Ninhydrin Test: Principle, Requirements, Procedure and Result
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.
- Take 1 ml test solution in dry test tube and 1 ml distilled water in another tube as a control.
- pour few drops of 2% ninhydrin in both the test tubes
- Keep the test tubes in water bath for 5 minutes.
- 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:
