Sample matrix effects on measured carbon and oxygen isotope ratios during continuous-flow isotope-ratio mass spectrometry

Research Article

Received: 13 March 2014 Revised: 8 August 2014 Accepted: 12 August 2014 Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2014, 28, 2259–2274

Sample matrix effects on measured carbon and oxygen isotoperatios during continuous-flow isotope-ratio mass spectrometry

Nicholas Paul Levitt*,†

NASA Astrobiology Institute, Department of Earth and Environmental Sciences, University of Kentucky, 121 WashingtonAvenue, Lexington, KY 40506, USA

RATIONALE: Continuous-flow isotope-ratio mass spectrometry (CF-IRMS) is frequently used to analyze CO2 found inmedia such as air, breath, and soil pore space gas with the aid of a sample preparation and transfer device such as aGasbench II. This study investigated the effect that matrices other than helium (He) have on the measured δ13C andδ18O isotope ratios of CO2.METHODS: Identical CO2 was added to sample vials with matrices of pure He, pure N2, or a 21:79 mixture of O2/N2 andanalyzed by a ThermoFinnigan DeltaPlus XP isotope-ratio mass spectrometer coupled to a ThermoFinnigan Gasbench II.Variables such as CO2 concentration, sample analysis sequence, and sample matrix removal (’blanking’) throughmanipulation of an injection and dilution open split were tested to identify systematic isotope ratio offsets betweenthe different matrix types.RESULTS: The process of blanking induced a δ13C and δ18O offset of ≤0.2‰ between otherwise identical populations ofCO2 samples in He. The 13C/12C and 18O/16O isotope ratios of CO2 sampled from pure N2 or a mixture of O2/N2 werefound to be within 0.1 to 0.2‰ of those of an identical CO2 sampled from a He matrix when N2 or O2/N2 was removedprior to transport to the mass spectrometer. The measured oxygen isotope ratios of CO2 sampled from N2 and O2/N2

varied by as much as 0.6‰ and 4‰, respectively, if matrix gas was not removed prior to ionization.CONCLUSIONS: Sampling CO2 from matrices similar to air does not significantly affect the measured 13C/12C and18O/16O isotope ratios of CO2 when a gas-handling procedure that includes the removal of matrix gas is utilized. Thisprocedure is much preferable to introducing matrix gas and potentially isobaric interference to the ion source. Copyright© 2014 John Wiley & Sons, Ltd.

(wileyonlinelibrary.com) DOI: 10.1002/rcm.7019

Continuous-flow isotope-ratio mass spectrometry (CF-IRMS)is used in a wide variety of fields. Researchers in the areas ofatmospheric science, biology, ecology, forensics, geoscience,and medicine all find use for this versatile technique.[1–6] This’on-line’ method was developed in the late 1980s and quicklygained popularity due to its automation capability and thehigh-precision analysis of large number of samples in a shorttime span compared with traditional dual-inlet IRMS.Although CF-IRMS is a ’gas source’ technique it is widelyused for the analysis of solid and liquid as well as gassamples.[7,8] Most continuous-flow analyses are facilitatedby a sample preparation and transfer device such as aGasbench II coupled to an autosampler. Conventionalpractice uses helium (He) as a carrier gas to transport analyteCO2 through the preparation device and ultimately to themass spectrometer. Analysis of carbonate minerals and

* Correspondence to: N. P. Levitt, NASA AstrobiologyInstitute, Department of Geoscience, University ofWisconsin-Madison, 1215 W Dayton Street, Madison, WI53706, USA.E-mail: [email protected]

† Current address: NASA Astrobiology Institute, Departmentof Geoscience, University of Wisconsin-Madison, 1215 WDayton Street, Madison, WI 53706, USA.

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dissolved inorganic carbon (DIC) calls for the sample vialheadspace to contain pure He before analyte CO2 is evolvedinto the gas matrix. Oxygen isotope ratio analysis of watersamples by CF-IRMS is achieved by equilibrating a lowconcentration CO2/He mixture prior to headspace sampling,which is followed by calculation back to δ18O water values.

Automated sample vial headspace sampling is achievedthrough the use of a double-holed needle connected to twocapillary tubes. One capillary tube delivers carrier gas to thesample vial while the second transports displaced analyteand matrix gas from the vial headspace to the samplepreparation device. Injecting He carrier gas into sample vialsdecreases the concentration of analyte CO2 but does notchange the sample matrix for applications that utilize He asa matrix gas. However, CF-IRMS is also applied to samplesfor which analyte CO2 is a minor constituent in other media.Most commonly, the alternative analyte matrix is a mediumsimilar in composition to air. Studies requiring analyses ofthis nature include samples of human and animal breath, soilpore space sampling, and atmospheric sampling for Keelingplots as well as ecological investigations.[9–18]

Early developers of CF-IRMS documented the need toseparate analyte CO2 from gases such as H2O, N2, O2, andN2O prior to introduction into the mass spectrometersource.[10,19,20] Schoeller and Luke[20] performed a series ofexperiments in which they added N2 or O2 to CO2 before

Copyright © 2014 John Wiley & Sons, Ltd.

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Figure 1. Schematic diagram of the Gasbench II open split(adapted from Figure 5-16 of the Finnigan Gasbench IIoperating manual[8]). In the left image the mass spectrometertransfer capillary is positioned to allow undiluted transfer ofsample CO2 and carrier/matrix gas. In the right image themass spectrometer transfer capillary is positioned to transferonly He and to “blank” gas being transported from thesample preparation portion of the Gasbench II.

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IRMS analysis. The researchers reported that "accuracy wasseverely degraded" for δ18O measurements during theseanalyses and they hypothesized that isobaric interference wasdue to the formation of nitrogen oxides (such as N2O or NO2

[NOx]) during ionization. These nitrogen oxides haveisotopologues including masses 44, 45, and 46. Of particularconcern for δ18O measurements are isotopologues of mass 46.Separation of CO2 from thematrix and other low-concentrationgases is commonly achieved by way of gas chromatographyand is one feature offered by preparation devices such as aGasbench II. After separation in a chromatography column,unwanted gases can be vented or redirected beforeintroduction into a mass spectrometer source. Often disposalof unwanted gases is through an open split in the gas transferline shortly before connection to the mass spectrometer. TheGasbench II device has the capability of diluting gasconcentrations entering the mass spectrometer source at thesample open split by a factor of greater than 100 in a processtermed ’blanking’.[8]

In the case of water, prevention is not always as easy asremoval through the open split. Memory effects and blankproblems are common with IRMS analyses involvingwater.[21,22] The source of these errors is from oxygenexchange between H2O molecules and analyte molecules aswell as from ion induced chemical reactions occurring in themass spectrometer source. Oxygen atom exchange mostaffects δ18O values while formation of HCO2

+ ions createsmainly the m/z 45 species that skew δ13C values. Water vaporcontamination sourced from samples is a major concern forthe type of biological and environmental work describedabove; however, it is not the only possible source of watercontamination during IRMS analyses. As a highly polarmolecule, water tends to readily adsorb to surfaces, and thisholds true for interior surfaces associated with massspectrometry.[23,24] Unless the water is actively removed bybaking or flushing, capillaries, valves, columns, and eventhe interior mass spectrometer walls can be heavily coatedwith water. Another potential source of water is impurecarrier gas used to transport fluid in gas handling devicessuch as the Gasbench II. Methods used to control waterinfluences include drying during sample collection,[10] waterremoval during sample handling,[8] and CO2 isolation andconcentration.[25,26]

One critical aspect of CF-IRMS analyses is the need tomaintain stable ion source conditions at all times.[27] Theaforementioned open split is integral in providing a continuousgas stream to the mass spectrometer inlet and ensuringconstant pressure in the source chamber. As illustrated in Fig. 1,the open split is open to the atmosphere and therefore held at aconstant pressure. Gas transfer to the source chamber at apressure of approximately 10�6 mBar from the open split isaccomplished through an unimpeded capillary tube. Thepressure gradient between the open split and the massspectrometer is such that flow is natural and continuous. Anexcess of He is always added to the open split to prevent airfrom diffusing to the transport capillary and being carried tothe mass spectrometer source. Analyte dilution or blanking isaccomplished by adjusting the mass spectrometer transportcapillary inlet location with respect to the He flushing capillary.Many studies have been performed to assess the precision

and accuracy of CF-IRMS analyses under the variety ofconditions associated with common analytical practices.

