ABSTRACT
Reversed-phase high-performance liquid chromatography (RPC) has been the preferred technique for the separation and purification of polypeptides on the analytical as well as preparative scale. Like all other chromatographic techniques, RPC utilizes the ability to manipulate the binding forces between sample molecules and a stationary phase. Although this manipulation in RPC is performed almost universally at acid pH in solvents containing various amounts of potential toxic or harmful organic solvents, the number of literature reports describing partial or total loss of biological activity is in no way overwhelming. The main steps in RPC purification of polypeptides are binding of the sample to the stationary phase, shorter or longer fixation period with increasing amount of organic solvents and, finally, elution from the column followed by an isolation procedure in order to obtain the sample free of mobile phase contamination. The chapter also provides a conclusive strategy for reversed-phase-based purification and isolation of polypeptides retaining full bioactivity.
