Preparative Chromatography - Introduction > Page 1

Introduction

Preparative chromatography can be a very ambiguous term and its meaning will often depend on the raison d'Ítre for its use. To the forensic chemist, preparative chromatography may mean the isolation of only a few microgram of material for structure elucidation by subsequent spectroscopic examination. To the biochemist, it may mean the isolation of a few milligrams of a substance required for assessing its physiological activity.In contrast, to the organicchemist, preparative chromatography will often mean the isolation of 5 or perhaps even 50 g or more of a pure intermediate for subsequent synthetic work (this can be particularly important in the separation of chiral mixtures). Thus, the amount of material that is separated does not necessarily determine whether the separation can be classed as preparative or not. However, all preparative separations involve the actual collection of an eluted component and does not merely comprise peak profile monitoring for quantitative estimation and elution time measurement. It is interesting to note that the technique of chromatography, originally invented by Tswett in the latter part of the nineteenth century, was not initially developed for analytical purposes, but for the isolation of some specific pigments from plant extracts. In fact, all the early applications of chromatograph were exclusively for preparative purposes and it was not until gas chromatography (GC) was introduced by Martin and Synge (1) was the technique used for analytical purposes. Even after the introduction of GC, liquid chromatography (then called column chromatography) was still used largely for preparative work. Liquid column chromatography evolved from a preparative procedure into an analytical technique during the late nineteen sixties, largely provoked by the development of high performance liquid chromatography (HPLC), which, in turn, was largely sparked off by the successful development of GC. Initially, column loads were increased for preparative purposes by increasing the dimensions of the column both in GC and in HPLC. However, this approach has distinct limitations.

If the column radius is increased, unless special packing techniques are employed, the packing procedure becomes inefficient and the packing itself unstable. In addition to maintain the optimum mobile phase velocity, the flow rate will need to be substantially increased and the consumption of mobile phase will eventually become economically impractical. Conversely, if the column length is increased, then the impedance to flow will become greater leading to high column pressures. If large column radii are employed, then the mechanical strength of the column system will limit the maximum permissible pressure. Consequently, lengthening the column will eventually require the particle diameter to be increased to provide adequate permeability. Increased particle diameter will, in turn, reduce the column efficiency, which may impair the resolution of the compounds of interest. It is seen that scaling up a chromatographic separation can be quite complex as many of the controlling factors are interacting and so the optimum parameters required by the separation are not easy to define. To understand the problems associated with preparative chromatography the factors controlling the loading capacity of a column need to identified.