Intramolecular carbon isotopic analysis of acetic acid by direct injection of aqueous solution

Organic Geochemistry 40 (2009) 195–200

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Intramolecular carbon isotopic analysis of acetic acid by direct injection of aqueous solution Burt Thomas *, Katherine H. Freeman, Michael A. Arthur Department of Geosciences, Penn State University, 801 Deike Building, University Park, PA 16802, USA

a r t i c l e

i n f o

Article history: Received 5 March 2008 Received in revised form 28 October 2008 Accepted 30 October 2008 Available online 6 November 2008

a b s t r a c t We report an improved method for determining the intramolecular carbon isotopic composition of acetate using direct injection of aqueous samples. The system builds upon prior work that established pyrolytic conditions for online analysis and represents a significant advance in that it requires minimal preparation for samples containing as little as 1 mM sodium acetate in aqueous solution. The technique is applicable for analysis of oilfield brines, culture samples, biological samples and natural porewaters. We demonstrate its accuracy by use of a stable isotope dilution series. We also show that addition of a base and cryogenic preconcentration may induce an isotopic effect on the carboxyl carbon. This isotopic fractionation does not appear to extend to the measured methyl carbon isotope value although it can significantly alter the measured isotopic composition of the whole molecule. Our preconcentration experiments demonstrate that the method is suitable for carbon isotopic measurements of acetate methyl carbon in natural samples at concentrations as low as 90 lM, considerably broadening potential applications. Published by Elsevier Ltd.

1. Introduction Acetate is a ubiquitous and rapidly transformed intermediate in natural and engineered anoxic systems. It is also a nearly universal carbon source for microbial respiration. In highly reducing anoxic environments, microbes rely on complex and often obligate syntrophic relationships that maximize energy yields and community growth rates (Schink, 1997). These syntrophic connections are easily severed, whether intentionally by inhibitors or antibiotics, or unintentionally by experimental conditions or other treatments. Thus, ex situ culturing methods and many in situ experimental techniques alter the state of the microbial system they are designed to probe (Conrad, 2005). Measurements of natural isotopic abundances avoid these pitfalls and provide critical tools for tracing carbon flow in complex natural systems. Whole acetate isotope measurement (Ferchaud-Roucher et al., 2006; Fey et al., 2004; Heuer et al., 2006) provides a powerful alternative in situ technique for studying acetate production and consumption processes without disturbing delicate microbial relationships. However, isotopic compositional differences within molecules can complicate interpretation of results of whole molecule isotopic analysis (Abelson and Hoering, 1961; Corso and Brenna, 1997; Deniro and Epstein, 1978; Monson and Hayes,

* Corresponding author. Tel.: +1 814 865 1178; fax: +1 814 863 7823. E-mail address: [email protected] (B. Thomas). 0146-6380/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.orggeochem.2008.10.011

1980). Acetate isotopic compositions observed from analysis of the whole molecule reflect an average composition of the methyl and carboxyl carbons, which can be quite different from each other (Dias et al., 2002a). Further, even if systematic isotopic differences between the methyl and carboxyl carbon of newly produced acetate record kinetic isotopic fractionations, there is ample evidence that exchange reactions between acetate carboxyl carbon and inorganic carbon can overprint original fractionation effects. Exchange of the acetate carboxyl carbon with dissolved inorganic carbon is observed in both high temperature hydrous pyrolysis reactions (Dias et al., 2002a; Felipe et al., 2005; Franks et al., 2001) and at low temperature in sediment culture (deGraaf et al., 1996; Valentine et al., 2004). In fact, early hypotheses of the important role of carbon monoxide dehydrogenase in methanogens were confirmed by the observation of carbon isotopic exchange between labeled inorganic carbon and acetate carboxyl carbon (Eikmanns and Thauer, 1984; Raybuck et al., 1991; Spormann and Thauer, 1989). Since the isotopic composition of acetate carboxyl may be influenced by dissolved inorganic carbon, it is difficult to interpret whole acetate isotopic information without separate measurements of acetate carboxyl. This study extends isotopic approaches to include intramolecular isotope analysis for low acetate concentrations found in soils and sediments and has immediate application in a wide variety of disciplines, including: environmental studies of marine and terrestrial sediments, human and animal colonic fermentation processes and industrial and municipal waste digestion.

