INTRODUCTION
Compound-specific radiocarbon analysis (CSRA) is a powerful tool to investigate carbon cycling and/or as a dating technique in paleoclimate reconstructions (Uchida et al. Reference Uchida, Shibata, Kawamura, Kumamoto, Yoneda, Ohkushi, Harada, Hirota, Mukai, Tanaka, Kusakabe and Morita2001; Rethemeyer et al. Reference Rethemeyer, Kramer, Gleixner, John, Yamashita, Flessa, Andersen, Nadeau and Grootes2005; Ohkouchi and Eglinton Reference Ohkouchi and Eglinton2008; Uchikawa et al. Reference Uchikawa, Popp, Schoonmaker and Xu2008; Kramer et al. Reference Kramer, Trumbore, Froberg, Dozal, Zhang, Xu, Santos and Hanson2010; Kusch et al. Reference Kusch, Eglinton, Mix and Mollenhauer2010; Douglas et al. Reference Douglas, Pagani, Eglinton, Brenner, Hodell, Curtis, Ma and Breckenridge2014; McIntosh et al. Reference McIntosh, McNichol, Xu and Canuel2015; Tao et al. Reference Tao, Eglinton, Montlucon, McIntyre and Zhao2015). The 14C content of individual compounds can be used to estimate residence times, identify carbon sources of organic matter, or establish chronologies if traditional dating materials (e.g. macrofossils, pollen, charcoal) are not available. However, the isolation of compounds from parent material (e.g. plant material, soil, lacustrine or marine sediments) involves chemical extractions and isolation procedures that result in carbon contamination. In addition, the target compounds are often present in low concentrations; thus, it is inevitable that the extracted quantities of carbon are often as little as tens of micrograms (μg), which amplifies the effect from carbon contamination. In CSRA, apart from carbon contamination derived from routine procedures of combustion and graphitization (corrected for by using internationally accepted 14C standards), carbon contamination is also derived from the chemical extraction and compound isolation, often achieved by preparative capillary gas chromatography (PCGC). In order to report accurate values from CSRA, efforts must be made to correct for carbon contamination derived from these procedures, hereafter referred to as extraneous carbon (Cex).
In order to correct for Cex, the amount and the 14C content of Cex must be determined by either using process blanks or process standards (materials processed in the same manner as unknowns at matching sizes) of known 14C content (Mollenhauer and Rethemeyer Reference Mollenhauer and Rethemeyer2009; Ziolkowski and Druffel Reference Ziolkowski and Druffel2009; Santos et al. Reference Santos, Southon, Drenzek, Ziolkowski, Druffel, Xu, Zhang, Trumbore, Eglinton and Hughen2010). The use of process blanks, known as the “direct method,” involves the processing of solvent only (no sample or standard). The difficulty with this approach is that the amount of carbon obtained is often too small (<10 µg C) for a reliable accelerator mass spectrometry (AMS) measurement. The use of process standards, known as the “indirect method,” aims to estimate the old (14C-free) and the modern (modern 14C content) component of Cex by using standard materials of modern 14C content and 14C-free, respectively. This approach assumes that the process standard has been diluted with a constant amount of Cex, which causes a deviation in its 14C content from its consensus (or in-house determined) value.
