Methyl sulfates as methoxy isotopic reference materials for δ13C and δ2H measurements

Article abstract of DOI:10.1002/rcm.8355

Rationale: Stable hydrogen and carbon isotope ratios of methoxy groups (OCH₃) of plant organic matter have many potential applications in biogeochemical, atmospheric and food research. So far, most of the analyses of plant methoxy groups by isotope ratio mass spectrometry have employed liquid iodomethane (CH₃I) as the reference material to normalise stable isotope measurements of these moieties to isotope–δ scales. However, comparisons of measurements of stable hydrogen and carbon isotopes of plant methoxy groups are still hindered by the lack of suitable reference materials. Methods: We have investigated two methyl sulfate salts (HUBG1 and HUBG2), which exclusively contain carbon and hydrogen from one methoxy group, for their suitability as methoxy reference materials. Firstly, the stable hydrogen and carbon isotope values of the bulk compounds were calibrated against international reference substances by high-temperature conversion- and elemental analyser isotope ratio mass spectrometry (HTC- and EA-IRMS). In a second step these values were compared with values obtained by measurements using gas chromatography/isotope ratio mass spectrometry (GC/IRMS) where prior to analysis the methoxy groups were converted into gaseous iodomethane. Results: The ²H- and ¹³C isotopic abundances of HUBG1 measured by HTC- and EA-IRMS and expressed as δ-values on the usual international scales are ?144.5 ± 1.2 mUr (n = 30) and ?50.31 ± 0.16 mUr (n = 14), respectively. For HUBG2 we obtained ?102.0 ± 1.3 mUr (n = 32) and +1.60 ± 0.12 mUr (n = 16). Furthermore, the values obtained by GC/IRMS were in good agreement with the HTC- and EA-IRMS values. Conclusions: We suggest that both methyl sulfates are suitable reference materials for normalisation of isotope measurements of carbon of plant methoxy groups to isotope–δ scales and for inter-laboratory calibration. For stable hydrogen isotope measurements, we suggest that in addition to HUBG1 and HUBG2 additional reference materials are required to cover the full range of plant methoxy groups reported so far.

Full text of DOI:10.1002/rcm.8355

Methyl sulfates as methoxy isotopic reference materials for δ13C and δ 2H
measurements
Markus Greule1*, Heiko Moossen2, Heike Geilmann2, Willi A. Brand2, Frank Keppler1,3*
1 Institute of Earth Sciences, Heidelberg University, Im Neuenheimer Feld 234-236, 69120
Heidelberg, Germany.
2 Max-Planck-Institute for Biogeochemistry, Hans-Knoell-Str. 10, 07749 Jena, Germany
3 Heidelberg Center for the Environment (HCE), Heidelberg University, D-69120 Heidelberg,
Germany
*Correspondence to: markus.greule@geow.uni-heidelberg.de and frank.keppler@geow.uni-
heidelberg.de
Abstract
RATIONALE: Stable hydrogen and carbon isotope ratios of methoxy groups (OCH3) of
plant organic matter have many potential applications in biogeochemical, atmospheric and
food research. So far, most of the analyses of plant methoxy groups by isotope ratio mass
spectrometry have employed liquid iodomethane (CH3I) as the reference material to
normalise stable isotope measurements of these moieties to isotope–δ scales. However,
comparisons of measurements of stable hydrogen and carbon isotopes of plant methoxy
groups are still hindered by the lack of suitable reference materials.
METHODS:We have investigated two methyl sulfate salts (HUBG1 and HUBG2), which
exclusively contain carbon and hydrogen from one methoxy group, for their suitability as
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methoxy reference materials. Firstl, the stable hydrogen and carbon isotope values of the bulk
compounds were calibrated against international reference substances by high temperature
conversion- and elemental analyser isotope ratio mass spectrometry (HTC- and EA-IRMS).
In a second step these values were compared with values obtained by measurements using
gas chromatography isotope ratio mass spectrometry (GC-IRMS) where prior to analysis the
methoxy groups were converted to gaseous iodomethane.
