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

本课题旨在通过建立简单、全面的二维高效液相色谱，提高高效液相色谱的峰容量。通过在二维（2d）上使用单片二氧化硅- c18柱，我们能够获得以下结果。(1)我们以氟烷基键合二氧化硅的颗粒填充柱作为第一维（1 -d）柱，C18-或五溴苄氧基丙基键合整体二氧化硅作为第二维（2 -d）柱，以反相反相模式证明了简单而全面的二维高效液相色谱的概念。(2)利用有机溶剂的选择性差异，采用C18硅胶整体柱在第1 - d和第2 - d进行了2D-HPLC分析。(3)二维高效液相色谱（2D-HPLC）在第1 - d采用填充聚合物颗粒的离子交换柱，在第2 - d采用硅胶- c18整体柱或硅胶整体毛细管柱进行多肽分离。这些系统的使用产生了(1)甲醇-水流动相的峰值容量高达1100，(2)碳氢化合物和苯衍生物的峰值容量分别为700或更高。离子交换反相2D-HPLC系统提供了(3)在第2 - d使用短整体硅胶柱的牛血清白蛋白酶解峰容量为700或更高，在第2 - d使用0.2 mmlD， 10 cm长的毛细管柱的牛血清白蛋白酶解峰容量为500左右。试图优化二维高效液相色谱条件，以显示在1小时内达到峰值容量大于2000的可能性。结果表明，流速、溶质保留因子和柱长是影响二维高效液相色谱系统峰容量的主要因素。整体硅胶柱具有高渗透性和高流动相线速度下的高效液相色谱特性，是二维高效液相色谱的最佳选择。

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.