【作者】 冯蕾；

【导师】 赵凤生；

【作者基本信息】 上海交通大学 ， 生物医学工程， 2009， 博士

【摘要】 随着生物工程上游技术的迅猛发展,采用分离技术集成化的研究思路开发新型生物分离技术日渐引起学术界重视。将电泳与色谱分离技术进行集成,即在色谱分离过程中引入电场以提高色谱的分离度或分离速度是近年来国内外液相色谱技术的一个重要发展方向。目前,国内外有关制备型电色谱系统的研究工作只有少数课题组进行,这些研究都以蛋白质和核酸等生物大分子为分离对象,使用的固定相主要是分离大分子常用的排阻凝胶、离子交换凝胶和羟基磷灰石等介质。而在生物活性物质中,除了蛋白质等生物大分子外,越来越多的极性小分子活性物质被发现,例如,多肽、多酚、寡核苷酸和水溶性抗生素等。因为极性强,多为水溶性,作为药品具有良好的生物相容性,但却给分离工程师带来难题。目前较成熟的制备技术,例如萃取技术和RP-HPLC(反相高效液相色谱)技术在非极性或弱极性小分子化合物的分离中有良好表现,并且易于放大,但对强极性小分子物质的分离效果很差(例如头孢菌素C和小分子寡核苷酸)。由于大部分强亲水性物质都具有可解离基团,可以用毛细管电泳或电色谱技术进行高效率的分离,但这些方法难以放大,不能制备微克级以上的样品。因此,开发用来分离极性小分子物质的制备型电色谱技术可以为这类物质的分离纯化提供新的制备手段,具有重要的实际意义。本课题在这样的背景下,首次提出并设计了一套适合分离极性或弱极性小分子物质的制备型电色谱装置,建立了各种适合分离或浓缩小分子物质的制备型电色谱技术,并对各种影响因素和分离机理进行了较全面的研究,揭示了通电对色谱分离过程的各种影响。以柱色谱(40 cm×0.6 cm I.D., 40 cm×1.2 cm I.D., 40 cm×2.0 cm I.D.)为基础设计并构建了制备型电色谱装置,基本解决了电色谱技术放大引起的主要问题,即电解气体进柱和焦耳热累积的问题,确定了装置的结构和实验操作流程。与文献报道的装置类似,在色谱柱两端各连接一个电极室以放置电极溶液和电极;但柱与电极室的连接方式及相对位置与文献报道的不同。文献报道的较成熟的装置都在电极室和柱连接处用排阻膜或凝胶条分隔,电极室的缓冲液和色谱柱的缓冲液分开。这样,用膜或凝胶条可以很好地阻挡电解气体进入色谱柱,同时电极缓冲液与外界循环又可以不断带走气体。由于以大分子样品为分离对象,样品不会穿过膜或凝胶条进入电极缓冲液而造成损失。显然,这些报道的装置不适合可以自由出入排阻膜或凝胶条的小分子物质的分离。本课题的电极室均设计为敞口,将制备型色谱柱的出口电极室改成内径约5 mm的T型三通管,放置在色谱柱侧下方,与柱的支管相连。经过改进的装置可以随时排走电解产生的气泡,不再需要膜或凝胶进行分隔。色谱流出液经过T型三通管电极室后全部进入在线检测器,不必担心小分子样品的泄漏。在冷却系统中,除了用文献报道的色谱柱夹套冷却和进柱缓冲液槽冷却外,还对出口T型电极室和连接管道等焦耳热聚集部位进行了全方位的冷却,确保可以施加较高电场(60~120 V/cm)(报道的制备型电色谱多用20~50 V/cm的低电场),分离过程稳定。在本论文的主体部分,分别论述了以常用于分离小分子物质的非极性大孔吸附树脂、可以与阴离子物质构成离子排斥色谱的阳离子交换凝胶和具有多孔结构的排阻凝胶为固定相,用设计的制备型电色谱装置分离几种小分子极性物质,包括茶多酚、单核苷酸和水溶性头孢菌素的研究结果。在装有聚苯乙烯型非极性DIAION HP20树脂的色谱柱上加反向电场(色谱柱入口加负极,出口加正极),可以从含有咖啡因的茶叶粗提物中部分分离茶多酚单体表没食子儿茶素没食子酸酯(EGCG)和表儿茶素没食子酸酯(ECG)。研究表明,色谱的主要影响因素——固定相颗粒度、色谱流速和缓冲液中乙醇浓度,电泳的主要影响因素——缓冲液pH值等对样品的保留时间都有明显影响,证明该分离系统实现了电泳与非极性吸附色谱的叠加。在分离过程中,电泳、电渗和色谱都对分离有一定作用。在通电过程中,电场的存在还通过影响样品在DIAION HP20树脂颗粒内的传质,影响样品的非极性吸附色谱行为。由于实验中使用的树脂颗粒内孔径较小(<30 nm),不足以引起颗粒内液体的电渗流动,导致带有样品的液体不易进入树脂颗粒内,传质减慢。尤其是EGCG和ECG,因为样品分子内的非极性基团的分布不对称,色谱保留时间受电场影响更明显。通过增大柱内径,保持柱长不变的方式对电色谱柱进行了放大,结果施加相同的电场,可有效减少放大后的焦耳热。放大后电泳仍然可与色谱作用叠加,但由于色谱柱的高径比减小,损失了部分柱效,降低了分离效果。在填充弱酸性阳离子交换葡聚糖凝胶的离子排斥色谱柱上加反向电场(色谱柱入口加负极,出口加正极),凝胶上的羧酸基团可以被正极电解产生的氢离子质子化,而且随着通电时间的延长,凝胶的质子化程度提高,结果茶多酚(EGCG和ECG)与咖啡因的分离度增加,直到被完全分离。本课题首次研究了在离子排斥色谱柱上施加电场的分离,结果表明出口电极的电解反应修饰了固定相,减弱了样品与固定相之间的离子排斥力,从而改善了样品的色谱行为,是电场影响分离的主要原因。目前关于制备型电色谱的研究还未见涉及该原理,该法可以为类似的多酚或弱酸物质的分离纯化提供参考。与在吸附色谱柱上通电分离茶多酚的情况相比,此时电场的作用机理截然不同,体现了电场影响样品色谱行为的不同方面。用分离生物大分子常用的排阻凝胶为固定相,通电分离亲水性小分子——单核苷酸混合物和头孢菌素混合物的研究,与前两种分离茶多酚的方法不同,这里使用的排阻凝胶不与或微弱地与样品发生吸附作用,所有样品在不加电场时的保留时间相同,丝毫不能被分离。排阻凝胶的主要作用是作为电泳填充介质,防止样品返混,并实现在线检测与收集。通电后,样品可以按照带电量差异被分离,因此该系统也可以看作制备型电泳系统。研究发现,凝胶内孔径越大,电泳分离效果越好。用恒组分缓冲液分离单核苷酸混合物时,根据电泳方向与液体流动方向一致还是相反,可以减小或延长样品的保留,并根据带电量分离样品。在此基础上,结合pH梯度缓冲液建立了等电聚焦电泳法,根据两性头孢菌素的等电点分离并浓缩了4种头孢菌素分子,分离时间比离子交换色谱法短,样品峰集中,缓冲液的轻微变化也不影响分离效果。等电聚焦电泳法用低浓度的乙酸和氨水溶液(0.01 mol/L)制作pH梯度,价格便宜,配制方便,不仅可以用于结构相似的小分子物质的细分和精制,还可以用于极性小分子物质的浓缩。综上所述,本课题主要针对极性小分子物质的分离建立了制备型电色谱装置及4种在色谱柱上施加电场的分离方法,主要使用水溶液分离极性或弱极性小分子物质,并且都用水溶解样品上样。4种施加电场的方法的分离机理各不相同,但都体现了电场提高样品分离度和洗脱浓度的优点。建立的各种方法适合分离可溶于水的极性小分子物质,可以与常用的分离亲脂性化合物的制备型反相高效液相色谱和硅胶柱-有机溶剂法互补,有广阔的应用前景和实际价值。更多还原

