Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic.
J Sep Sci. 2019 Feb;42(3):670-677. doi: 10.1002/jssc.201801085. Epub 2018 Dec 11.
Two-dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On-line coupling of a reversed-phase column with an aqueous normal-phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on-line two-dimensional liquid chromatography needs a capillary or micro-bore column providing low-volume effluent fractions transferred to a short efficient second-dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro-columns in fused silica capillaries with structurally different dimethacrylate cross-linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed-phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on-line an organic polymer monolithic capillary column in the first dimension with a short silica-based monolithic column in the second dimension provides two-dimensional liquid chromatography systems with high peak capacities. The silica monolithic C columns provide higher separation efficiency than the particle-packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed-phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.
二维液相色谱在单次运行中极大地增加了分离化合物的数量，理论上最多可达柱上不同选择性分离的峰的乘积。反相柱与水相正相（亲水相互作用液相色谱）柱的在线偶联可得到具有高峰容量的正交系统。快速在线二维液相色谱需要毛细管或微径柱，以提供低体积的洗脱馏分，转移到短高效二维柱中，以高流动相流速进行分离。我们在熔融石英毛细管中制备了具有不同结构的二甲基丙烯酸酯交联剂的聚甲基丙烯酸酯两性离子整体微柱。该柱提供双重保留机制（亲水相互作用和反相）。通过设定流动相组成，可以调整对各种极性物质类别的分离选择性。在第一维中将有机聚合物整体毛细管柱与第二维中的短硅胶整体柱在线偶联，提供具有高峰容量的二维液相色谱系统。硅胶整体 C 柱在第二维中使用高达 5 mL/min 的流速时，比颗粒填充柱具有更高的分离效率。减小硅胶整体柱的直径允许在最高操作压力下使用更高的流速，并从第一维（亲水相互作用）转移到第二维（反相）模式的更小的馏分体积，避免流动相兼容性问题，提高分辨率，增加峰容量和峰生产速率。
