Biuret Test: Principle, Reagent, Preparation & Result Interpretation

Principle Or Basis of The Biuret Test

The biuret test also referred to as Piotrowski’s test, is a chemical test used for detecting the presence of peptide bonds. Biuret test 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 sulphate. In the presence of an alkaline solution blue-colored copper II ions can form a complex with the peptide bonds (CO-NH group) since the peptide has unshared electron pairs in nitrogen and oxygen of water.

Four nitrogen atoms donate lone pairs to four covalent bonds with cupric ion resulting in the formation of a chelate complex. The reaction of the cupric ions with the nitrogen atoms involved in peptide bonds leads to the displacement of the peptide hydrogen atoms under the alkaline conditions. Once this complex has been formed, the solution turns from blue to purple. The deeper the purple color, the more the number of peptide-copper complexes that have been formed.

Since all proteins and peptides possessing at least two peptide linkage i.e tripeptide gives positive biuret test, the principle of biuret test is conveniently used to detect the presence of proteins in biological fluids.


Biuret reagent is an aqueous solution of potassium sodium tartrate treated with cupric sulfate and sodium hydroxide. In the presence of peptide bonds (protein), this blue solution will change color to pink-purple.

  • Rochelle salt/Potassium Sodium Tartrate (KNaC4H4O6.4H2O)- It acts as a chelating agent and stabilizes the copper II ions.
  • Potassium Hydroxide Solution (KOH) does not get involved in the reaction but provides the alkaline medium.
  • Hydrated Copper Sulphate. This provides the Copper III ions which form the chelate complex. Copper II ions give the reagent its characteristic blue color.

Preparation Of 1000ml Of Biuret Reagent

Biuret reagent is prepared by adding NaoH in Copper II Sulphate solution, making it alkaline.

Reagent Preparation Procedure

  • 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 acts as a chelating agent and stabilizes the copper II ions.
  • Take 400 ml of 2 molar sodium hydroxide
  • Mix both solutions in volumetric flask and make it final volume to 1000 ml by adding distilled water.

Biuret Test Procedure

  • Take 3 dry clean test tubes
  • Add between 1-2ml of the test solution, egg albumin and deionized water in the respective test tubes.
  • Add between 1-2 ml of Biuret reagent to all the test tubes.
  • Shake well and allow the mixtures to stand for at least 5 minutes.
  • Observe for any color change.

Result Interpretation

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)

Uses Of Biuret Test

  • It can be used to detect the amount of protein in the urine.
  • It is used in the quantitative determination of the total protein by spectrophotometric analysis.

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