RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2001; 15: 501±519

REVIEW Referencing strategies and techniques in stable isotope ratio analysis

Roland A. Werner and Willi A. Brand* Max-Planck-Institute for Biogeochemistry, P.O. Box 100164, 07701 Jena, Germany Received 5 December 2000; Revised 6 December 2000; Accepted 23 December 2000

SPONSOR REFEREE: T. B. Coplen, US Geological Survey, 431 National Center, Reston, VA 20192, USA

Stable isotope ratios are reported in the literature in terms of a deviation from an international standard d-values). The referencing procedures, however, differ from instrument to instrument and are not consistent between measurement facilities. This paper reviews an attempt to unify the strategy for referencing isotopic measurements. In particular, emphasis is given to the importance of identical treatment of sample and reference material `IT principle'), which should guide all isotope ratio determinations and evaluations. The implementation of the principle in our laboratory, the monitoring of our measurement quality, the status of the international scales and reference materials and necessary correction procedures are discussed. Copyright # 2001 John Wiley & Sons, Ltd.

Roughly 15 years ago a new and relatively economic educating students and also newcomers to the exciting field chromatographic method to measure stable isotope ratios of high precision stable isotope ratio measurements. In with high precision became commercially available. Carrier particular, we want to emphasize techniques for reliable gas or on-line isotope ratio analysis systems now cover a standardization in carrier gas or on-line techniques where an wide range of applications and have led to a dramatic accepted protocol for assigning d-valuesa on an internation- increase in the number of stable isotope ratio measurements ally accepted scale has not yet been defined. We feel worldwide. Although this has been a benefit to this field of particularly suited for this task because we were given the science as a whole, it has also created a number of problems. opportunity to start a large isotope ratio measurement We feel that the depth of knowledge that the few original facility from scratch two years ago and thus had to develop experts possessed has been diluted considerably over the all the techniques and protocols for such an endeavor years with the consequence that the overall precision and recently. reliability of isotope ratio values may have declined. In particular, the accuracy of reported d-values relative to an Stable isotope ratios and their international international standard may have suffered from the vast scales increase in analyses made. The stable isotope ratios measured most widely include On the other hand, such a large increase in the number of 13C/12C, 15N/14N, 18O/16O and 2H/1H 1or simply D/H). analyses offers the opportunity to establish fully automated Other less frequently measured ratios are inter alia 17O/16O, analysis sequences that include a proper referencing 34S/32S, 33S/32S. The common feature of these ratios is that strategy. This opportunity should enable the reliability of they can be determined using a few light gases 1CO2, CO, N2, isotope ratio value assignment to be improved. Although O2,H2, and SO2). Hence they share a common technology this has happened to a certain extent in many laboratories, termed 1Gas) Isotope Ratio Mass Spectrometry. referencing strategies have certainly not been unified The principle of the classical method is very easy to throughout the community. understand: Two gases are stored in containers connected This review will be of considerable value both in via capillaries to a switching unit, the `changeover valve'1 1Fig. 1). The latter serves to direct one of the gases to the ion source of an isotope ratio mass spectrometer 1IRMS)2,3,4,5,6 *Correspondence to: W. A. Brand, Max-Planck-Institute for Biogeochemistry, P.O. Box 100164, 07701 Jena, Germany. while the other gas flows to a waste vacuum line, and vice E-mail: [email protected] versa. The ion currents are measured separately from both E-mail: [email protected] gases and compared a number of times. The measured a The variation of stable isotope ratios in nature is small. The small relative difference in ion current ratios is then calculated differences are conveniently expressed as delta values 1d) in per mill [%] relative to an internationally agreed isotope ratio scale. deviation from a reference2,3,8 according to Table 1 is a compilation of the international stable isotope d‰%Šˆ Rsa=Rref À 1†Á1000 1† ratio scales in use together with the presently accepted

Rsa and Rref are the sample and reference isotope ratios, respectively. absolute ratios and their errors. When comparing samples

DOI:10.1002/rcm.258 Copyright # 2001 John Wiley & Sons, Ltd. 502 R. A. Werner and W. A. Brand

eous belemnite material was picked from the Pee Dee formation in South Carolina, hence `PDB' for Pee Dee Belemnite. The original material no longer exists. It has been replaced by assigning exact d-values 1both d13Cand d18Ob)to another carbonate 1NBS-19) relative to PDB.10,11 This new scale is termed `VPDB' 1Vienna PDB) in recognition of the role that the International Atomic Energy Agency 1IAEA), located in Vienna, has played in redefining the PDB scale. The IAEA played a similar role for the other d-scales that have a `V' preceding the original scale name.

Unfortunately, isotope ratios of CO2 must be determined using the molecular ion masses 44, 45 and 46. The 13C information comes in disguise. It is available from the 45/44 ion current only after subtracting the 7% contribution from 12C17O16O‡ 1see `Correction of isobaric interferences'). This complicates the exact comparability of d13Cvalues between different laboratories.

Oxygen High precision isotope ratio measurements of oxygen were

first made on CaCO3,O2 and H2O. Again it was Urey's Figure 1. Schematic representation of a dual inlet system group that developed these applications for paleoclimate featuring the ‘Changeover Valve’ as the classical referencing reconstruction.2,19,20,21 The two accepted international scales technique in Stable Isotope Ratio Mass Spectrometry. While the 1VSMOW and VPDB) have developed over time. A variety of gas in one volume is flowing to the mass spectrometer, the gas in carbonate 1including PDB) and water standards were used other goes to a waste line and vice versa. until 1961, when Harmon Craig proposed SMOW 1Standard Mean Ocean Water). SMOW isotopically represents the hydrosphere with the world oceans as the largest reservoir of with one of the standard materials, however, the errors are oxygen 1and hydrogen). Interestingly, the standard that generally considerably smaller owing to the fact that a Craig proposed was not available as a reference material. It relative measurement has far fewer sources of error than an had a d-value that was believed to represent average ocean absolute determination. water based on experience accumulated over time. It was precisely defined relative to the 1now exhausted) water Carbon standard NBS-1 that was almost 8% off the new d18O-scale The international scale for 13C/12Cstarted as a carbonate and 47.6% off the simultaneously defined dD-scale. Both laboratory standard for oxygen and carbon isotope ratios in Craig and the IAEA engaged in developing a reference the group of Harold Urey at the University of Chicago in the material with an isotopic composition of SMOW, which led early 1950s.7,8 Being representative for carbon in the litho- to the current VSMOW scale. This material presently is still sphere that precipitated from the world oceans, PDB was available in limited amounts from the IAEA in Vienna. A later proposed as the international reference material for the new batch of reference water matching the VSMOW scale as carbon and oxygen d-scale by Harmon Craig.9 The Cretac- closely as possible in all isotope ratios is currently being prepared. 18 16 O/ O ratios are measured using CO2 after appropriate b 18 13 d ONBSÀ19 À2.2% vs. VPDB;  CNBS À 19 ‡1.95% vs. VPDB conversion from the original material. Due to the fact that the

Table 1. International isotope ratio scales12

Isotope ratio International scale Accepted ratio [Â106] Error of ratio Error of ratio [%] Ref.

13C/12C VPDB 11180.2c Æ2.8 Æ2.5 Chang and Li13 18O/16O VSMOW 2005.2 Æ0.45 Æ0.22 Baertschi14 VPDB 2067.212 using ‡30.92% 17O/16O VSMOW 379.9 Æ0.8 Æ2.11 Li et al.15 VPDB 386.012 with ‡30.92% and  = 0.52 15 14 16 N/ N AIR-N2 3678.2 Æ1.5 Æ0.41 DeBievre et al. 2H/1H VSMOW 155.75 Æ0.08 Æ0.51 De Wit et al.17 34S/32S VCDT 44150.9 Æ11.7 Æ0.27 Ding et al.18

Isotope ratios are reported as d-values in per mill 1%) deviations from the origin of the respective international scale. Please note that for oxygen two internationally accepted scales coexist. c Chang and Li report a 13C/12Cvalue of 0.011202 for NBS-19. The d13C- value of NBS-19 is ‡1.95%, de®ning the VPDB scale. The previously established 13C/12CPDB ratio value 9 of 0.0112372 is 5% higher.

