Determination of tryptophan and indole substances by a colorimetric diazotisation method

Abstract

A colorimetric method for the determination of tryptophan in protein hydrolysates by its conversion into nitrosamine with nitrous acid followed by diazotisation with N-1-naphthylethylenediamine dihydrochloride has been studied. Selective nitrosation of tryptophan was best achieved at 20 to 35 °C using 1·0 to 1·2 M hydrochloric acid. Diazotisation was best achieved at 10 °C or below. Sodium chloride inhibited the nitrosation reaction to a considerable extent and, therefore, tryptophan standards should contain an amount of sodium chloride equal to the amount present in sample hydrolysates. Such standards are prepared from a portion of the sample hydrolysate pretreated with activated charcoal. Twenty common protein amino-acids other than tryptophan did not interfere in the colour development, but the method was found to be applicable to indole and its derivatives in addition to tryptophan. Compounds such as phenols and aromatic amines were found to interfere.