Evaluation of Gas Chromatographic Isotope Fractionation and Process

carbon analysis (CSRA). Nowadays, the excellent sensitivity of radiocarbon AMS allows measuring the 14C content of samples containing less than 20 µg...
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Anal. Chem. 2007, 79, 2042-2049

Evaluation of Gas Chromatographic Isotope Fractionation and Process Contamination by Carbon in Compound-Specific Radiocarbon Analysis Zdenek Zencak,† Christopher M. Reddy,‡ Emma L. Teuten,‡,§ Li Xu,| Ann P. McNichol,| and O 2 rjan Gustafsson*,†

Department of Applied Environmental Science (ITM), Stockholm University, 10691 Stockholm, Sweden, Department of Marine Chemistry and Geochemistry, and National Ocean Sciences Accelerator Mass Spectrometry Facility, Department of Marine Geology and Geophysics, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, and School of Biological Sciences, University of Plymouth, Plymouth, PL4 8AA, UK

The relevance of both modern and fossil carbon contamination as well as isotope fractionation during preparative gas chromatography for compound-specific radiocarbon analysis (CSRA) was evaluated. Two independent laboratories investigated the influence of modern carbon contamination in the sample cleanup procedure and preparative capillary gas chromatography (pcGC) of a radiocarbondead3,3′,4,4′,5,5′-hexachlorobiphenyl(PCB169)reference. The isolated samples were analyzed for their 14C/12C ratio by accelerator mass spectrometry. Sample ∆14C values of -996 ( 20 and -985 ( 20‰ agreed with a ∆14C of -995 ( 20‰ for the unprocessed PCB 169, suggesting that no significant contamination by nonfossil carbon was introduced during the sample preparation process at either laboratory. A reference compound containing a modern 14C/12C ratio (vanillin) was employed to evaluate process contamination from fossil C. No negative bias due to fossil C was observed (sample ∆14C value of 165 ( 20‰ agreed with ∆14C of 155 ( 12‰ for the unprocessed vanillin). The extent of isotopic fractionation that can be induced during pcGC was evaluated by partially collecting the vanillin model compound of modern 14C/ 12C abundance. A significant change in the δ13C and δ14C values was observed when only parts of the eluting peak were collected (δ13C values ranged from -15.75 to -49.91‰ and δ14C values from -82.4 to +4.71‰). ∆14C values, which are normalized to a δ13C of -25‰, did not deviate significantly (-58.9 to -5.8‰, considering the uncertainty of ∼ (20‰). This means that normalization of radiocarbon results to a δ13C of -25‰, normally performed to remove effects of environmental isotope * To whom correspondence should be addressed. E-mail: [email protected]. † Stockholm University. ‡ Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution. § University of Plymouth. | National Oceans Sciences Accelerator Mass Spectrometry Facility, Department of Marine Geology and Geophysics, Woods Hole Oceanographic Institution.

2042 Analytical Chemistry, Vol. 79, No. 5, March 1, 2007

fractionation on 14C-based age determinations, also corrects sufficiently for putative isotopic fractionation that may occur during pcGC isolation of individual compounds for CSRA. Isolation of individual compounds by preparative capillary gas chromatography (pcGC) combined with subsequent radiocarbon (14C) isotope analysis by offline accelerator mass spectrometry (AMS) was first reported in 1996 by Eglinton and co-workers.1 Since then, the combined use of these techniques has provided a powerful analytical tool able to achieve compound-specific radiocarbon analysis (CSRA). Nowadays, the excellent sensitivity of radiocarbon AMS allows measuring the 14C content of samples containing less than 20 µg of carbon. There is a multitude of CSRA applications in environmental, climate, biochemistry, and medicine fields, including the source apportionment of carcinogenic combustion products between biomass versus fossil fuel combustion.2-7 Further applications include the unequivocal differentiation of the origin of halogenated compounds between natural and industrialsynthetic formation8-10 and the compound-specific dating of biomarkers, recording past climate conditions in sedimentary archives.11-13 (1) Eglinton, T. I.; Aluwihare, L. I.; Bauer, J. E.; Druffel, E. R. M.; McNichol, A. P. Anal. Chem. 1996, 68, 904-912. (2) Reddy, C. M.; Xu, L.; O’Connor, R. Environ. Forensics 2003, 4, 191-197. (3) Lichtfouse, E.; Budzinski, H.; Garrigues, P.; Eglinton, T. I. Org. Geochem. 1997, 26, 353-359. (4) Mandalakis, M.; Gustafsson, O.; Alsberg, T.; Egeback, A. L.; Reddy, C. M.; Xu, L.; Klanova, J.; Holoubek, I.; Stephanou, E. G. Environ. Sci. Technol. 2005, 39, 2976-2982. (5) Currie, L. A.; Klouda, G. A.; Benner, B. A., Jr.; Garrity, K.; Eglinton, T. I. Atmos. Environ. 1999, 33, 2789-2806. (6) Mandalakis, M.; Gustafsson, O.; Reddy, C. M.; Li, X. Environ. Sci. Technol. 2004, 38, 5344-5349. (7) Kumata, H.; Uchida, M.; Sakuma, E.; Uchida, T.; Fujiwara, K.; Tsuzuki, M.; Yoneda, M.; Shibata, Y. Environ. Sci. Technol. 2006, 40, 3474-3480. (8) Teuten, E. L.; Xu, L.; Reddy, C. M. Science 2005, 307, 917-920. (9) Reddy, C. M.; Xu, L.; Eglinton, T. I.; Boon, J. P.; Faulkner, D. J. Environ. Pollut. 2002, 120, 163-168. (10) Reddy, C. M.; Xu, L.; O’Neil, G. W.; Nelson, R. K.; Eglinton, T. I.; Faulkner, D. J.; Norstrom, R.; Ross, P. S.; Tittlemier, S. A. Environ. Sci. Technol. 2004, 38, 1992-1997. 10.1021/ac061821a CCC: $37.00

