ANALYTICAL SCIENCES JANUARY 2013, VOL. 29
9
2013 © The Japan Society for Analytical Chemistry
Fast Measurement of Dissolved Inorganic Carbon Concentration
for Small-Volume Interstitial Water by Acid Extraction and
Nondispersive Infrared Gas Analysis
Takuroh NOGUCHI,*† Mayumi HATTA,* Toshiro YAMANAKA,** and Kei OKAMURA*
*Center for Advanced Marine Core Research, Kochi University, B200 Monobe, Nankoku, Kochi 783–8502,
Japan
**Faculty of Science, Okayama University, 3-1-1 Tsushimanaka, Okayama 700–8530, Japan
We developed a system for measuring the total dissolved inorganic carbon (DIC) concentrations in interstitial water and
hydrothermal fluid, which are hard to obtain in large volumes. The system requires a sample volume of only 500 μL, and
it takes only 150 s per one sample. The detection limit of this system was estimated to be 66.6 μmol/kg with repeated
analysis of CO2-free ultrapure water (n = 9). The precision of this nondispersive infrared (NDIR) system was ±3.1% of
the relative standard deviations (2σ) by repeated CRM batch 104 (n = 10). This result is much larger than the required
precision for oceanographic studies, but is comparable to a previous result of interstitial water analysis. An on-site trial
showed a significant DIC enrichment in interstitial water of hydrothermally altered sediment, and is considered to occur
by the mixing of hydrothermal fluid. This procedure will achieve carbon dioxide flux calculations from hydrothermal
activities, and will bring a more accurate feature on the global carbon cycle.
(Received August 10, 2012; Accepted October 12, 2012; Published January 10, 2013)
Introduction
The ocean is the largest reservoir of carbon on Earth, storing
about 50-times as much carbon as the atmosphere.1 Because
accurate measurements of the CO2 content of the ocean is
important for studying biogeochemical processes as well as for
evaluating the effects of increases in anthropogenic CO2
emissions on the global carbon cycle and climate change,
methods for accurate analysis of the carbonate system are
required.
The carbonate system can be described by four parameters:
pH, total alkalinity (AT), total dissolved inorganic carbon (DIC)
concentration, and CO2 fugacity (fCO2). Given any two of the
four parameters, the remaining two can be calculated from
thermodynamic constants.2 The analysis of fCO2 presents some
difficulties, particularly for deep seawater samples, in terms of
sampling and determination because it is hard to maintain the
in-situ water pressure during sample recovery. However, fCO2
has been accurately and continuously measured in surface
seawater in various environments (e.g., the open ocean and coral
reefs) for the purpose of estimating the air–sea CO2 flux.3–5
On-site pH is usually measured with pH electrodes, but the
values obtained by this method are affected by the variable ionic
strength of seawater. Therefore, colorimetric methods for
To whom correspondence should be addressed.
E-mail: [email protected]
T. N. present address: Marine Technology and Engineering
Center, Japan Agency for Marine-Earth Science and Technology
(JAMSTEC), 2-15 Natsushima-cho, Yokosuka, Kanagawa
237–0061, Japan.
†
measuring the pH, as well as AT, have been developed to improve
both the precision and accuracy.6–8 Coulometric methods for the
determination of DIC concentration in seawater have been
available since the mid-1980s, and among the four carbonate
system parameters, the DIC concentration has been measured
with the greatest accuracy and precision.9–11 Nondispersive
infrared (NDIR) gas analysis has also been used for the rapid
measurement of DIC concentration.12,13 The development of
accurate and precise measurement techniques has allowed the
distribution and behavior of anthropogenic CO2 in the ocean to
be clearly characterized.14
In contrast, the influence of hydrothermal activity and gas
hydrates on the seafloor (discovered in the 1970s),15 as well as
associated chemosynthetic organisms, on the supply of CO2 in
the ocean and on the global carbon cycle has not been fully
explored. Hydrothermal fluid and gas hydrates generally
contain large amounts of CO2 produced by volcanic activity and
underground diagenesis.
