1260 infinity chromatograph

The assessment of chemical composition of each extract (RE, LBE) was carried out on a 1260 Infinity chromatograph (Agilent, Santa Clara, CA, USA) consisting of binary pump, a column oven, and photo-diode array (PDA) detector over 55 min period. The separation was performed using a Kinetex XB C18 column (150 × 3 mm, 2.6 µm) (Phenomenex, Torrance, CA, USA). The mobile phase was 0.1% (v/v) FA in water (A) and 0.1% FA in acetonitrile (B). The separation was achieved by a gradient of 0–2 min 1% B; 2–20 min 1–20% B; 20–40 min 20–75% B; 40–45 min 75–95% B; 45–48 min 95% B; 48–49 min 95–1% B; 49–55 min 1% B. The flow rate was 0.5 mL/min and the column temperature was maintained at 25 ± 0.8 °C. The UV–Vis spectra was recorded from 190 to 540 nm with selective wavelength monitoring at 280 nm. Mass spectrometry (MS) detection was carried out on a 6230 MS/TOF mass spectrometer (Agilent, Santa Clara, CA, USA) equipped with an electrospray ionization source with Agilent Jet Stream thermal focusing. The parameters used for ionization source were set as follows: drying and sheath gas flow: 12 L/min; nebulizer: 35 psi; source temperature 350 °C; ion spray voltage 4500 V for the positive mode analysis. The data were collected in 115–1900 m/z range and processing was performed using Mass Hunter qualitative analysis software.

The monosaccharide composition of CPP 2–4 was determined by HPLC with precolumn derivatization. In brief, TFA (4 mL, 4 mol/L) was added to CPP 2–4 (4 mg), and the resulting product was hydrolyzed at 110 °C for 4 h to remove excess acid and then dissolved in distilled water (0.2 mL). NaOH solution (0.1 mL, 0.6 M) and PMP methanol solution (0.2 mL, 0.5 M) were added to the above solution, which was then vortexed and heated in a water bath at 70 °C for 100 min. Next, HCl (0.2 mL, 0.3 M) was added to neutralize the solution, followed by trichloromethane (1 mL). After mixing thoroughly, the mixture was centrifuged at 12,000 rpm for 10 min, and then trichloromethane (1 mL) was added to the supernatant. This operation was repeated three times. Finally, the supernatant was fixed to a volume of 2 mL and filtered through 0.22-μm microporous filter membrane and analyzed by HPLC (Agilent 1260 Infinity chromatograph) with a UV detector at 254 nm.
The analytical column was C₁₈ (5 μm, 4.6 mm × 250 mm, Agilent, USA) and operated at 30 °C. Mobile phase A was acetonitrile and mobile phase B was 0.1 mol/L phosphate buffer (pH 7.65). The gradient program was as follows: 0–28 min, 17% A; 28–40 min, linear gradient to 30% A; 40–45 min, 30% A; 45–50 min, linear gradient to 17% A. Elution was performed at a flow rate of 1.0 mL/min and the injection volume was 10 μL.

The purified lipopeptide fraction (PL₈₄) was characterized by reverse-phase high-pressure liquid chromatography (HPLC) with a 1260 infinity chromatograph (Agilent Technologies, Santa Clara, CA, USA) with a C18, 4-mm Zorbax, Agilent (100 × 4.6 mm) column. Samples (10 mL) of PL₈₄ and mycosubtilin standard (Lipofabrik, Lesquin, France) were analyzed at a flow rate of 0.3 mL/min with an isocratic elution using 60% solvent A (water) and 40% solvent B (acetonitrile) at 20 °C. Peaks eluting from the column were detected by their absorbance at 220 nm [43 (link)].