What is the importance of alkaline condition in biuret test? Biuret test aims to quantify the amount of protein in a given unknown sample. Biuret agent contains copper sulphate, sodium potassium tartrate and Sodium hydroxide. Coppper ions form the complex of purple colour. Sodium hydroxide is added to make the solution alkaline. Why is it needed to make the solution alkaline?

In the Biuret reaction, copper forms a complex with the nitrogen of the peptide bond, that looks like this (Structure 61 from the paper below):

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A strong base is necessary to provide the conditions for deprotonating these nitrogen atoms and have the conditions to form the complex. If you want to read this in detail, have a look at this publication:

* Coordinating properties of the amide bond. Stability and structure of metal ion complexes of peptides and related ligands