Research Article

Received: 3 March 2014 Revised: 9 June 2014 Accepted: 9 June 2014 Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2014, 28, 1821–1828

Evaluation of commercially available reagents as a referencematerial for intramolecular carbon isotopic measurements ofacetic acid

Keita Yamada1*, Makiko Kikuchi1, Alexis Gilbert2, Naohiro Yoshida1,2, Nariaki Wasano3,Ryota Hattori4 and Satoshi Hirano3

1Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku,Yokohama 226-8503, Japan2Earth-Life Science Institute, Tokyo Institute of Technology, Meguro-ku, Tokyo 152-8550, Japan3Central Laboratories for Key Technologies, Research and Development Division, Kirin Company Limited, 1-13-5 Fukuura,Kanagawa-ku, Yokohama 236-0004, Japan4Quality Assurance Department, Kirin Company, Ltd, 4-10-2 Nakano, Nakano-ku, Tokyo 164-0001, Japan

RATIONALE: Recent advances in analytical techniques for the intramolecular carbon isotopic ratio measurement of someorganic compounds have provided important information on carbon cycles in biochemistry, organic geochemistry andfood chemistry. These advances have made it necessary to prepare intramolecular isotopic reference materials (RMs)to use for inter-laboratory calibration and/or inter-calibration among different analytical methods.METHODS: We evaluated the feasibility of preparing RMs using commercially available reagents for intramolecularcarbon isotopic ratio measurement of acetic acid. The intramolecular carbon isotopic distribution of nine acetic acidand four sodium acetate reagents was determined with high precision using off-line pyrolysis combined with gaschromatography–combustion–isotope ratio mass spectrometry (GC-C-IRMS). We also evaluated the potential alterationin the isotopic signature of acetic acid reagents by evaporation.RESULTS: The intramolecular carbon isotopic distributions for the acetic acid and sodium acetate reagents weredetermined with a precision of better than 0.45‰. We found that the isotopic values of these reagents spanned the carbonisotopic range of acetic acid in biological and environmental samples. We also found that the isotope fractionationassociated with the evaporation of acetic acid occurs solely on the methyl position, the carboxyl position beingunaffected.CONCLUSIONS: These commercially available reagents will be used as RMs in the future for inter-laboratory calibrationand/or inter-calibration with another intramolecular isotopic measurement technique, namely quantitative 13C NMR.In cases where acetic acid is being used as a RM, its storage must be carefully controlled to prevent evaporation.Copyright © 2014 John Wiley & Sons, Ltd.

(wileyonlinelibrary.com) DOI: 10.1002/rcm.6964

Since the pioneering study of Abelson and Hoering,[1]

the potential of intramolecular carbon isotopic ratiomeasurement of organic compounds at natural isotopicabundance levels to provide a better understanding of carboncycles at various scales from cell metabolism to globalenvironments has been demonstrated.[2–7] However, thenumber of research projects using intramolecular carbonisotopic distributions has been limited due to the difficultiesinherent to the measurement. Conventionally, intramolecularcarbon isotopic distributions of organic compounds havebeen measured using off-line chemical and/or thermaldecomposition of the analyte into simpler molecules(hereinafter referred to as ’fragment molecules’) followed

* Correspondence to: K. Yamada, Interdisciplinary GraduateSchool of Science and Engineering, Tokyo Institute ofTechnology, 4259 Nagatsuta-cho, Midori-ku, Yokohama226-8503, Japan.E-mail: yamada.k.ag@m.titech.ac.jp

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by dual-inlet isotope ratio mass spectrometry (DI-IRMS)analysis of the CO2 produced from the fragmentmolecules.[5] This measurement requires milligram levelsof a pure target compound and cumbersome preparationprocedures. This shortcoming has prevented the developmentof intramolecular carbon isotopic ratio measurements,especially for environmental samples.