Different methods have been used to include process blanks or standard materials of known 14C age to assess Cex in studies involving CSRA. In a coastal sediments study, a mixture of commercially available compounds that ranged from 14C-free to modern 14C content was added to sea sand and used as a process standard (Santos et al. Reference Santos, Southon, Drenzek, Ziolkowski, Druffel, Xu, Zhang, Trumbore, Eglinton and Hughen2010). In a study of 14C analysis of phospholipid fatty acids (PLFA) extracted from mineral soil, two commercially available fatty acid methyl esters (FAMEs; nC18:0 and nC16:0) of modern 14C content were individually isolated by PCGC to determine the amount of 14C-free Cex added during PCGC isolation (Kramer et al. Reference Kramer, Trumbore, Froberg, Dozal, Zhang, Xu, Santos and Hanson2010). In 14C analysis of PLFA and n-alkanes extracted from ocean sediments, Druffel et al. (Reference Druffel, Zhang, Xu, Ziolkowski, Southon, dos Santos and Trumbore2010) used several approaches to determine modern and 14C-free Cex during PCGC isolation that included solvent only, a modern methyl stearate standard, and a 14C-free C22 n-alkane standard, and the assessment of the combined procedures of chemical extractions and PCGC was achieved by using blanks (no sample added). In the isolation of black carbon (BC), Ziolkowski and Druffel (Reference Ziolkowski and Druffel2009) used commercially available modern and 14C-free vanillin to determine carbon addition during PCGC isolation and BC reference materials from the BC Ring Trial (modern grass char, 14C-free hexane soot; Hammes et al. Reference Hammes, Schmidt, Smernik, Currie, Ball, Nguyen, Louchouarn, Houel, Gustafsson, Elmquist, Cornelissen, Skjemstad, Masiello, Song, Peng, Mitra, Dunn, Hatcher, Hockaday, Smith, Hartkopf-Froeder, Boehmer, Luer, Huebert, Amelung, Brodowski, Huang, Zhang, Gschwend, Flores-Cervantes, Largeau, Rouzaud, Rumpel, Guggenberger, Kaiser, Rodionov, Gonzalez-Vila, Gonzalez-Perez, de la Rosa, Manning, Lopez-Capel and Ding2007) and blanks (no sample added), to evaluate the chemical and PCGC isolation steps combined. Coppola et al. (Reference Coppola, Ziolkowski and Druffel2013) used a similar approach to assess Cex during isolation of BC using reference materials from the BC Ring Trial (modern grass char, wood char, and 14C-free hexane soot) in addition to NIST Standard Reference Material urban dust aerosol (SRM 1649a), marine sediment (SRM 1941b) added to wood char, and US Geological Survey Green River Shale. Tao et al. (Reference Tao, Eglinton, Montlucon, McIntyre and Zhao2015) used solvents-only through the entire sample preparation procedure and solvents spiked with compounds of 14C-free and of modern 14C content after PCGC isolation as process standards.
One of the challenges for CSRA is the lack of suitable process standard materials, i.e. materials of known 14C content, containing the compounds of interest and that can be subjected to the same chemical extractions and isolation procedures used on unknowns. Here, we present the potential of using single-year-growth grass as a modern process standard for the extraction and PCGC isolation of n-alkanes for 14C analysis. We started from the assumption that the 14C content of the grass leaf waxes, such as the long-chain n-alkanes (>C21), will be equal to the 14C content of the bulk grass, which is representative of the carbon fixed from atmospheric CO2 during one growing season (i.e. preceding collection). Our results showed that the n-alkanes extracted from the grass are indeed of modern 14C content similar to the bulk grass and thus can be suitable for the assessment of 14C-free Cex derived from sample preparation for CSRA. Grass material can be subjected to the same chemical extractions used on unknown samples (e.g. soils, sediments, plant matter) and has a similar composition to that of the unknowns (e.g. terrestrial material), thus constituting a good option as a process standard material.
MATERIALS AND METHODS
Grass Material
While any modern grass material could be used for the purpose described in this study, we took advantage of an earlier collection of grass near our facility from which a large stock of material is still available. Single-year-growth grass was collected locally (55.76°N, –4.18°W) in East Kilbride (EK), UK, near the NERC Radiocarbon Facility during the growing season of 1984 and stored in dry, cool, and dark conditions. The 14C content of the bulk grass, initially measured by liquid scintillation counting (LSC) at the NERC Radiocarbon Laboratory (n=2), is 1.2301±0.008 fraction modern, which agrees with atmospheric values reported for central Europe and the Northern Hemisphere during 1984 (Levin et al. Reference Levin, Kromer, Schoch-Fischer, Bruns, Münnich, Berdau, Vogel and Münnich1985; Hua et al. Reference Hua, Barbetti and Rakowski2013). For this study, we performed several 14C measurements by AMS of a subsample of this grass. Approximately 500 g of the grass was ground (using a new grinder to avoid cross-contamination) to pass a 500-µm mesh size, freeze-dried, and stored in an air-tight clean container. Three subsamples (~9 mg) were combusted to CO2 and converted to graphite (in replicates of three) following established protocols (Slota et al. Reference Slota, Jull, Linick and Toolin1987). Graphites from bulk combusted grass were sent to the SUERC AMS in East Kilbride and to the KECK CCAMS Facility at the University of California, Irvine (UCI), for analysis, with each facility measuring one or two graphites from each combustion. 14C concentration in this study is reported as fraction modern (F14C) according to international conventions (Stuiver and Polach Reference Stuiver and Polach1977; Reimer et al. Reference Reimer, Brown and Reimer2004). The average F14C value of all measurements by AMS (n=9) is 1.2224±0.0051. For the purpose of this study, we used all 14C measurements of the bulk grass available, including the two historical values obtained by LSC (Figure 1), to obtain the average bulk F14C value of the grass of 1.2238±0.0058 (n=11).
