ABSTRACT
This chapter aims to introduce to the researcher standard chromatographic conditions that should be first attempted in the separation of any peptide or protein mixture by size-exclusion, ion-exchange, reversed-phase (RPC) or hydrophobic interaction (HIC) chromatography. Separation of peptides or proteins by a mechanism based solely on molecular size occurs only when there is no interaction between the solutes and the column matrix. The tendency of protein fragments to maintain or reform a particular conformation as opposed to a random coil configuration in non-denaturing media will complicate retention time prediction. Strong cation-exchange chromatography is probably the most useful mode of high-performance ion-exchange chromatography for peptide/protein separations. Peptides and proteins may be removed from the ion-exchange sorbents by either gradient or isocratic elution. The excellent resolving power of RPC has resulted in it becoming the predominant HPLC technique for peptide and protein separations. Protein separations on HIC columns are typically carried out by employing linear decreasing salt gradients at neutral pH.
