On-Chip Coupling of Isoelectric Focusing and Free Solution Electrophoresis for Multidimensional Separations

We have developed an acrylic microfluidic device that sequentially couples liquid-phase isoelectric focusing (IEF) and free solution capillary electrophoresis (CE). Rapid separation (<1 min) and preconcentration (73×) of species were achieved in the initial IEF dimension. Using full-field fluorescence imaging, we observed nondispersive mobilization velocities on the order of 20 )mu)m/s during characterization of the IEF step. This transport behavior allowed controlled electrokinetic mobilization of focused sample bands to a channel junction, where voltage switching was used to repeatedly inject effluent from the IEF dimension into an ampholyte-based CE separation. This second dimension was capable of analyzing all fluid volumes of interest from the IEF dimension, as IEF was ʹparkedʹ during each CE analysis and refocused prior to additional CE analyses. Investigation of each dimension of the integrated system showed time-dependent species displacement and band-broadening behavior consistent with IEF and CE, respectively. The peak capacity of the 2D system was ~1300. A comprehensive 2D analysis of a fluid volume spanning 15% of the total IEF channel length was completed in less than 5 min.