Fraction modern (F14C) values (error bars denote AMS uncertainty) from separate measurements of the grass material (bulk combusted in amounts varying from 0.7 to 1.0 mg C). These include triplicate measurements by AMS of three independent combustions (each shown as black, gray, and white symbols; n=9), measured at the KECK CCAMS and SUERC AMS facilities as indicated by triangles and circles, respectively. Also included are two historical measurements by LSC measured at the NERC Radiocarbon Laboratory (NERC RCL; a sample size of 1 mg of carbon is used for plotting purposes). Dashed line shows average±standard deviation (1.224±0.006 fraction modern; n=11).
Extraction of n-Alkanes
Two independent extractions of n-alkanes from the grass material were carried out at Newcastle University and Rothamsted Research, hereafter “extraction 1” and “extraction 2,” respectively, using two different methods. Two extractions were performed in order to obtain an additional set of n-alkane fractions. Extraction 1 consisted of microwave-assisted solvent extraction (MARS 5, CEM Microwave Technology, UK) of ~24 g of grass material using 15 mL of dichloromethane (DCM):methanol (3:1). A blank (no sample, solvent only) was also processed in the same manner as the grass sample. Glassware was cleaned with Decon90 (Decon Laboratories, UK), rinsed with ultrapure water, dried in the furnace, then rinsed with solvents before use. Pipettes and vials were heated for 1 hr at 450°C. Approximately 1–2 g of grass were extracted in a single microwave vessel and extracts from multiple vessels were combined. The microwave program ramped to 70°C and was held for 5 min. Total extracts were centrifuged then the solvent decanted and dried down using a rotary evaporator and nitrogen stream. The solvent extract was redissolved and added to aluminium oxide (150 mesh) before being added to 5% activated silica gel 60 columns, which were used to elute the hydrocarbon fraction using hexane (four column volumes). Extracts were subsequently dried using a rotary evaporator and nitrogen stream. The total hydrocarbon fraction and blank were analyzed by gas chromatography/mass spectrometry (GC/MS) to check purity of the extracts. The GC column used was a 30-m length Hewlett-Packard (HP) 5 and temperature program used was 50°C for 2 min, then 5°C/min to 310°C for 21 min. Extraction 2 consisted of the Soxhlet extraction of ~12 g of grass. Glassware was cleaned by washing with critical detergent, rinsing in ultrapure water, then drying with acetone, before heating in a muffle furnace for 1 hr at 450°C. Grass sample was extracted for 24 hr using DCM:acetone (9:1 v/v) to obtain a total lipid extract (TLE). The solvent was removed using a rotary evaporator and nitrogen stream. The TLE was redissolved in DCM:isopropanol (2:1 v/v) and filtered over defatted cotton wool. Glass columns packed with dried activated silica gel 60 (120°C, >12 hr) were pre-eluted with hexane. The TLE was resuspended in hexane and applied to the column. The hydrocarbon fraction was eluted using hexane under positive pressure supplied by a stream of nitrogen. The solvent was evaporated under nitrogen at 40°C.
