Analytical chromatography was performed on a Waters Alliance e2695 system, equipped with a 2998 PDA detector. The columns used were Waters XBridge BEH C18 4.6 mm × 50 mm, 2.5 μm or Waters Symmetry C18 4.6 mm × 250 mm, 3.5 μm. Solvent A was water, solvent B acetonitrile, and solvent C water containing 1% of trifluoroacetic acid. The Waters XBridge column was used under gradient conditions as follows: flow rate, 1.5 mL/min; column temperature, 40°C; t=0 min 80% A/10% B/10% C; t=5 min 0% A/90% B/10% C; t= 6 min 0%A/100% B/0% C; run time 8 min; UV detection from 200 to 800 nm. The Waters Symmetry column was used under isocratic conditions as follows: flow rate 1.5 mL/min; column temperature, 35°C; t=0 min 35% A/55% B/10% C.
Semi-preparative chromatography was performed on a Waters Autopurification system composed of a Binary Gradient Module 2545 pump, a 996 PDA detector and a 2767 Sample Manager. Solvent A was water with 0.1% TFA, solvent B acetonitrile with 0.1% TFA. The column used was a Waters XBridge OBD C18 10 mm × 100 mm, 5 μm and the conditions were as follows: t=0 min 55% A/ 45%B; t= 5 min 55% A/45%B; t= 5.5 min 50% A/50% B; t=12 min 50% A/50% B; t=12.5 min 45%A/55%B; t=17 min 45% A/55%B; t=19 min 0% A/100% B; run time 25 min; UV detection from 200 to 800 nm.
Semi-preparative chromatography was performed on a Waters Autopurification system composed of a Binary Gradient Module 2545 pump, a 996 PDA detector and a 2767 Sample Manager. Solvent A was water with 0.1% TFA, solvent B acetonitrile with 0.1% TFA. The column used was a Waters XBridge OBD C18 10 mm × 100 mm, 5 μm and the conditions were as follows: t=0 min 55% A/ 45%B; t= 5 min 55% A/45%B; t= 5.5 min 50% A/50% B; t=12 min 50% A/50% B; t=12.5 min 45%A/55%B; t=17 min 45% A/55%B; t=19 min 0% A/100% B; run time 25 min; UV detection from 200 to 800 nm.