Digitally Controlled Electrophoretic Focusing

Proteins can be simultaneously separated and concentrated by applying a constant force and opposing this with a gradient in a second force. In this work, a constant hydrodynamic force is opposed by a gradient in the electric field which allows charged molecules to focus in order of their apparent electrophoretic mobilities. The electric field gradient is established and maintained using an array of electrodes whose voltages are individually monitored and adjusted by a computer-controlled circuit board. The computer-generated electric field gradient allows charged molecules to be focused without using a pH gradient. Since the proteins are not focused at their p I s, precipitates do not form, so focused concentrations in excess of 50 mg/mL are not unusual. In addition, since the field shape is dynamically controlled from the computer on a point-by-point basis, the field profile can be adjusted during a run to improve the resolution. In this paper, the column and controller are described together with experimental results and a model which illustrates the separating power and flexibility of this technique.