© 2007 American Chemical Society Published on Web 01/26/2007

Methodologies other than pcGC can, in some situations, be applied to isolate specific compounds in the high purity necessary for CSRA (e.g., preparative high-performance liquid chromatography was used to isolate carotenoids and retinoids from human plasma14 or to isolate biomarkers such as sterols, phytols, glycerol dialkyl glycerol tetraethers, and alkenones from sediment extracts15,16). Nevertheless, pcGC provides the best selectivity and allows isolation of semivolatile compounds amenable to gas chromatography from highly complex matrixes.1,17 Despite the broad spectrum of possible applications, the combined use of pcGC and offline AMS is, 10 years after its introduction, still highly experimental and far from being robust. Broadly accepted quality control criteria have not been established. One reason for this is the very demanding sample treatment necessary to isolate enough material from environmental matrixes for radiocarbon analysis. Two key putative sources of error are the contamination of the sample with compounds of either higher or lower 14C/12C isotopic composition18 and isotopic fractionation of the target analyte during pcGC. Contamination amenable to gas chromatography may be identified by a suitable purity analysis (mass spectrometry or flame ionization detection), while other carbon contamination may not be observed (e.g., contamination by nonvolatile, or polar compounds or by particulate organic carbon).18,19 Furthermore, 14C-labeled compounds are used in many biologically or environmentally oriented research fields, and often these compounds are also targets of CSRA (e.g., polycyclic aromatic hydrocarbons2,4,6,7,20). Contamination by very small amounts of these compounds is sufficient to cause an extreme error in the radiocarbon analysis, since the natural abundance of 14C relative to 12C is in the order of 1.2 × 10-12 (e.g., only 30 ag of 14C is sufficient to change the ∆14C value of a 25-µg C sample from +100 to +1100‰).21,22 Such contamination by 14C-labeled compounds cannot be detected by a normal purity control analysis. Isotopic fractionation is a well-known process in chromatography.23,27 Due to its higher separation efficiency, capillary gas chromatography can provide a better separation of isotopes than high-performance liquid chromatography.24-26 It is well known that (11) Uchida, M.; Shibata, Y.; Kawamura, K.; Yoneda, M.; Mukai, H.; Tanaka, A.; Uehiro, T.; Morita, M. Nucl. Instrum. Methods B 2000, 172, 583-588. (12) Eglinton, T. I.; BenitezNelson, B. C.; Pearson, A.; McNichol, A. P.; Bauer, J. E.; Druffel, E. R. M. Science 1997, 277, 796-799. (13) Slater, G. F.; White, H. K.; Eglinton, T. I.; Reddy, C. M. Environ. Sci. Technol. 2005, 39, 2552-2558. (14) Dueker, S. R.; Lin, Y. M.; Buchholz, B. A.; Schneider, P. D.; Lame, M. W.; Segall, H. J.; Vogel, J. S.; Clifford, A. J. J. Lipid Res. 2000, 41, 1790-1800. (15) Smittenberg, R. H.; Hopmans, E. C.; Schouten, S.; Sinninghe Damste, J. S. J. Chromatogr., A 2002, 978, 129-140. (16) Ohkouchi, N.; Xu, L.; Reddy, C. M.; Montlucon, D.; Eglinton, T. I. Radiocarbon 2005, 47, 401-412. (17) Mandalakis, M.; Gustafsson, O. J. Chromatogr., A 2003, 996, 163-172. (18) Mollenhauer, G.; Montlucon, D.; Eglinton, T. I. Radiocarbon 2005, 47, 413424. (19) McNichol, A. P.; Ertel, J. R.; Eglinton, T. I. Radiocarbon 2000, 42, 219227. (20) Reddy, C. M.; Pearson, A.; Xu, L.; McNichol, A. P.; Benner, B. A.; Wise, S. A.; Klouda, G. A.; Currie, L. A.; Eglinton, T. I. Environ. Sci. Technol. 2002, 36, 1774-1782. (21) Brown, K.; Tompkins, E. M.; White, I. N. H. Mass Spectrom. Rev. 2006, 25, 127-145. (22) Buchholz, B. A.; Freeman, S. P. H. T.; Haack, K. W.; Vogel, J. S. Nucl. Instrum. Methods B 2000, 172, 404-408. (23) Filer, C. N. J. Labelled Compd. Radiopharm. 1999, 42, 169-197. (24) Matucha, M.; Jockisch, W.; Verner, P.; Anders, G. J. Chromatogr. 1991, 588, 251-258.

