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A novel combination of CE-based separation techniques was used for the precise fractionation of ionic compounds from impurities. The combination of on-capillary concentration and separation using transient isotachophoresis, with multiple injections and a two-point detection system provided higher efficiency, and accuracy at a microliter-scale injection volume, than when CE was individually used for purification. In this paper, we present successful applications of the CE fractionation techniques for the purification of fluorescein, fluorescein-4-isothiocyanate, two fluorescent metal ion probes, and a fluorescein-modified DNA aptamer. The purity of the isolated fluorescent probes ranged from 95 to 99%. Such high purity could not be achieved using chromatographic purification techniques. With relatively low dilution factors of 6-9, the purified probe solutions were practical for use as purified stock solutions. In addition, the fluorescein-modified DNA aptamer purified by our method was successfully used in a thrombin binding assay. The method developed was useful for the purification of anionic fluorescent reagents to be of ultratrace analytical grade for use with CE-LIF., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The most common method to determine the EOF in CE is to measure the migration time for a neutral marker. In this study, 12 compounds (three novel and some previously used) were investigated as EOF markers in aqueous and nonaqueous BGEs. In the aqueous buffer systems (ammonium acetate, sodium phosphate, and sodium borate) the evaluation included a wide pH range (2-12). Two BGEs contained chiral selectors (sulphated-β-CD, (-)-diketogulonic acid) and one that contained a micellar agent (SDS) were included in the study. The majority of the evaluated compounds were found to migrate with the EOF in the water-based BGEs and are thus useful as EOF markers. However, in the SDS-based BGE only four of the compounds (acetone, acrylamide, DMSO, and ethanol) were found to be applicable. In the nonaqueous BGEs 11 markers (acetone, acetophenone, acrylamide, anthracene, benzene, 4-(4-methoxybenzylamino)-7-nitro-2,1,3-benzoxadiazole, benzyl alcohol, 2,5-diphenyloxazole, ethanol, flavone, and mesityl oxide) seemed to be functional as EOF markers. Even though several of the evaluated compounds can be used as EOF markers in the investigated BGEs, the authors would recommend the use of acrylamide as a general marker for UV detection. Furthermore, the four fluorescent markers (of which three were novel) gave RSD values equal to the other markers and can be used for the determination of the EOF in CE or microchip CE with fluorescence detection., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Instrumentation has been developed for the implementation of ultra-high voltage capillary electrophoresis (UHVCE) with potentials up to and exceeding 300 kV. Several separations have been used to demonstrate the utility of higher applied voltages for improving the resolution of peptide, protein, and nucleic acid separations. Previously reported instrumentation was limited to 120 kV and required submersion in a bath of transformer oil to prevent corona and high voltage arcing between the components of the instrument [Hutterer, 1999, 2000, 2005] [1-3]. A modular design that uses plastic dielectric materials to overcome these obstacles enabling simplified operation of the instrument in air is described here in detail. A forced air system developed to control the temperature of the instrument to within a few degrees over a range of 25-60 °C for use with ultra-high voltage capillary gel electrophoresis is also described. UHVCE instrumentation and its applications with UV absorption and laser induced fluorescence detection are further developed, and the first demonstration of UHVCE coupled to electrospray ionization-mass spectrometry is shown., (Copyright © 2012 Elsevier B.V. All rights reserved.)

An approach to performing in-line extraction employing functionalized magnetic particles for CE and microchip electrophoresis is presented. Silica-coated iron oxide particles were synthesized and used as the solid support. The particles were functionalized with octadecylsilane and used as reverse-phase sorbents for in-line SPE followed by electrophoresis. Magnets were used to locally immobilize these sorbents inside the capillary or microchip. Extraction, elution, and detection of the analytes were performed sequentially without interruption or need for sample handling. Mixtures of hydrophobic analytes were successfully extracted from solution using the synthesized magnetic sorbents. CE was able to extract and separate mixture of parabens within 10 min. In-line extraction was also carried out on a disposable PMMA microfluidic device with LIF detection. Electrophoretic separation of fluorescent dyes, Rhodamine 110 and SulfoRhodamine B, was completed in under a minute. The results demonstrated the feasibility of performing the in-line extraction/separation technique in a microchip platform enabling rapid analysis, low sorbent consumption, and increased analyte recovery (relative to the capillary format).

