Derivatization techniques for free fatty acids by GC

Anyone who has tried to analyze free fatty acids by GC realizes they have to derivatize these compounds. The inherently low volatility of free fatty acids and the carboxylic acid (-COOH) interaction with the siloxanes in the stationary phase. If not addressed, the first problem leads to late-eluting peaks and the latter results in peak tailing due to secondary retention mechanism (see Figure 1).

Figure 1: Peak tailing due to multiple retention mechanisms [1]. Retention mechanism 2 represents the interaction between -COOH group and siloxanes in the stationary phase.In this blog, I’ll go over the two main derivatization procedures I’ve seen most commonly used for free fatty acids – esterification with BF₃ in methanol and silylation with BSTFA or MSTFA [2].

Note: Both of these methods are moisture sensitive! If the sample is aqueous, the water needs to be removed. For example, water can be removed by drying the sample or lyophilization.

 

Derivatization with BF₃-methanol

The preferable method when working with free fatty acids is to generate FAMEs (fatty acid methyl esters) via esterification with methanol catalyzed by BF₃. This esterification needs only mild conditions (temperatures around 50-60 °C) and only about an hour of reaction time (can be tailored to application). Figure 2 shows a schematic of the reaction.

Figure 2: Schematic of esterification with methanol in presence of BF3.

General instructions

Starting with 1 mg/mL acid mixture in a solvent, such ACN

• Combine acid (100 µL) and derivatization agent (14% BF₃ in methanol; 50 µL; 10x molar excess) in autosampler vial
• Cap the vial, vortex for 10s and place to incubator or oven at 60 °C for 60 minutes (both temperature and time can be optimized depending on the analytes)
• After cooling down, add 0.5 mL of saturated NaCl water solution. Vortex for 10s
• Add 0.6 mL of hexane, vortex and wait for separation to take place
• Remove the supernatant into a new autosampler vial with a layer of anhydrous Na₂SO₄ and repeat another 0.6 mL hexane twice more.
• If the Na₂SO₄ gets wet, transfer the contents to a new vial with fresh Na₂SO₄
• Analyze on GC or GC-MS

Using this method we can achieve selective acid derivatization and clean MS (EI) spectra.

Derivatization with BSTFA or MSTFA

The second common method to derivatize acid is silylation. Commonly used reagents are BSTFA (N,O-Bis(trimethylsilyl)trifluoroacetamide) and MSTFA (N-Methyl-N-(trimethylsilyl) trifluoroacetamide) with addition of 1% TMCS (trimethylchlorosilane). This method generates trimethylsilyl (TMS) esters from the carboxylic group, however, it can also derivatize hydroxyl and amino groups, among others. Figure 3 shows the mechanism of the silylation of an OH group.

Figure 3: Mechanism of silylation with BSTFA

General instructions

Starting with 1 mg/mL acid mixture in an aprotic solvent, such ACN

• Combine acid (100 µL) and derivatization agent (BSTFA or MSTFA with 1% TMCS; 50 µL; 10x molar excess) in autosampler vial
• Cap the vial, vortex for 10s and place to incubator or oven at 60 °C for 60 minutes (both temperature and time can be optimized depending on the analytes)
• After cooling down add a solvent of choice (e.g. DCM)
• Analyze on GC or GC-MS

The advantage of this method is it can work for multiple functional groups, so it is helpful for the analysis of multiple analyte types in one run, e.g. acids and sugars. However, this might also lead to some derivatization artifacts in more complex samples. Moreover, in cases of MS analysis, we can possibly run into two problems – 1) long solvent delays are necessary to elute the BSTFA or MSTFA remains in the solution and 2) more complex EI spectra. The stability of TMS derivatives is also limited - the best results are achieved when analyzed within a week.

Resources:

[1] S. C. Moldoveanu and D. Victor, Derivatization Methods in GC and GC/MS, InTechOpen, 2018.

[2]D. R. Knapp, Handbook of analytical derivatization reactions, New York: John Wiley & Sons, 1979.