Ninhydrin Test

What is the 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 chemical reaction that generally occurs in a positive ninhydrin test is illustrated below.

Ninhydrin Test

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.

Ninhydrin Test Principle

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.

Ninhydrin Test Procedure

  • First, a 2% solution of ninhydrin must be prepared by dissolving 0.2 grams of ninhydrin in 10ml of either ethanol or acetone.
  • Now a 1% solution of the amino acid (analyte) in distilled water must be prepared. A few drops of the 2% ninhydrin solution must be added to this solution.
  • The test tube must be kept in a warm water bath for approximately 5 minutes.
  • The development of a deep blue/violet colour indicates the presence of amino acids.

Ninhydrin Test Result Interpretation

  • For ammonia, primary/secondary amines, and amino acids, deep purple colour is obtained.
  • For hydroxyproline and proline, a yellow colour is obtained.
  • For asparagine, brown colour is obtained.
  • If no colour change is observed, the analyte does not contain amino acids, amines, or ammonia.

Ninhydrin Test Results

An image detailing coloured solutions obtained in positive ninhydrin tests is provided above. To learn more about the ninhydrin test and other important chemical tests (such as the biuret test), register with BYJU’S and download the mobile application on your smartphone.

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