The Biuret Test for Proteins

The biuret test is the standard school test for protein. Add a few drops of blue reagent to a sample, and if protein is present, the solution turns lilac or purple within a few minutes. It’s simple, safe and needs no heating, and with a colorimeter it can even estimate how much protein a sample contains.

Purpose

• To detect protein in foods and solutions.
• To compare the protein content of different samples.
• (Extension) to estimate protein concentration using a colorimeter and a calibration curve.

Principle

The test detects peptide bonds, the –CO–NH– links that join amino acids in proteins (see the peptide bond).

Biuret reagent contains copper(II) ions in an alkaline solution (usually sodium or potassium hydroxide), often with potassium sodium tartrate to keep the copper dissolved. In alkaline conditions, the nitrogen atoms of neighbouring peptide bonds can donate electrons to Cu²⁺. Each copper ion binds to four nitrogen atoms from peptide bonds, forming a coordination complex that absorbs light differently from free Cu²⁺:

• free copper(II) in the reagent is blue;
• the copper–peptide complex is purple (violet), with an absorption peak near 540 nm.

A molecule needs at least two peptide bonds to form the complex. So:

• proteins and polypeptides give a positive result;
• tripeptides and longer give a positive result;
• single amino acids and dipeptides do not.

The test is named after biuret, a small molecule formed by heating urea, which also contains two –CO–NH– groups and gives the same colour. Biuret itself isn’t part of the reagent.

Because the colour depends on the number of peptide bonds, and every protein has many, the intensity of purple is roughly proportional to protein concentration, whatever the protein.

Equipment and materials

• Biuret reagent (or separate solutions: dilute sodium hydroxide, about 1 mol dm⁻³, and dilute copper(II) sulfate, about 0.1 mol dm⁻³)
• Test tubes and rack
• Pipettes or measuring cylinders
• Samples: egg-white solution (albumen), milk, gelatine solution, glucose solution, vegetable oil, distilled water (control), crushed foods such as bread, lentils, nuts or meat mixed with water
• Mortar and pestle, filter funnel and paper for solid foods
• (Extension) colorimeter and cuvettes, standard protein solution (e.g. bovine serum albumin, BSA, at known concentrations)
• Eye protection

Safety

• Wear eye protection. Sodium hydroxide is corrosive, even at around 1 mol dm⁻³, and can cause serious eye damage. Copper(II) sulfate is harmful if swallowed and an irritant.
• Wash any splashes off skin immediately with plenty of water.
• Don’t taste any food samples in the lab.
• Dispose of copper solutions according to school procedures (see waste disposal in the lab).
• Check allergen information if using nuts or other foods that may cause allergic reactions.

Method

Part A: testing foods

1. Prepare solid foods by crushing a small amount with a little distilled water, then filtering or letting the solids settle. Use the liquid.
2. Label a test tube for each sample, including a negative control (distilled water) and a positive control (egg-white solution).
3. Add 2 cm³ of each sample to its test tube.
4. Add 2 cm³ of biuret reagent to each tube and shake gently to mix.
   • If using separate solutions: add 2 cm³ of sodium hydroxide solution, then a few drops of copper(II) sulfate solution, mixing after each drop.
5. Leave for 5 minutes at room temperature. (No heating is needed.)
6. Record the colour against a white background.

Part B (extension): estimating protein concentration

7. Make a series of standard protein solutions, for example 0, 1, 2, 4, 6 and 8 mg cm⁻³ of BSA, by diluting a stock solution.
8. Mix equal volumes of each standard and biuret reagent, wait 20 to 30 minutes for the colour to develop fully, and measure the absorbance at about 540 nm (a green filter) in a colorimeter.
9. Plot absorbance against concentration to make a calibration curve.
10. Treat the unknown samples in exactly the same way, measure their absorbance and read their concentration from the graph.

The method is explained in more detail in colorimetry and calibration curves.

Results

Sample	Colour after 5 minutes	Protein present?
Distilled water (control)	Blue	No
Egg-white solution	Purple	Yes (high)
Milk	Lilac to purple	Yes
Gelatine solution	Purple	Yes (gelatine is almost pure protein)
Glucose solution	Blue	No
Vegetable oil	Blue (oil may separate)	No
Lentils (extract)	Lilac	Yes

A pale lilac indicates a small amount of protein; a deep purple indicates a lot.

Limitations

• Semi-quantitative by eye: colour judgement is subjective. A colorimeter is much better.
• Low sensitivity: the biuret test needs roughly 1 to 10 mg of protein per cm³ to give a clear colour, which makes it much less sensitive than laboratory methods such as the Bradford or BCA assays.
• Doesn’t identify the protein: it confirms the presence of peptide bonds, not which protein is present, and it can’t tell native from denatured protein (see protein denaturation).
• Insoluble proteins in solid foods may not dissolve well enough to react; grinding and mixing thoroughly helps.

Sources of error

• Unequal volumes of sample or reagent between tubes.
• Not waiting long enough for the colour to develop, especially in quantitative work.
• Coloured samples: juices or other coloured foods can mask the result. Use a blank of the sample plus alkali without copper to compare.
• Excess copper sulfate (when using separate solutions) makes everything look blue, hiding a weak purple.
• Ammonium salts and some other substances can interfere with the colour.
• Cloudy samples (fats, undissolved solids) scatter light and affect colorimeter readings. Filter or centrifuge first.

Putting food tests together

The biuret test is usually carried out alongside the other food tests:

Test	Detects	Positive result
Benedict’s	Reducing sugars	Brick-red precipitate on heating
Iodine	Starch	Blue-black
Emulsion (ethanol)	Lipids	Cloudy white emulsion
Biuret	Proteins	Purple

Running all four on the same food builds a simple nutritional profile. Milk, for example, is positive for protein, reducing sugar (lactose) and lipid, but negative for starch.

How professional labs measure protein

The biuret principle is still used in hospital analysers to measure total protein in blood serum, typically around 60 to 80 grams per litre in healthy adults, because it responds to all proteins roughly equally. For smaller amounts, researchers use more sensitive methods. The BCA assay builds on the biuret reaction: copper(II) reduced by the protein is detected with a second reagent that gives an intense purple. The Bradford assay uses a blue dye that shifts colour when it binds to proteins. The Kjeldahl method, used for foods, measures total nitrogen, which is then converted to protein using a factor, because proteins contain about 16% nitrogen by mass.

Key takeaways

• The biuret test detects peptide bonds, so it identifies proteins and polypeptides.
• Copper(II) ions in alkaline solution form a purple complex with the nitrogen atoms of peptide bonds; no heating is needed.
• At least two peptide bonds are needed, so single amino acids and dipeptides test negative.
• With standards and a colorimeter at about 540 nm, the test can estimate protein concentration.
• Sodium hydroxide is corrosive, so eye protection is essential. For more on proteins, read proteins: chains of amino acids.