ABSTRACT
The retention behavior of natural alpha-, beta-, and gamma-cyclodextrins on a porous graphitic carbon (PGC) stationary phase is investigated. Unusual retention properties for reversed-phase chromatographic conditions are observed with acetonitrile-methanol and water-methanol mixtures as mobile phases. It is assumed that the retention process is governed not only by the standard solvophobic effect but also by specific interactions described as "CD-PGC" effect. The retention factor versus the volumetric methanol fraction in the mobile phase show second-order curves expressing this double mechanism hypothesis. van't Hoff plots demonstrate the contribution of these two retention processes. The retention factor of each natural cyclodextrin is shown to depend on the mobile phase property to act as a proton acceptor, according to the solvent selectivity classification described by Snyder. The "CD-PGC" effect is interpreted as an equilibrium between different interactions: cyclodextrin-PGC stationary phase, London dispersion forces, and cyclodextrin-mobile phase hydrogen bonding. The balance of these interactions may monitor the orientation of the cyclodextrin molecule facing the carbon surface, which is therefore suspected to be the major parameter of this retention mechanism.
ABSTRACT
The adsorption step of human serum albumin on a reversed-phase support was analyzed by studying the "split-peak" effect in mass-overload conditions. This behavior is characterized by the occurrence of a first non-retained fraction and is described by an analytical expression in the case of a Langmuirian adsorption isotherm. The method was applied to determine the column loading capacity, the number of mass-transfer units and the apparent adsorption rate constant measured at a given flow-rate. The nature of the organic modifier influences the split-peak effect: it increases with the eluotropic strength of the organic solvent added to the buffer. Compared to the results with pure buffer, it is the association of two effects, the decrease of the column loading capacity and that of the apparent adsorption rate constant, which increases the split-peak effects observed when methanol and 2-propanol are added to the eluent. These results allow us to gain a better understanding of the role of the organic solvent in the elution behavior of proteins in reversed-phase high-performance liquid chromatography.