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two reaction furnaces. The effluent from each furnace is directed through a nafion dryer and, ultimately, to the ion source of the mass spectrometer via an open-split interface.

2. Method development 2.1. Method precedents Early techniques for measuring the intramolecular carbon isotopic composition of acetate employed liquid chromatography (LC) separation of acetate followed by offline closed-tube pyrolysis (Gelwicks and Hayes, 1990). An online technique for separately analyzing pyrolyzed methane and carbon dioxide was reported by Yamada et al. (2002) but requires prior concentration to be useful for complex natural samples. Dias et al. (2002a) reported the first use of a continuous flow technique for the combustion and pyrolysis of acetate, relying on solid phase microextraction (SPME) to separate the acids of interest from the solution matrix (Dias and Freeman, 1997). The SPME application of Dias and Freeman (1997) represented a significant advance because it allows both online separation and combustion or pyrolysis. However, the adsorption efficiency of SPME fibers depends directly on the concentration and inversely on the aqueous solubility of the analyte. The concentration of dissolved organic species that compete for SPME active sites also influences the efficiency. The solubility of acetic acid in water is so high that Dias et al. (2002a,b) found it impossible to obtain accurate analyses from samples in which the concentration of acetate was less than 600 lM. In common with other phase partitioning techniques, adsorption on SPME can also lead to small but measurable isotopic fractionations (Dias and Freeman, 1997). These factors ultimately limit SPME techniques to samples with high concentrations of acetate (ca. 1 mM) and low concentrations of competing solutes. Our new method eliminates sorption of acetate on SPME media but utilizes the pyrolytic analytical conditions of Dias et al. (2002a). It is the first procedure to use water as a solvent in gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-irMS) analysis of low molecular weight fatty acids. We report detection limits of 1 nM of acetic acid per injection, similar to the SPME detection limit reported by Dias et al. (2002a). Since it does not require equilibration with SPME fibers, it is both faster and appropriate for much smaller sample sizes and does not suffer from solute competition effects in samples high in dissolved organic carbon (DOC). We have also developed a method for pre-concentrating acetic acid from aqueous samples, further lowering the detection limit for larger samples with lower concentrations of acetate. A diagram of the system components is presented in Fig. 1. A polar phase GC capillary column separates analyte acid peaks from each other and from water. The chromatographic effluent is either directed to waste (i.e. during the elution of water) or to one of the

120kPa He Carrier

vent

600C Pd He

vent

- 40C Nafion Dryer

He

1020C, Ni,Pt

30m Nukol O2 H2

Finnigan MAT 252

Fig. 1. Schematic of combined GC-C/PY-irMS system. The carrier gas can be routed to either the pyrolysis furnanceor the combustion furnace by switching simultaneously the 4 port valves upstream and downstream of the furnaces.