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 501±519 Referencing techniques in IRMS 503 behavior of terrestrial 17O and 18O are closely linked via Sulfur 17 16 mass fractionation laws, O/ O ratios 1measured using O2 Sulfur is most commonly measured as SO2 gas on the gas) are analyzed only occasionally. molecular ion masses 64 and 66. Unfortunately, the isotopes

When carbonates or CO2 in air are measured, oxygen of oxygen directly interfere with the sulfur isotopes 1isobaric isotope ratio values are preferentially reported on the VPDB interferences) at these mass positions and thus cannot be or VPDBgas scale and this can lead to confusion when the measured independently 1the ion current at m/z 66 comprises 34 16 ‡ 32 16 18 ‡ scales are not stated explicitly. The reason for the coexistence S O2 and S O O ). Using SF6 as the measurement gas of two oxygen scales is that measurements of d-values avoids this problem, but preparation of SF6 requires fluorine against a chemically identical or similar reference can in gas as a reagent and preparing clean SF6 is difficult. Hence, principle be made with enhanced precision. VPDB has a SF6 has found only limited use. The international d-scale is 18 22 26 d O value of ‡30.92% on the VSMOW scale. The CO2 gas defined by VCDT 1Vienna CanÄ on Diablo Troilite) The 28 `developed' from hypothetical VPDB 1= VPDBgas)at25°C original CDT, adopted as the primary standard in 1960, 23,d using pure, water-free H3PO4 is 10.25% heavier; it has a was prepared from the FeS phase of a large iron meteorite d18O value of ‡41.5% on the VSMOW scale. It should be found at Meteor Crater, Arizona. Improved measurement noted that d-values can not be added or subtracted in a precision has revealed that CDT is not sufficiently homo- simple way when referring to different standards.e geneous27 to be continued as the primary reference material and, as a consequence, the CDT scale has been replaced with 34 Hydrogen VCDT. VCDT is defined by assigning a d S-value of À0.3% 1exactly) to IAEA-S-1 1formerly NZ-1, Ag S).26 It is worth The isotopes of hydrogen have the largest relative mass 2 noting that the absolute 34S/32S ratio in VCDT given in Table difference. Since most isotope fractionations in nature 1 differs from the previously established value 145004.5)28 for roughly scale with relative mass difference, the largest CDT by about À20%. This affects the correction for 18O variations in terrestrial isotope ratios are found for hydro- contribution 1see `Correction of isobaric interferences'). gen. Although the large mass difference makes the isotope signatures easier to detect, the low abundance of deuterium 1only about 150 ppm in ocean water) can create problems Referencing techniques in isotope ratio mass during high-precision analysis. Consequently, two interna- spectrometry and the principle of identical tional reference materials are in frequent use to cover the treatment natural range of isotope ratios for D/H. The basis of the Dual inlet system and changeover valve. The breakthrough in international scale is the same as oxygen, VSMOW. The other classical isotope ratio mass spectrometry was the dual inlet reference material is a water sample named SLAP 1Standard mass spectrometer, introduced by Urey in 19482 and Light Antarctic Precipitation) that is, relative to VSMOW, described by McKinney et al. in 1950.3 Despite significant more than 42% depleted in deuterium 1À428%). The improvements in electronics, vacuum system design and full difference between VSMOW and SLAP is used to correct computerization, the basic principles embodied in this mass for the nonlinear behavior of instrumentation and 1or) spectrometer still form the basis of modern stable isotope sample preparation during analysis. ratio mass spectrometers for high-precision analysis of clean analyte gas. Nitrogen A key feature of the McKinney instrument is the `change- 13 18 1 Unlike the d Cand d O values in atmospheric CO2, the over valve' 1Fig. 1), which allows continuous flows of 15 d N value of atmospheric N2 does not change within reference and sample gas to be alternated between the measurement precision over time or space.24,25 This is due to mass spectrometer and a waste line vacuum pump. The the fact that the atmospheric pool of nitrogen is by far the `changeover valve' is the classical referencing technique. largest of all nitrogen-bearing pools on earth and thus cannot Together with the dual variable volume 1or mercury piston) be altered significantly by known processes. It participates in reservoir system for introduction of sample and reference all natural processes as source and sink so that the net gases, it allows a direct comparison of ion currents and ion change over time is close to zero. Consequently, AIR-N2 has current ratios. Instrumental effects like temporal fluctua- been adopted as the international reference material for all tion of sensitivity or temperature drifts cancel almost nitrogen isotope ratio analyses. This does not imply that the completely. isotope ratios are easy to measure. From air, N2 must be In order to avoid isotopic composition changes of the gas isolated without isotopic fractionation for use as a primary in either reservoir over time due to molecular effusion into reference. For convenience 1and thus enable better data vacuum, the connection between the gas reservoirs and the comparability) the IAEA has made secondary standards `changeover valve' is made with thin capillaries 10.1±0.2 mm 1mostly ammonium and nitrate salts) available for direct use i.d.) of about 100 cm length that are crimped near the end.29 in sample preparation lines involving combustion. The hole right at the capillary crimp also constitutes a molecular leak. However, provided the pressure in the d More recently, a value of 10.44% has been reported by Kim and capillary is sufficiently high 1typically >50 mbar for hydro- O'Neil.52 gen and >15 mbar for other gases), the flow through the e The d-value for a sample 1sa) measured against a working standard capillaries is viscous, not molecular, and thus prevents the 1ws) on an international scale 1is) is given by isotopically enriched gas at the crimp from diffusing back 4 dsa=is ˆ dsa=ws ‡ dws=is ‡ dsa=ws  dws=is=1000 2† into the reservoirs. This viscous flow condition establishes a

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 501±519 504 R. A. Werner and W. A. Brand

Table 2. Measurement of two CO2 gas samples by different laboratories d13C[% VPDB]

Sample CIO INSTAAR CSIRO Heidelberg Scripps mean std. dev.

GS-19 À7.502 À7.517 À7.516 À7.539 À7.465 À7.508 0.0273 GS-20 À8.622 À8.624 À8.617 À8.635 À8.575 À8.615 0.0231

18 d O[% VPDB-CO2]

Sample CIO INSTAAR CSIRO Heidelberg Scripps mean std. dev.

GS-19 À0.193 À0.790 À0.617 À0.426 À0.126 À0.430 0.280 GS-20 À0.991 À1.580 À1.380 À1.191 À0.915 À1.211 0.274

lower limit to the minimum amount of gas that can be general conclusion can be reached. Several points need to be determined with high precision. Small amounts of gas may considered: be frozen into a small volume 1`microvolume') in front of the . The capillaries to the `changeover valve' are carefully capillary. Making this reservoir as small as is physically heated and the inlet system is conditioned until the ratios possible 1250 mL) and operating at 15 mbar results in a for the same gas, introduced to both reservoirs, is close to minimum sample size of about 4 bmLf gas. zero 1`zero enrichment test').g However, in the real world, Using modern dual inlet mass spectrometers it is possible the two gases are different and may even differ in their to obtain a precision of 0.01% for d13C1and similar values for trace gas impurities. Traces of water or other protonation other isotope ratios) by comparing the ion current ratios of agents can give rise to an isobaric interference of CO H‡ the gases in both reservoirs a number of times for several 2 1m/z 45) resulting from ion/molecule reactions in the ion seconds. The mean is calculated and such measurements are source. Similar protonation reactions are possible for other compared a number of times during the course of a day. gases resulting in H‡ or N H‡. However, precision is not the same as accuracy. On the next 3 2 . The isotopic composition of a sample and reference gas day the results may again be very precise. The mean, should not be very different; however, in reality, they however, may differ from the mean from the previous day, always will be and, as a consequence, balancing the major or from the previous week, month, or year. ion beam means that the minor beams will have different The situation is illustrated by results from a recent readings. If the response of the measurement channels is intercomparison of CO gas initiated by the Groningen 2 not perfectly linear 1and it never will be), this is a source of Centre for Isotope Research 1CIO).30 The ring test involved error that can vary with time. two gases 1GS-19 and GS-20) sent to five prominent . The measurement precision and results for d18O from CO laboratories for isotopic determination. The results from 2 gases depend critically on the nature and the cleanliness of the ring test are given in Table 2. the internal surfaces of the reservoir and the transfer The precision of the d13C-values is approx. 0.025% and capillaries. Not only can CO exchange oxygen with appears acceptable, whereas the d18O data show an 2 surface water, it may even exchange with other forms of unacceptably large scatter. This finding is attributed to loosely bound oxygen on the surface and hence cause different referencing methods or materials in the labora- uncontrolled isotopic fractionation. This may be different tories. If GS-19 is used as the reference 1d-value set to 0.0%) for the two capillaries and may change over longer time and GS-20 measured against it, the precision of the reported periods. differences improves to 0.009% for d13Cand 0.016 % for . Different mass spectrometers 1even from the same manu- d18O. Through direct referencing, the d13Cresults are facturer) can have several sources of error in determining a improved by a factor of 3 and the d18O situation improves given isotopic difference. This has been shown for almost 20-fold. hydrogen where the isotope ratio differences are often How can the precision that these mass spectrometers are large. It is also important for other gases like CO when the obviously able to deliver be conserved and converted into an 2 measured d-values differ by 10% or more and the required accuracy figure over long time periods? How can different accuracy is high. mass spectrometers deliver results that can be compared reliably at a precision level of 0.01%? It is common practice in laboratories that strive for the These questions require some discussion before a more highest long-term precision and accuracy to have sample and reference gas measured from the same 1sample) reservoir of the dual inlet system. The gas in the reference f 1bmL 1`bar microliter') is an amount of 1 mL gas at STP. volume serves as a temporary mediator between the g Please note that crimping of the capillaries will not necessarily result in different runs. Proceeding in this manner, sample and the same signal height for the same gas pressure on both sides. In fact, reference gas are handled in a highly comparable, almost when referencing through the sample line 1`IT principle'), it is more important to have a long lifetime for the standard gas and thus make the equal, fashion and, as a result, some 1but not all) of the errors crimp for the capillary tighter on this side. discussed above tend to cancel out.