2
13
RESULTS: The H- and C isotopic abundances of HUBG1 measured by HTC- and EA-
IRMS and expressed as δ-values on the usual international scales are -144.5±1.2 mUr (n=30)
and -50.31±0.16 mUr (n=14), respectively. For HUBG2 we obtained -102.0±1.3 mUr (n=32)
and +1.60±0.12 mUr (n=16). Furthermore, the values obtained by GC/IRMS were in good
agreement with the HTC- and EA-IRMS values
CONCLUSIONS: We suggest that both methyl sulfates are suitable reference materials for
normalization of isotope measurements of carbon of plant methoxy groups to isotope–δ
scales and for inter-laboratory calibration. For stable hydrogen isotope measurements we
suggest that next to HUBG1 and HUBG2 additional reference materials are required to cover
the full range of plant methoxy groups reported so far.
Introduction
The biospheric C1 methyl (CH3) pool of plant origin, including mainly lignin and pectin
methoxy groups (OCH3), comprises ca 2.5% of the total amount of carbon in plant biomass
[1]. It has been shown that plant methoxy groups have both distinct stable hydrogen (δ2H
values) and carbon (δ13C values) isotopic signatures [2]–[7]. Relative to the bulk biomass of
plants, a 13C depletion of up to 50 mUr was observed for methoxy groups, the largest carbon
isotope fractionation ever observed in the plant kingdom to date [8]. (Note that we follow the
[9]
suggestion by Brand and Coplen and express isotope δ-values in milli-Urey [mUr] (after
2
H.C. Urey [10]) instead of in per mil [‰]; 1 mUr = 1 ‰). On the other hand, a striking H
depletion of plant methoxy groups relative to source water has been observed with a uniform
apparent isotopic fractionation (~ -150 to -210 mUr), noted over a range of δ2H values for
meteoric water [6],[7],[11]–[17]. These particular δ2H and δ13C isotopic signatures have
considerable potential for use as tools for investigations in biogeochemical, atmospheric and
food research.
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For example, the information gained from the isotope signatures of plant methoxy groups can
be applied for climate reconstructions of various climate archives such as tree rings,
[18]–[23]
sediments and peat cores on time scales from the Anthropocene until the Eocene
Furthermore they are useful in food science for studies on the authenticity of vanillin
.
[17],[24]–
[26], in atmospheric sciences to investigate the origin and fate of C1 volatile organic
compounds such as methanol, chloromethane and bromomethane in the atmosphere
[3],[6],[8],[27],[28]
and in biochemical investigations to better understand bio-methylation
processes [29],[30]
.
Stable carbon and hydrogen isotope ratiosof methoxy groups from various plant origins have
been determined by site-specific natural isotope fractionation−nuclear magnetic resonance
(SNIF-NMR) and isotope ratio mass spectrometry (IRMS). The principle of measurement by
IRMS is the generation of gaseous iodomethane (CH3I) via the reaction of methoxy groups
with hydriodic acid (HI). This reaction was first used by Zeisel in 1885 to quantitatively
[31]
determine the methoxy content of wood
and it is commonly referred to as the ‘Zeisel
method’. Later this method was applied by Galimov et al and Krüger et al for measurements
of the δ13C values of biogenic methoxy groups [24],[32],[33]. In the last two decades the method
has been employed for the precise and rapid δ2H and δ13C analysis of methoxy groups by gas
chromatography isotope ratio mass spectrometry (GC/IRMS) [8],[34]–[36]
.
[25]
The reported δ2H and δ13C values of plant-related methoxy groups range from -149 mUr
to -405 mUr [22] and from -7.1 mUr [25] to -77.2 mUr [8], respectively. In addition the δ2H and
δ13C values of C1-groups from extraterrestrial matter such as meteorites (carbonaceous
chondrites) have been shown to be in the range of +1054 ± 626 mUr and +43.2 ± 38.8 mUr,
respectively [37]
.
Generally, stable isotope abundance analyses require reference materials to normalise
measured δ-values on the respective δ-scale. When selecting reference materials for IRMS
analyses the following criteria have to be considered [38]–[41]: isotopic homogeneity (to the
smallest amount to be analysed), (ii) unchanging stable isotopic composition over time, (iii)
chemical similarity to the samples to ensure that errors during preparation will tend to cancel
out (principle of identical treatment [39]), (iv) easy preparation, storage and handling, (v) a
single chemical compound (preferably), (vi) non-hygroscopic (especially important when
measuring hydrogen and oxygen isotopes), (vii) comprising only non-exchangeable hydrogen
(unless the non-exchangeable δ2H value is known), (viii) should not constitute a health risk
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when applied for measurements.
