The quantitative determination of proline and pipecolic acid with ninhydrin.

In recent years, a number of photometric methods for the quantitative determination of ar-amino acids have been introduced (1, 2). These methods, based on reaction with ninhydrin in aqueous solution, are less satisfactory for the determination of imino acids.1 Grassmann and von Atnim (3) described reaction products of ninhydrin with imino acids and have suggested that these might be of use for the determination of proline. More recently, Chinard (4) has described a method which is applicable to proline, ornithine, lysine, and hydroxylysine. No quantitative determination of the newly discovered pipecolic acid (5, 6) has been reported. The present method is based on the formation of a red color when proline and pipecolic acid react with ninhydrin in glacial acetic acid under nearly anhydrous conditions. The absorption spectra of the colored products correspond with those isolated by Grassmann and von Arnim (3). Most primary amino acids, hydroxyproline, and ammonia give little or no color under these conditions. The sensitivity is similar to that of the ninhydrin methods used for or-amino acids (1, 2). In principle, this method is similar to the one described by Chinard (4). The conditions for color development used here are different and offer the advantages of greater sensitivity, speed of color development, and applicability to various types of mixtures including protein hydrolysates. Early work yielded results which were variable owing to instability of the colored products. Attempts to stabilize the color by the addition of organic solvents, such as ethanol, methyl Cellosolve, or pyridine, decreased the color yield. Reducing agents, such as stannous chloride (1) and ascorbic acid, gave color increases on a few occasions, but these were not re-