title: Pulsed lasers versus continuous light sources in capillary electrophoresis and fluorescence detection studies: Photodegradation pathways and models.
abstract: Pulsed lasers are widely used in capillary electrophoresis (CE) studies to provide laser induced fluorescence (LIF) detection. Unfortunately pulsed lasers do not give linear calibration curves over a wide range of concentrations. While this does not prevent their use in CE/LIF studies, the non-linear behavior must be understood. Using 7-hydroxycoumarin (7-HC) (10-5000 nM), Tamra (10-5000 nM) and tryptophan (1-200 μM) as dyes, we observe that continuous lasers and LEDs result in linear calibration curves, while pulsed lasers give polynomial ones. The effect is seen with both visible light (530 nm) and with UV light (355 nm, 266 nm). In this work we point out the formation of byproducts induced by pulsed laser upon irradiation of 7-HC. Their separation by CE using two Zeta LIF detectors clearly shows that this process is related to the first laser detection. All of these photodegradation products can be identified by an ESI-/MS investigation and correspond to at least two 7HC dimers. By using the photodegradation model proposed by Heywood and Farnsworth (2010) and by taking into account the 7-HC results and the fact that in our system we do not have a constant concentration of fluorophore, it is possible to propose a new photochemical model of fluorescence in LIF detection. The model, like the experiment, shows that it is difficult to obtain linear quantitation curves with pulsed lasers while UV-LEDs used in continuous mode have this advantage. They are a good alternative to UV pulsed lasers. An application involving the separation and linear quantification of oligosaccharides labeled with 2-aminobezoic acid is presented using HILIC and LED (365 nm) induced fluorescence.
authors.affiliation: Picometrics Technologies, 478 Rue de la Découverte, Labège France.
authors.forename: Audrey
authors.initials: A
authors.lastname: Boutonnet
authors.affiliation: Picometrics Technologies, 478 Rue de la Découverte, Labège France.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Morin
authors.affiliation: Institut de Mathématiques, Université de Toulouse, Université Paul Sabatier, Toulouse CEDEX 04, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Petit
authors.affiliation: Laboratoire des IMRCP, UMR 5623, Université de Toulouse, Université Paul Sabatier, Toulouse CEDEX 04, France.
authors.forename: Patricia
authors.initials: P
authors.lastname: Vicendo
authors.affiliation: Laboratoire des IMRCP, UMR 5623, Université de Toulouse, Université Paul Sabatier, Toulouse CEDEX 04, France.
authors.forename: Véréna
authors.initials: V
authors.lastname: Poinsot
authors.affiliation: Laboratoire des IMRCP, UMR 5623, Université de Toulouse, Université Paul Sabatier, Toulouse CEDEX 04, France. Electronic address: couderc@chimie.ups-tlse.fr.
authors.forename: François
authors.initials: F
authors.lastname: Couderc
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods*; D007834:Lasers; D008027:Light; D008956:Models, Chemical; D055668:Photochemical Processes; D013050:Spectrometry, Fluorescence / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
keywords: Laser induced fluorescence; Photodegradation; Pulsed laser; Quantitative study