Spectroscopic Methods for Examining Retention Mechanisms within Individual Chromatographic Particles

Joel Harris, Department of Chemistry, University of Utah, Salt Lake City, U.S.A.
Jay Kitt, Department of Chemistry, University of Utah, Salt Lake City, U.S.A.
Justin Cooper, Department of Chemistry, University of Utah, Salt Lake City, U.S.A.

Recent breakthroughs in combining spectroscopy with optical microscopy are allowing chemical analysis to be carried out with unprecedented spatial resolution (in fL volumes) and sensitivity (at the single-molecule level).  In this talk, these tools will be applied to investigate interfacial chemistry that governs retention of molecules within individual reversed-phase chromatographic silica particles. Confocal-Raman microscopy can be used to interrogate sub-femtoliter volumes inside of single silica particles and report interface structured through vibrational spectra of solute molecules retained at the alkyl-chain/solution interface. This method is useful both for investigating retention mechanisms and for detecting solutes that are extracted from the surrounding solution and concentrated within the particle. As detection limits of fluorescence microscopy have reached the single-molecule level, imaging of single-molecule trajectories can be used to follow the transport of individual molecules within individual reversed-phase chromatographic particles.  This technique allows direct measurement of intraparticle molecular residence times, diffusion rates, and the spatial distribution of molecules within the particle. The observation of stuck molecule events provides direct evidence of the heterogeneity of molecular interactions with the chromatographic media, where the molecules held fixed by strong adsorption represent a major contribution to peak tailing in chromatographic elution.