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Sodium hydroxide: 10% solution in water
Copper sulphate: 0.5% solution in water
Compounds containing two or more peptide bonds give characteristics purple color when treated with dilute copper sulphate in an alkaline solution. The name of the test comes from the compounds Biuret, which is the simplest compound that gives a typical positive reaction by combining with CuSO4 in the presence of NaOH and is converted to Cu(OH)2
2NaOH + CuSO4 → Cu(OH)2 + Na2SO4
Cu(OH)2 + Compound with peptide bond → violet colored complex
Take two test tubes and label these as “T” (for test) and “B” (for Blank).
Take 2 ml of the protein solution in “T’ test tube and 2 ml of the distilled water in “B” test tube.
Add 2 ml of sodium hydroxide in both test tube.
Mix and add copper sulphate solution drop by drop, mix after each addition by inversion. Usually
4-6 drops of copper sulphate solution are sufficient to obtain good results.
A purplish- violet or pinkish-violet color indicates the presence of protein in “T” test tube. If no such color appears, add CuSO4 drop by drop until the test become positive or the color becomes blue, which indicates a negative test.
Peptone gives pink color and may require the addition of larger amount of copper sulphate solution than normal.
Gelatin may give a very faint color and therefore, control is required to avoid confusion.
Excess of copper sulphate should be avoided; otherwise, the blue color of cupric hydroxide will mask the violet color.
Magnesium sulphate and ammonium sulphate interfere with this test. Therefore, the test should not be carried out with solutions containing these salts.
The test may also be given by substances containing a peptide bond, whether it is in protein and non-protein compound, such as biuret and oxamide.