This situation is changing due to advances in analyticaltechnique. In 1997, Corso and Brenna introduced an on-linepyrolysis–gas chromatography–combustion–IRMS (GC-C-IRMS)system that allowed the measurement of intramolecular carbonisotopic distributions in organic compounds with nanogramlevels of target compound in mixtures.[8] This approach has beenapplied for relatively simple compounds such as acetic acid[9–13]

and ethanol.[14] As an alternative, quantitative isotopic 13CNMR spectroscopy has been developed for the determinationof intramolecular carbon isotopic distributions not only forsimple compounds like ethanol,[15,16] but also for relativelycomplex compounds such as vanillin[17] and glucose.[6]

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With these advances in analytical techniques, it is nownecessary to ensure the consistency of the data obtained bydifferent methods (e.g. IRMS and 13C NMR spectroscopy) orconfigurations (e.g. on-line and off-line pyrolysis IRMS) usedfor intramolecular carbon isotopic ratio measurements indifferent laboratories. The availability of isotopic referencematerials (RMs) is essential for that purpose. At present,however, no isotopic RMs exist for use with intramolecularcarbon isotopic ratio measurements.Here, we focus on the intramolecular carbon isotopic

distribution of acetic acid. Because of the ubiquity ofacetic acid, its intramolecular carbon isotopic distributionis important information for the study of carbon cyclesnot only in metabolic pathways, but also in naturalenvironments. Thus, in this study we evaluated thefeasibility of preparing RMs for intramolecular carbonisotopic ratio measurement of acetic acid usingcommercially available acetic acid and sodium acetatereagents.First, the range of isotopic ratio values of these reagents

was evaluated at molecular and intramolecular levels. Thiswas because, for precise and accurate measurement byIRMS, it is important to use RMs with isotopic ratios valuesthat span the isotopic range of the acetic acid beinganalyzed. Secondly, the magnitude of the isotopefractionation associated with the evaporation of acetic acidwas evaluated. This was because any loss of the acetic acid

Table 1. Molecular and intramolecular δ13C values for acetic acimaterials

IDPurity(%)

δ13Cbulk (‰)average ± SD

δ13Cmethylaverage ±

Acetic acidAAA 99.7 �32.26 ± 0.12 (n = 5)a �37.47 ±AAB 99.7 �22.32 ± 0.02 (n = 4)a �30.96 ±AAC 99.99 �20.62 ± 0.02 (n = 4)a �30.52 ±AAD 99.7 �35.86 ± 0.02 (n = 3)a �45.01 ±AAE 99 �21.16 ± 0.09 (n = 6)a �32.99 ±

�32.87 ±AAF 99.7 �35.94 ± 0.01 (n = 3)a �45.04 ±

�44.92 ±AAG 99.7 �35.78 ± 0.01 (n = 3)a �44.96 ±AAH 99.7 �35.79 ± 0.00 (n = 3)a �44.61 ±AAI 99.7 �35.79 ± 0.00 (n = 3)a �44.99 ±

Sodium acetateSAA 98.5 �31.88 ± 0.27 (n = 5)a �38.18 ±

�31.89 ± 0.32 (n = 7)b �37.89 ±SAB 98.5 �34.59 ± 0.08 (n = 5)a �42.13 ±

�34.90 ± 0.34 (n = 3)b �41.89 ±SAC 99.99 �11.00 ± 0.13 (n = 4)a �11.19 ±

�11.02 ± 0.10 (n = 3)b �11.54 ±SAD 98.5 �31.44 ± 0.14 (n = 4)a �36.96 ±

�31.39 ± 0.02 (n = 3)b �36.76 ±aDetermined using sealed tube combustion method.bDetermined using EA-IRMS.cDetermined using off-line sealed tube pyrolysis followed by GdDetermined using on-line pyrolysis-GC-C-IRMS.eDefined as δ13Cmethyl value determined by on-line pyrolysis-GCpyrolysis.

wileyonlinelibrary.com/journal/rcm Copyright © 2014 John Wil

from a bottle by evaporation during storage could lead toalteration of its isotopic ratio value at molecular andintramolecular levels.

EXPERIMENTAL

Chemicals

Nine acetic acid and four sodium acetate reagents werepurchased and designated as AAA (Wako Pure ChemicalIndustries, Osaka, Japan, Lot No.TPG7942), AAB (Aldrich,Milwaukee, WI, USA, Lot No.00612PR), AAC (Aldrich, LotNo.14002CU), AAD (Wako Pure Chemical Industries, Lot No.ALH4835), AAE (Sigma-Aldrich, St. Louis, MO, USA, LotNo.10610MH), AAF (Nacalai Tesque, Kyoto, Japan, Lot No.V8M7761), AAG (Kishida Chemical, Osaka, Japan, Lot No.J98199X), AAH (Junsei Chemical, Tokyo, Japan, Lot No.8I5027),AAI (Kanto Chemical, Tokyo, Japan, Lot No.007 W1087), SAA(Wako Pure Chemical, LotNo.WAP0333), SAB (KantoChemical,Lot No.411C2231), SAC (Aldrich, Lot No.04017MO), and SAD(Nacalai Tesque, Lot No.M8E8025), as presented in Table 1.IAEA-CH-6 (sucrose), IAEA-CH-7 (polyethylene) and USGS40(L-glutamic acid) were purchased as isotopic referencematerials. Sodium hydroxide (NaOH, 97%) and hydrochloricacid (HCl, 35–37%) were purchased fromWako Pure ChemicalIndustries. NaOH was heated under vacuum condition toremove water and any organic matter that it contained.

