Effect of the pH, the concentration and the nature of the buffer on the adsorption mechanism of an ionic compound in reversed-phase liquid chromatography: II. Analytical and overloaded band profiles on Symmetry-C 18 and Xterra-C 18

Abstract

In a previous report, the influence of the pH, the concentration, and the nature of the buffer on the retention and overloading behavior of propranolol (p K a = 9.45) was studied on Kromasil-C 18 at 2.75 < pH < 6.75, using four buffers (phosphate, acetate, phthalate, and succinate), at three concentrations, 6, 20, and 60 mM. The results showed that the propranolol cation was eluted as an ion-pair with the buffer counter-anion. A similar study was carried out with Symmetry-C 18 and Xterra-C 18. Two additional buffers, formate and citrate, were also used. Propranolol elution band profiles were recorded for a small (less than 1 μg) and a large (375 μg) sample size. The results are similar to those obtained with Kromasil and confirm earlier conclusions. The buffer concentration, not its pH, controls the retention time of propranolol, in agreement with the chaotropic model. The retention factor depends also on the nature of the buffer, particularly on its valence, and on the hydrophobicity of the basic anion. With the monovalent anions HCOO − (pH 3.75), H 2PO 4 − (pH 2.75), HOOC–Ph–COO − (pH 2.75), HOOC–CH 2–CH 2–COO − (pH 4.16), CH 3COO − (pH 4.75) and HOOC–CHCOOH–COO − (pH 3.14), at moderate loadings, and for the two larger buffer concentrations, the band profiles are well accounted for by a simple bi-Langmuir isotherm model (no adsorbate–adsorbate interactions). By contrast, these profiles are accounted for by a bi-Moreau isotherm model (i.e., with significant adsorbate–adsorbate interactions) with the bivalent anions −OOC–Ph–COO − (pH 4.75), −OOC–CH 2–CH 2–COO − (pH 5.61), HPO 4 2− (pH 6.75), and HOOC–CHCOO −–COO − (pH 4.77) and with the trivalent anion −OOC–CHCOO −–COO − (pH 6.39). The best values of the isotherm parameters were determined using the inverse method. The saturation capacity and the equilibrium constant on the low-energy sites increase with increasing buffer concentration, a result consistent with the formation in the mobile phase of a hydrophobic complex between the propranolol cation and the buffer anion. With bivalent and trivalent anions, adsorbate–adsorbate interactions are strong on the low-energy sites but they remain negligible on the high-energy sites. The density of the high energy sites is lower and the equilibrium constant on the low-energy sites are both higher with the bivalent and the trivalent buffer anions than with the univalent buffer anions. These results are consistent with the formation of a 2:1 and a 3:1 propranolol–buffer complex with the bivalent and the trivalent anions, respectively.