Measurement of position-specific 13C isotopic composition of propane at the nanomole level

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ScienceDirect Geochimica et Cosmochimica Acta 177 (2016) 205–216 www.elsevier.com/locate/gca

Measurement of position-specific 13C isotopic composition of propane at the nanomole level Alexis Gilbert a,⇑, Keita Yamada b, Konomi Suda c, Yuichiro Ueno a,c, Naohiro Yoshida a,b b

a Earth-Life Science Institute (WPI-ELSI), Tokyo Institute of Technology, Meguro, Tokyo 152-8550, Japan Department of Environmental Chemistry and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503, Japan c Department of Earth and Planetary Sciences, Tokyo Institute of Technology, Meguro, Tokyo 152-8551, Japan

Received 8 July 2015; accepted in revised form 24 January 2016; available online 1 February 2016

Abstract We have developed a novel method for analyzing intramolecular carbon isotopic distribution of propane as a potential new tracer of its origin. The method is based on on-line pyrolysis of propane followed by analysis of carbon isotope ratios of the pyrolytic products methane, ethylene and ethane. Using propane samples spiked with 13C at the terminal methyl carbon, we characterize the origin of the pyrolytic fragments. We show that the exchange between C-atoms during the pyrolytic process is negligible, and thus that relative intramolecular isotope composition can be calculated. Preliminary data from 3 samples show that site-preference (SP) values, defined as the difference of d13C values between terminal and sub-terminal C-atom positions of propane, range from
1.8‰ to
12.9‰. In addition, SP value obtained using our method for a thermogenic natural gas sample is consistent with that expected from theoretical models of thermal cracking, suggesting that the isotope fractionation associated with propane pyrolysis is negligible. The method will provide novel insights into the characterization of the origin of propane and will help better understand the biogeochemistry of natural gas deposits. Ó 2016 Elsevier Ltd. All rights reserved.

1. INTRODUCTION Stable isotopes at natural abundance have long been used to study geochemical problems. Applied to natural gas hydrocarbons (methane, ethane, propane, n-butane, i-butane), carbon and hydrogen isotopes
together with other indicators such as chemical composition
are used as tracers for the origins of hydrocarbons and their processes of formation or degradation (Bernard et al., 1978; Schoell, 1980; Clayton, 1991; Mango et al., 1994; Rooney et al., 1995; Whiticar, 1999; Tang et al., 2000). The isotopic

⇑ Corresponding author. Tel.: +81 3 5734 2887; fax: +81 3 5734

3416. E-mail address: [email protected] (A. Gilbert). http://dx.doi.org/10.1016/j.gca.2016.01.017 0016-7037/Ó 2016 Elsevier Ltd. All rights reserved.

signature of natural gas hydrocarbons is therefore a useful tool in studies aiming at, for instance, evaluating global budgets of hydrocarbons on Earth (e.g. microbial vs thermogenic), assessing thermal maturity of natural gas reservoirs or identifying geological sites for abiotic C-fixation and CH4 polymerization. However, a clear identification of their origin (e.g. thermogenic vs. abiotic) is often difficult due to overlap of chemical and isotopic signatures for different origins and/or to mixing of components from different origins. New lines of evidence would thus be helpful in order to constrain the origin of natural gas components and to understand the mechanisms responsible for their formation. New modes of isotopic substitution have been explored in the past decades. Among them, clumped isotope thermometry has recently been applied on methane

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in order to infer its formation temperature (Stolper et al., 2014). Although kinetic factors may complicate the interpretation (Wang et al., 2015), it appears to be a promising tool for gas-generation temperature. Intramolecular isotopic analyses, also referred to as position specific isotope analyses (PSIA), have been shown to provide unique information to constrain the origin of natural molecules: carbohydrates (Rossmann et al., 1991; Gilbert et al., 2012), ethanol (Gilbert et al., 2011), acetic acid (Rinaldi et al., 1974; Hattori et al., 2011), fatty acids (Monson and Hayes, 1982), n-alkanes (Gilbert et al., 2013b). The principle is based on measuring the isotope composition of a specific C-atom position in a given molecule; or in other words measuring the relative abundance of 13 C-isotopomers with a 13C atom at a different C-atom position. C3 and C4 alkanes (n-propane, i-butane, n-butane) have two singly substituted 13C-isotopomers: terminal and central (for instance for propane: 13CH3– CH2–CH3 and CH3–13CH2–CH3 are the terminal and central 13C-isotopomers, respectively). Although intramolecular 13C isotope distribution of C3–C4 hydrocarbons has not been measured until now, theoretical calculations of isotopic fractionation factors suggest that a considerable amount of information could be obtained from this measurement. Thermal cracking leads to C–C bond fissions in which 12C–12C bonds break faster than their heavy homologue 12C–13C. The terminal C-atom position of the newly formed hydrocarbon is thus depleted in 13C, the other positions remaining isotopically unchanged (e.g. Chung et al. (1988)). This is actually reflected in the trend of compound specific isotope composition of thermogenic hydrocarbons, for which low carbonnumber components are systematically depleted compared with high carbon-number ones (dC1 < dC2 < dC3. . .). Thermogenic hydrocarbons are thus expected to be 13C-depleted in the terminal C-atom position. It is worth noting that terminal C-atom is also expected to be 13C-depleted under isotope equilibrium conditions (Webb and Miller, 2014). In addition, as both kinetic and equilibrium fractionation factors are expected to decrease with increasing temperature (Tang et al., 2000; Webb and Miller, 2014), PSIA of hydrocarbons could be used as an indicator of the maturity of natural gas. Abiotic reduction of C1 compounds to methane and higher hydrocarbons, in contrast, leads to a reverse isotopic distribution (dC1 > dC2 > dC3. . .) (McCollom and Seewald, 2006; Fu et al., 2007; Proskurowski et al., 2008), suggesting a reverse intramolecular pattern for C3–C4 hydrocarbons, i.e. a 13C-enrichment in the terminal C-atom position (Sherwood Lollar et al., 2008). Measuring intramolecular isotopic composition relies either on measuring the concentration of different isotopomers simultaneously (e.g. 13CH3–12CH2OH and 12 CH3–13CH2OH in the case of ethanol) using quantitative Nuclear Magnetic Resonance (Tenailleau et al., 2004; Caytan et al., 2007) or on degrading the analyte into fragments whose isotopic composition would reflect those of specific C-atom positions of the starting material. For the latter, several means have been used: chemical/enzymatic reactions (Abelson and Hoering, 1961; Monson and Hayes, 1982; Rossmann et al., 1991), off-line pyrolysis