d and sodium acetate reagents prepared as isotopic reference

(‰)SD

δ13Ccarboxyl (‰)average ± SD

Δδ13Cmethyl(‰)e

0.32 (n = 4)c �27.05 ± 0.340.17 (n = 3)c �13.68 ± 0.170.27 (n = 3)c �10.72 ± 0.270.28 �26.71 ± 0.28 (n = 5)d

0.13 (n = 3)c �9.33 ± 0.16 0.120.40 �9.45 ± 0.38 (n = 10)d

0.28 (n = 3)c �26.84 ± 0.28 0.120.23 �26.96 ± 0.23 (n = 5)d

0.31 �26.60 ± 0.31 (n = 5)d

0.23 �26.96 ± 0.23 (n = 10)d

0.21 �26.59 ± 0.21 (n = 8)d

0.32 (n = 3)c �25.61 ± 0.45 0.290.40 �25.90 ± 0.23 (n = 3)d

0.25 (n = 4)c �27.66 ± 0.42 0.240.41 �27.90 ± 0.22 (n = 8)d

0.32 (n = 8)c �10.84 ± 0.34 �0.350.35 �10.49 ± 0.34 (n = 12)d

0.15 (n = 3)c �25.82 ± 0.15 0.200.39 �26.02 ± 0.39 (n = 4)d

C-C-IRMS.

-C-IRMS minus δ13Cmethyl value determined by off-line

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Notation

All reported carbon isotope ratios are expressed as δ13Cvalues relative to the international Vienna Pee Dee Belemnitestandard (VPDB). The δ13C value is:

δ13Csample ¼ Rsample=RVPDB� � � 1� � � 1000‰ (1)

where Rsample and RVPDB, respectively, denote the 13C/12Cratios in the sample and the VPDB standard.

Determination of molecular level δ13C values for acetic acidand sodium acetate

The molecular level δ13C values (hereinafter expressed as’δ13CBulk values’) for acetic acid and sodium acetate weredetermined using three methods; (i) sealed tube combustionfollowed by DI-IRMS, (ii) elemental analyzer (EA)-IRMS, and(iii) GC-C-IRMS combined with head space solid-phasemicroextraction (HS-SPME-GC-C-IRMS).

Sealed tube combustion followed by DI-IRMS

The δ13CBulk values for the acetic acid reagents weredetermined using the conventional off-line method consistingof sealed tube combustion followed by cryogenic separationof the resulting CO2 using a high vacuum line system andmeasurement of the purified CO2 by DI-IRMS. Briefly, about6 μL of acetic acid was combusted in an evacuated and sealedquartz tube with 2 g CuO as the oxidant at 850°C for 2 h.Measurements of δ13C values for the resulting CO2 gaseswere made using an isotope ratio mass spectrometer(Finnigan MAT 252; Thermo Fisher Scientific Inc., Bremen,Germany) with a dual-inlet system. The δ13CBulk values forthe sodium acetate reagents were also measured by thismethod. In that case, about 8 mg of sodium acetate wereused. Isotopic standardization was accomplished bycomparison with CO2 produced by the sealed tubecombustion of IAEA-CH-6 (�10.45‰) and IAEA-CH-7(�32.15‰) reference standards.[18]

EA-IRMS

The δ13CBulk values for the sodium acetate reagents werealso determined by EA-IRMS. The instrumentationconsisted of an elemental analyzer interface and an isotoperatio mass spectrometer (Finnigan MAT 252). Theelemental analyzer interface was a combination of anelemental analyzer (EA 1110, CE Instruments, Milan, Italy)and a flow control unit (ConFlo II™, Thermo FisherScientific Inc.). Isotopic standardization was accomplishedby two-point linear calibration of IAEA-CH-6 and USGS40(�26.39‰).[18]

