Biuret test: Principle, Requirements, Procedure and Result Interpretation

  • 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 inĀ  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.

Biuret test: Principle, Requirements, Procedure and Result Interpretation