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 501±519 Referencing techniques in IRMS 505

potential for isotopic fractionation is encountered in almost every detail of the reaction including the type of vessel, the temperature, the amount of reduction material, and the timing sequence of the reduction reaction. Hence, running reference water samples concurrently with unknown sam- ples in every batch is required in order to obtain reliable results.

Isotope ratio monitoring techniques Isotope ratio monitoring 1`irm') encompasses all techniques for high-precision determination of stable isotope ratios that employ a helium carrier gas for transferring the analyte gas as a transient into the isotope ratio mass spectrometer.31 The term `irm-GC/MS' was introduced by D.E. Matthews and J.H. Hayes in their pioneering paper in 1978.32 Popular acronyms in common use for specific irm couplings are GC/ C/IRMS 1GC/combustion/IRMS) and EA/IRMS 1elemental analyzer/IRMS). The techniques were originally developed to measure 13C/12Cand 15N/14N. More recently, D/H, 34S/32S and 18O/16O ratio determination has become available, accompanied by a host of new acronyms. In irm systems the sample gas leaves the appropriate Figure 2. Referencing technique in isotope ratio monitoring sample preparation and separation device as a peak applications. Helium enters a small glass tube through a fused- entrained in helium. The chromatographic separation not silica capillary. Another fused-silica ‘sniffing’ capillary (usually only ensures that the analyte gas is pure or coming from a 1000 mm long and 0.1 mm i.d.) connects to the ion source of the single precursor, it also separates the stable isotopes 1i.e. mass spectrometer. Reference gas pulses are generated by isotopomeric molecules) of the analyte to a certain extent. In moving a third capillary between two positions, one upstream and irm-GC/MS applications the separation often exceeds the other downstream from the sniffing point. 100 ms between e.g. the 13Cmoiety of the analyte, which elutes first, and its 12Ccounterpart. 31,33 Referencing can be carried out in a number of ways. The most common method is to inject pulses of reference gas The IT principle directly into or in parallel to the GCeffluent stream. 34 The We will refer to `Identical Treatment' of sample and time window or windows for the reference gas pulses must reference material as the `IT principle' for relative measure- be carefully selected in order to avoid interference with any ments. The IT principle, which does not apply exclusively to analyte material entering the mass spectrometer concur- the dual inlet technique, serves excellently for maintaining rently. Other methods include the admixture of reference long-term laboratory performance records for other isotope compounds to a GCmixture 1internal standards) or, less ratio measurement techniques as well 1see `Performance favorably, the direct comparison of ratios from chromato- charts'). gram to chromatogram. It is rare that the measurement gas is identical to the Figure 2 shows schematically an example of a reference original sample. Typically, a carbonate, a water sample, an gas injection mechanism. Inside a closed, thin 1<1 mm) glass organic compound, a piece of bone, soil, hair or a gas mixture tube, the effluent stream from the interface is fed in etc., is selected from which the measurement gas has to be continuously with a fused-silica capillary. Downstream a produced or extracted. During this transformation it is second fused-silica capillary acts as the transfer line to the critical to avoid isotopic alteration. In carbonate analysis or mass spectrometer. Usually, this capillary has an internal water equilibration the IT principle has been common diameter of 0.1 mm and a length of 1000 mm. Together with practice for a long time. Carbonates are effectively standar- the pressure drop from atmosphere to about 10À5 mbar, this dized by running carbonate reference material alongside the capillary allows for a flow of about 400 bmL per minute. The samples on a daily routine basis. Water samples are flow through long, thin capillaries under viscous flow equilibrated together with reference water standards in the conditions follows the law of Hagen-Poisseulle. For a given same temperature-controlled bath. Because the isotopic pressure difference the flow is linked to the 4th power of the fractionation occurring in the CO2 liberation or the isotope radius and linearily to the length of the capillary. Hence, transfer reaction to the gas phase is strongly temperature when doubling the radius, the flow will increase by a factor dependent, the reference material is subjected to the same of 16 while keeping the other parameters constant. The third reaction conditions. The isotopic fractionation cancels as capillary 1see Fig. 2) serves to inject pulses of reference gas. long as the chemical nature of sample and reference is Using a piston mechanism it can be switched between two closely comparable. The same argument applies to water positions: one upstream and one downstream from the reduction methods for hydrogen isotope ratio analysis. Here, sniffing point of the transfer capillary. Only the upstream irrespective of the specifics of the reduction process, the position will deliver reference gas to the sniffing point.

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 501±519 506 R. A. Werner and W. A. Brand

Figure 3. Graphical output of a irm-GC/MS system, in this case from a Gasbench II run (see d 18O determination). Three GC traces are monitored simultaneously with different sensitivity factors. The upper trace is an overview over the whole run. The middle window (‘45/44 Ratio Trace’) represents the instantaneous 45/44 ion current ratio. The first, flat-topped peak represents a post column injection of reference gas. For the sample peaks, please note the isotope swing going positive first which is indicative of the partial separation of the isotopomers on the GC column. Reference pulses do not show this behavior. At the flanks they exhibit a reaction to the fast changing intensity due to differences in the amplifier time constants.

Downstream the helium carrier will sweep away any effuent reference is pure gas. The principle governing long-term from capillary three. Figure 3 shows an example of a simple precision and, more importantly, accuracy requires that GCrun 1in d13Cmeasurement mode). The first, flat-topped sample and reference be identical in nature and follow the peak is a reference gas injection lasting for about 30 s. The same sample isolation and conversion pathway. Hence, a n- other peaks are from sample gas analyses. Their shape is heptadecane sample should be referenced with a n-hepta- typical for this type of gas chromatography. Please note the decane reference. variation of the instantaneous 145/44)-ratio along each GC Often gas chromatograms are very complex. The conver- peak going positive first due to the 13Cmoiety eluting earlier sion efficiency of n-heptadecane should be very close to n- than the pure 12Ccompound. hexadecane or n-octadecane. Even an n-C25 alkane should The principle of moving capillaries in the open split region be a valid reference point provided the stability of the mass has been utilized in a number of ways. Multiple reference spectrometer is high and the conversion unit reliably works gases can be introduced, the GCeffluent gas can be diluted close to 100% yield. Hence, even whole chromatograms can with helium further to cover a wider dynamic range35 or the serve as references in pursuit of the IT principle. In this case, transfer capillary may be switched into a clean helium reference gas pulses again take the role of a mere mediator cushion for `heartcutting' 1i.e. selecting or selectively between runs, very much as the standard gas in dual inlet discarding) GCpeaks. analyses. In principle, the number of reference runs is Other means of injecting reference gas pulses include determined by the precision required for the experiment in rotary valves equipped with an injection loop or carefully question. designed multiple valve systems. The drawback common to The workhorse in most modern isotope ratio laboratories all of these systems is that during switching a short is the combination of an elemental analyzer with isotope interruption of the continuous flow of carrier gas occurs ratio mass spectrometry 1EA-IRMS or irm-EAMS, sometimes which leads to problems in the background definition of the termed BSIA4 for bulk sample isotope analysis). As with irm- reference gas peaks and, hence, to reduced precision. GC/MS, the immediate or working referencing is commonly It is important to note that referencing solely relative to made through co-injected reference gas pulses into the reference gas pulses is an immediate violation of the IT effluent stream. Earlier commercial versions of this instru- principle introduced above. Sample peaks are generated mentation relied solely on comparing from sample to from original material going through a combustion/separa- sample, without reference gas peaks. However, precision tion or separation/combustion step whereas the 1working) of a single measurement is improved when comparing

