Ph.D. (Doctor of Philosophy)
Department of Chemistry and Biochemistry
Stereoisomers; Enantiomers; Chemistry; Analytic; Biochemistry
Biological systems are sensitive to the subtlest differences in the structure of chemicals they encounter, and discriminate between their isomeric forms at virtually all levels of interaction. As a consequence, structural isomers of exogenous compounds including stereoisomers can have extremely different effects on human health. The development of refined analytical techniques and selectors for the precise determination and purification of bioactive chiral and other structurally similar molecules is therefore of great interest. The research group of Drs. Oliver and Heike Hofstetter at Northern Illinois University in DeKalb, Illinois has previously raised antibodies that stereoselectively bind to α-amino acids and α-hydroxy acids, respectively. These antibodies have been used for enantiomer detection and separation in immunoassays, sensors, and chromatographic techniques. In this work, several random and site-specific protein immobilization strategies were employed to immobilize a monoclonal anti-L-amino acid antibody onto a silica support material. The resulting chiral stationary phases (CSPs) were comparatively evaluated using frontal affinity chromatography. Furthermore, enantiomer separation of free α-hydroxy acids was achieved using a monoclonal anti-D-hydroxy acid antibody immobilized onto a synthetic high flow-through chromatographic support. The resulting CSP was used for enantiomer separation of several aliphatic and aromatic α-hydroxy acids. The effect of the mobile phase parameters flow rate, temperature, pH, and ionic strength on the enantiomer separation of the model analyte mandelic acid was investigated. The same CSP was used for the determination of lactate enantiomers in human urine, which demonstrates the utility of such phases for the analysis of complex biological samples. In addition, the binding properties of a camelid anti-caffeine V[sub HH] antibody were investigated using noncompetitive and competitive ELISA. Immobilization of this antibody fragment onto a perfusion-type support material produced a column suitable for simultaneous separation of caffeine and its metabolites paraxanthine and theophylline.
Franco, Elliott Javier, "Preparation and characterization of antibody-based stationary phases for the separation of structurally related small molecules" (2008). Graduate Research Theses & Dissertations. 4485.
xiv, 132 pages
Northern Illinois University
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