Testing for biological molecules

Reagent. Benedict's solution — copper(II) sulfate in alkaline sodium citrate / carbonate.

Procedure. Add an equal volume of Benedict's reagent to ~2 cm³ of sample in a boiling tube. Heat in a water bath at approximately 80 °C for about three minutes. A water bath is used (not a Bunsen flame) for safety and reproducibility.

Positive result. Blue → green → yellow → orange → brick-red precipitate of copper(I) oxide, Cu₂O.

Chemistry. Reducing sugars donate electrons that reduce soluble blue Cu²⁺ to insoluble brick-red Cu⁺ (as Cu₂O). The free aldehyde group (or hemiacetal) on the open-chain form does the reducing.

Reducing sugars include: all monosaccharides (glucose, fructose, galactose) and most disaccharides — maltose and lactose. Sucrose is the major exception.

Semi-quantitative use. The deeper the brick-red colour (and the more precipitate), the higher the concentration of reducing sugar. Compare against known standards or use a colorimeter for a quantitative measurement.

Sucrose has its reducing aldehyde group locked inside its α-1,β-2 glycosidic bond, so it cannot reduce Cu²⁺ directly.

Procedure.

1. Take a fresh portion of sample.
2. Add dilute hydrochloric acid (or dilute sulfuric acid).
3. Heat in a water bath for ~5 minutes to hydrolyse any non-reducing sugar.
4. Cool. Neutralise with sodium hydrogencarbonate (or sodium hydroxide).
5. Repeat the Benedict's test.

Interpretation.

• Negative original + positive after hydrolysis = non-reducing sugar present.
• Negative both times = no sugar.

Why neutralise? Benedict's reagent precipitates in acid and the test will not work reliably. The mark scheme credits the neutralisation step explicitly.

Reagent. Iodine in potassium iodide solution (also written I₂/KI). The active species is the triiodide ion, $\text{I}_3^-$.

Procedure. Add a few drops of iodine solution to the sample on a white tile or in a test tube. No heating required.

Positive result. Orange-brown / yellow-brown solution → blue-black.

Mechanism. Amylose (one of the two starch polymers) coils into a helix with about six glucose residues per turn. Triiodide ions slot into the central channel of the helix and form a charge-transfer complex that absorbs strongly in the visible spectrum — appearing blue-black.

Selectivity. Cellulose has straight β-1,4 chains with no helix, so the test is negative. Glycogen gives a red-brown colour because its highly branched chains form only short helical segments.

Reagent. Dilute sodium hydroxide + dilute copper(II) sulfate (sometimes pre-mixed as ‘biuret reagent’).

Procedure. Add an equal volume of NaOH to the sample. Add a few drops of dilute CuSO₄. Mix gently. No heating required.

Positive result. Blue → violet / mauve / purple / lilac.

Mechanism. Cu²⁺ ions form a coordination complex with the nitrogen lone pairs of —CO—NH— peptide bonds. At least two peptide bonds are required, so dipeptides are usually negative but tripeptides upward give a positive result. Free amino acids give no colour.

Sensitivity. Useful semi-quantitatively — deeper purple = more protein. Often paired with a colorimeter using a green filter for accurate protein assays.

Procedure.

1. Add a few cm³ of ethanol to the sample and shake to dissolve any lipid.
2. Add an equal volume of distilled water. Shake gently.

Positive result. A cloudy milky-white emulsion forms.

Why it works. Lipids are non-polar and dissolve in ethanol but not in water. When water is added, the ethanol-water mixture cannot hold the lipid in solution. Tiny droplets of lipid come out of solution and scatter light, producing the milky appearance.

Limitation. The test does not distinguish triglycerides from phospholipids, and gives no quantitative information.

A colorimeter measures the absorbance (or percentage transmission) of a chosen wavelength of light by a coloured solution. It converts a colour change into a number, so two operators can produce the same reading.

Reducing-sugar protocol.

1. Prepare a serial dilution of glucose standards (e.g. 0.0, 0.2, 0.4, 0.6, 0.8, 1.0 g dm⁻³).
2. React each standard and the unknown with excess Benedict's reagent under identical conditions of volume, temperature and time.
3. Centrifuge each tube to remove the brick-red Cu₂O precipitate. Keep the supernatant — it contains the remaining blue Cu²⁺.
4. Choose the red filter (complementary to blue). Zero the colorimeter with distilled water.
5. Measure the absorbance of each standard.
6. Plot a calibration curve of absorbance (y-axis) against standard concentration (x-axis). Draw a smooth line of best fit.
7. Measure the absorbance of the unknown and read its concentration off the curve.

Why the trend? More reducing sugar → more Cu²⁺ used up → less Cu²⁺ remains → less blue colour → LOWER absorbance at 600 nm. The calibration curve therefore slopes downward.

Controls. Identical volumes and timings; tubes heated in the same water bath; same filter throughout; zero the colorimeter between samples if drift suspected.