Study And Application On New Micellar Electrokinetic Chromatography Method

Capillary electrophoresis (CE) is a relatively new separation technique driven by electric field and with capillary as separation channels. Since the pioneering studies in 1980s, it received much attention and is developed rapidly. Nowadays, CE has been widely used for routine analysis due to the advantages of higher separation power, short analysis time and consuming small volumes of sample and reagents. Introduced in 1984 by Terabe et al., micellar electrokinetic chromatography (micellar EKC) has become one of the most important modes of capillary electrophresis, since it can achieve the analysis of neutral analytes, not possible with capillary zone electrophoresis. The separation ability of MEKC was determined by the buffer components and properties. Because a lot of surfactants were used in the MEKC, so that it is widely used in biochemistry,medicine,food, et al. This dissertation focused on the development and application of MEKC methods for drugs, amino acids and protein. In this dissertation, we have carried out some original studies as following:(1) Reverse micellar solution was firstly used separation medium in CE, and a reverse micelle electrokinetic chromatography method was developed to study the separation and electrophoretic behavior of some phenolic and benzoic compounds.(2) A reverse micelle electrokinetic chromatography method was firstly used for separation and determination of alpinetin and cardamonin.(3) A MEKC method was developed for separation and determination of IgG,HSA and Lysozyme in serum.(4) A affinity electrokinetic chromatography with laser-induced fluorescence detection method was firstly developed for enantioseparation of glutamic acid and Aspartic acid derivatized with 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein.(5) A MEKC method with Ultra violet detector was developed for separation of catecholamine and 5-hydroxytryptamine in Toad Venom and Shanyao.This dissertation consists of six chapters.Chapter 1 In this chapter, the development of MEKC and its applications are reviewed from three aspects:(1) The fundamental of MEKC is summarized including retention factor; resolution; column availability; peak capacity; migration time window;(2) The development of pseudostationary phases in MEKC is presented. They include micellar pseudostationary phases (anionic, cationic, nonionic and zwitterionic), mixed micelles and polymeric pseudostationary phases;(3) Sensitive alternatives such as on-line preconcentration methds are introduced. The on-line preconcentration methods used field-amplified sample stacking, sweeping and high-salt sample stacking as sections,(4) Sensitive detection techniques such as MEKC-LIF and MEKC-MS fluorescence are introduced;Chapter 2 A novel reverse SDS micelle system was used as separation medium for the analysis of some phenolic and benzoic compounds. The optimum reverse SDS micelle system is formed with n-butyl chloride as continuous phase, 0.70 M sodium dodecyl sulfate (SDS) as the surfactant, W₀ (13.0), 14.0% (v/v) 1-butanol as the co-surfactant, 8.0% (v/v) acetonitrile, 1.5% (v/v) heptane, and a 0.06 M tris-(hydroxymethyl)aminomethane (Tris) buffer, pH 10.3, as dispersed phase. The electrophoretic behavior of the seventeen analytes in a novel reverse SDS micelle systemwas investigated. A satisfactory linear equation wasμ_app= -23.319pK₁ + 36.183, and thesquare of linear correlation coefficient R² was 0.9131 between the apparent electrophoretic mobilities and pK₁ of eleven phenolic compounds. A quantitative structure-apparent electrophoretic mobility relationship (QSAR) was developed for the apparent electrophoretic mobilities of seventeen phenolic and benzoic compounds based on the descriptors calculated from the structure alone.Chapter 3 A novel electokinetic capillary chromatography method, reverse sodium dodecyl sulfate (SDS) micelles as pseudo-stationary phase, was developed for separation and detection of alpinetin and cardamonin. In this work, reverse micelles (RMs) have been firstly introduced into background electrolyte for electrophoresis separation. The optimum reverse SDS micelle system was formed with n-butyl chloride as continuous phase, SDS (20.9%, w/v) as the surfactant, W₀ (13.0, water-surfactant molar ratio), 18.0% (v/v) 1-butanol as the co-surfactant, 8.0% (v/v) acetonitrile (ACN), 1.5% (v/v) heptane, and a 60 mol L-1 tris-(hydroxymethyl)aminomethane (Tris) buffer, as dispersed phase. Linear relationships (correlation coefficients: 0.9961 for cardamonin and 0.9991 for alpinetin) between the peak areas and concentration of the two compounds were obtained (5.0-350.0 mg/mL for cardamonin and 1.25-350.0 mg/mL for alpinetin). The detection limits (S/N = 3) for cardamonin and alpinetin were 0.19 and 0.14 mg/mL, respectively. The method was successfully applied for the quantification of alpinetin and cardamonin in Alpinia katsumadai Hayata and kuaiwei tablet with satisfactory recoveries in the range of 95.9-100.2%.Chapter 4 A new micellar electrokinetic chromatography with Ultra violet detection method was developed for the quantification of IgG,HSA and Lysozyme in serum. The optimum separation conditions were 10mM SDS;10mM phosphate; pH 6.5. The detection limits of IgG,HSA and Lysozyme 0.95μg/mL, 1.32μg/mL, 1.16μg/mL. Analysis results of samples proved that the method is applicable for the quantification of IgG and HSA in serum.Chapter 5 A MEKC with Ultra violet detection method was developed for separation of catecholamine and 5-hydroxytryptamine in Toad Venom and Shanyao. The optimum separation conditions were 20 mM borate; 120mM SC; pH 9.6, and 20.0% (v/v) methanol. Under the optimum conditions, four analytes was baseline separated in 9min. the method was successfully used to analyze the Toad Venom and Shanyao with satisfied results. Chapter 6 With 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein as derivatization reagent, a affinity electrokinetic chromatography with laser-induced fluorescence detection method was firstly developed for enantioseparation of glutamic acid and Aspartic acid. The parameters having predominant influence on the derivatization and separation were studied. The detection limits of D,L-Asp and D,L-Glu were 0.52 ng/mL; 0.50 ng/mL; 0.32 ng/mL; 0.29 ng/mL. The average recoveries were 99.6-102.5%. Analysis results of samples proved that the method is applicable for the quantification of amino acid injection.