2.3. Chromatographic conditions
Several chromatographic conditions were attempted. After
achieving the first baseline conditions on reversed mode, the effects
of several factors on the chromatographic separation were
studied, including EtOH content, buffer concentration, pH, column
oven temperature and flow rate. The optimized mobile phase was
EtOH/10 mM aqueous ammonium acetate buffer (92.5/7.5, v/v), pH
6.8, performed at isocratic mode with a flow rate of 1.0 mL min1
and the column oven set at 20 C. The pH adjustments were done
with pH meter HI 2210 pH/C bench meter from Hanna Instruments.
Volume of injection was 20 lL.