新型亲水作用色谱固定相的制备及色谱性能研究

【作者】 戴小军；

【导师】 卫引茂；

【作者基本信息】 西北大学 ， 化学， 2011， 博士

【摘要】 近年来,随着生物医药技术,食品安全和环境监测等研究领域的发展以及各种组学研究的深入,强极性和亲水性物质的分析迅速成为分析化学和生物化学领域的重要研究对象。由于对强极性物质的良好保留,亲水相互作用色谱(HILIC)越来越受到人们的重视。在HILIC中,针对溶质与固定相表面功能团之间的选择性作用力是决定溶质保留的事实,作者认为在HILIC固定相中引入结构新颖的功能基以及控制功能基密度是制备选择性固定相的关键。围绕这一问题,作者采用不同类型表面修饰方法,将功能基团键合在有机聚合物和硅胶表面,制备了五种固定相并研究了其色谱性能。全文包括六个部分：1.绪论介绍了HILIC的发展历史,溶质的保留特点和分离机理。综述了HILIC固定相及应用研究进展,对今后HILIC的发展趋势进行了展望。2.四唑键合硅胶固定相的制备及色谱性能使用“点击”化学的方法,制备了以四唑为功能基的新型HILIC固定相,并用小分子碱性化合物和苯甲酸类样品对其色谱性能进行了评价。发现碱基和核苷样品的保留随流动相中强洗脱剂水含量的增加而减少,符合亲水作用色谱模式。但是,将该固定相用于对苯甲酸类化合物的分离,却发现溶质保留由强氢键作用控制,阐述了这两类溶质保留行为差异的原因。3.磷酰胆碱改性聚合物色谱固定相的制备及色谱性能采用PGMA/EDMA微球与氯化胆碱和三氯氧磷反应,产物经水解后,制备了磷酰胆碱为功能基的新型HILIC固定相,并使用小分子极性溶质对其色谱性能进行了评价。发现所有溶质保留符合HILIC特征。用极性和碱性溶质验证了磷酰胆碱改性PGMA/EDMA微球表面带负电荷的结论。该结论对于不同溶质分离条件的建立具有指导意义。4.超支化聚缩水甘油醚亲水作用色谱固定相的制备及色谱性能以缩水甘油醚为单体,在硅胶表面阴离子引发聚合制备了超支化聚缩水甘油醚包覆的HILIC固定相,并研究了其色谱性能。发现溶质以何种机理与固定相作用还与溶质的结构有关。依据Hofmeister效应,分析了流动相中缓冲盐种类和浓度对溶质选择性的影响,发现正离子和中性化合物,在含kosmotrope盐的流动相中的保留强于使用chaotropic盐的情况。5.表面引发原子转移自由基聚合(SI-ATRP)法制备聚合物接枝型HILIC固定相及其分离性能依据溶质在HILIC上的保留机理,设计合成了新型单体甲基丙烯酸-2-羟基3-(4羟甲氧基-1,2,3-三唑)酯(HTMA)。利用SI-ATRP技术,将此单体聚合在PCMS/DVB微球表面,实现了聚苯乙烯微球的亲水性改性。用碱基、核苷样品以及酚酸、糖苷样品研究3种不同接枝量的HILIC固定相的分离性能。结果表明,固定相的极性和选择性与聚合物接枝链长有关,聚合物链越长,固定相的极性和选择性越好。这一结论对制备极性和选择性不同的固定相具有理论指导意义。所有溶质在固定相上的保留受静电作用和氢键作用控制。但对酚酸类溶质而言,静电吸附作用更为强烈,导致其洗脱需采用含三氟乙酸的乙腈-水流动相,说明该固定相可应用于中草药中酚酸类物质的选择性分离研究。6. SI-ATRP法制备的聚乙烯四唑接枝型色谱固定相及其色谱性能通过SI-ATRP法,在硅胶基质表面聚合丙烯腈,然后通过“点击”化学方法,制备表面修饰聚乙烯四唑的硅胶固定相。与普通方法合成的四唑硅胶相比,在新方法合成的固定相上,四唑基团含量提高数十倍。碱基和核苷样品,酚酸和糖苷类样品在此固定相上的保留均符合HILIC特征。在5-95%(v/V)之间改变流动相体系中水的比例,酚酸和糖苷类样品的保留值与流动相中水浓度存在“U”型曲线关系,体现出HILIC和RPLC对保留共同起作用的双机理。更多还原

【Abstract】 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.更多还原