1/3/2024 0 Comments Sandvox columnsThis is a useful safeguard because it also prevents inadvertant injection of acidic water into the GC/MS if the extraction was not done carefully.Ī. To provide a cleaner FAME sample, we recommend first cleaning up the extract on a short silica-gel column using hexane to elute the FAMEs. The hexane extract can now be concentrated into a GC vial and injected directly on the GC/MS. To maximize extraction yield, you can repeat the extraction with further 2 mL additions of hexane, if desired (note C).ĥ. Remove the hexane (top) phase and collect in a vial. This volume works well for biomass samples containing <100mg dry weight scale volume up as needed.Ĥ. Cap the tube tightly and heat at 100C for 10 minutes (note B). To each sample, add 2 mL of the methylation mixture (above) and 1 mL of hexane. The mixture needs to remain anhydrous, so limit exposure to air as much as possible.ģ. Failure to follow this procedure will result in spattering and/or boiling of the mixture, which should be avoided. Add acetyl chloride dropwise from a pipette, swirling the mixture as you add to prevent boiling. Be sure not to get any water around the vial top or cap, where it might drip into the methanol when it is opened. Prepare a mixture of 20:1 v/v anhydrous methanol/acetyl chloride as follows: transfer methanol into a clean 40mL vial, cap, and set to cool in an ice bath for ~5 minutes. We typically freeze-dry biomass overnight in the culture tubes used for extraction (note A). Samples must be thoroughly dried for this procedure. Keep the hood sash lowered while samples are heating, and wear safety glasses.ġ. Capped vials can sometimes burst while being heated, especially at the high temperature (100☌) used here. Be especially careful to keep acetyl chloride away from water. Always wear gloves and safety glasses, and add acetyl chloride to methanol slowly while keeping the mixture cold on ice. The reaction is highly exothermic, and can result in rapid boiling and/or explosions of an acidic solution. Acetyl chloride is very reactive with substances containing acidic protons, including water, methanol, etc. For those samples, we recommend overnight saponification followed by extraction. Note that the procedure is not recommended for plant materials, where hydrolysis of the more recalcitrant structural carbohydrates is required to get good lipid yields. The following procedure is our adaptation of the method published by Rodriguez-Ruiz et al (Biotechnology Techniques, Vol 12, No 9, September 1998, pp. For samples of bacterial biomass, the workup can be combined into a single rapid, quantitative step resulting in fatty acid methyl esters (FAMEs) ready for GC/MS or GC/IRMS analysis. Preparation of fatty acids from biological materials for GC/MS analysis is typically time consuming, requiring separate extraction, hydrolysis, and derivatization steps.
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