Organic Geochemistry 55 (2013) 22–25

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Organic Geochemistry

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d13C analysis of bulk organic matter in speleothems using liquidchromatography–isotope ratio mass spectrometry

Alison J. Blyth a,⇑, Yulia Shutova b, Colin Smith c

a WA-OIGC, Department of Chemistry, Chemistry and Resources Precinct, Curtin University, GPO Box U1987, Perth, WA 6845, Australiab Water Research Centre, School of Civil and Environmental Engineering, University of New South Wales, Australiac Archaeology Program, La Trobe University, Victoria 3086, Australia

a r t i c l e i n f o a b s t r a c t

Article history:Received 31 May 2012Received in revised form 5 November 2012Accepted 6 November 2012Available online 17 November 2012

0146-6380/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.orggeochem.2012.11.003

⇑ Corresponding author. Tel.: +61 (0)892669388; faE-mail address: alison.blyth@curtin.edu.au (A.J. Bl

The determination of d13C values in speleothems is of considerable importance in palaeoenvironmentalresearch, but has focussed solely on analysis of the carbonate. Here we demonstrate a new method foranalysing the d13C values of organic matter (OM) trapped in speleothems, utilising flow injection liquidchromatography–isotope ratio mass spectrometry (LC–IRMS). Developmental analysis using a homoge-nised speleothem powder showed that the method is robust, with repeated digests and analyses havingan average standard deviation of 0.1‰. Dilution tests with samples of 4–23 lg total organic carbon (TOC)show relatively small linearity effects, with the overall standard deviation across a peak response range of1700–9000 mV being 0.2‰.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Two principal carbon pools are present in speleothems: carbon-ate in the calcite and carbon in entrapped organic matter (OM).Conventional d13C analysis accesses only the former pool, whichis derived from carbonate dissolved from the bedrock, and trans-ported dissolved soil CO2 (Genty et al., 2001). The second pool, car-bon contained in OM, represents compounds derived from the soiland those derived from in situ cave organisms and has not beenstudied in speleothems because of methodological difficulties.Investigating the isotopic signal of this OM enables recovery of anew type of d13C record from speleothems, as well as helpingunderstand the controls on the calcite signal. Here we propose asimple method for analysing the stable carbon isotope ratio ofthe acid soluble OM, utilising liquid chromatography–isotope ratiomass spectrometry (LC–IRMS) in flow injection analysis mode.Depending on the acid and oxidant used, the system allows mea-surement of dissolved inorganic carbon (DIC; e.g. Brandes, 2009)in a liquid sample, or dissolved organic carbon (DOC; more cor-rectly non-purgeable OC, NPOC; e.g. Albéric, 2011). Advantages in-clude direct injection of samples from the acid digest withoutsubstantial off line wet chemistry and injection of small samplevolume (10–20 ll). Combined, these factors mean that the ap-proach uses calcite samples 6200 mg, considerably smaller thanthe samples needed for other organic analyses (1–20 g), allowingfor the first time records to be produced at a resolution comparableto inorganic isotope records.

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x: +61 (0)892662300.yth).

2. Material and method

2.1. Samples

Bulk calcite powder was obtained by milling cleaned lumps of alarge stalagmite to fine powder and mixing to homogeneity (Blythet al., 2006). A 5 g aliquot was weighed into a 10 ml vial and furthermixed by shaking.

2.2. Total OC (TOC)

TOC was measured using an Aurora 1030 wet oxidation TOCanalyser (OI Analytical, College Station, TX, USA), which allowedmeasurement NPOC concentration in a sample via high tempera-ture oxidation with sodium persulfate (Na2S2O8), following re-moval of DIC via addition H3PO4 and sparging (2 min) in astream of inert gas. The TOC analyser was attached to a 1088 rotaryauto-sampler (OI Analytical, College Station, TX, USA) compatiblewith 40 ml sample vials and equipped with a 10 ml syringe forsample injection. We dissolved 0.5, 1 and 1.5 g aliquots of homog-enised powder within the sample vials using 6–16 ml sonicatedultrapure HCl and topped the mixture up to 40 ml using sonicatedMilliQ water. Samples were left overnight to equilibrate beforeanalysis using the standard water sample TOC method describedabove.

