Article pubs.acs.org/est
Optimized Demineralization Technique for the Measurement of Stable Isotope Ratios of Nonexchangeable H in Soil Organic Matter Marc Ruppenthal,*,† Yvonne Oelmann,† and Wolfgang Wilcke‡ †
Geoecology, University of Tübingen, Rümelinstrasse 19-23, 72070 Tübingen, Germany Geographic Institute, University of Berne, Hallerstrasse 12, 3012 Berne, Switzerland
‡
S Supporting Information *
ABSTRACT: To make use of the isotope ratio of nonexchangeable hydrogen (δ2Hn (nonexchangeable)) of bulk soil organic matter (SOM), the mineral matrix (containing structural water of clay minerals) must be separated from SOM and samples need to be analyzed after H isotope equilibration. We present a novel technique for demineralization of soil samples with HF and dilute HCl and recovery of the SOM fraction solubilized in the HF demineralization solution via solid-phase extraction. Compared with existing techniques, organic C (Corg) and organic N (Norg) recovery of demineralized SOM concentrates was significantly increased (Corg recovery using existing techniques vs new demineralization method: 58% vs 78%; Norg recovery: 60% vs 78%). Chemicals used for the demineralization treatment did not affect δ2Hn values as revealed by spiking with deuterated water. The new demineralization method minimized organic matter losses and thus artificial H isotope fractionation, opening up the opportunity to use δ2Hn analyses of SOM as a new tool in paleoclimatology or geospatial forensics. addressed by Schimmelmann et al.15,16 in studies on the isotope ratio of nonexchangeable H (δ2Hn) of kerogen, but we are not aware of any similar work considering SOM, despite its importance in numerous ecosystem processes.17 Possibly, bulk δ2Hn values of SOM could be a valuable integrator of paleoenvironmental information and could complement information gained from the widely used δ2H values of lipid biomarkers6 or may be used for provenance assignment of soil samples. Dissolution of minerals using hydrofluoric acid (HF) has been in use for many years as a routine method to isolate and concentrate organic matter (OM) from sediments.18,19 This treatment, termed demineralization, has been applied to sediments in order to isolate kerogen20−22 or immature sedimentary organic matter23−25 and to soils in order to isolate SOM.26−28 Among the existing demineralization procedures, we identified three techniques which have been most widely used in the literature. These are (1) the HF-HCl technique,19 (2) the HF-BF3 technique,20 and (3) the HF-only technique, meaning that no reagents other than water are used for removal of carbonate and/or fluoride minerals before and after the HF treatment, respectively.29 The HF-BF3 technique has been proposed as a milder alternative to the HF-HCl technique which makes use of concentrated (6 M) HCl for dissolution of
1. INTRODUCTION Soon after the discovery of a characteristic spatial pattern of H isotope ratios in global precipitation in the 1960s,1,2 the pioneering works of Schiegl and Vogel3,4 demonstrated the usefulness of H isotope ratios of bulk sedimentary organic matter as a proxy of paleoclimate or geographical origin. Afterward, a large number of studies has shown the great potential of compound-specific H isotope ratios of lipid biomarkers in soil and sediment for deduction of paleoenvironmental information5,6 but studies utilizing the H isotopic composition of bulk soil organic matter (SOM) remain comparatively scarce. This has good reasons: (1) The δ2H value of a bulk soil sample is a mixture of organic matter H and mineral H of e.g., clay minerals and metal hydroxides. Because SOM and pedogenic minerals will likely differ in mean age,7 their H isotopic compositions reflect different climate signals. Furthermore, the size of H isotope fractionation between neoformed pedogenic minerals and water is different from that between biosynthesized organic compounds and water.8−10 Therefore, organic and inorganic H need to be separated in order to obtain unbiased measurements of the H isotope ratio of SOM. (2) Another difficulty is the presence of exchangeable H (usually bound to O, N, or S) in bulk SOM, which will equilibrate with atmospheric humidity and water in the laboratory and does not carry a meaningful isotope signal.11−14 Exchangeable H therefore has to be separated from nonexchangeable H by equilibration with water vapor of known H isotopic composition.11−14 Both problems have been © 2012 American Chemical Society
