Mixed self-assembled monolayers in chemical separations.

Chemical separations of many biomolecules and pharmaceuticals are limited by their electrostatic interaction with the surfaces of the separation medium. Mixed self-assembled monolayers of octadecyl and methyl chains organize into a dense, two-dimensionally cross-linked network over the chromatographic silica surface to reduce acid dissociation of the surface silanols. Molecular models predict that two-dimensional cross-linking is sterically possible for pure methylsiloxane monolayers, silicon-29 nuclear magnetic resonance measurements show that cross-linking predominates for mixed monolayers of primarily methylsiloxane, and chromatographic measurements confirm that electrostatic interactions are reduced when the monolayer is primarily methylsiloxane. Chromatographic separation of genetic variants of a highly charged protein, cytochrome c, demonstrates the promise of self-assembled monolayers in separations of biomolecules.

Electrostatic Interactions between Ru(bpy)(3)(2+) and Chromatographic Surfaces.

In HPLC, the zones of organic bases tail when silica-based stationary phases are used because the analytes and the surface are oppositely charged. In developing new stationary phases to achieve lower surface charge, a measure of surface charge is needed. The choice of a suitable analyte to quantitate electrostatic interactions is complicated by the acid-base equilibrium of the analyte itself and by nonelectrostatic interactions between the analyte and the surface, which alter the charge-induced tailing. This paper describes the study of the pH dependence of adsorption to isolate electrostatic interaction and the use of a cationic probe, tris(2,2'-bipyridine)ruthenium chloride (Ru(bpy)(3)(2+)), to sense surface charge without the complication of the probe's acid-base equilibria. The paper further describes the application of Gouy-Chapman theory to reveal the surface charge density. The results confirm that type A silica is considerably more acidic than type B silica and that horizontal polymerization makes type A silica perform as well as type B silica.