Uncoated fused silica capillaries

Spectrofluorimetry studies were conducted using a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Foster City, CA, USA). An excitation wavelength of 360 nm was used, followed by an emission scan from 365–700 nm. Excitation and emission slit widths were 5 nm; the scan rate was 300 nm/min; and the PMT voltage was 600 V. CE studies were conducted using a P/ACE MDQ CE System with 32Karat software (Beckman Coulter, Redwood City, CA, USA) or an Agilent G1600A CE System equipped with Chemstation software. Detection was performed by UV absorbance at 200 nm, or by laser-induced fluorescence (LIF) using a 375 nm diode laser with 5 mW output power (Oz Optics Ltd., Carp, ON, Canada) and 400 nm long pass filter (Omega Optical, Brattleboro, VT, USA), or a Picometrics LIF Detector (406 nm laser with 12.5 mW output power and 410 nm emission filter) for the Beckman-Coulter and Agilent CE systems, respectively. All CE experiments employed uncoated fused-silica capillaries (Polymicro Technologies, Phoenix, AZ, USA) with different lengths and inside diameters (as specified in the Results and Discussion section).

Fructose, glucose, and sucrose concentration was determined following the method described by Cebolla-Cornejo et al. (2012) (link) with some modifications using an Agilent 7100 capillary electrophoresis system (Agilent Technologies, Waldbronn, Germany). Prior to use, uncoated fused silica capillaries (67 cm total length, 60 cm effective length, 375 μm outside diameter, 50 μm internal diameter) from Polymicro Technologies (Phoenix, AZ, United States) were conditioned at 50°C with NaOH 1 N (5 min), NaOH 0.1 N, and MilliQ water (10 min). Before each working session, the capillary was rinsed for 30 min with the running buffer (20 mM PDC and 0.1% w/v HDM at pH 12.1). Between runs, the capillary was flushed with 60 mM SDS (3 min), MilliQ water (1 min) and the running buffer (2 min). Samples were diluted ½ with MilliQ water, filtered (0.2 μm) and then injected hydrodynamically at 6900 Pa during 30 s. Separations were performed at -25 kV and 20°C, with indirect detection at 214 nm.

All MEKC experiments were carried out with a P/ACE MDQ plus system (Sciex, Framingham, MA, USA). The electropherograms were recorded and analyzed with the 32 Karat Software (version 10.2). The uncoated fused silica capillaries (50 mm i.d., Polymicro Technologies, West Yorkshire, UK) of a total length equal to 60 cm × 50 µm were used during the study. The following rinsing procedures were applied before every working day: first rinsing with 0.1 M NaOH for 30 min, next ultrapure water for 10 min, and finally BGE for 30 min. Between analyses the capillary was conditioned with BGE for 2 min. The applied pressure for all rinsing operations was 345 kPa. The investigated isoxazolone derivatives were dissolved in BGE at concentrations of 100 µg/mL with the addition of quinine (micelles marker) and DMSO (EOF marker). Hydrodynamic injected mode (35 kPa for 5 s) was used to introduce samples into the capillary. The separation condition was as follows: voltage application of 20 kV with positive polarity and a constant temperature of 25 ± 0.1 °C. The separations were performed in duplicates. The BGE consisted of aqueous solution of 50 mM SDS and 120 mM HEPES/100 mM Tris buffer of pH 7.4. Detection was carried out at 200, and 250 nm with 8 Hz probing frequency. The logarithm of retention factor logk_MEKC was calculated by the equation proposed by Terabe and co-workers.