Electrophoretic separation and confocal laser-induced fluorescence detection at ultralow concentrations in constricted fused-silica capillaries

2003 ◽  
Vol 24 (11) ◽  
pp. 1737-1744 ◽  
Author(s):  
Anders Lundqvist ◽  
Daniel T. Chiu ◽  
Owe Orwar
Keyword(s):  
Fluorescence Detection ◽  
Laser Induced Fluorescence ◽  
Fused Silica ◽  
Confocal Laser ◽  
Electrophoretic Separation ◽  
Laser Induced Fluorescence Detection ◽  
Fused Silica Capillaries ◽  
Ultralow Concentrations ◽  
Silica Capillaries

Characterization of a Rugged, Open-Gap Flow Cell for Confocal Laser-Induced Fluorescence Detection in Capillary Electrophoresis

1998 ◽  
Vol 52 (2) ◽  
pp. 292-297 ◽  
Author(s):  
David L. Gallaher ◽  
Mitchell E. Johnson
Keyword(s):  
Capillary Electrophoresis ◽  
Electric Fields ◽  
Fluorescence Detection ◽  
Fluorescein Isothiocyanate ◽  
Flow Cell ◽  
Laser Induced Fluorescence ◽  
Confocal Laser ◽  
Laser Induced Fluorescence Detection ◽  
Gap Flow ◽  
Wide Range

Construction details and performance characteristics of an open-gap flow cell for fluorescence detection in capillary electrophoresis are described. The flow cell is created by separating two pieces of capillary by a small (90 μm) gap. The gap is surrounded with buffer and grounded, and the application of electric fields to both inlet and outlet capillaries causes the material in the inlet capillary to flow across the gap. The use of a simple confocal optical arrangement for laser-induced fluorescence detection allows straightforward application of the gap flow cell to detection in capillary electrophoresis. The signal-to-noise ratio is measured to be about a factor of 2 better than that for on-column confocal detection at nanomolar concentrations over a wide range of pinhole diameters. The detection limit for fluorescein isothiocyanate is in the low picomolar range. Detection of a simple mixture of amino acids that have been derivatized with fluorescein isothiocyan ate demonstrates the stability and utility of the gap. Increased tailing is observed with the gap cell, with average asymmetry of about 1.4 near the center of the gap. Dispersion characteristics as a function of position in the gap are interpreted as dilution of the analyte as it flows across the gap. Fortunately, resolution and theoretical plates, measured by using least-squares fitting, are not significantly different from on-column separations, in spite of the tailing.

scholarly journals Quantification of Riboflavin, Flavin Mononucleotide, and Flavin Adenine Dinucleotide in Human Plasma by Capillary Electrophoresis and Laser-induced Fluorescence Detection

1999 ◽  
Vol 45 (6) ◽  
pp. 862-868 ◽  
Author(s):  
Steinar Hustad ◽  
Per Magne Ueland ◽  
Jørn Schneede
Keyword(s):  
Capillary Electrophoresis ◽  
Human Plasma ◽  
Fluorescence Detection ◽  
Limit Of Detection ◽  
Sodium Dodecyl ◽  
Laser Induced Fluorescence ◽  
Fused Silica ◽  
Flavin Mononucleotide ◽  
Laser Induced Fluorescence Detection

Abstract Background: Riboflavin is the precursor of flavin mononucleotide (FMN) and FAD, which serve as cofactors for several redox enzymes. We have developed a capillary electrophoresis method for the determination of riboflavin and its two coenzyme forms in human plasma. Methods: Trichloroacetic acid-treated plasma was subjected to solid-phase extraction on reversed-phase columns. The analytes were separated by micellar electrokinetic capillary chromatography in uncoated fused- silica capillaries filled with borate buffer containing 50 mmol/L sodium dodecyl sulfate, methanol, and N-methylformamide. Native fluorescence was monitored at 530 nm, using an argon laser operating at 488 nm as excitation source. Results: The assay was linear over a concentration range of two orders of magnitude, and the limit of detection was far below physiological concentrations for all vitamers. The within-day and between-day coefficients of variation were 4–9% and 6–12%, respectively. The reference values (median, 5–95 percentiles) obtained by analyzing plasma from 63 healthy subjects were 8.6 nmol/L (2.7–42.5 nmol/L) for riboflavin, 7.0 nmol/L (3.5–13.3 nmol/L) for FMN, and 57.9 nmol/L (44.5–78.1 nmol/L) for FAD. Conclusions: Capillary electrophoresis with laser-induced fluorescence detection allows determination of all riboflavin vitamers far below physiological concentrations. The method may become a useful tool for the assessment of riboflavin status in humans.

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