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To do the biuret test, add sodium hydroxide then dilute copper(II) sulphate solution; purple shows protein is present. Free AQA notes and 14 questions answered.
The Biuret Test for Proteins is part of Proteins in A Level AQA Biology.
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To do the biuret test, add sodium hydroxide then dilute copper(II) sulphate solution; purple shows protein is present. Free AQA notes and 14 questions answered.
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Use this lesson to stay inside Proteins while connecting the detail back to the wider Biological molecules topic.
Read the core explanations from The Biuret Test for Proteins before testing yourself from memory.
Proteins are everywhere in biology — from enzymes catalysing reactions to antibodies defending against pathogens. But how do you actually confirm a protein is present in a sample? That is exactly what the biuret test does, and it is a core practical skill you need to know precisely for AQA A Level.
The test is elegant in its logic: it does not detect amino acids directly. Instead, it detects the peptide bonds that link amino acids together. This distinction matters enormously, as you will see when we look at what the reagent is actually reacting with.
Picture a protein molecule — a long chain of amino acids connected by peptide bonds. Each peptide bond contains a nitrogen atom bonded to a carbon atom (the –CO–NH– linkage). The biuret reagent exploits this structure directly.
The reagent contains copper(II) sulphate dissolved in sodium hydroxide solution. The sodium hydroxide creates an alkaline environment, and under these conditions the copper(II) ions ($\text{Cu}^{2+}$) form a coordination complex with the nitrogen atoms of the peptide bonds. This complex absorbs light in the yellow-green region of the visible spectrum, which means the colour we perceive is its complement — purple or violet.
The key insight here is that you need at least two peptide bonds (i.e., a tripeptide or longer) for the complex to form properly and produce a reliable positive result. A single amino acid has no peptide bonds, and a dipeptide contains only one peptide bond, which is generally insufficient for a positive result—so dipeptides typically give a negative or very weak result. In practice, the test works reliably with tripeptides, polypeptides, and proteins, which contain multiple peptide bonds along their chain.
Now that you understand what the reagent is reacting with, the procedure itself makes complete sense. There are two ways the reagent is applied in practice: as a pre-mixed biuret reagent, or by adding sodium hydroxide and copper(II) sulphate separately. AQA mark schemes accept both approaches, but the two-step method is more common in school laboratories.
Worked example: Carrying Out the Biuret Test
Describe the procedure for testing an unknown solution for the presence of protein using the biuret test.
Answer: A purple/violet colour indicates a positive result (protein present). A blue colour indicates a negative result (no protein detected).
Notice that the negative control is blue rather than colourless — this is because the copper(II) sulphate solution itself is blue. Students sometimes misread a negative result as a positive one if they have not seen the reagent colour before running the test.
Running controls alongside your test sample is not optional — it is essential for valid interpretation. A positive control (a solution of known protein, such as albumin) confirms the reagent is working correctly. A negative control (distilled water) confirms the purple colour is not an artefact of the reagent itself.
| Sample | Colour Observed | Interpretation |
|---|---|---|
| Distilled water (negative control) | Blue | No protein — confirms reagent baseline |
| Known protein solution (positive control) | Purple/violet | Protein present — confirms reagent is functional |
| Unknown sample — positive result | Purple/violet | Protein detected in sample |
| Unknown sample — negative result | Blue | No protein detected in sample |
The biuret test is qualitative — it tells you whether protein is present, not how much. If you needed to compare protein concentrations between samples, you would need a quantitative method such as a colorimeter measuring absorbance at a set wavelength, or a Bradford assay. The biuret test alone cannot distinguish between a sample with a small amount of protein and one with a large amount.
The biuret test sits within the broader AQA required practical on food tests. In an exam, you may be asked to describe the procedure, explain the chemistry, or evaluate the method. Each of these command words demands a different type of response, and confusing them is one of the most common ways students lose marks.
Evaluation questions at A Level ask you to weigh up the strengths and limitations of a method and reach a reasoned conclusion. The biuret test has clear advantages, but also genuine limitations that examiners expect you to discuss with precision.
When an exam question asks you to evaluate, you must do more than list points — you need to reach a conclusion. For example: the biuret test is a reliable and accessible qualitative screen for protein, but it would need to be paired with a quantitative method (such as colorimetry using a standard curve) if concentration data were required.
The biuret test appears in exam questions with a range of command words, and each one demands a different response structure. Practising the distinction between describe, explain, and evaluate will protect marks that students routinely lose by answering the wrong type of question.
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Move to the related lessons or back to the topic page when you need the wider sequence before exam-style practice.
To do the biuret test, add an equal volume of sodium hydroxide solution to 2 cm³ of the sample, then about 5 drops of dilute copper(II) sulphate solution, and mix. A purple/violet colour shows protein is present; if the solution stays blue, no protein is detected.
The biuret test is a chemical test that detects peptide bonds, and hence proteins, rather than amino acids directly. Sodium hydroxide and a few drops of copper(II) sulphate are added to the sample; a purple or violet colour shows protein is present, while blue is negative.
Biuret test results are read as purple or violet for protein present and blue for no protein, with no intermediate colour to interpret. Distilled water should stay blue as a negative control, and a known protein solution such as albumin should turn purple as a positive control.
Protein concentration can be measured with a quantitative method such as a colorimeter measuring absorbance at a set wavelength, using a standard curve, or a Bradford assay. The biuret test alone is only a qualitative screen for whether protein is present.
The biuret test detects peptide bonds, not amino acids directly. In alkaline conditions, copper(II) ions form a coordination complex with the nitrogen atoms of the peptide bonds, giving a purple colour. At least two peptide bonds are needed, so single amino acids and dipeptides typically give a negative or very weak result.
A positive biuret test gives a purple/violet colour, showing protein is present. A negative result stays blue, because the copper(II) sulphate solution itself is blue. Running distilled water as a negative control and a known protein solution as a positive control makes the result easier to interpret.
Sodium hydroxide is added in the biuret test to create the alkaline conditions needed for the copper(II) ions to form a coordination complex with the peptide bonds. Without this step the reaction does not occur, so no purple colour can form even if protein is present.
The biuret test is qualitative only, so it cannot show the concentration of protein, and it cannot identify which specific protein is present. Heavily coloured samples can make the purple colour hard to see, and dipeptides may give a weak or unreliable result. Colorimetry using a standard curve is needed for concentration.
The test for protein is the biuret test, which uses copper(II) sulphate in sodium hydroxide solution to detect peptide bonds. A purple or violet colour indicates protein is present, and a blue colour indicates no protein is detected.
Biuret A and B refer to the two-step method, in which sodium hydroxide solution and copper(II) sulphate solution are added separately instead of as a pre-mixed biuret reagent. About 2 cm³ of sodium hydroxide is added first, then about 5 drops of dilute copper(II) sulphate.
Single amino acids give a negative biuret test because they have no peptide bonds. A dipeptide has only one peptide bond and usually gives a negative or very weak result; the test works reliably with tripeptides, polypeptides and proteins, which have at least two peptide bonds.
A negative biuret test is blue because the copper(II) sulphate solution itself is blue. Students sometimes misread a negative result as positive if they have not seen the reagent colour before, which is why a distilled water control is run alongside.
Controls are used in the biuret test for valid interpretation. A positive control, such as albumin solution, confirms the reagent is working, and a negative control of distilled water confirms the purple colour is not an artefact of the reagent itself.
The biuret test is qualitative: it shows whether protein is present, not how much. It cannot distinguish a sample with a small amount of protein from one with a large amount, and it cannot identify which specific protein is present.