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 501±519 Referencing techniques in IRMS 507 standard and reference more closely in time. Data security is daily basis. Primary reference materials in general are in an issue when a single result relies on the measurement of short supply and their commercial availability is restricted. the whole sequence. For d13Canalysis using a combustion technique, NBS-22 In order to work under the rule of the IT principle 1À29.78%h vs. VPDB) is a valid reference material for almost individual sample results versus a gaseous working stan- any purpose. The secondary laboratory standard should be dard can be compared with results from reference material chosen according to the following criteria: that has passed the full sample line in the same sequence of . easy to handle during weighing or other preparatory steps. measurements. . preferably a pure single chemical compound. Day-to-day working environment for high- . isotopically homogenous down to the smallest amount precision isotope ratio analyses and generation of used during analysis. performance charts . stable and constant in isotopic content over a long time period. For liquids, in particular water, large quantities are In our laboratory we have implemented a number of routine advantageous in order to minimize evaporation losses. The measurement schemes for the following types of analyses: isotopic composition should be stable versus frequent use.i . d13Cfrom bulk solid 1and liquid) material 1irm-EAMS . easy to replace if exhausted. system) . chemically identical or close to the samples to be . d15N from bulk solid 1and liquid) material 1irm-EAMS measured. This point must be evaluated in terms of the system) corresponding analysis. It is probably easy to use some oily . dD from water samples 1Cr-reduction/dual inlet) material like NBS-22 for referencing almost any bulk 18 13 . d O from water samples 1CO2/H2O equilibration fol- combustion 1d C). However, using carbonates for referen- lowed by irm analysis) cing water samples is more difficult if not impossible in 13 18 18 36 . d Cand d O from CO2 in air samples 1cryogenic pyrolysis systems 1d O). separation/dual inlet, in development) . the isotopic composition should be in the range of the

. O2/N2 ratios in air samples 1changeover system with open samples to be measured. Isotopic differences can be split, in development) measured most precisely when small. Because most natural organic material is produced primarily through The first step in setting up routine protocols is the C -photosynthesis, d13Cvalues around À25% vs. VPDB establishment of effective laboratory standards that can be 3 are recommended for the laboratory standard. used and monitored on a daily basis. General rules for . non-hygroscopic. This is of particular importance for laboratory standards are difficult to fix, they largely depend oxygen and hydrogen isotope ratio analysis using high- on the type of analysis. temperature carbon reduction 1`pyrolysis') systems. The second step is the development of consistent analysis sequences, i.e. the decision how on a daily basis samples, reference material, and blanks 1if applicable) are positioned irm-EAMS in a sample carousel or autosampler. In this respect, We analyze any bulk material that can be combusted economic aspects, such as the number of samples per day quantitatively to CO2,N2 and H2O1‡ other oxides if and week, scheduling of routine maintenance, number of applicable) using the combination of an elemental analyzer samples per reactor, etc., are of importance and must be 1NA 1110, CE Instruments, Milan, Italy) with an isotope ratio considered with great care and separately for every type of mass spectrometer 1DELTA‡XL, Finnigan MAT, Bremen, analysis. In our laboratory we have tried to adjust the Germany). The coupling interface is a ConFlo II2, modified measurement cycles to the diurnal and weekly cycle of the in-house to a ConFlo III,35 which includes two open splits, human operators, to optimize throughput without sacrifi- one for the coupling, the other one for reference gas cing precision. introduction. The coupling split can be varied over a wide range from zero to 64-fold dilution of the effluent stream Selection of laboratory reference materials without causing detectable isotopic fractionation. Two

After having obtained the relevant primary reference reference gases 1mostly CO2 and N2, alternatively H2 and material from the IAEA or other agency, secondary or CO) can be actuated under computer control. H2 and CO laboratory standards must be prepared for every day use. reference gases are used when the system operates in high- The IAEA recommends not using the original material on a temperature `pyrolysis' mode with a Finnigan MAT TC/EA for D/H or 18O/16O isotope ratio analysis. The operation of h The previously recommended value of À29.74% was adjusted to the system generally follows a well-defined protocol. À29.78% by the 8th Advisory Group Meeting on Future Trends in Stable Samples, reference material aliquots, and blanks are Isotope Reference Materials and Laboratory Quality Assurance, IAEA, Vienna, Sept. 18±22, 2000. weighed and filled into the 32-position autosampler carousel i Carbonates can exchange oxygen isotopes with air-CO2. This effect according to a loading list 1Fig. 4). Please note that each list depends on grain size, humidity, nature of the carbonate, and frequency represents samples for three carousels 1we use 96-position of opening when the material is kept under inert gas. Water reference material can alter its isotopic composition through evaporation and sample trays for storing samples in a desiccator before transpiration through container walls. Hence, the frequency of opening measurement). It serves as input for the sequence informa- the reference water container can affect the isotopic stability. GC tion in the mass spectrometer control software. injection mixtures may change their isotopic composition and concen- tration via evaporation when low boiling point material is used as a The prefilled positions in the loading list are mandatory. reference. They are used in a post-run off-line evaluation on a

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 501±519 508 R. A. Werner and W. A. Brand

Figure 4. Typical loading list for sequential isotopic analysis of samples and reference materials in a prescribed fashion. The 96 positions (‘line’) on this list represent samples for three carousels with 32 positions each.

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 501±519 Copyright # 01Jh ie os Ltd. Sons, & Wiley John 2001 ai omn asSpectrom. Mass Commun. Rapid eeecn ehiusi RS509 IRMS in techniques Referencing 2001; 15 501±519 : Figure 5. Spreadsheet calculation of data from the irm-EAMS system showing the evaluation of the raw data to the final d-values on an international scale. See text for further explanations. 510 R. A. Werner and W. A. Brand

Figure 6. Performance Chart. d13C-values of a quality control standard as a function of measurement time. The scale is based on reference material that went through the same preparation channel as the QA standard. Each sequence of 32 samples measured holds one QA standard that is represented by a single point. standardized spreadsheet 1Fig. 5) for assigning the final d- is used to correct all data 1Eqn. 12). The data in column 13 are values on the respective international scale. the final d13C-values on the VPDB scale. The last two The spreadsheet evaluation37 starts with the original columns are average and precision of the selected Ali-j1 data. values directly transferred from the host computer 1`ISO- In principle, this procedure represents a one-point DAT' from Finnigan MAT). Columns 1 to 5 1Fig. 5) hold calibration of the d13C-values; our scale expansion factor is information about the sample including the sample name 1.000. For measuring larger differences in isotopic composi- and weight, the integrated peak area and the measured d13C- tion it will be necessary to include a scaling factor in the value based on the injected reference gas. The average peak calculations 1see Fig. 10 and the discussion in `Further area 1Vsec) and d-value 1%) of the blank measurementj are corrections'). used to correct the data for blank contribution applying a Not shown in Fig. 5 are further routine calculations of the simple mass balance correction. carbon elemental contents based on the measured peak area and the known carbon content of the reference material 1Ali- 13 13 13 15 d Ctot  areatot ˆ d Csa  areasa ‡ d Cblk  areablk 3† j1, 71.09% C). The d N analyses are made in a completely analogous fashion. The suffixes in Eqn. 13) refer to the total 1tot), sample 1sa) and blank 1blk) properties, respectively. Generation of performance charts Usually the correction is very small. The data in column 8 We use one dedicated sample position for determining a QA 1Fig. 5) represent the blank-corrected preliminary d13C- standard 1quality assurance), in this case Caf-j1 1a caffeine values 1%) when the reference gas is used for standardiza- sample from a `Traube synthesis' in larger supply), in every tion. The following columns denote the laboratory standards sample carousel we run. We have chosen this material 13 selected for further correction and final positioning of the because it is off the usual d Cvalues for C 3-plants by about data onto the VPDB scale. Ali-j1 1Acetanilide-Jena1) has been À20%. With this QA standard, which does not enter the determined directly versus NBS-22 oil and USGS24 graphite reference calculation, the long-term performance of the irm- a number of times and has been assigned a d13C-value of EAMS line has been monitored 1Fig. 6 for d13Cand Fig. 7 for À33.94% on the VPDB scale. The measured average d13C- d15N). value of the selected Ali-j1 samples in Fig. 5 is À34.01%. Figures 6 and 7 are representative for all measurements Thus, there is an offset of 0.07% to the accepted d-value that made on our irm-EAMS systems 1in total from three different mass spectrometers and two different interfaces) 13 j We found that the carbon blank in our laboratory mostly has a d13C- since we started routine analyses. We measured both d C value around À25% vs. VPDB. The major contributions are carbon in the and d15N from the same sample until April 1999. From the tin containers and memory in the combustion reactor. Often the blank 13 performance charts we noticed that the accuracy, especially area is so small that the d Cmeasurement is erroneous 1outside the 15 natural abundance range). In such cases we replace the wrong d13C-value for d N, was not satisfactory and needed improvement. of the blank by our average value, À25%. Careful analysis of the data indicated that the problems were