Isolation of Compounds and Preparation for 14C Analysis
All Pyrex™ glassware and GC vials were cleaned by using either Decon90 or soaking in 5M nitric acid overnight, rinsed with ultrapure water, and dried then heated for 1 hr at 450°C. U-traps for collection of isolated compounds (see below) were rinsed with DCM five times, dried in a fume hood overnight, and heated for 1 hr at 450°C. Quartz glassware was heated for 1 hr at 900°C the day before use (aluminium foil and tweezers were heated for 1 hr at 450°C). All clean glassware was kept in air-tight containers along with desiccant (Silica gel, Fisher Scientific) and CO2 adsorbent (BDH Laboratory Supplies) and was heated again if stored for several weeks.
Separation of compounds was performed with a HP 5890 Series II GC with a fused silica capillary column (Rxi-1ms Restek, 30 m length, 0.32 mm ID, 0.25 um thickness), equipped with a HP 7673 injector and HP 5972 mass selective detector (MSD). The GC temperature program for the separation of grass n-alkanes was 50°C for 2 min, then 10°C/min to 320°C and held for 5 min. The same temperature program but ramping to 250°C was used for isolation of the standard material docosane (see below). The injection volume was 2 µL splitless for all samples (injection volume limited by the use of a standard GC injector). Compounds were isolated using a Gerstel preparative fraction collector (PFC) interfaced to the HP GC/MSD in a setup similar to that used by Eglinton et al. (Reference Eglinton, Aluwihare, Bauer, Druffel and McNichol1996). Approximately 1% of the flow eluting from the GC column was diverted to the MSD and 99% was sent to the PFC. The transfer line and PFC oven were kept at the maximum GC temperature program in use. The PFC was equipped with six U-traps for collection of compounds and one trap for waste. Care was taken to collect the entire peak of the target compound to avoid isotopic fractionation (Eglinton et al. Reference Eglinton, Aluwihare, Bauer, Druffel and McNichol1996; Zencak et al. Reference Zencak, Reddy, Teuten, Xu, McNichol and Gustafsson2007). The U-traps for collection were kept at –10°C using a cooling system of 50%/50% mixture of glycol/water. To prevent cross-contamination, all samples were first injected 10 times and collected into U-traps, which were then replaced with clean traps to start the sequence of injections for trapping. The total number of injections for trapping varied from 200 to 325 (see below) and final data corrections accounted for this.
Trapped compounds were retrieved by rinsing the U-traps four times with 250 µL of DCM into a clean GC vial. An aliquot of 100 µL was taken for determination of purity and yield by GC/MSD. Compounds were then transferred to a clean quartz insert (45 mm long, 5 mm ID) and solvent was removed under a stream of ultra-high purity nitrogen. The quartz insert was handled with tweezers and kept inside a clean 4-mL GC vial during solvent removal, covered loosely with clean aluminium foil (perforated at the top) to keep the insert clean. Solvent was removed to dryness and ~100–150 mg of copper oxide (precleaned for 1 hr at 900 °C) was added to the quartz insert. The insert was then placed inside a quartz tube (270 mm long, 9 mm ID on one end and 3 mm ID on the other end) and the quartz tube was flame-sealed at the 9-mm-ID end. Tubes were evacuated to 10–5 Torr, flame-sealed, and combusted for 6 hr at 900°C followed by 8 hr at 700°C. These combustion temperatures were not chosen for any particular reason other than the convenience of combusting samples along with other samples in our facility (using ramped cooling to optimize purity of combusted gas). All samples in this study, including those not prepared via PCGC, were combusted using the same type of quartz tubes and same combustion temperatures. After combustion, CO2 was cryogenically purified and reduced to graphite using standard procedures (Slota et al. Reference Slota, Jull, Linick and Toolin1987). Graphite targets from isolated compounds were analyzed at the KECK CCAMS Facility at UCI, normalized to OXII primary standard and fractionation corrected to –25‰ by using the AMS δ13C. Data corrections for combustion and graphitization procedures were done following the “non-matching” method (Santos et al. Reference Santos, Southon, Griffin, Beaupre and Druffel2007) using internationally accepted 14C standards and in-house 14C-free materials. Data corrections for PCGC preparation (isolation and solvent removal) accounted for 14C-free Cex. Modern Cex was assessed for solvent removal and applied to PCGC too (see explanation in the following section). 14C-free Cex and modern Cex were evaluated using commercially available compounds of known 14C content (indirect method) as described in the following.