HS-SPME-GC-C-IRMS

The HS-SPME-GC-C-IRMS procedure for the determinationof δ13CBulk values is described elsewhere.[19] Briefly, analiquot of acetic acid or sodium acetate was diluted withsaturated saline water to about 0.2 mmol/L and adjustedusing HCl to a pH of 2, checked using a pH meter (TPX-90i,Tokyo Chemical Laboratories Co., Ltd, Tokyo, Japan). The10 mL solutions were enclosed in 20 mL SPME vials (GerstelGmbH and Co. KG, Mülheim, Germany). Acetic acid in the

Copyright © 2014Rapid Commun. Mass Spectrom. 2014, 28, 1821–1828

head space of the SPME vial was extracted on a SPME fiber(85 μm carboxen/polydimethylsiloxane (Carboxen/PDMS)StableFlex™; Supelco, Bellefonte, PA, USA) and injectedinto the GC-C-IRMS system. The HS-SPME conditions were30°C extraction temperature and 60 min extraction time.The GC-C-IRMS system consisted of a GC-combustioninterface (GC Combustion III; Thermo Fisher Scientific Inc.)and an isotope ratio mass spectrometer (DeltaplusXL; ThermoFisher Scientific Inc.). The GC-combustion interface consistedof a gas chromatograph (HP6890; Agilent Technologies Inc.,Palo Alto, CA, USA) equipped with a capillary column(Nukol, 30 × 0.32 mm i.d., 1.0 μm film thickness; Supelco).The GC conditions were as follows: splitless injection modefor 5 min; injector temperature, 220°C; oven temperatureprogram, 35°C for 5 min, raised from 35°C to 120°C at15°C min�1, maintained at 120°C for 10 min, then raisedto 190°C at 50°C min�1, and maintained at 190°C for8 min; the He flow rate was 1.5 mL min�1. The combustionfurnace was a microvolume oxidation reactor consisting of aceramic tube (Al2O3, 320 mm×0.5 mm i.d.) packed withCuO, NiO, and Pt wires, operated at 960°C. Isotopicstandardization was accomplished by comparison with thereference solution prepared using AAA, whose δ13CBulk value(�32.26± 0.12‰) was determined using sealed tubecombustion followed by DI-IRMS as described above.

Determination of intramolecular level δ13C values for aceticacid and sodium acetate

The δ13C values for the methyl and carboxyl groups(hereinafter expressed as ’δ13CMethyl value’ and ’δ13CCarboxyl

value’, respectively) of acetic acid and sodium acetate weredetermined using two methods; (i) off-line pyrolysis followedbyGC-C-IRMS and (ii) on-line pyrolysis-GC-C-IRMS combinedwith HS-SPME (subsequently denoted ’HS-SPME-GC-Py-GC-C-IRMS’).

Off-line pyrolysis followed by GC-C-IRMS

About 20 μmol of the acetic acid or sodium acetate reagentswere placed in a Pyrex® glass tube (7 mm i.d, 30 cm length)with NaOH, resulting in NaOH/ acetic acid or sodiumacetate ratios of about 50 (ranging from 37 to 62) mol/mol.The tube was evacuated, flame-sealed and then preheated at150°C for 1 h. After the preheating, the sealed sample washeated at 500°C for 3 h to produce CH4 from the methyl groupof the acetic acid or sodium acetate reagents. The CH4 wasinjected using a gas-tight syringe into a measurement systemused to determine the δ13C values of CH4

[20] in order todetermine the δ13CMethyl value. The measurement systemconsisted of an on-line CH4 extraction system, a GC-combustion interface (GC Combustion III), and a FinniganMAT 252 isotope ratio mass spectrometer. The on-line CH4

extraction system consisted of a syringe injection port, a columnpacked with magnesium perchlorate (20/48 mesh, Wako PureChemical Industries) and Ascarite II (8/20 mesh, ThomasScientific, Swedesboro, NJ, USA), a CH4 concentrationtrap which contained 1 g of Porapak Type Q (50/80 mesh,Waters Co., Milford, MA, USA) and a cryo-focus trap.The GC-combustion interface consisted of a HP6890 gaschromatograph equipped with a split/splitless injector and acapillary column (HP-PLOT Q, 30 m×0.32 mm i.d., 20 mm

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film; Agilent Technologies Inc.), a combustion furnace, aNafiondryer, and an open split. The CH4 injected was collected on theCH4 concentration trap at �196°C for 5 min through thecolumn packed with magnesium perchlorate and AscariteII to remove H2O and CO2. The trap was then warmedto room temperature (25°C) to liberate the CH4. The CH4

was collected on the cryo-focus trap at �196°C for 5 minand then injected into the gas chromatograph by warmingthe cryo-focus trap to room temperature (25°C). Isotopicstandardization was accomplished by comparison with alaboratory CH4 standard gas (1000 ppm CH4 in N2, TaiyoNippon Sanso Corp.), where the δ13C value (�39.8 ± 0.2‰)was calibrated against VPDB. The δ13CCarboxyl value wascalculated by use of the mass balance equation from theδ13CMethyl and the δ13CBulk values.