【Abstract】 The extensive progress of biotechnology and bioengineering requires the development of novel separation techniques. The combination of the existing techniques is an important way. In order to enhance the separative resolutions and efficiency, coupling liquid chromatography with electrophoresis by introducing an electric field into a column has been attempted at home and abroad recently. But up to now, only several groups are engaged in the research of preparative electrochromatography. In reported literatures, samples are confined to macromolecules, such as proteins and nucleic acids. Accordingly size-exclusion gels, ion-exchange gels and hydroxylapatite, which are commonly used for macromolecules, are chosen as the stationary phase. In the field of bio-active substances, besides macromolecules, a lot of small polar compounds with novel activities have been discovered, such as peptides, polyphenols, oligonucleotides and water-soluble antibiotics. These compounds have good biocompatibility due to their strong polarity and water-solubility, but these features induce some trouble to separation engineers. In the field of separation process, liquid-liquid extraction and RP-HPLC could be easily scaled, which have good performance for the separation of non-polar and weakly polar compounds, but they are not suitable for polar ones due to the poor retention (e. g. cephalosporin C and oligonucleotides). Since most polar compounds are chargeable, they could be well separated by capillary electrophoresis or electrochromatography. However the capillary-based methods could not be scaled, so that it is difficult to provide microgram-scale fractions. If preparative electrochromatography is explored, it may be of significance and novelty for the separation of small polar compounds. Therefore a novel kind of preparative electrochromatography apparatus was developed in this dissertation, which is especially suitble for small polar or weak polar compounds. Accordingly preparative electrochromatography techniques to enhance resolution and concentration of fractionated samples were designed and investigated. Experiments in terms of the influencing factors and the mechanisms were also investigated, which illustrated that the electric field had great effects on chromatographic performance.A novel preparative electrochromatography apparatus was constituted on the basis of column chromatography (40 cm×0.6 cm I.D., 40 cm×1.2 cm I.D., 40 cm×2.0 cm I.D.). Common problems of preparative electrochromatography, i. e. electrolytic bubbles and Joule heat, were alleviated. The construction and manipulation of this apparatus were established. Two electrode chambers containing electrode buffer and electrodes were separatedly connected with two column ends, which was similar to the reported apparatus, while the manner of their connection was newly proposed in this dissertation. In literatures, exclusive membranes or gel rods were put between the electrode chambers and the column in order to prevent bubbles from column, meanwhile electrode buffers were circulated with the reservoir to take the bubbles away. Apparently the reported apparatus are not suitable for small molecules, which may travel across the membranes or gel rods and lose in electrode buffer. Diverse from the reported apparatus, electrode chambers in this dissertation were open to free electrolytic bubbles. The