Correction for the Amount and 14C Content of Cex
Since this was our first assessment of the usefulness of grass n-alkanes as process standards, that is, whether their F14C values agree with the bulk F14C value of the grass, we corrected the F14C values of the grass n-alkanes for Cex derived from PCGC isolation and solvent removal (after correcting for combustion and graphitization). The chemical extraction procedure (prior to PCGC) was not evaluated (apart from processing a blank for GC/MS analysis, see Results and Discussion) since it is a relatively simple procedure that does not require derivatization; thus, it is unlikely to introduce as much Cex compared to PCGC isolation and solvent removal. It should be noted that evaluating the chemical extraction becomes relevant if there are co-eluting compounds in the reagents and solvents used in the extraction procedure and/or extensive chemical pretreatments are used (Ziolkowski and Druffel Reference Ziolkowski and Druffel2009; Coppola et al. Reference Coppola, Ziolkowski and Druffel2013). Our results showed that our extraction methods do not contribute co-eluting compounds (see Results and Discussion).
To assess Cex, we followed the indirect method by using commercially available compounds of modern 14C content and 14C-free as standard materials to estimate the 14C-free and modern components of Cex, respectively. The bulk F14C values of these compounds were measured in duplicate by combusting an amount equivalent to ~0.8 mg C of the unprocessed material following the described procedures. As a modern standard, we used docosane (C22 n-alkane, Aldrich, 134457, Lot# MKBJ6726V), bulk F14C value=1.059±0.003 (n=2), and as 14C-free standards we used adipic acid (Acros Organics, 102815000, lot# A0306460), bulk F14C value=0.0015±0.0001 (n=2), and vanillin (Sigma Aldrich, W310700), bulk F14C value=0.0022±0.0001 (n=2). To determine the amount of modern and 14C-free Cex derived from PCGC and solvent removal, different amounts of the compounds were dissolved in 1 mL DCM (usual volume in unknown samples) and subjected to these procedures before preparation for 14C analysis. The deviation in the F14C values of the standard materials measured after PCGC isolation and solvent removal from their bulk F14C values (measured on unprocessed standard materials) was used to estimate the amount of Cex (of modern or 14C-free content depending on the standards used; Table 1). The amount of Cex was estimated by mass balance using the formulae by Santos et al. (Reference Santos, Southon, Griffin, Beaupre and Druffel2007) adding an extra term for “dead carbon correction” to include our 14C-free Cex derived from PCGC isolation and solvent removal. Our modern component of Cex corresponded to the “modern carbon correction” term in the formulae by Santos et al. (Reference Santos, Southon, Griffin, Beaupre and Druffel2007). We estimated the amount of Cex as the mass of extraneous carbon needed to correct the F14C values of the processed standard materials to within 1σ of their bulk F14C value. We express Cex derived from PCGC isolation and solvent removal in µg C per minute, per 50 (1-µL) injections for consistency with published literature (Ziolkowski and Druffel Reference Ziolkowski and Druffel2009; Coppola et al. Reference Coppola, Ziolkowski and Druffel2013), although Cex values are unique to each laboratory and procedure. In the case of solvent removal, Cex is expressed as µg C (Table 1).
Materials and sample sizes used to assess extraneous carbon (Cex) added during PCGC isolation and solvent removal (14C-free only) and solvent removal (14C-free and modern 14C content).
(a) Average F14C value (n=2) of unprocessed material combusted in sample sizes of 0.6–0.9 mg C.
(b) Corrected for combustion and graphitization procedures only.
(c) Estimated by mass balance using the formulae in Santos et al. (Reference Santos, Southon, Griffin, Beaupre and Druffel2007) based on the deviation in the F14C value of each processed sample (corrected for combustion and graphitization) from the F14C value of unprocessed material (bulk F14C). This is the mass of extraneous carbon needed to correct the F14C values of the processed samples to within 1σ of the bulk F14C value. Uncertainty is estimated as 50% of the carbon mass.
(d) 14C-free or modern 14C component of Cex as evaluated.