Up until now most of the results reported in the literature from analyses of methoxy groups
by IRMS were obtained using liquid CH3I with a known isotope ratio as the reference
material. Iodomethane is a liquid at room temperature with a boiling point of 42°C and high
vapour pressure of 441 hPa (20°C). It is toxic (LD50, rat, oral: 76mg/kg) and probably also
carcinogenic. Moreover, when used as the reference material for the analysis of methoxy
groups CH3I is not treated in an identical manner to the analyte, as it is usually not heated at
130°C with HI. Furthermore, the narrow range of values of both δ2H and δ13C for industrial
CH3I do not span the full range of δ2H and δ13C values of methoxy groups found in the
environment. This is a substantial drawback when utilising CH3I as the reference standard, as
the isotopic composition of samples should be spanned by standards to account for scale
contraction during analysis. Finally, since δ-values are measured more precisely when the
differences between the sample and standard are small a wide range of standards is highly
desirable [38]. To date there is a lack of available isotope reference materials suitable for stable
isotope analysis for plant methoxy groups that fulfill the requirements listed above.
There is an urgent need to establish a suite of solid reference materials for the δ2H and δ13C
analysis of methoxy groups that ideally cover the whole measurement range of δ2H and δ13C
values reported for the environment so that they can be applied easily on a long-term basis
and be readily available for inter-laboratory comparison studies.
We investigated two salts of methylsulfuric acid (that exclusively contain carbon and
hydrogen from one methoxy group; for detailed chemical structures please see Figure 1) for
their suitability as stable isotope reference material for the δ2H and δ13C analysis of methoxy
groups. We first performed δ2H and δ13C measurements of the two compounds by high
temperature conversion (δ2H values) and elemental analyser (δ13C values) stable isotope ratio
measurements (HTC-/EA-IRMS) and calibrated them against international reference
substances. Next we determined the δ2H and δ13C values of the methoxy salts by
transforming the salts to CH3I using the Zeisel method and analysing them using continuous
flow (CF)-IRMS. Then, the data from both methods were compared. Furthermore, we tested
if there was hydrogen isotope exchange between the methyl groups of the methyl sulfate salts
and the water in humid air during storage. Finally, we discuss whether both compounds are
suitable reference materials for the determination of the δ2H and δ13C values of samples
containing methoxy groups.
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Material and Methods
Chemicals
Hydriodic acid (57 wt.% aqueous solution) was obtained from Acros (Thermo Fisher
Scientific, Geel, Belgium), methyl iodide (99.5%) was purchased from Sigma-Aldrich
(Seelze, Germany). Methyl sulfate sodium salt (CAS: 512-42-5) was obtained from Aldrich
(Seelze, Germany, Lot: #MKBT4673V) and potassium methyl sulfate (99%, CAS: 562-54-9)
was purchased from Acros (Thermo Fisher Scientific, Geel, Belgium; Lot: A0378638).
Hydrogen and carbon isotopic analyses at the Max Planck Institute for Biogeochemistry
The two methyl sulfate salts were first analysed in the stable isotope laboratory at the Max
Planck Institute for Biogeochemistry (BGC-IsoLab). The hydrogen isotopic signatures of the
salts were determined by High-Temperature Conversion-Isotope Ratio Mass Spectrometry
[42]
(HTC-IRMS). We refer the reader to Gehre et al.
regarding method details. In short,
samples and standards were weighed into Ag-capsules and placed in a Costech (Cernusco sul
Naviglio, Italy) Zero-Blank 50-position autosampler. The conversion to H2 gas was achieved
with a high-temperature furnace (Hekatech, Wegberg, Germany) held at 1430 °C that was
equipped with a glassy carbon tube inside a silicon carbide tube (tube in tube design) with a
[43],[44]
[45]
reverse feed He carrier gas flow rate of ~ 80 mL/min. Gehre et al
and Nair et al
have shown that the HTC reactor should be filled with chromium when measuring the δ2H
signature of heteroatom (N, S, F, Cl, Br, I) containing compounds. Chromium traps the
heteroatoms and thus facilitates a quantitative conversion of analyte to H2 gas, and
subsequent transport to the isotope ratio mass spectrometer [43]. Consequently, the analyses of
the methyl sulfate salts and associated standards were performed using a glassy carbon tube
filled with special chromium and glassy carbon chips. The HTC-furnace was coupled to a
Deltaplus XL isotope ratio mass spectrometer via a ConFlo III open-split interface (both
Thermo Fisher Scientific, Bremen, Germany).