Received: Revised: Accepted: Published: 949
August 29, 2012 November 30, 2012 December 18, 2012 December 18, 2012 dx.doi.org/10.1021/es303448g | Environ. Sci. Technol. 2013, 47, 949−957
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neoformed fluorides from the demineralized concentrates. Comparing their HF-BF3 technique to the HF-HCl technique, Robl and Davis20 state that their HF-BF3 method resulted in a more efficient removal of neoformed fluorides and hence more complete reduction of residual ash content. In contrast to the aforementioned methods, the HF-only technique, which is most widely used, does not include particular steps (other than washing with deionized water) for removal of neoformed fluorides from demineralized samples. This is to reduce alteration and loss of SOM because of strong acid treatment.28,30 However, leaving neoformed fluorides in the sample may introduce inorganic H to the sample, distort the results of elemental analysis, and might have detrimental effects to analytical equipment used (e.g., quartz glass combustion tubes,27 ceramic tubes, fused silica capillaries). While losses of OM by HF demineralization are often negligible for samples that have undergone some diagenetic alteration (e.g., shales), HF demineralization of soils and immature sediments can cause OM losses as high as 50−90% of total organic C.26,31−33 To overcome the problem of OM losses during HF demineralization, Gélinas et al.24 proposed a procedure to recover OM solubilized in HF involving pH adjustments, precipitation of dissolved metals and chromatographic desalting, which allowed them to increase recoveries of organic C (Corg) from 32 to 95% to 80−95%. Recently, Li and Jia34 proposed solid-phase extraction (SPE) using Bond Elut PPL sorbent (Agilent Technologies Inc., USA) to recover solubilized organic matter from HF in an attempt to separate organic from inorganic N in a marine sediment sample and reported a Corg recovery of 98%. Besides Bond Elut PPL, Oasis HLB sorbent (Waters Corp., USA) has been reported to yield good recoveries of soluble organic compounds.35,36 Our objective was to identify the demineralization technique that is best suited for isolation of SOM for δ2Hn analyses. Such a method should maximize the purity of the demineralized SOM concentrate in order to avoid detrimental effects of residual inorganic H on SOM δ2Hn determinations, but at the same time minimize SOM losses during HF treatment and leave the H isotopic composition of SOM largely unaltered. Unfortunately, those techniques offering the most efficient demineralization (HF-HCl and HF-BF3 technique) also pose the greatest risk of unwanted alterations of SOM because of acid hydrolysis. We therefore decided to explore a new demineralization approach which uses dilute (0.1 M) HCl to more gently dissolve neoformed fluorides after HF treatment and includes recovery of solubilized SOM via SPE using Bond Elut PPL sorbent.34 We compared our new HF-dilute HCl approach to the three most widely used procedures (HF-HCl, HF-BF3, and HF-only technique) with regard to reduction of residual ash content, Corg and Norg recovery, and alteration of soil organic matter C, N, and H elemental and isotopic composition. For recovery of solubilized OM, we compared two different SPE sorbents (Bond Elut PPL and Oasis HLB). Possible effects of HF treatment on δ2Hn of SOM were investigated by spiking of demineralization reagents with deuterated water. The tests were performed on topsoil replicates and a number of additional subsoil horizons of a Stagnosol, a Cambisol, and an Arenosol37 from southwest Germany, which vary considerably in texture, chemical properties, and SOM content.
