Date of Award

Spring 5-2000

Degree Type

Dissertation

Degree Name

PhD. Chemistry

Department

Chemistry and Biochemistry

Advisor

Nicholas H. Snow, Ph.D

Committee Member

Wyatt R. Murphy, Ph.D.

Committee Member

Yuri Kazakevich, Ph.D.

Committee Member

Richard Sheardy, Ph.D

Keywords

Chemistry, Capillary electrophoresis, Electrokinetic sampling

Abstract

Capillary electrophoresis has become a powerful separation technique in fields of biochemical separations, inorganic ions, and chiral separations. The technique has experienced exponential growth since the historic publication of Zone Electrophoresis in Open-Tubular Glass Capillaries by Lukacs and Jorgenson in 1981. However, the use of capillary electrophoresis as a primary research analysis tool still remains to be seen in many pharmaceutical laboratories. One of the main reasons for this is that it remains difficult to validate CE methods using the criteria of quantitation and accuracy posed by the current government regulating agencies, as developed for high performance liquid chromatography and gas chromatography methods.

This dissertation will examine the fundamental principles of capillary electrophoresis as they pertain to quantitative reproducibility of peak. areas with the primary focus on effects of electrokinetic sampling conditions. Data supporting longer sampling times and higher sampling voltages contributes to reproducibility relative standard deviation values of less the 2%. Injection plug lengths elucidated from sampling criteria are also evaluated.

Two models of predicting the injection plug length under the conditions of electrokinetic injection are contrasted. The first system employs standard electrokinetic sampling equations. The second model, developed by Otsuka and Terabe, predicts a maximum length above which a 5% variation in peak width may be observed. The combination of these models lead to the derivation of another expression describing the electrokinetic injection plug length. More accurate measurements of analyte mobility are shown through the use of this equation in a fashion similar to the graphical analysis of the standard Beer's Law plot.

Five different pre-injection conditions and four different quantitation techniques are evaluated as they affect the overall separation performance of caffeine and theophylline using capillary zone electrophoresis with electrokinetic injection. When quantitation methods such as internal standardization or internal area normalization are used, preinjection rinsing conditions are irrelevant.

With a better understanding of analyte mobility, the field of capillary electrophoresis analysis may be extended to the analysis of polymetallic complexes. Polymetallic complexes are of current interest in the literature due to their complexity and wide range of potential applicability. The first isomeric compound to be studied is [Ru(tpy)CI] 2(bpmi+ (tpy = 2,2':6"2"-terpyridine) (bpm = 2,2'-bipyrimidine). These isomers, which are of the same charge to mass ratio, were separated in approximately four minutes in a fused-silica capillary column with phosphate buffer ofpH 7.5 at an applied voltage of 20 kV followed by direct UV detection. An electrophoretic concentration step (stacking) was utilized in order to improve peak shape.

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