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

Optimization of operating conditions can allow themeasurement of carbon and oxygen isotope ratios to a precisionof ≤0.1‰.[28–31] Previous investigations have addressed samplecontainer preparation effects, sample container filling effects,sample container storage effects, and advanced samplecleaning/purification techniques to identify the best techniquespossible for a variety of applications.[9,11,12,29,32] However, thisauthor is not aware of a direct systematic investigation ofsample matrix influences on measured isotope ratios duringGasbench II sample handling. In cases where sample matrixmay affect the measured isotope ratios, analysis accuracy hasbeen assessed through comparison of working standardmeasured values[15,33,34] or by comparing unknown sampleresults against δ13C and δ18O values determined by alternativeanalysis methods such as dual-inlet IRMS.[31]

y & Sons, Ltd. Rapid Commun. Mass Spectrom. 2014, 28, 2259–2274

Sample matrix effects on C and O isotope ratios during CF-IRMS

This report outlines an investigation that directly comparesmeasured carbon and oxygen isotope ratios of identical CO2

sampled from matrices of different gases. Analysis sequenceswere run with sample vials containing CO2 in He and samplevials containing CO2 in either pure N2 or a 21:79 mixture ofO2 and N2. Experimental subsets explored variables such asanalyte concentration, open split blanking, and sampleanalysis sequence to evaluate individual variables that mayaffect the ultimate delta value determination.

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METHODOLOGY

The study presented here is similar to the analyses describedin the work of Schauer et al.[31] where an automated systemwas developed to perform gas handling and purificationbefore IRMS analysis of atmospheric CO2. The descriptionof their system mentions use of an open split but details ofpossible matrix effects are not discussed. The motivation forthe current investigation was to explore sample matrix effectswhen analyzing samples of CO2 contained in septum-cappedvials under typical Gasbench II (Thermo ElectronCorporation, Bremen, Germany) and CF-IRMS operatingconditions. An example of representative analyses that thiswork simulated includes the analysis of atmospheric CO2

for Keeling plot application by Tu et al.[9] In addition toexperiments directly comparing matrix effects, investigationof other variables was needed to elucidate first order samplematrix influences. The Gasbench II method used to prepareCO2 in matrices other than He is slightly different from themethod used for traditional He matrix samples. The majordifference between the two methods is the capillaryconfiguration in the open split.[8] Figure 1 depicts therelationship between the mass spectrometer transfer capillaryand the sample (and matrix) delivery capillary for typicalsamples in a He matrix (left) and during matrix blanking(right). More specifically, during normal analyses the massspectrometer transfer capillary is always in the ’down’position allowing constant He carrier gas and pulses of CO2

to flow to the mass spectrometer. During analysis of CO2 inmatrices other than He, the mass spectrometer transfercapillary will cycle between the ’up’ position when matrixgas elutes from the GC column and the ’down’ position whenCO2 elutes from the GC column. The open split is constantlyflushed with a separate He capillary to prevent aircontamination and open to the atmosphere in order tomaintain a constant pressure. When the mass spectrometertransfer capillary is in the ’up’ position it has retracted intoan inner tube of the open split above the outlet of the He flushcapillary. This configuration allows for high levels of matrix(or sample) dilution. In theory, since the open split is at aconstant pressure, the amount of gas (and therefore massspectrometer source pressure) does not vary based on themovement or location of the mass spectrometer transfercapillary.Modified open split flow dynamics required a full suite of

experiments exploring Gasbench II preparation methods inaddition to direct matrix replacement analyses. Secondaryanalysis conditions explored include CO2 concentrations,analysis sequence variation, and the physical configuration ofthe Gasbench II open split (i.e., Gasbench II method). This studyis designed to investigate specific types of matrices (i.e. He, N2,

Rapid Commun. Mass Spectrom. 2014, 28, 2259–2274 Copyright © 2014

and O2/N2). Matrix compositions between end-members andtrace impurities are not directly addressed in this work.However, a certain degree of interpolation may be possiblebased on the observations reported here.

EXPERIMENTAL

All analyses were performed with a ThermoFinniganDeltaPlus XP (Thermo Electron Corporation) isotope ratiomass spectrometer except for two sets of analyses, whichwere performed with a ThermoFinnigan MAT 253 (ThermoElectron Corporation) isotope ratio mass spectrometer. Thesame Gasbench II and autosampler equipment was used onboth instruments. All measurements were made incontinuous-flow mode. Each analysis set or analytical sessionconsisted of 96 sample vials. Eighteen of the sample vialscontained a working standard. The first three analyses of eachsession were of a working standard. The remaining workingstandards were distributed such that one was measured afterevery fifth sample in order to monitor mass spectrometerperformance. The remaining 78 sample vials were dividedinto two sets of 39. Each set of 39 vials either contained adifferent matrix gas, or was subjected to a different GasbenchII preparation sequence (’standard’ method vs ’blanking’method), or both. This analysis structure allowedcomprehensive testing of the primary variable (matrix) aswell as secondary variables (analysis sequence, CO2

concentration, and open split configuration). Theexperimental conditions and variables are outlined in Table 1and described in detail below. For a given experiment, a set of39 analyses was synthesized into a single data point andcompared with a single data point representing the other 39analyses performed during the same analytical session. In somecases, one or more of the 39 analyses were rejected due toobvious sample container leakage/contamination (i.e., verylow or noCO2 signal). The number of accepted analyses in eachanalytical session can be identified in the ’# of Samples’ columnof Supplementary Table S1 (Supporting Information).

The isotope composition of all the analyte CO2 wasdetermined by comparison with a monitoring gas. Themonitoring gas was taken from the same source throughoutthe entirety of the study and its isotope composition hadpreviously been determined against acid digestion analysisof National Institute of Standards and Technology(Gaitherburg, MD, USA) Reference Material 8544 NBS19Limestone (NBS-19). The monitoring gas was introduced intothe mass spectrometer three times before and two times aftersample injections. Five consecutive analyte CO2

measurements were averaged to determine the isotopecomposition for a given sample. Nominally measured valuesare reported in the Results and Discussion section (unlessspecified otherwise) without correction according toinstrument drift as observed by independent workingstandard measured values. In other words, the measuredsample values were not corrected for long-term instrumentdrift before data analysis. The relative differences betweenanalytically determined isotope compositions are of interestfor this study and therefore adjustment to ’true’ values isunnecessary. The independent working standard used in thisstudy was a 0.3% mixture of CO2 in He prepared by PurityPlus Specialty Gases (Lexington, KY, USA) and stored in a

John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm

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Table 1. Experimental subsets and conditions varied during this study

Experiment subset Variable(s) Session subset 1 samples Session subset 2 samples

Primary Matrix Matrix &Gasbench II Method

N2 or O2/N2 Matrix,Blanking Used

He Matrix,Blanking Not Used

Secondary Control Matrix N2 or O2/N2 Matrix,Blanking Not Used


Blanking Gasbench II Method He Matrix,Blanking Used


Concentration* Concentration 0.3% or 0.8% CO2in Sample Vial

0.3% or 0.8% CO2in Sample Vial

Sequence* Batch Sequence Groups of 5 or13 Like Matrices

Groups of 5 or13 Like Matrices

*Different CO2 concentrations and sample sequences were used in individual Matrix, Control, and Blanking analytical sessions.

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typical gas cylinder. While similar in isotope composition, theworking standard CO2 was not prepared from a subsample ofthe monitoring gas CO2 or vice versa. The isotope compositionof the working standard was determined following alternatinganalysis of the working standard and acid-digested NBS-19.The values of δ13C reported in this investigation are withrespect to the international standard Vienna Peedee Belemnite(VPDB) while the δ18O values are with respect to ViennaStandard Mean Ocean Water (V-SMOW).A separate set of analyses was performed to determine the

isotope composition of the O2 used as the matrix gas in theO2/N2 matrix experiments. Molecular oxygen was convertedinto CO2 following the procedure described by Barkan andLuz.[35] In this method, O2 is heated in a quartz glass reactiontube containing a graphite rod wrapped with platinum wire.An approximate temperature of 770 °C was used during O2

conversion to minimize oxygen adsorption to the graphitesurface and to minimize the production of CO. Alsofollowing Barkan and Luz,[35] the graphite reactor wasconditioned prior to analyses, degassed between each O2

conversion, and the reaction progress was monitored bypressure change. Aliquots of O2 were converted into CO2 inparcels of the order of 200 μmol. Newly formed CO2 wascollected in a liquid nitrogen trap connected to the graphitereactor. The reaction was allowed to proceed untilquantitative conversion was indicated by baseline pressureas measured by an capacitance diaphragm gauge (CDG-100;Agilent Technologies, Inc., Santa Clara, CA, USA) as well asby a thermocouple (model 536; Agilent Technologies).

