A method to measure the isotopic (13C) composition of dissolved organic carbon using a high temperature combustion instrument

Marine Chemistry 103 (2007) 318 – 326 www.elsevier.com/locate/marchem

A method to measure the isotopic ( 13 C) composition of dissolved organic carbon using a high temperature combustion instrument Susan Q. Lang ⁎, Marvin D. Lilley, John I. Hedges ✠ University of Washington, School of Oceanography, Box 355351, Seattle, WA 98195, United States Received 7 February 2006; received in revised form 3 October 2006; accepted 5 October 2006 Available online 4 December 2006

Abstract The stable isotopes of dissolved organic carbon (DOC) are a powerful tool for distinguishing sources and inputs of organic matter in aquatic systems. While several methods exist to perform these analyses, no labs routinely utilize a high temperature combustion (HTC) instrument. Advantages of HTC instruments include rapid analysis, small sample volumes and minimal sample preparation, making them the favored devices for most routine oceanic DOC concentration measurements. We developed a stable carbon DOC method based around an HTC system. This method has the benefit of a simple setup, requiring neither vacuum nor high pressures. The main drawback of the method is a significant blank, requiring careful accounting of all blank sources for accurate isotopic and concentration values. We present here a series of experiments to determine the magnitude, source and isotopic composition of the HTC blank. Over time, the blank is very stable at ∼ 20 ng of carbon with a δ13C of − 18.1‰ vs. VPDB. The similarity of the isotopic composition of the blank and seawater samples makes corrections relatively minor. The precision of the method was determined by oxidizing organic standards with a wide isotopic and concentration range (− 9‰ to −39‰; 18 μM to 124 μM). Analysis of seawater samples demonstrates the accuracy for low concentration, high salinity samples. The overall error on the measurement is approximately ± 0.8‰. © 2006 Elsevier B.V. All rights reserved. Keywords: Dissolved organic carbon; Stable isotopes; High temperature combustion

1. Introduction Stable carbon isotope ratios are a powerful tool with which to study the sources and dynamics of dissolved organic carbon (DOC). δ13C measurements provided early evidence that deep-sea marine DOC is relatively homogenous and has minimal input from terrestrial sources (Williams and Gordon, 1970; Eadie et al., 1978; Williams and Druffel, 1987). Stable carbon isotopes have ⁎ Corresponding author. E-mail address: [email protected] (S.Q. Lang). F Deceased. 0304-4203/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.marchem.2006.10.002

also been used to constrain organic inputs into rivers and estuaries (Stuermer and Payne, 1976; Sherr, 1982; Onstad et al., 2000; Raymond and Bauer, 2001) and as food web tracers (Rau and Hedges, 1979). Despite the utility of the measurements and the numerous methods that exist, routine analysis of DO13C is not performed in most laboratories. Existing methods for DO13C analysis require large setup times, high pressures, vacuum, or specialty equipment. In each, dissolved inorganic carbon is removed from the sample followed by oxidation of the DOC to CO2 which is then is purified and introduced to an isotope ratio mass spectrometer (IRMS) for isotopic

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Fig. 1. Schematic of oxidation and trapping system.

analysis. The analyses primarily differ in the method of oxidizing DOC. High energy ultraviolet irradiation is used most frequently (e.g. Williams and Gordon, 1970; Eadie et al., 1978; Williams and Druffel, 1987; Druffel et al., 1989, 1992; Bauer et al., 1998) since small blanks and the ability to oxidize large sample volumes make it also amenable for radiocarbon (14C) analysis. One method for stable carbon isotope measurement has been developed using a high temperature combustion (HTC) technique (Bauer et al., 1992) that requires trapping the CO2 onto a molecular sieve and recovering it at 550 °C under vacuum. Recently, an automated UVoxidation system has been developed that allows for smaller sample sizes and more rapid analysis (St-Jean, 2003). A supercritical oxidation method using a high temperature liquid chromatography instrument also exists (le Clercq et al., 1998). The majority of laboratories use HTC systems to oxidize DOC for routine concentration measurements since they allow for rapid analysis and small sample volumes (Sharp et al., 2002). To our knowledge, no laboratories are routinely using these HTC instruments to prepare DOC samples for stable carbon isotope analysis. We have developed a method based around a high temperature combustion (HTC) system. The advantages are that it utilizes equipment that many laboratories already have and does not necessitate a vacuum or high pressures. Measurements can be performed rapidly; samples can be analyzed in triplicate in 2–3 h. 2. Experimental The analysis consists of three steps: 1) sample oxidation with a modified HTC DOC instrument; 2) trapping the CO2 with liquid nitrogen (LN2); and 3) isotope ratio

