HILIC-based Bacterial Lipid Separation

Bacterial lipids were separated by a Waters UPLC (Waters Corp., Milford, MA, United States) as described previously (Hines K. M. et al., 2017 (link); Hines KM. et al., 2017 (link)). Briefly, hydrophilic interaction liquid chromatography (HILIC) was performed with a Phenomenex Kinetex HILIC column (2.1 × 100 mm, 1.7 µm) maintained at 40°C at a flow rate of 0.5 ml/min. The solvent system consisted of: A) 50% acetonitrile/50% water with 5 mM ammonium acetate; and B) 95% acetonitrile/5% water with 5 mM ammonium acetate. The linear gradient was as follows: 0–1 min, 100% B; 4 min, 90% B; 7–8 min, 70% B; 9–12 min, 100% B. A sample injection volume of 5 µL was used for all analyses.