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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...

A simplified version of the solvophobic theory is employed to reexamine a large set of retention data with nonpolar and weakly polar eluites in reversed phase chromatography (RPC) to test certain predictions by the theory and to clarify further the roles of the mobile and the stationary phase in the retention process. The free energy of retention in RPC is expressed in terms of the nonpolar surface area of the eluite, the pertinent interfacial tensions, and the energetics of eluite−stationary phase interactions in the gas phase. Within this framework changes in retention free energy per unit water accessible nonpolar surface area are evaluated in the entire range of the organic modifier concentration by using thermodynamic data for the retention of nonpolar eluites on alkyl silica bonded phase in gas chromatography and for the transfer of nonpolar solutes from the hydroorganic solvent to the gas phase. The normalized retention free energy parameters thus obtained according to the theory are in excellent agreement with those evaluated from experimental retention data using methanol, acetonitrile, tetrahydrofuran, and 2-propanol as organic modifiers in RPC. Furthermore, these parameters are found to be the same from column to column for a particular stationary phase and therefore characterize the RPC retention process. Analysis of experimental retention data obtained with various bonded phases indicates that the stationary phase chain length has a much smaller effect on the changes in selectivity of nonpolar aliphatic eluites than the mobile phase concentration. The results demonstrate the usefulness of the solvophobic theory for the evaluation of physicochemical parameters associated with retention of hydrocarbonaceous eluites in RPC and confirm the dominant role of the mobile phase in governing the retention and selectivity changes in RPC of nonpolar eluites.

Exothermodynamic relationships in reversed-phase and hydrophobic interaction chromatography, with the temperature, organic modifier or salt concentration, and carbon number of the eluite and of the stationary phase ligate as the operating variables, are classified and the links between various linear free energy relationships established. Starting from Martin's relationship based on group additivity, we arrive at two linear free energy relationships:  one between the logarithmic retention factors, κ, obtained on two different columns with eluites of close structural similarity and the other between κ and the logarithmic octanol−water partition coefficient. Molecular interpretation of classical enthalpy−entropy compensation is offered by the combination of van't Hoff's relationship with linear exothermodynamic relationships between thermodynamic quantities on one hand and properties of the eluite, the eluent, or the stationary phase on the other. Thus, the compensation temperature is expressed by the enthalpy and entropy changes per unit of the above properties. Furthermore, the criterion for the invariance of the compensation temperature is set within the framework of dual compensation. A generalized compensation model is developed to extend the concept of enthalpy−entropy compensation to phenomena involving compensation by any two chromatographic variables by drawing analogies between the dependence of κ on the reciprocal temperature and on other operating variables, such as the organic modifier concentration in the eluent. The existence of 12 different compensation parameters is revealed, each marking the common intersection point of linear plots of free energy versus variables of the retention process. The compensation model leads to 12 three-parameter equations, each describing the retention behavior in reversed-phase chromatography as a function of two chromatographic variables. The family of exothermodynamic relationships encompasses most characteristic features of chromatographic retention and is expected to facilitate the organization, interpretation, and prediction of retention data.

Reversed phase chromatography (RPC) is the most popular branch of HPLC for the analysis and purification of a wide variety of substances. Despite significant advances in both our knowledge and understanding of the fundamental principles governing the retention behavior in RPC, there is considerable debate in the literature regarding the mechanism of retention. This review addresses the theoretical foundation of the chromatographic technique, with an emphasis on thermodynamic and exothermodynamic treatment of retention equilibrium, as well as their implication on the mechanistic aspects of RPC retention. A unified and rigorous treatment based on the solvophobic theory is reviewed in terms of its ability to shed light on the physicochemical underpinnings of the retention in RPC, and to quantitatively predict the retention behavior of nonpolar compounds, acids and bases, and peptides and proteins. Also highlighted are areas of future challenges in the theory and practice of RPC, potentially leading to a better quantitative understanding and use of the popular technique. It is undoubtedly the unsung champion of the modern biological sciences, enabling the intricacies of cellular biology to be at last discriminated in detail. [2] Without the recent advances in HPLC, modern biology, functional genomics, and proteomics would not exist. The incredible power of HPLC to discern the molecular diversity of biological phenomena is largely attributed to its ability to distinguish mass differences as little as 1 Da in a macromolecule, such as proteins when coupled with mass spectrometry, [3,4] to separate proteins that differ by only a single amino acid, [5] to separate conformational isomers of a long chain polypeptide, [6] and to resolve the different tertiary conformational structures of DNA fragments. [7] The exquisite sensitivity, the speed, and the impressive resolving power of modern HPLC find application in various fields, such as pharmaceutical, food and clinical analysis, pollution control, downstream processing, measurement of physicochemical properties of drugs as well as the separation of peptides, proteins, and nucleic acids. At the heart of the HPLC revolution is the mode of separation that we are all familiar with-reversed phase chromatography (RPC). The seminal works of Horváth and co-workers [8 -11] have contributed significantly to the theory and practice of RPC, resulting in its wide acceptance by the scientific community as a high resolution separation technique of choice. It is estimated that approximately 80% of HPLC analysis is conducted in this mode. [12] The success of this technique is attributed to the employment of microparticulate alkyl-silica monomeric bonded phases, such as octadecylated silica, which offers high separation efficiency combined with unparalleled convenience, versatility and reproducibility. The use of bonded hydrocarbonaceous stationary phases having a variety of functional groups and a wide choice of hydroorganic eluents to modulate retention offer a broad range of operating con...