(Meinschein et al., 1974) on-line pyrolysis (Corso and Brenna, 1997; Yamada et al., 2002; Gauchotte et al., 2009; Gilbert et al., 2013a), and
more recently
ultra high resolution mass spectrometry (Eiler et al., 2013). Intramolecular isotopic composition of long chain hydrocarbons (C11–C31) has recently been measured using quantitative isotopic 13C Nuclear Magnetic Resonance (Gilbert et al., 2013b). Isotopic composition of the terminal and sub-terminal carbon atom positions of hydrocarbons allowed us to make a distinction between biogenic and thermogenic origins. Samples of presumed biological origin (C16–C31) shown a systematic alternation between position-specific isotope composition of odd and even compounds which is inherited from the alternation pattern of fatty acids due to acetyl-CoA polymerization (Monson and Hayes, 1980; Hayes, 2004). On the other hand, no alternation was observed for C11–C15 compounds and a depletion of ca. 11‰ was measured for the terminal C-atom positions, consistent with a thermogenic origin. However, the NMR approach suffers from low sensitivity (tens to hundreds mmol of compound is usually required). This would be problematic for the analysis of samples where typical volume mixing ratios of C3–C4 hydrocarbons are 0.01–1%. In addition, due to their high vapor pressure, measuring intramolecular 13C isotopic distribution of C3–C4 hydrocarbons using quantitative NMR would be difficult. Sensitive techniques dedicated to gaseous compounds analysis are thus more appropriate for this purpose. Here, we implement and evaluate an apparatus for the measurement of the intramolecular 13C isotopic composition of propane. The device is based on pyrolysis coupled to gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS), as used previously for ethanol (Gilbert et al., 2013a). Estimating the accuracy requires assessing that the fragments originate only from specific C-atom positions of the original material. Since standards with known 13C intramolecular composition are not available for propane, we use isotopically spiked propane samples to establish the extent of isotopic exchange associated with the process. The method is then used to determine natural abundance intramolecular 13C isotopic compositions of three propane samples of different origins. Finally, we discuss and evaluate how PSIA of propane could help with distinguishing between samples from different sources or processes. 2. MATERIALS AND METHODS 2.1. Samples 1-13C-Enriched propane (1-13C-enrichment >99% and purity >98%) was supplied by SI Science Co., Ltd, Saitama, Japan. The natural abundance propane sample (denoted subsequently as ‘‘Commercial 1”) was supplied by GL Sciences Inc., Tokyo, Japan (purity = 99.5%). The natural gas mixture denoted as ‘‘Commercial 2” (CH4, CO2, C2H6, C3H8, n-C4H10 and i-C4H10 each 1% in N2) was provided by GL Sciences, Japan. The natural gas standard (NGS-2) was provided by NIST (C3H8 = 1.3% v/v) (Hut, 1987).

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Six propane samples with different enrichments at CH3 position were prepared using gas-tight syringes by dilution of 1-13C-propane with natural carbon isotope abundance propane ‘‘commercial 1” (dilution factor >1000) in glass vials sealed with butyl rubber caps. The exact dilution factors do not need to be known for our purpose. 2.2. Determination of the molecular d13C value of propane samples The molecular 13C composition of propane samples (d CBulk) was determined using a gas chromatograph coupled with isotope ratio mass spectrometer (DeltaplusXP, Thermo Fisher Scientific) via a combustion furnace and an open split interface (GC Combustion III, Thermo Fisher Scientific). High purity helium was used as the carrier gas. The conditions of the GC oven were as follows: injector temperature 250 °C; split ratio 40:1 (for pure compounds) or 2:1 (for ca. 1% diluted compounds); flow rate 1.5 mL/min; oven temperature program 50 °C (maintained 1 min.) raised to 150 °C (maintained 10 min.) at a rate of 10 °C/min. The effluent was then introduced into a combustion furnace (ceramic tube packed with CuO, NiO, and Pt wires; operating at 960 °C) before being analyzed by the IRMS. d13C values were calibrated against NIST natural gas standard NGS-2 (Hut, 1987). 13

