The influence of organic modifiers on the structure of reversed-phase liquid chromatographic (RPLC) stationary phases has been a topic of considerable investigation. Retention of organic modifiers in the stationary phase has previously been determined by chromatographic measurements, and the polarity and heterogeneity of the resulting solvation environment has been studied using solvatochromic, fluorescent, and spin probes. In the present work, the composition and solvation environment of a stationary phase is investigated using confocal Raman microscopy, which allows the in situ examination of the solvation environment within individual chromatographic stationary phase particles without the use of probe molecules. The accumulation of organic modifiers in the stationary phase can be quantified, and the environment of the modifier in the stationary phase can be determined. Specifically, we have investigated the interactions of acetonitrile with C(18)-functionalized silica particles using confocal Raman microscopy, which enables the sampling of small (approximately 1 fL) volumes within individual 10 microm particles. Bare chromatographic silica was also studied in order to investigate the interactions of acetonitrile with surface silanols. The nitrile-stretching (nu(CN)) frequency of acetonitrile responds sensitively to the dipolarity of its local microenvironment. The populations of solution-phase and interfacial acetonitrile are thus spectroscopically distinguishable. Scattering from nu(CN) shows contributions from three different environments within a single RPLC chromatographic particle: acetonitrile in the interparticle mobile phase, C(18)-chain associated acetonitrile, and acetonitrile that is interacting with residual surface silanols. Data are presented quantifying these populations and characterizing their environments within single stationary phase particles.