(e) Unprocessed material dissolved in ~1 mL of dichloromethane and solvent evaporated under a stream of ultra-high purity nitrogen.
Modern Cex was only evaluated for solvent removal due to technical issues with the GC/MSD interfaced to the PCGC collector. We used the amount of modern Cex estimated for solvent removal as the amount of modern Cex for PCGC isolation. Nevertheless, the modern component of Cex derived from PCGC processing is generally less significant than the 14C-free component (Druffel et al. Reference Druffel, Zhang, Xu, Ziolkowski, Southon, dos Santos and Trumbore2010; Kramer et al. Reference Kramer, Trumbore, Froberg, Dozal, Zhang, Xu, Santos and Hanson2010; Coppola et al. Reference Coppola, Ziolkowski and Druffel2013). In addition, the modern F14C value of our grass material makes the evaluation of 14C-free Cex relatively more relevant.
RESULTS AND DISCUSSION
The distribution and relative abundance of n-alkanes extracted from the grass are shown in Figure 2 and were similar across the two independent extractions. The most abundant n-alkanes were C29 and C31 and these compounds were targeted for PCGC isolation. In addition, a group of compounds from extraction 2, consisting of C23-27+C33 n-alkanes, was also PCGC-isolated for 14C analysis (combined to obtain enough carbon for AMS analysis). The F14C values of the n-alkanes and the total n-alkane fraction (before PCGC isolation of individual n-alkanes) from each extraction are shown in Table 2 as “uncorrected” (corrected only for combustion and graphitization) and “corrected” for Cex derived from PCGC isolation and solvent removal. The F14C values of C29 and C31 n-alkanes were in agreement across the two extractions and they were within 1σ and 2σ, respectively, of the F14C value of the bulk grass (Figure 3). The grouped C23-27+C33 had a F14C value that was within 2σ of the bulk grass. The F14C value of the total n-alkane fraction from extraction 1 agreed with that of the bulk grass while for extraction 2 it was within 3σ of the bulk grass. The blank processed through the chemical extraction 1 and analyzed by GC/MS showed a clean extract and without compounds co-eluting with the n-alkanes. Although we did not evaluate the chemical extraction 2 with a blank, the difference in the F14C value of the total n-alkane fraction with respect to extraction 1 is likely due to a different overall composition of the total extract, e.g. varying trace amounts of compounds other than n-alkanes, (rather than co-eluting compounds, see below), which could be possible given differences in the protocols between the two extractions. Regardless, trace compounds other than the targeted n-alkanes are excluded during PCGC isolation and thus do not affect the 14C content of the target compounds.
Relative abundance of n-alkanes extracted from the grass material
Fraction modern (F14C) values of grass n-alkanes PCGC-isolated from two independent extraction methods. Also shown are the F14C values of the total n-alkane fraction (“Total extract”; before PCGC isolation of individual n-alkanes) from each method. The F14C value of the grass (bulk combusted; n=11) and standard deviation are shown as solid and dotted lines, respectively.
F14C values of n-alkanes extracted from the grass material before and after correction for extraneous carbon (Cex) added during PCGC isolation and solvent removal (excludes chemistry prior to PCGC).
(a) Corrected for combustion and graphitization only.
(b) Aliquot of the total n-alkane extract before PCGC isolation of individual n-alkanes.
