Rapid comprehensive two-dimensional separations of peptides via RPLC-optically gated capillary zone electrophoresis

Coupled-column two dimensional (2D) separation systems offer potentially high peak capacity and are amenable to automation. Reversed phase liquid chromatography (RPLC) and capillary zone electrophoresis (CZE) are complementary techniques well suited for use in a 2D system. Optically gated CZE is a means of performing rapid CZE analyses. In a 2D system, these rapid CZE analyses enable more frequent sampling of the RPLC separation and thus more freedom in control of the RPLC analysis conditions. Complete 2D separations can be done in the time usually required to do the RPLC analysis alone. With the present system, a complete 2D analysis can be done in under 10 min. Because the peak capacity of a 2D method is inherently high, some of the available peak capacity can be exchanged for speed of analysis. Acceleration of the RPLC elution gradient will decrease analysis time, but RPLC resolution will suffer. However, because it is a 2D system, some of the resolution lost in the RPLC can be regained in the second dimension CZE analysis. Here, the RPLC gradient was done over only 2 min, but CZE analyses done every 2.5 s restore some of the resolution lost in the rapid RPLC gradient.