analysis of the CO2 on an IRMS. The system is shown schematically in Fig. 1. Each step will be described in greater detail below. 2.1. The oxidation and CO2 trapping systems Samples were oxidized with a modified MQ-1001 high temperature combustion DOC analyzer (Qian and Mopper, 1996; Peterson et al., 2003). The instrument is typical of many newer HTC analyzers in that a small volume of sample water is automatically injected onto the head of a heated (750 °C) combustion column that is purged by a continuous flow of high purity oxygen carrier gas. Water vapor explosively formed from injection of 100–200 μL of sample is sequentially eliminated from the carrier gas stream by condensation in a sparger tube, removal through a selectively permeable Nafion® membrane, and finally, absorption onto Mg (ClO4)2. The dried gas stream is then passed through a LICOR 7000 non-dispersive infrared detector that measures the CO2 absorption and forwards the data to a chromatography data system for processing. The instrument was modified specifically for the δ13C method. First the gas stream was changed from high purity O2 to a high purity He/O2 mixture (75/25; 99.9995%, UHP grade; Matheson Tri-Gas). Pure oxygen gas (boiling point −183.0 °C) condenses when brought down to the LN2 temperatures (boiling point −195.8 C), and could lead to potentially dangerous situations and incomplete CO2 gas recovery. Changing the carrier gas to an He/O2 mixture allows for enough oxygen to be present as a reactant for sample oxidation while preventing oxygen condensation. Other HTC instruments, such as the Shimadzu, use a pure air carrier gas and may not require

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this alteration. The carrier gas stream is passed through a CO2 absorber (Alltech) before entering the MQ-1001 instrument. The second modification allows trapping of the CO2 gas. Just upstream of the detector, the carrier gas is redirected through a stainless steel tubing coil (1/8 [0.125] inch OD, 0.028 inch ID, 10 ft. long; Swagelok; Fig. 1). CO2 is condensed from the gas stream by immersing the coil in LN2. Once the CO2 is frozen within the tubing, valves on either end of the coil are closed and the entire loop is disconnected from the line. While the sample coil is removed, the gas stream is redirected through a bypass loop to the LICOR detector. The bypass tube allows the instrument to be used as a typical DOC analyzer even if the sample coil is removed from the system. The sample tube is placed in line before the LICOR detector to ensure the entire peak is trapped. If LN2 is not emplaced early enough, or is removed too soon, the LICOR will respond to the untrapped CO2. Minimizing the time the LN2 trap is in line is important in order to reduce the isotopic blank. The 3-way valves (Swagelok, SS-42XS4) that isolate the sample coil minimize atmospheric contamination thereby requiring less time to purge the system of atmospheric CO2 between samples. 2.2. Mass spectrometer After being removed from the HTC system, the sample coil is placed in line with an isotope ratio mass spectrometer (IRMS). The 13C content of the CO2 was measured using a Finnigan Delta Plus IRMS during 2002 experiments and a Finnigan MAT 251 IRMS during 2003 experiments. In both cases, each sample was trapped on a gas preconcentrator, which freezes the CO2 plug onto capillary tubing, and then releases the gas into a gas chromatograph with a PoraPlot-Q column to separate N2O from CO2 before entering the mass spectrometer. Reported here are the 13C/ 12C ratios expressed as per mil deviations from the international standard Vienna Peedee belemnite (VPDB): δ13Csample = (Rs / Rst − 1) × 1000 where Rs is the ratio of 13C/12C in the sample and Rst is the ratio of the VPDB standard. 3. Methods The following section describes the treatment of samples and standards discussed for the remainder of the paper. Consistent results were obtained when samples were processed in exactly the same fashion, hence the MQ-1001 analyzer was programmed to repeatedly inject 100 μL of sample every 5 min. This injection volume was used unless otherwise stated (e.g. during blank