2.3. On-line determination of intramolecular d13C composition in propane samples Propane samples (0.1 mL for commercial-1; 1.0 mL for NGS-2 and commercial-2) were introduced using a gastight syringe into an on-line pyrolysis system coupled with GC-C-IRMS, as described by Hattori et al. (2011). High purity helium was used as the carrier gas. A first GC column (HP-PLOT-Q, 30 m  0.32 mm i.d., 10 lm film thickness; Varian, CA, USA) was connected to a high temperature conversion furnace (deactivated fused-silica capillary column 0.25 mm i.d. inserted into a ceramic tube of 25 cm  0.5 mm i.d., operating at different temperatures) to pyrolyse propane. The pyrolytic fragments were separated on a second GC capillary column (HP-PLOT-Q, 30 m  0.32 mm i.d., 10 lm film thickness; Varian, CA, USA), and introduced into a combustion furnace (ceramic tube packed with CuO, NiO, and Pt wires; operating at 960 °C) before being analyzed by the IRMS (Delta XP, Thermo Fisher Scientific Inc., Waltham, MA, USA). The conditions of the first GC oven were as follows: injector temperature 250 °C; split ratio 40:1 (for pure compounds) or 2:1 (for ca. 1% diluted compounds); flow rate 2.5 mL/min; oven temperature program 50 °C (15 min) raised to 100 °C (10 min) at a rate of 10 °C/min; then raised to 150 °C (15 min) at a rate of 20 °C/min and kept at 150 for 15 min. The second GC oven was kept at 40 °C throughout the analysis. Once the C1 and C2 fragments from pyrolysis of hydrocarbons were eluted from the second column the temperature of the second GC oven was raised to 150 °C at 20 °C/min in order to elute unreacted hydrocarbons.

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The conditions were optimized in order to pyrolyse separately different compounds from a mixture of natural gas compounds (CH4, C2H6, C3H8, n-C4H10, i-C4H10) and to separate all the C1–C2 pyrolytic fragments (See Fig. S1 for a chromatogram obtained from a mixture of gases using the conditions described above). The fragments were identified based on similar retention times as pure gas samples. d13C values were calibrated against the NIST natural gas standard NGS-2 (Hut, 1987). The connections between the GC columns and the pyrolysis and combustion furnaces were made using a deactivated fused-silica capillary column (0.25 mm i.d.). 3. RESULTS 3.1. Fragmentation pattern as a function of temperature The aim of this study is to determine the site-specific isotope composition of propane from natural gas samples. Propane first has to be isolated from other natural gas constituents. It is then cracked into smaller fragments whose isotope composition ideally reflects the original sitespecific isotope composition of propane. Chromatographic conditions were optimized in order to be able to isolate and measure the d13C values of fragments from propane pyrolysis. Our experimental set up can resolve fragments arising from propane pyrolysis, even when the starting material was a mixture of natural gas components (sample ‘‘commercial 2” or NGS-2) (see Fig. S1 for a chromatogram). The amount of products formed by pyrolysis and their isotopic composition were investigated for temperatures ranging from 750 to 950 °C, corresponding to temperatures at which the minimal and maximal rates of propane degradation occurs, respectively (Fig. 1A and B). Detected pyrolysis products include CH4, C2H4, C2H6, and C3H6. The combined area of all fragments was 117 ± 4 Vs, similar to the area of propane without pyrolysis 118 ± 15 (pyrolytic temperature = 100 °C), suggesting other pyrolytic byproducts are minor. As expected, the amount of propane decreases with increasing pyrolysis temperature while that of other fragments increases (Fig. 1A). The isotopic composition d13C of propane increases with pyrolytic temperatures (Fig. 1B), consistent with a higher consumption of propane at higher temperatures. The d13C value of propylene follows the same trend as that of propane suggesting a common origin which is consistent with propylene arising from dehydration of propane (Layokun and Slater, 1979). The other fragments (CH4, C2H4, C2H6) have relatively constant d13C values within a range of about 2‰. At all pyrolysis temperatures methane is 13C-enriched compared with both ethane and ethylene, the latter being the most 13C depleted fragment (d13C values ranging from
37.6‰ to
39.8‰). 3.2. Fragmentation pattern as a function of temperature Isotopic compositions of fragments arising from pyrolysis of propane with different degrees of enrichment were measured at four different pyrolysis temperatures keeping other conditions identical. The relationship between d13C values

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A

100

mol%

80

CH4

60

C2H4 C2H6

40

C3H6 C3H8

20 0

700

750

800

850

900

950

1000

Temperature (°C)

-10.0

B

CH4 C2H4 C2H6 C3H6

δ13C (‰)

-20.0

C3H8

-30.0

-40.0

700

750

800

850

900

950

1000

Temperature (°C) Fig. 1. Temperature profile of propane pyrolysis using the apparatus described in Section 2. Plain circles: propane C3H8; open circles: propylene C3H6; plain squares: ethane C2H6; open squares: ethylene C2H4; triangles: methane CH4. (A) Relative molar concentration (mol %) calculated after the area of each fragment peak. (B) Isotopic composition d13C of each fragment. Error bars (standard deviations from the mean of 3 repetitions) are within the symbol.

Table 1 Slopes and associated uncertainties from the correlation between isotopic composition d13C of fragments from pyrolysis at different temperatures of propane samples. Propane samples used were those spiked with different amounts of 13C-1-propane (n = 6). Temperature

800 °C (n = 6)

850 °C (n = 6)

900 °C (n = 6)

950 °C (n = 4)

dC2H4 = f(dCH4) dC2H6 = f(dCH4) dC2H6 = f(dC2H4) dBulk = f(dCH4)

0.51 0.98 1.94 0.65

0.50 0.98 1.97 0.65

0.53 0.97 1.84 0.67

0.57 0.98 1.71 0.65

(±0.02) (±0.02) (±0.08) (±0.01)

of the fragments is a key parameter to evaluate the isotopic filiation between propane and its derived fragments (see Section 4.3 for a detailed explanation). The values of the slopes obtained by linear regression for the curves d13CC2H4 = f(d13CCH4), d13CC2H6 = f(d13CCH4), d13CC2H6 = f(d13CC2H4) and d13CBulk = f(d13CCH4) are reported in Table 1. In addition, Fig. 2 represents a graphical version of data in Table 1 for pyrolysis temperature of 850 °C (second column in Table 1). In all cases, the coefficient of determination r2 is above 0.99. The slope of the curve d13CC2H4 = f(d13CCH4) increases gradually with pyrolysis temperature from 0.51 to 0.57, while that of d13CC2H6 = f(d13CCH4) remains nearly the same (ca. 0.98) at all temperatures. The slope for d13CC2H6 = f(d13CC2H4) decreases with temperature. The

(±0.03) (±0.04) (±0.14) (±0.02)

(±0.02) (±0.04) (±0.12) (±0.01)

(±0.09) (±0.17) (±0.04) (±0.04)

slope for d13CBulk = f(d13CCH4) remains constant with temperature, with a value of 0.65. These data will be interpreted and exploited in Section 4 in order to evaluate the filiation between starting propane and the fragments arising from its pyrolysis.

