Gas chromatography of indole auxins

Abstract

For effective separation of indole auxins by gas chromatography it was necessary to divide them into acidic and neutral indoles. The neutral indoles were volatile enough to be chromatographed directly, but the acidic indoles needed to be esterified in order to be chromatographed. Most indole acids were successfully esterified with diazomethane or by BF 3 catalysis, but indolelactic acid could be esterified by diazomethane only. Esterification by diazomethane was preferred because this substance is a powerful esterifying reagent. Since the diazomethane was prepared as an ether solution, it had the additional advantage that after esterification the ether and excess diazomethane could be easily removed. The neutral indoles chromatographed well with a silicone substrate, SE-30 or SE-52, though some overlapping occurred with a 6 ft column. Excellent separations of indole esters were also obtained with the silicones, particularly SE-52, even on a 5 ft column. Other substrates which were effective to a certain extent included neopentyl glycol succinate and QF-1. Attempts to prepare acetal derivatives of indole aldehydes met with little success and it may be preferable to chromatograph indole aldehydes directly. The overlapping of neutral indole compounds was overcome by increasing the length of column. Higher efficiency was obtained and the longer column did not appear to destroy the compounds. Proper conditioning of the columns was necessary, especially for the more labile indoles, which could not be detected unless the column had been aged for a few days. Most efficient separation was obtained when the oven temperature was about 205°C, with the injector block at least 50° above that of the oven. Some prepurification of plant extracts was necessary before they could be applied to the gas chromatograph. However, sensitivity limitations made it difficult to detect the small amounts present in individual plants or plant parts. For larger quantities of material gas chromatography might be employed in the initial separation steps followed by spectrophotometric, or fluorometric analysis, or bioassay.