大管电泳及其多维分离系统的研究
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摘要
毛细管电泳(CE)作为当前分离效率最高的一种分离技术,在诸多领域得到了广泛的应用。但随着科技的快速发展,常常需要对目标物进一步深入研究,而CE样品用量小的特点便成为拓展其应用的技术瓶颈,正因为如此CE目前仅被局限于分析水平,很难实现各分离组分的进一步转移、收集甚至多维高分辨分析。
首先,为提高CE载样量,本文以大内径石英管(1.0 mm)为电泳分离通道,并将内制冷机制引入到大管电泳(WBE)中以消除电泳过程中产生的焦耳热。同时,借助中空纤维膜的支撑,将内制冷毛细管准确固定于大管轴向中心,以此强化电泳系统的散热性能,此法显著提高了WBE系统的分离效率。此外,本文还探索了大管电泳进样方式及大管电泳的应用。结果表明,大管电泳电进样是可行的,且WBE双侧压力进样可承载1.6μL左右的样品,如此大的载样量使其具备了作为半制备或制备的能力。
其次,高载样量预示着WBE可能实现样品转移再分析,为此,我们利用大管及毛细管内外径尺寸的差异,实现了WBE-CE转移系统的零死体积连接。在此基础上,利用场放大效应及独特的三电极模式,实现了大管电泳样品在线转移和再分离。与此同时,随着WBE-CE多维分析系统的搭建以及碱性磷酸酶催化对硝基苯磷酸二钠这一酶反应模型的验证,证明了WBE-CE多维分析系统可集分离、转移、收集、储存、离线及在柱酶反应于一体,该系统为生物样品的快速分离及深入分析搭建了实验平台。
第三,本文还利用大管电泳独立的进样系统,搭建了二维高效液相色谱(HPLC)-大管电泳系统。HPLC流出物的直接WBE分析预示着此二维分离系统避免了商品化离线HPLC-CE分析仪在样品收集、浓缩、复溶带来样品变质等潜在风险。同时,利用液相手动进样阀实现了HPLC-WBE二维在线联用,并设计了具多根进样毛细管的大管系统,通过延长空间间距缩短了连续WBE进样时间间隔,实现了二维连续分析。
总之,本文多种多维系统的成功搭建为广义的WBE提供了更为广博的适用空间,为WBE的发展提供了实验基础,为复杂样品的快速分析提供了实验平台。
CE is one of the most efficient separation technologies, which has been extensively accepted and acknowledged in many fields. With the technology development, further study of the target has become a popular trend. However, the small sample loading capacity of the CE has turned to be its technical bottleneck in expanding its application. Consequently, CE has been restricted in analysis level so that it is difficult for CE in further process, such as transfer, collection, and even analysis through multi-dimensional high-resolution system.
Firstly, for improvement the sample loading capacity, we adopted wide-bore tube with 1.0 mm id as separation channel, and introduced inner cooling mechanism into wide bore electrophoresis (WBE) to eliminate the Joule heat produced during the electrophoresis process. In WBE system, the inner cooling capillary was exactly fixed to the axial center of the wide-bore tube with the help of two hollow fiber membranes to strengthen the radiating efficiency. The result manisfested the method could improve the separation efficiency of the WBE. In addition, the sampling method based on WBE was explored and some samples were analyzed in WBE by hydrodynamic and electrokinetic injection. The results indicated the electrokinetic injection was feasible in WBE and the maximum sample loading amount was about 1.6μL by two-side hydrodynamic injection, which implied the WBE could be used as semi-preparing or preparing CE.
Secondly, the larger sample loading capacity entitled the wide-bore electrophoresis system could be taken on sample transfer and further analysis operations. Taking advantage of the disparity between the inside diameter of wide-bore tube and the outside diameter of capillary, the two dimensional WBE-CE transfer system was built and zero dead volume connection was realized. Some model compounds certified it was feasible and controlled when the special three electrodes mode was adopted and field amplification was introduced. On the basic of the WBE-TC system, a multi-dimensional WBE-CE analysis platform integrated separation, transferring, collection, storage, offline and online enzyme reaction was built up. The alkaline phosphatase reaction model certified its feasibility. The results established the experimental foundation for the study of the wide-bore microchip. The experimental platform can facilitate the biological samples rapid separation and further analysis.
Thirdly,another aim on establishing WBE system is to simplify two dimensional systems, especially HPLC-CE. WBE can directly separate and detect the effluent from HPLC, which avoided the sample deterioration during the collection, dried, or reconstitution processes in offline HPLC-CE. With the help of an injection valve, online two dimensional HPLC-WBE systems was built by coupling high performance liquid chromatography and the WBE system with large loadability and independent sampling system an and was called. Furthermore, the many sampling capillaries inWBE system design which can convert the space into time and shorten the interval in continue HPLC-WBE analysis. The current serial WBE analysis systems will broaden the application of electrophoresis technology, supply experimental basement for its development and made it possible for the analysis of complicated biological samples.