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 501±519 Referencing techniques in IRMS 511

Figure 7. Performance Chart: Same as Fig. 6 for d15N-values

15 caused by tailing of the CO2 peak into the nitrogen using irm-EAMS altogether. For d N determination we now chromatogram of the subsequent sample. Most materials trap CO2 in a chemical 1Ascarite) trap installed between the analyzed had an elemental content of 2% nitrogen and water trap and the GCcolumn of the elemental analyzer.

40% carbon. For analysis the CO2 had to enter the GC The performance or quality chart is also the perfect tool to column 1Porapak Q) and, due to the relatively large amount, monitor the status of the laboratory reference materials. Any was still present and declining steadily during the subse- contamination in either the laboratory standard or the QA ‡Á quent nitrogen analysis. CO2 ions undergo unimolecular standard will show up as a step. Slow long-term alteration decay in the ion source resulting in a 10% CO‡ contribution 1e.g. evaporative loss in water standards) will show as a drift. ‡Á at m/z 28 and 29 that interferes with N2 and has a very We recommend using such charts as a general tool for different isotopic signature. The background assignments of maintaining and proving analytical performance in every all peaks in the nitrogen chromatogram were affected and isotope laboratory for every type of analysis. hence precision and accuracy suffered. As a consequence, we In order to demonstrate the effect of the rigorous decided to stop simultaneous isotope ratio measurement implementation of the IT principle, we have compiled the

Figure 8. Performance Chart. d13C-values as in Fig. 6. Here, the scale is based on reference gas injections only. Please note the enhanced scatter of the data.

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 501±519 512 R. A. Werner and W. A. Brand

Figure 9. Performance Chart. d 15N-values as in Fig. 7. The scale is solely based on reference gas. Compared with Fig. 7 a larger scatter and some outliers are evident.

results of our QA standard based exclusively on our D/H determination from water reference gas 1Figs. 8 and 9). The data clearly exhibit a larger scatter than the data compiled including the IT calculations. Measurement of dD-values from water samples is carried out For both d15N and d13C, the precision has increased by about on a fully automated chromium reduction system at 900°C 50%. It is also worth noting that a number of marked outliers 1`H/Device', Finnigan MAT, Bremen, Germany) directly are present in Fig. 9 that are not seen in Fig. 7. These outliers coupled to the sample inlet of the dual inlet system of our might have escaped our attention if the IT principle had not MAT 252 isotope ratio MS.39 The samples, positioned in a GC been included in the measurement and evaluation. autosampler tray 1AS200, CTC Analytics, Switzerland), are

The composition of the N2 gas inside our 50-L high- transferred to the hot injection port via a gas-tight syringe pressure cylinders has not changed much over the reported 1Model 702, Hamilton, Switzerland). The quartz reactor time span. There is a slope of 0.04%/a in the solid line in Fig. 1Finnigan MAT) is filled with chromium powder 1100 mesh, 9 that is not present in Fig. 7. This slope probably represents Alfa Aesar, Karlsruhe, Germany). The reaction of water a change in the N2 reference gas. To explain the differences vapor with the hot chromium is almost instantaneous. The between the graphs we suspect a number of sources of error valve at the end of the reactor is opened after a preset time that all contribute to the observed dependence of the data: and the H2 gas is transferred to an intermediate equilibration volume. Again, after a preset equilibration time 1in order to . the pressure regulator on the tank is critical. Most avoid diffusive alteration of the isotopic composition of the regulators alter the isotopic composition of the dispensed hydrogen gas), the reactor valve is closed and the H gas is gas at least temporarily, the size of the alteration being 2 passed to the sample volume of the dual inlet for measure- dependent on the gas itself and on the surfaces inside the ment. The equilibration and transfer times are critical and regulator.38 The same applies to the second type of GC must be under computer control. regulators 1Porter) in the interface. The alteration for the We routinely run 60 analyses 120 h) in a sequence with isotopic composition of CO is in the sub-per mill range. 2 the laboratory reference water and the QA standard . The combustion and 1or) the reduction tube can be distributed in a prescribed loading list similar to that contaminated with previous sample material giving rise presented in Fig. 4. All data enter a spreadsheet for final to memory effects. calculation of dD-values 1Fig. 10). . The combustion efficiency may vary slightly thereby The data in column 3 1Fig. 10) are the original results as altering the isotopic composition. automatically transferred from the mass spectrometer PC. . The efficiency of the reduction tube for scavenging excess All samples are run in triplicate. The data treatment is more oxygen and for reducing nitrous oxides may change with elaborate owing to the fact that multiple effects must be time. accounted for. First, we apply a memory correction 1column . When diluting the CO effluent, a change in isotopic 2 4) to the dD-values against the working gas. The magnitude composition may occur due to diffusion effects.35 of the correction is 1% carry over from the previous sample As long as the reference material suffers from the same plus 0.2% from the sample before. The effect of the memory deficiencies during preparation, the errors will at least tend correction can be judged from the triplets where the to cancel out and the long-term data monitored by a QA difference to the precursor sample is large. standard will exhibit much better laboratory performance Next is the IT calculation, that is, replacement of the than with pure standard gas referencing. hydrogen gas reference by the laboratory standard water

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 501±519 Referencing techniques in IRMS 513

Figure 10. Spreadsheet calculation of data from the H/Device sample preparation line showing the evaluation of the raw data to the final d-values on an international scale. See text for further explanations.

samples in the run. The dD-value 1À66.45%) of WWj-1A 10) is calculated according to: 1working water-Jena1A) has been established by direct offset ˆ À66:45 À avg †= 1 ‡ avg =1000† 4† measurement against VSMOW and SLAP samples 1freshly wgas wwj1 wwwj1 opened ampoules) obtained from the IAEA in Vienna. Using This offset is used to correct the raw memory corrected this dD-value and the actually measured average of WWj- dD-values and to provide a preliminary positioning relative 1A, the apparent working reference gas offset 1À2.35%, Fig. to VSMOW 1Eqn. 12).

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 501±519 514 R. A. Werner and W. A. Brand

