Microscopic origins of band broadening in chromatography. Polarity distribution in C(18) stationary phase probed by confocal ratiometric imaging of Nile red.

Band broadening is a major factor that influences the efficiency and resolution of chromatographic separations. Studies of microscopic origins of band broadening, such as the micropolarity distribution of chromatographic stationary phase, can provide a better understanding of many chromatographic phenomena and retention behavior. In this work, we probe the chemical environments of C18 chromatographic stationary phase with quantitative confocal fluorescence microscopy under real reversed-phase liquid chromatography conditions. Ratiometric imaging of C18 interface is achieved by loading the stationary phase with a polarity-sensitive dye, Nile red, and optical sectioning with confocal microscopy. The results reveal that there are uniform micropolarity distributions inside individual chromatographic beads, but the polarity may differ between stationary-phase particles. The homogeneity of micropolarity of individual beads suggests that there are not any spatially large exposed silica sites beyond the optical resolution in C18 stationary phase. The strong adsorption sites are smaller in size than the optical resolution of a few hundred nanometers. The heterogeneity between chromatographic beads indicates that the interactions of Nile red with C18 bonded phase are different between beads. This contributes to the broad overall polarity distribution of the C18 stationary phase and can be one of the factors that cause band broadening in separations. With its high spatial resolution and optical sectioning capabilities, confocal fluorescence imaging is shown to be an ideal method to probe the chromatographic stationary phase. The distribution of micropolarity sheds light on the microscopic heterogeneity in chromatographic processes and its influence on chemical separations.