Recycling and screen-segmented column isotachophoresis, two free-fluid approaches for fractionation of proteins.

Recycling and screen-segmented column isotachophoresis (ITP), two approaches for the milligrams to grams preparative-scale purification of proteins, are discussed and compared. Recycling ITP was performed in a recycling free-flow focusing apparatus. In this process, fluid flows rapidly through a narrow channel and the effluent from each channel is reinjected into the electrophoresis chamber through the corresponding input port. The residence time in the cell is of the order of 1 s per single pass, which does not allow complete separation, so recycling is essential to attain the steady state. Immobilization of the advancing zone structure is obtained via a controlled counterflow. Thirty fractions of about 4 ml each are obtained. Column ITP was executed in a Rotofor apparatus and in a similar column operated vertically and without rotation. These instruments feature a screen-segmented annular separation space with twenty subcompartments of about 2 ml each. With both approaches, the collected fractions were analysed separately for conductivity, pH and UV absorbance. Selected fractions were characterized by analytical electrophoretic methods. Examples presented include the cationic and anionic ITP behaviour of model proteins, including bovine serum albumin, ovalbumin and ribonuclease A, and the ITP removal of the major impurities from a commercial ovalbumin sample. These examples revealed that the screen-segmented column is suitable for ITP protein purification and operates optimally in a horizontal rotating mode and without internal cooling. The recycling experiments showed that counterflow improves separation and the steady-state patterns are dependent on the fluid layer thickness in the separation cell but, with a given gap, essentially independent of applied current and recycling pump rate.