Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time II. Two-run assay procedures

By optimizing column temperature T and gradient time t(G), complex samples can often be separated by means of reversed-phase high-performance liquid c hromatography (RP-LC). Conclusions reached in Part I suggest that the compl ete separation of such samples will be difficult, however, when more than 1 5-20 components are present in the sample. An alternative approach is to ca rry out two separations with different conditions (T, t(G)) in each run. Th e combination of results from these two runs then allows the total analysis of the sample, providing that every sample component is adequately resolve d in one run or the other. Examples of this approach, carried out by means of computer simulation, are shown here for several samples of varying compl exity. Also considered is the ability of a single separation where T and t( G) are optimized to enable the separation and analysis of one or more indiv idual sample components from complex mixtures (e.g., drugs in animal plasma ), including the resolution of isomeric compounds from each other. (C) 1999 Elsevier Science B.V. All rights reserved.