Biuret test

Principle of Biuret test:

Biuret test is a general test for compounds having a peptide bond. Biuret is a compound formed by heating urea to 180° C. When biuret is treated with dilute copper sulfate in alkaline condition, a purple colored compound is formed. This is the basis of biuret test widely used for identification of proteins and amino acids.

This test is given by compounds containing two or more peptide bond (CO-NH group). Since all proteins and peptides possessing at least two peptide linkage ie. tripeptide gives positive biuret test.
The principle of biuret test is conveniently used to detect the presence of proteins in biological fluids.
Alkaline CuSO4 reacts with compounds containing two or more peptide bonds to give a violet colored product which is due to formation of co-ordination complex of cupric ions with un-shared electron pairs of peptide nitrogen and O2 of water.

Biuret reagent is prepared by adding NaOH in CuSO4 solution, making it alkaline.
To prepare 1000ml of Biuret reagent
- Take 1.5 gram of pentavalent copper sulphate (CuSO4) and 6 gram of Sodium potassium tartarate and dissolve them in 500 ml of distilled water
- **Sodium potassium tartarate is a chelating agent and it stabilize the copper ion
- Take 375 ml of 2 molar Sodium hydroxide
- Mix both the solution in volumetric flask and make it final volume to 1000 ml by adding distilled water.

Take 1 ml of test solutions in dry test tubes and in another tube take 1 ml distilled water as control.
Add 1 ml of biuret reagent to all test tubes, mix well.
Look for the development of blue colors

Biuret test positive:

Biuret test negative:

Presence of magnesium and ammonium ions interfere in biuret test. This can be overcome by using excess alkali.

Reference: