Synapt g2 s column

Reverse-phase HPLC analyses were performed using a C₁₈ column (Mightysil RP-18 GP 250-4.6, 5 μm) at a UV absorbance of 420 nm. The mobile phase consisted of solvent A (water with 0.1% trifluoroacetic acid [TFA]) and solvent B (acetonitrile). The program for HPLC was as follows: 10% acetonitrile at 0 min, 30% acetonitrile at 5 min, 50% acetonitrile at 10 min, 90% acetonitrile at 15 min, 70% acetonitrile at 18 min, and 10% acetonitrile at 30 min, with a flow rate of 1 mL/min. High-resolution quadrupole time of flight electrospray ionization-mass spectrometry (HR-QTOF ESI/MS) analysis was performed in the positive ion mode using an Acquity column (UPLC; Waters Corp., Billerica, MA, USA) coupled with a SYNAPT G2-S column (Waters Corp.).

S. lividan TK24 and other mutants (S. lividan pIBR25-thaA, S. lividan pIBR25) were cultivated in 50 ml of R2YE media supplemented with 15 μg/ml of thiostrepton at a temperature of 28°C for a duration of 5 days and extracted with a double volume of ethyl acetate. The organic layer was then concentrated using a rotary evaporator and the resulting concentrated sample was dissolved in a suitable amount of methanol, filtered, and subsequently examined through reverse-phase high-performance liquid chromatography (HPLC, Mightysil RP-18 GP 250-4.6, Japan), and mass analysis was performed by liquid chromatography-electron spray ionization/mass spectrometry (LC-ESI/MS) in the positive ion mode using an Acquity column (UPLC; Waters Corp., USA) coupled with a SYNAPT G2-S column (Waters Corp.). The sample was eluted using a solvent mobile phase gradient of 0.1%trifluoroacetic acid (TFA) in water and 100% acetonitrile (ACN) over a period of 0 to 12 min at a temperature of 35°C. The volume of the injected sample was 10 microliters.