Binding Behavior of Crystalline and Noncrystalline Phases: Evaluation of the Enthalpic and Entropic Contributions to the Separation Selectivity of Nonpolar Solutes with a Novel Chromatographic Sorbent

In this paper, we describe studies of the retention characteristics of nonpolar molecules with a novel liquidcrystalline, silica-supported, comb-shaped polymer chromatographic phase, Sil-ODA 18 . These results extend and amplify previous reports of the roles of enthalpic-and entropic-driven processes in the modulation of the selectivity of nonpolar and polar compounds in reversed-phase high-performance liquid chromatography (RP-HPLC). The investigations reveal that phase reorganization is the most important factor controlling selectivity enhancement with silica-supported, comb-shaped polymer phases as the temperature, T, of the system is varied. Moreover, these studies demonstrate that contributions from the stationary and the mobile phases can be independently fine-tuned to achieve enhanced selectivity via partition and/or adsorption binding processes. The relevant thermodynamic parameters, namely, the changes in enthalpy, entropy, and heat capacity for various nonpolar solutes with this comb-shaped polymeric sorbent, have also been determined using recently developed analytical procedures for the evaluation of nonlinear van't Hoff plots. These investigations into the thermodynamic properties of the comb-shaped polymeric sorbent in its ordered crystalline and noncrystalline states clearly delineate the differences in binding behavior compared to conventional types of monolayer n-alkylsilica sorbents and thus should facilitate wider application of this new class of reversed-phase sorbents in the separation sciences. Introduction Reversed-phase chromatography (RPC) is currently the most widely used of all of the high-performance liquid chromatographic (HPLC) modes of separations. The evaluation of the physicochemical basis of the retention mechanisms of different classes of solutes in RPC has received extensive attention, with the experimental results often interpreted in terms of the solvophobic model proposed by Horvath et al. 1,2 A central question pertaining to all RPC separations is, What drives the retention process? This question has been the subject of considerable debate and investigation since the concept of RPC was first used in 1950 as an analytical separation method by Howard and Martin. 3 Two primary RPC mechanisms can be considered, namely, the solvation/desolvation model, 1,2 whereby expulsion of solutes from a polar mobile phase dominates the free energy of transfer with nonpolar sorbents acting as receptive but passive surfaces, and the partitioning model, 4-6 where the stationary phase contributes in a much more significant way to the overall distribution process. On the basis of solvophobic considerations that encompass the solvation/desolvation model, Horvath and co-workers 1 have proposed that the interaction between the solute and the mobile phase provides the primary driving force. According to this model, retention in the high-performance modes of RPC can then be attributed to adsorption rather than partitioning processes between the solutes and the nonpolar sorbent. 1,2 In the solvation/desolvation model, the contributio

Adsorption behavior of multicomponent protein mixtures containing α1-proteinase inhibitor with the anion exchanger, 2-(diethylamino)ethyl-Spherodex

The equilibrium binding behavior of α1-proteinase inhibitor (α1-PI) in the presence of human serum albumin (HSA) has been determined in packed bed systems with the anion exchanger, 2-(diethylamino)ethyl (DEAE)-Spherodex. Experimental data derived for the individual proteins were compared with the corresponding data obtained from batch adsorption studies as well as studies in which mixtures of these two proteins were loaded at different concentration ratios onto columns of the same anion exchange adsorbent. The results confirm that α1-PI has a greater affinity for the anion exchanger, although competitive adsorption was observed as the inlet concentration of HSA was increased. Under these conditions, decreased binding capacities and lower dynamic adsorption rates were observed for α-PI with the DEAE-Spherodex anion exchange adsorbent. The results are discussed in terms of the influence which various contaminants that occur in multicomponent mixtures of proteins from human plasma can have on the equilibrium binding characteristics of a target protein with weak or strong ion exchange adsorbents under conditions approaching concentration overload in preparative chromatographic systems. These investigations have also addressed, as the first part of an iterative approach for the simulation of the adsorption behavior of multicomponent mixtures of human plasma proteins with ion exchange and affinity chromatographic adsorbents, the ability of noncompetitive and competitive Langmuirean models to simulate the adsorption of α1-PI in the presence of different concentrations of HSA to DEAE-Spherodex