EA Reference Materials

Our isotopic calibrations relative to primary reference materials relied on off-line methods whereby all listed organic RMs were quantitatively combusted overnight at 800 ºC in sealed ‘quartz’ ampoules to produce 100 % yields of carbon dioxide, nitrogen and water.

Water was quantitatively reduced to hydrogen via reaction with uranium metal. Each gas was cryogenically purified, collected separately, and measured isotopically in manual dual-inlet mode against gases derived from primary RMs. In contrast, on-line oxidative and reductive interfaces cannot guarantee 100 % yields of product gases.

Our laboratory’s comparison of on-line, TCEA-based δ2H values of aromatic coumarin and numerous n-alkanes indicated that coumarin does not perfectly fall on the correlation line comparing off-line versus on-line δ2H values. We surmise that aromatic hydrogen in coumarin has a slightly different response to TCEA pyrolysis conditions. Aromatic structures like coumarin may have a tendency to rapidly attach themselves to the vitreous carbon granules in the hot reactor before liberating all hydrogen atoms.

An incomplete hydrogen yield may entail some isotope fractionation that likely depends on temperature, flow rate, presence of heteroatoms N and O, and reactor conditioning.  Our observation underscores the need to optimize the match of reference materials to analytes.

The following is a short listing of available reference materials for EA applications with specific comments.

Available reference materials for EA applications


  • Acetanilides are available with different levels of 13C-enrichment (Schimmelmann et al., 2009, Nicotine, acetanilide and urea multi-level 2H-, 13C- and 15N-abundance reference materials for continuous-flow isotope ratio mass spectrometry. Rapid Communications in Mass Spectrometry 23:3513–3521. https://doi.org/10.1002/rcm.4277).

Indiana University purchased pure acetanilide and 15N-enriched acetanilide. The regular acetanilide #1was powdered using a large, industrial-strength blender. Aliquots of the regular acetanilide #1 and the 15N-enriched acetanilides were melted and subsequently powdered to prepare medium enriched and highly 15N-enriched acetanilides #2 and #3. The user is advised to finely powder the received acetanilides prior to utilizing sub-milligram aliquots.

  • L-Alanine Androstanes (5-a)


  • Benzene
  • Benzoic acids serve as reference materials for 18O/16O. Benzoic acid has several advantages over cellulose and many other oxygen-containing compounds:
  1. it has been reliably measured isotopically by conventional off-line methods and on-line TC/EA;
  2. it is chemically stable and inert, so that its organic oxygen inventory receives no significant addition from elemental atmospheric oxygen species via oxidation over time; (c) it shows no hygroscopicity that could add atmospheric water vapor via adsorption (unlike cellulose);
  3. it has been prepared in relatively large quantities with sufficient isotopic homogeneity;
  4. it shows no significant toxicity;
  5. it does not rapidly exchange isotopically with ambient atmospheric moisture.

The IAEA had commissioned the development of two isotopically different benzoic acid oxygen isotope standards (jointly developed by Drs. Willi Brand and Roland Werner at the Max-Planck-Institut für Biogeochemie in Jena, Germany, and by Arndt Schimmelmann at Indiana University; a pdf copy of the final report to the IAEA is available upon request). The artificial enrichment in 18O in one of the acids did not entail proportional enrichment in 17O.

The algorithm for calculating δ138C values hinges on the 17O abundance, and thus pyrolytically derived CO or CO2 from our 18O-enriched benzoic acid would yield incorrect δ13C values. The applicability of the 18O-enriched benzoic acid as a stable isotope standard is thus limited to 18O/16O. The use of benzoic acids for carbon isotope ratios was discussed by Coplen et al. (2006, New guidelines for δ13C measurements. Analytical Chemistry 78:2439-2441. https://pubs.acs.org/doi/10.1021/ac052027c). The 18O-enriched benzoic acid must not be used for calibration of 13C/12C unless exchange of benzoic-acid-derived carbon-oxides is achieved with excess oxygen via oxidative combustion (Willi Brand, personal communication). Oxygen stable isotope ratios were recently recalibrated (Brand et al., 2009, Comprehensive inter-laboratory calibration of reference materials for δ18O versus VSMOW using various on-line high-temperature conversion techniques. Rapid Communication in Mass Spectrometry 23, 999-1019. https://doi.org/10.1002/rcm.3958). The small supply of benzoic acids retained at Indiana University limits the availability to trial amounts only. Regular users must order their benzoic acids from the IAEA (IAEA-601 and IAEA 602). Each benzoic acid should be powdered by the user prior to use, in order to reduce isotope fractionation among individual crystals.

  • Biphenyl


  • Caffeines are available with different levels of 2H-, 13C- and 15N-enrichments.
  • Coumarin is valuable for hydrogen TC/EA because it has a remarkably high deuterium concentration.
  • Dibenzothiophene
  • Eicosanoic acid methyl esters, e.g., #Y, #Z1, #Z2, #Z3 EDTA
  • Glycines
  • Hexatriacontane, n-alkane C-36
  • Polyethylene powder
  • Phenanthrene
  • L-Phenylalanine
  • Phthalic acid


  • Starch from corn
  • Ureas

Ureas have a purity of ≥99.5 % and are available in 3 isotopic varieties (Schimmelmann et al., 2009, Nicotine, acetanilide and urea multi-level 2H-, 13C- and 15N-abundance reference materials for continuous-flow isotope ratio mass spectrometry. Rapid Communications in Mass Spectrometry 23:3513–3521. https://dx.doi.org/10.1002/rcm.4277). Urea #1 has natural 13C and 15N abundances. Urea #2 is moderately 13C and 15N-enriched, whereas urea #3 is strongly 13C and 15N-enriched.

The highest precision can be achieved when each urea is dissolved in water and suitable volumetric aliquots of stock solutions are micro-pipetted into EA capsules, followed by freeze-drying in a vacuum. We caution that urea crystals are known to adsorb smaller molecules, for example straight-chain n-alkanes. Off-line combustion experiments with urea stock solutions suggest that CO2 and/or methane can be trapped in crystallizing urea and shift bulk δ13C values.

  • Vacuum pump oils
  • L-valines