outlet electrode chamber in“T”-shape with each branch of 5 mm I.D. was put at the foot of the column and connected with the column branch, so as to free bubbles without the need of membranes or gel rods. The effluent out of column could pass through the outlet chamber and then flew to online detection without loss. Therefore the novel apparatus could accommodate the separation of small compounds. In the cooling system of this apparatus, besides column water jacket and the ice-water bath for the inlet eluent, the“T”-shape outlet chamber and the connecting tubes were also cooled, where the Joule heat was prone to accumulate. Thus higher electric field of 60~120 V/cm could be applied steadily, while most electric field used in literature was only 20~50 V/cm.In the main part of the dissertation, macroporous non-polar adsorption resin usually used for the separation of small molecules, cation-exchange gel which could compose anion-exclusion chromatography with anions, and porous size-exclusion gel were chosen as the stationary phase to separate small polar compounds, including tea polyphenols, mononucleotides and water-soluble cephalosporins.Tea polyphenol monomer EGCG and ECG could be partially separated from crude tea extract containing caffeine by applying negative electric field (cathode at column inlet, anode at outlet) on the column packed with non-polar adsorptive DIAION HP20 resin. By altering the chromatographic factors, including particle size, flow rate, ethanol concentration, and the electrophoretic factor, buffer pH, solute retention could be dramatically affected. This verified the successful combination of the chromatography and electrophoresis techniques. During the separation course, the mechanisms including electrophoresis, electroosmosis and chromatography could affect separation results. Besides, electric field could affect the chromatographic performance by influencing the mass transfer from mobile phase liquid into resin particles. The electroosmotic flow could not formed in the intraparticle of the resin due to its small pore size (<30 nm), thus mobile phase flow containing sample solutes could merely travel interpartically and mass transfer into intraparticles was hindered to some extent. Especially the retention of EGCG and ECG, whose adsorptive parts are asymmetrically distributed in molecule, was more likely to be affected. The electrochromatography was amplified by column diameter while the column length kept the same. Consequently Joule heat was not apparently increased, and the coupling of electrophoresis and chromatography was observed. However the separative performances were not as good as those obtained from smaller columns, due to the loss of partial column efficiency.When applying negative electric field (cathode at column inlet, anode at outlet) on an anion-exclusive chromatographic column packed with weakly acidic gels, the carboxy groups on resin could be protonated via the electrolysis of anode, and the protonation could be enhanced with the increase of electric field applying time. Consequently, tea polyphenols (EGCG and ECG) were completely separated from caffeine by applying electric