| Electrical gradients enhance protein separations in chromatography columns. The goal of this dissertation was to improve a system in which application of an axial electric potential across a liquid chromatography column separates proteins without resorting to a chemical gradient. The research was based on the hypothesis that the size and charge of proteins in a dextran gel column can be moderated using an electric potential so that differences in individual retention, and therefore resolution would be enhanced. Application of a one-dimensional differential material balance that incorporated electrophoretic mobilities predicted protein retention as a function of the charge/mass ratio of proteins and the pore size of an electrically neutral stationary phase. An energy balance incorporating the thermal properties of dextran and Tris-glycine buffers enabled the calculation of radial temperature profiles. This theoretical analysis led to identification of conditions resulting in stable column operation at electrical potentials of 50 to 125 V/cm. Heat transfer and autothermal effects were controlled and a constant temperature maintained in a 15 mm i.d. column for over four hours. These mechanistic models enable the specification of flowrate, pH, buffer composition, mobile phase temperature, stationary phase porosity; and the polarity, duration, and field strength of the applied electric potential required to achieve protein separations in electrochromatography columns. |
