Phenazine Analysis

The quantification of phenazines was performed using high-performance liquid chromatography (HPLC) with a C₁₈ column (Waters Corporation) equipped with a photodiode array UV/VIS detector (LC-168, Beckmann), which detected and quantified the PCA at 366 nm and PYO at 280 nm. Separation was achieved with a gradient of 0.1% trifluoroacetic acid (TFA) in acetonitrile (solution A) and 0.1% TFA in water (solution B) as mobile phase (with solution A at 15% for 2 min, 100% for 15 min, and 5% for 3 min) at a flow rate of 0.8 mL/min and a column temperature of 20°C (Kern and Newman, 2014). Analytical grade PCA (Princeton BioMolecular) and PYO (Cayman Chemical) were used as standard solutions of phenazines.