analysis). For each sample, at least 5 injections were made to condition the column before capturing a single CO2 peak by manually immersing the sample coil in LN2. After a single injection peak was captured, the gas flow was redirected through the bypass tube, the coil removed from the HTC system, and a new sample coil placed in line. To purge atmospheric CO2 from the system, at least three injections were made with the sample coil in line but not immersed in LN2 before capturing another peak Using this procedure, it took approximately 2–3 h and 20 mL of total sample (including rinses etc) to collect CO2 in triplicate for isotopic analysis. For each conditioning injection LICOR peak areas were recorded for DOC concentration determination. After the CO2 samples were captured in the coils, it was removed and attached to the preconcentrator of the IRMS. The short tubing ends on the sample coil that are open to the atmosphere were purged with helium for 10 min. The CO2 in the coil was then concentrated and injected into the IRMS. The timing of when to emplace and remove the dewar of LN2 was determined using a standard solution with a high DOC concentration (120 μM) and monitoring the LICOR output. This exact timing (approximately 2.5 min total) was used for each peak even though samples with smaller DOC concentrations could have been completely trapped with shorter immersion times. By keeping the timing exactly the same for all analyses, blanks originating from impurities in the carrier gas mixture were kept constant. 4. Results and discussion 4.1. Characterizing the size and isotopic composition of the blank One of the main drawbacks of high temperature combustion instruments is the presence of a large blank. Without proper correction for this blank, significant errors can be introduced into DOC concentration and isotopic values (Hedges et al., 1993). Generally, DOC analysis blanks are determined by injecting ‘carbon-free’ Millipore Milli-Q water and measuring the resulting peak area. Any organic carbon present in the Milli-Q water, however, will be incorrectly subtracted from sample peak areas. Distinguishing between a blank caused by organic material in the water used to make standard solutions versus instrument and reagent blanks can be difficult. Sources of instrument blanks can include carbon from column packing materials, tubing, leaks in the system, and organic impurities in the carrier gas (Tanoue, 1992). Instrument blanks for various HTC

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analyzers have been shown to be both volume dependent (varying with the total amount of water injected; Tanoue, 1992; Benner and Strom, 1993; Cauwet, 1994; Peterson et al., 2003) and time-dependent (varying with the time between injections; Tanoue, 1992; Qian and Mopper, 1996; Peterson et al., 2003). A series of experiments was performed to determine the source, magnitude, and isotopic composition of the blank. In the first experiment different volumes of MilliQ water were injected into the system (50, 100, 150 and 200 μL) and the resulting CO2 peaks trapped. The magnitude of the CO2 blank as measured with the LICOR detector was determined by averaging at least 15 injections and the results are shown in Fig. 2 (right axis). For each injection volume three CO2 peaks were individually trapped and then analyzed with the IRMS for isotopic composition. The magnitude of the CO2 peak injected into the IRMS is presented as an average of the triplicate samples (Fig. 2, left axis). For DOC concentration analysis, peak areas are measured against a steady background signal and any CO2 present in the carrier gas does not affect the peak area above background. Since samples for isotope analyses are cryogenically trapped, any CO2 present in the gas stream will be an additional source of blank. The magnitude of this carrier gas blank was determined by immersing the trapping coil in LN2 without injecting a sample (Fig. 2, left axis, ‘0 μL’). The amount of CO2 entering the injection column was also tested by placing the trapping coil immediately after the CO2 absorber and immersing it in LN2 (Fig. 1, coil location denoted as point A; Fig 2, ‘He/O2’). In both cases, the sample loop was immersed in LN2 for the same amount of time as

Fig. 2. Blank area response vs. μL of Milli-Q water injected. Area responses were measured on the IRMS (n = 3 for each data point) and on the LICOR 7000 (n = 15–25 for each data point). The “0 μL” postcolumn data point represents trapping the carrier gas without injecting a sample. The “He/O2” data point represents trapping the carrier gas just before the high temperature column (point A on Fig. 1).

