Testing for biological molecules
Introduced| English | Meaning |
|---|---|
| carbohydrate/ˌkɑːbəʊˈhaɪdreɪt/ | An organic compound such as a sugar, starch or cellulose, made primarily from carbon, hydrogen and oxygen. |
| lipid/ˈlɪpɪd/ | A member of a group of largely water-insoluble biological molecules, including fats, oils, phospholipids and steroids. |
| reducing sugar/rɪˈdjuːsɪŋ ˈʃʊɡə/ | A sugar capable of reducing a suitable oxidising reagent, producing a positive Benedict's or similar test under appropriate conditions. |
| emulsion/ɪˈmʌlʃn/ | A mixture in which droplets of one liquid are dispersed in another liquid that does not mix with it. |
| semi-quantitative/ˈsemi ˈkwɒntɪteɪtɪv/ | Providing approximate amounts, ranges or comparisons rather than precise numerical measurements. |
| acid hydrolysis/ˈæsɪd haɪˈdrɒləsɪs/ | acid hydrolysis |
What does a food-test result establish?
- Two clear solutions can contain different biological molecules. Test separate portions of each sample: one colour change cannot identify every molecule present.
- Carbohydrates 碳水化合物, lipids 脂质 and proteins need different tests. Record the observation first, then state the conclusion it supports.
The four food tests
| Test | Method on a separate sample portion | Positive observation |
|---|---|---|
| Benedict's | Add Benedict's solution; heat in a hot water bath | A coloured precipitate: green, yellow, orange or brick-red; detects reducing sugar 还原糖 |
| Iodine | Add iodine solution; no heating is needed | Orange-brown becomes blue-black; detects starch |
| Emulsion 乳浊液 | Shake the sample with ethanol; add the ethanol extract to water | A white, cloudy emulsion; detects lipid |
| Biuret | Add biuret reagent, or add alkali then a few drops of copper sulfate solution | Blue becomes lilac/purple; detects protein |
A negative Benedict's test remains blue. A positive result need not reach brick-red.
A sample is heated with Benedict's solution and turns brick-red. This shows the presence of:
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Heating with Benedict’s detects reducing sugars. A green, yellow, orange or brick-red precipitate can be positive; brick-red is not required for every positive result.
Orange-brown iodine solution turns blue-black. This detects:
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The iodine test detects starch — the orange-brown solution turns blue-black.
In the biuret test, a positive result for protein turns the solution from blue to ______.
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Biuret solution turns blue → purple when protein is present (no heating needed).
A positive emulsion test for a lipid gives a clear blue solution.
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A positive emulsion test gives a white, cloudy emulsion — not a colour change.
Match each test to the molecule it detects.
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Benedict's → reducing sugar; iodine → starch; emulsion → lipid; biuret → protein.
Describe how to test separate portions of a food sample for lipid and protein. Give a positive observation for each, and state which test needs ethanol.
For lipid, shake the sample with ethanol and add the ethanol extract to water: a white cloudy emulsion is positive. For protein, add biuret reagent (or alkali followed by a few drops of copper sulfate): lilac/purple is positive. Ethanol belongs to the lipid test; neither of these tests requires heating.
Semi-quantitative 半定量 Benedict's
- A semi-quantitative test estimates concentration by comparison with known standards. Under the same conditions, more reducing sugar usually produces an earlier first colour change.
- Prepare known concentrations and a water blank. Keep sample and reagent volumes, water-bath temperature and the definition of the first colour change the same; test the unknown by the same method.
Testing for non-reducing sugars
- Test one portion with Benedict's first. If it stays blue, heat a fresh portion with dilute hydrochloric acid: acid hydrolysis 酸水解 can split a non-reducing sugar into reducing sugars. Cool, neutralise the acid, then add Benedict's solution and heat in a water bath.
- A positive test after a negative initial result supports a non-reducing sugar in a sample known to contain sugars. Hydrolysis can also release reducing sugars from other carbohydrates, so use the sample context and a starch test; the result alone does not prove the sugar is sucrose.
Use a fresh portion for hydrolysis; do not add acid to the tube already containing Benedict's reagent. Neutralisation restores conditions for the second Benedict's test.
Testing for a non-reducing sugar
Step through the trick: a non-reducing sugar stays blue, so you hydrolyse it with acid, neutralise, then re-test.
A solution known to contain a sugar gives a negative Benedict’s test. Which next procedure tests for a non-reducing sugar?
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Acid hydrolysis can produce reducing sugars. Use a fresh portion; cool and neutralise before the heated Benedict’s test. A positive result does not identify sucrose specifically.
Put the non-reducing sugar test in order.
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The first test and hydrolysis use separate portions. Hydrolysis releases reducing sugars; neutralise before the second Benedict’s test.
A student adds acid directly to a tube already used for a Benedict’s test, then heats it again without cooling or neutralising. Describe the corrected procedure and explain why a second positive result would not identify sucrose specifically.
Use a fresh portion for acid hydrolysis, heat with dilute hydrochloric acid, cool and neutralise before adding Benedict’s and heating in a water bath. The initial Benedict’s test is on a separate portion. The result shows reducing sugar after hydrolysis, not the unique identity of its precursor; different carbohydrates can yield reducing sugars. Use the sample context, and check for starch if relevant.