2.1.1. Aqueous sample introduction We maintain the injected sample at ca. pH 2, well below the pKa of acetic acid (pKa 4.7). The low pH minimizes the precipitation of acetate salts in the injector, thus reducing memory effects. Because strong acids degrade the polyethylene glycol chromatographic media and corrode the metal catalysts in the combustion and pyrolysis furnaces, we selected oxalic acid as the pH-controlling reactant. It has a low pKa and is involatile under initial GC conditions. At low injection temperatures, it also forms poorly soluble salts, which decrease the interaction of acetic acid with the glass liner of the GC inlet for subsequent sample additions. At temperatures >145 °C, undissociated excess oxalic acid decomposes to CO2, H2O and formic acid. We find that, for sample pH values between 1.5 and 2, there is no detectable carryover of acetate into subsequent analytical blanks and peak areas suggest complete acetate delivery to the combustion or pyrolysis interface. It is important to monitor the gradual buildup of formate and oxalate salts and periodically remove these by clipping the front end of the column and by routine injector maintenance. Alternatively, column maintenance is significantly aided by the use of a 0.53 lm guard column in front of the analytical column (Traitler et al., 1988). A wide bore guard column capably captures salts and other nonvolatile components and can be periodically removed and rinsed with acetone prior to reinstallation. 2.1.2. GC conditions One microliter of aqueous sample is introduced at 50 °C into a flash on-column injector liner (Supelco, Bellefonte, PA) connected to an undeactivated silica guard column (5 m  0.53 mm) and a capillary GC column (Supelco, Bellefonte, PA; 30 m  0.25 mm ID, 0.25 lm film thickness, Nukol), with He as carrier gas. This injection system ensures complete sample transfer to the separation column. The injector temperature program is identical to the column ramp: 50 °C (hold 1 min) at 10 °C/min to 130 °C (hold 5 min). A final temperature ramp to 185 °C decomposes excess oxalic acid and prepares the column for the next injection. The volumetric column flow rate is ca. 1.75 ml He/min. Typical retention time for water is between 350 and 450 s. Column flow is directed to vent for ca. 600 s, then the 4 port valve is switched, directing acetic acid and subsequent analyte peaks to either the pyrolysis or combustion furnaces. 2.1.3. Drying efficiency Water in the GC effluent presents two significant challenges for accurate isotope irMS: (i) it is a notorious source of error for isotope ratio measurements because of its contribution to proton transfer reactions and (ii) it can carry ions that precipitate as salts during sample evaporation in the GC inlet, thereby degrading chromatographic performance. Despite significant peak tailing of water (which is expected), both the polar polyethylene glycol-based chromatographic phases we tested, Nukol (Supelco) and FFAP (J&W Scientific) provide adequate separation between water and acetic acid with 30 m columns and a moderate GC oven temperature ramp rate of 10 °C/min. This was true for all column diameters and phase thicknesses. A 4 port valve after the GC column allows the water solvent peak to be diverted to vent prior to the delivery of the analyte peak to the combustion or pyrolysis furnaces. We employ a standard counter-flow ‘Nafion’ water trap (Leckrone and Hayes, 1997) to remove any residual water from the carrier gas prior to transfer to the mass spectrometer via the opensplit. Signals related to water are lower than those observed in conventional combustion-based analysis. This may indicate that the

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use of water as the solvent significantly reduces background levels of hydrocarbons in the column effluent. 2.1.4. Acetic acid pyrolysis The combustion furnace is identical to that used in conventional irm-GCMS applications and is packed with wound Ni/Pt wires (Merritt et al., 1995). The pyrolysis furnace is composed of a ceramic alumina tube (0.5 mm ID) filled with four 0.05 mm OD palladium wires (Dias et al. (2002a)) and maintained at 600 °C. The furnace is constantly supplied with a small flow of H2 through a T junction between the column and the pyrolysis furnace. Excess H2 leads to protonation of CO2 in the ion source, which is observable via an elevated ion current for m/z 45. We find isotope ratio measurements of CO2 are accurate when H2 addition is below detection of protonated CO2. To determine the maximum H2 flow that does not affect mass-ratio measurements of CO2, we gradually increase the flow at the T junction with a low flow mass controller until the m/z 45/44 signal ratio begins to increase. The H2 flow is then decreased to a level just below detection of elevated m/z 45, usually much less than 0.01 ml/min. 2.1.5. Temperature optimization We determined the temperature for optimum pyrolysis yield by monitoring the production of CO2 from injection of known amounts of acetic acid. A maximum in yield was observed at 600 °C (Fig. 2). This temperature optimum is similar to that chosen by Dias et al. (2002a) and is consistent with earlier work (Maier et al., 1982). At temperatures < 550 °C, lower yields are accompanied by significant unpyrolyzed acetic acid, which was observed by monitoring m/z 60. At temperatures > 750 °C, CO becomes a significant decomposition product. The production of CO at higher temperatures may be a consequence of catalyzed decomposition of acetic acid to MeOH and CO (Doolan et al., 1986). 3. Analysis of method Acetic acid pyrolysis under the conditions presented is hypothesized to be a catalyzed decomposition on surface Pd-H groups (Maier et al., 1982) so, while H2 is not a reactant in the pyrolysis of acetate to CH4 and CO2, it is required for consistent pyrolysis of acetate. In addition to replenishing Pd-H surface sites, it is also possible that hydrogen plays a role in reducing trace oxygen from the carrier stream and preventing formation of PdO. In an early test of pyrolytic conditions, we found that preconditioning the catalyst with H2 was required to eliminate the oxidation of the acetate methyl group to CO2. Following overnight pure H2 treatment, a furnace can maintain pyrolytic catalysis for as long as 24 h in the absence of continued H2 addition. Occasional regeneration of the