Chemicals

The helium used as sample vial matrix gas was taken fromthe same tanks as supply the Gasbench II carrier gas. Ultra-pure (99.999%, UHP) grade He from Purity Plus SpecialtyGases was used for this purpose. The nitrogen and oxygenused as the matrix gas was pre-purified grade and supplied byScott-Gross Company, Inc. (Lexington, KY, USA). .The δ18Ovalue of the matrix O2 was determined to be �4.4±0.3‰. Asingle pre-purified tank of CO2 also supplied by Scott-GrossCompany was used as the analyte for all experiments involvingthe Gasbench II. The average isotope composition of the analyteCO2 gas taken from He matrix samples of all the Matrix andBlanking experiments over the course of this investigation was


determined to be δ13C=�10.8±0.1‰ and δ18O=2.1±0.2‰,as corrected by the working standard gas. The workingstandard was a 0.3% mixture of CO2 in He pre-prepared byPurity Plus Specialty Gases. The isotope composition of theworking standard CO2 was determined by calibration againstNBS-19 and found to have δ13C and δ18O values of�11.6±0.1‰ and 0.2±0.1‰, respectively. The gases utilizedduring this study were used as received and were not furtherpurified or assessed for levels of contamination.

Preparation of sample vials

Exetainers (5.9 mL, flat-bottomed Na glass, cat. #718W, LabcoLtd, Lampeter, UK) were used for all sample vials in thisinvestigation. Vials used for He versus N2 matrix experimentswere recovered from previous analyses involving the aciddigestion of carbonate minerals. That is, the interior of thevials had been exposed to ’103%’ H3PO4 (from 85% ACSreagent grade H3PO4, Fisher Scientific, Fair Lawn, NJ, USA)for more than 24 h and subsequently cleaned by rinsingresidual acid out of the vials and then soaking them for 24 hin fresh deionized (DI) water (NANOpure 18.2 MΩ, ThermoBarnstead, Dubuque, IA, USA) a minimum of three timesbefore drying in an oven at 50 °C for over 24 h. Blankingexperiments, where only He matrix was used, also employedacid digestion recovered vials. However, the vials used for theHe versus O2/N2 matrix experiments as well as all the controlexperiments were new and had not been exposed to H3PO4

before use. Any given vial in both sample vial sets was usedmultiple times over the course of this study; however, a newseptum was used for each new sample. New, unused vialswere employed to hold the 0.3% CO2 working standardduring all analyses. However, after an analysis the designatedworking standard vials were issued with new septa and re-used for working standard vials multiple times over thecourse of the investigation.

Sample vials were individually flushed with pure He,pure N2, or a 21:79 mixture of O2 and N2 for 13 min at aflow rate of 100 mL/min. In all cases, flushing wasaccomplished with the same type of double-holed samplingneedle (Part No. 113-7020, Thermo Electron) used duringheadspace sampling as described above. In the case offlushing, however, the displaced gas was vented to theatmosphere during matrix addition. A mixture of 21% O2



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and 79% N2 was created by combining a 21 mL/minstream of pure O2 and a 79 mL/min stream of pure N2.The oxygen and nitrogen flows were regulated byindividual mass flow controllers (model SLA5850, BrooksInstrument, Hatfield, PA, USA). The individual andcombined flows were verified by a soap film flow meter(model 0101-0113, Hewlett-Packard (HP), Palo Alto, CA, USA).CO2 was introduced into the sample vials via hand

injection by a gastight syringe (1700 Series, HamiltonCompany, Reno, NV, USA). The gas syringe was filled frompure CO2 contained in a small volume connected to the gasregulator of the source CO2 tank. The small volume wascapped by a septum maintained under positive pressure sothat atmospheric air could not leak into the volume andcontaminate the analyte CO2. Shortly preceding CO2 injectionthe sample vial septa were quickly punctured with open-ended needles to release excess matrix gas and bring thesample vial into pressure balance with the gas syringe. Thisallowed the preparation of the desired CO2 mixing ratio as apercent volume. Immediately before CO2 transfer to samplevials, the syringe and needle were flushed a minimum ofthree times with analyte CO2. Fully loading the syringeand discarding the CO2 accomplished the removal ofatmospheric components. The sample vials were locatedvery close to the source tank and filled sequentially with aminimum amount of time between injections. This process isbelieved to have prevented the introduction of atmosphericCO2 into the sample vials. This assumption is supportedby a general lack of air contamination peaks in un-blankedHe samples. Two concentrations were used during thisinvestigation. A volume of 18 μL or 47 μL was added to anindividual sample vial to prepare 0.3% or 0.8% CO2 samples,respectively.The 0.3% CO2 working standard was loaded into sample

vials in a manner similar to the matrix gas flushing processdescribed above. That is, the working standard gas wasdelivered by a double-holed needle to a sample vial for13 min at a flow rate of 100 mL/min while the content ofthe vial was allowed to escape to the atmosphere. Thisprocess allowed the full turnover of the vial and completeexpulsion of air.[29,30]

Filled sample vials were immediately loaded into theautosampler holding block followed shortly by initiation ofthe sampling sequence. This sample preparation processallowed the analysis of all samples within 24 h of CO2

introduction into the sample vials, thus minimizingcontamination, leak fractionation, and isotopic exchangeduring storage. The holding block was maintained at aconstant temperature of 25 °C during sampling.

Mass spectrometers and gas handling device

This investigation utilized a Finnigan DeltaPlus XP isotope-ratiomass spectrometer to determine most carbon and oxygenisotope ratios of gas-phase CO2. The mass spectrometer systemwas interfacedwith a Finnigan Gasbench II device andGC-PAL(CTCAnalytics, Zwingen, Switzerland) autosampler for samplepreparation and transfer. The differentially pumped massspectrometer was equipped with three Faraday cups coupledto resistors of 3×108, 3×1010, and 1×1011 Ω for measurementof the m/z 44, 45, and 46 ion beams. The Gasbench IIfunctionality details are described in the Gasbench II Operating


Manual.[8] Briefly, the Gasbench II manipulates gas forpurification and aliquoting prior to introduction into theionization chamber of a gas source mass spectrometer. For thisstudy, a double-holed capillary needle was used for sampleextraction and transfer to the Gasbench II device. Carrier He isflushed into a sample vial from one capillary of the needle whilesample and matrix gas are forced into a needle return capillary.During this study, analyte CO2 was sampled from butyl rubberseptum-capped exetainers before purification and separationfrom matrix gas by the Gasbench II device.

Prior to analyses, the mass spectrometer was tuned tobalance sensitivity and linearity. Monitoring of gas linearityanalyses was performed prior to each analytical session toconfirm linearity at or below 0.1‰ per volt over a voltage rangeof 900 mV to 5500 mV for both carbon and oxygen. The typicallinearity was of the order of 0.03‰ per volt for carbon and0.08‰ per volt for oxygen. The water level inside the massspectrometer was monitored periodically and confirmed to benear baseline levels (hundreds of mV for m/z 18) duringsessions reported here. Sample purification performed by theGasbench II device includes water removal by hygroscopicNafion tubes (Part No. 074–3390, Thermo ElectronCorporation) and separation of gases by a CP-Poraplot Q GCcolumn (27.5 m length, 0.32 mm i.d., #CP7551, Varian Inc., PaloAlto, CA, USA). The chromatographic parameters used duringthis studywere set according to theGasbench IImanufacturer’sspecifications.[8] The temperature of the GC column was heldconstant at 25 °C during all sample purification as well asbetween samples while a continuous stream of He gas passedthrough the column. The carrier He flow rate from the samplevial to the sample loading loop was set to 0.6 mL/min. Thevolume of the sample loop used during these experimentswas 100 μL. The gas-handling methods used during theseexperiments allowed for 40 s of sample loop loading and 30 sof sample injection. When utilizing blanking, the massspectrometer transfer capillary was in the blanking positionfor all but 25-s windows during analyte CO2 elution from theGC column. The duration and timing of transfer capillarymovement were determined by observing the timing of un-blanked chromatogram peaks (sample as well as matrix) andverified by monitoring Isodat (Thermo Electron) determinedpeak widths and positions during blanking and controlexperiments. The timing of transfer capillary movement willvary depending on the Gasbench II operating conditions. TheCP-Poraplot Q GC column was baked out overnight at 120 °Cwith constant He flowing through the column prior to theperiods of analytical sessions reported here.

