Utilisation of pH stacking in conjunction with a highly absorbing chromophore, 5-aminofluorescein, to improve the sensitivity of capillary electrophoresis for carbohydrate analysis
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Kazarian, A and Hilder, EF and Breadmore, MC, Utilisation of pH stacking in conjunction with a highly absorbing chromophore, 5-aminofluorescein, to improve the sensitivity of capillary electrophoresis for carbohydrate analysis, Journal of Chromatography A, 1200, (1) pp. 84-91. ISSN 0021-9673 (2008) [Refereed Article]
This study explores the use of pH stacking in conjunction with 5-aminofluorescein as a derivatization agent for the sensitive analysis of simple sugars such as glucose, lactose and maltotriose by capillary electrophoresis (CE). The derivatization agent was selected on the basis of its extremely high molar absorptivity, its compatibility with a 488 nm light-emitting diode (LED) and the fact that it has two ionizable groups making it compatible with on-line stacking using a dynamic pH junction. The influence of both acetic and formic acids at concentrations of 0.19, 0.019 and 0.0019 mol L-1 were investigated with regard to both derivatization efficiency and the ability to stack using a dynamic pH junction. Superior sensitivity and resolution was obtained in formic acid over acetic acid. Substantially lower peaks were obtained with 0.19 mol L-1 formic acid when compared to 0.019 and 0.0019 mol L-1 concentrations, which was confirmed by computer simulation studies to be due to the inadequate movement of the pH boundary for stacking. Further simulation studies combined with experimental data showed the separation with the best resolution and greatest sensitivity when the carbohydrates were derivatized with the 0.095 mol L-1 formic acid. Utilisation of stacking via dynamic pH junction mode in conjunction with LED detection enabled efficiencies of 150,000 plates and detection limits in the order of 8.5 × 10-8 mol L-1 for simple sugars such as glucose, lactose and maltotriose hydrate. The current system also demonstrates a 515 times improvement in sensitivity when compared to using a normal deuterium lamp, and 16 times improvement over other systems using LEDs. © 2008 Elsevier B.V. All rights reserved.
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