the 13C/12C ratio varies within the gas chromatographic signal.27 However, for 14C, the difference in retention behavior compared to 12C was only studied with isotopically labeled compounds,25 since current gas chromatography-mass spectrometry instruments are not sensitive enough to detect 14C isotopomers at natural levels. Recently, Holmstrand et al. showed that significant isotope fractionation of chlorine takes place during pcGC if the compound eluting from the column is collected only partially.28 Considering these results, it is reasonable to expect a potential for isotopic fractionation also for 14C/12C during pcGC if the isolated compounds are not collected completely. Such fractionation may then induce an error in the resulting 14C data. Especially when isolating compounds from very complex matrixes, the chromatographic separation may be difficult and narrow trapping time windows may be necessary to obtain an isolate of high purity. The aim of this study was to investigate whether current methodologies used to isolate compounds from environmental matrixes, in particular pcGC treatment, can lead to significant process contamination of the sample, resulting in a bias of the radiocarbon result. A further aim of this study was to investigate whether incomplete collection of the compound eluting from the capillary of the pcGC significantly diminishes the accuracy of CSRA, thereby potentially leading to erroneous conclusions. For this purpose, vanillin eluting from the pcGC was collected only partially (i.e., peaks were advertently split). The isotopic composition of “partial peaks” was compared with that of the unprocessed compound. EXPERIMENTAL SECTION Chemicals and Solvents. PCB 169 (crystalline, 99%) was obtained from Ultra Scientific (North Kingstown, RI). Vanillin (crystalline, 100% purity, of natural origin) was purchased from Sigma Aldrich (St. Louis, MO). Toluene (glass distilled) was obtained from Fluka (Buchs, Switzerland), water (HPLC quality) was from VWR International (Poole, United Kingdom), and diethyl ether (>99%, stabilized with 2,6-di-tert-butyl-4-methylphenol) was purchased from Sigma Aldrich. Silica gel (0.063-0.200 mm) was purchased from Merck (Darmstadt, Germany). Before use it was kept at 450 °C for 5 h and then deactivated with 10% water (w/w). Experimental Outline. To test for a positive carbon process contamination (i.e., with nonfossil and thus modern carbon with elevated 14C/12C ratio), 3,3′,4,4′,5,5′-hexachlorobiphenyl (PCB 169, a compound manufactured from fossil material and therefore containing no 14C) was dissolved in solvent, eluted through a silica column, and isolated by pcGC by two independent laboratories (Stockholm University (SU) and Woods Hole Oceanographic Institution (WHOI)). To evaluate for a negative carbon process contamination (i.e., with lower 14C/12C ratio than contemporary biosphere, e.g., fossil carbon from GC column bleed), a compound with modern 14C signal (natural vanillin) was employed. The δ13C and ∆14C of both isolated compounds were compared with their (25) Matucha, M. In Synthesis and Applications of Isotopically Labelled Compounds, 1994; Allen, J., Ed.; John Wiley & Sons Ltd.: Chichester, 1995; pp 489494. (26) Caimi, R. J.; Brenna, J. T. J. Chromatogr., A 1997, 757, 307-310. (27) Ricci, M. P.; Merritt, D. A.; Freeman, K. H.; Hayes, J. M. Org. Geochem. 1994, 21, 561-571. (28) Holmstrand, H.; Mandalakis, M.; Zencak, Z.; Gustafsson, O.; Andersson, P. J. Chromatogr., A 2006, 1103, 133-138.

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Figure 1. Collection windows used to harvest vanillin by pcGC. Collection of the whole vanillin peak (A) and of front and tail parts (B, C).

counterparts in the unprocessed compound. Furthermore, to evaluate the potential for 14C/12C isotopic fractionation during pcGC, the column eluate was collected in complementary ways (Figure 1). First the whole peak was collected. Then the first eluting half and the second eluting half of the vanillin were collected separately by diverting the flow into different traps of the preparative fraction collector. Finally, the front part, as well as the tail part of the peak were collected separately from the middle part of the eluting signal. The peak was “split” so that the resulting parts both had the same area. After AMS analysis, the isotopic composition (δ13C and ∆14C) of the isolates was compared with the isotopic composition of the unprocessed material. Sample Preparation. At Stockholm University, PCB 169 (1 mg) was dissolved in 200 mL of toluene and partitioned against 200 mL of water. The toluene phase was separated, evaporated to 1 mL, and eluted with 75 mL of toluene through a silica gel column (10-mm i.d., 10-cm height, deactivated with 10% water 2044