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Fig. 3. Composition of the total CO2 blank. We assume that the blank present in the He/O2 gas and post-column gas (“0 μL” injection) is present in the each total blank. With increasing water injection volume, the magnitude of the injection volume dependent blank also increases. This volume dependent blank may be due to organic matter in Milli-Q water or due to an instrument blank.

when trapping actual samples. The magnitude of these blanks could not be measured by the LICOR detector since they are part of the constant background; instead they are determined only for the IRMS. Surprisingly, there is a measurable CO2 blank in the He/O2 gas stream, even after passing through a CO2 absorber. An even more significant source of background CO2 appears to be continuously released from the combustion column, as is evident from the magnitude of the ‘0 μL’ blank. Fig. 3 presents the different blank sources as a function of injection volume. It is assumed that the pre-column He/O2 blank and the post-column ‘0 μL’ blank are always present when water is injected. There is also a blank that increases in size with increasing injection volume, contributing between 2%– 33% of the total signal. This volume dependent blank is not necessarily due to organic material in the Milli-Q water and may be intrinsic to the instrument. For instance, larger injection volumes may oxidize more impurities on the column packing materials. As stated above, this blank alone is typically measured and reported by DOC analysts. For a 100 μL injection, the peak area of the volume dependent blank (0.5 ± 0.06 mV min on the IRMS, 110 ± 10 mV min on the LICOR) is 5–10% of the total peak area of a deep Sargasso seawater standard (10.6 ± 0.5 mV min on the IRMS, 1050 ± 50 mV min on the LICOR). This percentage is similar to that found by Sharp et al. (2002), who report a 4–8 μM instrument blank with a Sargasso seawater concentration of 44 μM (i.e. 9–18%).

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Fig. 4. Isotopic composition of the total blank vs. μL of Milli-Q water injected.

The isotopic composition of the total blank ranges from − 19.2‰ to − 24.3‰ depending on the volume of water injected (Fig. 4). The δ13C of the blank becomes more negative with larger injection volumes, indicating there is more than one isotopically distinct source for the blank. Presumably, one source is a constant CO2 stream coming off the column with a relatively heavy isotopic composition and an additional source which is volume dependent and isotopically lighter. The δ13C of the He/ O2 is quite different than that of the ‘0 μL’ sample, indicating the He/O2 blank represents a third isotopically distinct source, whose signature is incorporated into the constant CO2 stream. Several attempts were made to isolate and minimize the sources of the blank. The steady ‘0 μL’ post-column blank could be explained by organic contamination in the gas stream that passes through the combustion column and is continuously oxidized. To test this, a stainless steel loop immersed in LN2 was placed after the CO2 absorber and prior to the column (Fig. 1, point A) to trap any contamination from the He/O2 tank not removed by the absorber. However, there was no significant change in the blank size. The addition of a hydrocarbon trap immediately after the CO2 absorber also did not reduce the size of the blank. Using a gas mixture of reported similar purity from another company (AirGas, Inc.) increased the size of the blank five-fold. The presence of the LN2 trap and hydrocarbon trap did not significantly decrease the size of the blank with the less pure gas. Either the traps were inefficient or impurities in the gas stream were so large that the traps were quickly overwhelmed. For that reason, we cannot rule out the presence of organic contamination in the gas stream.

