Figure 3.

HPLC and TLC analysis of fatty acids. (A) Fractionation of the p-bromophenacyl esters of fatty acids from the mc²-155 (solid line) or the 155NS1 mutant (dashed line) by HPLC. Cells (10 mg dry weight) were saponified and derivatized with p-bromophenacyl bromide as described in Materials and Methods. The fatty acid esters were dissolved in 100 μl of CH₂Cl₂, and 10 μl of this solution was applied to the HPLC column. Only fractions eluted between 18 and 34 min are shown. (B) TLC analysis of fatty acid methyl esters. Lanes: 1, methyl esters of mycolates purified from mc²-155; 2, esters of fatty acids from mc²-155; 3, esters of fatty acids from 155NS1. TLC was developed with hexane/ethyl acetate (9:1) as described in Materials and Methods. The weak spot underneath the mycolate ester spot in lane 2 contains mostly epoxy-mycolate, based on two-dimensional TLC analysis (not shown).