Salt-mediated retention of proteins in hydrophobic-interaction chromatography

Melander, Wayne R.; Corradini, Danilo; Horváth, Cs.

doi:10.1016/s0021-9673(01)91648-6

Cited by 276 publications

(122 citation statements)

References 8 publications

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“…The solvophobic theory is based on the association and solvation of the participating species and assumes that the molal surface tension increment of the salt determines solute retention (Melander and Horvath, 1977;Melander et al, 1984Melander et al, , 1989. In the solvophobic theory, it is believed that the cavity is formed and then closed on the stationary and mobile phases, which is not always valid if the salt has a strong interaction with the proteins.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of selectivity changes in HIC systems using a preferential interaction based analysis

Xia

Nagrath

Garde

et al. 2004

Biotech & Bioengineering

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It is well established that salt enhances the interaction between solutes (e.g., proteins, displacers) and the weak hydrophobic ligands in hydrophobic interaction chromatography (HIC) and that various salts (e.g., kosmotropes, chaotropes, and neutral) have different effects on protein retention. In this article, the solute affinity in kosmotropic, chaotropic, and neutral mobile phases are compared and the selectivity of solutes in the presence of these salts is examined. Since solute binding in HIC systems is driven by the release of water molecules, the total number of released water molecules in the presence of various types of salts was calculated using the preferential interaction theory. Chromatographic retention times and selectivity reversals of both proteins and displacers were found to be consistent with the total number of released water molecules. Finally, the solute surface hydrophobicity was also found to have a significant effect on its retention in HIC systems. B 2004 Wiley Periodicals, Inc.

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Section: Introductionmentioning

confidence: 99%

Evaluation of selectivity changes in HIC systems using a preferential interaction based analysis

Xia

Nagrath

Garde

et al. 2004

Biotech & Bioengineering

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“…The technique employs weakly hydrophobic stationary phases and the retention is modulated by varying the salt concentration in the aqueous mobile phase (3,4). The effect of salt on protein interactions in aqueous solutions (5)(6)(7)(8)(9) as well as on protein adsorption in HIC (5,10,11) has been extensively investigated, and the salt effect on HIC retention has been treated within the hermeneutics of the solvophobic theory (12)(13)(14) and Wyman's linked functions (15)(16)(17)(18).…”

Section: Introductionmentioning

confidence: 99%

Temperature effects in hydrophobic interaction chromatography.

Haidacher

Vailaya

Horváth

1996

Proc. Natl. Acad. Sci. U.S.A.

148

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The effect of temperature from 5°C to 50°C on the retention of dansyl derivatives of amino acids in hydrophobic interaction chromatography (HIC) was investigated by HPLC on three stationary phases. Plots of the logarithmic retention factor against the reciprocal temperature in a wide range were nonlinear, indicative of a large negative heat capacity change associated with retention. By using Kirchoff's relations, the enthalpy, entropy and heat capacity changes were evaluated from the logarithmic retention factor at various temperatures by fitting the data to a logarithmic equation and a quadratic equation that are based on the invariance and on an inverse square dependence of the heat capacity on temperature, respectively. In the experimental temperature interval, the heat capacity change was found to increase with temperature and could be approximated by the arithmetic average. For HIC retention of a set of dansylamino acids, both enthalpy and entropy changes were positive at low temperatures but negative at high temperatures as described in the literature for other processes based on the hydrophobic effect. The approach presented here shows that chromatographic measurements can be not only a useful adjunct to calorimetry but also an alternative means for the evaluation of thermodynamic parameters.Hydrophobic interaction chromatography (HIC) is widely used for the separation and purification of proteins in their native state (1, 2). The technique employs weakly hydrophobic stationary phases and the retention is modulated by varying the salt concentration in the aqueous mobile phase (3, 4). The effect of salt on protein interactions in aqueous solutions (5-9) as well as on protein adsorption in HIC (5, 10, 11) has been extensively investigated, and the salt effect on HIC retention has been treated within the hermeneutics of the solvophobic theory (12-14) and Wyman's linked functions (15)(16)(17)(18).Less progress has been made in elucidating the effect of temperature on retention in HIC. The observed increase in protein retention with temperature, for instance, has been ascribed to enhancement of hydrophobic interactions with increasing temperature due to temperature-induced conformational changes of proteins and concomitant increase in hydrophobic contact area upon binding to the chromatographic surface (16). So far the only thermodynamic study on the effect of temperature in HIC was carried out with aliphatic alcohols and carboxylic acids on octyl-agarose stationary phase with neat aqueous phosphate buffers (19) and hydro-organic mobile phases containing methanol or ethylene glycol (20). Since the salt concentration was very low in these studies, the conditions differed from those employed for protein separation in HIC. In order to shed light on the HIC retention behavior of proteins, it is necessary to understand first the physicochemical basis of HIC with simple molecules that undergo no significant conformational changes and to useThe publication costs of this article were defrayed in part by page c...

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“…Chaotropic agents improve solubility, but do so at the expense of stability [1,3]. The obvious tactic would seem to be exclusive deployment of known stabilizing agents, but many of the most effective ones are also strong precipitants, or 'salting-out' agents, as is the case with salts high up in the Hofmeister lyotropic series [1,[3][4][5][6][7][8][9][10]. Along with protein self-association, excessive concentrations of these agents can promote hetero-association with other surfaces.…”

Section: Introductionmentioning

confidence: 99%

An adaptation of hydrophobic interaction chromatography for estimation of protein solubility optima

Gagnon¹,

Mayes²,

Danielsson

1997

Journal of Pharmaceutical and Biomedical Analysis

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This study describes and adaptation of hydrophobic interaction chromatography (HIC) that can be used to estimate protein solubility optima. The method does not support determination of absolute, e.g. quantitative solubility, however it does provide a basis for identifying the salt concentration, pH, or additive concentrations that support the highest relative solubility. The magnitude of a given salt's effects are consistent with its ranking in the Hofmeister series. IgG in solutions of strong 'precipitating' salts exhibits a classical salting-in/salting-out curve, described by a solubility minimum at low ionic strength, increasing to a well-defined maximum, and then losing solubility with further elevation of salt concentration. The direct effect of pH on protein solubility, as well as its indirect effect via modification of the ionic equilibria of dissolved salts, can also be tracked. Cooperative effects of solubility modifiers such as amino acids can likewise be assessed. The technique can be a useful tool in the development of liquid formulations for protein pharmaceuticals. © 1997 Elsevier Science B.V.

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Salt-mediated retention of proteins in hydrophobic-interaction chromatography

Cited by 276 publications

References 8 publications

Evaluation of selectivity changes in HIC systems using a preferential interaction based analysis

Evaluation of selectivity changes in HIC systems using a preferential interaction based analysis

Temperature effects in hydrophobic interaction chromatography.

An adaptation of hydrophobic interaction chromatography for estimation of protein solubility optima

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