Introduction

Organic acids can be esterified with methanol (or other alcohols) to produce methyl-ester derivatives suitable for analysis by GC. The reaction can be carried out under acidic conditions using a number of different reagents. This method uses methanol (the reagent) and BF3 (an acidic catalyst), which are available premixed in the correct proportions from Alltech and other supply houses. The resulting methyl esters are stable indefinitely in neutral solvents, but can be hydrolyzed under strongly acidic or basic conditions. The byproducts of the reaction cannot be injected into the GC, so derivatized analytes must first be extracted from the reaction mixture.

Materials

• BF3/MeOH (premixed reagent)
• DCM, hexane
• organic-free water (note A)
• heating block (70C)

Warnings

• BF3 is toxic and corrosive. Always wear gloves and keep it in the hood. If you spill it on yourself, wash immediately with water.
• Capped vials have occasionally burst while being heated. Keep the heating block in the hood with the sash lowered while samples are being heated.

Procedure

1. Transfer sample to a 15mL culture tube using an appropriate aprotic solvent. If necessary, evaporate solvent to remove any methanol and redissolve in DCM. The sample should be carefully dried prior to this step.
2. Add 100uL of BF3/MeOH reagent to the sample. This amount is sufficient for a sample containing <100ug of organic acids. If your sample is bigger, you will need to scale up the amount of reagents being added, or take a smaller aliquot of your sample for the reaction.
3. Cap the vial tightly and heat at 70C for ~10 minutes. Although the exact time and temperature are not critical, avoid heating for much more than 30 minutes or the BF3 will begin to self-polymerize (a strong yellow color appears in the sample).
4. Add 2mL of water to the reaction mixture, then extract the methyl esters three times using 3mL of hexane. The solvent volume should be adjusted upward if your sample is larger (see step 2). Collect the organic extract in a 40mL VOA vial and dry by eluting through a pipette packed with anhydrous sodium sulfate (note B).
5. Evaporate the solvent down to a small volume in the TurboVap (note C), then transfer to a GC vial using DCM or hexane.

Notes

1. Water from a Milli-Q or other purification system is usually (but not-always) sufficiently organic-free for this purpose. If necessary, the water can be further cleaned by shaking it with DCM.
2. The drying step is added to this method as a precaution, in case any water/BF3 gets carried into the hexane on the pipette tip. Blowing down a sample that contains a trace of water/BF3 will likely hydrolyze the esters you just worked so hard to synthesize. You can omit this step -- at your own peril -- if you are very confident in your pipetting abilities. An easy way to make this step less painful is to pipette the hexane off the reaction mixture and right onto the sodium sulfate column, which is then collected in a VOA vial.
3. The methyl esters of short-chain fatty acids, like C12 and C14, are surprisingly volatile. Be careful not to evaporate samples completely to dryness or you may lose some of these shorter components through evaporation.
4. When using polar solvents like ether or DCM to extract the methyl esters from the BF3/water solution, we have occasionally had problems with traces of acid being carried onto the GC column, resulting in large amounts of column bleed and headache. We recommend using hexane as the extraction solvent to help avoid such problems