Fatty Acid Methyl Esters-Acidic Methanol

Use this new and IMPROVED method to make the fatty acid methyl esters.

Mild methanolysis/methylation and rapid methanolysis/methylation using concentrated HCl.

Work in the hood, wear protective clothing, googles and gloves. This procedure requires the use of strong acid.

This solution has been prepared: Commercial conc. HCl (35%, w/w; 9.7 ml) diluted with 41.5 ml of methanol to make 50 ml of 8.0% (w/v) HCl. This HCl reagent contains 85% (v/v) methanol and 15% (v/v) water derived from conc. HCl and is stored in a refrigerator.

1. Evaporate 2 mL of your lipid sample in a screw-capped glass test tube. (One 16.5 × 105 mm tube for neutral lipids and one tube for polar lipids)
2. Dissolve the dry lipids in 0.20 ml of toluene.
3. Add 1.50 ml of methanol
4. Add 0.30 ml of the 8.0% HCl/Methanol solution. The final HCl concentration is 1.2% (w/v) or 0.39 M, which corresponded to 0.06 ml of conc. HCl in a total volume of 2 ml.
5. Vortex the test tubes and heat at 100°C for 1 hour. (May work for cholesterol esters if extended to 1.5 hours.) Alternatively the reaction can be incubated at 45°C overnight (14 h or longer) for mild methanolysis/methylation.
6. Cool to room temperature
7. Extract FAMEs by adding 1 mL of hexane and 1 ml of water. Vortex and remove the organic layer.
8. Analyze the organic layer by GC, capillary column, FID detector.

Modified from:
Ichihara K, Fukubayashi Y. Preparation of fatty acid methyl esters for gas-liquid chromatography. Journal of Lipid Research. 2010;51(3):635-640. doi:10.1194/jlr.D001065.

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