New system for preparative electrochromatography of proteins.
Electrochromatography employs an axial electric field across a chromatographic stationary phase to separate proteins and other molecules based on differences in electrophoretic mobility. Because the separation is electrically driven, the need for additional chemical reagents is reduced. Two major impediments to scale-up of electrochromatography columns, removal of heat and electrolysis gases, have historically limited the diameter of packed columns to 2.5 cm ID with volumes of approximately 55 mL. We report a novel electrochromatography column that effectively removes electrolysis gases and minimizes heating. A vital component of this system is a new electrode design that couples a platinum gauze with an ultrafiltration membrane across both ends of the column. Use of a methacrylate base stationary phase enabled axial voltage gradients of 10 to 20 V/cm. Thermocouples inserted radially in the column at four axial positions showed that the flow of a 4 degrees C mobile phase coupled with heat conduction through the column walls controlled the temperature to 28 degrees C. The new column design, with dimensions of 3.81 cm ID x 38.1 cm long and bed volume of 400 mL, was demonstrated by separating mixtures of BSA and myoglobin. The column was operated in a horizontal position with radial sample injection and withdrawal at the ends of the packed bed. These experiments are a first step in demonstrating that scale-up of electrochromatography columns can be achieved by choosing appropriate flow rates, voltage gradients, and stationary phase.
Keim
C
C
Laboratory of Renewable Resources Engineering and Department of Agricultural and Biological Engineering, Purdue University, Lafayette, Indiana.
Ladisch
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M
D002845:Chromatography / Q000379:methods*; D004553:Electric Conductivity; D004566:Electrodes; D004586:Electrophoresis / Q000379:methods*; D009211:Myoglobin / Q000302:isolation & purification; D009995:Osmosis; D010649:Phenylalanine / Q000302:isolation & purification; D012710:Serum Albumin, Bovine / Q000302:isolation & purification; D013696:Temperature; D013997:Time Factors; D014462:Ultrafiltration
D009211:Myoglobin; D012710:Serum Albumin, Bovine; D010649:Phenylalanine