Chiral Chromatography is a specialized form of High-Performance Liquid Chromatography (HPLC) designed to resolve optical isomers — molecules that are mirror images but not superimposable. Since enantiomers often exhibit different biological activity, their separation and quantification are critical in the pharmaceutical, agrochemical, and life science industries.
This separation mode relies on chiral recognition, where analytes interact differently with a chiral stationary phase (CSP). These interactions are typically based on a combination of hydrogen bonding, dipole–dipole forces, π–π interactions, and steric complementarity.
Chiral chromatography can be performed under normal phase, reversed phase, polar organic, or supercritical fluid conditions, offering remarkable flexibility in method development and scaling.
The stationary phase is the key element in chiral chromatography, as it determines both selectivity and stability of the separation. Most chiral stationary phases (CSPs) are based on polysaccharide derivatives, such as cellulose or amylose carbamates, which are applied onto high-purity silica supports. Other selectors include proteins, cyclodextrins, or macrocyclic glycopeptides, each offering unique enantioselectivity profiles.
Two main types of chiral stationary phases are used: coated and immobilized. In coated phases, the chiral selector is physically applied to the silica surface, forming a uniform but non-covalent layer. These materials often provide excellent enantioselectivity and sharp peak shapes; however, they are sensitive to strongly polar or reactive solvents that can dissolve or damage the coating. In contrast, immobilized phases feature a covalent bond between the chiral selector and the silica surface. This permanent attachment significantly enhances solvent compatibility and mechanical robustness, allowing the use of a wider range of mobile phases — including reversed phase, polar organic, and supercritical fluid chromatography (SFC) conditions — without compromising stability.
The choice between coated and immobilized CSPs depends on the analyte properties and the solvent system requirements: coated materials offer maximum selectivity for traditional normal phase separations, while immobilized CSPs provide greater flexibility and durability across multiple chromatographic modes.
Depending on the CSP type and application, mobile phases in chiral chromatography may range from nonpolar systems (e.g., n-hexane with alcohol modifiers) to aqueous or polar organic systems (e.g., methanol, acetonitrile, ethanol). The correct solvent choice is crucial for maximizing enantioselectivity while maintaining analyte solubility and compatibility with the stationary phase. In SFC, supercritical CO₂ mixed with organic modifiers is often used to achieve high-efficiency separations.
Chiral recognition and retention are based on the formation of transient diastereomeric complexes between the analyte and the chiral selector. Differences in interaction strength — driven by hydrogen bonding, steric fit, dipolar interactions, and π–π stacking — result in distinct retention times for each enantiomer. The combination of phase chemistry, mobile phase composition, and temperature determines the strength and selectivity of these chiral interactions.