HS-SPME-GC-Py-GC-C-IRMS

The HS-SPME-GC-Py-GC-C-IRMS procedure for the deter-mination of the δ13CMethyl and δ13CCarboxyl values is describedelsewhere.[10] Briefly, aliquots of acetic acid and sodiumacetate were diluted with saturated saline water to about2 mmol/L and adjusted using HCl to pH 2. The 10 mLsolutions were enclosed in 20 mL SPME vials. Acetic acid inthe head space of the SPME vial was extracted on the 85-μmCarboxen/PDMS StableFlex™ SPME fiber (Supelco) andinjected into the on-line pyrolysis-GC-C-IRMS system. TheHS-SPME conditions were 30°C extraction temperatureand 60 min extraction time. The system for the onlinepyrolysis-GC-C-IRMS consisted of two gas chromatographs,two high-temperature furnaces, and an isotope ratio massspectrometer (DeltaplusXL). The first gas chromatograph(HP6890) was equipped with a capillary column (Nukol,30 m× 0.32 mm i.d., 1.0 μm film thickness; Supelco) toseparate acetic acid. The pyrolysis furnace (Al2O3,320 mm×0.5 mm i.d.) was operated at 1000°C to pyrolyzethe acetic acid. The second gas chromatograph (HP6890)was equipped with a capillary column (PoraPLOT Q,30 m× 0.32 mm i.d., 10 μm film thickness; Varian Inc.,Lake Forest, CA, USA) to separate fragment moleculesproduced by pyrolysis of the acetic acid. It was operated at35°C. A deactivated fused-silica capillary (0.32 mm i.d.)was used as the transfer line from the second GC columnto the combustion furnace operating at 960°C. The firstconditions for the first gas chromatograph were: injectionmode, splitless for 5 min; injector temperature, 220°C; oventemperature program, 60°C for 5 min, raised from 60°C to190°C at 15°C min�1, maintained at 190°C for 20 min, thenraised to 200°C at 10°C min�1, and maintained at 200°C for5 min; column flow rate, 0.8 mL min�1. The δ13C value ofCO2 derived from the on-line pyrolysis of acetic acid wasderived only from the carboxyl group.[9] Therefore, thissystem was able to determine the δ13CCarboxyl value, whilethe δ13CMethyl value was calculated from the δ13CBulk andδ13CCarboxyl values using isotopic mass balance. The methodwas applied to AAD, AAE, AAF, AAG, AAH, AAI, SAA,SAB, SAC and SAD. Isotopic standardization wasaccomplished by comparison with the reference solutionprepared using AAA, for which the δ13CCarboxyl value(�27.05 ± 0.34‰) was determined by off-line pyrolysisfollowed by GC-C-IRMS as described above.

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Laboratory evaporation experiment for the acetic acid reagent

To quantify the isotope effect associated with evaporation ofacetic acid, a laboratory evaporation experiment wasperformed in a static mode. For this experiment, a set of14 glass bottles (volume 2 mL each) was prepared. Each bottlewas weighed, filled with AAF and weighed again to obtainthe initial mass of acetic acid. The set of bottles was placedin a hermetic chamber with NaOHaq to remove the acetic acidevaporated into the atmosphere. The chamber was evacuatedfor a few minutes using a rotary pump until a pressure of0.06 MPa was reached, and then held at room temperature(22°C). The bottles were removed individually from thechamber after specific time intervals, after which the removedbottle was weighed and the proportion of residual acetic acid(%) determined. Using each residual acetic acid, 10 mL ofacetic acid solution was prepared in a 20-mL SPME vial asdescribed above. The δ13CBulk value was determined usingHS-SPME-GC-C-IRMS. The δ13CMethyl and δ13CCarboxyl valueswere determined using HS-SPME-GC-Py-GC-C-IRMS.

