COUNTERCURRENT GRADIENT CHROMATOGRAPHY - A CONTINUOUS FOCUSING TECHNIQUE

The continuous separation of proteins was performed in a countercurren t gradient chromatography (CGC system. A magnetically stabilized fluid ized 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 cour se 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 ve locities of the liquid and solid phases. We have performed the separat ion of myoglobin and human serum albumin (HSA) using this device and a chieved concentration factors of 1.75 for myoglobin and 1.2 for HSA. A mathematical model that has no adjustable parameters has been develop ed that predicts the focusing behavior and capabilities of the CGC sys tem. 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.