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.

Requirements for Biuret test:

• 1 % alanine, 5 % egg white (albumin)
• Biuret reagent
• Water bath
• Dry test tubes
• Pipettes

Biuret reagents:

• Copper sulfate (CuSO40
• Sodium hydroxide (NaOH)
• Sodium potassium tartarate (commonly known as Rochelle salt)

Preparation Biuret reagent

• 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.

Procedure for Biuret test

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

Result:

Biuret test positive:

• color changes to purple
• all peptides and protein give the test positive
• Histidine is the only amino acid that give biuret test positive.

Biuret test negative:

• No color change

Precautions:

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

Reference:

1. laboratoryinfo.com

Biuret test-Principle, Requirements, Reagents, Preparation, Procedure and Result

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