4. DISCUSSION 4.1. Preliminary considerations In order to get an accurate intramolecular isotope composition of propane from its pyrolytic fragments, two conditions must be achieved concomitantly:

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A

B

δ

δ

δ

δ

C

209

D δ

δ

δ

δ

Fig. 2. Relationships between isotopic composition d13C of pyrolytic fragments for propane samples with different 13C-enrichment in the terminal C-atom position at a pyrolytic temperature of 850 °C. The exact enrichment is not known. Error bar are standard deviations from the mean of 3 repetitions. Linear regression lines have been made according to a linear relationship. Parameters obtained from the linear regression are reported in Table 1. (A) d13CC2H4 = f(d13CCH4); (B) d13CCH4 = f(d13CC2H6); (C) d13CC2H4 = f(d13CC2H6); (D) d13CBulk = f(d13CCH4).

(i) isotope fractionation associated with the pyrolytic process must be known and repeatable; (ii) a filiation must be observed between fragments and propane. For instance, CH4 from pyrolysis of propane must arise only from the terminal (CH3) position of starting propane. Thus, any C-atom exchange between pyrolytic fragments must be avoided, or at least the extent of exchange must be known with sufficient accuracy so that a correction can be made. While the fulfillment of the latter condition is necessary to compare intramolecular 13C patterns of different propane samples, the former is necessary only to obtain absolute values of position-specific isotope composition. In other words, the isotope fractionation associated with the pyrolysis process does not need to be known in order to determine relative intramolecular isotope composition of propane. The subsequent discussion aims to evaluate these two points in the context of our study.

of ethylene C2H4 from the terminal and central C-atom positions of propane. The intramolecular isotopic composition could then be calculated from the isotopic composition of the fragments CH4 and C2H4. Yet, the process is indeed more complex. As shown in Fig. 1, CH4, C2H4 and C3H8 are detected, but other fragments are also observed at all pyrolytic temperatures. These fragments are ethane (C2H6) and propylene (C3H6) and have already been detected in previous works related to propane pyrolysis (Layokun and Slater, 1979; Khan et al., 2008). The presence of these fragments indicates that the pyrolysis process does not occur only through a single C–C bond reaction, but rather involves secondary reactions. This implies that potentially C-atoms can be exchanged between CH4 and C2H4 through secondary reactions, which would affect the accuracy of the method. Understanding the origin of the secondary fragments (C2H6 and C3H6) is the key to evaluating the accuracy of the method. Propylene C3H6 can be obtained by dehydration of propane, following the equation: C3 H8 ! C3 H6 þ H2

4.2. Origin of the pyrolytic fragments

ð2Þ

As stated above, getting accurate results from the present method would require propane to split into CH4 and C2H4 without isotope fractionation and through a single C–C bond breaking according to the following reaction scheme:

which does not create any path for the exchange of atoms from CH4 to C2H4. However, the presence of ethane C2H6 is more critical since it could be a reaction intermediate for the exchange of C-atoms between CH4 to C2H4. The C-atom exchange can occur in two ways; either from breaking down ethylene to methane through the formation of ethane:

C3 H8 ! CH4 þ C2 H4

C2 H4 þ H2 ! C2 H6 þ H2 ! 2  CH4

ð1Þ

In this case, the C-atom of methane would arise from the terminal C-atom position of propane and the two C-atoms

ð3Þ

or from dimerization of methane through ethane (the reverse of the reaction above).

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4.3. Evaluation of the isotopic filiation between propane and its pyrolytic fragments We herein evaluate potential C-exchange associated with pyrolysis using 1-13C-propane diluted with natural abundance propane. Let us assume a propane with a being the d13C value of the terminal carbon atom position (CH3) and b that of the central carbon atom position (CH2). a is a variable that depends on the amount of 13C spiked on the terminal C-atom position. b is considered as constant: given that the dilution is higher than 1000 times, b is not affected by the input of enriched propane. Assuming a simple model for the pyrolysis reaction where a single C–C bond dissociates, the d13C values of CH4 and C2H4 formed by thermal decomposition are: d13 CCH4 ¼ a þ e1

ð4Þ

d13 CC2H4 ¼ ða þ bÞ=2 þ e2

ð5Þ

where e1 and e2 are the overall isotopic fractionation factors associated with the formation and degradation of methane and ethylene, respectively, from their original C-atom positions in propane (CH3 and CH3–CH2, respectively). It is worth noting that the isotopic fractionation factor is assumed constant for all the samples under the same conditions. Writing d13CC2H4 as a function of d13CCH4 gives: 1 1 d13 CC2H4 ¼ d13 CCH4 þ ð
e1 þ bÞ þ e2 2 2