RESULTS AND DISCUSSION

δ13CBulk values for acetic acid and sodium acetate reagents

As Gelwicks and Hayes pointed out,[21] the determination ofδ13CBulk values for sodium acetate by sealed tube combustionfollowed by DI-IRMS can be inaccurate because thecombustion of sodium acetate yields not only CO2 and water,but also sodium oxide that captures CO2 as the combustiontube is cooled, leading to low CO2 yields and thus alteredδ13CBulk values. In this study, we determined the δ13CBulk

values for four sodium acetate reagents using both sealedtube combustion and EA-IRMS methods (Table 1). Althoughthe values obtained by the two methods are identical withinthe precision of the experiments, we adopted the valuesdetermined by EA-IRMS as the δ13CBulk values for sodiumacetate in this study. Acetic acid and sodium acetate reagentscould be prepared as isotopic RMs with δ13CBulk valuesranging from �11.02‰ to �35.94‰ with a precision of0.2‰ (Table 1).

δ13CMethyl and δ13CCarboxyl values for acetic acid and sodiumacetate reagents determined using off-line pyrolysis followedby GC-C-IRMS

The δ13CMethyl values of acetic acid and sodium acetate haveconventionally been determined by the measurement of theδ13C value of the CH4 produced from the methyl group ofacetic acid and sodium acetate by off-line pyrolysis withNaOH. For the off-line pyrolysis procedure, differentconditions (e.g., sample amounts, heating temperature, andtime) have been used by different researchers.[4,22,23] Theδ13C values of the CH4 formed have been measured in twoways: (i) CO2 converted off-line from CH4 is analyzed usingDI-IRMS and (ii) CH4 is analyzed directly using GC-C-IRMS,and thus does not require off-line conversion into CO2. Theformer can cause isotopic fractionation if the off-lineconversion is not quantitative. Therefore, in this study, off-line pyrolysis followed by GC-C-IRMS was selected toprepare the RMs for the intramolecular isotopic ratiomeasurement of acetic acid and sodium acetate.

ey & Sons, Ltd. Rapid Commun. Mass Spectrom. 2014, 28, 1821–1828

Reference material for intramolecular carbon isotopic measurements

Because the values determined by the off-line pyrolysisapproach will be subsequently regarded as accuratevalues, it is necessary to evaluate (i) the accuracy (truenessand precision) of the results obtained, and (ii) therobustness of the approach towards different pyrolysisconditions. The trueness of the results obtained can beevaluated via the yields of CH4 produced by pyrolysis: aquantitative yield of CH4 indicates no alteration in theisotopic signature from the starting material (methyl partof acetic acid).

Figure 1. Influence of pyrolysis conditionformed by off-line pyrolysis of acetic aci(sample SAC), as a function of pyrolysisstandard deviation (1SD).

Copyright © 2014Rapid Commun. Mass Spectrom. 2014, 28, 1821–1828

We therefore investigated different pyrolysis conditions(pyrolysis temperature, pyrolysis time and ratio of NaOH/acetic acid or NaOH/sodium acetate) with two differentstarting materials (acetic acid and sodium acetate) todetermine proper conditions for our setup. For each set ofconditions, the yield of CH4 and by-product formed, andthe δ13C value of CH4, were determined (Figs. 1 and 2).Overall, off-line pyrolysis with pyrolysis temperature higherthan 500°C, more than 3 h of pyrolysis time, and withNaOH/acetic acid or NaOH/sodium acetate ratios higher

s on the yield and δ13C value of CH4d (sample AAC) and sodium acetatetime (h). The error bar indicates ±1

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Figure 2. Influence of NaOH on the yield and δ13C value ofCH4 formed by off-line pyrolysis of acetic acid (sampleAAC) and sodium acetate (sample SAC), as a function ofNaOH/acetic acid or sodium acetate ratios (mol/mol).Pyrolysis temperature was 500°. The error bar indicates ±1standard deviation (1SD).

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than 50 mol/mol resulted in a quantitative yield of CH4, noformation of by-products and a precision (1SD) below 0.3‰for replicate measurement of the CH4 formed.The δ13CMethyl values of five acetic acid and four sodium

acetate reagents were measured using the pyrolysisconditions described above (Table 1). The δ13CCarboxyl valuewas calculated using a simple mass balance equation usingthe δ13CMethyl and δ13CBulk values. Compounds withintramolecular isotopic distributions ranging from �11 to�45‰ for the δ13CMethyl value and �9 to �28‰ for theδ13CCarboxyl value could be prepared as RMs with a precisionof better than 0.45‰. The isotopic values of these RMsspanned the isotopic range of acetic acid found in biologicaland environmental samples.[4,10,11,22] These RMs will beused in the future for inter-laboratory calibration and/orinter-calibration with other measurement techniques.We then examined the consistency between data obtained

from the off-line pyrolysis and on-line pyrolysis IRMS usingthese RMs.

