【作者】 谷从影；

【导师】 贾金平； 方能虎；

【作者基本信息】 上海交通大学 ， 环境科学， 2008， 博士

【摘要】 环境问题的日益严峻对色谱分离技术提出了更高的要求,毛细管整体柱是应用于微柱液相色谱中的一种新型色谱柱,具有制备简单、重现性好、多孔性优越、能实现快速分离等优点,但目前仍然处于发展初期,在新型基质研制、制备方法优化、理论基础研究以及应用方面还有待进一步发展。本文在以往研究的基础上,以环境生物大分子和农药为分离对象,制备了三种聚合物毛细管整体柱,并拓展了毛细管整体柱在环境生物大分子和农药分析方面的应用。论文的研究内容和主要结论如下:第一,新型聚苯乙烯-十八碳烯-二乙烯基苯(PS-OD-DVB)毛细管整体柱的制备及其在蛋白质分离中的应用研究。实验结果表明,以苯乙烯为单体、1-十八碳烯为功能单体、二乙烯基苯为交联剂单步制备的PS-OD-DVB毛细管整体柱具有高达85%的孔隙率、良好的渗透性、稳定的机械性能以及足够的柱容量。通过对比发现,PS-OD-DVB和聚苯乙烯-二乙烯基苯(PS-DVB)毛细管整体柱具有相似的蛋白质分离性能,可以在2.5 min内实现六种蛋白质的基线分离;对于多肽分离,由于C18长碳链的引入,PS-OD-DVB毛细管整体柱可以在9.0 min内实现血红蛋白α、β多肽链的基线分离。PS-OD-DVB毛细管整体柱制备简单、分离效果显著,有望应用于环境毒理学和环境医学中生物大分子的快速分离分析。第二,毛细管内径对整体柱的物理性能以及对分离蛋白质的色谱性能的影响。实验结果显示毛细管内径越小,柱床的微球簇和簇间孔隙越大,渗透性越大。从色谱基础理论入手,以动力学角度考察不同内径整体柱对蛋白质的色谱分离性能发现小内径毛细管整体柱的Van-Deemter方程的涡流扩散、分子扩散系数项较小,蛋白质传质效率较高。虽然小内径毛细管整体柱内形成的微球簇更大,但是柱效反而更高,这些实验结果与传统填充柱的实验现象相反。本部分所得的实验结果以及相关理论的初步探讨对整体柱的基础理论发展以及在分离生物大分子中的实际应用具有一定的指导意义。第三,聚甲基丙烯酸丁酯类毛细管整体柱在μ-HPLC和加压毛细管电色谱(pCEC)系统中分离微囊藻毒素(MCs)的新方法。该部分共包括三项内容:(1)聚甲基丙烯酸丁酯毛细管整体柱在μ-HPLC系统中分离MCs新方法的建立。优化条件后可以在9 min之内实现MC-LR、MC-YR和MC-RR的基线分离,并成功应用于巢湖水样中MCs的分离分析。(2)磺酸化聚甲基丙烯酸丁酯毛细管整体柱在μ-HPLC系统中分离MCs,实验表明该整体柱在μ-HPLC模式下具有疏水作用和离子交换混合分离机理,优化分离条件后可以在5 min内实现模拟水样中藻毒素的基线分离。(3)磺酸化聚甲基丙烯酸丁酯毛细管整体柱在pCEC系统中分离MCs,优化分离条件之后可以在6 min内等度洗脱条件下实现三种藻毒素的基线分离。与μ-HPLC相比分离柱效更高,还可以避免梯度洗脱,具有良好的重现性。总之,甲基丙烯酸酯类毛细管整体柱分离藻毒素可达到良好的分离度,与常用方法相比大大缩短了分析时间,充分显示了毛细管整体柱在快速分离环境生物样品方面的优势。第四,合成单体11-acrylamidoundodecanoic acid(AAUA),利用D-optimal实验设计和响应面(RSM)分析方法优化聚合混合物配比,制备了适用于分离烷基苯和烷基苯酮同系物的AAUA-EDMA毛细管整体柱。实验结果表明在最佳配比下制备的整体柱的渗透性和机械性能良好,CEC模式下分离性能稳定,整体柱制备的批次内和批次间重现性分别可以达到2.14%和2.92%以内。实验证明,这种软件辅助优化方法可以通过较少的实验来实现有限制性条件的多因素优化,除此之外,与普遍采用的固定其他因素变化单一因素的优化方法相比,避免了单纯的经验性优选,同时还可以考察各因素之间的相互作用。实验结果与模型预测结果误差在-4%~9%,证明这种方法在整体柱制备条件优化中的应用是成功的。另外还初步研究了AAUA-EDMA毛细管整体柱在CEC中分离氨基甲酸酯农药的可行性,在35 min内可以实现7种农药的完全分离。更多还原

