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Fatty Acid Analysis of Natural Lipids by GC/FID

Natural lipids contain a heterogeneous mixture of fatty acids attached to the sphingosine moiety. The fatty acid composition is dependent on several factors including species, location within the organism, age, and environmental conditions. Normal variations in the fatty acids include chain-length, hydroxylation, and unsaturation.

To determine the fatty acid components present in a lipid sample the fatty acid is converted to a fatty acid methyl ester. It can then be analyzed by GC/FID and the individual fatty acid methyl esters identified by comparison of the retention times to reference standards.

For a more thorough review of the preparation of fatty acid esters from lipids please visit the Lipid Library

Preparation of Methyl Esters from Glycerides and Phospholipids: Methanolic Base Hydrolysis

  1. Weigh 5 mg of dry sample to be tested into a reaction vessel.
  2. Add 1ml of hexane to the sample.
  3. Add 50 μl of 1M sodium methoxide in methanol.
  4. Mix thoroughly.
  5. Let sit at room temperature for 5 minutes.
  6. The sample is now ready to analyze by GC/FID.

Preparation of Methyl Esters from Phospholipids, Neutral Sphingolipids, Neutral glycosphingolipids, and Acidic Glycosphingolipids: Methanolic Sulfuric Acid

  1. Make a solution of 2% sulfuric acid in methanol. The 2% sulfuric acid/methanol solution must be made fresh before use.
  2. Add 1 ml of the sulfuric acid solution to 5-10 mg of dry lipid sample in a 15 ml reaction tube.
  3. Add 1 boileezer to the reaction mixture.
  4. Blanket with argon and seal with a Teflon-lined cap.
  5. Place in an equilibrated heating block as indicated below:
    1. Fatty acid to Methyl ester – 5 Minutes @ 80°C
    2. 2- or 3-Hydroxy fatty acids – 1.5 Hrs. @ 80°C
    3. Phospholipids – 2 Hrs. @ 70°C
    4. Glycolipids – 6 Hrs. @ 100°C
    5. Sphingomyelin – 2 Hrs. @ 100°C
  6. At the end of the desired time, remove from the heating block and let cool to room temperature. Do not open until cool!.
  7. Add 0.5 ml of DI water, which will stop the reaction.
  8. Add 4 ml of hexane and shake vigorously.
  9. Let set until two phases are visible. Note: Top hexane layer must be clear.
  10. Remove the top hexane layer and save.
  11. Repeat steps 7-9 two more times.
  12. Discard the bottom layer after fully extracted.
  13. Combine the three top layers and add a small amount of a mixture of sodium sulfate/ sodium bicarbonate, (4:1).
  14. Shake vigorously.
  15. Using a pipette, transfer the solvent layer to a 15mL reaction tube. Note: Be careful not to pull any of the sodium sulfate/sodium bicarbonate solids.
  16. Concentrate, with a stream of nitrogen, down to 1 ml.
  17. The sample is now ready to analyze by GC/FID.

  18. GC/FID Analysis of Fatty Acid Methyl Esters

    GC Conditions:*
    1. Column: SP-2330 or RTX-2330; 30 x 0.25mm x 0.2μm
    2. Oven: 200°C Isothermal
    3. Carrier Linear Velocity: helium at 20 cm/sec.
    4. Detector: FID, 250°C
    5. Injector: 250°C
    6. Split ratio: 100:1
    7. *These are general GC conditions for fatty acid methyl esters and may not separate all components of a sample.

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