The carbon isotopic composition of the methyl sulfates were measured on a Thermo Fisher
Deltaplus isotope ratio mass spectrometer coupled to a Carlo Erba 1100 CE Elemental
Analyser (Thermo Fisher Scientific, Rodano, Italy) via a ConFlo III open-split interface. The
measurement procedure has been described previously [46][39][47]. In brief, samples were
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introduced into the EA via an autosampler. The samples were combusted at 1020 °C in the
oxidation oven, and then reduced at 650 °C. Water was removed with a NafionTM and
magnesium perchlorate trap before the sample gas was introduced into the gas chromatograph
held at 80 °C, containing a HaysepQ filled 2m packed column (IVA-Analysetechnik,
Meerbusch, Germany). The He-carrier gas flow rate was 110 mL/min.
Generation of iodomethane from methyl sulfate salts
CF-IRMS measurements were conducted at Heidelberg University, Institute of Earth
Sciences, Heidelberg, Germany (HU). The δ2H and δ13C values of the sodium and potassium
methyl sulfate salts were measured as methyl iodide (CH3I), released upon treatment of the
[34],[35]
samples with hydroiodic acid (HI) using the method described by Greule et al
.
Hydriodic acid (HI) (0.25 mL) was added to the salt samples (hydrogen analysis: 5 mg;
carbon analysis: 2 mg) in a crimp glass vial (1.5-ml; IVA Analysentechnik). The vials were
sealed with crimp caps containing PTFE lined butyl rubber septa (thickness 0.9 mm; part #
IVA70311101, IVA Analysentechnik) and incubated for 30 min at 130 °C. After heating, the
samples were allowed to equilibrate at room temperature (22 ± 0.5 °C) for at least 30 min
before an aliquot of the headspace (10 – 90 µL) was directly injected into the respective
GC/IRMS system as described below using a gastight syringe (100 µL, SGE Analytical
Science, Melbourne, Australia).
CH3I standard preparation
For measurements made where only CH3I standard was added, crimp vials (1.5-ml; IVA
Analysentechnik) were first sealed and then 1-2 µL of liquid CH3I standard was injected into
each vial through the septum using a 10µL GC autosampler syringe (Hamilton Company,
Reno, NV, USA; part #80300). The vials were placed into an A200S autosampler (CTC
Analytics, Zwingen, Switzerland) (22 ± 0.5 °C, air conditioned room) and headspace gas (10-
90µL) injected into the GC/IRMS system using a gastight syringe following the equilibration
period.
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Hydrogen stable isotope analysis using GC/HTC-IRMS
The δ2H values of the CH3I were measured using an HP 6890N gas chromatograph (Agilent,
Santa Clara, CA, USA) equipped with an A200S autosampler, coupled to a DeltaPLUSXL
isotope ratio mass spectrometer via a thermo conversion reactor [ceramic tube (Al2O3), length
320 mm, 0.5 mm i.d., reactor temperature 1450 °C] and a GC Combustion III Interface
(ThermoQuest Finnigan, Bremen, Germany).
The gas chromatograph was fitted with a Zebron ZB-5MS capillary column (Phenomenex,
Torrance, CA, USA) (30m x 0.25mm i.d., df 1µm) and the following conditions were
employed: split injection (4:1), initial oven temperature at 30 °C for 3.8 min, ramp at 30
°C/min to 100 °C. Helium 5.0 was used as the carrier gas at a constant flow rate of 0.6
mL/min.
A tank of high purity hydrogen gas 6.0 (AlphagazTM 2 H2, Air Liquide, Düsseldorf,
+
Germany) was used as the working reference gas. The H3 factor was 2.27 ppm/nA.
[48]
All δ2H values were normalised by a two-point linear calibration
using the δ2H values of
two CH3I working standards relative to VSMOW. The δ2H values of the CH3I working
standards were calibrated against international reference substances (VSMOW2 [δ2HVSMOW
=
0.0 ± 0.3 mUr] and SLAP2 [δ2HVSMOW = -427.5 ± 0.3 mUr]) by HTC-IRMS at the BGC-
IsoLab. The calibrated δ2H values in mUr vs VSMOW for the two CH3I working standards
were −173.0 ± 1.5 mUr (n=9, 1σ) and −66.2 mUr ± 1.2 mUr (n=8, 1σ).
