Luna omega 5 μm polar c18 lc prep column

A LB overnight culture of P. aeruginosa PAO1 was diluted 100-fold into 300 mL of M9 medium (1% w/v 5X M9 Salts (BD Difco, Franklin Lakes, NJ), 1.5% w/v Bacto Casamino Acids with low iron and salt content (BD Difco), 1 mM MgSO₄, 1 mM CaCl₂) in a 2 L flask and grown aerobically for 24 h at 37 °C. Bacteria were then removed by centrifugation and filtration through a 0.22 μm membrane. The filtrate was incubated with 10% w/v amberlite XAD-4 resin (MilliporeSigma) at room temperature for 4 h with constant agitation. After rinsing the resin with copious amounts of water, pyoverdine was eluted in 50% methanol. This eluent was diluted in water to 15% methanol and loaded onto a Luna Omega 5 μm Polar C18 LC prep column (Phenomenex, Torrance, CA) for high-performance liquid chromatography on a 1220 Infinity LC system (Agilent Technologies, Santa Clara, CA). Pyoverdine was eluted from the column by a 0–100% methanol gradient across 4 h at a flowrate of 5 mL/min. Fractions were collected every other minute for pyoverdine content analysis (Fig. 5B). The fractions with the highest pyoverdine content were pooled. Methanol was evaporated using a SpeedVac vacuum concentrator. The final purified product was analyzed by HPLC on an analytical column to verify sample purity (Fig. 5C).