Heterogeneity of reversed-phase chromatographic surfaces: quenching of sorbed pyrene fluorescence

Abstract

Abstract : The influence of overlaying solvent on the environment and structure of polymeric C18, alkylated silica surfaces is studied using the fluorescence of pyrene as a probe. Intensity ratios of vibronic emission bands are used to determine changes in polarity around the probe sorbed to the C18 surface, while quenching of fluorescence by potassium iodide is used to correlate those changes to exposure of pyrene to the mobile phase. Two distinct regions of stationary phase behavior are identified. At high concentrations of methanol in the mobile phase, pyrene is partitioned or fully surrounded by alkyl chains which are apparently well solvated by methanol. At lower concentrations of methanol, the increase in surface polarity and onset of quenching by solution phase ions indicate that some fraction of sorbed pyrene is exposed to the surrounding solvent as the stationary phase volume collapses. Under these conditions, a significantly greater heterogeneity in the polarity of sorption environments is observed, where a fraction of the fluorescent probe molecules are partitioned into nonpolar domains which are inacessible to a sorbed polar quencher.