ð6Þ

According to Eq. (6), and if no C-exchange occurs during the pyrolytic process, the slope of the curve d13CC2H4 = f(d13CCH4) must be 0.50. Any deviation from this slope value would imply either that methane does not arise only from the terminal C-atom position of propane (slope < 0.5) or that C2H4 does not arise from an equal amount of CH3 and CH2 atoms (slope > 0.5). It is clear from the values presented in Table 1 that C-exchange occurs at pyrolysis temperatures above 900 °C (slope > 0.50). However, at temperatures below or equal to 850 °C, the slope is not significantly different from 0.50, implying that C-exchange between CH4 and C2H4 is negligible. In addition, the relationship between d13CCH4 and d13CC2H6 is ca. 1.0 over the pyrolytic temperature range tested, suggesting that methyl radicals combine to form C2H6 (Fig. 2B). The slope of the regression line between d13C values of C2H4 and C2H6 is ca. 2.0 (Fig. 2B) at temperatures below or equal to 850 °C but drops at temperatures above 900 °C (Table 1), indicating that C2H6 is subsequently dehydrogenated to C2H4. This implies that C2H4 arises not only from the equal contribution of methyl and

Table 2 Site-preference (SP) in ‰ values calculated for natural abundance propane samples. See text for details of calculation. Sample

Site preference SP (‰)

Commercial (n = 7) Commercial 2 (n = 8) NGS-2 (n = 3)


8.2 (±0.6)
1.8 (±0.4)
12.9 (±0.5)

methylene positions of the starting propane but also from an secondary contribution of methyl C-atoms at temperatures above 900 °C. This mechanism is actually supported by the curve d13CBulk = f(d13CCH4) (Fig. 2D). The bulk isotope composition of propane should equal the following mass balance equation: 2 1 d13 CBulk ¼ a þ b 3 3

ð7Þ

The slope of this curve should be 0.667 (=2/3) if CH4 arises only from the terminal C-atom position of propane. The values found here are in the range 0.65–0.67 (Table 2) and show no variation with temperature. This is consistent with methane C-atoms arising solely from the terminal C-atom of propane. To summarize, at temperatures below or equal to 850 °C, 2 molecules of methane (or more likely 2 methyl radicals) form one molecule of ethane (C2H6) which does not react further. At pyrolysis temperatures above or equal to 900 °C, ethane further dehydrates to form ethylene C2H4. At a pyrolytic temperature of 850 °C, the combined area of CH4 and C2H6 peaks equals 0.45 that of C2H4, whereas the expected value is 0.50. The slightly lower value can be explained by recombining processes consuming methyl radicals to form higher molecular weight hydrocarbons (including propane) that are difficult to constrain here. The origin of propylene (C3H6) can be assessed by comparing its isotope composition to that of starting propane: the difference between d13CC3H6 and d13CC3H8 is repeatable whatever the enrichment of the sample (Dd13C = 0.5 ± 0.4‰ at 850 °C; n = 6). Therefore, as already shown in previous works, propylene might arise from the dehydrogenation of propane, although a recombination from methyl radical and a C2 compound (ethylene, acetylene) cannot be fully ruled out. Fig. 3 summarizes the discussion above.

Fig. 3. Schematic view of the pyrolytic reactions occurring in the reactor of our device. The reaction arrows are based on experiments done with propane 13C enriched at terminal position (denoted ‘‘a”) at different levels (see text for more details). For clarity purposes only molecules that appear on the chromatogram are taken into account. In primary reactions, propane forms methane (CH4), ethylene (C2H4) and propylene (C3H6). Methane can react subsequently to form ethane (C2H6) that can react to form ethylene (C2H4) at temperatures above 900 °C (dotted line). Note that the cracking of propylene into methane and ethylene is only hypothetical and cannot be assessed nor dismissed using our approach.

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We therefore chose to work at a pyrolysis temperature of 850 °C since (i) C-exchange is negligible (ii) the amount of fragments makes the sensitivity higher than at 800 °C. At that temperature, one standard deviation of the mean d13C values of all the fragments is below 0.5‰. 4.4. Determination of site-preference (SP) values from d13C of pyrolytic fragments As demonstrated in Section 4.3, the method presented here is repeatable and a good filiation is obtained between original propane and its pyrolytic fragments. In order to obtain accurate SP values, one must know the isotopic fractionation factors associated with the formation of ethylene and methane from propane, e1 and e2 (e1 and e2 are isotope fractionation factors associated with the formation and degradation of methane and ethylene, respectively, from their original C-atom position in propane; see Section 4.3 and Fig. 3). For further calculation, we neglect these isotopic fractionation factors and consider e1 = e2 = 0. While being aware that this assumption may result in a systematic error to the calculation, determining these factors with sufficient accuracy appears too difficult at present. Sitepreference SP (in ‰) is defined as the difference in isotopic composition between a and b, and can be written as:
SP ¼ d13 CCH4
d13 CC2H4  2 ð8Þ Therefore, SP values of propane can be calculated using the isotope composition of methane and ethylene. Transformation of methyl radicals into ethane may alter the SP value calculated using Eq. (8). In fact, this reaction may be associated with isotope fractionation (e3; Fig. 3). Nevertheless, in contrast to e1 and e2, e3 can be calculated based on the amount of C2H6 formed and its isotopic composition. Using a mass balance calculation and the area of each peak, the original d13C value of CH4 (including atoms of C2H6) could be calculated:
d13 CCH4;original ¼ d13 CCH4  ACH4 þ d13 CC2H6  AC2H6 = ðACH4 þ AC2H6 Þ

ð9Þ

where A is the area of the fragment peaks. The SP value for a given sample can then be calculated using the following equation:
SP ¼ d13 CCH4;original
d13 CC2H4  2 ð10Þ Two samples from commercial sources were analyzed (‘‘commercial-1” and ‘‘commercial-2”; see Section 2), as well as an isotopic standard NGS-2 that is thermogenic in origin. Using the calculation above site preference values ranging from
1.8‰ to
12.9‰ could be calculated (Table 2). The standard deviation from the mean ranges between 0.4‰ and 0.6‰. Note that these SP values can only be considered as relative ones, since isotope fractionation factors associated with pyrolysis (e1 and e2, see Eqs. (4) and (5)) are not known.