2.3. NPOC analysis with LC–IRMS

Samples (200 mg) were digested in 2 ml of 3 M H3PO4 (Sigma,HPLC grade). H3PO4 was used in preference to HCl to avoid halide

A.J. Blyth et al. / Organic Geochemistry 55 (2013) 22–25 23

interference with the LC–IRMS oxidant (Albéric, 2011). After com-plete digestion of the calcite, aliquots of samples were transferredto 1.8 ml LC–MS vials and dissolved CO2 was removed under vac-uum (1 h) in a rotational vacuum concentrator. Tests showed thatvacuum treatment removed DIC to below measurement limit,without affecting the NPOC signal (Fig. 1). After vacuum treatment,samples were sealed with caps and transferred for analysis. Samplevials were not topped up to remove the head space, to avoid dilut-ing the sample. Experiments leaving vials on the bench uncappedfor 24 h and measuring DIC before and after showed no detectableredissolution of CO2.

Stable isotope analysis was carried out with a Thermo ScientificLC–IRMS instrument (consisting of an Accela autosampler and Acc-ela 600 pump attached to a Delta V plus isotope ratio mass spec-trometer via an LC-Isolink). Reagents and mobile phase weremade with MilliQ water, degassed (1 h) under vacuum with soni-cation and then constantly sparged with He. The analytical methodwas similar to that described by Albéric (2011). Analysis was inflow injection mode using a mobile phase of dilute H2SO4 (pH4.0–4.2; 100 ll 1:50 H2SO4 in 1 l of MilliQ water) at 300 ll min�1

and maintained at 20 �C using the column oven. For each run,10 ll of sample were injected using the autosampler and oxidationof the OC was achieved using a catalyst (1.28 M H3PO4 at20 ll min�1) and oxidant (0.13 M Na2S2O8 at 20 ll min�1). The oxi-dation reactor in the LC-Isolink was maintained at 99.9 �C. Runtime was 5 min, with measurements made relative to the secondof two 20 s reference gas pulses at the start of the run; three morereference gas pulses were used after the analyte peak had ap-peared, to check for drift over the run. The reference gas was cali-brated to �22.92‰ VPDB (Vienna Peedee Belemnite) using USGS-41 glutamic acid (+37.626‰ VPDB) as standard. As this is an en-riched standard to which we were restricted due to availability,the calibration was also checked against in-house amino acid stan-dards in the range �7.6‰ to �31.6‰, which gave satisfactory re-sults. Between analytical runs, H3PO4 blanks were run to helpclean the sample loop and reduce sample carry over. To preventbuild-up of solid CaPO4 in the needle, flushing used non-degassedmobile phase. Due to the presence of two in-line filters in the sys-tem, providing areas for salts to nucleate, build-up of CaPO4 withinthe instrument itself was not observed. However, as a precaution,in addition to the blank runs, we maintained water flow throughthe system at all times, and also subjected it to intermittent runsof H2SO4.

Tests for possible DIC interference in the degassed phosphatesolution and samples utilised the same methodology as above withthe exception that only the acid catalyst reagent was used (no oxi-dant) and the oxidation reactor was at 60 �C [similar to the methodof Brandes (2009)].

a

Fig. 1. Chromatogram (m/z 44) showing analyte peak for (a) DIC run and (b) NPOC run fsamples had been vacuum purged (30 min), and the DIC peak is equivalent to that seen fowas increased to 1 h, to ensure maximum removal of DIC.

3. Results and discussion

The samples had a mean (n = 6) TOC concentration of 114 lg/gcalcite or 0.011%, demonstrating the generally low abundance ofOM in stalagmites, and equating to a TOC of 23 lg in our 200 mgcalcite samples. However LC–IRMS showed a good measurablepeak response of 1500–9000 mV, even at 1/6 dilution of the origi-nal concentration (TOC of ca. 4 lg), indicating that the method caneasily handle low abundance samples. Blanks showed no measur-able contamination.

To test the repeatability of the technique, four separate digestsof powder (test stal a–d) were each analysed 5� (6� for test stal d).This tests both instrumental repeatability and the influence onrepeatability of the wet chemistry process. The consistency of themethod (Table 1) was excellent, with a mean d13C value of�19.9‰ and standard deviation (SD) of 0.1‰ across the 21 runs.These injections showed a peak response of 5000–9000 mV, thevariation indicating that, although robust for isotopes, the methodshould not be used for measuring the abundance of NPOC. The con-sistency of the isotopic values indicates that linearity is not an is-sue for samples within this range, although as it is known that bothprecision and accuracy are affected by changing sample abun-dance; to test the effect of this, the original digest of test stal bwas diluted into two new samples 1/3rd and 1/6th of the originalconcentration, and analysed 5� and 6� respectively. The 1/3rddilution show a peak amplitude of 2200–4200 mV, a mean d13C va-lue of �19.7‰ (SD 0.4‰). The SD was affected by the second injec-tion, which showed an abnormally high value of �19.1‰. For the1/6th dilution, the peak amplitudes were 1700–2100 mV, with anisotopic mean of �19.5‰ and SD 0.2‰. This indicates that lowersample size did not have a significant effect on the precision ofthe technique, but there was a slight trend towards slightly heaviervalues with lower peak amplitude (Fig. 2). However, when theanalyses were considered across the whole amplitude range(n = 32; peak amplitude 1700–9000 mV), the mean was �19.8‰

