Analysis of FAMEs - modified Bannon Method.
The procedure is rapid, accurate ( for common triglyceride oils ), and properly-prepared reagents last for years.
Method
10 - 100 mg of oil is accurately weighed into a glass 4ml screw cap vial, dissolved in 2.0 mLs of A/R ( or chromatography ) grade iso-octane ( aka 2,2,4-trimethylpentane ), add 100 ul of 2M methanolic potassium hydroxide, immediately mix by vigorous shaking for 30 seconds ( vortex mixer preferred ). Allow to stand to room temperature, mixing vigorously for 10-15 secs for each of the subsequent 6 minutes.
Add 1-2 drops of methyl orange indicator solution, and immediately add 2M aqueous hydrochloric acid dropwise, swirling gently after each addition. Mix more vigorously towards the endpoint ( usually about 80-100 uL ) to ensure the hydrocarbon phase contacts the acid. The indicator should remain at the endpoint colour for at least 1 minute after mixing.
Allow to stand for a couple of minutes to obtain a clear hydrocarbon upper phase, then immediately pipette an aliquot into a vial, diluting with solvent or internal standard solution, as required. The final concentration depends on your method, but I injected ~ 10 mg/mL solution into split injection.
The advantage is that you can process about 6 - 8 samples at once, taking only about 10 minutes, even if you don't have a vortex mixer handy. It also can be used for short chain acids ( dairy lipids have C4:0 ). I used a 100ul pipettor to dispense the KOH, pasteur pipette for the indicator drops, and the pipetter again for the HCl.
The 2M alcoholic KOH, 2M HCl, and methyl orange should be prepared as per standard methods ( eg AOAC, AOCS, Vogel ), and the KOH filtered through glasswool to remove insolubles. Stored in the fridge, and used straight from the fridge, they will last for years.
The essential parameter of the method is the mixing, you must ensure the immiscible liquids mix intimately and frequently during the reaction. If you don't get a sharp methyl orange endpoint, either you haven't mixed well, or your sample contains polar lipids or other material that reacts slowly - use another method. Longer time or above ambient temperatures are not better, mix well, and complete within 8 - 10 minutes, and ensure the upper phase is clear and bright before removing your sample.
In the second paper cited below, the authors subsequently claim the method is unsuitable for longer chain acids, however I had no problems in client method correlation studies, and believe that they didn't emphasise the importance of regular mixing during the reaction, however, YMMV.
Original Method
" Analysis of Fatty Acid Methyl Esters with High Accuracy and Reliability. IV. Fatty Acids Containing Four or More Carbon Atoms ".
Cecil D. Bannon, John D. Craske, Audrey E. Hilliker
JAOCS, Vol 62, no. 10. ( October 1985 ) p. 1501-1507.
Subsequent Repudiation
" Limitations of Ambient Temperature Methods for the Methanolysis of Triacylglycerols in the Analysis of Fatty Acid Methyl Esters With High Accuray and Reliability ".
John D. Craske, Cecil D Bannon, and Lynette M. Norman
JAOCS, Vol 65, no.2 ( February 1988 ), p. 262-266.
Please keep having fun,
Bruce Hamilton
