Effect of the sample solvent on band profiles in preparative liquid chromatography using non-aqueous reversed-phase high-performance liquid chromatography

Abstract

If a solvent with a higher elution strength than the mobile phase is used to dissolve poorly soluble samples in preparative chromatography, significant deformation of the band profiles occurs, especially when the column is overloaded. Eventually, band splitting may take place. This behavior was observed in non-aqueous reversed-phase chromatography of cholesterol and other low-polarity sample compounds dissolved in a non-polar solvent, such as dichloromethane, at concentrations exceeding their solubilities in the mobile phase, a higher polarity solvent such as acetonitrile. In this chromatographic system, the dependence of the band deformation and of its splitting on the volume and concentration of the sample, the composition of the mobile phase and the column temperature were investigated. A model taking into account the dependence of the solubility of the sample components in the sample solvent and in the eluent and of the equilibrium isotherm of these components between the stationary and mobile phases on the composition of these two solvents (eluent and sample solvent) was worked out. The possible formation of supersaturated solutions was also considered in the model. This model permits the computer simulation of the band profiles for a single compound or for mixtures dissolved in a solvent different from the mobile phase. The results of the simulations are in qualitative agreement with the behavior observed experimentally.