30

25

Area (V*s)

20

catalyst under high H2 partial pressure at 900 °C for 3 h is sufficient to both replenish the pyrolytic capacity of old reactors that have begun to oxidize and to precondition new reactors. Although isotope ratio measurements are degraded by high quantities of H2, its presence in the furnace is critical for maintaining reducing conditions, to strip low amounts of contaminant oxygen from the column effluent and to provide H for absorption on to the Pd catalyst. In the absence of catalyst and at temperatures >1000 °C, other studies have reported two competing acetic acid unimolecular decomposition pathways. One reaction produces CH4 and CO2 and the other results in dehydration to ethenone (ketene) and water (Mackie and Doolan, 1984). With the Nafion chiller turned off, we determined that the Dias et al. (2002a) catalytic pyrolysis method produces no appreciable ethenone, observed as m/z 42, and no other significant mass fragments apart from m/z 16 (CH4) and m/z 44 (CO2). Our observations are consistent with Dias et al. (2002a) and confirm the completeness of the catalyzed pyrolysis of acetic acid to CH4 and CO2 under these conditions. 3.1. Accuracy and precision We demonstrated the accuracy of our intramolecular measurements through analysis of a labeled isotope dilution series. To create the dilution series, a 1 mM solution of labeled 1-13C sodium acetate (99%; Sigma Aldrich, St. Louis, MO) was mixed with a 1.000 mM solution of homogenized unlabeled laboratory grade sodium acetate that also serves as an isotopic internal standard (Sigma Aldrich, St. Louis, MO). The final dilution series represents an approximate 100‰ range in d13C values of acetate carboxyl. The intramolecular isotopic composition of the internal standard was determined independently from separate measurements of both whole acetate and the methyl carbon. Whole acetate d13C values of the powdered internal standard were determined using conventional EA-irMS. The isotopic composition of the methyl group of the internal standard acetate was determined by first liberating it as CH4 via closed-tube pyrolysis (Gelwicks and Hayes, 1990). This CH4 was then transferred with a gas-tight syringe and isotopically analyzed using combustion irm-GCMS (Sowers et al., 2005). These internal standard measurements are presented in Table 1. The expected mole fraction of 13C, 13F, for each sample in the dilution series can be calculated from the independently determined offline d13C values of both carboxyl carbon and total acetate if the mole fraction of labeled acetate is known for each solution. We use the relationship, 13RIS = 13RStd(dIS/1000 + 1) to convert offline d values to isotopic ratios where the standard ratio is the internationally accepted 13RVPDB = 0.0112372. We complete the conversion of delta notation to 13F, with the relation, 13FIS = 13 RIS/(1 + 13RIS)). The expected 13F for each subsequent dilution is determined by applying separate mass balance relations for the acetate carboxyl and the whole molecule. For the carboxyl mass balance, 13 FC ¼ 13 F ISC ð1  XÞ þ ðXÞ13 F Label where X is the mole fraction of labeled acetate and 13 F Label is the mole fraction of label at the carboxyl position. For the whole molecule, 13 F Whole ¼ 13 F ISwhole ð1  XÞþ 13 F Labelwhole ðXÞ. Conveniently, since 99% 1-13C acetate consists of equal parts carboxyl carbon with 13F  0.99 and

15 area of mass 44 response (V*s) 2° polynomial fit

10

5

0

Table 1 Results of offline and online analyses of the internal standard acetate.

Offline 300

400

500

600

700

T (ºC)

Fig. 2. Temperature optimum for catalyzed pyrolytic production of CO2. A second order polynomial fit through the data illustrates a broad optimum at 600 °C.

Online

Internal standard

d13C

n

Std. Dev.