Carbon stable isotope analysis using GC/C-IRMS
The δ13C values of CH3I were measured using the same analytical system as described for
hydrogen measurements but with the difference that the gas chromatograph and the mass
spectrometer were coupled by an oxidation reactor [ceramic tube (Al2O3), length 320 mm, 0.5
mm i.d., with Cu/Ni/Pt wires inside (activated by oxygen), reactor temperature 960°C] and
different GC conditions were employed: split injection (10:1), initial oven temperature at 40
°C for 3.8 min, ramp at 30 °C/min to 100 °C. Helium was used as carrier gas at a constant
flow rate of 1.8 mL/min.
A tank of high purity carbon dioxide 4.8 (Kohlendioxid ISO-TOP, Air Liquide) was used as
the working reference gas.
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All the δ13C values were normalised by two-point linear calibration using the same CH3I
working standards described above, relative to VPDB. The δ13C values of the CH3I working
standards were calibrated against international reference standards (NBS 19 [δ13CVPDB = 1.95
± 0.05 mUr] and NBS 22 [δ13CVPDB = -30.03 ± 0.04 mUr]) by EA-IRMS at BGC-IsoLab. The
calibrated δ13C values in mUr vs VPDB for the two CH3I working standards were -70.04 ±
0.13 mUr (n=4, 1σ) and -60.72 mUr ± 0.05 mUr (n=7, 1σ).
Hydrogen isotope exchange experiments
To test if there was hydrogen exchange between the two methyl sulfate salts and water in
humid air during storage, both salts were stored over a vessel containing water of known δ2H
value in a desiccator under vacuum for three weeks (100 % humidity). The exact δ2H value of
the water (vapour) did not need to be known (exactly) because the aim of this experiment was
to see if the post‐experiment δ2H values of either compound or even both had changed
significantly. Both sulfate salts dissolved shortly after the desiccator was evacuated due to
hygroscopicity. Therefore, after the desiccator was vented the vessel containing water was
exchanged for a vessel containing drying agent (silica gel) to allow both salts to re-
crystallize. Subsequently both salts were analyzed at HU (GC/TC-IRMS) and the δ2H values
before and after the storage experiment were compared.
Results and discussion
The stable hydrogen and carbon isotope ratios of sodium methyl sulfate (HUBG1) and
potassium methyl sulfate (HUBG2) were measured using two different analytical techniques
(HTC- and EA-IRMS and CF-IRMS). While the bulk material of the methyl sulfates was
pyrolysed (δ2H values) and oxidised (δ13C values) during HTC- and EA-IRMS analysis, CH3I
(δ2H and δ13C values) was analysed using CF-IRMS following its formation by reaction of
the methyl sulfates with HI. The results, which are shown in Table 1, are compared and
discussed in detail below.
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Stable hydrogen isotope results
The δ2HVSMOW values for hydrogen of HUBG1 and for HUBG2 measured by HTC-IRMS are
-144.5 ± 1.2 mUr (n=30) and -102.0 ± 1.3 mUr (n=32), respectively. The errors shown
represent the combined uncertainties which were calculated by error propagation containing
contributions from the precision of replicate measurements, the bias of experimental
processes, the uncertainty of δ-values in reference materials used to fix and normalize the δ –
scale and the algorithms applied to correct and normalize the data [38]. The complete
propagated uncertainty of the methyl sulfates UHUBG1/2 is calculated as:
푈퐻푈퐵퐺1/2 = 푢퐶2푅푀 + 푢퐻2푆 + 푢퐻2푈퐵퐺1/2
√
where uCRM indicates the uncertainty of the in-house standards analysed against primary
reference standards, uHS indicates the measurement uncertainty of the in-house standards, and
uHUBG1/2 indicates the measurement uncertainty of the methyl sulfates HUBG1 and HUBG2.
All uncertainties are multiplied by the Student’s factor (t) at a 90 % confidence limit to
account for the limited number of analyses.
The methyl sulfate values measured at BGC-IsoLab were normalised to the SMOW/SLAP
isotopic δ scale using a two point calibration based on two in-house reference standards, Pet-
J1 (polyethylene powder; -78.2 ± 0.4 mUr) and Jena-Pristane (Pristane; -362.6 ± 0.5 mUr).
Both standards are calibrated against VSMOW2 (0 ± 0.3 mUr) and SLAP2 (-427.5 ± 0.3
mUr). IAEA-CH7 (-100.3 ± 2 mUr) was interspersed throughout the samples and used as a
quality control.
