New methods for bioanalytical chemistry

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This thesis focuses on the development and application of new methodologies and instrumentation involving two potentially powerful techniques: Chemiluminescence--a spectroscopic technique in which the generation of fluorescence from the electronically excited state of the molecule is provided by a chemical reaction; and Capillary electrophoresis--a separation technique in which small ions as well as large biomolecules can be separated with high efficiency within a narrow capillary under the influence of high voltage.

A chemiluminescence detection system was described for the sensitive and reproducible detection of three dansyl-amino acids separated on thin-layer chromatographic plates involving the peroxyoxalate chemiluminescence (CL) reaction. Further study in this area was concentrated on the investigation of those parameters which may affect the analytical application of peroxyoxalate chemiluminescence to thin-layer chromatography using a stopped-scan, kinetic system.The observation of light emission from bilirubin in an organic solvent as a result of the peroxyoxalate chemiluminescence reaction is reported for the first time. The visible spectrum of bovine serum in the blue-green region was obtained using the present bioanalytical method and was briefly compared with those obtained from absorption and fluorescence methods.

Four major bilirubin species in serum were separated by micellar electrokinetic chromatography (MEKC) with sodium dodecyl sulfate (SDS) in sodium tetraborate-boric acid buffer at pH 8.5. Due to the solubilization of the serum proteins by the SDS micelles, serum samples were injected directly into a fused silica capillary and complete separation of the four bilirubin species was accomplished within ca. 10 min. without extensive sample pretreatment.Capillary zone electrophoresis was evaluated as a bioanalytical method for the speciation of aluminum in aqueous solution using indirect UV absorption detection.

The feasibility of employing peroxyoxalate chemiluminescence (PO-CL) detection in capillary electrophoresis (CE) was demonstrated using a two-step approach for the CE separation and dynamic elution (elution under pressure) of the analytes. Also, an improved whole capillary scanning device was constructed which permitted the scanning of capillaries.