field. Studies on this novel method revealed that the electrolytic reaction in electrode chambers was the main cause of the chromatographic performance alteration. The gel at the column bottom was protonated due to electrolysis, thus the anion-exclusive interactions with polyphenols were weakened and the resolutions were improved. It is the first time to study this separative method, which is referable for the preparation of polyphenols or other weakly acidic compounds. The separation mechanisms by applying electric field on anion-exclusive chromatography and adsorptive chromatography were different, which illustrated that the electric field could affect the chromatographic performance from different aspects.Porous size-exclusion gels, which are commonly used to separate macromolecules, were first attempted to separate small polar mononucleotides and cephalosporins with electric field. Different from the above methods for tea polyphenols, gels here could slightly or not adsorb sample solutes, thus all solutes result in the same retention and could not be separated without electric field. The gels here functioned as electrophoretic media to prevent the convective effects and realize the online detection and fractionation. Solutes could be partially separated on the basis of their charges when applying electric field. Actually, this could be taken as a preparative electrophoresis system. It was discovered that the bigger the pore of the gel, the better the performance of electrophoresis. Electrophoresis with isocratic eluent could apparently diminish or enhance the retention when the direction of electrophoresis was the same or opposite to hydrodynamic flow. On this basis, isoelectric focusing electrophoresis of four zwitterionic cephalosporins was established by using pH gradient eluent. Consequently, the separative time was much shorter and sample peaks were much more concentrated than the results by ion-exchange chromatography. Resolution by isoelectric focusing electrophoresis was less affected when slightly altering the buffer pH. Moreover, the pH gradient buffers were made of dilute acetate and ammonia solution (0.01 M), which is cheap and easy to be composed. The established novel isoelectric focusing electrophoresis could be used to refine and concentrate small polar compounds.Conclusively, in this dissertation a preparative electrochromatography apparatus especially for the separation of small polar compounds, and four separation methods with applying electric field were designed and studied. In the experiments, aqueous buffers were used to separate samples in aqueous solution, including small polar and weakly polar compounds. The separative mechanisms of every established method were different, but all the separations revealed that applying electric field on chromatography could enhance resolution and fractionated sample concentration. These methods are suitable for the separation of small polar compounds, and are complemented with RP-HPLC and silica-solvent technique for non-polar compounds. These methods are promising and valuable in the investigation of novel small polar or weakly polar compounds.更多还原