Methyl EA combustion Carboxyl by difference Carboxyl by pyrolysis Combustion Methyl by difference

27.3 33.6 39.9 39.8 33.5 27.3

3 3 3 5 5 5

0.4 0.2 0.4 0.5 0.7 0.7

13

FIS

0.010673679 0.010742965

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Table 2 Results of dilution series calculations and measurements. Std. Dev. Pyr.

n pyr.

39.8 27.1 14.0 7.8 62.1

0.7 0.5 1.1 0.9 5.2

3 4 4 3 2

Mean 13 Fpyr

Mean d13C comb. (‰)

Std. Dev. Comb.

n comb.

0.0106753 0.0108144 0.0109582 0.0111984 0.0117943

33.3 26.5 20.2 7.6 15.5

0.7 0.7 0.6 0.9 1.8

2 3 3 3 3

13 methyl carbon with F = 0.01, it follows that 13 F Labelwhole ¼ :99=2 þ :01=2 which simplifies to 0.50. The results of these dilution series calculations along with measured mean values of dilution series samples are in Table 2. Fig. 3 presents the expected and measured 13F values for pyrolysis and combustion measurements of the dilution series. Two to four duplicate analyses were performed for each serial dilution. Both whole acetate and carboxyl values closely correspond to the 1:1 identity line, confirming that this method accurately reproduces the expected isotopic content of the carboxyl carbon and the whole compound. Alternatively, average measured values for all dilution series samples can be presented in the familiar delta notation, where d13C = 1000  (13Rsample13Rstd)/13Rstd), and these values can be compared with the expected 13F of the carboxyl in labeled dilutions (Fig. 4). Separate regression lines for combustion and pyrolysis clearly match the independently determined offline d13C values. Since the serial dilution contains 13C label at the carboxyl site only, the slope of the combustion line is expected to be half that of the pyrolysis line. The slope ratio from the two dilution curves, acetate total:acetate carboxyl, is approximately 0.48, which is very close to the expected result, 0.50. The slightly lower value of the slope ratio may be significantly impacted by random errors in isotope measurements associated with the most isotopically enriched dilutions which are several tens permil enriched relative to the reference gas. The precision of the method is estimated by five repeat intramolecular analyses of the internal standard presented in Table 1, and is within ±0.7‰ (1 r, 95% confidence interval) for both pyrolysis and combustion measurements in the natural abundance range of d13C.

4. Preconcentration procedure and analysis The method described in the previous Section requires 1 mM sodium acetate solution for a 1 ll injection. This is in the high

Measured

13

F

0.0116 0.0114 0.0112 13

Mean F pyrolysis 13 Mean F combustion 1:1 line

0.0110 0.0108

0.0108

0.0110

0.0112

Expected

0.0114

0.0116

Mean 13 Fcomb

X (mole fraction labeled acetate)

13

Expected Fcarb

Expected 13 F whole

######## ######## ######## ######## ########

0 0.0001301 0.0002603 0.0005205 0.001041

0.0106737 0.0108011 0.0109285 0.0111834 0.0116932

0.0107430 0.0108066 0.0108703 0.0109976 0.0112523

60 Pyrolysis slope = 99230

40

Online combustion and pyrolysis values 13

δ C whole

20

13

δ C carboxyl Combustion slope = 47711

0

13

IS B5 B4 B3 B2

Mean d13C pyr. (‰)

δ C (‰) vs. VPDB

Sample

Internal standard values offline whole offline carboxyl

-20

-40 0.0108

0.0110 13

0.0112

F carboxyl

Fig. 4. Measured combustion and pyrolysis d13C values compared to the expected 13 F of the carboxyl. The horizontal lines reflect independently determined values of internal standard acetate whole and acetate carboxyl. Linearity confirms the robust nature of this analysis across a range of isotopic content while close correspondence between the offline determined values with the online regression line confirms accuracy. Vertical error bars represent 1 r 95% confidence intervals for multiple injections.

