ABSTRACT
Ion-exchange chromatography is a popular technique for the analysis and purification of proteins and peptides. High-performance ion-exchange chromatography (IEC) is more attractive than low pressure techniques because it yields better resolution than classical column methods in 10 to 20% of the time with equivalent or higher loading capacities and recoveries. The IEC packings which are currently available contain ionic functional groups grafted onto a base material composed of silica or a polymer which is suitable for high-performance liquid chromatography. Most of the surface area of a porous IEC support is within the pores and it is generally inversely related to the pore diameter. Salt composition, pH, gradient slope, additives, and temperature can all determine the effectiveness of an IEC procedure. The pH of a protein provides a guideline to selection of both an appropriate IEC column and an initial pH for the mobile phase. The critical effects of pH in IEC necessitate its control during a gradient for reproducibility.
