On a separation voltage polarity switching transient capillary isotachophoresis method for higher sample loading capacity and better separation performance

Limited sample loading capacity is one of the major reasons that prevents the utility of capillary electrophoresis (CE) as a routine separation method as compared to liquid chromatography (LC). In our previous study, a separation voltage polarity switching transient capillary isotachophoresis (PS-tCITP) was proposed. Both sample loading capacity and separation resolution could be improved by using a single PS-tCITP instead of routine transient capillary isotachophoresis (tCITP). In this study, a detailed investigation on optimization strategy of PS-tCITP method was performed systematically. A possible mechanism of the sample preconcentration in multiple PS-tCITP was first proposed to better understanding the multiple PS-tCITP process. Several optimization experiments were then performed, including single PS-tCITP, paused PS-tCITP and multiple PS-tCITP, sequentially by using a mixture of five peptides. By selecting an optimum polarity switching time, sample loading capacity of 100% capillary volume could be achieved in a single PS-tCITP. Introducing an additional pause between each polarity switching in a single PS-tCITP further improved the separation resolution. Experimental results showed a baseline separation of five selected peptide standards at 100% sample loading volume by using a 100 min pause in a single PS-tCITP. To further improve separation efficiency while still maintaining a 100% sample loading volume, a multiple PS-tCITP technique was developed through this study. Compared to the separation performance of the optimal single PS-tCITP at 100% sample loading volume with 10 min pause, the separation window was improved 54% and peak capacity was improved 48% in the optimal four PS-tCITP with the same sample loading volume and pause.