Determination of δ13CMethyl and δ13CCarboxyl values of aceticacid and sodium acetate by HS-SPME-GC-Py-GC-C-IRMS

Off-line pyrolysis requires cumbersome preparation and it isnot adapted to low sample amounts. To reduce thecomplicated preparation and work with lower sampleamounts, new methods based on on-line pyrolysis coupledwith GC-C-IRMS have been developed. Dias et al.[11] firstreported the use of IRMS combined with an on-line pyrolysisfurnace (consisting of an Al2O3 tube filled with palladiumwires and operating at 600°C with a small flow of H2) forthe determination of the intramolecular isotopic distribution

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of acetic acid in complex aqueous solutions. They confirmedthe accuracy of the technique using isotope-dilutionexperiments and subsequently demonstrated the capabilityof this technique for analyses of natural samples, particularlyacetic acid generated via hydrous pyrolysis of an oil-pronesource rock.[13] Furthermore, Thomas et al.[12] improved themethod using direct injection of aqueous samples into thesystem. They demonstrated that this method enableddetermination of the intramolecular isotopic distribution ofacetic acid in natural samples at concentrations as low as1 mmol/L (90 μmol/L when using a pre-concentrationsystem) with a precision of 0.7‰.

In 2002, our laboratory also reported an on-line pyrolysis-GC-C-IRMS technique for the determination of theintramolecular isotopic distribution of acetic acid.[9] Thebasic concept of this technique is the same as that of Diaset al.[11] except that the pyrolysis furnace consisted of ahollow Al2O3 tube operating at 1000°C, as designed byCorso and Brenna.[8] Recently, the method combined withHS-SPME injection (HS-SPME-GC-Py-GC-C-IRMS) wasapplied to acetic acid in vinegar and we demonstrated itseffectiveness for the intramolecular isotopic measurementof acetic acid in complex matrices such as food materials,with a precision of 0.4‰ at a concentration of about1 mmol/L.[10]

As mentioned above, linearity tests have been conductedby Dias et al.[11] and Thomas et al.,[12] although using13C-enriched acetic acid and dilution experiments for theon-line pyrolysis coupled with GC-C-IRMS. Here, weevaluated the linearity using RMs whose δ13C values atnatural abundance were determined using the off-linemethod. The advantages of using natural abundancesamples such as RMs are (i) the ease of handling, (ii) loweruncertainties on their values because no isotopic dilution isnecessary, and (iii) the ease of distribution and sharing ofthese RMs with different laboratories. The comparison ofthe measurement results for acetic acid and sodium acetatereagents obtained using off-line pyrolysis followed byGC-C-IRMS with those obtained by HS-SPME-GC-Py-GC-C-IRMS showed good correlation, fitting perfectly a 1:1 trend(Table 1). Furthermore, the mean difference in the δ13CMethyl

values for six samples was �0.10 ± 0.23‰ (1SD). Similarresults were obtained for the δ13CCarboxyl values because ofthe symmetrical calculation. This confirms the isotopiclinearity of measurements by HS-SPME-GC-Py-GC-C-IRMS,ranging from �11 to �45‰ for the δ13CMethyl value and from�9 to�28‰ for the δ13CCarboxyl value. It also emphasizes theusability of RMs of known isotope composition at theintramolecular level for evaluating the accuracy of on-linepyrolysis methods.

Isotope effect associated with the evaporation of acetic acid

A prerequisite in designing RMs for intramolecular isotopicmeasurements is that attention must be paid to potentialisotope fractionation associated with leakage from the bottleduring storage. To the best of our knowledge, however, thereare currently no data about the intramolecular isotope effectsassociated with the evaporation of acetic acid. We thereforeconducted a laboratory evaporation experiment for the aceticacid reagents.

