Principle:

• This test is a general test and thus given by all amino acids. This test is due to a reaction between a amino group of free amino acid and ninhydrin. Ninhydrin is a powerful oxidizing agent and its presence, amino acid undergo oxidative deamination liberating ammonia, CO2, a corresponding aldehyde and reduced form of ninhydrin ( hydrindantin). The NH3 formed from a amino group reacts with another molecule of ninhydrin and is reduced product ( hydrindatin) to give a blue substance diketohydrin ( Ruhemanns complex). However,  in case of imino acid like proline and hydroxyproline, a different product having a bright yellow color is formed. Asparagine, which has a free amide group, reacts to give a brown colored product.

Requirements

• test solution: 1 % alanine, 1 % proline , 1% asparagines
• 2 % ninhydrin in acetone
• Water bath
• Dry test tubes
• Pipettes

Procedure

i. Preparation of 2% ninhydrin solution:

• weight 0.2 gm of Ninhydrin and dissolve in 10ml of acetone or ethanol

ii. Preparation of test solution:

• prepare 1% amino acid solution in distilled water.

1. Take 1 ml test solution in dry test tube and 1 ml distilled water in another tube as a control.
2. pour few drops of 2% ninhydrin in both the test tubes
3. Keep the test tubes in water bath for 5 minutes.
4. Look for the development of blue or violet color.

**But in the case of proline and hydroxyproline, yellow color will develop instead of blue color. Similarly, asparagine will give brown color.

Result:

Ninhydrin Test: Principle, Requirements, Procedure and Result

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