Scale up of electrochromatography

Craig Alan Keim, Purdue University

Abstract

Electrochromatography employs an axial electric field across a chromatographic stationary phase to separate molecules based on differences in electrophoretic mobility. Although electrochromatography columns have existed for over 40 years, ineffective removal of heat and electrolysis gases have historically limited the scale-up of these columns to 2.5cm ID with sample volumes of less than 0.5mL. Several new electrode designs were tested that employed either agarose or polyacrylamide gels. In all cases, the gel based electrodes suffered from excessive heating in the gel. However, an ultrafiltration membrane and platinum gauze electrode design reduced both electrolysis gas formation and heating in the electrode. To study column-heating effects, a new column was designed with thermocouple ports at four axial positions. In each of these ports specially sheathed thermocouples were used to measure the temperature at the radial center of the column. A transient two-dimensional temperature model was developed to predict the temperature rise in the column. From both model prediction and experimental data, it was determined that heating in the electrochromatography column could be substantially reduced by using a non-conductive methacrylate base stationary phase with a low conductivity (3.9mM tris-47mM glycine) buffer. The resolving power of this new column (3.81cm ID x 38.1 cm long) was demonstrated by separating mixtures of BSA and myoglobin. Careful selection of operating conditions yielded a separation of 19.5mL of a mixture containing BSA and myoglobin on a 400mL column. Further scale up of the separation experienced problems with excessive heating at the column inlet. The most likely cause of this heating was dissolved CO2 that entered the column with the sample. However, decreased column heating and increased sample loading was achieved if an injection of 0.1M NaCl was made after the initial protein sample injection. Using a second salt injection, a baseline separation of 49mL of bovine serum albumin and myoglobin was achieved. Lower overall column temperature and higher resolution were obtained over traditional electrochromatography operation. Based on the heat transfer model, this column could be scaled to a 10.8cm ID x 38.1cm long column capable of processing over 390mL of sample per run.

Degree

Ph.D.

Advisors

Ladisch, Purdue University.

Subject Area

Chemical engineering|Agricultural engineering

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