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w/w), which had been conditioned with 20 mL of toluene. The solvent was evaporated to dryness, and the sample was dissolved in 2 mL of toluene and processed by pcGC. Vanillin crystals were dissolved in diethyl ether and diluted to a concentration of 200 ng/µL prior to injection in the pcGC system. At WHOI, PCB 169 (1 mg) was dissolved in 250 µL of n-hexane and eluted with 20 mL of hexane/DCM (9:1) through a silica gel column (5-mm i.d., 10-cm height, 100-200 mesh, fully activated silica). The solvent was reduced to ∼0.5 mL by rotary evaporation and then exchanged into isooctane (final volume of ∼750 µL). This extract was treated by pcGC as described below. The so obtained isolate was further eluted through a silica column (0.5mm i.d., 5-cm height, 100-200 mesh, fully activated silica) to remove eventual column bleeding. All glassware and silica gel were baked out at 450 °C for 4 h before use. Preparative High-Resolution Gas Chromatography. The pcGC system used at Stockholm University for the PCB 169 and for the vanillin experiments consisted of a 6890N gas chromatograph equipped with a flame ionization detector (FID) and a 7683 Series injector, all from Agilent Technologies (Palo Alto, CA), combined with a cold injection system (CIS) and a preparative fraction collector (PFC) from Gerstel GmbH (Mu¨lheim an der Ruhr, Germany). The vial containing the sample was kept at 6 °C during the entire pcGC procedure to limit the evaporation of solvent from the vial during the 3-5 days needed to process the sample by pcGC, thus preventing problems caused by variation of peak width and retention times due to increasing sample concentration. The CIS injector was operated in “solvent vent” mode, with vent flow and vent pressure adjusted to 60 mL/min and 5 psi, respectively. The solvent venting time was 0.1 min. The temperature of the inlet was set to 90 °C for 0.1 min and then increased to 270 °C at a rate of 12 °C/s, kept isothermal for 2.5 min, and increased again to 300 °C with 5 °C/s when PCB 169 was collected, whereas it was set to a starting temperature of 30 °C when vanillin was processed (the other parameters were not changed). The splitless time was 2 min and the injection volume was 5 µL in all experiments. A “megabore” fused-silica capillary column (60-m length, 0.53-mm i.d.) coated with 0.5 µm of VF-5MS (cross-linked 5% phenyl methylpolysiloxane, Factor Four, Varian, Walnut Creek, CA) was used with helium as carrier gas at a constant flow of 6.8 mL/min. The pcGC oven temperature programs were as follows: 85 °C isothermal for 2.1 min, then 15 °C/min to 300 °C, and isothermal for 10 min to harvest PCB 169, and 30 °C isothermal for 2.1 min, then 6 °C/min to 200 °C, and isothermal for 2 min to harvest vanillin. Approximately 1% of the flow eluting from the capillary column was diverted to the FID, and the other 99% was sent to the PFC. The temperature of the FID was kept constant during all injections at 300 °C. Air and hydrogen flows in the FID were 400 and 40 mL/min, respectively, with nitrogen as makeup gas at 45 mL/min. The PFC switch temperature, as well as the transfer line temperature, was kept constant at 300 °C through all injections when PCB 169 was processed and at 250 °C when vanillin was processed. The traps were kept at 6 °C by the PFC cooling unit. Each sample was repeatedly injected, with the number of injections ranging from 100 to 412 (see Table 1). For PCB 169, the trapping time window started 3 s before and ended 3 s after the elution of the compound.

Table 1. General Description of the Samples Obtained from pcGC Experiments and Results of the Individual Compound Isolation

sample name

no. of injections

collected amt (µg)

total yield (%)

reproducibility of retention time (rsd %)

description

PCB 169 SU PCB 169 WHOI vanillin-2: whole peak vanillin-4: whole peak vanillin-5: first half of peak vanillin-6: second half of peak vanillin-7: middle part of peak vanillin-8: outer parts of peak

412 270 100 100 220 220 160 160

920 650 103 86.8 108 90.8 78.6 59.8

92 54 81 87 49 41 49 38

0.10 0.04 0.08 0.03 0.04 0.04 0.11 0.11

sample prepared at SU sample prepared at WHOI whole peak collected whole peak collected first half of peak collected second half of peak collected middle part of peak collected outer parts of peak collected

