Confocal Raman microscopy for in situ detection of solid-phase extraction of pyrene into single C18-silica particles

Solid-phase extraction (SPE) is a well-known method for separating and preconcentrating analytes prior to ex situ detection and quantification. Despite advances in the miniaturization of solid-phase extraction, the technique has not evolved to meet the needs for detection in small-scale microfluidic devices. Although successful efforts have led to smaller-scale extractors that allow preconcentration from small volumes, detection within correspondingly small volumes has remained out of reach because of analyte dilution during a postextraction transfer or "wash-off" step prior to detection. In this work, successful extraction into subpicoliter collection volumes is accomplished by using a single chromatographic particle as an extractor; wash-off dilution is completely avoided by using confocal Raman microscopy for in situ detection within the single-particle collection phase. Specifically, micromolar concentrations of pyrene in methanol/water solution were equilibrated with an individual C18-functionalized silica particle, and Raman spectra were acquired from a small confocal sampling volume (∼1 fL) within the particle interior. By comparing the Raman scattering intensity of the pyrene with that of the C18 chains in the stationary phase, it was possible to quantify the equilibrium coverage of pyrene relative to the C18 chains. Spectroscopic isotherms were measured to determine the saturation surface coverage of pyrene relative to C18 chains and to study how the preconcentration equilibrium can be controlled by the source-phase solution composition. For extraction from aqueous solutions having the lowest methanol concentration (5% by volume), the preconcentration factor for pyrene into the particle was found to be 4.8 (±0.8) × 10(4), which allowed detection of pyrene extracted from a 10 nM solution into a 0.52-pL particle volume.