Lipid analysis by thin-layer chromatography

A volume of 200 µL of lipid sample, approximately 5% of the lipid extract from 50-mL bacterial culture, was transferred to an Agilent glass sample vial, dried for 10 min in vacuum, and resuspended in 30 µL of 4:1 chloroform/methanol. Then a glass spotting capillary tube was used to spot the sample 1.8 cm above the bottom edge of an EMD Millipore TLC plastic sheet cut to a height of 10 cm. After drying in air at an ambient temperature of 21 °C for 20 min, the TLC sheet was placed into a TLC chamber pre-equilibrated with a mixed solvent of chloroform/methanol/water (70:25:4) or chloroform/methanol/water/acetic acid (70:25:3:1). After 25 min of TLC development, the TLC sheet was removed from the TLC chamber and dried for 20 min at ambient temperature. For 2D-TLC analysis, the dried TLC sheet was rotated 90 degrees and placed into a TLC chamber pre-equilibrated with a mixed solvent of chloroform/methanol/water/acetic acid (70:25:4:1), developed for another 25 min, and dried for 20 min. The dried TLC sheet was sprayed with 0.02% primuline (Sigma-Aldrich) solution in 80:20 acetone/water, and then dried in air at ambient temperature for an hour. The fluorescent image was recorded with a Syngene G:BOX system. The silica gels at each representative fluorescent band were lifted with a metal spatula and suspended in 200 µL chloroform in an Agilent glass sample vial and stored at −20 °C. 400 µL methanol was added to the suspension right before MS analysis. After shaking by hand for 10 s followed by sedimentation of silica gel for 2 min, the clarified sample was injected into the mass spectrometer for analysis. MS scan, precursor scan, neutral loss scan, and MS/MS spectra were acquired to assign lipid composition in each TLC fluorescent band. After baseline subtraction, fluorescence intensities of lipid bands on thin-layer chromatograms were integrated using GelAnalyzer version 2010a.