Doctoral Dissertations

Date of Award

12-2002

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Chemistry

Major Professor

Michael Sepaniak

Committee Members

Doug Gilman, Ben Xue, Cynthia Pereson

Abstract

It is well known that poor quantitative reproducibility substantially limits the practical implementation of capillary electrophoresis (CE) separations in chemical analysis. The principal sources of variance in observed peak areas are irreproducible flow rate which influences on-column detector response, and inconsistent injection volume or amount. In addition, current efforts in our laboratory to assess sources of quantitative variance for separations of dansylated amino acids using an automated CE system are presented and related when appropriate to the body of existing knowledge on this important topic. A comparison of different injection methods, the effect of random changes in electroosmotic flow (EOF), and choice of certain peak integration parameters in terms of peak area reproducibility are presented. An approach is presented to optimize conditions for capillary electrophoresis separations of multi-analyte enantiomeric-pairs (D- and LDansyl (Dns)-amino acids) that involves the rational use of combinations of cyclodextrins (CDs) as enantio-selective running buffer additives. Migration data is experimentally obtained for a range of concentrations for native CDs used individually and employed to determine inclusion constants for the Dns-amino acids of interest. Simplex methods are then employed for the first time to optimize conditions for the separation of amino acid enantiomers. The validity of this approach is demonstrated for separations of five Dns-amino acids enantiomers using y- and f3-CDs at various concentrations. Extending the dual-CD approach to other CDs and increasing the number of CDs beyond two should be possible. To this end, preliminary experiments are performed by using several available single-isomer, derivatized CDs (individually) to determine if they have potential for further studies. It was found that molecular mechanics modeling is useful in interpreting those cases where low inclusion constants likely contributed to the ineffectiveness of the CDs. Finally, it is important in CE to have solute identification capabilities for unknown mixtures and because of the variable electroosmotic flow, which causes irreproducible migration times. A counter flow arrangement for surface enhanced Raman Spectroscopy was developed for this purpose and colloidal silver is added externally since it can affect efficiency when inserted in the running buffer.

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