In every 60-sample sequence we measure RWB-j1 1re- ference water B-Jena1) as a QA standard for generating the performance chart associated with the D/H procedure. Figure 11 shows the data as a function of measurement time. The overall precision of 0.83% vs. VSMOW is certainly acceptable. There is a tendency to more negative values at the end of the sequence that deserves further attention. During the short period of measurements this is not likely to be caused by a relative drift of our laboratory working waters. However, most of the internal precisions measured are better than 0.8% 1see also Fig. 10). We attribute this finding to the fact that we do not determine the SLAP/ VSMOW scaling factor for every sequence in contrast to ‡ frequent measurement of the H3 factor. We started by using water samples with about 60% difference for implementing this correction. This difference turned out to be too small in relation to the measurement precision, leading to erroneous scaling factors. On the other hand, measuring water with about À400%dD in the same sequence affects the memory Figure 11. Performance Chart. d2H-values of the quality control correction and the three samples following this negative standard RWB-J1 as a function of measurement time. water would have to be discarded. The potential for improving the performance chart precision to about 0.5% The next columns constitute a two-pass drift correction. is within reach using the technique described. The drift correction is necessary because the hydrogen reference gas in the dual inlet changes isotopic composition over time 1a complete 60-sample sequence takes about 20 h). Equilibration/irm-analysis for d18O This is due to the fact that length and crimping of the determination 18 capillaries are a compromise between CO2 and H2 work. To The O abundance in water samples is usually measured avoid back diffusion of isotopically altered gas into the with a modified CO2/H2O equilibration technique. With a variable volume, the capillaries would have to be twice as syringe we inject 400 mL water aliquots into 10-mL glass long for H2. Alternatively, a different crimp on reference vs. containers 1`Exetainer') topped with a septum. Before water sample side could be used so that the reference could be injection the glass vials are filled with a mixture of 0.5% CO2 operated at a much higher pressure. The magnitude of the in helium.k Equilibration takes place inside a 96-position drift depends on the filling state of the reference bellow autosampler rack held at 30°C1Æ0.1°C) that is part of the irm volume at the beginning of the sequence. We have observed interface 1GasBench II, Finnigan MAT). We operate the drifts of 3±5% in 20 h when starting with a full reservoir. autosampler rack in two separate stages holding 48 sample Less reference gas results in larger drifts. Because the drift tubes each. While one sequence of 48 samples is measured, should follow an exponential function, we have implemen- the other batch of 48 samples is allowed to equilibrate for ted our analysis sequence with three reference water roughly 20 h. With this loading scheme we manage to have measurements in positions 34 to 36 and three more at the the system analyze samples almost continuously, i.e. 6 days end of the sequence. The results for these reference water per week. injections are used to correct the reference gas drift. A fine Following equilibration, the sample vial is analyzed by adjustment is made in column 13 1Fig. 10) that is only piercing the septum with a double wall needle. The needle necessary for making the data mathematically consistent has a feed 1He) and an exit 1sample CO2 in He). The flushing after drift correction 1in the example shown the data are rate is approximately 0.3 mL/min. The sample gas flows identical). The last correction involves scaling according to over a Nafion2 dryer and then through an injection loop the observed scale contraction.40 On average we have from which the GCrun 1Poraplot Q) is started. From a single measured À425% instead of À428% for SLAP vs. VSMOW, sample tube we inject about ten times. The ten CO2 peaks are hence all data are corrected by multiplying the difference evaluated isotopically and the results are transferred to a from WWJ-1A by 1.007. The last two columns are the final spreadsheet for further data evaluation. dD-values on the VSMOW/SLAP scale and their precision. The measurement sequence is assembled as before, i.e. following a rigid structure determined by a dedicated k Usually pure CO2 gas is used for equilibration and a mass balance loading list. Hence, the evaluation follows a similar pattern: correction according to: 18 a mean d O is calculated for all CO2 peaks from a single dtrue ˆ dmeas ‡ axgas dmeas À dgas†=xw 5† sample. The d-values are referenced at first to co-injected

18 CO2 standard gas peaks that serve as a mediator between the is applied 1a = exchange fractionation factor, dgas is the d O-value of the added CO2 gas before equilibration and xgas and xw are the mole samples. WW-J1A water samples strategically located in the fractions of gas and liquid, respectively). 48-sample sequence again serve to position the preliminary In our case, due to the small amount of CO in the gas phase, the 2 d18O-values onto the VSMOW scale. As in deuterium correction can be neglected down to about 50 mLH2O. Equation 15) also applies to the equilibration of hydrogen gas with water. analysis, a light reference water sample is used to establish

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 501±519 Referencing techniques in IRMS 515

overall precision of 0.14% also includes the error of the reference water determination in the respective analysis sequence.l

Isotopic analysis of CO2 in air The isotopic composition of CO2 in air is important for constraining the sources and sinks of this important green- 13 house gas in the atmosphere. The average d C-value of CO2 in the atmosphere has remained fairly constant over millions of years at about À6.5% vs. VPDB. During the last 200 years anthropogenic input from fossil fuel burning has lead to a decline with an average d13C-value of about À8% at present. The decline continues at a rate of about 0.03%/a. The seasonal cycle of d13Cvaries with latitude and is a function of photosynthesis as well as human activity. At the South Pole the size of the cycling is about 0.05% 1peak to peak) and increases to a maximum of 0.8% at high northern latitudes.41 These rather small isotopic signatures comprise multiple components including fossil fuel burning. Their measure- Figure 12. Performance Chart. d18O-values of the quality control ment requires a high precision and accuracy analytical setup. standard RWB-J1 as a function of measurement time. Analysis is Our extraction line is schematically depicted in Fig. 13. made using an equilibration/irm-GC/MS system (GasBench II, From a Multiport valve the air passes through a capillary Finnigan MAT). with a crimp, over a water trap at À70°Cand a trap kept at

À196°C. Here, CO2 is frozen out at a pressure of about 400 mbar. The air is pumped through the pumping lines of the final scale normalization so that SLAP returns a value of the dual inlet system of our MAT 252 mass spectrometer.42 À55.5% vs. VSMOW. Sample CO is measured directly from the sampling Figure 12 shows the long-term d18O-performance of our 2 reservoir via a crimped capillary to the `changeover valve'. RWB-J1 QA reference water. Each point corresponds to a The system is under full computer control for reliable timing single RWB-J1 sample in a 48-sample sequence. Apart from and unattended operation. The precision of the system is the scatter of the individual sample measurements, the aimed at and close to 0.01%d13Cvs. VPDB and 0.025 %d18O l If the reference material has been determined ten times in the sequence vs. VSMOW. It is very difficult to routinely prepare CO2 and the precision of the measurement is 0.1% then thep isotopic value of from the primary reference materials 1carbonates) across the reference is determined with an error of t .10.1%)/ 10 = 0.063%. The different laboratories at such levels of accuracy. Hence, the Student factor t 12.0 in our case) re¯ects the statistically low number of measurements. atmospheric CO2 isotope ratio scales are refined scales that

Figure 13. Schematic layout of the automated extraction system (‘BGC-Airtrap’) for high precision measurement of

CO2 isotope ratios in air.

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 501±519 516 R. A. Werner and W. A. Brand

