Optimizing separations in reversed-phase liquid chromatography by varying pH and solvent composition |
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Authors: | P. J. Schoenmakers N. Mackie R. M. Lopes Marques |
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Affiliation: | (1) Philips Research Laboratories, P.O. Box 80000, 5600 JA Eindhoven, The Netherlands;(2) Present address: Royal Dutch/Shell Laboratory, Badhuisweg 3, 1031 CM Amsterdam, The Netherlands;(3) Present address: Department of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, G1 1XL Glasgow, Scotland;(4) Present address: Centro de Technologia Química e Biológica, Quinta do Marguês, 2780 Oeiras, Portugal |
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Abstract: | Summary An interpretive optimization procedure in which pH can be one of the variables is presented with the emphasis on optimizing separations. When varying the pH in reversed-phase liquid chromatography the retention of ionogenic solutes will change. Thus, the selectivity between ionogenic and neutral solutes or between ionogenic solutes mutually can be optimized. However, pH also greatly affects the efficiency (plate count) and peak shape (asymmetry). Optimum selectivity (i.e. large differences in retention times) may be observed under conditions where peaks are broad and asymmetrical. Thus, it is essential to simultaneously consider retention, peak width and peak shape and their effects on separation (effective resolution) in pH-optimization studies. A procedure in which this is done is presented and applied to optimizing the separation of a synthetic mixture of selected pharmaceuticals. After initial experiments to establish the parameter space (boundaries for pH and binary methanol — water composition), twelve experiments are performed according to a 3×4 experimental design. At each loaction the retention, peak height, peak area and peak symmetry are recorded for each solute. These data are then used to build models for each of the four characteristics and for each solute. From this set of models the response surface, describing the quality of separation as a function of pH and composition, can be calculated. A variety of optimization criteria (quantifying quality of separation) can be used. The optimum corresponds to the highest point on the response surface. |
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Keywords: | Column liquid chromatography Reverse phase Optimization |
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