Graduate Thesis Or Dissertation

A new general method for the optimization of HPLC ternary of pseudo-quaternary mobile phases and the separation of two new metabolites of nefopam from greyhound urine

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  • A new general method is developed for the optimization of HPLC ternary or pseudo-quaternary mobile phases which are represented by the trilinear coordinate system. This method can predict the global optimum of the mobile phase composition. The global optimum composition along each edge of the triangle and the corresponding selectivity factor of the worst-separated peak pair(s) are used in this method. This method is named the weighted pattern comparison optimization method (WPCO) and is applicable for both known and unknown samples. The WPCO method is simpler than those currently in use. The WPCO method was tested by using 68 literature data sets whose separation response surfaces are different. Results of the WPCO method agree with the results obtained by the minimum α plot method and by the grid search method, and do so with substantially fewer experimental measurements. Compared with the 5% (in eluent composition) step size grid-search procedure, the WPCO method using the same step size reduces the experimental work by 75%. For further reducing the experimental work, the original WPCO method is simplified. In an ordinary HPLC separation, the separation factor and resolution are approximately proportional to the logarithm of the selectivity factor. Based on this, the separation factor replaces the logarithm of selectivity factor in the original WPCO method. This further reduces the experimental work and avoids the error introduced in the measurement of the column dead volume. The simplified WPCO method has been tested in the normal-phase and reversed-phase chromatography separation cases. The simplified WPCO method has been tested by using 27 literature data sets whose separation response surfaces are different. Results of the simplified and original WPCO methods are nearly identical when the capacity factors of the solutes of the worst-separated peak pairs are greater than 5. When the capacity factors are less than 5, the simplified WPCO method is satisfactory in less complex, less critical applications. Two new metabolites of nefopam have been separated from greyhound urine. In the separation process, flash chromatography is used for cleaning up and preseparating the samples in a single step. Compared with other techniques, experimental work is reduced. The structure of one of the newly discovered metabolites is determined using MS and NMR. The most probable structure of the other metabolite is determined using MS. The main metabolic pathways at different doses in greyhounds are studied.
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