摘要
毛细管电动微分离方法包括毛细管电泳(CE)和毛细管电色谱(CEC)。本文针对CE和CEC在生物物质如蛋白质、氨基酸等分离应用中存在的问题,提出了相应的解决方法。
首先针对蛋白质CE中蛋白质尤其是碱性蛋白质与管壁硅羟基非特异性吸附这一问题,制备了新型的聚乙烯醇涂层毛细管柱。制得的管柱可有效屏蔽蛋白质与管壁的非特异性吸附,分离柱效一般在600 000理论板/米以上,且具有较好的分离结果重现性。例如,对三种碱性蛋白质进行100次分离得到的保留时间的相对标准偏差均小于1.0%。
提出了两端进样蛋白质CE方法。该方法可在中性pH值的条件下,通过一次CE操作,同时分离带有正电荷和负电荷的蛋白质。此外,进一步提出了电渗泵辅助式蛋白质CE方法,该方法同时分离带正电荷、负电荷和不带电的蛋白质。
针对开管CEC(OTCEC)低相率、低配基容量这一缺点,本文提出了采用触须式聚合物作为OTCEC固定相的设想,研制了金属螯合触须式聚合物涂层管柱,并将其用于氨基酸及嘌呤类衍生物的微分离。与单层配基修饰管柱相比,触须柱的金属离子螯合量提高了近900倍,可显著增加分析物的保留因子和分离度。
为建立适用于修饰各种色谱配基的触须式聚合物涂层管柱,本文研制了以甲基丙烯酸缩水甘油酯为接枝聚合单体的管柱制备方法。制得的管柱内壁含有大量环氧基,可用于修饰各种色谱配基。以苯丙氨酸为修饰配基,将制得的苯丙氨酸触须柱用于苯类化合物、氨基酸混合物以及蛋白质混合物的微分离。
针对毛细管电动微分离方法检测灵敏度较低的缺点,制备了阴离子触须式聚合物接枝涂层管柱,并将其用于蛋白质在线萃取、富集分离。阴离子触须柱可有效的增加蛋白质的吸附容量,提高富集倍数,对肌红蛋白的富集倍数高达1500倍。该富集方法还可用于蛋白质混合物的富集分离。
Capillary electro-kinetic microseparation method includes capillary electrophoresis (CE) and capillary electrochromatography (CEC). The thesis focuses on solving the problems meet in the separations of biological molecules such as proteins, amino acids and so on with CE and CEC.
A novel method for the fabrication of poly(vinyl alcohol) coated capillary column was developed to avoid the irreversible adsorption between proteins and capillary inner wall. The prepared column can promise high protein separation efficiency (generally higher than 600,000 plates/m), and excellent reproducibility. For example, relative standard deviations for the migration time of three basic proteins were smaller than 1.0% within 100 injections.
A“both-ends-sample-injections”method was designed to simultaneous separation of both negatively and positively charged proteins with the running buffer of a neutral pH. Besides, electroosmotic pump-assisted protein CE was also developed for the separation of cationic, anionic and neutral proteins in a single run.
To solve the problem (low phase ratio and low ligand capacity resulting in poor resolution) in open tubular CEC (OTCEC), tentacle-type polymer was designed as the OTCEC stationary phase. A tentacle-type metal-chelating polymer coated capillary column was introduced and used for the separation of amino acids and purine derivatives in this thesis. Immobilized metal capacity of the prepared tentacle-type column was nearly 900 times higher than that of mono-layer metal-chelating group modified one. Besides, the tentacle-type capillary can obviously increase the retention factor and resolution of analytes.
To prepare a tentacle-type polymer coating easily functionalized with different ligand, a new method for grafting glycidyl methacrylate to the capillary inner wall was thus developed. The prepared polymer coated column could be easily modified with different chromatographic ligand by a ring-opening reaction of the epoxy groups. In this study, phenylalanine was selected as the ligand. Benezene derivatives, amino acids as well as protein mixtures were then separated, respectively.
To improve the detection sensitivity in capillary electrokinetic microseparation methods, a novel negatively charged tentacle-type polymer-coated capillary column was fabricated and applied for on-line extraction and preconcentration of proteins. The prepared tentacle-type polymer coating can efficiently increase the protein adsorption capacity, thereby increasing the sensitivity enhancement factor. In this work, over 1500-fold sensitivity enhancement was realized for myoglobin. The developed method also promised for enrichment and separation of protein mixture.
引文
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