During operation of the Gasbench II device, a carrier gasstream passes through an open split prior to transfer to themass spectrometer. Figure 1 depicts different configurationsfor the open split system used during this study. Undernormal He matrix and carrier gas conditions, the capillaryleading to the mass spectrometer is at the bottom of the opensplit and the GC column effluent passes undiluted to theionization source chamber. However, the open split isdesigned such that sample dilution or blanking can occur ifthe position of the final transfer capillary is adjusted. Sampledilution is achieved by allowing extra He to mix with sampleand carrier gas prior to leaving the open split through thetransfer capillary. Blanking is the process of blowing sampleor matrix gas out of the open split and preventing the GCcolumn effluent from being transported to the mass


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spectrometer ionization source chamber. Blanking is at theextreme end of dilution and is accomplished by allowingthe GC column effluent capillary to fully retract above theHe dilution capillary in the open split.As mentioned previously, a ThermoFinnigan MAT 253

isotope ratio mass spectrometer was used for two sets ofcontinuous-flow experimental sets coupled to the sameGasbench II as for all other continuous-flow experiments.These experiments have ID numbers of 20130817 and20130819 as indicated in Supplementary Table S1 (SupportingInformation). In addition, the same MAT 253 was used indual-inlet mode to determine the oxygen isotope compositionof CO2 derived from O2. The MAT 253 is equipped with threeFaraday cups coupled to resistors of 3 × 108, 3 × 1010, and1× 1011 Ω for measurement of m/z 44, 45, and 46 ion beamsas well as three additional Faraday cups coupled to resistorsof 1 × 1012 Ω for additional CO2 isotopologue m/z valuemeasurements. In dual-inlet mode, the MAT 253 was tunedwith a preference to sensitivity. An electron energy of100 eV and an accelerating voltage of 9.5 kV were usedduring measurements. Ion beam ratios were measured at26-s integration times for 8 cycles between sample andreference gas. The intensity of the m/z 44 ion beam wasadjusted to 12 V for a pressure of 60 mbar in each bellow.As with the continuous-flow analyses, Isodat software wasused to calculate δ18O values for dual-inlet measurements.The Isodat software program (ISODAT 3.0, Gas Isotope

Ratio MS software, Isodat Acquisition version 3.0, ThermoElectron Corporation) was used to control the massspectrometer, autosampler, and Gasbench II utilized duringthis investigation. Batch sample processing was organizedby establishing an analysis sequence consisting of a collectionof analysis methods. Isodat is supplied with a variety ofmethods for use with multiple Gasbench II applications.Included among these methods is a procedure which utilizesthe blanking functionality of the Gasbench II open split toremove most (effectively all, dilution factor >100) O2 andN2 from the GC column effluent prior to mass spectrometertransfer while allowing CO2 to proceed unaltered.Specifically, sample purification is achieved through samplealiquoting at the eight-port switching valve (ValcoInstruments, Inc., Houston, TX, USA), separation of CO2,O2, and N2 in the GC column, and repeated repositioning ofthe mass spectrometer transport capillary in the open split.Gasbench II methods which sample the vial headspacecontaining CO2 in a pure He matrix do not vary the positionof the capillary in the open split as potential isobaricinterference-forming compounds are not present andtherefore do not need to be removed.Isodat software with default settings was used to

determine the chromatogram peak areas and these areas weresubsequently converted to delta values according to IAEArecommendations.[36] The following default values for peakfitting parameters were used in the Isodat control softwarefor DeltaPlus XP data processing: start slope = 0.2 mV/s, endslope= 0.4 mV/s, peak minimum height = 50 mV, peakresolution = 20%, and maximum peak width= 180 s. Asdetailed below, some CO2 peaks could not be resolved fromtailing matrix voltages during MAT 253 analyses. The peak-fitting parameters for the MAT 253 were modified whereneeded to exclude matrix voltage, and set to startslope = 1.0 mV/s, end slope = 0.4 mV/s, peak minimum


height = 50 mV, peak resolution = 20%, and maximum peakwidth = 180 s. This modification was only needed for thefirst and sometimes the second CO2 peak for a set of fivesample CO2 injections. Most CO2 peaks identified duringMAT 253 analyses utilized the default peak-fittingparameters. Modified peak-fitting parameters for the firstpeak(s) did not noticeably change delta values comparedwith the rest of the CO2 peak delta values.

Experimental plan and variables

Table 1 lists the variables explored during this study. Mostexperiments compared the primary variable of gas matrix(referred to as ’matrix’ experiments). That is, samples ofCO2 in a He matrix are directly compared with identicalCO2 in a matrix of either pure N2 or a 21:79 mixture of O2

and N2. During these experiments the He matrix samples(and working standards) were handled by the Gasbench IIwith the standard method while other samples experienceda method utilizing blanking to prevent N2 or O2/N2 fromentering the mass spectrometer source. Secondary conditionswere also varied during these experiments. These conditions,as outlined in Table 1, include Gasbench II gas-handlingprocedures (i.e., blanking vs not blanking), CO2

concentrations, and CO2 sample container batch sequence.The secondary effects (δ13C and δ18O offsets produced bysecondary conditions) proved to be minimal compared withprimary experiments and serve to verify differences observedbetween CO2 analyzed from different gas matrices.

The ultimate goal of this investigation was to determine thepresence of a matrix effect and to evaluate the suitability ofthe movable open split to eliminate this effect. However,CO2 samples originating from matrices other than He aregenerally not handled in exactly the same manner as theirHe counterparts. The modified analyte purification procedureis used due to the necessity of removing potential isobaricinterference-forming compounds. As outlined above, mobilephase blanking requires a different Gasbench II preparationsequence from analysis of CO2 in He with no otherconstituents. Therefore, a series of experiments wasperformed to determine the effect of blanking on themeasured isotope composition. This set of analyses is referredto as the ’blanking experiments’. For these analyses, He wasalways used as the matrix gas and only the Isodat methodcontrolling the Gasbench purification was varied. Theblanking procedure was identical to that used for the analysesof N2 and O2/N2 during matrix experiments.

A third set of analyses was performed on N2 and O2/N2

matrix samples with the standard Gasbench II Isodat method(i.e. not blanked to remove N2 or O2/N2). These analyses aretermed the ’control experiments’ and serve as frame ofreference for samples that experience isobaric interferenceduring measurement. During these experiments analyte CO2

was separated from N2 and O2 in the chromatographycolumn as in all analyses. Chromatographic separationestablished pulses of CO2 and N2 or O2/N2 that exited theGC column and were introduced into the mass spectrometersource sequentially. The temporal gap in gas-phase speciesprevented major direct isobaric interference and thechromatogram peaks were separated sufficiently duringDeltaPlus XP analyses to be characterized according to thepeak-fitting parameters described above. Control



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experiments performed with the MAT 253 were lessstraightforward, however. Due to the enhanced sensitivityof the MAT 253 compared with the DeltaPlus XP, tailing ofO2/N2 matrix peaks into CO2 peaks prevented the use ofthe same peak-fitting parameters for both the MAT 253chromatograms and the DeltaPlus XP chromatograms. (Seethe last paragraph of the previous section, Massspectrometers and gas handling device, for more details.)For all other cases, un-blanked N2 and O2/N2 matrix sampleswere analyzed in batches with He matrix samples in the sameway as experiments exploring other secondary conditions(variables). In this way, the results of control experimentsare directly comparable with those from other subsets ofexperiments.Two different CO2 concentrations were used during matrix,

blanking, and control sub-experiments. Concentrations of0.3% or 0.8% were prepared as a volume ratio of CO2 injectedto total sample vial volume. For a given set of 78 analysesonly 0.3% or 0.8% samples were used. A minimum of threesets of 78 analyses for both 0.3% and 0.8% CO2 wereperformed for each of the matrix, blanking, and controlexperiments. All analyses were performed alongside thesame 0.3% CO2 in He working standard.The final secondary variable introduced during analyses was

the sample analysis sequence. As outlined above, one criticalparameter carefully controlled by Gasbench II flow dynamicsis the static conditions under which ionization takes place inthe source of the mass spectrometer. For the sake of betterunderstanding how changing the sample matrix affects theGasbench II dynamics as well as the mass spectrometer sourceconditions, two different analysis sequences were used for allexperiment subsets. While every sixth analysis was of aworking standard during all experiments, regular samples(He, N2, or O2/N2 matrix) were grouped in series of five orthirteen samples of identical matrix. For example, analysissequences that grouped samples of identical matrix into seriesof five followed this pattern: working standard, five He matrixsamples, working standard, five N2 matrix samples, repeat.Analysis sequences that grouped samples of identical matrixinto series of thirteen followed this pattern: working standard,five He matrix samples, working standard, five He matrixsamples, working standard, three He matrix samples, two N2

matrix samples, working standard, five N2 matrix samples,working standard, five N2 matrix samples, working standard,one N2 matrix sample, four He matrix samples, workingstandard, etc. If working standards are not considered, thetwo sequences are simply alternating groups of five matricesof identical composition or thirteen matrices of identicalcomposition. Both experiment subsets included 39 He samples,39 matrix samples, and 18 working standard samples. Bothsample sequenceswere used aminimum of three times for eachdifferent set of experimental conditions.It is worth noting that the carbon and oxygen isotope

composition of the analyte CO2 was not varied during thisstudy. It is plausible that the magnitude of potential matrixeffects could depend on the isotope composition of CO2 beingremoved from sample vials. However, this has not beenshown to be the case for CO2 sampled from a He matrix.Therefore, the focus of this study is on other variables thatmay cause differences in measured δ13C and δ18O values.Further discussion regarding CO2 isotopic composition isfound in the Oxygen isotope considerations section below.


