Fatty acids composition

Safety measures

• When preparing the Sulfuric acid - do it in the chemical hood (with lowered glass) with safety glasses and thick gloves
• Pour the acid into the methanol in cooled (with ice) bucket
• The work should be conducted in chemical hood
• When dealing with hot equipment and sulfuric acid - wear thick gloves

Equipment

• The work should be conducted on the flask with the fat remains from the fat content
• Heating sleeves
• Condensation system (with ice!!!) and the long condenser
• 50 ml measuring flasks with plug
• Syringe with needle
• Filters (pink - 20 ul)
• Glass pipets
• Brown veils and cups

Materials

• Double distilled water (DDW)
• Methanol (LCMS grade)
• Sulfuric acid (3%) in methanol
• Petroleum ether

Protocol

1. Add to the flask with the fat remains 30 ml of acidic methanol
2. Heat the flask in condensation system for 1 hr
3. Cool the flask and transfer the content to 50 ml measuring flask
4. Clear the flask with 2 ml petroleum ether and transfer the liquid to the 50 ml flask
5. Add 10 ml of DDW, plug and shake well
6. Add water until the liquid reach the 50 ml mark
7. Wait 10 minutes for phase separation
8. Transfer the upper phase to brown veil
9. Filter the sample using syringe and filter
10. Plug the veil until running the GCMS
11. Before the first run in the GCMS Calibration curve should be done and samples should be diluted sample dilution