ABSTRACT
Ion-exchange chromatography (IEC) is a flexible and powerful analytical tool for aiding purification process development, performing in-process monitoring, and documenting final product quality. The primary retention mechanism in IEC is simple: electrostatic interactions. Anion exchangers carry a positive charge. Negatively charged molecules are retained; the more strongly electronegative, the more strongly they are retained. The opposite is true for cation exchangers. In either case, retained molecules can be eluted in order of increasing retention. The most common method of elution is by means of a salt gradient in which an increasing concentration of dissolved ions eventually outcompete the bound analyte for the support. Weakly bound analytes elute first, followed by analytes that are more strongly bound. A less common method of elution is to suspend the attraction between the column and the bound analyte by changing the charge on the analyte. This is accomplished by changing the pH of the mobile phase. In the case of amphoteric molecules such as proteins, for example, reducing the pH reduces negative charge and increases positive charge, thereby weakening the attraction to anion exchangers. The opposite is true for cation exchangers.
