| The role of hydrophobic interactions in two popular branches of modern high performance liquid chromatography, namely hydrophobic interaction chromatography (HIC) and reversed phase chromatography (RPC), is examined by employing thermodynamic and exothermodynamic analysis, and comparing the results with those of other processes driven by the hydrophobic effect. HIC retention data obtained with small compounds yields nonlinear van't Hoff plots indicative of significant heat capacity effects. The standard enthalpy and entropy changes evaluated from the retention data by using Kirchoff's relations are large and positive at low temperatures and negative at high temperatures. The results parallel those of calorimetric studies on other processes based on the hydrophobic effect and suggest that chromatographic measurements can not only be a useful adjunct to calorimetry but also an alternate means for the evaluation of thermodynamic data associated with hydrophobic interactions. The molecular aspects of HIC retention are investigated by molecular area correlations within the hermeneutics of the solvophobic theory and by enthalpy-entropy compensation (EEC). Thermodynamic quantities per unit nonpolar water-accessible surface area predicted by the theory employing readily available physicochemical data are in excellent agreement with the corresponding experimental values. The analysis of EEC reveals a triad of isoenergetic, isoenthalpic and isoentropic temperatures that are invariant for HIC retention, protein folding as well as the dissolution of nonpolar gases and solids in water, strongly indicating the mechanistic similarity of such apparently disparate processes at the molecular level. A unified framework of the solvophobic theory is presented for the treatment of RPC retention, oil-water partitioning and adsorption on activated charcoal from dilute aqueous solution. This approach clarifies the roles of the mobile and the stationary phase in determining the selectivity in RPC, sheds light on the energetic similarities between such processes driven by the hydrophobic effect and elucidates the relative significance of adsorption and partition mechanism in determining the energetics of RPC retention. The selectivities predicted by the solvophobic theory for weakly polar and nonpolar eluites agree with those evaluated from a large body of experimental RPC retention data using various organic modifiers. Analysis of RPC retention data obtained with various bonded phases and mobile phase concentrations reaffirms the dominant role of the mobile phase in governing the changes in retention and selectivity of nonpolar eluites. The free energy changes per unit nonpolar water-accessible surface area associated with octanol-water and hexadecane-water partitioning, retention in RPC as well as adsorption on activated charcoal from dilute aqueous solution are found to be similar and agree well with experimental data. The result is in contradistinction to the predictions by some other popular theories based on the lattice approach and exposes their shortcomings in distinguishing between partition and adsorption mechanisms in RPC. A clear distinction between partition and adsorption in RPC of nonpolar eluites, therefore, does not emerge from pure thermodynamic analysis. Finally, a classification of exothermodynamic relationships is presented that encompasses most characteristic features of chromatographic retention and is expected to facilitate the organization, interpretation and prediction of RPC retention data. |
