Precise Spectrophotometric Measurement of Seawater pHT with
an Automated Apparatus using a Flow Cell in a Closed Circuit
by
Shu SAITO, Masao ISHII, Takashi MIDORIKAWA and Hisayuki Y.
INOUE*
Geochemical Research Department*

Present affiliation: Graduate School of Environmental Earth Science,
Hokkaido University
閉流路中のフローセルを用いた分光光度法自動分析装置による海水の高精度 pHT 測定
斉藤 秀、石井雅男、緑川 貴、井上（吉川）久幸*（地球化学研究部）（* 現 北海道大学地球環境科学研究院）

Preface
Since the Industrial Revolution, huge amounts of carbon dioxide have been released
into the atmosphere as a result of human activities. The increase of anthropogenic CO2 in theatmosphere is thought to cause global warming that gives rise to climate change. About
one-third of the anthropogenic CO2 has been absorbed by the oceans. When dissolved inseawater, CO2 exists in the form of carbonic acid. Therefore, the uptake of anthropogenic
CO2 by the world’s oceans leads to acidification of the oceans. Ocean acidification is of great
concern because it can seriously affect marine ecosystems. Therefore, it is necessary tomonitor the pH of seawater, which is a measure of ocean acidification.
Measurements of pH have long been used in the field of chemistry. However, auniversally accepted method of measurement, which includes the standards for measurementsof seawater samples, has not yet been established. Consequently, comparison of pH dataobtained from different sources is problematic. Furthermore, pH measurements collectedfrom different cruises or other locations must be directly comparable so that ocean
acidification may be detected.
For these reasons, the present study was initiated with financial support from theresearch programme “Global Carbon Cycle and Related Mapping based on Satellite
Imaginary Programme” of the Research Promotion Fund from the Ministry of Education,
Culture, Sports, Science and Technology, Japan, as well as the research programmes“Observational Study on the Seasonal and Interannual Variability and its Mechanisms of the
Carbon Dioxide System at the Air-Sea Interface and in the Upper Layer of the Ocean” and“Observational Study on the Variability of Carbon Cycle in the Ocean, I and II” of the
Meteorological Research Institute, Japan Meteorological Agency’s research fund.
In this technical report, a new spectrophotometric technique for the measurement of
pHT, pH as measured on the total hydrogen ion concentration scale, is described. This
technique provides pHT values for seawater with repeatability better than 0.002 and withcomparability of 0.003. By applying the technique to oceanic waters, we will be able todetect seawater acidification as well as obtain new information on ocean carbon cycles.

December 2008

Katsumi Hirose
Director of Geochemical Research Department
Meteorological Research Institute序文

産業革命以来の人類活動の結果、膨大な量の二酸化炭素が大気中に放出されてきた。
大気中における人類起源の二酸化炭素の増加は気候変動を引き起こす地球温暖化の原因と考えられている。大気中に放出された人類起源二酸化炭素のかなりの量が海洋に吸収される。即ち、人類起源の二酸化炭素のおよそ三分の一は海洋に吸収されている。一方、一旦溶液（海水）に溶解した二酸化炭素は酸（炭酸）として挙動する。海洋へ多量の人類起源の二酸化炭素の溶入は、海洋の酸性化もたらす。さらに、海洋の酸性化は海洋生態系に重大な影響が及ぶのではないかと懸念されている。従って、酸性化の尺度である海水の pH を監視することは緊急の課題である。
化学の分野では古くから pH の測定が実施されてきた。しかし、pH 測定の分野で高精度のトレーサビリティの確保された pH 測定システムはまだ確立されていない。海水の酸性化を検出するためには、高精度であるばかりでなく異なった航海や異なった観測機関で pH の測定値のコンパラビリティ（比較可能性）が確保されることが重要である。その理由の一つは、海洋の二酸化炭素吸収により引き起こされる pH 変化が小さく長期に及ぶ観測が必要なためである。
以上のことを背景に、本研究は「炭素循環に関するグローバルマッピングとその高度化に関する国際共同研究（科学技術庁振興調整費）」のサブテーマである「海洋表層における炭素フラックスと一次生産に関する研究」の一環として始まり、続いて気象研究所の経常研究「大気・海洋間及び海洋表層における炭酸系の季節・経年変動とそのメカニズムの解明に関する観測的研究」、「海洋における炭素循環の変動に関する観測的研究 I、II」の一環として行われた。
本報告では、pHT（全海水水素イオン濃度）を定義すると共に、新しい分光学的方法による pHT の測定が紹介される。この方法では、pH 単位で 0.002 以下の繰り返し精度で、また 0.003 のコンパラビリティで高精度の pHT の測定を実施することができる。
海水の pH に対してこの分析法を適用すれば、海洋の炭素循環に新しい知見を加える
ばかりでなく海洋の酸性化に対する直接の証拠を提供することができるであろう。

