Stacking and separation of fluorescent derivatives of amino acids by micellar electrokinetic chromatography in the presence of poly(ethylene oxide)

A new approach for the analysis of large-volume naphthalene-2,3-dicarboxaldehyde (NDA) derivatives of amino acids by micellar electrokinetic chromatography (MEKC) in conjunction with a purple light-emitting diode-induced fluorescence detection is described. In order to optimize resolution, speed, and stacking efficiency, a discontinuous condition is essential for the analysis of NDA-amino acid derivatives. The optimum conditions use 2.0 M TB (pH 10.0) buffer containing 40 mM sodium dodecyl sulfate (SDS) to fill the capillary, deionized water to dilute samples, and 200 mM TB (pH 9.0) containing 10 mM SDS to prepare 0.6% poly(ethylene oxide) (PEO). Once high voltage is applied, PEO solution enters the capillary via electroosmotic flow and SDS micelles interact and thus sweep the NDA-amino acid derivatives having smaller electrophoretic mobilities than that of SDS micelles in the sample zone. When the aggregates between SDS micelles and NDA amino acid derivatives enter PEO zone, they are stacked due to decrease in electric field and increases in viscosity. Under the optimum conditions, the concentration and separation of 0.53-μL 13 NDA-amino acid derivatives that are negatively charged has been demonstrated by using a 60-cm capillary, with the efficiencies 0.3-9.0 × 10⁵ theoretical plates and the LODs at signal-to-noise ratio 3 ranging from 0.30 to 2.76 nM. When compared to standard injection (30-cm height for 10 s), the approach allows the sensitivity enhancements over the range of 50-800 folds for the derivatives. The new approach has been applied to the analysis of a red wine sample, with great linearity of fluorescent intensity against concentrations (R² > 0.98) and the RSD (three repetitive runs in one day) values of the migration times for the ten identified amino acids less than 2.8%.