Electromigration of Charged Analytes Through Immiscible Fluids in Multiphasic Electrophoresis

Multiphasic buffer systems have been of greatest interest in electrophoresis and liquid-liquid electrotransfer; this study extends that foundation by exploring the interplay of the geometric and viscous properties of an interleaving oil layer on the electrotransfer of a charged analyte from an aqueous solution into a hydrogel. We utilized finite element analysis to examine two complementary configurations: one being electrotransfer of a charged analyte (protein) in an aqueous phase into a surrounding hydrogel layer and another being electrotransfer of the protein from that originating aqueous phase-through an interleaving oil layer of predetermined viscosity and thickness-and into a surrounding hydrogel layer. Results indicate that the presence of an oil layer leads to increased skew of the injected peak. To explain this difference in injection dispersion, we utilize Probstein's framework and compare the Péclet (Pe) number with the ratio between length scales characteristic to the axial and radial dispersion, respectively. The formulation assigns electrotransfer conditions into six different dispersion regimes. We show that the presence or absence of an interleaving oil layer moves the observed peak dispersion into distinct electrotransfer regimes; the presence of an oil layer augments the electrophoretic mobility mismatch among the different phases, resulting in a five-fold increase in Pe and a six-fold increase in the ratio between the axial to radial dispersion characteristic lengths. We further show that oil viscosity significantly influences resultant injection dispersion. A decrease in oil-layer viscosity from 0.08 to 0.02 Pa s results in a >100% decrease in injection dispersion. Our theoretical predictions were experimentally validated by comparing the electrotransfer regimes of three different mineral oil samples. We show that lowering the oil viscosity to 0.0039 Pa s results in an injection regime similar to that of the absence of an oil layer. Understanding of the impact of electrotransfer of charged species across multiple immiscible fluid layers on peak dispersion informs the design of multiphasic electrophoresis systems.