Atmospheric CO2 could enter the system through small leaks or gas permeable tubing and contribute to the blank. All gas and water lines were converted to gas impermeable stainless steel, brass, or PEEK tubing. The one exception was the clear Teflon sample injection loops. We visually monitor the loop during analysis to ensure the water sample is properly drawn through the loop and that no gas bubbles are in the line. Switching to Peek tubing would have prevented this visual check. Despite these efforts, it is likely that atmospheric CO2 contributes to the blank to some degree. The packing material of the column has often been suggested as a probable source of the blank (Bauer et al., 1993; Benner and Strom, 1993; Perdue and Mantoura, 1993; Skoog et al., 1997; Chen et al., 2002; Peterson et al., 2003). The columns used here are packed with quartz beads, cupric oxide, and sulfix®. Few other instruments use quartz beads; platinized alumina is more common (e.g. most Shimadzu instruments). Despite differences in packing materials, most HTC instruments demonstrate similar behaviors in terms of their blanks. For instance, newly installed columns universally have large blanks that decrease over time and with an increasing number of injections (e.g. Benner and Strom, 1993). Another common observation is that the magnitude of the blank increases with increasing wait time between injections (Tanoue, 1992; Qian and Mopper, 1996; Chen et al., 2002; Peterson et al., 2003). Previous experiments with the MQ-1001 showed that removing the quartz beads significantly decreased the size of the blank while removing the cupric oxide and sulfix had little effect (Peterson et al., 2003). If the quartz beads are a primary source of the blanks observed here, other instruments may have blanks with different isotopic

Fig. 5. The area response and isotopic composition of 50 μL, 100 μL, 150 μL, 200 μL injections of Sargasso Sea reference water (Hansell, 2001).

S.Q. Lang et al. / Marine Chemistry 103 (2007) 318–326 Table 1 Comparison of blank corrected δ13C values for Sargasso Sea reference seawater computed using two different blanks correction methods; with and without the inclusion of the injection volume dependent blank as determined by the injection of Milli-Q water Injection volume

Measured IRMS area a

Measured δ13C a

Corrected δ13C using ‘0 μL’ blank b

Corrected δ13C using total blank c

(μL)

(mV)

‰ (VPDB)

‰ (VPDB)

‰ (VPDB)

50 100 150 200

6.9 (0.9) 10.6 (0.3) 16.2 (0.5) 21.4 (1.1)

− 21.8 (0.1) − 22.0 (0.3) − 21.9 (0.4) − 20.8 (0.2) Average

− 23.0 − 22.7 − 22.3 − 21.0 − 22.2 ± 0.9

− 23.5 − 21.8 − 21.9 − 20.3 − 21.9 ± 1.3

a

Standard deviations of three injections reported in parenthesis. Measured values corrected for only the “0 μL” blank. Assumes that the volume dependent blank is due to organic matter in the Milli-Q water and should not be subtracted. c Measured values corrected for the total blank measured at each injection volume using Milli-Q water (Figs. 2, 4). Assumes the volume dependent blank is due to an instrument blank.

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While the Sargasso Sea injection volume experiment was not able to distinguish between the two blank sources, it did demonstrate that the total blank corrections are relatively small, ranging between 0‰ to 0.7‰ for injection volumes greater than 50 μL. Additionally, the results suggest that for typical seawater DO13C concentrations the volume dependent blank makes up a small enough portion of the total blank, and is similar enough to seawater isotopic compositions, that it will not greatly change the corrected values. We will take this issue up again when discussing the isotopic composition of other seawater samples. 4.2. Testing the accuracy of the measurements

b

composition due to their different packing material. Measuring this would be an interesting test for the ultimate source of the HTC blank, which seems to be of similar magnitude in a wide variety of instruments (Sharp et al., 2002). A major question is whether the volume dependent blank was the result of organic matter in the Milli-Q water or intrinsic to the instrument. To address this problem, the above experiment was repeated by injecting varying amounts (50, 100, 150, and 200 μL) of Sargasso Sea reference water (Hansell, 2001). The seawater area response was 2.5 to 5.4 times larger than that of the blank and the δ13C values ranged from − 20.8‰ to − 22.0‰ (Fig. 5). If the volume dependent blanks are intrinsic to the instrument, and not the result of carbon in the Milli-Q water, then the entire blank measured at each injection volume should be used to correct the measured Sargasso Sea isotope measurements. For instance, the 100 μL injection of Sargasso Seawater (10.6 mV min, − 22‰) should be corrected assuming a blank of area 3.1 mV min, and δ13C = − 22.4‰. Alternatively, the volume dependent blanks could be the result of organic matter in the Milli-Q water. In that case, every injection volume should be corrected only for the CO2 present in ‘0 μL’ post-column blank (2.7 mV, − 19.9‰). Table 1 shows a comparison of correcting the measured Sargasso Sea values each way. The average corrected isotopic values for the four seawater injection volumes (− 21.9‰ and − 22.2‰) do not differ significantly from each other. Both are similar to measurements made for Sargasso Sea deep water (N 800 m) by UV oxidation methods (− 20.0 to − 21.3‰; Druffel et al., 1992).

