Lab Review part 3

Amino acid and Protein tests

  1. Biuret test:

This test is utilized to identify if peptide bonds present in proteins. It is specific for identification of peptide bonds in proteins and not amino acids. Due to intensity in colour change, one is able to quantitatively estimate proteins via the number of peptide bonds present in the solution.

Biuret_testThe reaction: Biuret is a compound that is formed when urea is heated. Biuret reacts with Cu2+ ions to form a violet/purple complex in which four biuret molecules complex with one cupric ion. Proteins mimic this formation and thereby yield this violet/purple complex when they react with Cu2+ ions. Biuret reagent is a solution composed of potassium hydroxide, hydrated copper (II) sulphate and potassium sodium tatrate that is utilized to stabilize the cupric ions in the solution. The dipeptide bonds in proteins react with the Cu2+ ions to form the complex. The lone pairs present on the Nitrogen of the peptide linkage bonds with the cupric ions in the reagent to create a violet purple colour change. The assessment of the concentration of the complex formed may be evaluated via spectrophotometry. This will determine the concentration of dipeptide bonds present in the solution and hence quantify the proteins present.

biuret polypeptideThe results: Biuret test will result in the formation of violet-purple complex/ colour change to indicate the presence of two or more dipeptide bonds present in the protein. The darker the colour, the more dipeptide bonds present and hence the larger the protein present.

biuret reactionThe Lab results: Albumin will react with the Biuret’s Reagent to form a purple colour change due to the formation of the complex of the dipeptide bonds with the cupric ions. The heated urea will give the same results since the heated urea forms the biuret compound that reacts with the cupric ions present in the reagent to form the coloured complex. When the urea is heated, it releases ammonia gas in the formation of the biuret and this is an irritant to the eyes and nose.


This test requires that more than large volumes of protein be available to bind in order to give a positive result. Therefore, more than two peptide bonds must be available to complex with the cupric ions.

  1. Ninhydrin test:

This test is utilized to identify amines in particular alpha amino acids present in the solution. It is commonly used to detect the lysine in fingerprints.

ninhydrin equationNinhydrin 1The reaction: The alpha amino group of the free amino acids react with ninhydrin to form a blue to purple colour change. Ninhydrin reagent is a powerful oxidizing agent that causes the oxidative deamination of the alpha amino acids to form aldehydes and reduced aldehyde forms of ninhydrin. In the process ammonia and carbon dioxide is released. The ammonia formed in this process then reacts with the ninhydrin as well as the reduced products to form a diketohydrin. This diketohydrin forms a blue-purple coloured substance. However, some amino acids such as the imminoacid proline will react with ninhydrin differently to form a bright- yellow colour change. Similar results may be observed with hydroxyproline.

ninhydrin on filter paperThe results: Alpha amino acids react with ninhydrin to form a blue to purple colour change. Some immino derivatives such as proline and hydroxyproline will react with ninhydrin to form a yellow colour change.






  • Chemistry 3251 Lab Manual. Department of Chemistry, Lakehead University. 1998. Experiment 5: Protein analysis.

  • Principles of Biochemistry, 2nd ed. Hortan, Moran, Ochs, Rawn, Scrimgeour. Prentice-Hall ©1996
  • Wikipedia. Biuret test. 2013

  • Demonstration Experiment on Video. Biuret Reaction- Protein in egg albumin by Peter Keusch.

  • Wikimedia. Biuret Test.

  • Department of Chemistry, University of Calgary. Chapter 27: Amino acids, peptides and proteins. Ninhydrin Test. By Dr Ian Hunt.

  • Wikipedia. Ninhydrin, 2012.

  • Dr Neil Glagovich. Department of Chemistry. Ninhydrin test: Amino acids.

  • Proteins, peptides and Amino Acids.

  • Morgellons Research: Proteinaceous Complex Identified. By Clifford Carnicom.

  • Essentials of Biotechnology by Ulhas Patil, Kalyani Muskan. Ninhydrin Test.


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