Displacement chromatography using the UNO continuous bed column as a stationary phase
Displacement separations of biopolymers are even more restricted in regard to the upper applicable limit of the flow rate than elution chromatographic ones, when conventional columns packed with porous particles are used. Bio-Rad has recently introduced a new column type, the UNO column, which contrary to conventional HPLC columns is not packed with particles but consists of a continuous porous polymer rod. The plate height of this continuous bed column was found to be nearly independent of the flow rate within the investigated range (0.01-4.5 mL/min, 1.56-701.6 cm/h). The strong ion exchanger columns UNO Q1 (7 x 35 mm, 1.3 mL) and UNO Q6 (12 x 53 mm, 6 mL) were investigated as stationary phases for protein displacement chromatography at elevated flow rates. With the UNO column, displacement separations of alpha-lactalbumin and beta-lactoglobulin (displacer: poly(acrylic acid), Mw 5100) were still possible at flow rates that were 1 order of magnitude higher than those applicable to conventional columns of similar dimensions. In fact the flow rate was limited by the necessity to collect the protein-containing fraction fast enough for an adequate monitoring of the separation rather than by a loss in resolution. While the UNO Q1 column did not allow for the full development of the displacement train, 50 mg of protein could be separated using the UNO Q 6 column. Recoveries were well over 95% in this case. More than 50% of the alpha-lactalbumin was collected in pure and concentrated form (concentration by a factor of 2). The steric mass action model was used to optimize the displacer concentration.
Laboratoire de Biotechnologie Cellulaire, Institut de Genie Chimique IV, ETH Lausanne, 1015 Ecublens, Switzerland.
S
Vogt
R
Freitag