolysis and neutralisation	Brick-red precipitate (after hydrolysis)
Starch	Iodine in KI solution	Yellow-brown → blue-black
Lipids	Emulsion test	Cloudy white emulsion
Proteins	Biuret test	Blue → lilac/purple

All these tests are qualitative — they tell you whether a substance is present, not precisely how much. Quantitative adaptations exist using colorimetry or reagent strips.

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2. Benedict's Test for Reducing Sugars

Principle

Reducing sugars (e.g., glucose, fructose, maltose, lactose, galactose) have a free aldehyde or ketone group that can reduce Cu²⁺ ions in Benedict's solution to Cu⁺, forming an insoluble brick-red precipitate of copper(I) oxide (Cu₂O).

Benedict's reagent (blue, contains Cu²⁺)
          +
Reducing sugar (RCHO)
          ↓  heat
Cu₂O (brick-red precipitate) + oxidised sugar

Procedure

1. Place approximately 2 cm³ of the sample solution into a test tube.
2. Add an equal volume of Benedict's solution (blue).
3. Heat in a water bath at 80–90 °C for 3–5 minutes.
4. Observe any colour change.

Interpretation

The intensity of colour and the amount of precipitate depend on the concentration of reducing sugar:

Colour	Approximate reducing sugar concentration
Blue	None
Green	Very low (trace)
Yellow	Low
Orange	Medium
Brick-red	High

Exam Tip: The test is semi-quantitative — the colour intensity reflects the amount of reducing sugar, but the test is not very precise. For accurate quantitative results, use colorimetry.

Quantitative Benedict's Test Using a Colorimeter

For reliable quantitative results:

1. Prepare a series of standard solutions of glucose at known concentrations (e.g., 0, 2, 4, 6, 8, 10 mg/cm³).
2. Perform Benedict's test on each standard, ensuring identical volumes, timings and temperatures.
3. Filter the Cu₂O precipitate and measure the absorbance of the remaining solution using a colorimeter with a red filter (complementary to blue).
4. As more Cu²⁺ is reduced, the blue colour fades — absorbance of red light decreases as [glucose] increases.
5. Plot a calibration curve of absorbance against glucose concentration.
6. Perform the test on the unknown sample under identical conditions and read its concentration from the calibration curve.

An alternative quantitative method centrifuges or filters the Cu₂O precipitate, dries it, and weighs it — the mass of precipitate is proportional to the reducing sugar concentration.

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3. Testing for Non-Reducing Sugars

Sucrose is the classic non-reducing sugar (both anomeric carbons are locked in the glycosidic bond). It gives a negative Benedict's test directly. To detect non-reducing sugars:

Procedure

1. Perform the Benedict's test on the sample — if it is negative, proceed.
2. Take a fresh sample and add dilute hydrochloric acid (e.g., 2 cm³ of 1 mol dm⁻³ HCl).
3. Heat in a water bath at 80 °C for 5 minutes — this hydrolyses glycosidic bonds into the component monosaccharides.
4. Neutralise with sodium hydrogencarbonate (NaHCO₃) until fizzing stops and the solution is no longer acidic (test with litmus or universal indicator). This step is critical — Benedict's reagent only works in alkaline conditions.
5. Repeat Benedict's test on the neutralised sample.
6. A positive result (brick-red) now indicates the original sample contained a non-reducing sugar.

Exam Tip: Common mistakes in this test include forgetting to neutralise after hydrolysis (Benedict's reagent is destroyed by acid), and not running a control showing the sample gives a negative direct Benedict's test first.

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4. Iodine Test for Starch

Principle

Iodine (in potassium iodide solution, forming the triiodide ion I₃⁻) binds to the helical structure of amylose in starch. The iodine molecules fit inside the helix and form a blue-black charge-transfer complex.

Procedure

1. Place a drop of the sample on a white tile or in a test tube/well of a spotting tile.
2. Add 1–2 drops of iodine in potassium iodide solution (often called "iodine solution" — yellow-brown).
3. Observe the colour.

Results

Colour	Interpretation
Yellow-brown (no change)	No starch
Blue-black	Starch present
Red-brown	Glycogen present (shorter helical segments)
Purple	Amylopectin (branched, partial helices)

The test is not quantitative in its standard form. The intensity of the blue-black colour can be measured with a colorimeter for semi-quantitative results.

Applications

• Testing leaves for starch after photosynthesis (leaf first decolourised in boiling ethanol to remove chlorophyll).
• Monitoring starch digestion in enzyme investigations (e.g., amylase activity — starch gradually disappears as the blue-black colour fades to yellow-brown).

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5. Biuret Test for Proteins

Principle

In the Biuret test, Cu²⁺ ions (in alkaline solution) form a coloured coordination complex with peptide bonds. The complex has a characteristic lilac/purple colour. Because the test detects peptide bonds, it is positive for all peptides of three or more amino acids.

Procedure