In addition, recent molecular
phylogenetic studies have indicated that a large amount of
biomass exists a few cm to a few km below the ground widely
(sediment to upper oceanic crust), and that hydrothermal and
gas hydrate fields are concentrated areas of microbial activity.16,17
Therefore, analysis of the carbonate system in hydrothermal
fluids and interstitial water in hydrothermal and gas hydrate
fields is important for estimating its influence on the global
carbon cycle. However, collecting samples of these fluids in
sufficient volume is difficult, which limits our ability to analyze
the carbonate system in such fluids. Even in porous surface
sediments, only 10 – 20 mL of interstitial water can generally be
Adapting the
collected from 30 – 40 mL of sediment.
coulometric analysis procedure for measuring the DIC
concentration described in the standard operation procedure of
10
Fig. 1 Schematic diagram of the system used for measuring the DIC
concentration.
the United States Department of Energy is difficult for smallvolume samples.11 NDIR detection has been reported to afford
the same level of accuracy and precision as coulometric
detection, which suggested the possibility for reducing the
sample volume.18 Our objective in this study was to modify a
reported rapid DIC analysis procedure, which involves CO2
extraction with phosphoric acid and NDIR detection, for use
with hydrothermal fluid and interstitial water samples. Here,
we report on the development and evaluation of a method for a
rapid analysis of DIC concentrations in small samples. We also
describe the use of the modified method for onboard DIC
analysis at Wakamiko submarine crater in Kagoshima bay.
Experimental
Reagents
We prepared sodium carbonate standard solutions by diluting
a 0.05 mol/L sodium carbonate solution for quantitative analysis
(f = 1.003; Wako Pure Chemicals, Japan) with CO2-free
ultrapure water. The density of the commercial 0.05 mol/L
sodium carbonate solution was 1.00254 g/mL at 25°
C, as
measured with a density/specific gravity meter (DA-650, Kyoto
Electronics Manufacturing Co., Japan). Prior to use, the
ultrapure water was stripped of CO2 with a stream of G1-grade
N2 gas (<0.1 ppm CO2) for 10 min. Phosphoric acid (8.5%) for
acidification of the samples was prepared by 10-fold dilution of
analytical-reagent-grade 85% phosphoric acid (Wako Pure
Chemicals, Japan) and stored in glass bottle. The 8.5%
phosphoric acid solution was also purged with a stream of
G1-grade N2 gas for 10 min to strip the CO2. Water vapor in the
gas stream exiting from the sample container was trapped with
a column of magnesium perchlorate.
Instruments
A schematic diagram of the analytical system (including the
CO2 gas extractor and detector) is shown in Fig. 1. Teflon
tubing was used to carry gases and reagents; 500 μL of a sample
solution was pipetted to a 2.5-mL polycarbonate vial with a
1000-μL micropipette (Finnpipette, Thermo, Finland), and was
then attached onto a polytetrafluoroethylene pedestal (Fig. 1).
After attachment of the polycarbonate vial, the residual CO2 gas
in the headspace of the vial was purged with N2. Once N2 gas
flow was stopped, 0.5 mL of 8.5% phosphoric acid was added
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JANUARY 2013, VOL. 29
Fig. 2 (a) NDIR chromatogram in three different N2 carrier gas flow
rates (140 mL/min (solid circle), 160 mL/min (solid square), and
180 mL/min (solid triangle)). (b) Calibration curves for three different
sample volumes (200 μL (solid circle), 500 μL (solid square), and
1000 μL (solid triangle)). The dash line shows 95% confidence lines
of each calibration carve for 3 times of repetition.
by means of a 1.0-mL plastic syringe, and then N2 bubbling was
started (the flow rate was fixed at 160 mL/min). The extracted
CO2 was detected using a NDIR gas analyzer (LI-820, Li-COR)
over the course of 150 s. Validations on the sampling volume
and the flow rate of N2 gas are discussed in following section,
“Measurement conditions”. To evaluate the accuracy of the
NDIR detector, we connected a coulometer (CM5012, UIC Inc.)
to the gas-out line from the NDIR to measure the CO2
concentration in the gas stream.
Results and Discussion
Measurement conditions
First, we evaluated the influence of the N2 flow rate (140, 160,
or 180 mL/min) on the NDIR chromatogram in the case of
500 μL of a 2000 μmol/kg Na2CO3 standard solution (Fig. 2a).
At all three flow rates, the peak area for the DIC fell within the
150 s detection interval for 500 μL of a solution with a DIC
concentration of 2000 μmol/kg. However, when we analyzed
more than the DIC concentration of a 3000 μmol/kg solution at
a flow rate of 140 mL/min, the peak tailing extended beyond the
detection interval (data not shown). In addition, the peak area
decreased with increasing the N2 flow rate (Fig. 2a). Increasing
the flow rate above 160 mL/min permitted more-rapid analysis,
but the higher flow rate reduced the peak area, and thus increased
the relative measurement error. Therefore, we fixed a flow rate
of 160 mL/min for the remainder of the experiments in this
study. Slight fluctuations in the N2 gas flow rate influenced the
counting efficiency of NDIR detection. Therefore, it is important
for precise analysis to control the flow rate carefully, for
example, by introducing a mass flow controller to the system.