For vanillin, the trapping time windows were set as shown in Figure 1. The retention time was frequently checked, and the trapping windows were adjusted as necessary. The isolated samples were rinsed from the glass traps 5 times with 200 µL of dichloromethane. The pcGC system used at WHOI to process the PCB 169 sample consisted of a 6890 gas chromatograph equipped with a FID and a 7683 Series injector, all from Agilent Technologies, combined with a CIS4 and a PFC from Gerstel GmbH. The CIS injector was operated in “solvent vent” mode, with vent flow and vent pressure adjusted to 80 mL/min and 20 psi, respectively. The solvent venting time was 0.3 min, and the split vent time was 2 min. The temperature of the inlet was set to 35 °C for 0.3 min, then increased to 320 °C at a rate of 12 °C/s, and kept isothermal for 35 min. The injection volume was 5 µL in all experiments. A megabore fused-silica capillary column (50-m length, 0.53-mm i.d.) coated with 1 µm of CP-Sil 5CB (dimethylsiloxane, Chrompack) was used with hydrogen as carrier gas at a constant flow of 8.6 mL/min. The pcGC oven temperature program was as follows: 80 °C isothermal for 2.0 min, then 20 °C/min to 280 °C, and isothermal for 4 min. Approximately 1% of the flow eluting from the capillary column was diverted to the FID and the other 99% was sent to the PFC. The PFC switch temperature, as well as the transfer line temperature, was kept constant at 320 °C through all injections when PCB 169 was processed. 13C and 14C Analysis. The isolated compounds were sent to the U.S. National Ocean Sciences AMS (NOSAMS) facility of the Woods Hole Oceanographic Institution (Woods Hole, MA) for carbon isotope analysis. The solutions containing the pcGC isolates were transferred into precombusted quartz tubes, the solvent was evaporated under a nitrogen stream, and ∼100 mg of copper oxide was added. The tubes were evacuated on a vacuum line (10-3 Torr) while immersed in a 2-propanol/dry ice slush (-78 °C), flame-sealed, and combusted at 850 °C for 5 h yielding carbon dioxide, water, and other combustion products. The tubes were reconnected to the vacuum line, and the carbon dioxide was isolated and purified through a series of cold traps and quantified by manometry. About 10% of the carbon dioxide was kept for δ13C analysis by isotope ratio mass spectrometry. The remaining 90% were reduced to graphite according to standard procedures.29,30 Targets of graphite were pressed and mounted on target wheels for 14C analysis by AMS. (29) Pearson, A.; McNichol, A. P.; Schneider, R. J.; Von Reden, K. F.; Zheng, Y. Radiocarbon 1998, 40, 61-75.

Calculations. Results of 13C isotope analysis are reported as δ13C according to the following equation:

δ13C )

(

(13C/12C)sample (13C/12C)std

)

- 1 × 1000‰

(1)

where the standard used for reporting δ13C values is the Vienna Pee Dee Belemnite (VPDB).31 Radiocarbon data are reported as fraction modern (Fm) according to the following equation:

Fm )

S-B M-B

(2)

where B, S, and M represent the 14C/12C ratios of the blank, the sample, and the modern reference, respectively. The 14C/12C ratio of the “modern reference” is defined as 95% of the radiocarbon concentration of NBS Oxalic Acid I in AD 1950 normalized to δ13CVPDB of -19‰. Results were calculated using the internationally accepted modern 14C/12C ratio of (1.176 ( 0.010) × 10-12. In addition, we also report the results as defined by Stuiver and Polach32 as the relative difference between the 14C/12C ratios of the absolute international standard (base year 1950) and sample corrected for the age of the sample:

δ14C ) (Fm‚eλ(1950-x) - 1) × 1000‰

(3)

where λ is 1/(true mean-life) of radiocarbon (corresponding to 1/8267) and x is the year of collection (in this case 2005). The application of this age correction allows obtaining the same results for two measurements of the same sample performed years apart (thus compensates for the radioactive decay of 14C after the collection of the sample). Since the 14C/12C ratio is also affected by isotopic fractionation in nature, radiocarbon data used for dating measurements are usually corrected to the ∆14C value they would have if they had an original δ13C of -25‰ in order to ensure comparability between (30) McNichol, A. P.; Gagnon, A. R.; Jones, G. A.; Osborne, E. A. Radiocarbon 1992, 34, 321-329. (31) Coplen, T. B. Geochim. Cosmochim. Acta 1996, 60, 3359-3360. (32) Stuiver, M.; Polach, H. A. Radiocarbon 1977, 19, 355-363.

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different samples. This is done for Fm with the following equation:

( )

25 1000 Fmδ13C ) Fm δ13C 1+ 1000 1-

2

(4)

Similarly, δ14C results can also be corrected for natural isotopic fractionation prior to sample collection according to the following equation:32

∆14C )

(( )( ) ) 25 δ C 1000 1+ 1000 δ13C 1+ 1000 14

1-

2

-1 )

(Fmδ13C‚eλ(1950-x) - 1) × 1000‰ (5)

It should be noted that results reported in the literature are usually normalized to δ13C of -25‰ according to eqs 4 and 5. The reported uncertainties are the larger of the internal error (statistical error calculated using the number of counts measured from each AMS target) and external error (error calculated from the reproducibility of individual analyses of a given target). A detailed discussion about reporting of 14C data can be found elsewhere.32 RESULTS AND DISCUSSION Quality Control. Although radiocarbon AMS measurements are well established and AMS facilities follow routine quality control, CSRA is still an immature analytical technique and quality control criteria are not well established for the whole process. One of the most severe issues is contamination of the sample by so-called “hot”, 14C-labeled, compounds, which are used in many research applications. It is possible to control whether laboratories have a high 14C level by performing swab tests.22 For example, NOSAMS recommends checking sample-submitting laboratories using such swab tests. The facilities where the samples discussed in this work were prepared were all tested with swab tests within 3 months of sample preparation. All tests showed natural amounts of 14C. Although this is not a guarantee for uncontaminated samples, it is a prerequisite for quality work. Other procedures commonly employed to minimize the risk of contamination in CSRA include confining the sample preparation to special rooms, potentially with full “clean-room” methodologies, heating of dedicated glassware to 450 °C, and the use of disposable laboratory equipment.22 Another prerequisite for accurate results is the use of optimized pcGC instrumentation allowing maximal recoveries and highly reproducible chromatography.18 In this work, the retention times of the chromatographic signals varied by 0.1% (relative standard deviation) for PCB 169 and by e0.14% for vanillin over several days (see also Table 1), which provided sufficiently stable and reproducible chromatographic conditions for preparative work. Evaluation of Background Contamination. The results for the fossil PCB 169 indicate the absence of any significant hot/ modern 14C contamination of the samples processed at either SU or WHOI, since the ∆14C values obtained for both samples did not significantly differ from the ∆14C measured for the two unprocessed PCB 169 samples (Figure 2 and Table 2). The sample 2046 Analytical Chemistry, Vol. 79, No. 5, March 1, 2007

