ABSTRACT
Liquid chromatography is a dynamic technique for separation of molecules, both analytically and in preparative scale, using liquid as the mobile phase. For separation of proteins and peptides, liquid chromatography is used in various modes depending on the nature of ligands immobilized onto the stationary phase. Ligands on ionexchange resins are charged in a broad pH range. If the resin ligand is positively charged the mode of operation is called anion-exchange chromatography, while cation-exchange chromatography is applied with negatively charged ligands, and
ion-exchange chromatography is basically used to separate proteins based on their overall or local difference in charge. In analytical liquid chromatography, loading of the chromatographic column is very low to obtain the best separation of the distinct molecules present in the sample, and various elution gradients and modifi ers are applied to achieve maximum resolution. In preparative liquid chromatography, high column loading is applied to get high productivities and good process economy, and a separation is developed to obtain optimal purity and yield of the target protein or peptide. Gradients and modifi ers are applied in the simplest way possible to obtain the desired purity and yield. The nature of gradients in ion-exchange chromatography is linked to the mode of operation, e.g., salt or pH and occasionally organic solvent, displacers, or other modifi ers to increase selectivity in bind-and-elute or fl ow-through modes of operation. These operational modes may be combined with each other and with displacement effects, and the transition between the two modes of operation is not well defi ned.
