Compound Specific Isotope Analysis (CSIA) by GC-C-IRMS

The SIF provides routine ¹³C isotope analysis of fatty acid methyl esters (FAMEs) using GC-combustion isotope ratio mass spectrometry (GC-C-IRMS).

Fatty Acid Methyl Ester (FAME) Analysis

Stable isotope ratios of ¹³C in fatty acid methyl esters (FAMEs) are measured using a Thermo GC/C-IRMS system composed of a Trace GC Ultra gas chromatograph (Thermo Electron Corp., Milan, Italy) coupled to a Delta Plus Advantage isotope ratio mass spectrometer through a GC/C-III interface (Thermo Electron Corp., Bremen, Germany). FAMEs dissolved in hexane are injected in splitless mode and separated on a J&W DB-5 column (30 m X 0.25mm ID, 0.25 micron film thickness). Once separated, FAMEs are quantitatively converted to CO₂ in an oxidation reactor at 950°C. Following water removal through a nafion dryer, CO₂ enters the IRMS. Delta ¹³C values are corrected using working standards composed of several FAMEs calibrated against NIST standard reference materials.

Column Selection: the DB-5 is suitable for most FAME mixtures. The highly polar SGE BPX70 column has better separation of cis- and trans- isomers, which is useful for analysis of methyl oleate (18:1n9c) and methyl linoleate (C18:2n6c). These are separated by less than 15 seconds on the DB-5. However, BPX70 is not suitable for samples that include C19:0 internal standards, because it does not sufficiently separate C19:0 from C18:2n6c.

Analysis of Other Compounds

It is also possible analyze other organic compounds, such as cholesterol, pheromones, flavors, etc. Generally, if a compound of interest can be analyzed by GC or GC-MS, we should be able to analyze its ¹³C isotope ratio using a specific GC column and oven program. For volatile organic compounds in water, samples in headspace vials can be analyzed by manual injection using SPME fiber. Please contact Bill Holmes regarding these and other possible applications.

Pricing

Sample Preparation

Submit FAME samples in standard GC vials (volume: 2 mL O.D. 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 concentration (~0.5 micrograms C per microliter) are acceptable for samples having <10 major peaks. Large FAME peaks are 50-150 ng C and the smallest detectable peaks are ~2 ng C. 

Shipping

Samples may be shipped with solvent (pentane, hexane, acetone, ethyl acetate, etc). If you use inserts in the vials, evaporate the solvent completely before shipping. Solvent may leak from the inserts due to agitation during shipping.

Always include a Compound Specific Analysis Order Form and Sample List when submitting 13C FAME or other compound specific samples to the SIF. This form helps us track your samples, and ensures that your samples are handled properly and analyzed correctly. Please e-mail a copy of the completed forms to sif@ucdavis.edu.

Contact information
UC Davis Stable Isotope Facility CSIA Lab
Department of Plant Sciences
One Shields Avenue, Mail Stop 1
Davis, CA 95616
USA

Phone:(530) 752-8100, Fax: (530) 752-4361
E-mail: sif@ucdavis.edu

Supplies

Tips

1) Avoid Over-tight Caps

We’ve received samples that leaked from vials because caps were over-tightened, causing the septa to get pinched out of the lids. Turn caps until snug, but not so far that the septa start to pucker.

2) Use Internal Standards

We recommend using an internal standard (e.g., C19:0) if you intend to calculate concentration from peak area.

3) Inspect Chromatograms for Small Peaks and Overlapping Peaks

Please inspect your data. We provide data for all detectable peaks and let you decide which data points to keep and which ones to discard, rather than performing this step for you. In the results file, we make note of small peaks for which isotope results are less accurate, but may be of interest. Very small peaks sometimes have suspicious values that should be ignored. Look for overlapping peaks. The isotope ratio may not be accurate for a small peak riding on a large peak, but the reading for the large peak is likely acceptable. As large peaks overlap each other, the readings for each approach the average of the two. The reason is the IRMS only measures CO2 and it cannot discern CO₂ from either peak.

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 the 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 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

Updated June 18, 2009