As explained earlier, the F14C values of the grass n-alkanes shown in Table 2 and Figure 3 were corrected for Cex derived from PCGC and solvent removal (Table 1) for the purpose of our initial assessment of their 14C content. We can also use the uncorrected F14C values of C29 and C31 n-alkanes isolated from the grass to estimate Cex derived from the entire sample procedure (chemical extraction + PCGC + solvent removal), assuming that the grass n-alkanes have the same 14C content of the bulk grass (our initial assumption) and thus any deviation represents Cex (14C-free) added during the entire sample procedure (assuming addition of modern Cex during PCGC isolation is relatively insignificant; Druffel et al. Reference Druffel, Zhang, Xu, Ziolkowski, Southon, dos Santos and Trumbore2010; Kramer et al. Reference Kramer, Trumbore, Froberg, Dozal, Zhang, Xu, Santos and Hanson2010; Coppola et al. Reference Coppola, Ziolkowski and Druffel2013). Our estimates show that ~0.91±0.46 to 1.3±0.65 µg C per minute, per 50 (1-µL) injections is derived from the entire sample preparation procedure versus 0.75±0.38 derived from PCGC isolation and solvent removal only (Table 3). The difference between these two estimates would suggest some contribution from the chemical extraction of grass n-alkanes. However, this contribution is likely small as the GC/MS analysis of the chemistry blank from extraction 1 revealed a clean chromatogram (dominated only by column bleed), showing that the extraction method 1 can produce clean extracts and free of co-eluting compounds. Although we did not evaluate extraction 2 in the same way, the similarity in the value of Cex between the two extraction methods (Table 3) suggests that extraction 2 also produces n-alkanes free of coeluting compounds. Since the extraction of n-alkanes does not require extensive processing or the use of derivatization (which adds carbon and requires an additional correction; Eglinton et al. Reference Eglinton, Aluwihare, Bauer, Druffel and McNichol1996; Ziolkowski and Druffel Reference Ziolkowski and Druffel2009), we should not expect the correction for Cex due to the prior chemical extraction alone to be significant relative to the correction due to PCGC isolation and solvent removal. GC column bleed, on the other hand, can contribute carbon (14C-free) to target compounds during PCGC isolation and this could explain the small difference in the estimated Cex values between PCGC and the entire procedure. Relatively greater GC column bleed occurs with later elution times; thus, the C31 n-alkane may be affected to a greater extent by column bleed relative to C29 (Figure 2) and both of these compounds may receive more column bleed relative to docosane (C22 n-alkane), which elutes the earliest. Given that docosane was used to estimate Cex derived from PCGC and the grass n-alkanes were used to estimate Cex from the entire procedure, the small differences in the estimated Cex values (Table 3) could be due to the effect of different degrees of GC column bleed on each compound rather than the chemical extraction of grass n-alkanes.
Estimation of the 14C-free component (F14C=0) of extraneous carbon (Cex) derived from PCGC and solvent removal procedures (using docosane) and derived from the entire sample preparation procedure (chemistry + PCGC + solvent removal, using the grass) for the isolation of n-alkanes.
(a) PCGC-isolated as indicated in Table 1.
(b) The 14C component of the carbon added (Cex) during sample processing.
(c) Estimated by mass balance using the formulae in Santos et al. (Reference Santos, Southon, Griffin, Beaupre and Druffel2007) based on the deviation in the F14C values of PCGC-isolated fractions (as in Table 1, corrected only for combustion and graphitization) from the bulk F14C value. This is the mass of 14C-free extraneous carbon needed to correct the F14C values of the fractions to within 1σ of the bulk F14C value. Uncertainty is estimated as 50% of the carbon mass.
(d) Estimated by mass balance using the formulae in Santos et al. (Reference Santos, Southon, Griffin, Beaupre and Druffel2007) based on the deviation in the F14C value of the n-alkane fraction (as in Table 2, “uncorrected”) from the bulk F14C value of the grass material. This is the mass of 14C-free extraneous carbon needed to correct the F14C values of the fractions to within 1σ of the bulk grass F14C value. Uncertainty is estimated as 50% of the carbon mass.
We compared the effect of correcting for PCGC + solvent removal versus correcting for the entire sample procedure on the F14C values of the grass n-alkanes, namely C31 and the group C23-27+C33. To correct for the entire procedure, we used the Cex values based on the C29 n-alkane (matching its value to the bulk grass), which are 0.93±0.47 and 0.91±0.46 µg C per minute, per 50 (1-µL) injections for extraction 1 and 2, respectively (Table 3). This correction brings the F14C value of C31 to within 1σ of the grass value for extraction 1, but it does not make much difference to the F14C value of C31 from extraction 2 (Figure 4). A similar effect is observed on the correction of the F14C value of the combined C23-27+C33. Again, this may be due to different amounts of 14C-free Cex added to each compound derived from GC column bleed at different elution times; therefore, the use of a single Cex value based on the C29 n-alkane does not fully correct the F14C values of compounds that elute relatively later. The estimated correction factors Cex based on the F14C value of C31 n-alkane (matching to the F14C value to the bulk grass) are 1.3±0.65 and 1.00±0.50 µg per minute, per 50 (1-µL) injections for extraction 1 and 2, respectively, which are slightly higher than those estimated based on C29 (Table 3). We did not estimate the correction factor based on the grouped n-alkanes collected from extraction 2 as the combined collection time is naturally much longer than the collection time needed for single compounds and thus artificially reduces the value of Cex, which is normalized to time. We collected this group of n-alkanes to have enough carbon for an AMS measurement and be able to compare their combined 14C content to the 14C content of the bulk grass despite their much lower abundance.
