Potential of Alternative Solvent Systems in Reversed-Phase Liquid Chromatography

The partitioning of a neutrally charged molecule between the mobile phase and the stationary phase is influenced by several factors, with the two primary factors being the composition of the mobile phase and the chemistry of the stationary phase. The starting point of the majority of reversed-phase liquid chromatography method development steps in the experimentation of acetonitrile-water or methanol-water binary solvent systems with a typical C18 stationary phase. When the desired separation is not satisfactory a different stationary phase chemistry is considered.  Before sorting to a different stationary phase chemistry for a desired chromatographic separation not very many analytical chemists consider altering the partitioning of a neutrally charged molecule between the mobile phase and the stationary phase by considering the solvent system outside of acetonitrile-water or methanol-water binary solvent systems with a typical C18 stationary phase.

There are several ways to alter reversed-phase liquid chromatography selectivity on a given stationary phase, e.g., a C18 phase. One less considered approach is an alternative organic mobile phase modifier than then acetonitrile and methanol, such as tetrahydrofuran, ethanol, isopropanol, and acetone. Intermolecular interactions responsible for chromatographic retention can be altered on a given stationary phase with the substitution alternative solvents tetrahydrofuran, ethanol, isopropanol, and acetone in place of acetonitrile or methanol. This in turn would result in a reversed-phase liquid chromatographic system with different selectivity and separation properties.

The second less considered approach would be to use ternary solvent systems instead of typical binary solvent systems acetonitrile-water or methanol-water. Ternary solvents in reversed-phase liquid chromatographic systems offer greater flexibility in exploring the potential of selectivity space on a given stationary phase. Greater options alter the partitioning process of a neutral molecule and the stationary phase resulting from blends of organic solvent mixtures in ternary solvent systems. Acetonitrile-methanol-water is perhaps the most widely considered ternary solvent system in reversed-phase liquid chromatography. In addition to acetonitrile-methanol-water, ternary solvent systems tetrahydrofuran-isopropanol-water, acetonitrile-tetrahydrofuran-water, methanol-tetrahydrofuran-water have been reported in the literature. In general ternary solvent systems can explore the reversed-phase liquid chromatography selectivity to the same extent as using a stationary phase with different selectivity.