and the SD 0.2‰. This is an acceptable level of error and indicatesthat the method is sufficiently robust to apply to time series sam-ples, provided analytical repeats are run.

A major issue for future work in the field is the identification ofthe precise OM fraction measured using this technique. It is estab-lished that it is the NPOC, not the TOC that is measured, acceptingloss of volatile purgeable compounds during preparation. Equally,any interaction of the OM with the H3PO4, or any precipitation ofsolid during the wet chemistry process could introduce bias. Inthe case of interaction with the acid, we observed during earlymethod development that samples rerun on later days showedan increase in isotopic value. This did not occur consistently, butwas seen frequently enough to be of concern. As redissolution of

b

rom a method development test sample. Square peaks represent reference gas. Ther the reagent blanks. Prior to preparation of the test stal samples, vacuum purge time

Table 1d13C results with mean and standard deviation (SD) for all stalagmite digests.

Stalagmite digest Amplitude (mV) of m/z 44 peak d13C (‰) Digest mean (‰) Digest SD (‰)

Test stal a 6777 �19.8 �19.9 0.16823 �19.96732 �20.06824 �19.96711 �19.9

Test stal b 9068 �20.0 �19.9 0.19054 �20.05785 �19.75074 �19.88087 �19.8

Test stal c 5302 �19.9 �20.0 0.15176 �19.95144 �20.05063 �20.05054 �19.9

Test stal d 6941 �19.8 �19.9 0.17093 �19.96968 �19.96947 �20.06993 �20.06592 �19.9

Test stal b 1/3 4064 �19.8 �19.7 0.44219 �19.12218 �19.93968 �19.83796 �20.1

Test stal b 1/6 2104 �19.5 �19.5 0.22001 �19.62061 �19.52030 �19.31968 �19.51747 �19.8

All digests (avg.) �19.8 0.2

δδ

Fig. 2. Scatter plot showing change in d13C of the NPOC with peak amplituderesponse.

24 A.J. Blyth et al. / Organic Geochemistry 55 (2013) 22–25

CO2 from the atmosphere had been experimentally tested and ru-led out, we hypothesise that the changes were due to prolongedexposure of the OM to H3PO4. However, the problems did not occurduring the first 24 h after digestion, and we therefore consider that,as long as samples are prepared and run in small batches, with theanalysis taking place within 24 h of digestion, this should not be anissue. The question of precipitation bias is more serious, and moredifficult to investigate. It is well established that humic acids inparticular will precipitate in acidic solution, a fact exploited intheir extraction (e.g. van Beynen et al., 2001). We would thereforeexpect humic-rich samples to precipitate some compounds in thiscontext. The extent to which this is a problem for the technique de-pends on whether the precipitation of compounds is consistent be-tween heterogeneous samples, and establishing this should be a

focus of future work. If it is consistent, then the bias would not af-fect the results of the technique, as long as it is noted that the en-tity is measured in each case is only the acid soluble NPOC, nottotal bulk OM.

4. Conclusions

The study demonstrates a simple and robust new method forthe study of acid soluble OM in small (6200 mg) calcite samplesfrom speleothems (or indeed any other CaCO3 medium). Worknow needs to focus on understanding the controls on the signal(vegetation, microbial degradation, soil turnover, in situ and exter-nal inputs, chemical bias) in order to ensure the utility of the pal-aeoenvironmental d13C records which can now be recovered.

Acknowledgements

The study was funded by an AINSE Research Fellowship to A.J.B.,ARC Future Fellowship to C.S. (FT0992258) and NERC ResearchGrant NE/G016925/1 to A.J.B. The stalagmite sample was collectedon the Ethiopia Venture 2000 expedition. An anonymous revieweris thanked for constructive comments to improve the manuscript.

Associate Editor—S. Schouten

References

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