Fraction modern (F14C) values of n-alkanes (triangles and squares) and total n-alkane extract (circles) from (a) extraction 1 and (b) extraction 2. F14C values before and after correction for extraneous carbon (Cex) added during sample preparation are shown as open and closed symbols, respectively. Correction for Cex added during PCGC isolation and solvent removal (excludes chemical extraction, Table 1) is shown in triangles and correction for Cex added during the entire sample procedure (chemical extraction + PCGC + solvent removal, based on C29 n-alkane isolated from the grass, Table 3) is shown in squares. The total n-alkane extract was corrected for Cex derived from solvent removal. The F14C value of the grass (bulk combusted; n=11) and standard deviation are shown as solid and dotted lines, respectively.
Further in support of the effect from GC column bleed, the difference in 14C content among the grass n-alkanes does not seem to be related to sample size. Lower uncorrected 14C content would be expected with smaller sample sizes due to greater effect from 14C-free carbon on samples <100 µg C (Santos et al. Reference Santos, Southon, Drenzek, Ziolkowski, Druffel, Xu, Zhang, Trumbore, Eglinton and Hughen2010). Although our data corrections accounted for this sample-size effect, we note that the PCGC isolated sample size of C31 n-alkane matched that of C29 or was bigger, yet had relatively lower 14C content across the two extractions (Table 2). Thus, greater GC column bleed (14C-free) at a later elution time seems to explain the relatively lower 14C content of C31 n-alkane and to some extent that of the grouped C23-27+C33 (Figure 3). Taking this into account, when using the grass material as a modern n-alkane process standard, it may be advisable to choose the grass n-alkane that has an elution time closest to the elution time of the unknown compound to be corrected for Cex. Table 3 shows that the Cex value estimated for a given compound is similar across the two extractions, which supports this approach.
CONCLUSIONS
C29 and C31 n-alkanes were the most abundant n-alkanes in our modern grass and have F14C values that are within 1σ and 2σ of the F14C value of the bulk grass (1.224±0.006), respectively, thus constituting a good choice of compounds using the grass material as a process standard. Based on our results and our PCGC setup, 25 g of ground and homogenized grass material was sufficient to obtain enough carbon from C29 and C31 n-alkanes for the tests and PCGC isolation presented here. The chemical extraction of the grass n-alkanes did not seem to contribute much extraneous carbon relative to PCGC isolation. The F14C values of the grass C29 and C31 n-alkanes were corrected for 14C-free extraneous carbon derived from PCGC isolation, using commercially available docosane, which has a relatively earlier elution time. Our results suggest small differences may exist among the size of 14C-free blank of the individual compounds, including the different grass n-alkanes, related to different elution times and associated with contribution from GC column bleed. Therefore, when using the grass material as an n-alkane standard, it may be advisable to choose the 14C-free blank of the grass n-alkane that has an elution time closest to the elution time of unknowns. The use of the grass n-alkanes as process standards allows for the determination of the 14C-free component of carbon addition during preparation of similar sample materials (e.g. terrestrial plant material) for 14C analyses. Based on these results, other compounds of interest in CSRA (e.g. alkanoic acids, lignin phenols) could also be explored using modern grass as process standards.
ACKNOWLEDGMENTS
Thanks to Paul Donohoe and Berni Bowler for GC/MS analytical support at Newcastle University. This work was supported by the NERC Radiocarbon Facility NRCF010001.