RESULTS AND DISCUSSION

Supplementary Table S1 (Supporting Information) includes thereduced comparison data collected during this study. Table 2includes averaged experimental data that is fully covered inSupplementary Table S1. Composite δ13C and δ18O offsetvalues represent 73–78 individual analyses. In each analysisset, the total number of non-working standard analyses wasdivided into two groups of 36–39 exetainers each. The twosubsets varied by one or more of the variables presented inTable 1. Individual analyses in a given subset were averagedand the difference between the ’varied’ conditions and the’typical’ conditions were used to gauge the influence on themeasured isotope ratios (’Difference between sample sets’column of Supplementary Table S1 and Table 2). The standarddeviation of 36–39 analyses as well as the difference betweenthe standard deviation of each subset is also included inSupplementary Table S1 as a way to gauge possible precisiondegradation introduced by the variables being tested. Averagesand standard deviations of same-type experiment subsets arepresented in Table 2. However, the offset (’difference’) standarddeviations presented in the far right category column of Table 2are calculated from the δ18O and δ13C difference columns ofSupplementary Table S1. Figure 2 graphically depictsinformation presented in the difference category (far right setof columns) of Table 2.

Significant differences in the measured δ13C and δ18Ovalues for identical CO2 were observed between samples thatused Gasbench II blanking to remove matrix gas and samplesthat were analyzed without matrix removal. Other devicesthat are comparable with the Gasbench II do not include anopen split with an inner tube that allows for the process ofblanking. According to the results and observations outlinedbelow, significant matrix effects may influence the measuredisotope ratios of impure samples if processes such as blankingor advanced chromatography are not employed.

The following sections discuss the probable formation ofisobaric interferences that influence the measured isotoperatios of sample CO2. The isobaric interferences are likelyNOx products derived from matrix gas. It is important topoint out that the NOx compounds discussed below areformed in the source of the mass spectrometer during gas-phase chemical reactions. N2O is a minor component of theatmosphere and is present when environmental samples arecollected for CO2 isotope ratio analyses. The presence ofN2O in environmental samples presents similar but slightlydifferent challenges. Specifically, atmospheric N2O can beseparated chromatographically from CO2 but may alsoparticipate in intermolecular rearrangements in the ionsource, as with N2 and O2. These challenges are addressedelsewhere[10,25,26] and are not directly related to this study.

Blanking experiments

Samples of CO2 from a He matrix which were processed withthe Gasbench II blanking method were measured to haveslightly higher average δ13C and δ18O values than samples thatwere processed by the typical Gasbench II method. Basedon Table 2, it appears that for lower concentration samplesthe Gasbench II blanking method may result in slightlyhigher isotope ratios being obtained when all other factorsare equal.


5

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N. P. Levitt

wileyonlinelibrary.com/journal/rcm Copyright © 2014 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2014, 28, 2259–2274

2266


226

The possible cause of positive offset between blanked andnon-blanked samples is not readily apparent. The timing ofGasbench II gas processing/transfer events was carefullychecked before the experimental campaign to ensure thatthe mass spectrometer transfer capillary was in position toallow the full transfer of each CO2 aliquot after exiting theGC column. It is very unlikely that the beginning or end ofindividual CO2 aliquots was lost or not transferred to themass spectrometer. The open split is designed to ensure aconstant stream of gas to the mass spectrometer source andtherefore a constant source pressure.[27] Considering this,pressure changes due to variation in fluid dynamics duringmass spectrometer transfer capillary movement are also nota likely explanation. The offsets for carbon and oxygen areapproximately the same under a given set of conditions. Bythe 2:1 (O:C) mass dependent fractionation rule-of-thumb,[32]

it does not appear that a single physical process is causing theoffsets between blanking and non-blanking. Elsig andLeuenverger[27] performed tests to identify sources of carbonand oxygen fractionations associated with open splits andoccurring in mass spectrometer ion sources. These workersidentified diffusion differences between reference gas opensplits and sample gas open splits that can cause shifts in δvalues. The sample open split conditions during CO2 elutionand transfer to the mass spectrometer should be identicalbetween blanked and non-blanked samples in this study asthe movement of the transfer capillary during blankinganalyses only occurs before and after CO2 travels throughthe open split. Taking this into consideration, it does not seemlikely that CO2 diffusion differences between the referenceopen split and sample open split explain the offsets observedin blanking experiments. In any event, as the observed offsetsare minimal, the source of apparent isotope ratio discrepanciesis not of major concern to this study. Further investigation ofthis phenomenonwould also include a comparison of blankingeffects on CO2 samples of different isotope compositions.Of course, factors other than the configuration of the sample

open split come into play for most of the analyses utilizing theGasbench II blanking method. These experiments wereperformed as a baseline from which to compare the primaryand secondary variables described below. The 0.2‰ or loweroffsets observed for blanking are generally consistent withoffsets observed during N2 matrix experiments and may bemasked by negative deviation O2/N2 matrix effects. Controlexperiments show the greatest offsets between variedconditions and typical conditions. These effects appear toovershadow any offset induced strictly by blanking. Thespecifics of matrix and control experiments are described indetail below.

Matrix experiments

The results of the N2 versus He matrix experiments do notshow a significant difference between mean isotope ratios.The results of the He versus O2/N2 matrix experiments aresimilar to those from experiments in a pure N2 matrix. Mostmatrix experiment results are in the range of instrumentprecision limits. However, approximately half of themeasured 13C/12C ratios for O2/N2 matrix samples arearound 0.2‰ higher than those of He matrix samplesanalyzed during the sample session and this may beindicative of meaningful matrix effects. The results of the


matrix experiments are very similar to the differencesobserved between the blanked and un-blanked samplesanalyzed in the blanking experiments. This observationsuggests that varying the GC column parameters may notinfluence the measured isotope ratios when blanking ofmatrix gas is employed.

The significance of blanking during analyses that transportCO2 from a matrix other than He can be best highlighted bycomparing matrix experiments with control experiments.The only difference between matrix experiments and controlexperiments is that N2 or O2/N2 was removed from thesample stream by the Gasbench II blanking method duringmatrix experiments while the typical Gasbench II processingmethod (non-blanking) was used for all analyses in thecontrol experiments. The N2 and O2/N2 matrix samples thatwere not processed by Gasbench II blanking did not undergoremoval of matrix gas prior to GC column effluent transfer tothe mass spectrometer source. In these analyses CO2 andmatrix gas experience ionization and subsequent transportto the mass analyzer. Figure 3 compares the chromatogramsof He matrix analyses (top panel) and N2 matrix analyses(bottom panel) for two individual samples in the 20130418sample set. Both samples were processed by the typicalGasbench II method and not blanked before transfer to themass spectrometer. In the top panel (Fig. 3(a)), the chromatogrambegins with three monitoring CO2 pulses followed by fivesample CO2 pulses starting at approximately 300 s. The topchromatogram endswith two finalmonitoringCO2 pulses. Also,barely visible in the ratio chromatogramof Fig. 3(a) is evidence ofair contamination preceding the CO2 sample peaks. These’peaks’ are analogous to N2 matrix peaks discussed belowand are identified in Fig. 3(a) for CO2 vs matrix peakidentification/location purposes.

The chromatogram in the bottom panel (Fig. 3(b)) is similarto that in the top panel (Fig. 3(a)) but five distinct additionalpeaks are present. These additional peaks are at much lowerintensity and do not decrease in intensity with temporalprogression as is the case with sample CO2. The additionalpeaks correspond to pulses of N2 leaving the Gasbench II GCcolumn and entering the mass spectrometer source. The iondetection configuration used during all analyses onlymonitored m/z values of 44, 45, and 46. Isotopologues of N2

have molecular weights of 28, 29, and 30 and therefore the’nitrogen’ peaks in Fig. 3(b) do not correspond to ionizedmolecular nitrogen. Instead, the ’nitrogen’ peaks (or ’air’ asthey are sometimes referred to in the literature[29]) result fromisobaric interference probably formed during ionization in themass spectrometer source. Evidence for specific speciesproduced during ionization is lacking. However, Schoellerand Luke[20] speculated during similar experiments that NO2

is the primary CO2 isobar formed during nitrogen ionizationin the presence of oxygen-containing compounds. For thisreason the ’extra’ peaks will be referred to as ’matrix’ herein.

Control experiments

Matrix peaks and CO2 peaks are resolved in chromatogramsduring DeltaPlus XP analyses regardless of the composition ofthe isobaric interference. Default Isodat peak-fitting parametersexcluded matrix peaks for isotope-ratio calculations andsuccessfully identified CO2 injection peaks. Average δ13C andδ18O values determined during N2 matrix control experiments


7

Figure 2. Measured isotope ratio offsets from typical He matrix and carrier gas operating conditions. The first row includesoxygen values. The second row includes carbon values. The first column depicts experiments with CO2 in a He matrix only(green, all bars). The second and third columns depict experiments with a N2 matrix (blue, left bar of two bar sets) andO2/N2 matrix (red, right bar of two bar sets). The offsets in (a) and (b) are due to the process of blanking. The remainingsub-figures include experiments comparing matrix effects when isobaric interferences are removed during blanking [(c) and(d)] and not removed [(e) and (f)]. Each bar in a given sub-figure represents unique experiment conditions. Experimentalconditions are 1) 0.3% CO2, sequence of 5 samples, 2) 0.3% CO2, sequence of 13 samples, 3) 0.8% CO2, sequence of 5 samples,and 4) 0.8% CO2, sequence of 13 samples. Error bars are 1 standard deviation of 2–5 sets of approximately 78 analyses.

