2.1.4—Biuret test for proteins

The Biuret test is a qualitative test for proteins. In alkaline conditions, copper(II) ions react with peptide bonds and produce a lilac or purple colour when a suitable protein is present.

1. Add sodium hydroxide or potassium hydroxide solution to the sample to make it alkaline.
2. Add a few drops of blue copper(II) sulfate solution, or use Biuret reagent containing both components.
3. Mix and observe against a white background: a blue-to-lilac or purple change indicates protein; no change from blue is negative.

The alkaline solution provides the required conditions for copper(II) ions to interact with peptide bonds. The resulting copper–peptide complex changes the observed colour from blue towards lilac or purple, linking the result to the protein’s peptide-bond structure.

The test detects at least two peptide bonds, so free amino acids and dipeptides give a negative result even though they contain nitrogen. It is a qualitative test, not an exact protein assay; keep reagent amounts, sample volume and observation background consistent when comparing samples.