The oxidation efficiency of the MQ-1001 was tested by analyzing a series of organic standards with a range of isotopic compositions (− 38.6‰ to − 9.1‰) and DOC concentrations (18 to 125 μM) representing possible seawater values. Solid standards were ground with a mortar and pestle to homogenize the sample. The isotope ratios of the solids were determined by direct combustion using an Elemental Analyzer/Finnegan Delta-Plus IRMS (Table 2). These same solids were then added to Milli-Q water to make standard solutions in the desired concentrations. The solutions were analyzed in the same fashion as seawater samples, as described in the Methods section. One simplification when analyzing standards was in the treatment of the blank. The solid standards were dissolved in the same Milli-Q water used for blank analysis and any organic material present in the water should be the same for all samples. For that reason, we used the value of the Milli-Q injection water blank. The magnitude of the blank could be determined using either the LICOR peak areas or the IRMS peak areas. Approximately 30 DOC injections were made for every three CO2 peaks trapped and run on the IRMS, and therefore the calculated reproducibility was more robust when using the LICOR peak areas. This option was not available when discussing the blank's source since the ‘0 μL’ peak area could only be determined on the IRMS. The standard materials were run on a different MQ-1001 column than the previous experiments, and the magnitude and isotopic composition of the blank were determined to be 285 mV min (LICOR peak area) and −18.1‰. An area of 285 mV min as measured on the LICOR represents 20 ng of carbon. Since the majority of this blank does not result from water injection, it would not be appropriate to convert this value into a concentration. The blank corrected δ13C values were systematically ∼ 1‰ heavier than the values as determined on the

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Table 2 Comparison of oxidation of standards by direct and high temperature combustion MQ-1001 high temperature combustion Compound

Direct combustion (‰ VPDB)

Chloroacetic Acid −38.6 Thiourea Ascorbic Acid Pthalic Acid Glucose

−22.2 −18.6 −26.6 − 9.1

a

DOC concentration b

Measured δ13C c

Blank corrected δ13C d

Fractionation corrected δ13C e

(μM)

(‰ VPDB)

(‰ VPDB)

(‰ VPDB)

18.4 44.9 81.3 124 119.8 100.4 73.1 113.2

−26.2 (0.1) −30.9 (0.4) −33.5 (0.5) −34.4 (0.2) −20.7 (0.1) −17.7 (0.2) −23.9 (0.5) −10.9 (0.5)

−37.6 −37.7 −37.8 −37.3 −21.2 −17.6 −25.7 −9.4

− 39.0 − 39.0 − 39.2 − 38.7 − 21.9 − 18.3 −26.6 − 9.8

a Isotopic values of standards were determined by direct combustion using an Elemental Analyzer/Finnegan Delta-Plus IRMS. Analysis error is ±0.4‰. b Reproducibility of multiple DOC injections is 2–5% depending on concentration. c Standard deviations of three injections reported in parenthesis. d Measured δ13C corrected using the total blank. e Blank corrected values differed systematically with direct combustion values (y = 0.9678 × −0.056; r2 = 0.998), indicating the carbon fractionates at some point. Numbers here are corrected for this fractionation.

