Study On The Enantioseparation Of Amino Acids And Enzyme Kinetics By Ligand-exchange Capillary Electrophoresis

Chiral recognition has attracted more and more attention in capillary electrophoresis (CE) technology researches. Therefore, development and exploration of new chiral selectors and new enantiomeric separation methods become a very important and key task. Although various chiral selectors have been obtained and chiral metal complexes have been introduced, the later would be a widely used type of chiral selecting syetems in CE and have not been well explored. However, few stable and efficient chiral ligand exchange CE systems (CLE-CE) are available for choice until now. Additionally, rare investigations have been developed for inspecting the enzymatic reaction and on-line labeling by using the CLE-CE method for the analysis of biosamples. Thus, the dissertation was designed to have an insight into the problems. The main results are as follows:In the introductory chapter, the applications of CE in chiral separation (including on-column labeling technique and enzymatic reaction research technique) and the main chiral selectors were reviewed. Subsequently, the study proposal of this dissertation was presented.In chapter two, a new CLE-CE method for the enantioseparation of D,L-amino acids (AAs) was developed with Zn(II)-L-amino acylamides as chiral selectors. The results showed that apart from the effect of the concentration of the complexes on the resolution, the buffer pH had a sharp influence on resolution. The employed chiral ligands also exhibited different enantioselectivities and enantiomer migration orders.In chapter three, a novel on-column labeling method of D,L-AAs by using dansyl chloride as the labeling reagent was explored combining with the CLE-CE technique. The results displayed that the samples introducing model, the reaction time and the amount of D,L-AAs introduced were seriously impacted on the on-column labeling yield and the enantioresolution.In chapter four, the use of Zn(II)-L-ornithine complex as a chiral selecting system for the enantioseparation of D,L-AAs was studied. Interestingly, this new method was shown to be applicable to measure the enzyme catalytic activity in the incubation of D,L-AAs with D-AA oxidase (DAAO) and determine the enzyme kinetic constants, lending insight into potential enzyme mechanism.In chapter five, a new strategy for the enantioseparation of D,L-AAs employing CLE-CE with Zn(II)-L-valine complex as a chiral selector in the presence of cyclodextrin was designed. Furthermore, the expanding enzymatic use of LAAO to incubate with different L-AAs had allowed understanding of the substrates'specificity.