Fatty Acid Methyl Ester (FAME) Sample Preparation

Total concentration of FAMEs in samples should range from 1 to 4 µg C/µL, for typical samples having 10-20 major peaks and 20+ minor peaks. Lower total concentrations (~0.5 µg C/µL) are acceptable for samples having fewer than 10 major peaks. Moderate to large FAME peaks are 50-200 C/µL, and the smallest detectable peaks are ~2 ng C, on-column.

Submit samples in standard GC vials (volume: 2 mL OD x length: 12 x 32 mm) with screw caps (9-425 or 10-425). Use inserts for low-concentration samples. With inserts, we can inject up to 4 µL from as little as 30 µL solvent. Use vials with a write-on patch or use clear tape and permanent marker to label vials. Do not wrap vials with opaque marking tape – this makes it difficult to view liquid level. Thick tape causes vials to stick in the autosampler tray. If shipping samples in solvent, please do not overtighten your vial caps. Samples can leak out of vials because over-tightened septa get pinched out of the lids. Turn caps until snug, but not so far that septa start to pucker.

We recommend using an internal standard (e.g. c19:0) if you intend to calculate efficiency of extraction. Please avoid the use of short-chain FAMEs as internal standards (e.g. c11:0, c12:0, c13:0), as we use these compounds as internal standards for our determination of the total amounts of each FAME (total µg as C) and the calibration of isotope-ratio measurements.

Derivatization

The SIF also offers derivatization services for the methylation of fatty acids from sample extracts. If extraction is required, please contact us.

Briefly, FAMEs derivatization is performed in 2 mL borosilicate vials with PTFE-lined phenolic caps and threads wrapped with Teflon tape. All samples are derivatized alongside a δ13C- and δ2H-calibrated mixture of fatty acids, as well the two quality assurance reference material extracts. Extract, in chloroform, is added to each vial, plus 20µL of the c19:0 acid internal standard (~20 mg in 10 mL of chloroform) and 800 µL of freshly prepared methylation reagent (concentrated sulfuric acid 1.5% in anhydrous methanol). Heptane is added to a final volume of 1mL, vials are firmly closed, vortexed, and placed on a heating block at 100ºC for 1 hour. After removing the vials from the block and allowing to cool to room temperature, 300 µL of 1M NaCl and 300 µL of heptane are added, vials are vortexed, phases separated, and the upper organic phase transferred to an amber GC vial. The extraction with heptane is repeated twice and the heptane fractions are pooled. If samples are low in FAMEs content (e.g. soil, plants, or sediments), the derivatives may be carefully dried under a gentle stream of nitrogen, re-dissolved in a reduced volume of heptane, and transferred to a vial with an insert. Samples are stored at -20ºC until analysis.

Correct for Derivatization of Fatty Acids
When preparing fatty acid methyl esters, one C is added to each fatty acid molecule. Thus, when we measure the δ13C of FAMEs, the measured value includes a small contribution from the carbon found in your methanol. This contribution is easily accounted for, if you also know the δ13C value of the methanol. If you include a 2-mL GC vial of the methanol used for esterification along with your project, we will analyze the δ13C of your methanol for you. There is no additional charge for this service.
You can do the δ13C-methanol correction using either δ13C or atom-%13C. Here we show the correction using atom-%. The equation below can be rearranged to solve for fatty acid atom-%13C, given the number of C atoms in the fatty acid molecule (#C), measured atom-%13C of FAME (FAME At%), and measured atom %13C of the methanol (MeOH At%):

#C x FA At% + 1 C x Me At% = (#C+1) x FAME At%

FA At% = ((#C+1) x FAME At% - 1 x Me At%)/#C

Example: Linoleic Acid(C18:2n6c, CAS# 60-33-3)

Methanol atom-% 13C = 1.06272 (delta 13C = -39.20)

Methyl Linoleate (FAME) atom-% 13C = 1.07366 (delta 13C = -29.20)

Linoleic Acid atom-% 13C = ((18+1) x 1.07366 – 1 x 1.06272)/18 = 1.07427 (δ13C = -28.64)

In this case, the correction made only a small difference in the resulting value. However, the isotopic value of methanol can vary among sources and batches. We’ve seen batches as low as -65‰.