Gradient Elution Moving Boundary Electrophoresis with Channel Current Detection

Abstract

Gradient elution moving boundary electrophoresis (GEMBE) is a recently described technique for electrophoretic separations in short (1-3 cm) capillaries or microchannels. With GEMBE, the electrophoretic migration of analytes is opposed by a bulk counterflow of separation buffer through the separation channel. The counterflow velocity is varied over the course of a separation so that analytes with different electrophoretic mobilities enter the separation channel at different times and are detected as moving boundary, stepwise increases in the detector response. The resolution of a GEMBE separation is thus dependent on the rate at which the counterflow velocity is varied (rather than the length of the separation channel), and relatively high resolution separations can be performed with short microfluidic channels or capillaries. In this paper we describe an implementation of the GEMBE technique in which a very short (2.5-3.5 mm) capillary or microchannel is used as both the separation channel and a conductivity detection cell. Because the channel is so short, only a single moving boundary "step" is present in the channel at any given time, and the measured current through the channel can therefore be used to give a signal comparable to what is normally generated by more complicated detector arrangements. A theoretical description of the new technique is given along with simulation and experimental data relevant to the optimization of the method parameters such as channel length, counterflow acceleration, and applied field strength. A key theoretical prediction is that although this technique is expected to be a factor of 10 or 20 slower than conventional capillary zone electrophoresis, separation times of the order 1 s or less can still be achieved, making it applicable for ultrahigh-throughput analyses when implemented in a multiplexed format.