Investigation: Identifying Biochemical Compounds in Foods

The food detective

A food scientist at a company in Guelph is handed an unlabelled white powder from a production line. Is it powdered milk, flour, icing sugar or protein powder? A few drops of the right reagents, and a hot-water bath, can answer that in about ten minutes. The same tests, scaled up and automated, check that a "sugar-free" or "high-protein" label is honest.

In Grade 11 you learned which reagent tests for which nutrient. In this investigation you will plan and carry out the tests like a Grade 12 biochemist: understanding why each one works, using controls, and judging how much you can trust your results.

The chemistry behind each test

Benedict's test (reducing sugars). Benedict's solution contains blue Cu²⁺ ions in an alkaline solution. When heated with a sugar that has a free aldehyde group (or a ketone, like fructose, that can convert to an aldehyde in hot alkaline solution), the sugar reduces Cu²⁺ to Cu⁺, and the Cu⁺ forms copper(I) oxide, Cu₂O, a brick-red solid. The sugar is oxidized. This is a redox reaction. The more reducing sugar, the more precipitate: the colour goes green → yellow → orange → brick red, so the test is semi-quantitative.

Why is sucrose negative? In sucrose, the glycosidic bond joins carbon 1 of glucose to carbon 2 of fructose, which are exactly the carbons that would form the aldehyde or ketone. With both locked up, there is no free reducing group. If you first hydrolyze sucrose (boil with dilute hydrochloric acid, then neutralize the acid with sodium hydrogen carbonate, because Benedict's must be alkaline), you get glucose and fructose, and the test becomes positive. Notice that this one procedure uses hydrolysis, neutralization and redox.

Iodine test (starch). Iodine in potassium iodide solution forms chains of iodine and iodide that slip inside the amylose helix. That complex absorbs light differently and looks blue-black. Glucose, sucrose and cellulose give no colour change (the solution stays yellow-brown); glycogen, with its short branched chains, gives only a red-brown colour.

Biuret test (protein). In the alkaline Biuret reagent, Cu²⁺ ions bind to the nitrogen atoms of peptide bonds, forming a violet complex. At least two peptide bonds (a chain of three amino acids or more) are needed, so free amino acids give a negative result.

Sudan IV test (lipids). Sudan IV is a red dye that is non-polar: it dissolves in lipids, not water. Shake it with a food in water and any lipid floats to the top as a red-stained layer. The grease-spot test is a simple alternative: lipids leave a translucent spot on brown paper that does not dry away.

Planning the investigation

Question: Which biochemical compounds are present in a set of foods (for example, potato, milk, egg white, apple juice and table sugar), and how do they compare?

Equipment (A1.2): test tubes and rack, 10 mL graduated cylinder or dropping pipettes, hot-water bath (a beaker of water on a hot plate, about 80 to 100 °C), test-tube holder, mortar and pestle, distilled water, the four reagents, labels, and a timer.

Procedure outline:

1. Crush solid foods with a little distilled water to make a suspension. Use the same volume of each sample (for example, 2 mL) and the same volume of reagent in every tube.
2. For each test, include a negative control (2 mL distilled water) and a positive control (glucose solution for Benedict's, starch suspension for iodine, egg albumin for Biuret, vegetable oil for Sudan IV).
3. Benedict's: add 2 mL of reagent, heat in the water bath for the same time (for example, 3 minutes) for every tube, then compare colours against a white background.
4. Iodine: add 2 to 3 drops. Biuret: add 2 mL, mix, wait 2 minutes. Sudan IV: add 3 drops, shake, let stand.
5. Record colours immediately in a table.

Safety (A1.4): wear goggles throughout. Biuret reagent contains sodium hydroxide (corrosive; rinse skin with lots of water). Benedict's tubes are hot: use a holder, point the tube away from people, and never heat a stoppered tube. Iodine stains skin and clothes. Sudan IV is usually dissolved in ethanol, which is flammable: keep it away from flames. Check the WHMIS labels and Safety Data Sheets and follow your teacher's disposal instructions. These reagents are for a school lab only: never use them at home.

Evaluating your results (A1.8)

These tests are qualitative, so judging error matters:

• Colour judgement varies between people and lighting. Compare against your controls on a white background, and have a partner record independently.
• Unequal volumes or heating times change Benedict's colours. Keep them the same in every tube.
• Coloured foods (grape juice, beet) can hide a colour change. Dilute them or use a filtered extract.
• Detection limits: a negative result may mean "too little to detect", not "none". Skim milk still contains a trace of fat.
• Interference: a food rich in protein can give a slightly misleading Benedict's colour, and the Biuret test can be masked by a strongly coloured food.

To make the Benedict's test more quantitative, run a standard series (glucose solutions of known concentration, such as 0.1%, 0.5%, 1% and 2%) and match each food's colour to the nearest standard. A colorimeter, which measures how much light passes through a sample, removes the human judgement entirely.