Compound-Specific
Stable Isotope
Analysis


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UC Davis Plant Sciences


Fatty Acid Methyl Ester (FAME) Sample Preparation

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.

Total concentration of FAMEs in samples should range from 1 to 2 micrograms C per microliter, for typical samples having 10-20 major peaks and 20+ minor peaks. Lower total concentrations (~0.5 micrograms C per microliter) are acceptable for samples having fewer than 10 major peaks. Large FAME peaks are 50-150 ng C, and the smallest detectable peaks are ~2 ng C. 

Correct for Derivatization of Fatty Acids
When preparing fatty acid methyl esters, one C is added to each fatty acid molecule. Thus, the isotope ratios we measure include the C from your methanol. FAME 13C results can be corrected if you know the isotope ratio of the methanol. Send a 2-mL GC vial of your methanol along with your samples, and we will analyze the methanol in nano-pure water as a DOC sample.


Make the correction using either the 13C/12C ratio 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 (Me 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 (delta 13C = -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 per mil.

Example chromatogram








 
e-mail: sif@ucdavis.edu | phone: 530-752-8100 | fax: 530-752-4361
UC Davis Stable Isotope Facility | Department of Plant Sciences
One Shields Avenue | Davis, California, 95616 | USA