Achiral, Chiral, and Protein Separations with Molecular Micelles Using Chromatographic Techniques

Abstract

In this dissertation, several chromatographic techniques were developed for separation of proteins as well as achiral and chiral compounds. Firstly, polyelectrolyte multilayer (PEM) coatings were used to separate chiral analytes and proteins of pharmaceutical and biomedical interests. Coating polymers used in PEM coatings are instrumental in analyte separation. Therefore, the effect of changing four different cationic polymers, (poly-L-lysine, poly-L-ornithine, poly-L-lysine-serine, and poly-L-glutamic acid-lysine) and three anionic polymers (sodium poly (N-undecanoyl-L-leucyl-alaninate) (poly-L-SULA), sodium poly (N-undecanoyl-L-leucyl-valinate) (poly-L-SULV) and sodium poly (undecylenic sulfate) (poly-SUS)) were investigated. The simultaneous effects of cationic polymer concentration, number of bilayers, temperature, applied voltage, and pH of the background electrolyte on the separation on these analytes were analyzed using a Box Behnken experimental design. In addition, the influence of NaCl on the column reproducibility was investigated. Secondly, mixed mode separation using a combination of micellar electrokinetic chromatography (MEKC) and PEM coatings was used for the separation of achiral and chiral analytes. In this study, it was observed that achiral and separations using MEKC and PEM coatings individually resulted in partial resolution of 8 very similar aryl ketones and 5 chiral compounds when the molecular micelles (sodium poly (N-undecanoyl-L-glycinate) (poly-SUG)) and poly-L-SULV were used. However, when mixed mode separation was introduced, baseline resolution was achieved for all analytes. In the last study of this dissertation, PEM coatings were constructed using molecular micelles in open tubular capillary electrochromatography (OT-CEC) and gradient elution moving boundary electrophoresis (GEMBE) for protein separations. In OT-CEC, proteins were detected using both ultra violet (UV) and laser induced fluorescence (LIF) detection, while only LIF detection was used with the GEMBE technique. The effects of bilayer number, type of molecular micelle as well as pH of the background electrolyte on the separation of 6 acidic proteins were analyzed using ultra violet (UV) detection in OT-CEC. In addition, internal diameter and the effective length of the capillary were studied to investigate their influence on protein separations with LIF detection. High resolution protein separations were achieved using PEM coatings, therefore, these conditions were also used for protein separations with the GEMBE technique.