Ninhydrin [I] generates a blue-purple color on reacting with free amino groups in amino acids, peptides, or proteins. It has a molecular weight of 178.14 and occurs as colorless or faint yellow crystals. Ninhydrin, also called 2,2-dihydroxy-1,3-indanedione, is synthesized from ethyl phthalate [II] and methyl sulfoxide [III] (Scheme 1):
1. Ninhydrin Reaction
When a solution containing amino acids, peptides, or proteins is boiled with ninhydrin under mildly acidic conditions, an intense blue-purple color with an absorption maximum at 570 nm develops In the original instruments for automatic amino acid analysis, a protein sample that has been hydrolyzed with 6 N HCl at 110°C for 24 h to amino acids is separated with an ion-exchange chromatographic column, and the eluate is monitored at 570 nm after being developed with the ninhydrin reaction. To detect peptides or amino acids on filter paper or other solid supports, the paper is dipped in or sprayed with ninhydrin solution in acetone.
2. Protein Detection by Ninhydrin Method
A solution containing 20 to 500 |ig protein is dried on a filter paper. Nonprotein compounds that might react with ninhydrin are eluted by washing with a trichloroacetic acid solution. Then the proteins are hydrolyzed with Ba(OH)2 solution at 120°C for 10 min, and the amino acids released are measured by the ninhydrin reaction (1). Because the amount of protein present is determined by the amino acids produced, this is an accurate method suitable for all proteins. (Scheme 2):
Amino acids, such as proline and hydroxyproline, develop color with an absorption maximum at 440 nm.