N. P. Levitt

2268

are all negative relative to those of He matrix samples analyzedduring the same analytical sessions. The average δ13C valuesare of the order of �0.1‰ while the average δ18O values rangefrom �0.6 to �0.4‰. The measured 13C/12C ratios determinedduring control experiments do not exhibit significant deviationsdepending on the matrix. However, compared with matrixexperiments the deviations are slightly more meaningful.Absolute differences between matrix and control experimentsare found to deviate by approximately 0.2‰. While this is abovethe analytical precision of the instrument, the seemingly trivialnet difference may be due to two different processes affectingthe measured ratios in opposite directions. Specifically, theblanking effect appears to shift delta values in a positive direction(see blanking experiments) and introduction of matrix gas intothe source appears to shift delta values in a negative direction.ForN2matrix control experiment analyses the net deviation fromHe analyses is minimized by opposing drivers. Regardless, it ismost convenient to use only one Gasbench II processing methodfor a given sample. Considering the peak tailing observed forsamples analyzed on the MAT 253 and significant offsets formost control experiments, blanking is themore desirablemethodwhen processing samples with matrices other than He.The CO2 peaks are also fully resolved in the case of the O2/N2

matrix experiments only utilizing the typical Gasbench IIprocessing method when analyzed with the DeltaPlus XP. Thematrix peaks for these experiments are of the same size andintensity as those in the pure N2 experiments. Two analyticalsessions (20130817 and 20130819) utilizing a MAT 253 forexperiments comparing He matrix versus O2/N2 matrix wereconducted. These two experiments are identified in


Supplementary Table S1 (Supporting Information). One sampleset was of 0.3% CO2 in sequences of five matrices of identicalcomposition exetainers. The other sample set consisted of 0.8%CO2 in sequences of 13 matrices of identical compositionexetainers. Due to the sensitivity of MAT 253 spectrometerscompared with DeltaPlus XP instruments, the initial andsometimes second matrix peaks were identified by Isodataccording to the default peak-fitting parameters cited aboveduring initial processing of the chromatograms. These initialmatrix peaks did not satisfy the end slope criterion by the timethat the CO2 eluted from the GC column, however. This ’peaktailing’ resulted in the combination of the initial matrix peakand the following sample CO2 peak into one ’peak’ for Isodatisotope ratio calculations. In order to preclude the classificationof matrix ion intensity as a ’peak’, chromatograms werereprocessed with a change in the start slope criterion to1 mV/s for the first and sometimes the second of five injectedCO2 samples. For later injected CO2 samples, the precedingmatrix gas had been diluted to the point that the normal peak-fitting parameters did not recognize the matrix voltage as a’peak’ andmodified parameters were not needed. Themodifiedpeak-fitting setting was only used for O2/N2 matrix samplesanalyzed on the MAT 253 and did not noticeably change themeasured isotope ratios compared with subsequent CO2 peaks.In fact, the standard deviations of five carbon or oxygen isotoperatio measurements for single samples reprocessed in thismanner were lower for Sessions 20130817 and 2010819 thanfor the analogous experiments performed on the DeltaPlus XP.As an alternative to modifying the slope criteria, the first twopeaks could be excluded from evaluation, or peak detection


Figure 3. Chromatograms (ratio or intensity vs time) for 0.8% CO2 in a (a) He and (b) N2 matrix. A very small amount ofcontamination (air) is detected in the ratio chromatogram of the He matrix sample and identified for CO2 vs NOx peak locatingpurposes. The top chromatogram in each set depicts the ratio of m/z 45/44 and 46/44 while the bottom chromatogram in eachset depicts the contemporaneousm/z signal intensities form/z 44, 45, and 46. Both analyseswere performedwithoutmatrix blanking.


226

could in some cases be changed from the default m/z 44 signalsto m/z values of 45 or 46.The two sample sets analyzed on the MAT 253 are

significantly different from the analogous sets analyzed onthe DeltaPlus XP. Table 2 displays the difference between theaverage O2/N2 matrix subsets and the He matrix subsets.While the difference is significant, the difference originatesfrom the O2/N2 matrix analyses and not from the He matrixanalyses. The effect can be most readily observed whencomparing O2/N2 matrix values with average He matrixvalues. These values have been corrected for instrumentoffsets as determined by the working standard gas for moremeaningful comparison and are included in SupplementaryTable S1 (Supporting Information). The matrix effect, asindicated by high standard deviation of O2/N2 matrixaverages, is clearly different between the two differentinstruments. While comparison of results between the twodifferent instruments is informative (see below), the inclusionof MAT 253 data with DeltaPlus XP derived averages is notconducive to understanding matrix effects duringcontinuous-flow analyses with a single instrument. For thesereasons, the averaged values presented in Table 2 onlyinclude measurements collected on the DeltaPlus XP.As opposed to He vs pure N2 control experiments, the

results of the He vs O2/N2 control experiments do show a


significant difference (>1‰) between the measured carbonand oxygen isotope ratios. The implications and possibleexplanations of these results are discussed in the Backgroundinfluences and Oxygen isotope considerations sections below.

Concentration and sequence experiments

The concentration of CO2 in exetainers is more challenging tocontrol for air-type samples than for carbonate mineral-derived CO2. As an attempt to widen the applicability of thisstudy, two CO2 concentrations were used in all experimentsconducted during this project. The results of experimentsoutlined in Table 2 are discussed above with respect toblanking, matrix, and control experiments. These analysissets all include 0.3% CO2 and 0.8% CO2 experiments andprimary (matrix) or secondary (Gasbench II processing)variables are compared between analyte concentrations ofthe same value. However, these datasets also includeimportant information about measured isotope ratios whenthe analyte concentration is compared within a givenexperimental set of analyses. The most significantconcentration comparisons related to the objective of thisstudy are the results from matrix experiments. The values ofthe 0.3%CO2 and the 0.8%CO2 experiments are approximately


9

N. P. Levitt

2270

within analytical uncertainty for almost all matrix experiments,the exception being δ18O values for analyte sequences of 13samples for O2/N2matrices. For these experiments, the oxygenisotope composition offsets were found to be �0.2‰ lower for0.8% CO2 than for 0.3% CO2. The magnitude of this differenceis small but potentially meaningful. Possible explanations forcontrol experiment offsets (background and matrix O2 isotopecomposition, discussed below) may also apply to the >0.2‰difference between these two analyte concentrations. However,for most matrix experiments conducted during this study theconcentration of CO2 in the sample vial does not significantlyinfluence the measured carbon and oxygen isotope ratios.Minimal concentration effects are consistent with linearityquality control analyses performed prior to all analyticalsessions. These results indicate that when the N2 or O2/N2

matrix gas is removed from the sample stream by the GasbenchII sample purification processes, instrument linearity is consistentbetween He matrix samples and N2 as well as O2/N2 matrixsamples.CO2 concentration differences also did not translate to

significant differences in isotope ratio measurements duringblanking experiments. Exetainers with concentrations of0.3% CO2 in He showed a slightly higher offset ofapproximately 0.1‰ in most cases. As with matrixexperiments, this offset is of the order of the instrumentprecision limits and the analytical uncertainties of theaveraged values in Table 2 overlap when compared betweenCO2 concentrations – the possible exception being theaverage δ18O difference between 0.3% and 0.8% duringsequences of 13 samples. This difference is probably abovethe instrument precision limits at 0.2‰. The analyticaluncertainty of the 0.3% and 0.8% CO2 dataset averages alsodoes not overlap between sequences of 13 samples.Insignificant CO2 concentration effects are observed when

control experiments are taken into account for mostexperiments. All the carbon and oxygen average CO2

concentration offsets in the N2 or O2/N2 matrix samples weremeasured to have a lower isotope ratio than those in the Hematrix samples. The concentration effect is observed only toa limited degree in experiments that compared N2 matriceswith He matrices. While differences in carbon isotope ratiooffsets are approximately the same as in the N2 matrix controlexperiments, the CO2 concentration effect is very pronouncedfor measured oxygen isotope ratios in O2/N2 controlexperiments. Possible explanations for the significantdifference in element trends are outlined in the Backgroundinfluences and Oxygen isotope considerations sections below.Sample number sequence appears to have little effect on

measured isotope ratios for most experiments. Matrixexperiment differences are slightly higher than blankingexperiments but average value differences were all lowerthan 0.1‰ between sample sequences of the same numberand pCO2 concentrations of the same value, as outlined inTable 2. Control experiments show the biggest differencesin average values but only for sequences of 13 comparedwith similar samples analyzed in sequences of 5 for 0.3%CO2 in O2/N2 matrices. Under the same matrix conditions,samples of 0.8% CO2 analyzed in sequences of 13 sampleswere found to have lower δ13C values by 0.1‰ than likesamples analyzed in sequences of 5. All other differencesobserved in control experiments were at or below 0.1‰when comparing sequences.


