Capillary Electrophoresis, Amino Acids, The Chiral Separation Of Dipeptides The Class Bceoc Derivatives

Capillary electrophoresis (CE) is a kind of separation technique, which has been rapidly developed in recent years. It has many advantages including high performance, high speed and small sample consumption, and can be used to separate many compounds such as amino acids, chiral drugs, vitamins, pesticides, inorganic ions, organic acids, dyes, peptides and proteins, and even whole cells and virus particles. It has been widely used not only in chemistry and biochemistry but also in life science, medical drugs, environmental science and many other fields.CE has been proved to be a powerful tool for the chiral separation, and has been used successfully for separating many types of chiral enantiomers. It has demonstrated its particular advantages, especially in the enantioseparation of amino acids, peptides and pharmaceutical drugs. In addition, CE has many varieties of flexible separation modes and can offer simple method, minimal sample volume requirements with almost no organic waste.Chemical derivation has been developed in the chiral separation of aminoacids and dipeptides, as these biological molecules have no or weak ultraviolet absorption and the low contents cannot be detected directly by UV detection. At present, the widely used derivatizations are o-phthalaldehyde (OPA) 9-fluorenylmethyl chloroformate (FMOC-Cl) 6-Aminoquinolyl-N-hydroxy-succinimidylcarbamate (AQC), but these reagents have also been reported some various shortcomings in their application, such as short detection wavelengths, poor stability of reagents and serious interference in the determination of biological samples.In this thesis, a new labeling reagent has been synthesized and applied to the chiral separation of amino acids and dipeptides by capillary electrophoresis. The thesis consists of three chapters.Chapter one:The developments of capillary electrophoresis (CE) and the applications in life science, medical drugs, environmental science and many other fields have been simply introduced. At the same time, the significances of chiral separation and the basic knowledge of chiral separation by capillary electrophoresis are briefly introduced. Then the applications of CE to the chiral separation of amino acids and dipeptides are reviewed.Chapter two:The applications of l,2-benzo-3-4-dihydrocarbazole-9-ethyl chloraformate (BCEOC) for the chiral separation of amino acids by CE are investigated. The principal contents of this chapter consist of three parts.2.1 A simple and rapid method for the chiral separation of derivatized amino acids (AAs) using BCEOC as derivatization reagent by capillary zone electrophoresis (CZE) has been developed. Under the CZE mode, the chiral separation conditions have been studied and optimized. In optimal conditions, seven pairs of AAs derivatives were separated within 15 min with a resolution of > 1.2.2.2 Under the micellar electrokinetic chromatography (MEKC) mode, thechiral separation of BCEOC-AAs derivatives haas been studied. On the basis of optimization of some main factors for the chiral separation such as the concentration of running buffer, pH value, the concentration of surfactant reagents and organic additives etc. 13 pairs of AAs derivatives are separated within 30 min with a good baseline resolution of > 1.0. The method is simple and rapid.2.3 The chiral separation difference of BCEOC-AAs derivatives between capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) has been compared and summarized. The results indicate that micellar electrokinetic chromatography (MEKC) mode is superior to capillary zoneelectrophoresis (CZE) for enantioseparation of amino acids. Chapter three:The applications of l,2-benzo-3-4-dihydrocarbazole-9-ethyl chloraformate (BCEOC) for the chiral separation of dipeptides by CE are investigated. The principal contents of this chapter consist of three parts.3.1 Under the capillary zone electrophoresis (CZE) mode, the chiralseparation of derivatized dipeptids using BCEOC as labeling reagent with DAD detection has been studied. On the basis of optimization for CZE separation, the optimum separation conditions are followed: the separation voltage 15kV, column temperature at 25 °C, 60mmol/L tris- (hydroxymethyl) aminomethane-phosphate (tris-H3PO4, pH 9.0), 20mmol/L sodium dodecyl sulphate (SDS), 3% acetonitrile(v/v), 1.8 mmol/L /?-CD and 2.4 mmol/L SDC. Under the optimized conditions, 8 pairs of dipeptide enantiomers were separated with a good baseline resolution.3.2 Under the micellar electrokinetic chromatography (MEKC) mode, the chiral separation of BCEOC-dipeptide derivatives has been studied. Under the optimized separation conditions, 15 pairs of dipeptide enantiomers were separated using BCEOC as derivatization reagent within 25 min with the resolution between 2.85 ( Giy-Trp ) ~ 35.29 ( Gly-Ser ). This method is simple and rapid, and likely to find numerous applications in CE system for peptide chiral separation.3.3 The chiral separation difference of BCEOC-dipetide derivatives between capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) has been compared and summarized. The elution orders of enantiomers are also evaluated. The results indicate that micellar electrokinetic chromatography (MEKC) mode is much superior to capillary zone electrophoresis (CZE) for dipeptid enantioseparation with higher resolution andperformance.