Comprehensive Multidimensional Separations of Biological Samples using Capillary Electrophoresis coupled with Micro Free Flow Electrophoresis

Abstract

Micro free-flow electrophoresis (μFFE) is a continuous separation technique in which analytes are streamed through a perpendicularly applied electric field in a planar separation channel. Analyte streams are deflected laterally based on their electrophoretic mobilities as they flow through the separation channel. The continuous nature of µFFE separations makes it uniquely suitable as the second dimension for multidimensional separations. The focus of this work is the development of coupling capillary electrophoresis (CE) to µFFE as a high speed two-dimensional (2D) separation platform, followed by an investigation of orthogonality of the two techniques, and finally a novel label-free detection method for µFFE separations. A new µFFE device was fabricated and coupled to CE via capillary inserted directly into the µFFE separation channel. High peak capacity separations of trypsin digested BSA and small molecule bioamines demonstrated the power of CE × µFFE. Since both methods rely on electrophoretic mobility to separate, an investigation on the orthogonality of the two techniques was carried out. µFFE can operate in many different separation modes to increase the orthogonality CE × µFFE. Lastly, fluorescent labeling of the analytes can cause the sample to lose its dimensionality affecting 2D separation peak capacity and coverage. A novel absorption detector was studied to demonstrate the first ever label free absorption detection on a µFFE device. A separation was performed on visible dyes and their detection limits quantified.