Countercurrent gradient chromatography (CGC): Continuous multicomponent separations using induced gradients.

The growth of industrial biotechnology has had a major impact on the research and development of both analytical and preparative focusing techniques. Pioneering techniques such as isoelectric focusing are being modified to overcome the past 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 developing new focusing systems. We have developed a new focusing technique called countercurrent gradient chromatography (CGC) that is able to continuously focus solutes such as proteins and amino acids in a stable pH gradient without a high power requirement or the need of special chemicals. CGC is a continuous focusing device that uses countercurrent flow of a solids and a liquid phase combined with an imposed gradient to focus solutes in an ordinary chromatography column. Countercurrent flow allows for the continuous exchange of H$\sp+$ ions between a solid ion exchange resin and a liquid buffer resulting in the formation of a time invariant or stable pH gradient. Proteins can then be separated near their isoelectric points in these stable pH gradients; the proteins remain in dynamic equilibrium with the solids and liquid phases and are continuously focused near their isoelectric points along the gradient. The continuous separation of proteins was performed using CGC. Stable pH gradients were formed in the system in less than 10 minutes and remained stable throughout the course of the separation experiment ($>$2 hours). 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 solids phases. The separation of various proteins (myoglobin, human serum albumin, ovalbumin, lysozyme, and bovine serum albumin) using this device was performed and under ideal conditions, a concentration factor of $\sim$5 times was achieved. A mathematical model that has no adjustable parameters has been developed that predicts quite well the focusing behavior and capabilities of the CGC system. From the model, the optimum phase velocities, particle diameters, and equilibrium parameters necessary for achieving high purity and high concentrations were determined.PhDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/104608/1/9542821.pdfDescription of 9542821.pdf : Restricted to UM users only