Development and application of capillary electrochromatography using modular instrumentation.

Electrophoretic separations have been demonstrated for over a century resulting in methods being devised to separate a variety of compounds, mainly of biological origin. Only in the past twenty-five years has capillary electrophoresis (CE) emerged as a viable technique, with a variety of different separation methods being reported. One draw back of CE is its inability to separate neutral compounds, hence alternative methods have been developed to facilitate this. This study investigated Capillary Electrochromatography (CEC), one of the techniques that can be used to separate neutral compounds, in which a capillary column is packed with a stationary phase designed for liquid chromatography. Separation is determined by interactions between the solutes and the stationary phase, with the flow being driven by electroosmosis.Initial work involved the development of an in-house packing method for CEC columns. The method developed, which was a pressure driven system using a Shandon HPLC packer, proved to be successful. The reliability of the retaining frit and the nature of the packing material were major factors in column performance. Once the column fabrication process had been developed, the experimental conditions for CEC in the Prince Technology CE instrument were optimised. The results showed that in many respects the system responded as a traditional LC system would, with changes in buffer compositions, stationary phase and, in this case, EOF etc. all producing definite and reproducible changes in the separation of the test mixture. Variations in sample loading technique were investigated and a simple method developed to improve the peak efficiencies and resolution of analytes, by focussing them on the head of the column.Once the experimental conditions were established, a series of applications were undertaken with differing results. The applications included studies of a series of polyaromatic Hydrocarbons (PAH), a test mixture of small molecules, with different polarities, prostaglandins and nicotine metabolites. Separation of the PAH and test mixtures were successful and corroborated some of the observations made while studying the experimental conditions for CEC. Separation of the prostaglandin mixture was unsuccessful; this was not totally unexpected due to the similarity in their structures. Study of the nicotine metabolites allowed a comparison of CZE with CEC, however due to limited availability sample the work was not fully completed. Despite this, the study did indicate that both methods showed promise, but required further development.