METHODS USED FOR CHARACTERIZING REFERENCE MATERIALS

Introduction: The widespread adoption of relative stable isotope-ratio measurements in organic matter by diverse scientific disciplines has been at odds with the dearth of international organic stable isotopic reference materials (RMs). Only two of the few carbon (C) and nitrogen (N) organic RMs, namely L-glutamic acids USGS40 and USGS41 provide an isotopically contrasting pair of organic RMs to enable essential two-point calibrations for δ-scale normalization (Qi et al., 2003: http://dx.doi.org/10.1002/rcm.1219), both available from the U.S. Geological Survey (USGS) and the International Atomic Energy Agency (IAEA) (Coplen, 1996: http://dx.doi.org/10.1016/0016-7037(96)00263-3; Coplen et al., 2006: http://dx.doi.org/10.1021/ac052027c). Numerous stable isotope laboratories have resorted to questionable practices, for example by using ‘CO2, N2, and H2 reference gas pulses’ for isotopic calibrations, which violates the principle of identical treatment of sample and standard (i.e., organic unknowns should be calibrated directly against chemically similar organic RMs) (Werner and Brand, 2001: http://dx.doi.org/10.1002/rcm.258), or by using only 1 anchor instead of 2 for scale calibration. Indiana University began developing in-house RMs in the late 1990s and began international distribution around 2000. Most of our RMs originate from in-house efforts, sometimes in collaboration with other institutions. In 2011, the U.S. National Science Foundation (NSF) funded an initiative of 10 laboratories from 7 countries to develop jointly much needed new organic RMs. The selection of targeted RMs attempted to cover various common compound classes of broad technical and scientific interest. Compromises were accepted to approach the ideal of chemically-pure substances with high chemical stability, lack of toxicity, and affordable price of raw materials. Some of our new RMs can be used for compound-specific isotopic measurements in conjunction with liquid and gas chromatographic interfaces.

Methods: The Biogeochemical Laboratories at Indiana University, originally in collaboration with Woods Hole Oceanographic Institution (J. M. Hayes, former director of NOSAMS) and Caltech (A. L. Sessions), began the development of isotope reference materials in 1998. We first established the purity of n-alkanes and n-alkanoic acid esters by GC-MS, followed by multiple off-line measurements of 2H/1H and 13C/12C ratios for each compound. Multiple analyses for each compound were performed via conventional combustion of milligram-amounts of individual compounds in quartz ampoules and cryogenic purification of combustion gases in a vacuum line. Water was converted to elemental hydrogen in contact with uranium, followed by collection of hydrogen gas using a Toepler pump.  Gas yields and atomic H/C ratios were routinely monitored manometrically for quality control.  Hydrogen and carbon isotopic ratios were determined using Finnigan MAT 252 and Delta Plus XP mass-spectrometers at Indiana University.  The hydrogen isotopic calibration employed the conventional normalization to VSMOW (zero ‰) and SLAP (-428 ‰) according to Coplen (1996). The same analytical strategy was used in later years to develop additional reference materials, in part in collaboration with Caltech (A. L. Sessions), K. Freeman's group at Pennsylvania State University, and others. Calibration for carbon isotope ratios relied on stable isotope standards NBS 19 and LSVEC (both carbonates were digested in 100% phosphoric acid at controlled temperatures). Carbon isotope ratios are reported relative to the VPDB scale where NBS 19 and LSVEC are defined as exactly +1.95 and -46.6 per mil, respectively (Coplen et al., 2006). Calibration for nitrogen was performed using international nitrogen isotope standards IAEA-N-1 and IAEA-N-2  (combusted and processed in the same way as our reference materials, according to the "principle of identical treatment"). In addition, we used USGS40 and USGS41 to take advantage of their larger isotopic ranges. Starting in 2013, we additionally used VSMOW2 and SLAP2.

Coplen T.B. (2011) Guidelines and recommended terms for expression of stable-isotope-ratio and gas-ratio measurement results. Rapid Communications in Mass Spectrometry 25 (17), 2538-2560. http://dx.doi.org/10.1002/rcm.5129
Coplen T.B., Brand W.A., Gehre M., Gröning M., Meijer H.A.J., Toman B., Verkouteren R.M. (2006) New guidelines for δ13C measurements. Analytical Chemistry 78 (7), 2439-2441.  http://dx.doi.org/10.1021/ac052027c
Qi H., Coplen T.B., Geilmann H., Brand W.A., Böhlke J.K. (2003) Two new organic reference materials for δ13C and δ15N measurements and a new value for the δ13C of NBS 22 oil. Rapid Communications in Mass Spectrometry 17, 2483-2487. http://dx.doi.org/10.1002/rcm.1219
Werner R.A., Brand W.A. (2001) Referencing strategies and techniques in stable isotope ratio analysis. Rapid Communications in Mass Spectrometry 15, 501-519. http://dx.doi.org/10.1002/rcm.258

Brand WA, Coplen TB, Vogl J, Rosner M, Prohaska T (2014) Assessment of international reference materials for isotope-ratio analysis (IUPAC Technical Report). Pure and Applied Chemistry 86 (3), 425-467. http://dx.doi.org/10.1515/pac-2013-1023