2. MATERIAL AND METHODS 2.1. Samples. We used soil samples from two grasslands in the Hunsrück mountains and from a mixed forest near the city of Mainz in southwest Germany. The Hunsrück soil samples were taken from a Leptic Cambisol (pH = 5.2, clay content = 20 ± 2 wt.%) on Permian basaltic andesite and a Haplic Stagnosol (pH = 4.8, clay content = 35 ± 3 wt.%) on Devonian shale. The forest soil sample was taken from a Calcaric Arenosol (pH = 5.8, clay content = 5 ± 1 wt.%) on a Pleistocenic sand dune. Table S1 in the Supporting Information summarizes additional selected properties of the soil samples. For each site, four topsoil replicates were collected at a distance of ca. 100 m from each other. In addition, all horizons of the soils were sampled at each site from the wall of a pit. Whole soils were used for comparison of demineralization techniques. To evaluate the effect of demineralization treatment on H elemental and isotopic mass balance, A horizon soil samples were used because only the A horizons yielded enough demineralized SOM concentrate for all analyses. Two samples of pure kaolinite (KGa-1b Kaolin, low-defect, Washington County, Georgia, USA) and montmorillonite (STx-1b Ca-rich Montmorillonite, Gonzales County, Texas, USA) powder were purchased from the Clay Minerals Society (Chantilly, USA) to test the success of clay mineral dissolution during demineralization treatment. 2.2. Sample Processing and HF Treatment. Soil samples were dried at 50 °C, and visible roots were removed prior to grinding in a ball mill. For demineralization, 0.5 ± 0.01 g of soil were weighed into 50 mL centrifuge tubes, and 40 ± 1 mL of demineralization reagent (either HF solution or HF-HCl mixture) was added. After shaking overnight for approximately 14 h at room temperature, the suspension was centrifuged at approximately 3000 G, and the HF supernatant was kept for later recovery of solubilized organic matter. The long demineralization time might increase the risk of fluoride precipitation, but some abundant minerals in soil (e.g., iron oxides/hydroxides or quartz) need several hours to dissolve in HF at room temperature.24,33,38 We therefore chose a time that would be sufficiently long for these minerals to dissolve. In order to identify the demineralization technique that causes highest purity of the demineralized SOM concentrate and least solubilization and alteration of SOM, we compared four different demineralization protocols: (1) HF-HCl technique: Mixture of 40 vol.% HF and 6 M HCl (1/1 vol./vol.), followed by centrifugation, decantation, and resuspension of the sample in 4 M HCl, followed three times by centrifugation, decantation, and resuspension in deionized water.19 (2) HF-BF3 technique: HF (20 vol.%), followed by centrifugation, decantation, and resuspension in HF-BF3 solution, followed three times by centrifugation, decantation, and resuspension in deionized water.20 (3) HF-only technique: HF (20 vol.%), followed four times by centrifugation, decantation, and resuspension in deionized water.26 (4) HF-dilute HCl technique: HF (20 vol.%), followed three times by centrifugation, decantation, and resuspension in 0.1 M HCl, followed once by centrifugation, decantation, and resuspension in deionized water. Furthermore, we compared two SPE sorbents for recovery of solubilized SOM from the HF supernatant: 950
dx.doi.org/10.1021/es303448g | Environ. Sci. Technol. 2013, 47, 949−957
Environmental Science & Technology
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Figure 1. Mean residual mass (a), mean Corg concentration (b), mean Norg concentration (c), mean Corg recovery (d), mean Norg recovery (e), mean C/N ratio (f), mean δ13C value (g), and mean δ15N value (h) of demineralized SOM concentrates using different demineralization techniques (n = 11 per group except for c, e, f, and h, where n = 10). Different letters (a, b, c, d) indicate significant differences among groups. All error bars represent standard errors.