For comparison, the δ2H values of HUBG1 and HUBG2 analyzed by GC/TC-IRMS
(measured as CH3I generated by the ‘Zeisel-method’) are -143.2 ± 3.3 mUr and -102.5 ± 3.2
mUr. These values are in very good agreement with the values obtained by HTC-IRMS. This
excellent data agreement might be explained by the fact that the CH3I working standards used
to normalise the results of the GC/TC-IRMS measurements at the laboratory of Heidelberg
University were previously calibrated at BGC-IsoLab (see Material and Method section), the
same laboratory where the HTC-IRMS measurements of HUBG1 and HUBG2 in this study
were conducted. Moreover, the two CH3I working standards applied to correct the δ2H values
for scale-compression for two-point normalisation cover a range of -66.2 to -173.0 mUr and
thus include the δ2H values of HUBG1 and HUBG2 (-143.2 mUr and -102.5 mUr).
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The above provided errors for GC/TC-IRMS measurements represent the combined
uncertainties which were calculated by error propagation including the same contributions as
mentioned for the EA-IRMS measurements. These are somewhat higher than the HTC- and
EA-IRMS measurements (3.3 versus 1.2 mUr, and 3.2 versus 1.3 mUr, respectively, for
HUBG1 and HUBG2). However, the precision of the replicate measurements (‘external
precision’ including chemical replication uncertainty) of δ2H values is 1.5 and 1.0,
respectivel,y for HUBG1 and HUBG2. This is in the same range as previously reported for
substances such as vanillin, pectin and wood [34]
.
Exclusion of hydrogen exchange between methoxy groups and ambient water during
storage
The ‘hydrogen-exchange-experiment’ (for detailed description refer to Material and Methods
section) and GC/TC-IRMS measurements for both HUBG1 and HUBG2 before and after the
experiment were all performed at HU. After a time period of three weeks where both sulfate
salts were stored over water of known isotopic signature (δ2HVSMOW = -59.5 mUr) the stable
hydrogen isotope ratios did not change (Table 2). This unambiguously indicates that there
was no hydrogen exchange between ambient water and the methyl group of the sulfates
during the storage experiment. This is in agreement with previous considerations and
measurements that esterified methyl groups are considered to be chemically stable in that the
hydrogen atoms of the methoxy moiety do not exchange with those of plant water during
ongoing metabolic reactions in the plant [11][34]. This experiment shows that the methyl sulfate
salts can be reliably utilised as reference materials for normalization of stable hydrogen
isotope data of organic matter containing methoxy groups if the ‘Zeisel method’ is employed
for formation of CH3I. Nevertheless, if the δ2H values of methyl sulfate salts are measured by
HTC-EA-IRMS it is mandatory to carefully remove any adsorbed water prior to analysis.
Stable carbon isotope results
The δ13CVPDB-LSVEC values for HUBG1 and HUBG2 measured by EA-IRMS are -50.31 ± 0.16
mUr (n=14) and +1.60 ± 0.12 mUr (n=16), respectively. The errors shown were calculated in
the same way as those provided for the hydrogen isotopic ratios. They represent the
combined uncertainties containing the same contributions as mentioned for the hydrogen
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measurements above. The methyl sulfates measured at the BGC-IsoLab by EA-IRMS were
calibrated to the VPDB scale using the reference material IAEA-603 (+2.46 ± 0.01 mUr) and
the in-house standard as the second scale anchor (Acetanilide; -30.06 ± 0.1 mUr), which was
calibrated against the two international secondary standards NBS 22 (-30.03 ± 0.04 mUr) and
LSVEC (-46.6 mUr). As of June 2018 IUPAC no longer recommends the use of LSVEC as a
scaling standard for the VPDB scale (Press release; http://iupac.org/standard-atomic-weights-
of-14-chemical-elements-revised). LSVEC has previously been shown to be an inadequate
δ13C isotopic standard [49],[50]. Over time, LSVEC carbon atoms exchange with atmospheric
CO2 carbon atoms. This causes a gradual contamination and 13C-isotopic fractionation of
LSVEC. The BGC-Isolab is currently involved in an effort to produce a replacement standard
for LSVEC. The envisaged replacement is USGS44, a Merck high purity CaCO3 with a
preliminary value of -42.15 ± 0.05 mUr, available at https://isotopes.usgs.gov. It is likely that
USGS44 will replace LSVEC in the future. Therefore, BGC-IsoLab also analysed HUBG1
and 2 using USGS44 as a second scale anchor. The reader should note that the provided
USGS44 value is a preliminary value and may change slightly upon its final publication.
