Effect of mobile phase and ligand arm on protein retention in hydrophobic interaction chromatography

Thermodynamic analysis of the interaction between proteins and solid surfaces: application to liquid chromatography

“…The binding strength of the hydrophobic interactions between proteins or polypeptides and a sorbent can be adjusted by many factors, for instance, the use of salts (or solvents) having different molal surface tension increment values, the use of different salt (or solvent) concentrations [37][38][39] and variation in the hydrophobicity of the sorbents. 23,24,28,32,[40][41][42] Because hydrophobic interactions contribute relatively minor exothermic heat to the system the free energy change upon binding of a protein or polypeptide to a sorbent in hydrophobic interaction chromatographic processes such as RPC or HIC has frequently been assumed to be governed by the corresponding entropy change. 23,24,28 In PBC, the van der Waals forces can be considered as a 'positive contribution' to the adsorption, meaning that the protein can be purified by carefully adjustment of the strength of van der Waals forces alone or in combination with other interactions.…”

Equilibrium-Based Modeling of Chemical Sorption on Soils and Soil Constituents

“…Lagaly et al (1983) speculate that if sufficiently close-packed chains exceeding a critical length exist, then the resulting strong aggregation of the ordered water molecules around the chains exhibit a discontinuity with the random orientation of the bulk water; that is, if an ordered structure is formed, then hydrophobic behavior will dominate even if an organic monolayer coverage is not complete. Additionally, hydrophobic interaction chromatography (HIe) research shows that the chain length of the hydrophobic solid phase affects the partitioning of organic compounds in the mobile phase (Schmuck et al, 1986).…”

The Column in Liquid Chromatography

“…During separation in HIC proteins are induced to bind to a weakly hydrophobic stationary phase using a buffered mobile phase of high ionic strength and then selectively desorbed during a decreasing salt concentration gradient. These conditions are minimized in HIC by using stationary phases of lower hydrophobicity together with totally aqueous mobile phases, in general, since solvent strength is controlled by varying ionic strength rather than by increasing the volume fraction of an organic modifier [320,[322][323][324][325][326][327]. In RPC multiple peaks, peak distortions and changes in the elution volume of proteins result from conformational unfolding of proteins on the bonded phase surface as a consequence of the high interfacial tension existing between the mobile and the bonded stationary phases.…”

“…Mobile phase parameters that have to be optimized are the salt concentration, salt type, slope of the salt gradient, pH, addition of surfactant or organic modifier and temperature [320][321][322]325,[328][329][330][331][332][333][334][335]. In the absence of specific binding of the salt to the protein molecule and at relatively high salt concentration in the mobile phase, retention increases linearly with the salt molality and at constant salt concentration with the molal surface tension increment of the salt used in the aqueous mobile phase.…”