measured δ13C values to the VPDB scale, and reference materials USGS-40 and IAEA-N-2 were used for normalization of measured δ15N values to the AIR-N2 scale.42,43 Measurement accuracy of IRMS analyses based on routine measurements of interspersed samples (n = 40) of sulfanilic acid (Merck KGaA, Germany) during the 3 months measurement period was ±0.1 ‰ for δ13C and ±0.2 ‰ for δ15N. 2.4. TC/EA-IRMS Analyses. The δ2 H values were determined on a vario PYRO cube EA (Elementar Analysensysteme, Germany) coupled to an IRMS (Isoprime, GV Instruments, United Kingdom). The H3+ factor varied between 6.18 and 6.30 ppm nA−1 over the 3 months measurement period. Two internal laboratory water standards were used for normalization of measured δ2H values to the VSMOW-SLAP reference scale. The internal laboratory water standards had been calibrated directly against VSMOW2 and SLAP2, using GISP as a measurement accuracy check,43,44 and had δ2H values of −268 ± 1 ‰ (n = 58) and +113 ± 1 ‰ (n = 26), respectively. We sealed liquid samples in tin capsules using end cutting pliers and analyzed liquid along with solid samples in the same carousel run, applying the so-called “packet dropping” technique,45 similar to the approach of Qi et al.46 The tin capsules used did not have a measurable H blank and also a small amount of entrapped air in the sealed capsules did not create a measurable H blank. The H isotope reference material IAEA-CH-7 with a certified δ2H value of −100.3 ± 2.0 ‰ was included in every carousel run as a permanent measurement quality and normalization accuracy control and yielded a δ2H value of −102 ± 2 ‰ (n = 80). 2.5. Water Steam Equilibration, Mass Balance, and Error Calculations. To eliminate the effect of isotopically exchangeable H on δ2 H measurements, samples were equilibrated in an equilibration device with two waters of known H isotopic compositions (−268 ± 1 and +113 ± 1 ‰). Details of the equilibration procedure and a schematic graph of
(a) Bond Elut PPL technique: SPE using 200 mg Bond Elut PPL cartridges (Agilent Technologies Inc., USA).34 (b) Oasis HLB technique: SPE using 200 mg Oasis HLB cartridges (Waters Corp., USA). SPE sorbents used for solubilized SOM recovery protocols (a) and (b) were preconditioned with acetone and methanol and equilibrated with 0.1 M HCl (one cartridge volume each). After sample addition, the sorbents were washed with 0.1 M HCl, and the extracted organic material was eluted with methanol followed by acetone (one cartridge volume each). Solubilized SOM recovered in this way from the HF supernatant was combined with the demineralized particulate SOM concentrate. The combined concentrates were dried at 40 °C and disaggregated in an agate mortar. To test possible effects of HF treatment on δ2Hn values of demineralized SOM concentrates, we prepared HF and HCl with a batch of deionized water having a δ2H value of +896 ± 3 ‰ and demineralized the samples with the spiked reagents. All chemicals used were of analytical grade. 2.3. EA-IRMS Analyses. Concentrations of total C (TC) and total N (TN) were determined with an Elemental Analyzer (EA) (vario EL III, Elementar Analysensysteme, Germany). C concentrations measured after combusting the soil samples for 4 h at 550 °C in a muffle furnace39 were assumed to be inorganic C (Cinorg), and Corg concentrations were calculated as the difference of TC and Cinorg. The δ13C and δ15N values were determined on a vario EL III EA (Elementar Analysensysteme, Germany) coupled to an Isotope Ratio Mass Spectrometer (IRMS) (Isoprime, GV Instruments, United Kingdom). In order to determine δ13C values of Corg for samples containing Cinorg, 15 mL of sulfurous acid (4 wt.%) was added to 1 g of sample, and the acid was vaporized overnight at 50 °C on a hot plate.40 This procedure allows efficient dissolution of carbonates while largely preserving OM composition.40,41 Reference materials IAEACH-6 and IAEA-CH-7 were used for normalization of 951
dx.doi.org/10.1021/es303448g | Environ. Sci. Technol. 2013, 47, 949−957
Environmental Science & Technology
Article
pure montmorillonite (STx-1b Ca-rich Montmorillonite, Gonzales County, Texas, USA) using the HF-only technique (white precipitate remained), whereas dissolution was virtually complete (residual mass