We next evaluated the effect of the sample volume by
preparing calibration curves for sample volumes of 200, 500,
and 1000 μL with 830, 1910, 2880, and 3750 μmol/kg Na2CO3
standard solutions at 160 mL/min of N2 flow (Fig. 2b). The
dash lines indicates the 95% confidence intervals of each
calibration curve, and the error bars show the standard deviation
(2σ) of each standard solution. The fact that the calibration
curve for each volume shows a good correlation with the
standard concentration at the constant flow rate indicates that
the detector was not saturated. The calibration curve of 1000 μL
had a larger variation than that of 200 and 500 μL. There is no
significant difference between the calibration curves of 200 μL
and 500 μL, but 200 μL of a sample might be more effective
ANALYTICAL SCIENCES JANUARY 2013, VOL. 29
Fig. 3 Comparison of the NDIR and coulometric results for sodium
carbonate standard solutions at four DIC concentrations. The black
solid circle shows the average values of CRM batch 104 measured by
the NDIR and coulonmeter for 10 repetitions. The error bar shows
2 times of the standard deviation (2σ) of each detector. The grayish
solid square and error bar show the certificated value and error.
during sample handling (e.g. evaporation and/or pipetting) than
500 μL. Therefore, we fixed that 500 μL of a sample would be
used for the remainder of the experiments in this study.
Precision and accuracy
The limit of quantitation for the procedure, which we
calculated by multiplying the standard deviation of the results of
repeated analyses of blanks (n = 9) by 10, was 66.6 μmol/kg.
To estimate the precision and accuracy of our method, we
conducted repeated measurements (n = 10) with a certified
reference material (CRM batch 104; DIC concentration,
2020.10 ± 0.38 μmol/kg; supplied by A. Dickson, Scripps
Institute of Oceanography, USA). Figure 3 shows a comparison
of NDIR with the coulonmetric detection of CRM batch 104.
The DIC concentrations of the CRM determined with the NDIR
and the coulometer were 2033 ± 63 μmol/kg and
2018 ± 99 μmol/kg, respectively.
The relative standard
deviations (2σ) of the NDIR and coulometric values were
±3.1% and ±4.9%, respectively. The results obtained with both
detectors were within the range of the certificated concentration.
The relative standard deviation of the NDIR results was slightly
lower than that of the coulometric results. The precision and
accuracy obtained with this method did not satisfy the
requirements for global carbonate cycle estimation, because the
precision and accuracy on DIC analysis in open ocean seawater
require ±1 and ±2 μmol/kg, respectively.2 However, the DIC
concentration in interstitial water is generally up to an order of
magnitude higher than that in seawater. For example, one
reference shows the isotopic composition and concentration of
total inorganic carbon on interstitial water.19 They showed that
the analytical precision ranged approximately a few %, but it is
sufficient precision to discuss the geochemical feature within
the sediment (e.g. microbial activity and decomposition of
organic materials). We suspected that the major source of
measurement error to be caused by sample quantification with a
1000 μL micropipette, which had a reproducibility of 1 – 1.5%;
11
Fig. 4 Location of Wakamiko crater in Kagoshima bay.
however, we chose the micropipette for sample injection for
reducing the measurement time because of the simple and easy
handling. In the case of more precise and accurate analysis, we
have to choose some other alternative sample injection system,
which should have better reproducibility. An alternative
candidate of sample injection with good reproducibility is
sample loop injection (500 μL), which achieves less than a
0.1% relative standard deviation. The use of sample loop
injection can be expected to reduce the measurement error to a
few μmol/kg, which should result in sufficient accuracy and
precision for not only interstitial water, but also oceanographic
and limnologic samples. In future work, we hope to improve
the precision by using a sample loop system for sample injection
instead of a micropipette.
On-site analysis at Wakamiko submarine crater
We conducted the DIC analysis on interstitial water and
hydrothermal fluid from Wakamiko submarine crater in
Kagoshima bay during the NT12-08 cruise in 2012 (Fig. 4).