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Fractionation factors associated with pyrolysis have been determined for ethanol (Gilbert et al., 2013a), although in that case a calibration could be made by comparison with an existing method (oxidation to acetic acid and subsequent decarboxylation). There is currently no method for determining intramolecular isotope composition of propane, which makes it difficult to assess the accuracy of our method. Nevertheless, an estimation of SP value can be made based on existing theoretical models. Chung et al. (1988) developed a theoretical model to account for isotopic composition of natural gas hydrocarbons (C1–C4) formed by thermal cracking. Thermal cracking of hydrocarbons is expected to be associated with isotopic fractionation (normal isotope effect) since C–C bonds are broken during the process (Tang et al., 2000). Natural gas plot (Chung et al., 1988) correlates the d13C values of thermogenic natural gas hydrocarbons versus the inverse of the carbon chain length nc. The linear relationship obtained reflects the fact that isotopic fractionation associated with thermogenic cracking is position-specific, namely it is significant only in the terminal C-atom position of the hydrocarbon formed. This model is consistent with the calculations made by Tang et al. (2000) that show that the isotopic fractionation from higher hydrocarbons (thermogenic cracking) is negligible for C-atom positions not involved in C–C bond breaking (secondary isotope effect) compared with that for C-atom positions directly involved in C–C bond breaking (primary isotope effect). The isotope fractionation at the bulk level (average isotope composition of all C-atom positions in the hydrocarbon considered) is thus higher for methane and decreases as a function of the carbon chain length nc in the hydrocarbon considered. This eventually gives rise to a linear relationship between 1/nc and d13C values (Chung et al. (1988)): d13 CBulk ¼ d13 Cm =nC þ d13 Cp  ðnC
1Þ=nC

ð11Þ

which rearranges to: d13 CBulk ¼ ðd13 Cm
d13 Cp Þ=nC
d13 Cp

ð12Þ

where nc is the carbon chain length of the hydrocarbon; d13Cbulk is the isotope composition of the hydrocarbon, d13Cm is the isotope composition of the C-atom position affected by the primary fractionation factor and d13Cp is the isotope composition of the C-atom positions unaffected by the primary isotope fractionation. Plotting d13Cbulk as a function of 1/nc thus gives a linear trend with a slope equals to d13Cm
d13Cp and an intercept equal to d13Cp. Site preference is defined as the difference in isotope composition d13C between central and terminal atom position. Using the notation above: SP ¼ a
b ¼ ðd13 Cm þ d13 Cp Þ=2
d13 Cp ¼ ðd13 Cm
d13 Cp Þ=2

ð13Þ

4.5. Toward absolute SP values

Therefore, the slope of the natural gas plot is twice the value of site-preference. The NGS-2 is thermogenic in origin (Hut, 1987) and follows the rules stated above, with the following equation:

Estimating fractionation factors associated with pyrolytic processes is necessary to obtain absolute SP values.

d13 C ¼
28:5  1=nC
16:6 ðr2 ¼ 0:992Þ

ð14Þ

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The SP value calculated by this mean is
14.3‰, comparable with that obtained using our method (
12.9‰). The values obtained using the method presented here are thus consistent with previous theoretical models. We recognize that this consistency is only the first step toward obtaining accurate SP values. Further work is currently ongoing and aims to assess the accuracy of the method presented here. The 3 samples analyzed here have SP values spanning a range of ca. 11‰. These preliminary data show that differences between samples are well beyond the precision of the method and that these differences can be used in order to better characterize the origin of hydrocarbons. While analysis of samples with different biogeochemical history is necessary to evaluate the contribution of position-specific isotope analyses for characterizing origins of hydrocarbons, estimations of intramolecular propane isotope composition can be made based on experimental studies and theoretical models. 4.6. Expected site-preference of propane from different biogeochemical processes Speculating on the usefulness of propane SP values based solely on the samples presented in this work would be uncertain, especially since two of them are of unknown origin. Nevertheless, predictions of intramolecular isotopic composition of propane can be made based on fractionation factors found in the literature and/or on mechanisms involved. In the paragraph below, we try to evaluate, at least qualitatively, the expected SP value of propane formed or degraded through diverse pathways. The pathways considered here are thermogenic and abiotic formation, as well as biodegradation. We admit that other pathways contribute to propane sources and sinks, such as biological formation (e.g. Hinrichs et al. (2006)), although predicting SP values for these pathways would be uncertain since too little is known about the mechanisms and/or the fractionation factors associated. 4.6.1. Thermogenic formation Tang et al. (2000) calculated kinetic isotope effects for homolytic C–C bond breaking of hydrocarbons giving rise to radicals, following the reaction scheme: R–CH2 –CH2 –CH2 –CH3 !  CH2 –CH2 –CH3

