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Qualitative Test of Protein and Amino Acid
Ninhydrin Test
The ninhydrin test is a chemical test which is used to check whether a given analyte contains amines or α-amino acids. In this test, ninhydrin (a chemical compound with the formula C9H6O4; IUPAC name: 2,2-dihydroxyindane-1,3-dione) is added to a test solution of the analyte. The development of a deep blue colour indicates the presence of ammonia, primary/secondary amines, or amino acids in the analyte.
The amino group belonging to a free amino acid undergoes a chemical reaction with ninhydrin, which behaves as an oxidizing agent. When exposed to ninhydrin, the amino acid undergoes oxidative deamination, resulting in the liberation of CO2, NH3, and an aldehyde along with hydrindantin (which is a reduced form of ninhydrin).
Now, the ammonia goes on to react with another ninhydrin molecule to form diketohydrin (which is also known as Ruhemann’s complex). This complex is responsible for the deep blue colour. When the analyte contains Imino-acids like proline, a yellow coloured complex is formed. When asparagine is used, the colour of the resulting complex is brown.
The amino group of the analyte forms a compound similar to diketohydrin. This compound has a deep blue colour, often referred to as Ruhemann’s purple.