Ninhydrin Test: Detection of Amino Acids | Chemca

Organic Chemistry

Ninhydrin Test: Detection of Amino Acids

By Chemca Editorial Team • Last Updated: January 2026 • 8 min read

The Ninhydrin Test is a general chemical test used to detect $\alpha$-amino acids, peptides, and proteins. It is highly sensitive and is widely used in biochemistry and forensic science (for developing latent fingerprints).

1. The Reagent

Ninhydrin is chemically 2,2-dihydroxyindane-1,3-dione. It is a stable hydrate of indane-1,2,3-trione.

Structure: A benzene ring fused to a five-membered ring containing two carbonyls and a gem-diol group at C2.

2. General Reaction & Observation

When an $\alpha$-amino acid is heated with Ninhydrin, it undergoes oxidative deamination. The amino acid reacts with two molecules of Ninhydrin to form a characteristic Deep Blue or Purple colored compound.

$$ \text{Amino Acid} + 2 \text{ Ninhydrin} \xrightarrow{\Delta} \underbrace{\text{Ruhemann's Purple}}_{\text{Deep Purple Complex}} + CO_2 + R-CHO + 3H_2O $$

Key Product: The purple pigment formed is called Ruhemann's Purple.

3. Detailed Mechanism

The reaction involves a series of condensation, decarboxylation, and hydrolysis steps.

Step 1: Condensation

The amino group of the amino acid condenses with the central carbonyl group of ninhydrin to form a Schiff base (Imine).

Step 2: Decarboxylation

The imine is unstable and undergoes decarboxylation (loss of $CO_2$) to form an amine intermediate.

$$ \text{Imine} \xrightarrow{-CO_2} \text{Intermediate} $$

Step 3: Hydrolysis

Hydrolysis of the intermediate yields an Aldehyde ($R-CHO$) and 2-Aminoindane-1,3-dione (Reduced Ninhydrin/Amine).

Step 4: Final Condensation

The amine intermediate condenses with another molecule of Ninhydrin to form the final conjugated system known as Ruhemann's Purple (Diketo-hydrindylidene-diketo-hydrindamine).

$$ \text{Ninhydrin} + \text{Reduced Ninhydrin-Amine} \rightarrow \text{Ruhemann's Purple} $$

4. Crucial Exceptions: Proline

The Yellow Result

Proline and Hydroxyproline are imino acids (secondary amines). They lack a primary amino group.

They react with Ninhydrin but cannot form the intermediate required for the purple color. Instead, they form a Yellow colored adduct.

Primary Amino Acids: Purple/Blue Color.
Proline (Secondary): Yellow Color.
Asparagine: Often gives a Brown color.

5. Applications

Chromatography: Used as a locating agent (spray) in Thin Layer Chromatography (TLC) and Paper Chromatography to visualize amino acids.
Forensics: Used to develop latent fingerprints on porous surfaces (paper). It reacts with the amino acids present in sweat residues.
Quantitative Analysis: The intensity of the purple color is proportional to the concentration of amino acids (measured at 570 nm).

Ninhydrin Quiz

Test your concepts on Amino Acid detection. 10 MCQs with explanations.

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