Ninhydrin Test Key words Ruhemann's purple ,proline

Ninhydrin Test

Ninhydrin (2,2-Dihydroxyindane-1,3-dione) is a chemical used to detect ammonia or primary and secondary amines. When reacting with these free amines, a deep blue or purple color known as Ruhemann's purple is evolved. Ninhydrin is most commonly used to detect fingerprints, as amines left over from peptides and proteins (terminal amines or lysine residues) sloughed off in fingerprints react with ninhydrin.

Ninhydrin can also be used to monitor deprotection in solid phase peptide synthesis (Kaiser Test). When the growing peptide chain is deprotected, a ninhydrin test yields blue. If the next peptide residue is coupled then the test is colorless or yellow.
Ninhydrin is also used in amino acid analysis of proteins: Most of the amino acids are hydrolyzed and reacted with ninhydrin except proline; Also, certain amino acid chains are degraded. Therefore, separate analysis is required for identifying such amino acids that either react differently or don't react at all with ninhydrin. The rest of the amino acids are then quantified colorimetrically after separation by chromatography.
A solution suspected of containing the ammonium ion can be tested by ninhydrin by dotting it onto a solid support (such as silica gel); treatment with ninhydrin should result in a dramatic purple color if the solution contains this species. In the analysis of a chemical reaction by thin layer chromatography (TLC), the reagent can also be used. It will detect, on the TLC plate, virtually all amines, carbamates and also, after vigorous heating, amides.
When ninhydrin reacts with amino acids, the reaction also releases CO2. The carbon in this CO2 originates from the carboxyl carbon of the amino acid. This reaction has been used to release the carboxyl carbons of bone collagen from ancient bones[3] for stable isotope analysis in order to help reconstruct the palaeodiet of cave bears.[4]
A ninhydrin solution is commonly used by forensic investigators in the analysis of latent fingerprints on porous surfaces such as paper. Amino acid containing fingermarks, formed by minute sweat secretions which gather on the finger's unique ridges, are treated with the ninhydrin solution which turns the amino acid finger ridge patterns purple and therefore visible. [
The carbon atom of a carbonyl bears a partial positive charge, so the central carbon of a 1,2,3-tricarbonyl is less stable and more electrophilic than a simple ketone. In most compounds, a carbonyl is more stable than the dihydroxy (hydrate) form. However, ninhydrin is a stable hydrate of the central carbon because this form does not have the destabilizing effect of adjacent carbonyl partial-positive centers. Indane-1,2,3-trione reacts readily with nucleophiles.
Note that in order to generate the ninhydrin chromophore, the amine is condensed with a molecule of ninhydrin to give a Schiff base. Thus only ammonia and primary amines can proceed past this step. At this step, there must also be an alpha proton (H* in the diagram) for Schiff base transfer, so an amine adjacent to a tertiary carbon cannot be detected by the ninhydrin test. The reaction of ninhydrin with secondary amines gives an iminium salt, which is also coloured, and this is generally yellow-orange in color.

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Procedure

Add about 2 mg of the sample to 1 mL of a solution of 0.2 g of ninhydrin (1,2,3indanetrione monohydrate) in 50 mL of water. The test mixture is heated to boiling for 15-20 sec; This reaction is important not only because it is a qualitative test, but also because it is the source of the absorbing material that can be measured quantitatively by an automatic amino acid analyzer. This color reaction is also used to detect the presence and position of amino acids after paper chromatographic separation.