Three combustion reactions in a single EA/IRMS acquisition as a
strategy to save helium and energy
The global energy shortage crisis, helium (He) shortages and rising
prices worldwide have forced academic institutions and industries to
reduce their consumption and look for alternatives1.
The excessive He consumption and operating time of continuous He-flow
instruments using high-temperature combustion or pyrolysis reactors
linked to isotope ratio mass spectrometers further complicate this
problem. Such challenges should be addressed. The stable isotope ratios
of carbon (δ 13C relative to Vienna Pee Dee
Belemnite limestone), nitrogen (δ 15N relative
to molecular nitrogen in air) and sulfur (δ 34S
relative to Vienna Cañon Diablo Troilite) in bulk samples determined by
elemental analysis and isotope ratio mass spectrometry (EA/IRMS) have
been proven to be a remarkable tool to investigate processes in
biogeochemical and geochemical cycles. For δ 34S
measurements, an alternative instrumental approach, operating with less
He than EA/IRMS, is multicollector inductively coupled plasma/mass
spectrometry (i.e., with He-Ar or Ar alone as the aerosol
carrier)2. However, to date, there is no alternative
to bypass EA/IRMS for the analysis of C and N isotopes. A large number
of EA/IRMS δ 13C andδ 15N measurements with high He consumption are
routinely being used in a wide range of research and industrial
applications in fields including geochemistry, biology, pharmacy,
medicine, food control, food authentication, archaeometry, and forensic
sciences. Most He is recovered as a byproduct from natural gas
accumulations, and the conventional reserves are becoming scarce or
logistically unavailable, raising concerns about He shortages and price
increases in the scientific research community3,4.
While replacing fossil fuels with alternative or renewable energy may be
a way to realize sustainable power sources,5 managing
the He supply will be more challenging. He used as carrier gas in gas
chromatography (GC) methods can be replaced with H2 when
using flame ionization detection (GC/FID) or with N2 in
some GC/MS assays. Replacing He with another gas (i.e.,
H2, N2, O2) in EA/IRMS
or TC-EA/IRMS would be impossible because the gas could be the analyte
and otherwise would provide a high background signal. Here, I present a
strategy to cope with He shortages in EA/IRMS.
Not all industrial or academic applications require both C and N
isotopes, and the material to be analyzed may contain very little N
(e.g., lipids, carbohydrates, hydrocarbons, most geological materials),
which makes methods analyzing δ 13C andδ 15N in the same acquisition unsuitable.
Previously, I published a method for analyzing liquid samples (e.g.,
ethanol, wine) containing volatile analytes using two sample-capsule
combustion reactions in a single EA/IRMS acquisition6.
This procedure was shown to reduce the time of stay of the
liquid-containing tin capsule remained in the cavity of the autosampler
on the sampler slide, thus reducing its residence time in the
autosampler drum and the risk of sample loss by evaporation. This
optimized EA/IRMS method was only used to analyze C-rich samples with
very low N content. Three 20 s pulses of CO2 reference
gas with a known δ 13C value were followed by
the CO2 peaks resulting from two successive EA
combustion reactions. In this method (hereafter called 2×EA/IRMS), the
time required to reach the CO2 peak center and inject
the reference gas pulses is used to measure the13C/12C ratio of the
CO2 produced by two samples or calibration standards.
Here, improvements of the 2×EA/IRMS method for both C and N isotope
analysis are presented, then extended to include three EA combustion
reactions (i.e., 3×EA/IRMS method), and finally validated with a set of
international reference materials (RMs) of different matrices and
covering a broad range of δ 13C andδ 15N values and C/N molar ratios (Table 1).
This contribution will help overcome difficulties in the supply of He
tanks, energy shortages, and dramatic increase in the prices of He
bottles and energy.
Here, modifications of the EA conditions and acquisition methods
previously presented in Spangenberg and Zufferey6 are
adopted. The system consisted of a Carlo Erba 1108 elemental analyzer
fitted with an AS-200LS pneumatic autosampler with a 49-sample drum
(Fisons Instruments, Milan, Italy) and a ConFlo III continuous flow
open-split interface linked to a Delta V Plus isotope ratio mass
spectrometer (Thermo Fisher Scientific, Bremen, Germany). Aliquots of
calibration standards and RMs were weighed in tin capsules. They were
combusted at 1020 °C in a quartz reactor (450 mm × 18 mm i.d., 2 mm
wall) filled in house with 40 mm quartz wool, followed by 60 mm of
silvered cobaltous/cobaltic oxide
(Ag-Co3O4), 10 mm quartz wool, 120 mm
chromium oxide (Cr2O3), and 5 mm quartz
wool at the top. EA sonsumables fwere obtained rom Säntis Analytical,
Teufen, Switzerland. At the time of the combustion, a 60 s
O2 pulse (30 mL min–1) was introduced
into the He carrier flow of 60 mL min–1, which is a
significantly lower flow rate than the conventionally used rate of 80 mL
min–1. A quartz insert (210 mm × 13 mm i.d., wall 2
mm, with 5 mm quartz wool at the bottom) was used to collect the ash and
residues from the tin capsules and replaced after 100–150 combustion
reactions. The reactor packing could generally be used for the
combustion of 800–1200 samples. The combustion-produced gases (i.e.,
CO2, N2, NOx and
H2O) were carried by the He through a reduction
reactor—filled from bottom upward with 40 mm quartz wool, 50 mm copper
oxide (CuO), 280 mm copper grains, 10 mm quartz wool, 50 mm copper
oxide, and 10 mm quartz wool at the top—at 640 °C to remove excess
O2 and reduce nonstoichiometric nitrous products to
N2. The gases were then carried through a glass column
(110 m × 12 mm i.d., 2 mm wall) filled with anhydrous magnesium
perchlorate (Mg(ClO4)2) to remove water.
