Ion chromatography (IC) is a high-performance liquid chromatography (HPLC) instrumental technique used in USP test procedures such as identification tests and assays to measure inorganic anions and cations, organic acids, carbohydrates, sugar alcohols, aminoglycosides, amino acids, proteins, glycoproteins, and potentially other analytes.

As dictated by the nature of the analyte, IC has been applied to all aspects of the manufacturing and disposition of pharmaceutical products, including characterization of active ingredients, excipients, degradation products, impurities, and process streams. The following sample types are among those that have been analyzed: raw materials, intermediates (including media and culture broths), bulk active ingredients, diluents, formulated products, production equipment cleaning solutions, and waste streams. The technique is especially valuable for ionic or ionizable (in the mobile phase) analytes that have little or no native UV absorbance. The ability to couple the ion-exchange separation with numerous detection strategies, e.g., pulsed amperometric detection (PAD), expands IC applications to instances where analyte-specific detection strategies can provide the required degree of sensitivity or specificity. Utilization of such strategies allows IC applications to be implemented on appropriately configured HPLC systems. Additionally, ion-exclusion separations and pulsed amperometric detection expand the range of application of IC to aliphatic organic acids as well as to nonionic analytes of significant pharmaceutical interest including alcohols, alditols, carbohydrates, and amino acids. The wide dynamic range of the methodology makes it applicable for the quantification of trace contaminants as well as major product components.

Because IC typically uses dilute acids, alkalis, or salt solutions as the mobile phase, and does not use an organic solvent, IC does not require the purchase of costly organic solvents and hazardous disposal of the waste effluent. The effluent can be disposed of after appropriate neutralization (to ~pH 7) and, when necessary, after dilution with water.

IC allows separation using ion exchange, ion exclusion, or ion-pair approaches. IC separations are based on differences in charge density of the analyte species, which in turn depend on the valence and size of the individual ionic species to be measured. Separations are also performed on the basis of differences in the hydrophobic character of the ionic species. IC is typically performed at ambient temperature. As with other forms of HPLC, IC separations are based on varying capacity factors and typically follow the Knox equation. Ion chromatography is a technique complimentary to the more commonly used reversed-phase and normal-phase HPLC and to atomic absorption and ion-coupled plasma (plasma spectrochemistry) techniques in pharmaceutical analysis.

IC instruments closely resemble conventional HPLC instruments. Typical components include an autosampler, a high-pressure pump, an injection valve with a sample loop of suitable size (typically 10 to 250 µL), a guard column, an analytical column, an optional suppressor or other forms of a post-column reaction system, a flow-through detector, and a data system ranging in complexity from an integrator to a computerized data system (Figure 1).

Because mobile phases generally consist of dilute acids, alkalis, or salt solutions, the components in contact with the mobile phase and the sample are typically made from inert materials, such as polyetheretherketone. Conventional HPLC systems also may be used provided that their components are compatible with the mobile phase and injected sample solutions. A metal-free system should be used for trace metal analysis. Following suitable preparation, the sample is introduced via the injection valve. After the optional chemical suppression or other post-column reaction on the column effluent, the analyte species are detected using conductivity, amperometry, UV/VIS, or other detection modes. Because IC uses a predominantly ionic mobile phase, a suppressor is often necessary prior to conductometric detection, although nonsuppressed conductometric detection has been successfully used in pharmaceutical analysis.

Typically sample preparation for IC includes dilution or filtering through a 0.45-µm filter, or both. Under certain circumstances, samples may require removal of undesirable species through solid-phase extraction (SPE) techniques. For example, a highly alkaline sample can be neutralized by having it pass through an SPE cartridge packed with cation-exchange material in the acidic form.

Conductometric detection requires high purity water (generally, resistivity greater than 18 megohm-cm) and high-purity chemicals for the preparation of the mobile phase. For ion-pair separation with UV detection, water and mobile phase components of low UV absorbance should be used.

For ion exchange, the retention time of ions increases with a decrease in the ionic strength and valency (charge) of the mobile phase components. For example, at equimolar concentrations of sodium hydroxide or sodium carbonate mobile phase, capacity factors (k¢) for anions are smaller with sodium hydroxide as the mobile phase than with sodium carbonate as the mobile phase. Some mobile phases, such as sodium hydroxide, can absorb ambient carbon dioxide, resulting in its composition change and often in baseline artifacts. In this instance, care should be taken to prevent absorption of carbon dioxide by the sodium hydroxide mobile phase.

For ion exclusion, capacity factors of organic acids increase with an increase in ionic strength or concentration of mineral acids but decrease with the increase of the column temperature. Because permeation volume remains constant, these effects are usually small. Addition of a solvent such as acetonitrile shortens the retention of organic acids.

Like other HPLC techniques, IC systems are calibrated by plotting peak responses in comparison with known concentrations of a reference standard, using either an external or internal standardization procedure.