Zorbax sb c18 reverse phase column

For antibiotics production, spore suspensions were inoculated into liquid SP medium and cultured at 28 °C for 24 h as seed culture in shake flask (220 revolutions per minute, rpm), and then 30 ml of seed culture was transferred to 3 L of SP in a 5 L fermentor (BIOTECH-5JG, BX-BIO). BIOTECH-FCS software was used to control the equipment and collect data. Air was sparged into the fermentor to supply oxygen at four times atmospheric pressure, and the rotor speed was 400 rpm. After fermentation for 5 days at 28 °C the culture broth of ΔwblA was filtered by Pyrex Buchner funnel with a fritted disc (pore size 40–60 mm). Then the supernatant was extracted by separatory funnel with equal volume of chloroform for three times at room temperature. Chloroform extract was evaporated to dry. The resulting sample was re-dissolved in methanol and then separated on Sephadex LH-20 as mentioned above. Active fractions were collected and purified by semi-preparative HPLC equipped with ZORBAX SB-C18 reverse phase column (9.4 × 250 mm, 5 μm, Agilent) by linear gradient elution as mentioned above.
MS analysis was performed on LTQ Orbitrap hybrid mass spectrometer (Thermo-Fisher) equipped with a Dionex Ultimate 3000 nano-flow system and a nano-electrospray ion source. NMR spectra were recorded on a 500 MH_Z Bruker spectrometer using CDCl₃ as the solvent.

The chemical constitutes of HKC were analyzed on a ZORBAX SB-C18 reverse-phase column (4.6 mm × 250 mm, 5 μm, Agilent Technologies Inc., Santa Clara, CA, USA) performed on an Agilent 1260 Series HPLC system and the absorptions were monitored by a VW detector at the wavelength of 254 nm. The column was eluted with a gradient concentration of acetonitrile with trifluoroacetic acid (TFA) at a flow rate of 1 mL/min using the following program. Solvent A: 0.1% TFA in ddH₂O, solvent B: acetonitrile, 0–5 min, 6% B; 5–30 min, 6–17% B; 30–40 min, 17% B; 40–60 min, 17–40% B.

The sampled liquid was centrifuged, and the supernatant was used for further analysis. The concentrations of quinoline and 2-hydroxyquinoline were analyzed by the use of a high-pressure liquid chromatography (HPLC) system (Agilent; Zorbax SB-C₁₈ reverse-phase column, 150 by 4.6 mm, 5-μm pore size). The mobile phase was a methanol solution with a volume ratio of 60:40 (methanol/water) used at a flow rate of 1.0 ml/min. Quinoline and 2-hydroxyquinoline were both detected at a 225-nm wavelength. The injection volume was 20 μl, and the column temperature was 30°C. The nitrate concentration was measured using a PXJ-1B ion meter (Jiangfen, China) with a pNO₃⁻¹ nitrate ion-selective electrode (Tianci, Shanghai).