- Biuret is a compound formed by heating urea at 1800
- It is the result of the condensation of 2 molecules of urea. The peptide bonds in Biuret give a positive result for the test hence the reagent is named so.
- Biuret test is a general test for compounds (proteins and peptides) having two or more peptide (CO-NH) bonds.
- Histidine is the only amino acid that gives Biuret test positive.
Principle:
- When biuret is treated with dilute copper sulfate in alkaline medium, a purple colored compound is formed. It is believed that the color is due to the formation of a copper co-ordinated complex or chelate complex.
- Cupric ions or Cu (II) ions form a violet-colored chelate complex with unshared electron pairs of peptide nitrogen and oxygen of water.
- The chelate complex absorbs light at 540 nm and hence appears violet. The color change from blue to violet indicates the presence of proteins.
- The greater the number of peptide bonds in a protein, the greater the color intensity. Hence, the color change is from blue to pink if the concentration of peptide bonds is low like in short-chain peptides.
- The principle of biuret test is conveniently used to detect the presence of proteins in biological fluids.
Requirements:
- Biuret reagent i.e. mixture of Hydrated Copper sulphate, Potassium hydroxide solution and Potassium sodium tartrate
- Test tubes (clean and dry)
- De-ionized water (negative control)
- Albumin or egg white (positive control)
Procedure:
- Take 3 clean and dry test tubes.
- Add 1-2 ml of the test solution, egg albumin and deionized water in the respective test tubes.
- Add 1-2 ml of Biuret reagent to all the test tubes.
- Shake well and allow the mixtures to stand for 5 minutes.
- Observe for any color change.
Result interpretation:
| S.N. | Observation | Interpretation |
| 1. | No color change (solution remains blue ) | Proteins are not present |
| 2. | The solution turns from blue to violet (deep purple) | Proteins are present |
| 3. | The solution turns from blue to pink | Peptides are present (Peptides or peptones are short chains of amino acid residues which contain less number of peptide bonds) |
*Presence of magnesium and ammonium ions interfere in biuret test. This can be overcome by using excess alkali.