Author: Erin Joan Ennis

Publisher:

Published: 2016

Total Pages: 636

ISBN-13:

In the method development process, it is imperative not only to be able to achieve a successful separation of all components of interest (all components fully resolved in a single experiment), but also to be able to achieve those separations without sacrificing time or chromatographic efficiency. Experimental optimization, both in the laboratory and in computational approaches, allows for the thorough development of chromatographic and electrophoretic techniques to separate compounds of interest. An experimental method was developed to improve electrophoretic separations of pharmaceutical compounds, a stochastic approach was developed to evaluate the likelihood of achieving separations for competitive techniques, and a computational approach was developed to optimize experimental parameters to increase the likelihood of achieving complete separation of complex samples. Nonaqueous capillary electrophoresis (NACE), an organic-solvent based capillary electrophoresis technique, was developed for the separation of pharmaceutical enantiomers without the addition of chiral selectors. In the interest of fully investigating the ability of the background electrolyte to separate enantiomers (when the organic solvent was enantiopure) the aqueous content, identity and concentration of organic solvent, and degree of enantioseparation were thoroughly examined in terms of selected chromatographic figures of merit for a variety of analytes and analyte mixtures. A chiral NACE background electrolyte (BGE) containing cyclodextrin was also developed to improve the non-visual enantioseparation achieved with only a chiral solvent to a visual enantioseparation with the chiral NACE BGE and additives. Two approaches were developed for the unbiased prediction of the probability of a successful separation of a given number of components for chromatographic and electrophoretic techniques. For high-performance liquid chromatography (HPLC), the stochastic approach eliminated the positive bias in previous studies by redefining the separation space and allowing all components in a separation to be considered in the prediction. The HPLC stochastic approach was applied to both gradient and isocratic separations, the latter of which had not been considered previously. The probability calculations were applied to sequential elution liquid chromatography (SE-LC) to facilitate an accurate comparison of the likelihood of separation in HPLC and SE-LC. For capillary electrophoresis separations, a computational approach was utilized to determine the effect of electroosmotic flow (EOF), migration distance, and electric field on the probability of a successful separation of a given number of components. A factorial design was utilized to examine the probability of success in the total separation space and in a time-restricted separation space for a given peak capacity and number of components. The individual and factorial design analysis allows for the full optimization of experimental parameters to achieve successful separations in capillary electrophoresis. The combination of experimental and computational approaches for chromatographic and electrophoretic techniques encompass many of the goals of analytical method development and provide new pathways to predict the feasibility of a separation and optimize experimental conditions.