Hydrophobic interaction chromatography separates proteins based on differences in surface hydrophobicity. Proteins are loaded under high-salt conditions that promote hydrophobic interactions with the stationary phase and eluted by decreasing the salt concentration, which weakens the hydrophobic effect.
Principle
At high salt concentrations, water molecules are ordered around the salt ions (salting-out effect), reducing the availability of water to solvate hydrophobic patches on protein surfaces. This forces hydrophobic regions on proteins to interact with hydrophobic ligands immobilized on the chromatography medium. As the salt concentration is reduced during elution, water becomes more available, hydrophobic interactions weaken, and proteins elute in order of increasing surface hydrophobicity. Binding depends on the type and concentration of salt, with ammonium sulfate, sodium sulfate, and NaCl being common choices arranged in the Hofmeister series according to their salting-out strength.
Selectivity and Resolution
HIC selectivity is orthogonal to ion exchange chromatography and size exclusion chromatography. Resolution depends on ligand type (alkyl — butyl, octyl, phenyl), ligand density, the slope of the salt gradient, and temperature. Linear gradients of 10–20 column volumes typically provide the best resolution. Step elution is used for process-scale capture. Phenyl ligands offer both hydrophobic and aromatic interactions, providing distinct selectivity from straight-chain alkyl ligands.
Column Media
Common HIC resins include butyl- and octyl-Sepharose (GE Healthcare), Phenyl and Butyl Toyopearl (Tosoh), and Macro-Prep t-Butyl and Methyl HIC supports (Bio-Rad). Resins vary in hydrophobicity: butyl > octyl for alkyl chains at equivalent density. Particle sizes of 30–100 µm balance resolution and flow properties for preparative work.
Buffer Considerations
Loading buffers contain 1–2 M ammonium sulfate or 3–4 M NaCl. Elution uses the same buffer without salt. Lowering the pH or including ethylene glycol further reduces hydrophobic interactions for tightly bound proteins. Detergents should be avoided as they interfere with hydrophobic binding. Samples are often adjusted directly by adding solid salt or by diluting with concentrated salt stock solutions.
Method Development
Start with a small screen column (1 mL) testing two or three resin chemistries at two salt concentrations. Load 5–10 mg of protein per mL of resin. Wash with 5 column volumes of loading buffer. Elute with a 10 column volume linear gradient to zero salt. Collect 1 mL fractions and analyze by SDS-PAGE. Optimize the gradient slope for the target separation.
Applications
HIC is widely used in biopharmaceutical manufacturing as an intermediate purification or polishing step. It effectively removes aggregates, clipped isoforms, and host cell proteins following a capture step such as Protein A or IMAC. HIC is particularly effective at removing hydrophobic contaminants including endotoxins, DNA-bound proteins, and virus particles. It is commonly coupled with FPLC systems for automated method execution.
