The growth of industrial biotechnology has had a major impact on the research and development of both analytical and preparative focusing techniques. Pioneering methods such as isoelectric focusing are being modified to overcome the limitations of batch mode operation, scale up difficulties, high power requirement, and excessive heat generation. present advances in focusing techniques have been made by either overcoming the limitations of standard techniques or by investigating new focusing systems. Here we review the background and history of isoelectric focusing and discuss several new focusing techniques including recycle isoelectric (RIEF), counteracting chromatographic electrophoresis (CACE), and countercurrent gradient chromatography (CGC).

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.