Development of comprehensive two-dimensional HPLC for high-performance separations in the future

本研究的目的是通过开发简单而全面的二维高效液相色谱仪来提高高效液相色谱仪的峰容量。通过在第二维（2nd-D）采用整体式二氧化硅-C18柱，我们能够获得以下结果。(1)我们证明了简单和全面的二维高效液相色谱的概念，在反相-反相模式，通过使用氟烷基键合的二氧化硅颗粒填充柱作为第一维度（1st-D）柱，和C18-或五溴苄氧基丙基键合的整体硅胶作为2nd-D柱。(2)通过利用有机溶剂提供的选择性差异，使用整体式硅胶C18柱在1st-D和2nd-D处进行2D-HPLC。(3)本文报道了用高分子颗粒填充的离子交换柱和硅胶C18整体柱或硅胶毛细管整体柱进行2D-HPLC分离多肽的方法，结果表明：（1）在甲醇-水移动的相中峰容量达1100，（2）烃类和苯衍生物的峰容量分别达700以上。离子交换-反相2D-HPLC系统提供（3）对于BSA消化物，在2nd-D处使用短整体硅胶柱的峰容量为700或更大，并且对于BSA消化物，在2nd-D处使用0.2mm ID、10 cm长的毛细管柱的峰容量分别为约500。尝试优化2D-HPLC条件以显示在1小时内达到大于2000的峰容量的可能性。流速、溶质保留因子和2nd-D处的柱长被证明是2D-HPLC系统的峰容量的主要因素。整体硅胶柱被认为是最适合的二维高效液相色谱柱的基础上，他们的高渗透性和高性能在高的线速度的移动的相。

The objective of this research project was to increase peak capacity of HPLC by developing simple and comprehensive two dimensional (2D) HPLC. By employing a monolithic silica-C18 column at the second dimension (2nd-D), we were able to obtain the following results.(1) We proved the concept of simple and comprehensive 2D-HPLC in a reversed-phase-reversed-phase mode by using a particle-packed column of fluoroalkyl-bonded silica as a first-dimension (1st-D) column, and C18- or pentabromobenzyloxypropyl-bonded monolithic silica as a 2nd-D column.(2) By utilizing the difference in selectivity provided by organic solvents, 2D-HPLC was carried out using monolithic silica C18 columns both at 1st-D and at 2nd-D.(3) 2D-HPLC was carried out by using an ion-exchange column packed with polymer particles at 1st-D and a monolithic silica-C18 column or a monolithic silica capillary column at 2nd-D for peptide separations.The use of these systems produced (1)peak capacity of up to 1100 in methanol-water mobile phase, (2)peak capacity of 700 or greater for hydrocarbons and benzene derivatives, respectively. The ion-exchange-reversed-phase 2D-HPLC system provided (3)peak capacity of 700 or greater for BSA digest with a short monolithic silica column at 2nd-D, and peak capacity of about 500 for BSA digest by using a 0.2 mmlD, 10 cm long capillary column at 2nd-D, respectively. Optimization of 2D-HPLC conditions were attempted to show the possibility of achieving peak capacity of greater than 2000 in one hour. The flow rate, retention factors of solutes, and column length at 2nd-D were shown to dominate the peak capacity of a 2D-HPLC system. Monolithic silica columns were found to be best suited for 2D-HPLC based on their high permeability and high performance at high linear velocity of mobile phase.