Step width, spacing, and resolution in gradient elution moving boundary electrophoresis. Part 1. Theory and comparison with zone electrophoresis.

Gradient elution moving boundary electrophoresis (GEMBE) is a recently described technique for electrophoretic separations in short capillaries or microchannels. With GEMBE, the electrophoretic migration of analytes is opposed by a bulk counterflow of separation buffer. The counterflow velocity is varied over the course of a separation so that analytes with different mobilities enter the separation channel at different times and are detected as moving boundary, step-wise increases in the detector response. The resolution of a GEMBE separation is thus dependent on the rate at which the counterflow velocity is varied, and relatively high-resolution separations can be performed with short microfluidic channels or capillaries. In this paper, a theoretical description of GEMBE is presented that can be used for calculation of expected resolution and for optimization of GEMBE separations. A comparison is made with CZE, a conventional electrophoretic separation technique for which the theoretical understanding is mature. The results indicate that the electric field strength and separation channel length are important parameters for both CZE and GEMBE. However, with GEMBE, the counterflow acceleration is also available as a parameter that can be easily adjusted for optimization of the trade-off between resolution and separation time. This allows for optimization of GEMBE separations by making changes only to the software controlling the apparatus rather than to the hardware of the apparatus itself. Further comparison of the theoretical descriptions of GEMBE and CZE indicates that the time required to achieve a desired resolution is equivalent for the two techniques.