Biuret Test For Protein-Definition, Principle, Procedure, Results, Uses

Biuret Test For Protein Definition

What is Biuret Test?

Numerous amino acids combine to produce the complex molecules known as proteins. The carboxyl and amino groups of the amphoteric electrolytes known as amino acids serve as an acid and a base, respectively. Such ions remain electrically neutral and therefore do not travel in an electric field, since they possess one positive as well as one negative charge. Dipeptide is formed when two amino acids form a link known as a peptide bond. By removing a water molecule, a connection is formed between an amino group from one amino acid and a carboxyl group from another.

The peptide bond is formed as a result of the condensation reaction. Tripeptides consist of three amino acids connected by two peptide bonds; as the chain becomes longer, it is known as a polypeptide. When urea is heated to 1800 degrees, two urea molecules condense to create the chemical known as biuret. Biuret is called Biuret because the peptide bonds inside it provide a positive test result. For compounds (proteins and peptides) containing at least two peptide (CO-NH) bonds, it is regarded as a generic test.

Biuret Test For Protein – Objectives

  • To identify the protein present in the solution.
  • To establish that the peptide bond exists.

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

Utilizing a chemical analysis known as the Biuret test, peptide bonds may be discovered in a sample. It relies on the biuret reaction, wherein alkaline copper sulphate induces a peptide structure that has at least two peptide bonds to produce a violet colour. In the presence of an alkaline solution, the blue-coloured copper II ion may mix with the peptide bonds due to the unshared electron pairs in the nitrogen and oxygen of the peptide. The amide nitrogen (=NH), carbonyl oxygen (>C=O), and Cu2+ ion of the peptide bond combine to form the colourful coordination complex.

After this complex forms, the colour of the solution changes from blue to purple. The quantity of peptide-copper complexes grows as the purple tint becomes darker. The reaction occurs with any molecule containing at least two H2N-C, H2N-CH2, H2N-CS, or analogous groups linked directly or indirectly by a carbon or nitrogen atom. Most likely, six neighbouring peptide linkages are coupled to a single copper ion through coordinate bonds. The intensity of the colour is proportional to the number of peptide bonds in the responsive protein molecule and the number of protein molecules in the reaction system.

Biuret reagent is a solution of potassium sodium tartrate, sodium hydroxide (NaOH), or potassium hydroxide (KOH). To chelate the cupric ions in the solution and preserve their solubility in alkaline solutions, potassium sodium tartrate is added. The alkaline environment is supplied by sodium hydroxide and potassium hydroxide.

Biuret Test For Protein – Procedure

  1. Take three dry, clean test tubes.
  2. Egg albumin, deionized water, and 1-2 mL of the test solution should all be added to the appropriate test tubes.
  3. Each test tube should receive 1-2 mL of the Biuret reagent.
  4. Give the mixed ingredients a good shake and let them rest for five minutes.
  5. Watch for any variations in colour.

Biuret Test Results

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

Biuret Test Uses

  • It may be used to figure out how much protein is present in the urine.
  • Spectrophotometric analysis may be used to quantitatively determine total protein using the biuret reaction with protein.


  • Shrestha B (2002). Practical biochemistry and biotechnology. First edition. 99933-665-1-X.
  • Satyanarayana U and Chakrapani U (2006). Biochemistry. Uppala Author-Publisher Interlink. Third edition. Page no. 43-67.
  • https://study.com/academy/lesson/what-does-biuret-test-for-method-equation.html.
  • 2% – https://www.slideshare.net/jammalavamsikrishna/amino-acids-71579203
  • 2% – https://www.sciencedirect.com/topics/medicine-and-dentistry/biuret
  • 2% – https://www.sciencedirect.com/science/article/pii/0009898175903514
  • 2% – https://peptidesciences.com/information/peptide-bonds/
  • 10% – https://onlinesciencenotes.com/biuret-test-principle-requirements-procedure-and-result-interpretation/
  • 1% – https://quizlet.com/18722031/biology-chapter-5-flash-cards/
  • 1% – https://microbiologyinfo.com/benedicts-test-principle-composition-preparation-procedure-and-result-interpretation/
  • 1% – https://brainly.in/question/716138
  • 1% – http://amrita.olabs.edu.in/?sub=79&brch=17&sim=205&cnt=1
  • <1% – https://education.jlab.org/qa/charges_01.html
Spread the love

Leave a Comment

Your email address will not be published. Required fields are marked *