Biuret Test for Protein

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Last Updated on May 5, 2020 by Sagar Aryal

What does Biuret test for?

Proteins are the complex compound formed by thousands of amino acids. Amino acids are amphoteric electrolytes having carboxyl and amino groups that act like acid and base. It has one positive charge and one negative charge, hence these ions are electrically neutral and do not migrate in the electric field. The two amino acids are linked together with the help of a bond called peptide bond and it yields dipeptide. The bond is formed between the α amino group of one amino acid and the α carboxyl group of another amino acid with the removal of a water molecule. The formation of the peptide bond is referred to as the condensation reaction. Similarly, three amino acids linked together by two peptide bonds are called tripeptide, and as the chain elongates, it is called a polypeptide. Biuret is a compound formed by heating urea at 1800 which results in 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. It is considered as a general test for compounds (proteins and peptides) having two or more peptide (CO-NH) bonds.


  • To detect the protein in the given solution.
  • To demonstrate the presence of the peptide bond.

Biuret Test Principle (How does the biuret test work?)

A Biuret test is a chemical test used to determine the presence of a peptide bond in a substance. It is based on the biuret reaction in which a peptide structure containing at least two peptide links produces a violet color when treated with alkaline copper sulfate. In presence of an alkaline solution, blue-colored copper II ion can form a complex with the peptide bonds since the peptide has unshared electron pairs in nitrogen and oxygen of water. The colored coordination complex is formed between Cu2+ ion and carbonyl oxygen (>C=O) and amide nitrogen (=NH) of the peptide bond. Once this complex has been formed, the solution turns from blue to purple. The deeper the purple color, the higher is the number of peptide-copper complexes. The reaction occurs in any compound containing at least two H2N-C, H2N-CH2-, H2N-CS- or similar groups joined together directly or through a carbon or nitrogen atom. One copper ion is probably linked to 6 nearby peptide linkages by co-ordinate bonds. The intensity of the color is directly proportional to the number of the peptide bonds present in the protein molecule that is reacting and also the number of the protein molecules present in the reaction system.

The Biuret reagent is a solution composed of sodium hydroxide (NaOH) or potassium hydroxide (KOH), hydrated copper (II) sulfate, and potassium sodium tartrate. Sodium hydroxide and Potassium hydroxide provide the alkaline medium and potassium sodium tartrate is added to chelate and thus stabilize the cupric ions in the solution or to maintain their solubility in alkaline solution.

Biuret Test 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.

Biuret Test Results

Biuret Test for Protein



No color change, i.e., the solution remains blueProteins are absent (negative biuret test)
The solution turns from blue to deep purpleProteins are present (positive biuret test)

Biuret Test Uses

  1. It can be used to detect the amount of protein in the urine.
  2. Biuret reaction with protein is applicable to the quantitative determination of total protein by spectrophotometric analysis.


  1. Shrestha B (2002). Practical biochemistry and biotechnology. First edition. 99933-665-1-X.
  2. Chemistry Learner (
  3. Satyanarayana U and Chakrapani U (2006). Biochemistry. Uppala Author-Publisher Interlink. Third edition. Page no. 43-67.

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