Figure 2. ∆14C values measured for the vanillin and PCB 169 samples indicating absence of process contamination by carbon. See Table 1 and Table 2 for sample description.

from the WHOI laboratory may have contained slightly more 14C than the other samples, but the difference is not significant considering the uncertainty of the AMS analysis. Finally, the δ13C values observed for the processed samples were also consistent with those measured for the unprocessed samples. This also suggests that no further fossil carbon was added to the samples during the cleanup procedure. However, it must be noted that the volumes of solvents used to simulate the extraction and isolation procedure were much smaller than what may be usual in certain real applications, where up to 10-15 L of solvent has been reported to be used for a single sample.4,6,8,10 Although contamination by 14C-labeled compounds is the “worst-case” scenario in a laboratory dedicated to 14C analysis, contamination by fossil C is even more likely since most, if not all, of the chemicals used have fossil origin. Indeed, the ∆14C of the vanillin sample processed by pcGC was ∼100‰ more than the ∆14C of the unprocessed compound. A review of the analytical process showed that the employed diethyl ether was stabilized with 2,6-di-tert-butyl-4-methylphenol. This stabilizer was separated from the vanillin during pcGC, but biased the measurement of the “unprocessed” vanillin, which had been dissolved in the same solvent. Knowing the concentration of the stabilizers in the solvent and the exact amount of solvent used, it was possible to correct the ∆14C value of the unprocessed vanillin (see footnote of Table 2 for more details). This case is a very good example of the attention to details, such as the presence of stabilizers in solvents, which is necessary to perform CSRA. The corrected results of the radiocarbon-modern vanillin indicated the absence of any fossil C contamination in the sample processed by pcGC (Table 2). Although no sample cleanup was performed for this sample, contamination by column bleeding was a priori conceived as quite plausible. Encouragingly, the result indicates that bleeding of the stationary phase of modern capillary columns did not significantly contaminate the isolates at the applied oven temperature (∼180 °C). Furthermore, this result is

Table 2. Results from 13C and 14C Isotope Analysis of the PCB 169 and Vanillin Samples Prepared at Stockholm University and at the Woods Hole Oceanographic Institution and of Unprocessed PCB 169 and Vanillin Reference Samples Showing Absence of Process Contamination by Carbon laboratory preparation

sample

seal and purificationa

yield (µmol C)

δ13C (‰)

CTC (NOSAMS green line) CTC (NOSAMS green line) elemental analyzerCO2 trapping system

24.3

-27.52 ( 0.1

-995 ( 20

26.6

-27.55 ( 0.1

-996 ( 20

28.9

-27.50 ( 0.1

-996 ( 20

22.4

-27.67 ( 0.1

-985 ( 20

5.4

-32.03 ( 0.1

155.3 ( 12.4b

5.2

-31.64 ( 0.1

164.7 ( 20.6

pcGC

unprocessed PCB 169 ref 1

none

not used

unprocessed PCB 169 ref 2

none

not used

PCB 169 SU

SU NOSAMS

CTC (Fye 105 line)

vanillin-1 (unprocessed ref)

dissolved in solvent, purified with silica gel, evaporated (SU) dissolved in solvent, purified with silica gel, evaporated (WHOI) dissolved in solvent

not used

vanillin-2: whole peak

dissolved in solvent

SU

CTC (NOSAMS green line) CTC (NOSAMS green line)

PCB 169 WHOI

∆14C (‰)

a CTC, closed tube combustion. b Corrected for residue of solvent stabilizer. ∆14C of vanillin can be easily calculated from the ∆14C measured for vanillin + stabilizer, the amounts of stabilizer and vanillin present in the sample, and knowing that the ∆14C of the stabilizer is -1000‰. Equation used for the correction: ∆14C(vanillin + stabilizer) ) (∆14Cvanillin × mg of vanillin - 1000‰ × mg of stabilizer)/(mg of vanillin + mg of stabilizer).