Hydrothermal fluid and associated gas venting at Wakamiko
crater was discovered using a ROV Hakuyo in 1977.20 Venting
gas was mainly composed of carbon dioxide (77 – 92 vol.%),
and also contained methane (5 – 20 vol.%), nitrogen (2 – 7 vol.%),
and hydrogen sulfide (0.1 – 1.3 vol.%). The observed maximum
temperature of hydrothermal fluid was 215°
C, and hydrothermal
petroleum and a high concentration of poly-aromatic
hydrocarbon (PAHs) were discovered in a sediment sample,
which originated by the interaction between the high-temperature
hydrothermal fluid and the sediment.21,22 We collected one
hydrothermally altered sediment core (1366-MB) and one
less-altered sediment core (1363-MB) by a push corer using a
ROV Hyper Dolphine of JAMSTEC (Japan Agency for
Marine-Earth Science and Technology). Immediately after
recovery of the sediment cores, approximately 30 – 40 cm3 of
sediment blocks were sub-sampled at 5 cm intervals. Interstitial
water was squeezed from each sediment block through a
titanium mesh, paper filter, and a 0.45-μm membrane disc filter
and collected in a 3-mL glass vial. About 300 mL of
hydrothermal fluid was collected by a ROCS (rotary clean
12
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JANUARY 2013, VOL. 29
Fig. 5 Vertical profiles of the DIC, total alkalinity, ammonium, and silica of interstitial water
squeezed from 1366-MB (well-altered) and 1363-MB (less-altered) sediment cores.
seawater sampler, Nichiyu Giken Kogyo Co. Ltd., Japan) from a
hydrothermal vent at Wakamiko crater.23 Hydrothermal fluid
was also filtered through a 0.45-μm membrane disc filter, and
collected in a 3-mL glass vial. The total alkalinity was measured
by Gran plot titration with a pH electrode (PHC2401, Radiometer
Inc., France). Ammonium and silica analysis of interstitial
water samples were conducted using traditional colorimetric
procedures (indophenol method for ammonium and molybdenum
blue method for silica).24 Figure 5 shows vertical profiles of the
DIC, total alkalinity, ammonium, and silica in interstitial water
samples collected from Wakamiko crater. The 1366-MB core
(well-altered) contained higher DIC, total alkalinity, ammonium,
and silica concentration than the 1363-MB core (less-altered),
and each concentration was increased with the sediment depth
increasing. The results for the total alkalinity, ammonium,
and silica agree with a previous study.25 The DIC concentration
in interstitial water fluctuated from 2000 to 12000 μmol/kg, and
the venting hydrothermal fluid reached to 20000 μmol/kg (this
DIC value of hydrothermal fluid is not described in Fig. 5). The
vertical profile of DIC was correlated with alkalinity, ammonium,
and silica; it is suggested that the mixing of ambient seawater
and high temperature hydrothermal fluid had occurred in the
sediment.
The significantly high DIC concentration in
hydrothermal fluid seems to be supplied from the volcanic gas
and organic material decomposition beneath the seafloor. Our
result concerning this on-site analysis shows the distribution and
behavior of the DIC in the interstitial water and hydrothermal
fluids. It is expected that we will be able to detect the DIC flux
from not only high-temperature hydrothermal fluid venting, but
also low-temperature diffusive flow, which it will allow the
more accurate estimations on global CO2 flux.
Conclusions
We developed a rapid procedure for DIC analysis in
small-volume (500 μL) fluid samples. One measurement took
only 150 s, and the fluctuation of the N2 gas flow rate caused a
variation of the NDIR detection efficiency. The detection limit
of this system was estimated to be 66.6 μmol/kg with repeated
analysis of CO2 free ultrapure water (n = 9). The average DIC
concentration and measurement error was 2033 ± 63 μmol/kg,
which was calculated with repeated CRM batch 104 (certificated
DIC value: 2020.10 ± 0.38 μmol/kg), and the relative standard
deviation (2σ) was ±3.1%. This result is much larger than the
required precision for accurate oceanographic studies, but it is
comparable to a previous report on interstitial water analysis.
The analytical precision will be improved by replacing the
sample-injection system from the micropipette to the sample
loop for future oceanographic studies.
Hydrothermally altered sediment core (1366-MB) has a
significant enrichment of DIC in interstitial water, influenced by
the hydrothermal fluid input. In the hydrothermal field at
Wakamiko crater, CO2 supply from volcanic gas and thermogenic
CO2 (decomposition of organic materials) may have caused DIC
enrichment in the hydrothermal fluid. This procedure will be
able to detect the DIC flux from not only high-temperature
hydrothermal fluid venting, but also the low-temperature
diffusive flow, which will achieve a more accurate feature on the
global carbon cycle.
Acknowledgements
This study was supported by a Grant from the Ministry of
Education, Culture, Sports, Science and Technology, Japan
(“Development of new tools for the exploration seafloor
resources”). We acknowledge H. Kimoto and T. Suzue of
Kimoto Electric Co. developing the DIC analyzer. We also
thank the officers and crew of the R/V Natsushima, the crew of
the ROV Hyper Dolphine, and the scientists who participated in
the NT12-08 cruise of the R/V Natsushima (Japan Agency for
Marine-Earth Science and Technology) for water sample
collection. We also thank for M. Utsumi in Univ. Tsukuba for
the ROCS sampling arrangement and preparation.
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