Background influences

The analysis of variable CO2 concentrations and analysissequences allowed the exploration of background effects.Recent studies indicate that small amounts of residual gasforming a background (or pressure baseline) in the source ofthe mass spectrometer can affect the measured isotope ratiosof an analyte being introduced.[37,38] A background effect islike a carryover (or ’memory’) effect[20,30] but may bedifferent in the source of interference. Carryover from theprevious sample is interference by a similar analyte. Abackground could result from a similar or dissimilar species.Isobaric interference could originate from a chemicallyproduced derivative (gas-phase reaction product) with a likemass to the analyte of the ’new’ sample. According to Elsigand Leuenberger,[27] a fluctuating background level inside amass spectrometer may originate from non-uniformintroduction of molecules and adsorption/desorptionprocesses occurring on the walls of the source housing. Afluid background of this type could alter the area under achromatograph peak and ultimately influence the calculatedisotope ratios.

Another factor to take into consideration when evaluatingthe possibility of elevated backgrounds is analyte zonebroadening during chromatography.[39] During a typicalsample analysis five aliquots of analyte CO2 and matrix gasare introduced into the GC column for separation andsubsequent transport to the mass spectrometer. Betweenanalyte injections He is introduced into the GC column tomaintain constant pressure and provide continuous flow tothe mass spectrometer. The theory of chromatographicrandom-walk mechanism and mobile phase-stationary phasetransfer[40] allows for the possibility of a small amount ofanalyte and (non-He) matrix gas from zone-broadened peaksremaining in the GC column after most of the injected gas haseluted from the column. The molecules investigated in thisstudy (i.e. CO2, O2, and N2) follow physicochemicalproperties described by individual partition coefficientswhich may or may not be linear with concentration.[40] Inany case, a small amount of analyte or matrix gas maypreferentially partition to the stationary phase in the GCcolumn packing material between injections of a givensample or between different samples. If retention is additivefor multiple injections of a given sample or for a sequenceof several like samples, the CO2 or matrix gas concentrationmay ’build up’ in the GC column. This can readily occur forpolar molecules such as H2O and is primarily addressed withthe use of Nafion tubing before and after the GC column inthe Gasbench II preparation device. In addition to continuousH2O removal through Nafion tubing, the Gasbench II operatingmanual[8] suggests that the user should periodically "bake out"the GC column to remove accumulated compounds.

The possibility of water influences should be considered,regardless of probable removal by Nafion tubing. As outlinedin the Introduction, water molecules can influence the carbonand oxygen isotope ratio measurements by exchangingoxygen atoms with CO2 molecules or acting as a protondonor resulting in the formation of HCO2

+ ions in the massspectrometer source. Oxygen isotope exchange betweenwater and CO2 can possibly occur during sample storage,transport to the mass spectrometer, or ionization in the massspectrometer. All samples reported in this study were



227

analyzed within 24 h of preparation in order to minimize thepotential for oxygen isotope exchange. Still this amount oftime could be sufficient for isotope exchange. This is not verylikely, however, considering relatively stable or non-systematic changes (drifts) in δ18O measurements over thecourse of all analytical sessions. In addition to the body ofanalyses as a whole, small temporal drifts in sample CO2

values can also be compared with drifts in monitoring gasisotope ratios. Monitoring gas CO2 would not be subject tooxygen isotope exchange with H2O molecules during samplevial storage or transport through the GC column, eight-wayvalve, etc. When present, slight drifts in the δ18O and/orδ13C values of the monitoring gas observed during individualanalytical sessions appear to be similar to sample CO2. Thissuggests that CO2 spending longer periods of time (up to24 h) did not exchange oxygen atoms with water molecules.Possible oxygen exchange occurring in the mass spectrometersource is discussed below in the "Oxygen isotopeconsiderations" section. Protonation of CO2 will be addressedhere. As specified above, the He used for sample vial matrixgas was of UHP grade. It is not likely that water wasintroduced from the He tank into the sample vials. Therefore,the similarity between blanking (He matrix only) and matrixexperiments (includes N2 and O2/N2 matrices) suggests thatprotonation due to the presence of water was also notoccurring during ionization following matrix experimentblanking.CO2 concentration was used to investigate background

effects due to residual gas elution from the GC column. Inthese experiments, varying the amount of analyte movingthrough the system may increase or decrease any backgroundinduced by CO2 eluting outside main analyte zones.Alternatively, higher concentrations of analyte used in 0.8%CO2 samples may be influenced less by source backgroundsdue to a higher signal-to-background ratio. Sample sequencewas also used to investigate background effects related to thenature of other variables being tested in a given analyticalsession. For blanking experiments, Gasbench II processingwhich discarded eluent He gas between analyte zones wasvirtually identical to Gasbench II processing which allowedall GC column eluent to be transferred to themass spectrometersource. Both CO2 concentration and analyte sequence effectswere absent from this set of experiments. These observationssuggest that measureable amounts of CO2 were not retainedin the GC column between injections of a given sample orbetween different samples. In other words, peak broadenedCO2 did not carry over and was not eluted outside its originalaliquot when only He was used as a matrix gas.No trend is also observed between sample CO2

concentration or sample analysis sequence and difference inaverage measured isotope ratios for matrix experiments. Hereagain, the GC column appears to adequately resolve CO2 andmatrix peaks while the Gasbench II blanking methodeffectively prevents transfer of matrix gas to the massspectrometer in order to prevent significant amounts ofmatrix (or ’tailing’ CO2) from influencing the measuredisotope ratios of analyte CO2. It does not appear thatbackground issues related to CO2 concentration or sampleanalysis sequence influence the measured isotope ratios forcases where blanking is utilized. In addition, the process ofblanking matrix gas does not appear to disturb the baselineof the chromatogram based on qualitative observations.


While the absence of blanking and introduction of matrix N2

or O2/N2 into the mass spectrometer source is the clear factorinfluencing measured isotope ratio offsets in controlexperiments, subtle CO2 concentration and/or sample sequenceeffects should also be evaluated. Analysis sequence differencesinfluence N2 matrix experiments by approximately 0.1‰ andO2/N2 matrix experiments by up to 0.2‰ for both carbon andoxygen isotope ratios. The δ13C values differ by approximately0.1‰ when comparing pCO2 in control experiments for allanalysis subsets except one. CO2 concentration has a moresignificant influence on the δ18O values during controlexperiments, however. The largest differences between averageδ18O values when comparing 0.3% CO2 samples and 0.8%CO2 samples were observed in O2/N2 matrix experiments.These differences are greater than 1‰ for both analysis sequenceexperimental sets. For these experiments, the lower CO2

concentration is affected to the highest degree and un-blanked0.3% CO2 analyzed from O2/N2 matrix gas was found todeviate from He matrix samples by the largest margins in theentire study. Comparatively, δ18O offsets between 0.3% CO2

and 0.8% CO2 were only �0.2‰ and �0.1‰ for sequences of5 samples and 13 samples, respectively. While the backgroundappears to be affected in most control experiments, themeasured isotope-ratio offsets are more closely related to CO2

concentration than analysis sequence.

Oxygen isotope considerations

Gonfiantini et al.[24] performed a series of isotopologue ionintensity measurements designed to assess atomic scramblingduring mass spectrometric analysis. Prior to the introductionof CO2 into the mass spectrometer, the workers mixed it withan isotope composition of natural abundance with CO2

enriched in 13C, 17O, and 18O so that the distribution of isotopesamong themixture ofmoleculeswas not at equilibrium.Duringanalysis, the workers observed CO2 isotopologue ion intensitypatterns that indicated significant redistribution of isotopestoward equilibrium or random assemblage. The observationsreported by Gonfiantini et al. suggest that source chemistry isa significant factor to consider when analyzing mixtures ofgases by IRMS. Under the conditions studied in the presentwork, control experiments indicate that measured oxygenisotope ratios for CO2 transferred from a matrix other than Heare more susceptible to error than measured carbon isotoperatios when matrix gas is not removed prior to analysis.However, these experiments alone do not provide evidencefor the root cause of this error. Higher offsets associated withO2/N2 matrices than with pure N2 matrices suggest matrixO2 is responsible for the differences but the exact mechanism(s) remain elusive. Possible explanations include (1) reactionsbetween O2 and CO2 which partition 16O from O2 into CO2 or(2) formation of nitrogen oxide isobaric interferences duringionization which disproportionately change m/z 46 comparedwith m/z 45, resulting in calculated δ18O values which areshifted to a higher degree than calculated δ13C values.

