Phenylalanine ninhydrin reaction

Six to eight standard dilutions in an appropriate concentration range for each amino acid are prepared 2 ml of amino acid solution and 2 ml of buffered ninhydrin are mixed in a test tube, heated in a boiling water bath for 15 min, cooled to room temperature, and 3 ml of 50% ethanol added. The extinction is read at 570 nm (or 440 nm for proline) after 10 min. Standard plots of concentration versus absorbance are drawn for each amino acid. The scraped layer corresponding to each spot is extracted with 70% ethanol in a known minimum volume, and ninhydrin reaction is performed followed by spectrophotometry. The concentration of unknown samples is read from the standard plots. TLC/den-sitometry was used to determine 0.5 mg/L of phenylalanine in blood serum as an indicator of phenylketonuria (181). 

Yields sometimes drop when an old bottle of borane-dimethyl sulfide is used. Reaction progress can be monitored by thin layer chromatography (silica gel, eluting with 10 10 1 chloroform-methanol-concentrated ammonium hydroxide). Any remaining phenylalanine stains heavily when exposed to ninhydrin (Rf 0.35). If phenylalanine is detected after 5 hr of reflux, an additional 10 ml (0.10 mol) of borane-dimethyl sulfide is added via syringe, and the solution is heated at reflux for 1 additional hr. 

Udenfriend and co-workers showed that phenylacelal-dehyde, which is formed by oxidative decarboxylation of phenylalanine by ninhydrin, reacts with excess of ninhydrin and with a primary amino group to give a fluorescent product [228]. The elucidation of this mechanism and of the structure of the fluorescent reaction product [229] led to the synthesis of fluorescamine [230]. 

Steiner et al. (1998) used HPTLC to analyze amino acids in water conditioned by several medically important snails—Biomphalaria glabrata, Helisoma trivolvis, and Lymnaea elodes. Snail-conditioned water (SCW) provides information value (in the form of pheromones) to attract larval trematode parasites. The SCW samples were dried with air and reconstituted in 10% -propanol and then applied to cellulose HPTLC plates and developed with n-propanol-water (7 3). Amino acids were detected with ninhydrin reagent and the resulting color sample zones were compared to known standards. The amino acids present in SCW (h/ p values and color reactions with ninhydrin given in parentheses) were as follows an unknown (7, purple), aspartic acid (21, purple), serine (29, purple), alanine (40, purple), tryptophan (51, purple), valine (58, purple/orange), phenylalanine (64, light blue), and leucine (69, purple). The above amino acids were detected in the SCW of all the snails, except that phenylalanine was not detected in Lymnaea elodes. 

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