There is a continuing drive in microfluidics to transfer microchip systems from the more expensive glass microchips to cheaper polymer microchips. Here, we investigate using polyelectrolyte multilayers (PEM) as a coating system for PMMA microchips to improve their functionality. The multilayer system was prepared by layer-to-layer deposition of poly(diallyldimethylammonium) chloride and polystyrene sulfonate. Practical aspects of coating PMMA microchips were explored. The multilayer buildup process was monitored using EOF measurements, and the stability of the PEM was investigated. The performance of the PEM-PMMA microchip was compared with those of a standard glass microchip and a PEM-glass microchip in terms of EOF and separating two fluorescent dyes. Several key findings in the development of the multilayer coating procedure for PMMA chips are also presented. It was found that, with careful preparation, a PEM-PMMA microchip can be prepared that has properties comparable--and in some cases superior--to those of a standard glass microchip.

This paper describes the use of two-beam line-confocal detection geometry for measuring the total mobility of individual molecules undergoing continuous-flow CE separation. High-sensitivity single-molecule confocal detection is usually performed with a diffraction limited focal spot (approximately 500 nm in diameter), which necessitates the use of nanometer-sized channels to ensure all molecules flow through the detection volume. To allow for the use of larger channels that are a few micrometers in width, we employed cylindrical optics to define a rectangular illumination area that is diffraction-limited (approximately 500 nm) in width, but a few micrometers in length to match the width of the microchannel. We present detailed studies that compare the performance of this line-confocal detection geometry with the more widely used point-confocal geometry. Overall, we found line-confocal detection to provide the highest combination of signal-to-background ratio and spatial detection efficiency when used with micrometer-sized channels. For example, in a 2 microm wide channel we achieved a 94% overall detection efficiency for single Alexa488 dye molecules when a 2 microm x 0.5 microm illumination area was used, but only 34% detection efficiency with a 0.5 microm-diameter detection spot. To carry out continuous-flow CE, we used two-beam fluorescent cross-correlation spectroscopy where the transit time of each molecule is determined by cross-correlating the fluorescence registered by two spatially offset line-confocal detectors. We successfully separated single molecules of FITC, FITC-tagged glutamate, and FITC-tagged glycine.

Phosphatidyl inositol 4,5-bisphosphate (PIP2) and phosphatidyl inositol 3,4,5-trisphosphate (PIP3) labeled with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY FL) on the acyl chain or a phosphatidyl ethanolamine head group were separated by CE with LIF detection. Several methods and capillary-coating procedures were tested for the separation of these phosphatidyl inositol phosphates (PIPs) at 20 degrees C. Separation of the PIPs in less than 20 min with excellent resolution was achieved using a buffer containing sodium deoxycholate (SDC), 1-propanol, MgCl2 and the polymer coating reagent, EOTrol LR. The efficiency of the optimized method was as high as 1.3x10(5) plates. The dependence of the separation on the concentration of 1-propanol, SDC, and MgCl2 was determined. The separation of PIP2 and PIP3 was primarily due to differential binding of the lipids to Mg2+ rather than to different solubilities in the micellar phase. The role of the SDC was to prevent adsorption of the hydrophobic lipids to the capillary wall and thus enhance the efficiency. The fluorescent PIPs are of value for both in vitro and in vivo assays of phospholipid metabolism. In particular, the use of these lipids with the optimized capillary-based separation will be of utility for drug screening as well as cell-based assays.

This paper presents a fabrication of polymer microchips with homogeneous material technique due to surface treatment by plasma before sealing. UV laser photoablation was used for fast prototyping of microstructures, and oxygen plasma was used as a surface treatment for both the microfabricated substrate and the polymer cover. It was found that with an oxidative plasma treatment, successful bonding could be achieved without adhesive material between polymer sheets substantially below the glass transition temperature of the polymer. Homogeneous polyethylene terephthalate (PET) microstructures were characterized by scanning electron microscopy (SEM) and analyzed by X-ray photoelectron spectroscopy (XPS) surface analyses after different surface treatments. The electroosmotic flow characteristics including the velocity and the stability over 20 days have been tested and compared to composite channels, in which the cover presents a polyethylene (PE) adhesive layer. Capillary zone electrophoresis in both homogeneous and composite microanalytical devices were then performed and compared in order to evaluate the separation efficiency. In preliminary experiments, a plate height of 0.6 microm has been obtained with homogenous microchannels. The surface analysis pointed out that the surface chemistry is of prime importance for the performance of microfluidic separation.