Confocal Raman Microscopy for Probing the Interior of Individual Chromatographic Silica Particles to Interrogate Interfacial Structure and Function

Description:
In development of stationary phase materials, it is critical to understand how their interface structure relates to their functioning as separation media for selective retention. To that end, we have adapted confocal Raman microscopy to examine the interior surfaces of porous chromatographic silica particles, reporting structural information on the stationary phase and its interactions with solvent and solute molecules. The spatial resolution of confocal microscopy can selectively probe interfaces inside an individual porous particle, yielding quantitative information on populations of molecules on its interior surfaces. The measurement is in situ so that interface structure can be monitored as solution and solute composition are varied. We have applied this methodology to study reversed-phase separations, to quantify interfacial populations of surfactants and solutes in ion-interaction retention, and to determine the role of n-alkyl chain order in shape selectivity of reversed-phase PAH separations. We have recently extended these investigations to affinity separations of peptides and proteins. Using wide-pore silica to immobilize selective ligands or deposit phospholipid bilayers, we quantify the retention of biomolecules while probing changes in the interface or ligand structure upon binding. Quantifying the population of a signaling peptide, GLP-1, as it associates with a model lipid bilayer membrane, reveals that the maximum peptide coverage is only 1/5th of a full monolayer. Saturation coverage, therefore, is not limited by the molecular size but by the capacity of the lipid bilayer to accommodate the peptide at high interfacial concentrations, where Raman spectra reveal increases in lipid acyl-chain order that provide free volume for the peptide. We have also studied the structure of DNA aptamers immobilized on porous silica surfaces and used for selective separations of proteins; we investigate the influence of the aptamer folding on protein association.

Speaker: Joel Harris - University of Utah - Chemistry