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Reference material for intramolecular carbon isotopic measurements

The results of the laboratory evaporation experiment areshown in Fig. 3, in which the δ13CBulk, δ13CMethyl andδ13CCarboxyl values for the remaining acetic acid are plottedas a function of the fraction, f, of the remaining acetic acidin each experimental bottle. Evaporation of acetic acid leadsto 13C enrichment of the remaining acetic acid at themolecular level, as shown in the trace of the δ13CBulk value.It is particularly interesting that this enrichment is apparentlymostly caused by 13C enrichment in the methyl group and notin the carboxyl group of the remaining acetic acid.The 13C enrichment of the remaining acetic acid at the

molecular and intramolecular levels can be modeled by theRayleigh equation. Thus, the apparent isotope effectassociated with evaporation of acetic acid was estimatedbased on an equation which is derived from rearrangementof Rayleigh equation as shown below:

ln 10–3� δ13CRemaining þ 1� �

¼ ln 10–3�δ13CInitial þ 1� �þ 10–3�εlnf

(2)

where δ13CRemaining and δ13CInitial stand, respectively, for themeasured and initial δ13C values for the bulk, methyl andcarboxyl carbons in the acetic acid, f is the remaining fractionof acetic acid, and ε denotes the enrichment factor of the

Figure 3. Molecular and intramolecular δ13C values forremaining acetic acid from evaporation experiments plottedas a function of the fraction, f, of the remaining acetic acid.The error bar indicates ±1 standard deviation (1SD).

Copyright © 2014Rapid Commun. Mass Spectrom. 2014, 28, 1821–1828

increment of the product (acetic acid vapor) to the substrate(remaining acetic acid) simultaneously. The fractionationfactor α is defined as:

α ≡ RProduct;i=RRemaining (3)

where RProduct,i and RRemaining are the isotope ratios of theincrement of product (acetic acid vapor) to the substrate(remaining acetic acid) at the same time. The relationshipbetween α and ε is defined as:

α≡1þ 10–3� ε (4)

From Eqn. (2), regression of ln(10�3 × δ13CRemaining + 1) on10�3lnf yields a straight line with slope ε.

The apparent isotope effects (ε) with evaporation of aceticacid for the bulk, methyl and carboxyl carbons werecalculated, respectively, as �0.97, �1.97 and 0.01‰ withregression coefficients of 0.98, 0.99 and 0.00. The resultsindicate that a mass loss of acetic acid from the storage bottleof the order of 20% will lead to an increase by 0.2‰ and 0.4‰of the δ13CBulk and δ13CMethyl values, respectively, which canbe considered as a limit, given the precision of the method.This implies the need for robust storage and quality controlif an acetic acid reagent is used as an RM.

Although finding a clear explanation for this trend isbeyond the scope of this paper, it is noteworthy that theisotope fractionation associated with the evaporation of aceticacid is located solely on the methyl position. The resultspresented here differ significantly from those observed byBotosa et al.[24] for ethanol. They reported that distillation ofethanol leads to 13C enrichment of the vapor fraction at boththe molecular and intramolecular level, the δ13C value of themethyl position of ethanol being less sensitive to distillationthan that of the methylene position.

According to previous evaporation experiments of volatileorganic compounds, such as benzene and toluene, themagnitude and trend of isotope fractionation at molecularlevel seem to be dependent on the type of evaporation:non-equilibrium and equilibrium evaporation.[25,26] Therefore,different trends of isotope fractionation could be observed foracetic acid at the molecular and intramolecular levels fordifferent types of evaporation.

182

CONCLUSIONS

In this study we evaluated the feasibility of preparing isotopicRMs for the intramolecular carbon isotopic ratiomeasurement of acetic acid using commercially availableacetic acid and sodium acetate reagents. Based on off-linepyrolysis followed by GC-C-IRMS, for which the optimumpyrolysis conditions were determined for our setup,intramolecular carbon isotopic distributions for nine aceticacid and four sodium acetate reagents were determined witha precision of better than 0.45‰. The isotopic values of thesereagents ranged from �11 to �45‰ for δ13CMethyl values andfrom �11 to �28‰ for δ13CCarboxyl values and spanned theisotopic range of acetic acid in biological and environmentalsamples. These reagents will be used as RMs in the futurefor inter-laboratory calibration and/or inter-calibration withanother intramolecular isotopic measurement technique,

wileyonlinelibrary.com/journal/rcmJohn Wiley & Sons, Ltd.

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namely, quantitative 13C NMR. As an illustration, using theRMs prepared here, consistency between data obtainedfrom off-line pyrolysis followed by GC-C-IRMS and fromHS-SPME-GC-Py-GC-C-IRMS was confirmed within thenatural 13C abundance range for both the methyl and thecarboxyl positions of acetic acid. Where acetic acid is beingused as an RM, its storage must be carefully controlled toprevent evaporation, which induces alteration of its isotopicsignature at both the molecular and the intramolecular level.

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