Simultaneous Separation of Negatively and Positively Charged Species in Dynamic Field Gradient Focusing Using a Dual Polarity Electric Field

Abstract

Dynamic field gradient focusing (DFGF) utilizes an electric field gradient established by a computer-controlled electrode array to separate and concentrate charged analytes at unique axial positions. Traditionally, DFGF has been restricted to the analysis of negatively charged species due to limitations in the software of our voltage controller. This paper introduces a new voltage controller capable of operating under normal polarity (positive potentials applied to the electrode array) and reversed polarity (negative potentials applied to the electrode array) for the separation of negatively and positively charged analytes, respectively. The experiments conducted under normal polarity and reversed polarity illustrate the utility of the new controller to perform reproducible DFGF separations (elution times showing less than 1% run-to-run variation) over a wide pH range (3.08 to 8.5) regardless of the protein charge. A dual polarity experiment is then shown in which the separation channel has been divided into normal polarity and reversed polarity regions. This simultaneous separation of negatively charged R-phycoerythrin (R-PE) and positively charged cytochrome c (CYTC) within the same DFGF apparatus is shown.