New Approaches For Micro-separation And Micro-Preconcentration Of Biomolecules

With the rapid development of life science, miniaturizing of the analysis system has been an important task for the development of analytical chemistry. In the miniaturized system, high detection sensitivity, high resolution and high sample throughput are needed to reduce the analysis time, analysis cost and reagent consumption, especially for the complicated sample system, such as biological sample. In recent years, micro-scale separation and perconcentration based on capillary technique have attracted extensive research interest. This work mainly focuses on the preparation and application of monolithic materials in the high efficient separation, as well as the development of new methods for on-line preconcentration in capillary electrophoresis, which are suitable for the analysis of biomolecules.Proteins play very important roles in fields of food, medicine, biology and so on. Development of high resolution and high sensitivity approach for protein analysis has become a hot spot in modern pharmaceutical analysis, analytical chemistry, and life science. This task is more challenging for glycoproteins, since one glycoprotein can have many isoforms with slightly different physicochemical properties due to the glycosylation. Monolithic column, which is recognized as the fourth generation separation medium, has several significant advantages such as easy preparation, high column efficiency and high permeability. Monolithic capillary column-based nano-flow liquid chromatography has become an important tool for the structural identification of proteins. However, the current monolithic columns are only applicable to separate some standard protein mixtures with great differences. A novel polymer monolithic capillary column was prepared by a simple thermally-initiated polymerization using tris (2,3-epoxypropyl) isocyanurate (TEPIC) and tri (2-aminoethyl) amine (TAEA) as monomers. Residual epoxide groups in the monolith were post-modified by ring-opening reaction with ammonia aqueos soulution to provide amine functionalities for anion exchange chromatography. The micrometer-size throughpores and the hydrophilicity of this kind of weak anionic exchange (WAX) column were very suitable for the analysis of macromolecules. Isoform separations of several intact glycoproteins were achieved. The monolithic capillary exhibited good WAX selectivity. It can be further developed as a promising tool for isoform profiling of glycoproteins.On the other hand, it is another issue for the analysis of glycoproteins that many important glycoproteins are expressed naturally in very low abundance. Capillary electrophoresis (CE) has been the workhorse for the high-resolution separation of glycoproteins. Due to the limited light path, however, CE is associated with poor detection sensitivity when it is equipped with a UV absorbance detector, which is a general configuration on commercial CE instruments. On-line sample precocentration is a simple and effective way to enhance the detection sensitivity. We presented a combined strategy for on-line preconcentrating glycoforms of glycoproteins, which coupled two individual on-line preconcentration techniques, i.e., large volume sample stacking (LVSS) and reversed pH junction (RPHJ). LVSS allowed for compressing a large injected sample volume into a narrow sample zone, contributing to the main sensitivity improvement, while RPHJ coordinated the preconcentration process to improve the resolution. This strategy was verified with recombinant human erythropoietin, a typical glycoprotein, as the test analyte, and the effects of experimental conditions were investigated. It was found that there was a compromise between sensitivity enhancement and resolution for on-line preconcentration of glycoprotein glycoforms.Like glycoprioteins, many other cis-diol-containing compounds (CDCCs), such as nucleosides and catecholamines, also play important roles in many biological processes. However, some of them are present in very low abundance. A new on-line preconcentration technique called borate complexation-assisted field-enhanced sample injection (BCA-FESI) was proposed for preconcentrating CDCCs in CE. The principle relies on further amplification of the difference in the electrophoretic mobility of CDCC in sample matrix and background electrolyte (BGE) through complexation of CDCC with borate in a sample matrix of basic pH and dissociation of the complex in a BGE of acidic pH. Meanwhile, CDCC and borate ions electro-injected into the capillary were finally in neutral state, which maintained the pre-filled low conductivity zone and thus allowed for longer injection time. The concentration factor was nearly2000folds.