range of natural samples in surficial environments, which typically range between 5 lM at oxic interfaces to a few mM at depth in some sediments. We tested a lyophilization method for concentrating more dilute solutions using synthetic samples of three different sample matrices: distilled water, filtered humic acid and unfiltered raw bog mud collected from the catotelm of Bear Meadows Bog, Centre County, PA. Each matrix solution contained <15 lM acetate, to which our internal standard acetate was added to a final concentration of 80–95 lM acetate, which is more than one order of magnitude below the detection limit of the pyrolysis system. Unamended matrix samples were reserved to be analyzed as blanks. We then filtered the synthetic samples and blanks sequentially through a qualitative GFF filter and 0.2 lm polycarbonate syringe filter (Pall Life Sciences). To the filtered samples we added varying amounts of 1 mM NaOH prepared from reagent grade NaOH solution in order to identify pH artifacts upon lyophilization. The pH of each synthetic sample was measured on a 1 mL subsample with a standard laboratory pH meter and another 1 ml subsample was taken for acetate determination using ion chromatograph. Aliquots (9 ml) of the matrix samples and blanks were then frozen and preconcentrated by lyophilization in a polypropylene centrifuge tube. The freeze-dried sample was resuspended in an appropriate volume of 18.2 MX distilled deionized (DDI) water and titrated to pH 2 with solid oxalic acid. The pH of the solution was verified by dropping 10 ll aliquots of sample on to low range pH paper (J.T. Baker). The acidified sample was then transferred to a new 1.5 ml centrifuge tube for storage at 20 °C prior to analysis.

13

F

Fig. 3. Comparison of means of measured and expected values of 13F. The measured carboxyl and whole acetate values are plotted for each analysis of the dilution series. Correspondence with the 1:1 line indicates good overall accuracy in the range of natural abundance values.

4.1. Preconcentration results The chemical and carbon isotopic properties of preconcentration test samples are presented in Table 3. Synthetic matrix blanks,

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Table 3 Results of preconcentration tests (OH 1, OH 2 and OH raw) corresponding to two different levels of base addition following sample filtration and one in which base was added to raw unfiltered synthetic samples (synthetic samples without isotope data did not contain sufficient acetate for isotope analysis). Sample

Test

Vol. (mL)

Sample matrix

Acetate internal standard spike (lM)

GF filter

.2 lm filter

1 M NaOH added (lL)

pH prior to freeze dry

[Acetate] before freeze dry (lM)

2FDT1 2FDT2 2FDT3 2FDT4 2FDT5 2FDT6 2FDT7 2FDT8 2FDT9 2FDT10 2FDT11 2FDT12 2FDT13 2FDT14 2FDT15 2FDT16 2FDT17 2FDT18 2FDT19

Blank Blank Blank Filter control Filter control Filter control Base control Base control Base control OH1 OH1 OH1 OH2 OH2 OH2 OH OH OH High

10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Raw Raw Raw pH

DDI HA POM DDI HA POM DDI HA POM DDI HA POM DDI HA POM 10 10 10 10

0 0 0 80 80 80 80 80 80 80 80 80 80 80 80 DDI HA POM HA

n n n n n n y y y y y y y y y 0 0 0 0

n n n n n n y y y y y y y y y n n n n

0 0 0 0 0 0 0 0 0 450 450 450 575 575 575 n n n n

6.5 4.2 4.0 5.5 4.4 4.0 5.5 4.5 4.4 10.1 9.8 9.2 10.2 10.0 9.5 575 575 575 1013

0.7 15.7 0.5 85.8 94.4 84.4 86.3 94.6 85.4 79.5 92.5 80.8 83.8 92.3 78.4 10.2 9.6 1.1 11.5

a

Measured d13C whole acetate

Measured d13C acetate carboxyl

Calculated d13C acetate methyl

n

Std. Dev.

