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Explain the Principle or Theory of biuret method?
If a strongly alkaline solution of Biuret is heated with very dilute copper sulphate, a violet colour is obtained. The substances which contain two carbamyl (-CONH2) groups joined together either directly or through a single carbon or nitrogen atom and those which contain two or more peptide links give a blue or purple coloured complex with alkaline copper solution.
Proteins respond positively since there are pairs of -CONH2 groups in the molecule. The reaction is due to coordination of cupric anion with the unshared electron pair of peptide nitrogen and the oxygen of the water. A coordination complex is formed which is purplish coloured and can be colorimetrically measured. When protein solution is made strongly alkaline with NaOH or KOH and very dilute CuSO4 is added, a purplish to pink violet colour is obtained, the intensity of the colour depending upon the concentration of protein or the number of peptide bonds undergoing the reaction.
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