Countercurrent gradient chromatography: A continuous focusing technique

Abstract

The continuous separation of proteins was performed in a countercurrent gradient chromatography (CGC) system. A magnetically stabilized fluidized bed (MSFB) was used to establish true countercurrent contact of a solid resin with a liquid buffer. STable pH gradients were formed in the system in less than 10 min and remained stable throughout the course of the separation experiment (>2 h). The shape of the pH gradient, which ultimately controls the resolution and purity of the separation, can be controlled by making simple adjustments in the interstitial velocities of the liquid and solid phases. We have performed the separation of myoglobin and human serum albumin (HSA) using this device and achieved concentration factors of 1.75 for myoglobin and 1.2 for HSA. A mathematical model that has no adjustable parameters has been developed that predicts the focusing behaviour and capabilities of the CGC system. Using the model, we have estimated the optimum phase velocities, particle diameters, and equilibrium parameters necessary for achieving high purity and high concentrations. (c) 1995 John Wiley & Sons, Inc.

Effect of Hydrodynamic and Magnetic Stabilization on Fluidized-Bed Adsorption

“…Magnetic stabilization of fluidized beds has also been used to reduce axial dispersion and enhance separations (19 − 29). The use of a magnetic field has been shown to limit particle motion in systems containing both a single magnetic support and a mixture of magnetically susceptible and nonmagnetic support material (19, 30 − 31). The mixed‐bed system, though, typically segregates (the two particle types form two distinct layers) when the field is removed.…”