Retention Thermodynamics in Hydrophobic Interaction Chromatography

Abstract

This paper is a review of the thermodynamics of retention in hydrophobic interaction chromatography (HIC) with mildly hydrophobic stationary phases and aqueous salt solutions usually employed in protein purification. Since the role of salt in HIC has been well documented, our focus was to investigate the temperature effect on the retention behavior in HIC and to compare the results with those obtained for other processes driven by the hydrophobic effect. Using nonpolar dansyl amino acids as model compounds, retention data obtained on three stationary phases yielded nonlinear van't Hoff plots in the temperature range from 5 to 50°C. Thermodynamic analysis of the data revealed significant heat capacity effects. The enthalpy and entropy changes were large and positive at low temperatures, decreased with increasing temperature, and became negative at high temperatures. The results parallel those of calorimetric studies on other processes based on the hydrophobic effect, such as dissolution in water of nonpolar liquids, gases, and solids as well as protein folding. Thermodynamic parameters from HIC measurements also confirmed the existence of certain exothermodynamic relationships, such as enthalpy-entropy compensation and molecular area correlations. In order to examine at the molecular level the energetics of HIC retention as well as the dissolution of nonpolar gases in water, the pertinent thermodynamic parameters were expressed in terms of nonpolar molecular area and interfacial tensions by employing the solvophobic theory. It was found that these expressions from HIC and dissolution data are nearly identical, thus confirming the mechanistic identity of the two processes.

A Review of the Thermodynamics of Protein Association to Ligands, Protein Adsorption, and Adsorption Isotherms

“…However, there are numerous papers where the authors claim to be able to calculate the Gibbs energy change of adsorption from the distribution coefficient [21][22][23][24]. The utilized expression, DG 0 = -R T ln K, is from Vailaya and Horvath [25]. Here, K, according to Vailaya and Horvath, is "the equilibrium constant, K, for the distribution of the eluite between the bulk mobile phase and the stationary phase" and DG 0 is "the Gibbs free energy change associated with eluite transfer from the mobile to the stationary phase."…”

Adsorption of plasmid DNA on anion exchange chromatography media

“…Haidacher et al [24] and Vailaya and Horvath [25] applied the van't Hoff equation to hydrophobic interaction chromatography (HIC) and thereby evaluated temperature effects in HIC. The equilibrium constant is described as…”

The effects of ligand chain length, salt concentration and temperature on the adsorption of bovine serum albumin onto polypropyleneglycol–Sepharose

“…4). Following the approach of Horváth and co-workers of incorporating the dependence of ∆H°, ∆S°, φ and ∆C°P on temperature, the quadratic equation [equation (5); Vailaya and Horváth, 1996] was used. The best-fit coefficients obtained using Table Curve 2D, a nonlinear least squares fitting program (AISN, 1989(AISN, -1994) that employs the Leventhal-Marquardt method, are summarized in Table 1; characterizations obtained with these coefficients are shown as solid curves in Fig.…”

“…Applying equation (4) to appropriate capacity factor data, the change in the binding of ions (∆ν + + ∆ν − ) and the number of water molecules released (∆ν 1 ) can be estimated. Vailaya and Horváth (1996) used the van't Hoff plots to treat the energetics of the retention in HIC. Assuming variation of ∆C°P, they derived the following expression termed the 'quadratic equation':…”

“…Linear HIC has been used as a tool for investigating the mechanisms underlying the hydrophobic interaction process (Wu et al, 1986;Geng et al, 1990;Haidacher et al, 1996;Vailaya and Horváth, 1996;Perkins et al, 1997). As long as the injected amounts of the sample components are kept sufficiently low, adsorption can be completely characterized by retention-factor behavior (Guiochon et al, 1994).…”