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A two-dimensional thin-layer chromatographic method was developed which allowed separation of the different mycobacterial mycolic acids as methyl esters. Dichloromethane (once) and petroleum ether:acetone (95:5, v/v twice or three times) were used as solvents. Alkaline saponification of freeze-dried cells followed by methylation of the mycolic acids using iodomethane gave satisfactory results, whereas methylation using boron trichloride-methanol complex or trans-esterification through direct acid methanolysis was found to degrade epoxy-mycolates. The chromatographic method developed here is rapid and informative, and should prove valuable in routine mycobacterial differentiation.
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