ð15Þ

Formation of propyl radicals from a higher hydrocarbon is associated with a normal isotope effect, namely 12C tends to react faster than 13C. Calculated primary kinetic isotope effects (13k/12k) for the C-atom position where the radical is formed range from 0.956 at 300 K to 0.985 at 600 K. Secondary kinetic isotope effects for other C-atom positions of the radical range from 0.991 to 0.995 (for the second position) and from 0.993 to 0.998 (for the third position) for temperatures of formation of 300 and 600 K, respectively. The fractionation factors for terminal and central positions of propane at 300 K are thus 0.975 (average of first and third position of the propyl radical) and 0.991, respectively. Assuming a starting hydrocarbon with an homogeneous 13C isotopic distribution (all C-atom

positions have the same d13C values), site-preference for propane formed at 300 K would thus be (0.975
0.991)  1000 =
16‰. This value becomes
3‰ for formation temperature of 600 K. The range of values embraces the SP values measured on C11–C15 alkanes by NMR (ca.
11‰; (Gilbert et al., 2013b)), as well as the SP values obtained using the present method (
12.9‰ for NGS-2; Table 2). Thermodynamic equilibrium calculations using DFT have been used to calculate isotope fractionation factors associated with the following exchange: CH3 –13 CH2 –CH3 $ 13 CH2 –CH2 –CH3

ð16Þ

13

Preference for the central C-isotopomer (on the left in Eq. (16)) was shown at temperatures ranging from 300 to 600 K, leading to site-preference values ranging from
14.4‰ at 300 K to
2.7‰ at 600 K (Webb and Miller, 2014). Overall, thermogenic formation of propane tends to favor the formation of the central 13C-isotopomer, leading to negative SP values. Qualitatively, the SP value of propane would be dependent on temperature (the higher the temperature, the higher the SP value), making it a potential indicator of temperature of formation. Site-preference would also depend on the accumulation history of natural gas. Following a Rayleigh distillation model (i.e. in case all thermogenic propane is accumulated), d13C values of the terminal position of propane should follow (Rooney et al., 1995): d13 Cterm ¼ d13 C0
eterm

ð1
F Þ  lnð1
F Þ F

ð17Þ

where d13Cterm is the isotopic composition of the terminal C-atom position of propane; d13C0 is the isotopic composition of the starting material from which propane is evolved; eterm is the fractionation factor associated with the terminal C-atom position of evolved propane; F is the extent of the reaction, namely how much propane is formed compared with the starting material. The same equation can be derived for the central C-atom position of propane. Using Eq. (17) and e values calculated by Tang et al. (2000), C-isotope composition for the central and terminal C-atom position of propane can be calculated. The resulting SP values are shown in Fig. 4 for different temperatures and for different degrees of maturity (extent of thermal cracking, F). Globally, increasing maturity leads to an increase in SP value. The latter eventually approaches 0 when the source material is totally exhausted. Propane SP values could then be an indicator of temperature of formation and/or maturity of natural gas reservoirs (see Fig. 5). 4.6.2. Abiotic formation Compared with thermogenic process, fewer models are derived to account for isotope effects related to abiotic polymerization of methane to higher hydrocarbons. Sherwood Lollar et al. (2008) derived a model accounting for the bulk isotopic composition of CH4 and C2+ hydrocarbons (ethane, propane, butane) produced by abiogenic polymerization, following the reaction sequence: CH4 þ CH4 ! CH3 –CH3 þ H2

ð18Þ

A. Gilbert et al. / Geochimica et Cosmochimica Acta 177 (2016) 205–216 F

to thermogenic gases. Reaction (19) – and in a more general way, formation of higher hydrocarbons – is associated with negligible isotope fractionation and propane isotope composition is expected to follow a simple isotopic mass balance: 1 2 d13 CC3H8 ¼ d13 CCH4 þ d13 CC2H6 3 3

Fig. 4. Expected propane site-preference (SP) values as a function of the extent of thermal cracking (F) from a given source. SP values were calculated based on kinetic isotope effect data at different temperatures from Tang et al. (2000) and on the Rayleigh fractionation model described in Rooney et al. (1995) (see Section 4.6 for details). The d13C value of the source material is assumed to be 0‰ and to be homogeneous for all carbon atom positions. The calculation implies that all the propane formed is accumulated.

Fig. 5. Conceptual diagram showing expected site preference (SP) values and bulk d13C values for propane formed or degraded through different pathways. Green and orange zones represent abiotic and thermogenic propane, respectively. Black line represents d13C and SP change from a low maturity level to a high maturity level at 300 K (dashed line) and 600 K (plain line). Data are those used in Fig. 4, assuming a source with a homogeneous intramolecular isotope distribution and a d13C of
30‰. Blue and red arrows (‘‘T” and ‘‘C”) represent biodegradation which first step involves the terminal and central C-atom positions of propane, respectively (see Section 4.6 for a detailed explanation), assuming a starting propane with a SP value of 0‰ and a d13C value of
50‰. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

CH4 þ CH3 –CH3 ! CH3 –CH2 –CH3 þ H2

213

ð19Þ

According to the model, isotope fractionation associated with ethane formation is the only fractionating step in the system. The isotope effect is normal (12C reacts faster than 13 C) so that ethane is 13C-depleted compared with methane. The average isotope fractionation between methane and ethane in Kidd Creek natural gas samples is 0.9967 ± 0.0006: d13C values for ethane are 3.3‰ lower than those of methane. This leads to a typical abiotic pattern where methane is 13C-enriched compared with ethane, in contrast

