Ninhydrin test
Detection of all amino acids, peptides and proteins
Ninhydrin (triketohydrindene hydrate) is used to assess amino acid qualitatively and quantitatively in ninhydrin reaction. All amino acids having α amino group, when heated in excess of ninhydrin give a purple coloured product. Only proline and hydroxyproline- amino acids molecules which contain a secondary amino group produce yellow condensation product. The colour intensity is proportional to the concentration of amino acid i.e. ammonia from the amino groups.
Image source: https://byjus.com/chemistry/ninhydrin-test/
Proteins also contain free amino groups on the alpha carbon and can react with ninhydrin to produce a blue-purple product. That is why the reaction with ninhydrin is used for the quantitative analysis of amino acids and free amino groups in proteins. It can be used qualitatively (e.g. for chromatographic visualization) or quantitatively (e.g. for peptide sequencing). Ninhydrin is most commonly used as a forensic chemical to detect “fingerprints”, as amines left over from proteins sloughed off in fingerprints react with ninhydrin giving a characteristic purple color.
Besides amino acids, other complex structures such as peptides, peptones and proteins also react positively when subjected to the ninhydrin reaction. Ammonium salts, amino sugars and ammonia also give positive ninhydrin test result. In order to obtain the reliable quantitative results the test solution should not contain the compounds mentioned above.
Principle:
Reaction of amino acids with ninhydrin leads to their decarboxylation, deamination (formation of CO2 and ammonia) and formation of aldehyde which has one carbon atom less in its structure. The ninhydrin undergoes reduction to form hydrindantin. The reduced ninhydrin condenses with ammonia and non- reduced ninhydrin molecule which leads to the formation of the violet-blue condensation product called Ruhemann’s purple.
Reaction:
Reagents:
Preparation of ninhydrin solution
- Weigh 0.2 g. of ninhydrin and dissolve in 10ml of acetone or ethanol.
Preparation of amino acid solution
- Prepare 1% amino acid solution in distilled water
Procedure:
- Take 1 ml of the sample solution in a test tube, pour few drops of ninhydrin solution.
- Heat the tube at 95⁰C for 2-5 mins or heat in the water bath and observe the development of purple colour.
- The development of violet-blue colour confirms the presence of amino acid.
Result interpretation:
Positive test: Blue-purple and yellow reaction products positively identify free amino groups on amino acids and proteins.
Negative test: No change (absence of amino acids and proteins)
References:
- https://vlab.amrita.edu/?sub=3&brch=63&sim=1094&cnt=1
- http://www.biologydiscussion.com/proteins/qualitative-and-quantitative-tests-for-amino-acids-and-proteins/13065
- https://byjus.com/chemistry/ninhydrin-test/
- http://www.chem.ucalgary.ca/courses/351/Carey5th/Ch27/ch27-3-3.html
- http://dept.harpercollege.edu/chemistry/chm/100/dgodambe/thedisk/food/ninhy/ninhy.htm
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