Boutonnet Audrey, Morin Arnaud, Petit Pierre, Vicendo Patricia, Poinsot Véréna, Couderc François
Picometrics Technologies, 478 Rue de la Découverte, Labège France.
Institut de Mathématiques, Université de Toulouse, Université Paul Sabatier, Toulouse CEDEX 04, France.
Anal Chim Acta. 2016 Mar 17;912:146-55. doi: 10.1016/j.aca.2016.01.036. Epub 2016 Jan 28.
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.
脉冲激光广泛应用于毛细管电泳（CE）研究中，以提供激光诱导荧光（LIF）检测。不幸的是，脉冲激光在很宽的浓度范围内不能给出线性校准曲线。虽然这并不妨碍它们在CE/LIF研究中的使用，但必须了解其非线性行为。使用7-羟基香豆素（7-HC）（10 - 5000 nM）、Tamra（10 - 5000 nM）和色氨酸（1 - 200 μM）作为染料，我们观察到连续激光和发光二极管能得到线性校准曲线，而脉冲激光给出的是多项式曲线。在可见光（530 nm）和紫外光（355 nm、266 nm）下均观察到了这种效应。在这项工作中，我们指出了脉冲激光照射7-HC时诱导产生的副产物的形成。使用两个Zeta LIF检测器通过CE对它们进行分离，清楚地表明这个过程与第一次激光检测有关。所有这些光降解产物都可以通过电喷雾负离子/质谱（ESI⁻/MS）研究来鉴定，并且至少对应两种7-HC二聚体。通过使用Heywood和Farnsworth（2010年）提出的光降解模型，并考虑7-HC的结果以及在我们的系统中荧光团浓度不恒定这一事实，有可能提出一种LIF检测中荧光的新光化学模型。该模型与实验一样表明，使用脉冲激光很难获得线性定量曲线，而连续模式下使用的紫外发光二极管具有这一优势。它们是紫外脉冲激光的良好替代品。本文展示了一个应用，即使用亲水作用色谱（HILIC）和发光二极管（365 nm）诱导荧光对用2-氨基苯甲酸标记的寡糖进行分离和线性定量。
