Role of the buffer in retention and adsorption mechanism of ionic species in reversed-phase liquid chromatography: I. Analytical and overloaded band profiles on Kromasil-C18

The influence of the pH, the concentration, and the nature of the buffer on the retention and overloading behavior of propranolol (pKa=9.25) on Kromasil-C18 was studied at 2.75<pH<6.75, using four buffers (phosphate, acetate, phthalate, and succinate), at three concentrations, 6, 20, and 60 mM. The propranolol band profiles were recorded for three sample sizes, less than 1 μg and 375 μg (sample less concentrated than the buffer), and 7500 μg (band more concentrated than the buffer). Results showed that 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 the valence of the basic anion. At moderate loading, the band profiles are well accounted for by a simple bilangmuir model (no adsorbate–adsorbate interactions) with the monovalent anions H2PO4− (pH 2.75), HOOCPhCOO− (pH 2.75), HOOCCH2CH2COO− (pH 4.16) and CH3COO− (pH 4.75), and by a bimoreau model (significant adsorbate–adsorbate interactions) with the bivalent anions −OOCPhCOO− (pH 4.75), −OOCCH2CH2COO− (pH 5.61) and HPO42− (pH 6.75). The isotherm were determined using the inverse method. The results show that both the saturation capacity and the equilibrium constant on the low-energy sites increase with increasing buffer concentration, a result similar to that observed with neutral salts. For bivalent anions, the adsorbate–adsorbate interactions are much stronger on the low than on the high energy sites. The density of high energy sites is lower and the equilibrium constant on the low energy sites are higher with bivalent than with univalent anions. These results are consistent with the formation of a propranolol–buffer (2:1) complex with bivalent anions.