Hydrophobic and Reversed Phase Chromatography (HIC/RPC)

Key Insight

Hydrophobic interaction chromatography (HIC) and reversed phase chromatography (RPC) both utilize hydrophobic stationary phases but differ in ligand density and character. RPC, introduced in 1950, employs highly hydrophobic stationary phases with bonded alkyl ligands, requiring hydroorganic mobile phases, where retention occurs through discrete interactions between non-polar ligands and hydrophobic patches on biomolecules. In contrast, HIC uses less dense, less hydrophobic ligands with aqueous kosmotropic salt solutions as the mobile phase. Common to both, protein binding is promoted by high mobile phase surface tension, and elution by low surface tension.

The hydrophobic interaction mechanism is driven by the thermodynamic unfavorability of non-polar substances dissolving in water, causing water restructuring and a positive entropy change, which minimizes surface area at the hydrophobic phase. In HIC, retention is promoted by kosmotropic salts, and the retention factor 'ln k'' linearly relates to salt concentration, akin to protein salting-out, explainable by solvophobic theory. For high salt concentrations, this simplifies to 'ln k' = lnk0' + S * m', where 'S' is the sensitivity coefficient. In RPC, retention is modulated by water-miscible organic solvents like acetonitrile; elution involves increasing the organic modifier concentration, described by 'ln k' = lnk0' - Sφ'.

RPC typically has much higher ligand densities than HIC, leading to greater hydrophobicity and significant conformational changes in its bonded alkyl ligands, which profoundly affects selectivity based on ligand density and type. While small molecules exhibit a smooth relationship between 'k'' and organic modifier volume fraction in RPC, larger peptides and proteins show very steep curves. HIC purification usually requires feed dissolved in high-salt buffer for binding, sometimes necessitating removal of chaotropic agents. RPC often bypasses extensive feed conditioning as proteins strongly bind aqueous samples. Elution in HIC involves decreasing salt concentration, whereas RPC uses increasing organic modifier concentration, up to 95% for regeneration. Both methods face concerns of potential protein destabilization and conformational changes upon interaction with hydrophobic surfaces, though HIC generally presents milder conditions than RPC.