首先,为提高CE载样量,本文以大内径石英管(1.0 mm)为电泳分离通道,并将内制冷机制引入到大管电泳(WBE)中以消除电泳过程中产生的焦耳热。同时,借助中空纤维膜的支撑,将内制冷毛细管准确固定于大管轴向中心,以此强化电泳系统的散热性能,此法显著提高了WBE系统的分离效率。此外,本文还探索了大管电泳进样方式及大管电泳的应用。结果表明,大管电泳电进样是可行的,且WBE双侧压力进样可承载1.6μL左右的样品,如此大的载样量使其具备了作为半制备或制备的能力。
其次,高载样量预示着WBE可能实现样品转移再分析,为此,我们利用大管及毛细管内外径尺寸的差异,实现了WBE-CE转移系统的零死体积连接。在此基础上,利用场放大效应及独特的三电极模式,实现了大管电泳样品在线转移和再分离。与此同时,随着WBE-CE多维分析系统的搭建以及碱性磷酸酶催化对硝基苯磷酸二钠这一酶反应模型的验证,证明了WBE-CE多维分析系统可集分离、转移、收集、储存、离线及在柱酶反应于一体,该系统为生物样品的快速分离及深入分析搭建了实验平台。
第三,本文还利用大管电泳独立的进样系统,搭建了二维高效液相色谱(HPLC)-大管电泳系统。HPLC流出物的直接WBE分析预示着此二维分离系统避免了商品化离线HPLC-CE分析仪在样品收集、浓缩、复溶带来样品变质等潜在风险。同时,利用液相手动进样阀实现了HPLC-WBE二维在线联用,并设计了具多根进样毛细管的大管系统,通过延长空间间距缩短了连续WBE进样时间间隔,实现了二维连续分析。
总之,本文多种多维系统的成功搭建为广义的WBE提供了更为广博的适用空间,为WBE的发展提供了实验基础,为复杂样品的快速分析提供了实验平台。
CE is one of the most efficient separation technologies, which has been extensively accepted and acknowledged in many fields. With the technology development, further study of the target has become a popular trend. However, the small sample loading capacity of the CE has turned to be its technical bottleneck in expanding its application. Consequently, CE has been restricted in analysis level so that it is difficult for CE in further process, such as transfer, collection, and even analysis through multi-dimensional high-resolution system.
Firstly, for improvement the sample loading capacity, we adopted wide-bore tube with 1.0 mm id as separation channel, and introduced inner cooling mechanism into wide bore electrophoresis (WBE) to eliminate the Joule heat produced during the electrophoresis process. In WBE system, the inner cooling capillary was exactly fixed to the axial center of the wide-bore tube with the help of two hollow fiber membranes to strengthen the radiating efficiency. The result manisfested the method could improve the separation efficiency of the WBE. In addition, the sampling method based on WBE was explored and some samples were analyzed in WBE by hydrodynamic and electrokinetic injection. The results indicated the electrokinetic injection was feasible in WBE and the maximum sample loading amount was about 1.6μL by two-side hydrodynamic injection, which implied the WBE could be used as semi-preparing or preparing CE.
Secondly, the larger sample loading capacity entitled the wide-bore electrophoresis system could be taken on sample transfer and further analysis operations. Taking advantage of the disparity between the inside diameter of wide-bore tube and the outside diameter of capillary, the two dimensional WBE-CE transfer system was built and zero dead volume connection was realized. Some model compounds certified it was feasible and controlled when the special three electrodes mode was adopted and field amplification was introduced. On the basic of the WBE-TC system, a multi-dimensional WBE-CE analysis platform integrated separation, transferring, collection, storage, offline and online enzyme reaction was built up. The alkaline phosphatase reaction model certified its feasibility. The results established the experimental foundation for the study of the wide-bore microchip. The experimental platform can facilitate the biological samples rapid separation and further analysis.
Thirdly,another aim on establishing WBE system is to simplify two dimensional systems, especially HPLC-CE. WBE can directly separate and detect the effluent from HPLC, which avoided the sample deterioration during the collection, dried, or reconstitution processes in offline HPLC-CE. With the help of an injection valve, online two dimensional HPLC-WBE systems was built by coupling high performance liquid chromatography and the WBE system with large loadability and independent sampling system an and was called. Furthermore, the many sampling capillaries inWBE system design which can convert the space into time and shorten the interval in continue HPLC-WBE analysis. The current serial WBE analysis systems will broaden the application of electrophoresis technology, supply experimental basement for its development and made it possible for the analysis of complicated biological samples.
引文
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