Interfacing microchip isoelectric focusing with on-chip electrospray ionization mass spectrometry.

In this work, we demonstrate the interfacing of microchip capillary isoelectric focusing (cIEF) with online mass spectrometric (MS) detection via a fully integrated, on-chip sheath flow electrospray ionization (ESI) emitter. Thanks to the pH-dependent surface charge of the SU-8 polymer cIEF can be successfully run in native SU-8 microchannels without need for surface pretreatment prior to analysis. On the other hand, the inherent electroosmotic flow (EOF) taking place in SU-8 microchannels at high pH can be exploited to electrokinetic mobilization of the focused pH gradient toward the MS and no external pumps are required. In addition to direct coupling of a cIEF separation channel to an ESI emitter, we developed a two-dimensional separation chip for two-step, multiplex cIEF-transient-isotachophoretic (tITP) separation. In this case, cIEF is performed in the first dimension (effective L=20mm) and tITP in the second dimension (L=35mm) followed by ESI/MS. As a result, the migration order is affected by both the pI values (cIEF) and the intrinsic electrophoretic mobilities (tITP) of the sample components. The selectivity of the separation system was shown to be different from pure cIEF or pure ITP, which allows at best for baseline separation of two compounds with nearly identical pI values. The repeatabilities of the migration times of the two-step cIEF-tITP separation were 3.1-6.8% RSD (n=3). Thanks to the short separation channel, relatively short focusing times of 60-270s (depending on the applied focusing potential) were sufficient for establishment of the pH gradient and cIEF separation of the sample components, yielding total analysis times (including loading, focusing, and mobilization) well below 10min.