elemental analyzer (Table 2, ‘blank corrected δ13C’). Plotting measured against known values gave a line with slope of 0.9678 and intercept of − 0.056 (r2 = 0.998), indicating that the carbon was fractionated at some point, possibly during DOC oxidation or CO2 trapping. When the blank corrected δ13C values were corrected for this fractionation, the final values compared closely to known values (Table 2, fractionation corrected δ13C). 4.3. Seawater measurements As a final test of the HTC method the δ13C of DOC in marine samples was measured. Deep Sargasso Sea refer-

ence water was run every other day to test the stability of the two instruments (Table 3). DOC concentrations were similar to what has been previously measured by the MQ-1001 HTC instrument (48.9 ± 2.2 μM vs. 46.1 ± 1.3 μM (Peterson et al., 2003), though slightly higher and more variable. The measured isotopic composition of the reference water (− 21.7‰) did not significantly change over this period of time (Table 3). The new method was also used to measure δ13C in seawater samples from a time series station in the eastern North Pacific (Station M) (Table 3; Bauer et al., 1998). With the current method, stable carbon isotopes varied between −20.9‰ and −22.2‰ for depths of 25, 250, 450,

Table 3 Seawater analysis DOC concentration a

Measured δ13C b

Corrected δ13C c

Reported DOC concentration d

Reported δ13C d

(μM)

‰ (VPDB)

‰ (VPDB)

(μM)

‰ (VPDB)

Sargasso Sea reference water

46.1 51.0 48.1 50.6

Station M 25 m Station M 250 m Station M 450 m Station M 700 m

67.7 46.7 39.9 38.2

−20.1 (0.2) −20.2 (0.1) −19.8 (0.7) −20.0 (0.1) Average −20.3 (0.4) −20.3 (0.3) −19.4 (0.6) −19.5 (0.4)

−21.9 −21.9 −21.3 −21.6 −21.7 −21.7 −22.2 −20.9 −20.9

69.5 ± 4.3 (n = 6)

−21.5 ± 0.9 (n = 5)

43 ± 1.5 (n = 6) 39.2 ± 1.2 (n = 6)

−21.1 ± 0.4 (n = 6) −20.8 ± 0.4 (n = 4)

Sample

a

Reproducibility of multiple DOC injections is 2–5% depending on concentration. Standard deviations of three injections reported in parenthesis. c Measured δ13C corrected using the total blank and calculated fractionation factor (Table 2). Based on a Monte-Carlo type analysis, the error for these corrected values is ±0.8‰. d As reported in Bauer et al. (1998). Presented here are averages of samples taken between 1991–1993 and analyzed using a UVoxidation method. Each ‘n’ consists of 4–6 individual samples taken from the most similar depth during that time period. b

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and 700 m. Over the same depths, reported isotopic values determined using the UV oxidation method (Bauer et al., 1998) are almost identical, ranging between −20.0‰ and −22.3‰. The results of the seawater analysis demonstrate both the reproducibility and the accuracy of this new analytical technique. Measured isotopic values were first corrected using the total blank and then for isotope fractionation. We used a Monte-Carlo type analysis to determine that error of in the δ13C measurement is approximately ±0.8‰. This brute force error analysis works by repeatedly picking random values with in a set error range for the blank correction, the fractionation correction, and the correction of the raw IRMS isotopic ratios to a standard gas. The final isotopic composition is calculated from these randomly generated numbers. This output is recorded and the process is repeated until the output converges on a single mean and standard deviation. Using the ‘0 μL’ injection gas blank instead of total blank changed the calculated values by approximately 0.5‰ which is within the precision of the δ13C values. An error of ±0.8‰ is larger than that of the UV oxidation method (±0.2‰). Since most open ocean DOC falls into a tight δ13C range (−18 to −23‰; Bauer, 2002), the coupled HTC-IRMS method may be best suited for systems in which the isotopic ratios of DOC are adequately distinct, such as in coastal, groundwater, or hydrothermal environments. 5. Conclusions The effectiveness of linking an HTC instrument to an IRMS for stable carbon isotopic analysis of DOC, even under challenging situations such as saline solutions and low concentrations, was demonstrated. The results of seawater analysis by this method are comparable to those obtained using the UV oxidation method. However, for the system to be accurate, a careful accounting of the blank must be performed, as we demonstrate here. The HTC blank is stable in both concentration and isotopic composition. It is composed of an ever-present instrument blank and an injection volume dependent blank. At injection volumes of 100 μL, the volume dependent blank is 17% of the total bank. Conveniently, the δ13C value of the blank is similar to that of deep seawater DOC which minimizes isotopic corrections. The δ13C of seawater DOC can be accurately measured with an error of ± 0.8‰ using the new HTC method. Acknowledgements John was the primary force behind this work and the loss of his guidance, insights and encouragement are

