2.3. Analytical conditions
For both studies involving design of experiments, i.e., factor screening and method optimization by response surface methodology, chromatographic conditions were the same.
Mobile phase A was a solution of 10 mM ammonium formate adjusted to different pH with formic acid, and ethanol 96% (95/5,v/v).
Mobile phase B was composed of 10 mM ammonium formatein ethanol 96% with the same proportion of formic acid as mobile phase A to avoid an ionic concentration gradient. The gradient pro-file consisted in an initial hold of 1 min at 21% of organic phase B followed by a linear ramp from 21% to 36% of mobile phase B, and 6 min of equilibration. For all experiments, the sample injection volume was kept at 2 l.
Experimental designs were used to vary simultaneously gradient slope, pH of mobile phase A, flow rate, and column temperature.