As outlined above and in the Introduction, the chemistryoccurring during ionization in a mass spectrometer source isstill not well understood and therefore difficult to interpretby considering the isotope compositions of materials used.However, the δ18O value of CO2 injected into exetainers hasbeen determined to be 2.1 ± 0.2‰ based on working standardcorrected He matrix analyses during matrix and blanking


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2272

experiments. As outlined above, the δ18O value of O2 used asmatrix gas has been determined to be �4.4± 0.3‰.Considering the relative abundance of 18O between the twomaterials only, it is possible to qualitatively evaluate δ18O shiftsto more negative values based on oxygen isotope exchangebetween CO2 and O2. A simple mass balance mixingcalculation gives δ18O values of �4.3‰ and �4.2‰ for 0.3%and 0.8% CO2, respectively, if the oxygen atoms were fullyand randomly exchanged between CO2 and O2 duringionization. This scenario does not account for any changes inCO2 ionization efficiency or the possibility of isobaricinterferences. While the measured δ18O values of CO2 in O2/N2

matrix experiments approach �4‰ for 0.3% CO2 subsets,samples with 0.8% CO2 have measured δ18O values closer to�2.7‰. These measured values and CO2 concentration trendsdepicted in Fig. 2 agree with an oxygen isotope exchange model.However, extensive oxygen isotope exchange would need tooccur to fully account for the observed shifts.Alternatively, nitrogen- and oxygen-containing isobaric

interferences which form/contribute to background levelscan be considered when evaluating possible sources ofmeasurement offsets. Nitrogen from N2 matrix experimentsmay bond with oxygen dissociated from CO2 molecules toformN2O orNO2 (NOx) in the source of themass spectrometer.A direct O2-N2 reaction mechanism seems more likely to formnitrogen oxide compounds for experiments comparing O2/N2

matrices. This scenario seems likely considering the magnitudeof offsets observed in O2/N2 matrix control experiments. Inaddition, the area of CO2 peaks in control experimentchromatograms also supports the formation of isobaricinterference compounds during O2/N2 matrix experimentscompared with pure N2 matrix experiments. The instrumentconditions were not consistent enough from one analysissession to the next to allow full quantification of CO2 peak areainfluences. However, average CO2 peak areas for m/z values of44, 45, and 46 for O2/N2 matrices were measured to be 13 to25% larger than those in corresponding N2 matrix experimentsunder the same CO2 concentration conditions. Figure 3 depictsthe chromatograms of 0.3%CO2 from an un-blankedHematrixand 0.3% CO2 from an un-blanked N2 matrix. The NOx peak isclearly visible in the N2 matrix chromatogram (Fig. 3(b)).However, peak identification settings for all experiments weresuch that the matrix peak areas were not measured. Uponinspection, the matrix peak areas formed during un-blankedN2 analyses were not noticeably smaller (compared with thereference CO2 peaks) than the matrix peaks formed duringun-blanked O2/N2 analyses.Based on the peak area observations presented above, it might

be suspected that all control experiments should have CO2 peakareas that are larger than the He matrix CO2 peak areas.However, this is not the case. Samples with both N2 and O2/N2

matrices are found to have CO2 peak areas that are 8 to 26%lower than those of He matrix samples analyzed during thesame analytical session and containing the same (0.3%) CO2

concentration in the sample container. This is intuitive if limitedsource ionization capacity is taken into account. For samples ofCO2 in He, only CO2 is ionized. However, background O2

and/or N2 will be ionized at the expense of CO2. Not all ionizedO2 or N2 will form isobaric interferences. Therefore, the netnumber of m/z 44, 45, or 46 ions will decrease when CO2 isionized in the presence of background O2 and/or N2. Lovelockdescribes this phenomenon eloquently in his work.[41]


Neither the oxygen exchange nor NOx formation isotoperatio alteration mechanism can be clearly identified as causingthe observed isotope composition shifts reported here.Probably a combination of oxygen isotope exchange betweenCO2 and O2 and isobaric interference accounts for inaccurateisotope ratio measurements during CO2 analysis in matrixgas other than He. It is possible that the contribution fromthese two processes could be qualitatively determined if anumber of CO2, O2, and N2 gases with different ranges inisotope composition were analyzed in a manner similar tothe experiments reported here and elsewhere.[24] As end-member examples, consider the following: changes in CO2

isotope composition due to atomic exchange would beminimized if analyte CO2 and matrix O2 of the same oxygenisotope composition were introduced into the massspectrometer source together. For the NOx possibility, if N2

and O2 of relatively heavy isotope compositions were usedfor the matrix gas, the potential to form m/z 45 and 46 isobarsand alter the measured CO2 δ

13C and δ18O values would begreater. Regardless of the mechanism, control experimentsclearly indicate that Gasbench II blanking is critical whenattempting to analyze CO2 from matrices other than He bythe continuous-flow technique.

CONCLUSIONS

Experiments exploring the effect of matrix blanking wereconducted on sample vials containing identical CO2 undera variety of different conditions. Matrix experiments onlyincluded CO2 in a He matrix. The parameter varied inthese experiments was the blanking gas-handlingprocedure that removes impurities when present. Thirteensets of approximately 78 analyses indicate that the δ13Cand δ18O values of blanked samples were both within0.2‰ of those of samples that did not experience blankingunder all conditions studied. Based on these observations,and considering the instrument precision under normalcontinuous-flow conditions (~0.1‰), it may be concludedthat the gas-handling procedure required for blankingdoes not significantly change the measured carbon oroxygen isotope ratios of CO2. A second set of experimentswas conducted utilizing the blanking gas-handlingprocedure to remove N2 or a 21:79 mixture of O2/N2 fromanalyte CO2 prior to analysis. Analyses of CO2 added to aN2 or O2/N2 matrix were compared with analyses ofidentical CO2 added to a He matrix. The results of thesematrix experiments were almost identical to those fromexperiments evaluating the blanking procedure. The δ13Cand δ18O values of blanked N2 and O2/N2 matrix sampleswere again both within 0.2‰ of those of CO2 sampledfrom a He matrix. Failure to remove matrix N2 or O2/N2

resulted in significant δ13C and δ18O value deviationsespecially for the oxygen isotope ratios of CO2.

Sample sequence and sample concentration sub-experimentsas well as the measured isotope ratio of matrix O2 gas wereused to evaluate sample vial sampling conditions andinstrument operating conditions leading to offsets betweenCO2 analyzed from different matrices. Background levels ofisobaric interference were not found to be sources of isotope-ratio offsets for analytical sessions that utilized blanking toremove matrix gas other than He. However, control



experiments that did not utilize blanking and allowed theintroduction of N2 or O2/N2 into the mass spectrometer didprovide evidence of isobaric interference which may have ledto measured isotope ratio offsets up to �4‰ in some cases.The exact mechanism of δ value discrepancies cannot bedetermined by the methods used in this study. However,independent determinations of the oxygen isotope ratios ofCO2 used to produce experiment samples and O2 used as theexperiment sample matrix offer evidence for another source ofcontrol experiment offsets. The measured oxygen isotope ratiosof CO2 sampled from an O2/N2 matrix were determined tohave δ18O values between those of the source CO2 and matrixO2. This finding may indicate that oxygen isotope exchangereactions between CO2 and O2 are occurring in the source ofthe mass spectrometer prior to mass-to-charge ratiomeasurements. Probably a combination of oxygen isotopeexchange between CO2 and O2 and isobaric interferenceaccounts for inaccurate isotope ratio measurements duringCO2 analysis with matrix gas other than He.Considered as awhole, this study confirms that a sample vial

sampling procedure including the removal of potential isobaricinterference-formingmatrix gas does not significantly affect themeasured carbon or oxygen isotope ratios of analyte CO2. Theresults further indicate that a gas stream experiencing blankingof matrix gas other than He is preferable to introducingpotential isobaric interference-producing compounds to theion source. Analysts utilizing the blanking functionality of aGasbench II coupled to CF-IRMS for atmospheric-type samplesshould feel confident that the measured carbon and oxygenisotope ratios are probably not being influenced by samplematrix or CO2 purification processes.

AcknowledgementsThe author thanks Dr Suvankar Chakraborty for instrumentmaintenance assistance and insightful discussions. Threeanonymous reviewers are acknowledged for their helpfuland constructive comments on an earlier version of thismanuscript. This work was supported by NASAHeadquarters under the NASA Earth and Space FellowshipProgram – Grant NNX12AN84H – as well as the NASAAstrobiology Institute.

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Sample matrix effects on measured carbon and oxygen isotope ratios during continuous-flow isotope-ratio mass spectrometry