The dried N2 and CO2 were separated in a
chromatographic column (3 m × 4 mm i.d. stainless steel column packed
with Porapak® QS 50/80 mesh) at 80 °C, and carried
into the source of the mass spectrometer for measurement of the isotope
ratios of C and N. The GC device was heated 10 °C higher than the
routinely used value of 70 °C (IDYST, University of Lausanne), to
improve the separation of the N2 and CO2peaks with the shortest possible retention times after reducing the He
carrier flow. Separate EA/IRMS analytical sequences with optimized
sample aliquot sizes (depending on the C/N ratio of the sample) were
used for the determination of δ 13C and δ15N values. The15N/14N ratios were measured in
CO2-free gas, obtained by connecting a column (110 m ×
12 mm i.d., 2 mm wall) filled with soda lime before the water trap.
Three reference gas (N2 or CO2, both
≥99.999 purity) pulses were injected into the He carrier gas before the
sample peak eluted from the gas chromatograph. The 3×EA/IRMS methods for
C- and N-isotope analysis were essentially the same; the conditions
after the peak center included three activations of the elemental
analyzer (at 45–50 s, 280–285 s, and 515–520 s), no He-dilution,
three reference gas pulses (at 20–40 s, 70–90 s, and 120–140 s), and
an acquisition end time of 900 s. The 2×EA/IRMS method had only the
first two activations of the elemental analyzer and an acquisition end
time of 700 s (Figure 1). Sample aliquot sizes were optimized to obtain
signals with the integrated peak area (total area in Vs) of the major
isotopes (m /z 44, 45, and 46 for CO2 andm /z 28, 29, and 30 for N2) within ±30 %
of the total area of the reference gas peaks. Isotopic data were
acquired and processed using ISODAT 3.0 software (Thermo Fischer
Scientific). The sample peaks were evaluated by assigning aδ -value to the second reference peak. Normalization of the
measured raw δ -values to the international isotope reference
scale, VPDB-LSVEC lithium carbonate (hereafter VPDB) for C and
air-N2 for N, was performed by 3-point linear
calibrations with certified RMs and well-calibrated in-house standards.
The samples and calibration standards were weighed in 3.3 × 5 mm tin
capsules. The analytical sequences consisted of two sets of three
calibration standards measured in duplicate with the 2×EA/IRMS method at
the beginning and end of each run, with typically 40 to 60 sample
capsules measured with the 2×EA/IRMS or 3×EA/IRMS method between the
calibration standards. The tin capsules combusted in the same adquision
conatined aliquots odf the same or different sample or standard. The
autosampler needed to be refilled during the runs. The δ -values
were reported using the milliurey (mUr) —a synonym of the no longe
acceptable unit per mil (‰)— complying with the International System
of Units.7
The validation of the developed procedures (2×EA/IRMS and 3×EA/IRMS) was
based on replicate (n = 4–6) C and N isotope analysis of RMs
and laboratory standards performed during eight analytical sessions
(August-September 2023). The results are reported in Table 1 as the mean
± 1 standard deviation (SD). The RMs and laboratory standards covered a
wide range of δ 13C values from –43.69 to
–0.67 mUr (n =17) and δ 15N values
between –4.52 and 50.137 mUr (n =8). The accuracy of the
proposed procedures was assessed by the agreement between the measured
and the accepted/recommended isotope δ -values. The differences
between the 3×EA/IRMS values and the acceptedδ 13C values (0.04 ± 0.07 mUr) were similar to
those for 2×EA/IRMS (0.01 ± 0.07 mUr). The highest differences were for
the NBS22 Oil RM. Similar results were obtained for δ15N, with the differences between the 3×EA/IRMS values
and the accepted values (–0.02 ± 0.11 mUr) comparable to those between
the 2×EA/IRMS values and the accepted values (–0.02 ± 0.10 mUr). The
average precision (1 SD values) of the 2×EA/IRMS and 3×EA/IRMS values
was essentially the same (0.06 ± 0.03 mUr and 0.06 ± 0.02 mUr forδ 13C; 0.09 ± 0.03 mUr and 0.09 ± 0.05 mUr for
the δ 15N). The reproducibility values,
estimated from the SD values, are within the range of the uncertainties
of accepted values for RMs and laboratory standards (Table 1). Finally,
the proposed procedures were validated with unknown samples from leaves,
stems, and roots of C3 and C4 plants
(i.e., grapevine and corn) and decarbonated soil fractions. The mean of
the δ 13C and δ 15N
values from the 2×EA/IRMS and 3×EA/IRMS analyses (n = 5) are in
good agreement with the values obtained by conventional EA/IRMS
(n = 4), with analytical errors of ±0.05 forδ 13C and ±0.10 forδ 15N.
In summary, the proposed adaptation of the routinely used EA/IRMS method
significantly reduces the He carrier consumption and analysis time per
sample by adding second and third combustion cycles. The conventional
600 mL He consumption and 600 s instrument time become 350 mL He and 300
s (2×EA/IRMS) or 300 s and 300 mL He and 300 s (3×EA/IRMS). The accuracy
and precision of the δ 13C andδ 15N values remain unaffected. Both methods
save He, save energy, save reference gases (CO2,
N2), and O2, while reducing the analysis
and instrumental times by ca. 50 %. In conclusion, the approach
described here is particularly suitable for academia or industry and
research, where cost-effective high throughput and accurate and precise
replicate analysis of C and/or N isotopes is needed. Additionally, the
method benefits students and researchers by reducing the turnaround time
and providing significant cost savings to the laboratory in terms of
helium and energy.