Fast Electrophoretic Separation Optimization Using Gradient Micro Free-Flow Electrophoresis

Abstract

The continuous nature of micro free-flow electrophoresis (μ-FFE) was used to monitor the effect of a gradient of buffer conditions on the separation. This unique application has great potential for fast optimization of separation conditions and estimation of equilibrium constants. COMSOL was used to model pressure profiles in the development of a new μ-FFE design that allowed even application of a buffer gradient across the separation channel. The new design was fabricated in an all glass device using our previously published multiple-depth etch method (Fonslow, B. R.; Barocas, V. H.; Bowser, M. T. Anal. Chem. 2006, 78, 5369-5374, ref 1 ). Fluorescein solutions were used to characterize the applied gradients in the separation channel. Linear gradients were observed when buffer conditions were varied over a period of 5-10 min. The effect of a gradient of 0-50 mM hydroxypropyl-β-cyclodextrin (HP-β-CD) on the separation of a group of 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) labeled primary amines was monitored as a proof of concept experiment. Direct comparisons to capillary electrophoresis (CE) separations performed under the same conditions were made. Gradient μ-FFE recorded 60 separations during a 5 min gradient allowing nearly complete coverage across a range of HP-β-CD concentrations. In comparison, 4 h were required to assess 15 sets of conditions across the same range of HP-β-CD concentrations using CE. Qualitatively, μ-FFE separations were predictive of the migration order and spacing of peaks in CE electropherograms measured under the same conditions. Data were fit to equations describing 1:1 analyte-additive binding to allow a more quantitative comparison between gradient μ-FFE and CE.Micro free-flow electrophoresis (μ-FFE) is an analytical separation technique used to separate a continuously flowing stream of charged analytes. 2,3 A thin sample stream is introduced into a planar separation channel with buffer running in parallel. An electric field is applied perpendicularly across the separation channel, and charged analytes are deflected laterally based on their electrophoretic mobility. Thus far, μ-FFE separations have been limited to simple separations of fluorescent dyes, 4-6 fluorescently labeled amino acids, 2 and fluorescently labeled proteins. 3,7 μ-FFE's larger-scale, preparative counterpart has proven useful in separating a range of analytes, including cells, 8,9 cellular components, 10-14 and proteins. [15][16][17] In the near future, μ-FFE could be useful in analysis or micropreparative separations of the same analytes.Recently several researchers have investigated a variety of fabrication methods for μ-FFE devices to improve their performance. [4][5][6][18][19][20] In early designs two major problems were encountered. Inefficient removal of electrolysis products, manifested as bubbles, from the electrode channels degraded separations. 4 Also, channel designs used to minimize the effects of the bubbles reduced the electric field applied in the separation channel. 5,6 These probl...

Reassemblable quasi‐chip free‐flow electrophoresis with simple heating dispersion for rapid micropreparation of trypsin in crude porcine pancreatin

“…In 2006-2010, Bowser and coworkers conducted a series of fundamental studies on the flow controlling [24], band and resolution optimizations [25], mitochondria mobility determination [26], and aptamer equilibria measurement [27]. A lot of works have demonstrated that C-FFE could be excellently used for the analyses of mitochondria [26], aptamers [27], and proteins [28] as well as the estimation of equilibrium constants [29]. Nonetheless, few of the works were reported on the micropreparation of biosample via C-FFE.…”

Recent developments in electrophoretic separations on microfluidic devices

“…The Bowser group from the University of Minnesota has made several contributions to FFE since 2008 [10][11][12]22]. In one study, Fonslow and Bowser used a microchip with varying depths to study the effects of a buffer concentration gradient on separations [10] and in another study, to separate mitochondria [11].…”

“…In one study, Fonslow and Bowser used a microchip with varying depths to study the effects of a buffer concentration gradient on separations [10] and in another study, to separate mitochondria [11]. In the first article, a concentration gradient in cyclodextrin was created, and the effect on amino acid separation was examined, as well as an efficient determination of the ideal separation conditions [10].…”

“…Various processes, such as FFE, are well known on the preparative-scale, whereas others, such as IEF, have been well characterized for analytical-scale separations. There have been many innovative areas of development on the smaller scale, including taking advantage of hydrodynamic counterflow [6,7], different channel designs [8], and applying electric fields perpendicular to the flow of the sample [9][10][11][12][13].…”

Recent advances in microfluidic technologies for biochemistry and molecular biology

“…Free flow electrophoresis is a continuous separation technique that utilizes hydrodynamic flow and an applied electric field perpendicular to the flow to separate analytes in flowing streams (54). Recent developments of this method include studies by Bowser and co-workers on the use of free flow electrophoresis in the separation of mitochondria and primary amines (56)(57)(58). Zalewski et al demonstrated synchronized, continuous flow zone electrophoresis on a microfluidic device for the first time (59).…”