ð20Þ

The mechanism consists of one insertion of a methane molecule into an ethane molecule. The added C-atom is derived from methane and will form one of the terminal C-atom positions. This step is assumed to be nonfractionating, thus the isotope composition of this position will be that of methane. The other terminal C-atom position arises from ethane. Overall, the isotopic composition of the terminal C-atom position will be the average of ethane and methane isotope composition. The isotopic composition of the central C-atom position will be that of ethane. For instance, Kidd Creek samples have d13C values of
34.1‰,
37.4‰ for methane and ethane, respectively (average of Kidd Creek samples from Sherwood Lollar et al. (2008)). Propane formed from the reaction of methane and ethane should have a pattern with d13C values of
34.1‰,
37.4‰ and
37.4‰ for the three atom positions. Since propane is a symmetrical molecule, the d13C value for the terminal C-atom position is the average of the d13C of the first and third positions, leading to a propane with d13C values of
35.8‰ and
37.4‰ for the terminal and central C-atom positions, respectively. The calculated SP value is then +1.6‰. Hence, if the assumptions made above are correct, PSIA of propane can be valuable to distinguish propane formed through abiotic polymerization (positive SP value) from that produced thermogenically (negative SP value; see above). We admit that this may not be true for all geological settings, and that the SP value may vary according to the parameters of the reaction (e.g. temperature, catalyst) as observed for bulk d13C and d2H values (McCollom, 2013). Measuring SP values of propane from different geological settings and from abiotic polymerization experiments under different conditions could be useful to build refined models accounting for mechanisms and isotope fractionation associated with abiotic polymerization reactions. 4.6.3. Biodegradation Living organisms can grow on hydrocarbons as a carbon source, using sulfate or nitrate as an energy source, for instance. The degradation can occur with or without oxygen (aerobic and anaerobic, respectively; for a review, see Rojo (2009)). Aerobic biodegradation proceeds through the oxidation of propane to the corresponding propanol; terminal C-atom oxidation is the major pathway, although central C-atom oxidation has also been reported (Shennan, 2006). In anaerobic conditions, the first step is fumarate addition either on the terminal or central position. The addition occurs mainly on the central C-atom position but addition to the terminal position has also been shown in the case of propane (Jaekel et al., 2014; Musat, 2015).

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In both cases, normal isotopic fractionation factors have been measured, i.e. propane left behind is 13C-enriched compared to starting propane. Depending on the mechanism involved, the remaining propane will be 13C-enriched either on the terminal or on the central C-atom position compared with the starting material. From a quantitative point of view, bulk isotope fractionation factors associated with propane aerobic biodegradation were measured as
10.8‰ (Bouchard et al., 2008) and
4.8‰ (Kinnaman et al., 2007). These values become
33‰ and
13‰ (3-fold the bulk value) when considering a single reactive C-atom position in the process of biodegradation (Bouchard et al., 2008). Recently, Jaekel et al. (2014) measured isotopic fractionation associated with anaerobic biodegradation of propane by sulfate reducing bacteria. Isotopic fractionation factor at the reactive position varied between
7.9‰ and
25.3‰. They have also shown that the major activation mechanism was fumarate addition on the terminal C-atom position of propane, although ca. 30% of the fumarate addition was made on the terminal C-atom position. Therefore, while bulk d13C values are commonly used to detect biodegradation of hydrocarbons, PSIA of hydrocarbons could go one step further and help identifying the mechanism of biodegradation. This would be especially useful for environmental studies aimed at deciphering the mechanisms of hydrocarbon degradation which currently use combined isotopic determination (2H vs 13C; e.g. Jaekel et al., 2014). Biodegradation could complicate the interpretation of the data regarding SP values. For instance, a thermogenic propane with a negative SP value could experience a biodegradation with an attack on the terminal C-atom position, which tends to enrich SP values. The SP value of propane experiencing that process would increase and could be similar to the expected abiotic signature (positive SP values). Vice versa, a propane sample from abiogenic polymerization with a positive SP value could be degraded via an attack on the central C-atom position: the SP value would decrease and would eventually resemble thermogenic ones. We also admit that other processes such as diffusion, adsorption and evaporation may complicate the interpretation of the data. Especially, adsorption and evaporation processes have been shown to fractionate isotopes at specific positions (Botosoa et al., 2008; Julien et al., 2015). The method presented here can be used in all the geological or experimental contexts discussed above and will be used in future work in order to explore the veracity of the hypotheses made above regarding sources and sinks identification of propane. 5. CONCLUSION This work aims at evaluating on-line pyrolysis coupled with GC-C-IRMS for the determination of the sitespecific 13C isotope composition of propane. Results obtained using isotopically enriched samples suggest that carbon exchange is minor at pyrolysis temperatures below 900 °C. The difference of d13C values between fragments (CH4, C2H4, C2H6) reflects the original site-preference of propane with an additional contribution from isotopic

fractionation factors associated with propane pyrolysis. Further work is currently ongoing, aiming at assessing these isotope fractionation factors. The method developed here presents several features that make it particularly adapted for geochemical applications: (i) natural gas can be injected directly without preliminary isolation of propane, (ii) required amount of propane injected is around 400 nmol, which is promising for geochemical applications; in addition, the system can be implemented with preconcentration lines (e.g. purge-and-trap systems) in order to increase the sensitivity, (iii) site-specific 13C isotope composition of higher hydrocarbons (butane and pentane isomers) are potentially accessible using the same device. The present method enables the measurement of 13C site-preference in propane, a feature that is governed by parameters such as mechanisms and temperatures of formation, as well as biodegradation. Here, we make this parameter available with sufficient accuracy and sensitivity which is a key step towards a better characterization of biogeochemical processes related to hydrocarbons formation and degradation. This method is not meant to be used alone but rather in combination with conventional methods (e.g. d2H vs d13C; CH4/C2+ vs d13CCH4) or more recent ones, such as clumped isotope thermometry of methane (Stolper et al., 2014; Wang et al., 2015). ACKNOWLEDGEMENTS A. Gilbert thanks the Grant-in-Aid for Young Scientists (B) (15K17774) and the Grant-in-Aid for Scientific Research (S) (23224013), MEXT, Japan, for financial support. Y. Ueno is partially supported by the STAR program from Tokyo Tech, Japan. The authors are particularly thankful to Lucy Kwok for linguistic assistance.

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