‡ use air samples and air in high-pressure cylinders as H3 on the mass 3 channel where dD is measured and the reference material. Consequently, the referencing in our isobaric 12C18O17O‡Á ion current at the mass 45 position 13 system is made by a combination of permanently attached where d Cfrom CO 2 is determined. Here we intend to reference air 1Multiport valve positions 1 and 2) as well as air summarize some of the more important corrections. Further in 1-L flasks that are exchanged routinely with other information for in-depth treatment is given in the cited laboratories. The major problem here derives from the fact literature. that gases are volatile. To keep them as reliable references requires a hierarchy of reference gases and a strategy that automatically detects mutual drifts in order to take correc- 17O correction for d13C determination tive action. Without internal referencing high-precision 13 Using CO2 gas, d Ccannot be measured independent of the measurement and international comparability of data would oxygen isotope ratio unless the mass spectrometer used not be possible. provides a working mass resolution of >52000. CO2 gas contains a number of species with different masses 1`iso- O2/N2 ratios in air topomers'). All combinations of stable isotopes are present, Complementary to the increase of CO2 in the atmosphere some of them very low in abundance. The list of isotopomers 12 16 16 13 16 16 there is a decrease of O2. For every carbon atom burnt from includes the major species C O O1m/z 44), C O O and 12 17 16 12 16 18 fossil fuel one O2 molecule is lost to CO2.O2 has sources and C O O1m/z 45), C O O1m/z 46), as well as the minor 13 17 16 12 17 17 13 17 17 sinks that differ greatly from those of CO2. Thus, by studying species C O O and C O O1m/z 46), C O O, 13 18 16 12 17 18 12 18 18 the decrease of O2, i.e. measuring the O2/N2 ratio with high C O O and C O O1m/z 47), C O O and precision, a great deal can be learned about the global carbon 13C17O18O1m/z 48) and 13C18O18O1m/z 49). When working cycle including the partitioning of sinks for CO2 between the in the natural abundance range of the isotopes the contribu- oceans and the land biosphere. tions of the minor species is small enough to be neglected. We have built a mass spectrometric inlet system for The 17O moiety at m/z 45, however, has a 7% contribution to m measuring O2/N2 ratios with a precision at the 5 perMeg the mass 45 ion current and must always be corrected for level that is capable of monitoring small changes in atmo- when determining d13C-values. 17 spheric O2 concentration. The system includes a 16-connec- The square-root formula for correcting the O contribu- tion Multiport valve and an open split which is fed tion to the 45/44 ratio is sometimes referred to as the Craig alternately from a sample and a reference gas, both switched correction. Harmon Craig9 assumed a tight relationship on and off from a common transfer point, to a Delta‡XL between 17O and 18O with a fractionation coefficient l of 0.5 isotope ratio mass spectrometer 1Finnigan MAT, Bremen, exactly and devised a simple correction expression: Germany). Referencing is made in a very similar fashion to 13 45 18 that for CO2 in air by measuring versus an air reference and d C ˆ 1:0676 dmeas À 0:0338d O 6† implementing a multiple referencing hierarchy system. There is no international scale for this kind of work. The numerical values in this expression are the ratio terms Currently, consistency in the data is achieved by matching 45R/13R and 17R/1213R) of the reference gas evolved from the the scales of longer-term records measured by different PDB standard as determined or used by Craig. As explicitly laboratories. An intensive cooperation to overcome these pointed out in the paper these numerical values do not apply limits to the comparability of data is under way by a group of to other standards and must therefore be used with great laboratories involved in this type of analysis. care.n Moreover, the average relation of 17O and 18Oon 18 18 l 17 17 earth 1Craig equation: 1 Rsa/ Rst) = Rsa/ Rst) cannot be Correction of isobaric interferences described exactly using a fractionation factor l of 0.5. A review of referencing in isotope ratio measurements Instead, 0.51643 seems to be closer to the correct value for would not be complete without a discussion of the necessary l.o The debate about the exact relationship found on earth is corrections to the raw data. The precise measurement of ongoing with new experimental values of 0.524 and 0.528 isotope ratios is often hampered by interfering ion currents reported for l.44,45 from other species hitting the same Faraday cup detectors. PDB has been superceded by VPDB and the absolute ratios Among the most prominent examples are the contribution of of the reference scale have been refined. In order to keep literature data consistent, Allison et al.46 have compiled a recommendation for the 17O correction that keeps the 0.5 m 1 perMeg is 0.001% in d-units. It corresponds to a concentration fractionation factor and provides exact values for all ratios change of 0.2 ppm O2 in air. n The numerical values in Eqn. 16) may be treated as variables. In this involved. On the other hand, Santrock et al.47 have shown in case, a set of water equilibration experiments with CO2 gas that has 13 aH2O/CO2 equilibration experiment that data for d Cwere identical carbon but different oxygen isotope ratios and vice versa can be 18 made to determine the values of these variables. A correction based on not independent of the d O-values of the equilibration these values will result in correct d13C-values irrespective of the exact water when using the Craig equation. The correction knowledge of the oxygen isotope fractionation factors 1T.B. Coplen, proposed includes a fractionation factor of 0.516 and an private communication). o If the kinetics of the reactions causing isotopic fractionation are iterative correction procedure because the exact equations governed entirely by the respective zero point energy differences it can cannot be solved analytically for 13C. The dispute is not yet be shown that  =[1m )À0.5 À 1m )À0.5]/[1m )À0.5 À 1m )À0.5], with m 16 17 16 18 settled. Whatever correction algorithm is chosen 1the mass being the reduced mass. Using exact masses for a C-O bond the result is  = 0.5273 which represents an upper limit for kinetic processes. In spectrometer software should provide a choice), it is equilibrium processes, the value for  can be as high as 0.531.43 important to make sure that the reference gas isotope ratios

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 501±519 Referencing techniques in IRMS 517 used in the calculations are consistent with the d13C-values as possible in order to apply a valid correction and keep assigned to the primary reference material. track of the details.

Please note that the correction is only valid for CO2 from 18 34 terrestrial sources, not for tracer studies. In addition, there is O correction for d S determination using SO2 gas 17 a strong O anomaly in ozone in the stratosphere that affects As for carbon isotope ratio analysis, the ion currents of SO2 34 other trace gases including CO2 sampled from the atmos- are influenced by the oxygen isotopes. In order to extract d S phere. the 32S16O18O‡Á contribution to the mass 66 ion current 1about 8.3%) needs to be taken into account. Unfortunately, ‡ H3 correction there is no independent measurement of the other oxygen Inside the ion source of the mass spectrometer ions are isotope, 17O, which would enable a strategy similar to that produced from neutral molecules m by 70 eV electron impact for carbon isotope ratios. Instead, the established correction according to: aims at keeping the oxygen isotopes in the sample and the reference SO identical. In this case, the measured d66-values m ‡ eÀ ! m‡Á ‡ 2eÀ 7† 2 can be converted to d34S-values according to:48 The molecular radical cation m‡Á is produced in a variety d34S ˆ66d ‡ 218R=34R  66d À Dd18O† 10† of exited states. If the internal energy is high enough, meas meas molecular ions may decompose in a unimolecular decay In Eqn. 110) 18R/34R refers to the isotope ratio values of the reaction resulting in a daughter ion and a neutral 1mostly a standard gas and Dd18O is the 18O difference between sample radical) giving rise to the observed mass spectrum. Another and reference SO . Since theses values cannot be measured process can take place when ions within the ion source hit a 2 independently in a simple way the expression 18R/34Ris neutral molecule. In this case, secondary ions may be formed usually computed using the ratio values of VSMOW via ion/molecule reactions. The formation of H‡ follows 3 118R = 0.0020052, see Table 1) and VCDT 134R = 0.0441509) such a mechanism: and Dd18O is set to zero. Hence, the correction simply ‡Á ‡ Á 66 34 H2 ‡ H2 ! H3 ‡ H 8† involves multiplying dmeas with 1.091 for obtaining d S. The simplification of Eqn. 110) applies strictly only when The reaction constant of Eqn. 18) is proportional to the the oxygen in both sample and reference SO2 are isotopically ‡Á 18 number of both H2 and H2. For a given sensitivity of the identical 1Dd O = 0) and equal to VSMOW and the sulfur is mass spectrometer, the number of H2 molecules is propor- isotopically equal to VCDT. Hence, this can only be regarded ‡Á tional to the H2 ion current, hence: as a first-order correction. Fortunately, small errors in the adopted ratios do not alter the analytical result significantly. ‰H ‡Šˆk ‰H ‡Š2 9† 3 2 A difference in 18O content in terms of d18O, however, ‡ ‡ translates into an error of the analytical result that is 9% of and the ratio [H3 ]/[H2 ] is a linear function of the mass 2 ion ‡ the Dd18O-value. current 1= k  [H2 ]). The proportionality constant k is called ‡ Due to the `stickiness' of SO on surfaces, further the `H3 factor'. It is conveniently expressed in 1ppm/nA) 2 49 units. corrections are necessary and recommended. A systematic In practice, the ion source is tuned to a large acceleration scaling factor of about 1.035 between sulfur measurements field across the ionization volume in order to minimize the using SF6 and others using SO2 has been observed by many time for the ion/molecule reaction 18) and hence suppress laboratories. The simplest overall correction is scaling the ‡ ‡ measured and first-order corrected SO data with known H3 production. The H3 factor is measured by observing the 2 mass 3 ion current as a function of mass 2 ion intensity; a isotopic compositions of reference material prepared to- linear function is fitted through the data points. The gether with the unknown samples. For sulfides, IAEA-S-2 correction involves a simple point-to-point subtraction of a 1‡22.66% VCDT) and IAEA-S-3 1À32.30% VCDT) provide linear portion of the mass 2 ion current from the signal good references for the scaling exercise. For analyzing observed at mass position 3. This is usually performed using barium sulfates, IAEA-SO-5 1‡0.49% VCDT) and IAEA- the 1clean) working reference gas. In particular, when helium SO-6 1À34.18% VCDT) will serve the purpose. Please note h carrier gas techniques are used, there may be other sources of that the latter two values are provisional. non-linearity of m/z 3/2 including isobaric interference of For irm techniques, where the SO2 reference gas comes He2‡ ions at m/z 2 that should be studied and evaluated from a high-pressure cylinder, calculation of data should be 66 separately or traces of hydrocarbons that constitute a made exclusively in terms of dmeas-values up to the point ‡ where the reference is a sample processed exactly as the different source for H3 ions. ‡ other samples 1identical treatment again). Only then is the There are other means of correcting the contribution of H3 to the measured 3/2 ion current ratio.40 The simplest oxygen isotope signature similar enough to use the first- 50 correction is no correction at all which will obviously result order correction given above. in differences between reference and sample that are too small. Provided there are several isotopically known Further corrections samples interspersed with the analyte material, a correction Drift corrections can be deduced from the results of these that may fully cover Duringanalysisofaseriesofmeasurementsoneoftenobserves the requirements. As a general guideline, however, we feel a drift of the results as a function of time 1or sample number). that all effects should be studied and quantified as precisely Drifts can have multiple causes including isotopic change of

