Capillary Electrophoresis Separation Of Amino Acid And Dipeptide Study

High performance capillary electrophoresis (HPCE or CE) is a powerful separation technique, which has been developed on the basis of traditional electrophoresis and chromatography. It has many advantages including high separation efficiency, high speed, and small sample consumption and so on. So it can be widely used not only in the field of analytical chemistry, but also in life science, pharmacy, environmental science and so forth.CE has been proved to be a powerful separation technique, which has been used successfully for separating many types of substances. It has demonstrated its particular advantages, especially in the separation of amino acids, peptides and chiral pharmaceutical drugs. In addition, CE has many varieties of flexible separation modes, versatile separation buffers and low consumption of buffers.Currently, ultraviolet detection on line is the most widely used detection method. However, as most amino acids and dipeptides have no or weak UV absorption, the low contents cannot be detected directly by UV detection. The research and applications of derivatization reagents can not only overcome some limitations of instruments, but also play an important effect on separation, enrichment, and detection of samples. At present, the widely used reagents are 9-fiuorenylmethyl chloroformate (FMOC), o-phthalaldehyde (OPA) and dansyl chloride(DNS), 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. Therefore, labeling of the analytes with new reagents has been widely adopted to alter the chemical physical properties of target compounds.The paper consists of three chapters. Chapter one:The basic theories and development of CE have been simply elucidated. The application of CE separation for amino acid, peptides and some chiral substances,is briefly reviewed. Chapter two:The separations of amino acids and dipeptides by CE were investigated. The principal contents of this chapter consist of two parts.2.1 A new method for the separation of amino acids by micellar electrokinetic capillary chromatography (MECC) using l,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) as derivatization reagent has been developed. The effect of several key factors on the separation was studied. Under the proposed conditions, a good baseline resolution for 14 amino acid derivatives was achieved.2.2 A new method for the separation of CEOC-dipeptide derivatives by MECC using 9-(2-carbazole) ethyl chloroformate (CEOC) as derivatization reagent has been developed. The effect of several key factors such as buffer concentration, SDS concentration, pH value, and organic modifier on the separation was observed. Under the optimal conditions, 16 dipeptide derivatives were resolved with a satisfactory result.Chapter three:The applications of 9-(2-carbazole) ethyl chloroformate (CEOC) for the chiral separation of amino acids and dipeptides by CE were elucidated. This chapter consists of two parts.3.1 Two simple and rapid methods for the chiral separation of amino acid enantiomers using CEOC as derivatization reagent by capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MECC) with diode array detector at 235 run have been developed. Based on cooperative effect of binary chiral selectors, /?-cyclodextrin (/?-CD) and sodium deoxycholate (SDC) as chiral selectors for chiral separation of amino acids were, evaluated. The optimal molar ratio of /?-CD to SDC for the chiral separation of each amino acid enantiomer was obtained. Under CZE and MECC separation modes, eight and ten pairs of amino acid enantiomers were, respectively, separated with a goodbaseline resolution. At the same time, the effect of some important parameters on the chiral separation was also investigated.3.2 Many small peptides are essential compounds in biological systems. So, quick and precise chiral determination of them is of great significance. In this paper, direct chiral separation of the derivatived dipeptides using CEOC as labeling reagent by capillary micellary electrokinetic chromatography (MECC) with /?-cyclodextrin (/?-CD) and sodium deoxycholate (SDC) as chiral selectors has been developed. The chiral separation in combination with the binary selectors can enhance the selectivity and resolution in comparison with the single P-CD or SDC. The molar ratio of binary chiral selectors, the buffer concentration of T11S-H3PO4, organic modifier and pH were studied and optimized. The complete chiral separation for 14 dipeptide derivatives was obtained.