34.5

40.3

28.7

2a

0.9

33.1

39.2

27.1

2a

0.2

30.7 31.0 31.1 30.5 30.6 29.2 n.a. 16.2

35.6 35.6 34.5 33.1 32.8 30.0

25.8 26.4 27.6 28.0 28.3 28.5

1 1 1 1 1 1

25.0

Denotes n = 2 for pyrolysis only.

filter controls, base controls and synthetic matrix samples of DDI water, humic acid (HA) and raw mud (POM) are noted. Intramolecular isotopic values were only possible for samples with pH > 5.5 prior to lyophilization since acetate in the form of acetic acid is lost upon vacuum distillation. Filter controls allow us to demonstrate an absence of analytical artifacts due to filtration on acetate concentration; however, we discovered an important concern when using NaOH to elevate the pH of synthetic samples prior to lyophilization. For samples with pH P 11, there was an increase in the acetate concentration in mixtures using both bog mud and bog HA. This was true for both filtered and unfiltered samples, which likely rules out an intracellular microbial source. Since the OH control samples do not seem to show an acetate dependence on OH addition at lower levels, we rule out the possibility that the released acetate is derived from the OH solution itself. We hypothesize that the observed acetate increase results from acetate desorption from the humic matrix or ester hydrolysis of acetoxyl groups of dissolved and particulate organic matter. Similar sorption behavior is described by Hordijk et al. (1994) for acetate on iron oxides. Our preconcentration procedure for dilute acetate samples unexpectedly resulted in significant isotopic fractionation of the

δ13C (‰) vs. VPDB

-25

Methyl measured via offline techniques

-30 13

δ C carboxyl 13

δ C methyl

-35 Carboxyl measured via offline techniques

-40

-45

4

6

8

10

12

pH

Fig. 5. Measured d13C of preconcentration test samples. When treated with OH, at high pH, the carboxyl carbon becomes significantly enriched relative to the known value of the internal standard. This enrichment does not extend to the methyl carbon.

acetate carboxyl (Fig. 5). The origin of the isotopic fractionation is unclear; however, we hypothesize that the difference between the observed and the expected carboxyl values is either a function of exchange between the carboxyl group and inorganic carbon during the lyophilization of very alkaline samples or due to an isotopic fractionation of sorption. When we compared calculated and expected isotopic values for methyl carbon in our standard, we found no effect of preconcentration. We are therefore confident that the effects of base addition and lyophilization do not extend to the acetate methyl carbon. The mechanism and specific conditions that favor this apparent carboxyl exchange reaction are the focus of continued investigation. 5. Conclusions Isotopic exchange of dissolved inorganic carbon (DIC) with the acetate carboxyl carbon is a recognized natural and laboratory phenomenon and is of growing interest for interpretation of whole acetate isotopic signatures in nature. Our refined method targets acetate in natural aqueous samples for separate measurement of the carbon isotopic compositions of the carboxyl and the whole molecule, without the need for offline extraction or derivatization. The technique requires minimal modification of an existing irmGCMS system. For acetate concentrations of 1 mM or greater, as commonly observed in deep porewater systems, oilfield brines, biological samples and microbial culture samples, no preconcentration is necessary. For high DOC samples, we suggest a target pH of ca. 7.0 as sufficient to target ‘free’ acetic acid and to minimize the concern for isotopic fractionation during preconcentration. Additional analytical protocols should be devised to target sorbed or ester-linked acetate. Our experiments demonstrate the acetate methyl isotopic composition is unaffected by lyophilization for purposes of preconcentration. However, freeze drying under highly basic conditions appears to induce an isotopic fractionation of the acetate carboxyl. Many prior investigations use either basic sample pretreatment, basic LC separation techniques, or base addition and preconcentration (Blair and Carter, 1992; Conrad et al., 2002; Fey et al., 2004; Gelwicks et al., 1994; Krzycki et al., 1987; Londry and Des Marais, 2003). In the light of these results, we suggest a cautious interpre-

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tation of measurements of whole acetate isotopic composition. Our improved method for intramolecular carbon isotopic analysis of acetic acid will significantly aid efforts to probe the processes that determine the heterogeneity and biological and abiological controls on the isotopic composition of acetate in nature. Acknowledgments The work was supported by a grant from the ACS Petroleum Research Fund, PRF-44729-AC2, to MA and BT, and by initial seed funding to BT from the Penn State Biogeochemical Research Initiative for Education (BRIE) sponsored by NSF-IGERT Grant DGE9972759. We would like to thank D. Walizer for technical assistance and P. Polissar and R.F. Dias for helpful comments and discussion during method development. The manuscript benefited greatly from insightful reviews by J.M. Hayes as an anonymous reviewer. Associate Editor—S. Schouten

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