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 501±519 518 R. A. Werner and W. A. Brand the reference gas in the dual inlet system during an analysis value for Z mostly ranges between 0.001 and 0.01. In some sequence, build up of water or other contaminants during cases 1when the ion source is rather tight, the sensitivity is analysis, changing conditions of the mass spectrometer for high and the sputtered surface area is large) the Z correction 13 instance following a fresh pump down of the vacuum system, can be as large as 1% for d Cin CO 2 when the isotopic deterioration of ion source conditions and many more. Many difference exceeds 40%. of these causes can and should be fixed by changing the An alternative and recommended correction for cross conditions of the analysis. However, this can only be contamination is scaling to precisely known 1large) differ- accomplished reliably when the overall design of the ences of reference materials as is routinely done in hydrogen analytical sequences allows easy detection and provides and oxygen isotope ratio analysis by scaling to the SLAP/ means for correction of the drifting results. The design rules VSMOW difference. Besides hydrogen and oxygen isotope are simple: A sufficient number of identical samples are ratio determination this scale normalization has proven interspersed with the analyte samples and used to correct the mandatory for sulfur 1measured as SO2) and might also turn drift. For an example of drift correction please refer to the out to be required for carbon, in particular when the spreadsheet in the hydrogen isotope analysis section 1Fig. 10). measured isotopic differences are large and 1or) the precision requirements are high. Linearity corrections Similar to drift in time is a drift with size, i.e. the measured Reporting of experimental data ratio is a function of the size of the sample 1or a reagent). Isotope ratio data often enter into a multidimensional puzzle Commonly observed size or linearity effects are the from which further conclusions are drawn. This is the case in dependence of the measured ratio of the analyte gas on the almost any carbonate or precipitation study; it is obvious size of the major ion beam 1`pressure effect'), the observed also in CO2 in air analyses. Sometimes isotope ratio data are fractionation of hydrogen isotopes as a function of the valued by other researchers in a way that the original author amount of reduction reagent used, or the dependence of had not anticipated. As a consequence, isotope ratio data measured d18O-values on temperature during an equilibra- reported in the scientific literature should be presented tion experiment. The first and obvious choice is to eliminate together with the necessary context that accompanies the the causes of the linearity problems by improving tempera- pure number. Wherever the source of the scale that was used ture stability or reducing the dwell time of the ions in the for referencing experimental values to an international high-pressure region of the ion source. This is often standard may have arisen from, the procedure should and successful for reducing the size of the effect. However, a must be described. small fraction will almost always remain and thus will have For instance, most combustion analyses are calibrated to be corrected for. The general correction strategy is similar with NBS-22 oil using a published d13C-value of, for to the one described above: A sufficient number of known example, À29.74% on the VPDB scale. This value, however, samples are measured together with the analyte samples. In has been revised a number of times; the latest revision to this case the amount of material is varied in order to detect À29.78% happened only recently.h Thus, when published the size of the required linearity correction. data are compared with other data reported vs. VPDB based on a different value or even based on a different material, Correcting cross contamination in dual inlet adjustments can be made, provided the basis of the scale is measurements %-correction) clear. In addition to the final d-values, precision 1 1s, Following a switching action of the `changeover valve' from standard deviation) as well as sample preparation and one gas to the other a delay time 1`idle time') is necessary to measurement techniques should accompany the data in await complete exchange of the gases in the ion source. order to judge the relative merits of published data. Depending on the gas this can be rather short 14 s) or it may Similarly, for reporting d2H-values of substances other take up to several minutes for a complete replacement. Gases than water, it is recommended that the author's measured 2 like H2,N2,SF6 or N2O have little surface activity and, hence, d H of NBS-22 oil, NBS-30 biotite, IAEA-CH-7 polyethylene are pumped away fast whereas SO2 and CO2 can take foil, or other internationally distributed reference material be considerably longer to exchange completely. The time reported, as appropriate to the analytical method. Please depends on the material of the sputtered surfaces, on the note that normalization to the À428% difference of SLAP sensitivity of the instrument, on the tightness of the ion and VSMOW, as is mandatory for water analysis, requires source design and other parameters. The true d-value can be the availability of new internationally distributed reference calculated from the measured value according to:51 materials. The normalization procedure should be stated in the author's report. d ˆ d = 1 À 2Z À Zd =1000† 11† true meas meas For reporting d18O-values of substances other than water with Z describing the cross contamination affecting the or carbonates 1including the measurement of pure CO2 gas), background of the subsequent measurement. 1Please note it is recommended that the author's measured d18O of NBS- that Eqn. 14) in Ref. 51 is cast in absolute terms of d, not in% 28 quartz, NBS-30 biotite, NBS-127 barium sulfate, atmo- `units'). Meijer et al.51 also suggested a correction strategy spheric oxygen or other internationally distributed reference which comprises 11) experimental determination of the cross material be reported, as appropriate to the analytical contamination parameter Z by introducing natural isotope method. The d18O scale should be normalized such that the abundance and enriched gas followed by 12) application of d18O of SLAP reference water is À55.5% VSMOW exactly,

Eqn. 111) to the raw analytical results. For CO2, the numerical and so stated in the author's report.

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 501±519 Referencing techniques in IRMS 519 CONCLUSIONS 19. Epstein S, Buchsbaum R, Lowenstam HA and Urey HC. Bull. Geol. Soc. Am. 1951; 62: 417. We have described some of the recently developed tech- 20. Epstein S and Mayeda T. Geochim. Cosmochim. Acta 1953; 4: niques for referencing stable isotope ratio values in routine 213. 21. McCrea JM. J. Chem. Phys. 1950; 18: 849. operation in our laboratory. The quality control for long- 22. Coplen TB, Kendall C and Hopple J. Nature 1983; 302: 236. term accuracy is built into the daily cycles. Performance or This paper recommends the use of ‡30.91% for VPDB vs. quality charts help detect errors in high-precision referen- VSMOW. However, NBS-19, the carbonate which now de®nes the VPDB scale, was measured and reported as cing early and provide means for correction. It is common À2.19% vs. PDB. Correction of this value to À2.2% leads to practice in many laboratories to treat sample and reference the revised value of ‡30.92% vs. VPDB. material in an identical manner. We propose to adopt this 23. Friedman I and O'Neil JR. US Geological Survey Professional Paper 11977); 440-KK. practice as the general principle guiding the positioning of 24. Junk G and Svec H. Geochim. Cosmochim. Acta 1958; 14: 234. measured isotope ratios on international scales. 25. Mariotti A. Nature 1983; 303: 685. 26. Coplen TB and Krouse HR. Nature 1998; 392: 32. 27. Beaudoin G, Taylor BE, Rumble III D and Thiemens M. Acknowledgements Geochim. Cosmochim. Acta 1994; 58: 4253. We would like to thank Beate Rothe, Stefan Braeunlich and 28. MacNamara J and Thode HG. Phys. Rev. 1950; 78: 307. Heike Geilmann for their excellent performance of most of 29. Halsted RE and Nier AO. Rev. Sci. Instrum. 1950; 21: 1019. 30. Available: http://www.cio.phys.rug.nl/HTML-docs/Ver- the analyses described. The students and post-docs in our slag/97/report_95-97.htm. institute have generously provided samples and weighed or 31. Brand WA. J. Mass Spectrom. 1996; 31: 225. filled them in for measurement. Elizabeth W. Sulzman and 32. Matthews DE and Hayes JM. Anal. Chem. 1978; 50: 1465. 33. Hayes JM, Freeman KH, Popp BN and Hoham CH. Org. Charles B. Douthitt have made helpful comments and Geochem. 1990; 16: 1115. corrections to the draft manuscript. We are grateful to T. B. 34. Merritt DA, Brand WA and Hayes JM. Org. Geochem. 1994; Coplen for his critical and very helpful comments to the first 21: 573. 35. Werner RA, Bruch BA and Brand WA. Rapid Commun. Mass draft of this manuscript in a difficult time and for promoting Spectrom. 1999; 13: 1237. publication by acting as a sponsor referee for this paper. 36. Kornexl BE, Werner RA and Gehre M. Rapid Commun. Mass Spectrom. 1999; 13: 1248. 37. Spreadsheets are the perfect tool for establishing the data REFERENCES analysis schemes and procedures. 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Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 501±519