Browsing by Subject "Micro Free Flow Electrophoresis"

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    Comprehensive Multidimensional Separations of Biological Samples using Capillary Electrophoresis coupled with Micro Free Flow Electrophoresis
    (2017-12) Johnson, Alexander
    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.
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    Enhancing Micro Free Flow Electrophoresis: Detection, Application, And Fabrication
    (2023-04) LeMon, Matthew
    Micro Free Flow Electrophoresis (µFFE) is a separation technique where analytes are moved through a planar separation channel via pressure driven flow. Analytes streams are deflected laterally within an electric field applied perpendicular to the flow and separated from one another based on differences in their electrophoretic mobility. Notably, µFFE is capable of continuously separating analytes making it uniquely useful for a range of applications such as multidimensional separations, microscale sample purification, and continuous on-line monitoring. This work focuses on improving the sensitivity of laser induced fluorescence (LIF) detection via modifying the laser alignment, exploring new device designs for rapid on-line buffer exchange, and developing a novel fabrication technique for hot embossing µFFE devices in cyclic olefin copolymer (COC) with the goal of expanding µFFE’s use in analytical workflows. A new laser alignment for LIF was explored, focusing the laser light through the side of the device instead of spreading it into a line and reflecting it down onto it. Improvement in the limit of detection (LOD) for fluorescein was obtained in a glass µFFE device using this alignment; however, it was found to be incompatible with 3D printed acrylonitrile butadiene styrene (ABS) due to excessive scattering of the laser light. New µFFE device designs were developed and modeled using Multiphysics modeling software to optimize a design for rapid on-line buffer exchange. This was performed with the intent of interfacing incompatible separation modes with electrospray ionization mass spectrometry (ESI-MS) to develop novel multi-attribute analysis techniques for the assessment of therapeutic monoclonal antibodies (mAbs). Lastly, a novel fabrication technique for µFFE was developed to hot emboss µFFE devices in COC utilizing a poly jet 3D printed master mold. The performance of devices produced this way were tested via a separation of three fluorescent dyes. Their LODs quantified and compared to similar ABS and glass µFFE devices.