摘要
毛细管整体柱(Capillary monolithic column)是一种用有机或无机聚合法在毛细管内进行原位聚合形成的分离介质,以其制备简单,无需柱塞,通透性好,分离快速高效等优点,成为备受关注的液相色谱固定相。硼酸亲和色谱(Boronate affinity chromatography)是以取代硼酸为配基的亲和色谱模式,主要用于含有顺二羟基化合物,比如糖蛋白、糖肽和核苷酸的分离与富集。随着蛋白质组学、代谢组学和糖组学等研究的兴起与发展,硼酸亲和色谱,尤其是基于纳米材料和整体材料的硼亲和分离介质,受到越来越多的重视。
本研究制备了一种新型的聚(VPBA-co-PETA)硼酸亲和毛细管整体柱,并通过实验设计软件(DOE)优化了其合成条件。随后以微径液相色谱(μHPLC)和加压毛细管电色谱(pCEC)为平台,考察了其分离机理和保留行为,搭建了硼酸亲和色谱/反相液相色谱二维分离平台(μ-BAC/RPLC 2D system),并将聚(VPBA-co-PETA)整体柱用于中药活性成分的富集。
本论文共分为五章,主要内容包括:
第一章分别从方法、原理、应用等方面综述了毛细管整体柱和硼酸亲和色谱的相关研究背景,以及硼酸亲和毛细管整体柱的研究现状。同时介绍了毛细管液相色谱和加压毛细管电色谱的理论依据,总结了其应用进展。
第二章介绍了新型硼酸亲和毛细管整体柱的制备、优化和表征。以4-乙烯基苯硼酸为单体,季戊四醇三丙烯酸酯为交联剂,偶氮二异丁腈为引发剂,乙二醇和二甘醇为致孔剂,经原位聚合制备了聚(VPBA-co-PETA)毛细管整体柱。以单体、交联剂、致孔剂和引发剂的用量为4种因素,最大吸附量、柱效和保留时间为三个考察标准,利用实验设计软件(DOE)优化了其合成条件,验证了正交实验最优结果,得到了整体柱合成的最佳配比为单体7.32 mg,交联剂6 mg,致孔剂100μL,引发剂1 mg。根据优化结果制备整体柱,利用SEM扫描电镜表征其结构和外部形态,利用傅里叶红外光谱对固定相的键合基团进行表征,并验证其性能。结果表明优化条件下合成的整体柱固定相空隙分布均匀,含有硼酸、苯环、羟基等集团,通透性良好,最大吸附量和柱效也有所提高。
第三章对聚(VPBA-co-PETA)毛细管整体柱的分离机理进行了探讨。在最佳条件下合成整体柱,在微径液相色谱(μHPLC)和加压毛细管电色谱(pCEC)实验中考察了分离特性,结果表明,整体柱固定相表面的硼酸基团在碱性条件下能特异性吸附邻苯二酚、腺苷等含有邻二羟基结构的化合物,具有亲和色谱的特性;同时由于交联剂的性质使其具有反相分离机理,在含有20%有机相的条件下能够将极性不同的苯系物分开。
第四章根据合成的固定相具有亲和作用和反相作用的双重机理,在聚(VPBA-co-PETA)毛细管整体柱上建立了硼酸亲和色谱/反相液相色谱(μ-BAC/RPLC)二维分离平台。考察了流动相酸碱性以及盐浓度对分离情况的影响,应用μ-BAC/RPLC二维分离系统在28 min内分离6种化合物。
第五章主要介绍了将聚(VPBA-co-PETA)毛细管整体柱应用于中药蒲公英和刺果卫矛提取物的分离和富集。先摸索标准对照品的液相色谱条件,将提取物直接稀释进样;再用硼酸亲和整体柱对提取物进行分离,收集酸性条件下的洗脱物,再用高效液相色谱法进行对比验证。结果证明硼酸亲和整体柱能够有效富集咖啡酸、绿原酸等含有邻二羟基基团的小分子,为中药质量检测提供了便捷有效的手段。
Capillary monolithic column as liquid chromatography stationary phase has attracted great attention from researchers due to its merits of easy preparation without frit, excellent permeability, high efficiency and low column pressure. Moreover, the result can be repeated and displayed easily, and achieve quickly sample separation, so it has been applied in capillary electrochromatography (CEC) and micro-high performance liquid chromatography (μHPLC). Boronate affinity chromatography (BAC) is an important tool for specific capture and separation of cis-diol containing compounds such as glycoproteins, RNA and carbohydrates. With the development of proteomics, metabonomics, glycomics and other research, boronic affinity chromatography, especially those based on nano-materials and monolithic materials, gets more attention.
In this study, a Poly(VPBA-co-PETA) boronate capillary monolithic column was synthesized, characterized and utilized in the mode of both affinity and reversed phase chromatography. In order to optimize the properties, the orthogonal experiment was designed by the DOE software. We establishedμ-BAC/RPLC 2 dimensional separation platform with affinity/reversed separation mechanism. Then, the Poly(VPBA-co-PETA) capillary monolithic column was applied to enrich small molecules contained cis-dihydroxyl group in the traditional Chinese medicine.
This thesis has five chapters.
The first chapter described the background of Capillary monolithic column and Boronate affinity chromatography from methodology, theory and applications, respectively. The outlook of capillary electrochromatography and micro-high performance liquid chromatography separation were summarized, where the development of boronate affinity monolithic column was also outlined.
Chapter 2 includes the preparation, characterization and applications of the poly(VPBA-co-PETA) monolithic capillary column. A boronate functionalized monolithic capillary column was synthesized, characterized and utilized in the mode of both affinity and reversed phase chromatography. The polymer was prepared using 4-vinylphenylboronic acid as the functional monomer, pentaerythritol triacrylate as crosslinker, azobisisbutyronitrile as trigger, ethanediol and diethylene glycol as porogenator. In order to optimize the properties, the orthogonal experiment was designed by the DOE software. The optimum preparation conditions were monomer (VPBA, 732 mg), cross-linking agent (PETA, 6 mg), trigger (AIBN, 1 mg) and pore-foaming agent (diethylene glycol/ethanediol = 2:8). It can be also seen that the monolithic bed exhibited a well-distributed open channel network and good permeability. The breakthrough capacity and the column efficiency are also improved.
In Chapter 3, the column with the best proportion was characterized and applied in capillary high performance liquid chromatography (μHPLC) and pressurized capillary electrochromatography (pCEC). The results suggested the column persists affinity chromatography character because the boronate group located at the stationary phase could specifically absorb compounds containing cis-dihydroxy functionality at alkaline conditions; because the properties of PETA, the column also persists RP separation mechanism and separate different benzene series with 20% ACN in H2O.
In Chapter 4, we established the BAC/RPLC 2 dimensional separation platform with affinity/reversed separation mechanism. The two types of chromatographic modes can be performed consecutively on the same column during one complete run, making it a 2-D chromatography. We also examined the influence of pH and the concentration of salt to the separation. After optimization, we separated 6 compounds in 28 min on the 2-D chromatography system.
Chapter 5 focused on the applications of Poly(VPBA-co-PETA) in the separation and enrichment of traditional Chinese medicines, Herba Taraxaci and Thorn fruit of Euonymus extract. The extract was separated onμHPLC and the fractions at acidic conditions were collected and examined by RP chromatography. The contract of the chromatograms before and after enrichment on the boronate affinity column showed the column can be used to enrich small molecules contained cis-dihydroxyl group efficiently, such as caffeic acid and chlorogenic acid.
引文
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