Ion-exchange chromatography separates molecules based on their respective charged groups. Ion-exchange chromatography retains analyte molecules on the column based on coulombic (ionic) interactions. The ion exchange chromatography matrix consists of positively and negatively charged ions. Essentially,  molecules undergo electrostatic interactions with opposite charges on the stationary phase matrix. The stationary phase consists of an immobile matrix that contains charged ionizable functional groups or ligands. The stationary phase surface displays ionic functional groups (R-X) that interact with analyte ions of opposite charge. To achieve electroneutrality, these immobilized charges couple with exchangeable counterions in the solution. Ionizable molecules that are to be purified, compete with these exchangeable counterions, for binding to the immobilized charges on the stationary phase. These ionizable molecules are retained or eluted based on their charge. Initially, molecules that do not bind or bind weakly to the stationary phase are first to be washed away. Altered conditions are needed for the elution of the molecules that bind to the stationary phase. The concentration of the exchangeable counterions, which competes with the molecules for binding, can be increased, or the pH can be changed to affect the ionic charge of the eluent or the solute. A change in pH affects the charge on the particular molecules and, therefore, alter their binding. When reducing the net charge of the solute's molecules, they start eluting out. This way, such adjustments can be used to release the proteins of interest. Additionally, concentration of counterions can be gradually varied to affect the retention of the ionized molecules, thus separate them. This type of elution is called gradient elution. On the other hand, step elution can be used, in which the concentration of counterions are varied in steps. This type of chromatography is further subdivided into cation exchange chromatography and anion-exchange chromatography. Positively charged molecules bind to cation exchange resins, while negatively charged molecules bind to anion exchange resins. The ionic compound consisting of the cationic species M+ and the anionic species B− can be retained by the stationary phase.
Cation exchange chromatography retains positively charged cations because the stationary phase displays a negatively charged functional group:
Anion exchange chromatography retains anions using positively charged functional group:
Note that the ion strength of either C+ or A− in the mobile phase can be adjusted to shift the equilibrium position, thus retention time.
The ion chromatogram shows a typical chromatogram obtained with an anion exchange column.