Biuret test

Objectives:
To determine the presence of proteins and peptide bonds in the solution.
Materials and Methods:
2mL each of Albumin, Casein and Gelatin were placed in clean labeled test tubes. An equal volume of 10%
NaOH solution was then added to each test tube and the solutions were thoroughly mixed. To the resulting
mixture, 0.5% CuSO
4
was added drop wise until a pinkish or violet color was visible.
Results:
Solution Results
Albumin Magenta-colored solution
Casein Eggplant purple solution
Gelatin Royal Violet solution



Discussion:
The Biuret test is dependent on the ability of Cu (II) to form violet-colored chelate complexes with peptide bonds. It could be
considered as a qualitative and a quantitative test since the amount violet-colored chelate can be measured using a
spectrophotometer. Measurement and comparison is made possible by the fact that the intensity of the color produced is
dependent on the number of peptide bonds reacting, the more protein in the solution the greater the intensity of its color. This is in
accordance with the Beer-Lambert law which states that absorbance of the sample is directly proportional to the concentration of
the species.
The Biuret reagent is a mixture of: Hydrated Copper sulphate which provides the Cu (II) ions which form the chelate complex.
The Cu (II) ions also gives the reagent its characteristic blue color, Potassium hydroxide solution which does not participate in the
reaction but provides the alkaline medium needed and Potassium sodium tartrate which works to stabilize the chelate complex.
Note: This test yields negative for single amino acids and dipeptides because the formation of a chelate complex requires at least
two peptide bonds.





Conclusion:
The Biuret test is a test used for the detection of proteins through reactions with peptide bonds. In the
experiment all three solutions were positive for peptides but to varying degrees.
Reference:
Biuret Test for Protein. (2013). Retrieved August 21, 2014, from http://brilliantbiologystudent.weebly.com/biuret-test-for-
protein.html