Table 3. Results from

13C

sample vanillin-3: unprocessed vanillin-4: whole peak vanillin-5: first half of peak vanillin-6: second half of peak vanillin-7: middle part of peak vanillin-8: outer parts of peak

and

14C

PCGC

Isotope Analysis of Vanillin after Different pcGC Fractionation Schemesa δ13C (‰)

14C

Fm

δ14C (‰)

14C

Fmδ13Ccorr

∆14C (‰)

no

-31.63 ( 0.1

0.9873 ( 0.0146

-19.2 ( 14.5

1.0009 ( 0.0148

-5.8 ( 14.7b

whole peak collected first half of peak collected second half of peak collected middle of peak collected outer parts of peak collected

-31.70 ( 0.1

nac

na

na

na

-15.75 ( 0.1

1.0114 ( 0.0172

4.71 ( 17.1

0.9925 ( 0.0169

-14.1 ( 16.8

-49.91 ( 0.1

0.9312 ( 0.0174

-74.9 ( 17.3

0.9807 ( 0.0183

-25.8 ( 18.2

-37.28 ( 0.1

0.9237 ( 0.0192

-82.4 ( 19.1

0.9474 ( 0.0197

-58.9 ( 19.6

-24.86 ( 0.1

0.9879 ( 0.0172

-18.7 ( 22.7

0.9876 ( 0.0228

-18.9 ( 22.7

a δ13C, δ14C, and ∆14C results are given in per mil measured relatively to VPDB and NBS Oxalic Acid I, respectively. For 14C isotope analysis, the results are also presented as Fmδ13Ccorr and Fm (relative to NBS Oxalic Acid I), which is used to calculate the ∆14C values. b Corrected for residue of solvent stabilizer. ∆14C of vanillin can be easily calculated from the ∆14C measured for vanillin + stabilizer, the amounts of stabilizer and vanillin present in the sample, and knowing that the ∆14C of the stabilizer is -1000‰. Equation used for the correction: ∆14C(vanillin + stabilizer) ) (∆14Cvanillin × mg of vanillin - 1000‰ × mg of stabilizer)/(mg of vanillin + mg of stabilizer). c na, not available as sample was lost during graphite target preparation. An equivalent sample is vanillin-2 (Table 2) and its corresponding unprocessed reference is vanillin-1 (Table 2).

consistent with the findings of McNichol et al., who showed that pcGC isolation does not alter the ∆14C of natural phenols (among these also vanillin).19 Therefore, this experiment not only shows the absence of fossil C contamination but it proves that correct ∆14C values are obtained if the whole peak is collected. Isotope Fractionation during pcGC. In the second part of this work, the extent of 14C/12C isotopic fractionation induced during pcGC was investigated. Vanillin was again selected as model compound, since it can be purchased as a certified natural product and thus has a modern 14C/12C ratio (a compound containing fossil carbon, thus no 14C, would obviously not be adequate to investigate 14C/12C isotopic fractionation). This property made vanillin a useful reference substance even though it is not an optimal compound for chromatography on the stationary phase normally used in pcGC. However, the fronting of the chromatographic signal (Figure 1) does not reduce the validity of this experiment. On the contrary, tailing and fronting chromatographic signals can often not be avoided and need to be dealt with in real applications. In such cases, the operator may

be tempted not to collect the tailing or fronting part of the signal to avoid contamination from other compounds eluting close to the target analyte. The aim of the experiment was to evaluate to what extent 14C results can be biased by partial collection of the eluting peak. Obviously, complete collection of the eluting compounds should always be attempted for CSRA measurements. The results obtained for 13C and 14C isotope analysis of the vanillin isolates indicated a significant variation in the δ13C values when only a part of the eluting chromatographic signal was collected, as also reported by Eglinton et al.,1 whereas the δ13C of the completely collected vanillin was consistent with that of the unprocessed compound (Table 3). It is possible to apply a simple mass balance calculation for the obtained split-peak samples. In this way, total δ13C values of -31.32 (by combining samples vanillin-5 and vanillin-6) and -31.91‰ (by combining samples vanillin-7 and vanillin-8) are obtained. The consistency of these values with the δ13C observed for the unprocessed compounds (-31.63‰) indicates that the PFC instrumentation Analytical Chemistry, Vol. 79, No. 5, March 1, 2007

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Figure 3. δ14C and ∆14C values measured for the vanillin samples in the pcGC isotope fractionation experiments.