However, if necessary, users of HUBG 1 and 2 can easily recalculate the values of HUBG1
and 2. The preliminary δ13C values of HUBG1 and HUBG2, calibrated against IAEA-603
and scaled to USGS44 (-42.15 ± 0.05 mUr) are -50.17 ± 0.08 mUr and +1.60 ± 0.05 mUr,
respectively (cf. Table 1).
Comparable with the measurement of the δ2H values, the δ13C values of HUBG1 and
HUBG2 were also measured by GC/TC-IRMS after conversion to CH3I by the ‘Zeisel-
method’. The δ13C values are -47.73 ± 0.16 mUr for HUBG1 and +4.42 ± 0.19 mUr for
HUBG2. These data show an offset of 2.58 mUr and 2.82 mUr, respectively, from the δ13C
values measured at BGC-IsoLab. This offset can be explained by the fact that the results of
GC/TC-IRMS measurements at laboratory of Heidelberg were normalised using the same
two CH3I working standards which were used for normalisation of the stable hydrogen
measurements. However, the range of δ13C values of the CH3I working standards (-70.04
mUr and -60.72 mUr) is narrow and unfortunately does not span the isotope range of the
methyl sulfates (-47.73 mUr and +4.42 mUr) and thus these standards are not ideal to correct
for scale-compression. This probably caused the differences in the δ13C values measured by
the two laboratories. However, we note that in each instance the offsets in the δ13C values for
HUBG1 and 2 are more positive by nearly the same value. This may indicate that when liquid
CH3I is applied as a reference material (1-2µL of liquid CH3I is injected into a 1.5-mL vial
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and a fraction of headspace gas injected into the GC/IRMS system; for details refer to the
method section) to normalize samples prepared by the “Zeisel-Method” other factors causing
13C fractionation might be involved. Thus, again this would highlight that it is important to
follow the principle of identical treatment of reference materials and samples [39]. The
adoption of HUBG1 and 2 as reference standards would facilitate following the IT Principle
when analysing methoxy groups.
The above provided errors for GC/TC-IRMS measurements represent the combined
uncertainties which were calculated by error propagation including the same contributions as
mentioned for the EA-IRMS measurements. These are in the same range as the EA-IRMS
measurements (0.16 versus 0.16 and 0.19 versus 0.12 for HUBG1 and HUBG2, respectively).
Conclusion
There is a lack of suitable reference materials to reliably normalise stable carbon and
hydrogen isotope measurements of plant methoxy groups. In order to rectify this situation we
conducted investigations with two methyl sulfate salts to determine if they could be adopted
for this purpose. Both salts were calibrated against international stable isotope reference
materials by HTC- and EA-IRMS to be δ2HVSMOW = -144.5 ± 1.2 mUr and δ13CVPDB = -50.31
± 0.16 mUr for HUBG1 and δ2HVSMOW = -102.0 ± 1.3 mUr and δ13CVPDB = +1.60 ± 0.12 mUr
for HUBG2, respectively. Quantities of each salt ranging between 0.52 and 0.65 mg for δ13C
values and between 6.91 and 7.10 mg for δ2H values were used for single EA-IRMS
measurements.
Using a different analytical approach where the methyl groups of the methyl sulfate salts
were converted to CH3I by reaction with HI and subsequent analysis of the generated CH3I
using GC/IRMS we obtained δ2HVSMOW values of -143.2 ± 3.3 mUr (HUBG1) and -102.5 ±
3.2 mUr (HUBG2) using sample amounts of ~5mg of each salt. These results are in excellent
agreement with the values measured by HTC-IRMS. The precision (‘external precision’
including chemical replication uncertainty) for δ2H values of replicate measurements (n=14)
obtained by GC/IRMS is ±1.5 mUr and ±1.0 mUr for HUBG1 and HUBG2, respectively.
To measure the stable carbon isotope ratios of methoxy groups using GC/IRMS,
approximately 2mg of each salt is required. The results show an offset of about 2 to 3 mUr
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from the EA-IRMS measurements which might be explained by the lack of available suitable
reference materials required to correct for scale compression for GC/IRMS analysis. The
precision of the replicate measurements of δ13C values obtained by GC/IRMS is ±0.21 mUr,
and ±0.40 mUr, for HUBG1 and HUBG2, respectively.
