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Biuret Test: Identification of Proteins

By Chemca Editorial Team • Last Updated: January 2026 • 7 min read

The Biuret Test is a general chemical test used to detect the presence of peptide bonds. It is commonly used to identify proteins and peptides in a solution. The reaction produces a characteristic violet or purple color.

1. The Reagents

The test uses Biuret Reagent, which consists of:

• Copper(II) Sulfate ($CuSO_4$): Provides the $Cu^{2+}$ ions (Blue).
• Sodium Hydroxide ($NaOH$): Provides the alkaline medium essential for the reaction.
• Sodium Potassium Tartrate (Rochelle Salt): (Optional but common) Used to stabilize the copper ions and prevent precipitation of copper hydroxide.

2. Principle & Mechanism

In a strongly alkaline solution, the nitrogen atoms in the peptide bonds deprotonate. The $Cu^{2+}$ ions form a coordination complex (chelate) with four nitrogen atoms from at least two peptide chains (or different parts of a long chain).

$$ \text{Protein/Peptide} + Cu^{2+} \xrightarrow{OH^-} \underbrace{\text{[Cu(Peptide)]}^{2-}}_{\text{Violet Complex}} $$

Color Change: Blue (Reagent) $\rightarrow$ Violet / Purple (Positive Result).

Note: Short peptides may give a pinkish color, while long proteins give a deep purple.

3. Structural Requirement

Minimum Two Peptide Bonds

A positive Biuret test requires the presence of at least two peptide bonds ($-CO-NH-$) directly joined or separated by a single carbon or nitrogen atom.

Therefore:

• Proteins & Polypeptides: Positive.
• Tripeptides: Positive (2 peptide bonds).
• Dipeptides: Negative (Only 1 peptide bond), except for Histidine dipeptides.
• Amino Acids: Negative (No peptide bonds).

4. Origin of the Name "Biuret"

The test is named after the compound Biuret ($H_2N-CO-NH-CO-NH_2$), which gives a positive result. Biuret is formed by heating Urea.

$$ 2 \underbrace{NH_2-CO-NH_2}_{\text{Urea}} \xrightarrow{\Delta} \underbrace{NH_2-CO-NH-CO-NH_2}_{\text{Biuret}} + NH_3 \uparrow $$

Since the Biuret molecule contains two amide linkages separated by a nitrogen, it mimics the peptide backbone and coordinates with copper to give the violet color.

5. Comparison: Ninhydrin vs. Biuret

Feature	Ninhydrin Test	Biuret Test
Target Group	Free $\alpha$-amino acids	Peptide Bonds (Proteins)
Mechanism	Oxidative Deamination	Complexation (Chelation)
Reaction Type	Irreversible Chemical Rxn	Reversible Coordination
Sensitivity	Very High (Fingerprints)	Moderate (Quantitative Protein)

Biuret Test Quiz

Test your concepts on Protein detection. 10 MCQs with explanations.

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