Make standards of known concentration
- Keep the final volume the same. The fraction of stock solution in the final mixture equals the required concentration divided by the stock concentration; add water to reach the final volume.
- Worked: to make $20\ \text{cm}^3$ of $0.40\%$ solution from $1.0\%$ stock, use $(0.40\div1.0)\times20=8.0\ \text{cm}^3$ stock and $12.0\ \text{cm}^3$ water. Mix and label it before testing.
Calculate the volume of 0.50% stock reducing-sugar solution and the volume of water needed to make 15.0 cm³ of a 0.20% standard. Show the dilution calculation.
The stock fraction is 0.20 ÷ 0.50 = 0.40. Use 0.40 × 15.0 = 6.0 cm³ of stock; add 15.0 − 6.0 = 9.0 cm³ water. Mixing gives the required 15.0 cm³ final volume.
Estimate without inventing precision
| Reducing sugar concentration / % | Time to first colour change / s |
|---|---|
| 0.10 | 96 |
| 0.20 | 64 |
| 0.40 | 32 |
| Unknown | 48 |
- In this example, 48 seconds lies between the 0.20% and 0.40% standards, so the unknown is estimated to lie between those concentrations. This table is teaching data, not laboratory results.
- Do not assume time is proportional to concentration or claim an exact midpoint. Test closer standards around the estimate and repeat measurements. For a final-colour method, heat every tube for the same time and compare with colour standards instead.
Teaching data: 0.05%, 0.10% and 0.20% standards first change colour at 110 s, 76 s and 44 s respectively. An unknown changes at 60 s using the same volumes and bath. Give its supported concentration range, two conditions that must be controlled, and two ways to improve the estimate.
The unknown lies between 0.10% and 0.20%: 60 s lies between 76 s and 44 s. Control sample volume, Benedict’s volume, bath temperature and the colour-change endpoint (any two). Test closer concentration standards within the estimated interval and repeat measurements to calculate means. These data do not justify an exact concentration from a presumed straight-line relationship.
Controls, repeatability and safe handling
- A known positive checks that the method and reagent work; a water blank checks for contamination or a reagent change without the target molecule. Use clean, labelled tubes and separate portions for different tests.
- Carry out these tests with teacher supervision and eye protection. Ethanol is flammable: keep it away from flames and do not heat the emulsion test. Handle hot tubes with a holder; cool hydrolysed samples before neutralising as directed by the teacher.
A water blank gives a white cloudy emulsion after ethanol and water are added. An unknown gives the same observation. Can the unknown result be accepted as evidence for lipid? Explain what should happen next.
The blank should remain clear. A positive-looking blank suggests contamination or a problem with the reagents or method, so the unknown result is unreliable. Use clean apparatus and fresh reagents, repeat the blank and include a known lipid positive control. Interpret the unknown only once the controls behave as expected.
Separate the observation from the conclusion
- “Iodine became blue-black” is an observation; “starch is present” is the supported conclusion. A negative result means the substance was not detected under the test conditions, rather than proof that not one molecule is present.
- A positive Benedict's result identifies reducing sugar, not glucose alone. If reducing sugar was already present before hydrolysis, a second positive result alone cannot establish that a non-reducing sugar was also present.
You've got it
- Use separate portions: heated Benedict's for reducing sugar, iodine for starch, ethanol then water for lipid, biuret for protein.
- Estimate concentration using standards tested under matching conditions; give a supported range and improve it with closer standards and repeats.
- For non-reducing sugar, compare an initial test with hydrolysis of a fresh portion, followed by cooling, neutralisation and a heated Benedict's test.
- State what was observed before interpreting it; keep the conclusion within what the test can establish.
A sample gives a blue-black colour with iodine and remains blue after a correctly heated Benedict’s test. A positive reducing-sugar control works. State two supported conclusions and explain why “the sample contains no carbohydrate” is wrong.
Different tests detect different classes of molecules. A negative reducing-sugar test does not overturn a positive starch test.
An unknown changes colour sooner than a 0.10% reducing-sugar standard but later than a 0.30% standard, under matching conditions. Which estimate is supported?
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A shorter time indicates more reducing sugar under the calibrated conditions. The observations place the unknown between the standards; they do not prove a linear relationship or an exact midpoint.
In a teacher-supervised laboratory, carry out the four food tests on separate sample portions, with a water blank and known positive controls. Then prepare reducing-sugar standards and estimate an unknown by time to first colour change or by final colour after a fixed heating time. Record actual methods, observations, controls and your supported estimate. If equipment is unavailable, write “practical pending”; do not invent results.
Results depend on the actual samples. Use labelled separate portions, the correct reagents and supervised safety precautions. A results table should identify the sample/test and record observations separately from conclusions. Record standard concentrations and times with units in headings, or the fixed heating time and colour matches used. Keep conditions the same, repeat readings where possible and state a supported range. Record a time beyond the chosen observation limit as “more than [limit]”, not an invented exact value. A teacher must check the handling and observations; this online reflection cannot certify practical competence.