Method for characterization of selectivity in reversed-phase liquid chromatography : V. Calibration of the retention scale for chromatographic systems with low concentrations of organic solvents in the mobile phase

Abstract

The method for the characterization and prediction of selectivity and absolute retention in reversedphase liquid chromatography based on two indices (a lipophilic and a polar one) has been previously applied to chromatographic systems with mobile phases containing 50% and more of one or two organic solvent(s). Because of a high retention, the homologues series of n-alkylbenzenes is not suitable for the direct calibration of the retention scale in mobile phases with lower concentrations of organic solvents, and the extrapolation of the calibration parameters based on the data for this series in mobile phases with low water content yields predicted retention data that are subject to significant errors. To circumvent this inconvenience and to expand the application possibilities of the above method to mobile phases with higher concentrations of water, three homologous series were tested as potential candidates for calibration standards: 3- n-alkyl-6-methyluracils, 3-alkoxycarbonyl-2-pyrazolines and alkan-2-ones. All three homologous series tested were found to be suitable substitutes for n-alkylbenzenes as calibration standards in mobile phases containing 25–50% methanol in water. The selection of suitable calibration standards for acetonitrile—water mobile phases is more difficult because of a curvature of the dependence of log k′ on the concentration of acetonitrile at low concentrations of this solvent in aqueous mobile phases. Here, the alkoxycarbonyl pyrazolines and alkan-2-ones proved useful as calibration standards in mobile phases containing 30–50% acetonitrile. The agreement between the predicted and experimental k′ was within 10% or better of the experimental k′.