allows a very accurate and quantitative collection of the eluting compounds. The observed inverse δ13C fractionation pattern was consistent with what is usually observed during gas chromatography coupled to online 13C-CSIA where heterogeneity of the 13C/ 12C ratio is observed over the gas chromatographic signal. In these applications, the heavy carbon isotopomers elute slightly earlier than the lighter isotopomers, since carbon isotopes show an inverse isotope effect under the commonly applied gas chromatographic conditions. Some other elements (e.g., chlorine) show a normal isotope effect during pcGC; thus, the lighter isotopomers elute before the heavy ones.28 The 14C results obtained for vanillins 3-8 showed a significant offset compared to the results obtained for vanillin in the first experiment (vanillin samples 1 and 2 in Table 2). However, the two sets of samples should not be compared since they were prepared more than 1 year apart and slightly different procedures were followed (different solvents were used). Furthermore, the combustion of vanillin samples 3-8 was not done at NOSAMS; thus, these samples did not have a blank correction for the combustion process. Interestingly, the δ13C results were similar for the two sets of samples, which means that if contamination is responsible for the offset its source had a similar 13C signature. Furthermore, the secondary reference standards analyzed for their 14C composition at the AMS facility with both batches of samples had correct ∆14C values. This indicates that the bias was caused before the reduction of the CO2 to graphite. The difference appears to be systematic and not related to pcGC since it affected both the samples prepared by pcGC and the unprocessed sample in equal measures. This underlines how important it is to process a reference standard of known isotopic composition to ensure the quality of CSRA data. This is currently done for the combustion to CO2, the preparation of the graphite target, and AMS measurement, but these reference standards do not cover the whole sample preparation process. Including a nonfossil reference control for the whole sample preparation process should be considered for future CSRA works. The control compounds should be selected to be similar in properties to the target analytes and will thus vary for different applications. Fortunately, the discrepancy between the two data sets does not affect the outcome of this experiment since the bias appears to be systematic. Hence, it is possible to verify whether incomplete collection of the eluting compound affected its isotopic composition. The results showed a variation for Fm as well as for δ14C (Table 3 and Figure 3). An inverse isotope effect was observed for 14C as was expected from the 13C data. Due to the high sensitivity of AMS, this is, to our knowledge, the first time that 2048 Analytical Chemistry, Vol. 79, No. 5, March 1, 2007

isotopic fractionation has been reported for 14C during capillary gas chromatography when natural abundance of 14C were injected. Theoretically, the observed δ14C fractionation should be twice as large as the δ13C fractionation. Within the uncertainty of the measurement, this trend was also observed for the here reported data. For example, for the vanillin samples 5 and 6, the difference between the δ13C values was ∼35‰, whereas the difference between the δ14C values was ∼70‰ (Table 3). However, it is important to note that radiocarbon data are usually reported after a correction of isotopic fractionation occurring in nature in order to not affect estimates of radiocarbon-based ages. This means that the results obtained from the AMS measurement (Fm or δ14C, which is the common reporting format) are corrected, using eqs 4 and 5, to the value the samples would have if they had a δ13C of -25‰. The resulting Fmδ13C and ∆14C values obtained in this study, by partially collecting the eluting vanillin, did not show a significant variation for all samples, except vanillin-7 (Table 3 and Figure 3). According to these results, the typical correction of 14C data to a δ13C of -25‰ also compensates satisfactorily for 14C isotope fractionation during sample preparation (e.g., in pcGC). Furthermore, isotope fractionation during pcGC does not appear to be an important source of error in comparison to the current precision of radiocarbon analysis of such small samples. This is particularly encouraging since it ensures the validity of Fmδ13C and ∆14C results previously obtained by combining pcGC and offline AMS measurements. CONCLUSIONS The CSRA results obtained by two independent laboratories for a fossil standard after sample cleanup, pcGC, and AMS were in good agreement and did not show any trace of contamination from 14C-labeled or natural-abundance 14C-containing compounds. Similarly, CSRA results for a 14C modern reference indicated the absence of any significant fossil carbon contamination from the pcGC (e.g., column bleeding) and associated handling. Nevertheless, continuous efforts are recommended to ensure a radiocarboncontamination-free working environment. Moreover, distinct isotopic fractionation of 13C/12C and 14C/12C was observed during megabore preparative capillary column chromatography. However, this fractionation can be corrected mathematically for the 14C/ 12C ratio using data processing commonly applied to avoid incorrect radiocarbon age determinations from environmental isotope fractionation. Nevertheless, harvesting of the whole signal is recommended, particularly if δ13C data (for which isotopic fractionation is not corrected) are used in combination with ∆14C for interpretation of the results. This work, in combination with previously reported experiments, allows us to make some recommendations for pcGC-based CSRA: (a) work should be performed in a clean-laboratory environment isolated from facilities using 14Clabeled compounds; (b) care should be applied to minimize contamination of the sample with other carbon (e.g., dust, solvent stabilizers, plasticizers from storage containers and lids, etc.), especially after the target compound was isolated by pcGC; (c) achieve a good chromatographic separation of the target compound from other compounds present in the sample extracts (if this is not possible, selectivity should be increased by an enhanced sample cleanup); (d) if the sample extract contains a compound

of known isotopic composition (e.g., PCB), its radiocarbon content should be measured, as well as quality control. ACKNOWLEDGMENT We gratefully acknowledge the financial support from the Swedish Foundation for Strategic Environmental Research (MISTRA Idesto¨d, contract 2002-057), the Swedish Research Council (VR contracts 629-2002-2309 and 621-2004-4039), the US-NSF (Cooperative Agreements OCE-0228996, OCE-0221181, and

OCE-0550486), and the Camille and Henry Dreyfus Foundation, Inc. Skillful technical assistance in parts of the laboratory analyses by Maria Unger is appreciated. Comments by three anonymous reviewers helped to improve the manuscript.

Received for review September 27, 2006. Accepted December 1, 2006. AC061821A

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