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deeply felt. We thank E. Druffel for providing Station M samples. P. Quay and D. Wilbur provided invaluable advice and assistance with IRMS operations. This work would not have been possible without the expertise and skills of M. Peterson. Careful comments by two anonymous reviewers greatly improved this manuscript. This work was supported by NSF/LExEn grant OCE0085615 to J. I. H. and a NDSEG fellowship to S. Q. L. References Bauer, J.E., 2002. Carbon isotopic composition of DOM. In: Hansell, D.A., Carlson, C.A. (Eds.), Biogeochemistry of Marine Dissolved Organic Matter. Academic Press, New York. Bauer, J.E., Druffel, E.R.M, Williams, P.M., 1992. Recovery of submilligram quantities of carbon dioxide from gas streams by molecular sieve for subsequent determination of isotopic (13C and 14 C) natural abundances. Am. Chem. Soc. 64, 824–827. Bauer, J.E., Occelli, M.L., Williams, P.M., McCaslin, P.C., 1993. Heterogeneous catalyst structure and function: review and implications for the analysis of dissolved organic carbon and nitrogen in natural waters. Mar. Chem. 41, 75–89. Bauer, J.E., Druffel, E.R.M., Wolgast, D.M., Griffin, S., Masiello, C.A., 1998. Distributions of dissolved organic and inorganic carbon and radiocarbon in the eastern North Pacific continental margin. DeepSea Res. Part II 45 (4–5), 689–713. Benner, R., Strom, M., 1993. A critical evaluation of the analytical blank associated with DOC measurements by high-temperature catalytic oxidation. Mar. Chem. 41, 153–160. Cauwet, G., 1994. HTCO method for dissolved organic carbon analysis in seawater: influence of catalyst on blank estimation. Mar. Chem. 47, 55–64. Chen, W., Zhao, Z., Koprivnjak, E., Perdue, M., 2002. A mechanistic study of the high temperature oxidation of organic matter in a carbon analyzer. Mar. Chem. 78 (4), 185–196. Druffel, E.R.M., Williams, P.M., Robertson, K., Griffin, S., Jull, A.J.T., Donahue, D., Toolin, L., Linick, T.W., 1989. Radiocarbon in dissolved organic an inorganic carbon from the central North Pacific. Radiocarbon 31 (3), 523–532. Druffel, E.R.M., Williams, P.M., Bauer, J.E., Ertel, J.R., 1992. Cycling of dissolved and particulate organic-matter in the open ocean. J. Geophys. Res. Oceans 97, 15639–15659. Eadie, B.J., Jeffrey, L.M., Sackett, W.M., 1978. Some observations on stable carbon isotope composition of dissolved and particulate organic-carbon in marine environment. Geochim. Cosmochim. Acta 42 (8), 1265–1269. Hedges, J.I., Bergamaschi, B.A., Benner, R., 1993. Comparative analyses of DOC and DON in natural waters. Mar. Chem. 41 (1–3), 121–134. Hansell, D.A., 2001. The good, the bad and the ugly: how good are my DOC results? U.S. JGOFS Newslett. 11 (2), 14–15. le Clercq, M., van der Plicht, J., Meijer, H.A.J., 1998. A supercritical oxidation system for the determination of carbon isotope ratios in marine dissolved organic carbon. Anal. Chim. Acta 370, 19–27. Onstad, G.D., Canfield, D.E., Quay, P.D., Hedges, J.I., 2000. Sources of particulate organic matter in rivers from the continental USA: lignin phenol and stable carbon isotope compositions. Geochim. Cosmochim. Acta 64 (20), 3539–3546. Peterson, M.L., Lang, S.Q., Aufdenkampe, A.K., Hedges, J.I., 2003. Dissolved organic carbon measurement using a modified hightemperature combustion analyzer. Mar. Chem. 81, 89–104.

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