| Because hydrophilic interaction chromatography (HILIC) has sufficient retention of strongly polar compounds, the interest in the technique in the last years has been promoted by growing demands for the analysis of polar drugs, metabolites and biologically important compounds in proteomics, glycomics and clinical analysis. Although the number of commercially available columns designed specially for HILIC is growing, there is still not a versatile stationary phase like C18 in RPLC. These special separation materials for HILIC demonstrate good selectivity and reproducibility for separation of polar compounds but still cannot meet the requirements of separating various types polar sample. Even though the reports about HILIC are increasing rapidly, the retention mechanism is short of systematic research because different types of separation materials for HILIC have different retention characteristics and separation selectivity. In our opinion, preparation of new HILIC stationary phase with novel functional groups is one of the ways to solve these problems, and also, try some new preparation methods to improve bonding density of the functional groups is another way to solve these problems.The dissertation includes the following six chapters:1. IntroductionThis chapter presents the history and retention mechanism of HILIC and provides a comprehensive review on the trends of the stationary phases, mobile phase and applications in HILIC. From the viewpoint of the author, the development tendency for HILIC is also presented.2. Tetrazole-functionalized silica for HILIC of polar solutesIn this chapter, tetrazole-functionalized stationary phase was prepared with nitrile-modified silica by an ammonium-catalyzed (3+2) azide-nitrile cycloaddition reaction. The prepared column showed high efficiency for the tested nucleobases/nucleosides and aromatic carboxylic acids but with a quiet different selectivity. The separation for nucleobases/nucleosides might be carried out in acetonitrile-water mobile phase while for the tested carboxylic acids only methanol/water mobile phase could give the good separation in a reasonable separation time (less than 40 min). The retention mechanism of the column was investigated by the models used for describing partitioning and surface adsorption through adjustment ratio of water in the mobile phase, and by the influence of salt concentration, buffer pH, and temperature on the retention of solutes. The results illustrated that the surface adsorption through hydrogen bonding dominated the retention behavior of nucleobases/nucleosides and carboxylic acids. From the separation ability, the tetrazole-functionalized stationary phase could become a valuable alternative for the separation of the compounds concerned.3. Preparation of phosphorylcholine modified polymer beads and their use in HILICIn this charpter, phosphorylcholine-functionalized monodisperse hydrophilic poly (glycidylmethacrylate-co-ethylenedimethacrylate) beads was prepared via reacting with phosphoryl chloride and cholinehydrochloride. The prepared stationary phase was used for HILIC mode in the separation of polar compounds. A typical HILIC mechanism was observed at higher organic solvent content (>65%). The effects of chromatographic parameters such as organic solvent concentration, buffer pH, buffer ionic strength were investigated. The results revealed that the content of organic solvent had the most influence on the retention of the analytes. The effect of buffer pH and salt concentration indicated that both hydrophilic interactions and electrostatic interactions contributed to the retention of the charged analytes, and the surface charge (zeta-potential) of the phosphorylcholine modified polymer beads is negative.4. Hyperbranched polyglycidol-functionalized silica for HILIC of polar solutesA surface-initiated polymerization was used to synthesize covalently linked hyperbranched polyglycidol brushes on the surfaces of silica gel via anionic polymerization of glycidol. The prepared stationary phase was used for HILIC mode in the separation of polar compounds. A typical HILIC mechanism was observed at higher organic solvent content, the retention mechanism is closely related to the structure of solutes in the same mobile phase. The specific ion effects on the retention was investigated by using different buffer salts in Hofmeister series, the results illustrated that the retention time of base and neutral compounds in the buffer containing kosmotropic anions is grater than that in chaotropic anions.5. Hydrophilic modification of polystyrene-based beads via surface-initiated atom transfer radical polymerization for the use in hydrophilic interaction chromatographyA one-step procedure to hydrophilize monodisperse poly (chloromethyl-styrene-co- divinylbenzene) beads has been presented with 2-hydroxy-3-[4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl] propyl 2-methylacrylate (HTMA) as monomer by surface initiated atom transfer radical polymerization (SI-ATRP). The length of the grafted poly (HTMA) chain was varied via controlling the ratio of HTMA to initiator on the surface of the beads. Using these grafted beads as the stationary phase in hydrophilic interaction chromatography, good separation was obtained for nucleobases/sides in the mobile phase of acetonitrile-water and for phenolic acids and glycosides with addition of trifluoroacetic acid (TFA) into acetonitrile-water. The retention time and selectivity of solutes showed a positive relationship with the length of the grafted poly (HTMA) chain. The strong retardation of phenolic acids and glycosides was attributed to the electrostatic attractive and hydrogen bonding between solutes and triazole group in the grafted poly (HTMA) chain. The retention of the tested solutes was also depended on the concentrations of the organic modifier and salt in the mobile phase. Because of the simplicity of the modification of polystyrene microspheres through ATRP, the new beads is expected to not only increased its hydrophilic and can be used as stationary phase in HILIC, but also can act as a useful building block to develop new stationary phases for other chromatographic mode.6. SI-ATRP prepared 5-vinyltetrazole functionalized silica gel for HILIC of polar solutesA well-defined polymer of acrylonitrile on the surface of silica gel were prepared by SI-ATRP, followed by a "click chemistry" reaction with sodium azide and ammonium chloride to yield polymeric materials with 5-vinyltetrazole units. Compared with the traditional methods in chapter 2, the SI-ATRP technique could improve the bonding dentisty of tetrazole group on scores of times. A typical HILIC mechanism of nucleobases/nucleosides, phenolic acids and glucosides was observed at higher content of acetonitrile (>85%, v/v) in the mobile phase. The retention of phenolic acids and glucosides was investigated on the column in buffered aqueous acetonitrile mobile phases (5-95%, acetonitrile). The column show mixed retention mechanism:reversed phases in highly aqueous mobile phases and normal phases (HILIC) in mobile phases with high concentration of acetonitrile, showing characteristic U-shape retention versus mobile phase composition plots.
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