Why ninhydrin is used for estimation of amino acids

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If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. The ninhydrin reaction, one of the most important method of detecting amino acids, both technically and historically, has been conventionally used to detect their microgram amounts.

When amino acids with a free alpha amino groups are treated with an excess of ninhydrin, they yield a purple colored product. Under appropriate conditions, the color intensity produced is proportional to the amino acid concentration.

The primary amino groups react with ninhydrin to form the purple colour dye now called Ruhemann's purple RP was discovered by Siegfried Ruhemann in Iminoacids like proline, the guanidino group of arginine, the amide groups of asparagine, the indole ring of tryptophan, the sulfhydryl group of cysteine, amino groups of cytosine and guanine, and cyanide ions also react with ninhydrin to form various chromophores that can be analyzed.

Primary amines also react with ninhydrin, but do not liberate of CO2. Several other convenient reagents are available which can react with the alpha amino group to form colored or fluorescent derivatives.

These include fluorescamine, dansyl chloride, dabsyl chloride, etc. In the quantitative estimation of amino acid using Ninhydrin reagent, the absorbance of the Ruhemann's purple formed by the reaction at nm is measured.

For imino acids, the absorbtion happens at nm. The principle behind the colorimetric estimation is given below:. Unknown compounds may be identified by their characteristic absorption spectra in the ultraviolet, visible or infrared regions. Enzyme-catalysed reactions frequently can be followed by measuring spectrophotometrically the appearance of a product or disappearance of a substrate. Light can be categorized according to its wavelength.

Figure 1 shows the relationship between the wavelength of light and the common types of electromagnetic radiation. Light in the short wavelengths of to nm is referred to as ultraviolet UV. Light in the longer wavelengths of to nm is referred to as near infrared near IR.

Visible light falls between the wavelengths of and nm. In addition, imines such as pipecolic acid and proline, the guanidino group of arginine, the amide groups of asparagine, the indole ring of tryptophan, the sulfhydryl group of cysteine, amino groups of cytosine and guanine, and cyanide ions also react with ninhydrin to form various chromophores of analytical interest. Since its discovery, extensive efforts have been made to apply manual and automated ninhydrin reactions as well as ninhydrin spray reagents to the detection, isolation, and analysis of numerous compounds of interest across a broad spectrum of disciplines.

These include agricultural, biochemical, clinical, environmental, food, forensic, histochemical, microbiological, medical, nutritional, plant, and protein sciences. This reaction is unique among chromogenic reactions in that at pH 5.