Preparation of Indole Extracts from Plants for Gas Chromatography and Spectrophotofluorometry

Abstract

Indole compounds have demanded increasing attention from plant physiologists in recent years since it has become evident that most naturally occurring plant auxins and their precursors are indolic (4). A recent review by Stowe (13) of the simple indoles found in plants clearly demonstrates that the investigator must be prepared to work with a relatively large number of these substances which are diverse in their molecular structure. Critical evaluation of naturally occurring plant auxins has been hampered because of the minute concentrations in which they nearly always occur. These difficulties are compounded by the fact that many indole derivatives exhibit some degree of lability with most conventional laboratory techniques, while a few are degraded under even the mildest conditions that can be usefully employed. Bioassays have generally been used to overcome the problem of low concentration, but bioassays at best are tedious and time consuming, and are probably seldom better than semi-quantitative. Furthermore, bioassays respond only to certain indoles, whereas other indoles without biological activity may be at least as important to a particular problem. Spectrophotofluorometry is a highly sensitive technique that can quantitatively detect nanogram (10-9g) amounts of many fluorescing substances (2). Stowe and Schilke (14) have recently shown that a number of simple indoles can be detected by this method in submicrogram amounts. Since activation and fluorescence spectra generally show broad rather than sharp peaks, other fluorescing substances can interfere with an analysis, even though the activation and fluorescence maxima may not be identical with that of the compound the investigator wishes to measure. Still other compounds may quench fluorescence. For successful spectrophotofluorometry of a substance it is generally necessary for a fairly high level of purification to be effected. A promising way to obtain much of the necessary purification of indoles prior to spectrophotofluorometry is gas-liquid chromatography (14). A number of acidic and neutral indoles have been assayed with this technique. However, preliminary purification of the extract is generally necessary before gas chromatography can be successfully applied. The major part of this paper will be concerned with methods for preparing plant extracts for gas chromatography and spectrophotofluorometry. It will then be shown that the method has been successfully applied to 2 different plant extracts to show the presence of 2 well known indoles whose existence other investigators have previously established by classical methods.