The results regarding stable carbon isotope analysis might have a substantial impact on the
δ13C values of plant methoxy groups that have been previously reported
[7],[12],[14],[19],[25],[26],[35],[51]. Most of the δ13C values of wood methoxy groups that have been
measured in the past (~ranging from -20 to -30 mUr) were normalised with CH3I reference
materials with very negative δ13C values (~ -60 to -70 mUr) may need to be corrected by 2 to
3 mUr towards more negative values. For example, these corrections would bring the δ13C
values of wood methoxy groups presented in the studies of Gori et al [12] and Mischel et al [14]
closer to the respective δ13C values of plant cellulose and whole wood.
An additional experiment clearly showed that the hydrogen atoms of the methyl sulfate salts
do not exchange with hydrogen from atmospheric water vapour. Thus the storage and
preparation of HUBG 1 and 2 standards are much easier than the storage and preparation of
CH3I, which evaporates easily, and is unstable, releasing I2 when in contact with air.
Furthermore, both sodium- and potassium methyl sulfate are much less harmful than CH3I,
which is thought to cause cancer (Sigma Aldrich SDS sheet 2018). This is another important
and practical reason to find a replacement for CH3I. Furthermore, using methyl sulfates for
generation of CH3I is advantageous because these standards allow the user to follow the IT
Principle.
The δ13C values determined for HUBG1 and HUBG2 span a relatively wide range of δ13C
values (-50 to +2 mUr) thus covering most of the natural δ13C values of terrestrial plant
methoxy groups that have been reported so far. The respective δ2H values of HUBG1 and
HUBG2 cover a narrow range on the δ-scale for δ2H value measurements of methoxy groups
that lie in the range of ~ -100 to -150 mUr. Therefore, in future work we aim to prepare
several alternative wood reference materials from subtropical and temperate regions that
ideally span a range of δ2HVSMOW values from around -200 to -350 mUr.
Following the recommendations for selecting reference materials for IRMS analysis as
outlined in the introduction we recommend that the two methyl sulfate salts HUBG1 and
HUBG2 should be adopted as suitable reliable reference materials to normalise the δ13C
values of methoxy groups. For stable hydrogen isotope measurements of plant methoxy
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groups we suggest that next to HUBG1 and HUBG2 additional reference materials are
required to cover the full range of δ2H values of plant methoxy groups reported so far.
Moreover, the two investigated methyl sulfates might also serve as reference materials for
bulk measurements of the δ13C and δ2H values of organic matter.
Acknowledgments
This study was supported by the German Science Foundation DFG (KE 884/6-3, KE 884/8-2)
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Table 1. Stable hydrogen and carbon isotope values of HUBG1 and HUBG2 analysed by HTC- and EA-IRMS
(MPI-BGC) and CF-IRMS (HU-BGC).
HUBG1
HUBG2
MPI-BGC1
δ2HVSMOW
HU-BGC2
δ2HVSMOW
MPI-BGC1
δ2HVSMOW
HU-BGC2
δ2HVSMOW
n
n
n
n
-144.5 ± 1.2 mUr
30
-143.2 ± 3.3 mUr
14
-102.0 ± 1.3mUr
32
-102.5 ± 3.2 mUr
14
δ13CVPDB
n
δ13CVPDB
n
δ13CVPDB
n
δ13CVPDB
n
-50.31 ± 0.16 mUr
14
-47.73 ± 0.16 mUr
35
+1.60 ± 0.12 mUr
16
+4.42 ± 0.19 mUr
35
-50.17 ± 0.08
mUr3
14
---
---
+1.60 ± 0.05 mUr3
16
---
---
1 IsoLab at the Max Planck Institute for Biogeochemistry, Jena, Germany
2 Heidelberg University, Institute of Earth Sciences,
3 values calibrated against IAEA-603 and scaled to USGS44
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Table 2. Results of the ‘hydrogen-exchange-experiment’ measured by GC-HTC-IRMS (HU).
HUBG1
HUBG2
δ2HVSMOW
-142.4 ± 1.4 mUr
15
-100.1± 1.5 mUr
15
n
↓ ↓ ↓ ↓ ↓
↓ ↓ ↓ ↓ ↓
storage in desiccator for 3 weeks (under vacuum, over water*)
initialδ2H value of the water = -59.5 mUr
↓ ↓ ↓ ↓ ↓
-142.9 ± 0.9 mUr
15
↓ ↓ ↓ ↓ ↓
-100.8 ± 1.4 mUr
15
δ2HVSMOW
n
* water with a 2H signature significantly different from that of both
HUBG1 and HUBG2
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Figure 1: Information on the investigated methyl sulfates.
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