Derivatization of Fatty acids to FAMEs

GC can be used to analyze fatty acids either as free fatty acids or as fatty acid methyl esters. The primary reasons to analyze fatty acids as fatty acid methyl esters include:

• In their free, underivatized form, fatty acids may be difficult to analyze because these highly polar compounds tend to form hydrogen bonds, leading to adsorption issues. Reducing their polarity may make them more amenable for analysis.
• To distinguish between the very slight differences exhibited by unsaturated fatty acids, the polar carboxyl functional groups must first be neutralized. This then allows column chemistry to perform separations by boiling point elution, and also by degree of unsaturation, position of unsaturation, and even the cis vs. trans configuration of unsaturation.

The esterification of fatty acids to fatty acid methyl esters is performed using an alkylation derivatization reagent. Methyl esters offer excellent stability, and provide quick and quantitative samples for GC analysis. The esterification reaction involves the condensation of the carboxyl group of an acid and the hydroxyl group of an alcohol. Esterification is best done in the presence of a catalyst (such as boron trichloride). The catalyst protonates an oxygen atom of the carboxyl group, making the acid much more reactive. An alcohol then combines with the protonated acid to yield an ester with the loss of water. The catalyst is removed with the water. The alcohol that is used determines the alkyl chain length of the resulting esters (the use of methanol will result in the formation of methyl esters whereas the use of ethanol will result in ethyl esters). The following typical esterification procedure (using BCl₃-methanol) is intended as a guideline. It may need to be altered to meet the needs of a specific application.

• Samples can be derivatized neat or after dissolving in solvent. If appropriate, dissolve sample in a nonpolar solvent (such as hexane, heptane, or toluene). If the sample is in an aqueous solvent, first evaporate to dryness then use neat or dissolved in an organic, non-polar solvent.
• Weigh 1-25 mg of sample into a 5-10 mL micro reaction vessel.
• Add 2 mL BCl₃-methanol, 12% w/w. A water scavenger (such as 2,2-dimethoxypropane) can be added at this point.
• Heat at 60 °C for 5-10 minutes. Derivatization times may vary, depending on the specific compound(s) being derivatized.
• Cool, then add 1 mL water and 1 mL hexane.
• Shake the reaction vessel (it is critical to get the esters into the non-polar solvent).
• After allowing the layers to settle, carefully transfer the upper (organic) layer to a clean vial. Dry the organic layer by either a) passing through a bed of anhydrous sodium sulfate during the transfer step to the clean vial, or b) adding anhydrous sodium sulfate to the clean vial then shaking.
• To determine the proper derivatization time, analyze aliquots of a representative sample using different derivatization times. Plot peak area (y-axis) vs derivatization time (x-axis). The minimum time to use is when no further increase in peak area is observed with increasing derivatization time (where the curve becomes flat).
• If it is suspected that complete derivatization is never achieved, use additional reagent or re-evaluate temperature.
• It is important to prepare a reagent blank, along with the samples, to identify any issues that may arise.

It is important to use only high quality derivatization reagents, to ensure that no artifacts are present during analysis. Additionally, only derivatization reagents with low moisture should be used, as the esterification reaction will be hindered by the presence of water. The storage conditions of derivatization reagents should be strictly adhered to, as some are susceptible to degradation during long-term storage.