Ion-exchange chromatography, or IEC, is a technique for separating ions based on their affinity for the stationary phase. The stationary phase is a cross-linked polymer resin with covalently attached ionic functional groups. The functional groups can be either positively charged (cation exchangers) or negatively charged (anion exchangers). A cation exchanger consists of a polymeric anion and active cations, while an anion exchanger is a polymeric cation with active anions. The choice of stationary phase depends on the analytes' nature and the desired separation.

The mobile phase, typically an aqueous buffer, flows through the stationary phase, and the pH of the buffer plays a crucial role in determining the retention time of the solutes. From the equilibration to wash steps, pH is selected to influence the type of ion exchanger used and the degree of binding affinity of the analyte to the exchanger. The larger the difference between the isoelectric point (pI) of the analyte and the pH of the buffer, the stronger the target species binds to the stationary phase. Adjusting the pH allows the ionic interactions between the analytes and the stationary phase to be controlled, leading to their differential elution.

Certain challenges can arise when analytes have similar charges under the given pH conditions, leading to poor resolution. Additionally, high ion concentrations in the mobile phase can contribute to high background conductivity, which interferes with detection. One solution is using an ion-suppressor column to remove interfering ions from the mobile phase.

The selectivity of IEC is determined by the type of exchange site used, either strong or weak, and the degree of cross-linking in the resin. Strong exchange sites have a higher affinity for ions and provide stronger interactions, resulting in more selective separations. Additionally, the degree of cross-linking affects the porosity and permeability of the resin, which in turn influences the separation efficiency.

Resins must meet specific requirements such as negligible solubility, ion diffusion rate, chemical stability, and higher density than water when swollen. Detection methods include UV/Vis absorbance or indirect detection if solutes do not absorb in the UV/Vis range.

IEC has wide applications in water analysis, biochemistry, protein purification, and the analysis of various compounds such as amino acids, nucleotides, and pharmaceuticals. It enables the separation of charged analytes based on their affinity for the stationary phase, providing a powerful tool for purifying, analyzing, and characterizing complex mixtures.