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峰容量来提高高效液相色谱的峰容量。通过在第二维(2-D)使用整体SiO_2-C18柱，我们能够获得以下结果。(1)通过使用氟烷基键合硅胶的颗粒填充柱作为一维(1-D)柱，以及C18或五溴苄氧丙基键合整体硅胶作为第二维柱，我们以反相-反相模式证明了简单而全面的2D-HPLC的概念。(2)通过利用有机溶剂提供的选择性的差异，使用硅胶C18整体柱在1-D和2-D进行了2D-HPLC分析。(3)在1-D使用填充聚合物颗粒的离子交换柱，在2-D使用整体硅胶-C18柱或整体硅胶毛细管柱进行多肽分离。这些系统的使用产生了(1)在甲醇-水流动相中的峰容量高达1100，(2)烃类和苯衍生物的峰容量分别达到700或更大。离子交换-反相2D-HPL C系统对BSA消化的峰容量为700或更大，用短的整体硅胶色谱柱在2-D，用0.2 mm×10 cm长的毛细管柱在2-D时，BSA的峰容量约为500。对2D-HPLC条件进行了优化，以显示在一小时内达到大于2000的峰容量的可能性。结果表明，流速、溶质保留因子和二维柱长度是影响2D-HPLC峰容量的主要因素。整体硅胶柱因其高渗透性和高流动相线速度下的高性能而被认为是最适合于2D-HPL的柱。

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.