Thin Layer Chromatography-Based Separation of Mycolic Acid Variants: A Method to Separate Mycobacterial Cell Wall Lipids Based on Polarity

JoVE Encyclopedia of Experiments

Biologische Technieken

0 weergaven • 3:26 min • July 8th, 2025

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The mycobacterial cell wall contains different variants of mycolic acids - long-chain fatty acids with conserved alpha chains. Structural differences exist in their beta chains, resulting in the variations in the polarity of different mycolic acids.

To separate the variants using thin layer chromatography, or TLC - a liquid chromatography technique - take mycobacterial cell wall lipids in the desired organic solvent. Spot the lipids near the base of a TLC plate precoated with a thin layer of adsorbent material, which functions as the stationary phase.

Place the plate inside a jar containing an organic solvent - the mobile phase - ensuring the liquid level is below the sample spots to prevent premature diffusion of the mycolic acids.

During the run, the mobile phase rises through the tiny pores of the adsorbent layer on the plate via capillary action. Eventually, the mycolic acids dissolve in the solvent and travel upward.

More polar mycolic acid variants are transiently adsorbed by the polar stationary phase via intermolecular forces, hindering their upward movement. Less polar mycolic acids remain in the non-polar mobile phase and migrate further.

The differences in migration result in the variants of mycolic acid separating into discrete bands.

Upon completion, spray the dried plate with a phosphomolybdic acid stain. Heat the plate to reduce the stain in the presence of the lipids, imparting a dark green color to the bands.

To analyze the samples by TLC, cover one wall of the TLC chamber with a piece of filter paper. Add Vaseline on the chamber corners to seal the chamber. Decant the solvent mixture over the filter paper, and add the remaining volume of the solvent to the bottom of the TLC chamber. Close the TLC chamber for at least 20 minutes to saturate it

Meanwhile, dissolve the lipids in the glass tube in 200 to 1,000 microliters of chloroform, and use a capillary glass tube to apply 10 microliters of the lipid-chloroform suspension directly onto the TLC plate. After allowing the sample to dry for five minutes, insert the plate into the saturated TLC chamber, and allow the mobile phase to run through the TLC.

When the solvent reaches 1 centimeter from the upper end of the plate, place the plate under laminar flux until the silica is completely dried. Then, spray the dried plate with 15 to 20 milliliters of 10% Molybdatophosphoric acid hydrate in ethanol, until the plate is bright yellow, and heat the plate for two to five minutes at 120 degrees Celsius.

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Laatst bijgewerkt: 15 augustus 2026