A Note on Overpressure Thin-Layer Chromatography of Ecdysteroids
It is now clear that the ecdysteroid family of compounds is very large and comprises at least 150 structurally related compounds. This diversity imposes severe problems for the use of chromatographic methods in the isolation and identification of ecdysteroids. We have, therefore, been interested in the evaluation of new chromatographic systems for these compounds, such as supercritical fluid chromatography ([1–3] and Morgan et al., this volume), and new developments which promise increased resolution for more traditional methodologies such as thin-layer chromatography (TLC). In the case of TLC two methods have recently been introduced, namely automated multiple development (AMD) and overpressure TLC (OPTLC or OPLC), which offer the prospect of enhanced resolution compared to normal TLC. In AMD the plate is repeatedly redeveloped in a solvent which is allowed to migrate a little further up the plate with each development. With each development a reconcentration of the components occurs resulting in high resolution and very sharp bands. Preliminary results for the use of AMD with ecdysteroids have been described elsewhere . In the OPLC technique the plate is held under an inert, impermeable membrane under hydrostatic pressure (typically ca. 25 bar) and solvent forced through the layer using a high pressure liquid chromatography (HPLC) pump. As the flow of solvent through the layer can be controlled and is no longer dependent on capillary action, plates can be developed very rapidly. In addition solvent can effectively be run off the end of the plate allowing the use of mobile phases of relatively low eluotropic strength to obtain increased resolution.
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