【Abstract】 Environmental problems have been posing a great challenge to separation technology. Microflow or nanoflow chromatographic methods have attracted strong interest. While there is a lot of problems when comes to the particulate-packed capillary columns which are the traditional columns used in HPLC. The appearance of capillary monolithic columns put forward a completely significantly alternative for traditional packed capillary column. But its development is still in the very beginning step. Therefore, novel monolithic columns were developed and their applications have been studied. The main contents include the following four parts:1 . A new poly (styrene-octadecene-divinylbenzene) (PS-OD-DVB) monolithic column was simply prepared by in-situ polymerization of styrene, DVB and octadecene with DMF and decanol as porogens in one step. It was found that this kind of monolithic column had a total porosity of 85%, perfect mechanical intensity and adequate loading capacity. Compared with PS-DVB monolithic column, PS-OD-DVB monolith showed similar ability for the fast separation of six proteins in 2.5 min, and both the PS-DVB and PS-OD-DVB monolithic columns have good durability for high pressure and have good reproducibility after repeated injections. Batch-to-batch reproducibility of column fabrication was good with RSD below 9.5%. Moreover, the experimental results indicated that the PS-OD-DVB monolith showed higher loading capacity and provided better resolution for the separation ofαandβchains of hemoglobin than the PS-DVB monoliths because of the importing of C18 chain. These results make it reasonable to believe that this kind of column is promising in fast and highly effective separation of protein and has great potential for separation of complicated bio-macromolecule samples.2. Eeffect of inner diameter of capillary monolithic column on separation of macromolecules inμ-HPLC. PS-DVB monolithic columns with different ID from 100μm to 320μm were prepared and used for separation of standard proteins. The effects of the ID on various column parameters were studied. It was found that the smaller the column diameter is, the higher the permeability. As to the separation efficiency, it was evident that more theoretical plates can be achieved in less time using the smaller diameter columns. It is interpreted that the interstitial structure of the pores formed during the polymerizing of the polymer matrix depends a lot on the ID of the monolithic column, and the monolithic matrix in-suit polymerized in column with smaller diameter consist of larger clusters and through pores. From the Van Deemter equation simulated for each column, it was found with the increase of the column ID, there is an overall increasing trend for the parameters for eddy dispersion, longitudinal infusion and mass transfer. The monolithic column with samller ID which has larger clusters and through pores gives better performance for protein sparation. These results and discussion will be helpful in practical use of monolithic column for separation of biomacromolecules and development of the theory of the monolithic column.3. Two kinds of polymethacrylate-based monolithic columns, prepared with or without 2-acrylamido-2-methyl-1-propanesulfoni acid (AMPS) in fused silica capillary, were used for rapid analysis of three cyanobacterial toxins (MC-LR, MC-YR and MC-RR) inμ-HPLC and pCEC systems. Under the optimal gradient elution conditions, polybutylmethacrylate monolithic column inμ-HPLC allows the separation of these toxins in less than 9 min with good reproducibility, recovery and satisfied LODs. For the column prepared with AMPS, introduction of sulphonic bands led to a mixed retention mechanism inμ-HPLC for separation of MCs. After optimizing the separation conditions, this method could separate three MCs in 5 min. In pCEC system, the supplementary high pressure could effectively suppress the bubble formation and shorten analysis time. Under the optimized conditions, three MCs could be baseline separated in less than 6 min in an isocratic elution mode. Compared with MCs separation inμ-HPLC, pCEC collude improve the efficiency obviously and gradient elution could also be avoided. pCEC is a flexible and versatile solution to method optimization studying, and there is a great potential that this system could be used as a fast separation tool for routine use in microcystin monitoring.4. A new monolithic column was prepared by a home-made monomer 11-acrylamidoundodecanoic acid (AAUA) and EDMA. The optimization of the preparation was carried out with the assistant of experimental design software. A D-optimal design was performed to evaluate the effect of preparation mixture (concentration of crosslinker, monomer and progens) and optimize the preparation solutions. This method allows one to obtain appropriate data that can be analyzed to study the individual effects and the interaction effects of several factors as well as to determine the optimum conditions through a relatively smaller number of experiments. It was found that the experimental results were in a good aggrement with the predicted value from the software, which proved that this optimization method is successful. The optimized column could provid satisfactory separation of alkylbenzene and alkyl phenyl ketone homologous with good reproducibility. Separation of seven N-methylcarbamates (NMCs) pesticides was also preliminarily studied and a satisfied result was achieved.更多还原