Column chromatography screening methods

Although classical column chromatography over chiral stationary phases has been used for several years, HPLC has until recently been regarded mainly as an analytical tool. However, methods have now been developed and many papers have been published on the various applications of particular stationary phases in preparative HPLC, which may be the method of choice when the preparation of small amounts of material are required for screening purposes, for use in biological assays, or as standards in purity assays. The use of chromatographic techniques may be quicker and less risky than custom synthesis and resolution and can guarantee material to a defined specification. 

High-pressure liquid chromatography (HPLC) formats that use reverse-phase analytical columns dominate the bioanalytical field. Isocratic elution formats are used for traditional LC-MS/MS bioanalysis, since these separations do not require additional time for column reequilibration. However, gradient elution is a more effective approach for cassette analysis in highthrough-put screening methods for discovery applications because of its versatility for the separation of compounds that have a wide variety of lipophilicity. 

Instead of columns, chromatography separations can be carried out in thin layers. This method is often used for screening experiments and qualitative analysis because the laboratoiy equipment is less expensive but more flexible. 

Th. Reuvers, E. Perogordo and R. Jimenez, Rapid screening method for the determination of diethylstilbestrol in edible animal tissue by column liquid chromatography with electrochemical detection, J. Chromatogr., 1991, 564, 477-484. 

Maskarinec and Harvey [61] have described a method for the screening of solid wastes and sludges for organic compounds. The technique involves sequential extraction with acidic, basic and neutral media and the extracts are all analysed directly by gas chromatography with fused silica columns. Results achieved using the technique are discussed. The method is compared with traditional Soxhlet extraction. 

To apply a screening approach to proactive method development, analyses of selectivity samples under a variety of mobile phase conditions are conducted on different HPLC columns. HPLC columns should be as orthogonaT as possible and variations in solvent composition should be designed to maximize the probability of selectivity differences. Alternate separation techniques, such as ion exchange chromatography (IC), supercritical fluid chromatography (SFC), or capillary electrophoresis (CE) may also be used to obtain orthogonality. 

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