Preparation of Indole Extracts from Plants for Gas Chromatography and Spectrophotofluorometry.

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 dlifficulties 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.