betadex and 7-fluoro-4-nitrobenzo-2-oxa-1-3-diazole

The continuous nature of micro free-flow electrophoresis (mu-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 mu-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-beta-cyclodextrin (HP-beta-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 mu-FFE recorded 60 separations during a 5 min gradient allowing nearly complete coverage across a range of HP-beta-CD concentrations. In comparison, 4 h were required to assess 15 sets of conditions across the same range of HP-beta-CD concentrations using CE. Qualitatively, mu-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 mu-FFE and CE.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; 4-Chloro-7-nitrobenzofurazan; Amino Acids; beta-Cyclodextrins; Buffers; Electrophoresis; Ethanolamines; Fluorescein; Fluorescent Dyes; Kinetics; Models, Theoretical; Viscosity

A capillary zone electrophoresis method with laser induced fluorescence detection for the chiral separation of highly fluorescent enantiomeric derivatives of d/l-Serine from 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-d/l-Serine) was developed and optimized. Enantiomeric separation of NBD-d/l-Serine was accomplished by using 40 mM hydroxypropyl-beta-cyclodextrin (HP-beta-CD) contained in 100 mM borate buffer, pH 10.0. A 70 cm (effective length of 50 cm) uncoated fused-silica capillary at a voltage of 15 kV was used for the separation. The optimized electrophoretic conditions were subsequently applied to the analysis of d-Serine in rat brain, and satisfactory analytical results with respect to accuracy were obtained. This assay showed acceptable precision, with linearity in the d-Serine concentration range of 0.2-20.0 microM. The limit of detection for d-Serine was 3.0 x 10(-7)M.

Topics: 4-Chloro-7-nitrobenzofurazan; Animals; beta-Cyclodextrins; Brain Chemistry; Electrophoresis, Capillary; Fluorescence; Lasers; Male; Rats; Serine; Stereoisomerism