2008 年 12 月

廣瀬勝己
地球化学研究部長

Abstract
We constructed a new automated apparatus for the precise spectrophotometricmeasurement of the pH of seawater samples. This method uses m-cresol purple as an indicatordye. The new apparatus was capable of continual onboard measurement at intervals of 10
minutes as well as bottle sample measurement. The measured pH values (on a total hydrogenion concentration scale, pHT) for certified reference materials (CRMs) used as standards for
total dissolved inorganic carbon (TCO2) and total alkalinity (TA) analyses agreed with thepHT values calculated from the certified TCO2 and TA values within ±0.003 when the
carbonic acid dissociation constants proposed by Lueker et al. (2000) were used in thecalculations. The pHT values of surface seawater measured at sea during various cruises
coincided with the values calculated from the partial pressure of carbon dioxide (pCO2) andTCO2 within ±0.020. The repeatability of pHT measurements for a single cruise was ±0.0002(n = 32) for onboard measurements and ±0.002 for bottle sample measurements. The larger
value for bottle sample measurements resulted from an uncertainty of ±0.0011 (n = 32) causedby bottling of seawater samples and from a perturbation of 0.0012 caused by the addition of asaturated HgCl2 solution as a bactericide. Data for onboard measurements and for bottle
sample measurements agreed within the error limits. The reproducibility of several sets ofmeasurements taken during different cruises over several years as examined by themeasurement of CRMs using different batches of dye solutions was ±0.003 (1ı, n = 34). We
obtained stable pHT values (±0.002, n = 24) for samples of surface seawater that had beensterilized with HgCl2, sealed in borosilicate glass bottles, and stored. The stability of thevalues enabled us to analyse the pHT of seawater samples at a laboratory on land aftercollecting the samples during cruises.

概要指示薬色素として m-クレゾールパープルを使用する分光光度法により海水の pH を
高精度に自動分析する装置を我々は新たに開発した。新装置は洋上での連続測定と共に容器試料の測定を 10 分間隔で行うことが可能である。全炭酸濃度（TCO2）と全ア
ルカリ度（TA）分析用の認証参照物質（CRM）の pH（全海水水素イオン濃度、pHT）
分析結果は、Lueker ら（2000）によって提唱された炭酸の酸解離定数を用いて TCO2と TA の認証値から計算した pHT 値と±0.003 の範囲で一致した。洋上観測においては、
表面海水の pHT 分析値は海水中の二酸化炭素分圧（pCO2）と TCO2 から計算した pHT
値と±0.020 の範囲で一致した。単一航海における pHT 測定の繰り返し性（repeatability）
は、連続測定については±0.0002（n = 32）、容器試料測定については± 0.002 だった。
容器試料測定のばらつきがより大きいのは、海水の瓶詰めで生じる不確かさ（±0.0011、
n = 32）と殺菌のために添加された飽和塩化水銀（II）溶液による摂動（0.0012）の結果である。連続測定と容器試料測定の結果は測定誤差範囲内で一致した。異なるバッ
チの色素溶液を使用して CRM 測定によって見積もった数年間の異なる航海間の測定の再現性（reproducibility）は、標準偏差 0.003（n = 34）だった。海水試料を塩化水銀（II）で殺菌してホウケイ酸ガラスびんに密封した場合、表面海水試料を保存してもその pHT は安定していた。これによって、航海後に陸上実験室で海水の pHT を分析することが可能になった。

Contents
1 Introduction ······································································································································· 1-2
2 Methods and materials ······················································································································ 2-9
2.1 Principles ····································································································································· 2-4
2.2 Apparatus ····································································································································· 4-5
2.3 Preparation of dye solution ·········································································································· 6
2.4 Perturbation caused by addition of dye solution ·········································································· 6-7
2.5 Correction for the temperature deviation ····················································································· 7-8
2.6 Seawater samples ························································································································· 8
2.7 Certified reference materials (CRMs) and working standards ···················································· 9
3 Onboard measurements ···················································································································· 9-10
4 Measurements of bottle samples taken at depth ··············································································· 10-15
4.1 Effects of bottling and headspace ································································································ 11-12
4.2 Perturbation of pHT caused by addition of saturated HgCl2 solution ·········································· 13
4.3 Storage of samples ······················································································································· 13-14
4.4 Uncertainty in pHT measurements of bottle samples ··································································· 15
5 Consistency of measured and calculated pHT values for CRMs andcomparability of pHT data among several cruises ······························································· 15-16
6 Procedure to measure pHT with high comparability and precision ··················································· 16-17
7 Discussion ········································································································································· 17-20
7.1 The standard deviation of pHT in reference materials ·································································· 17-18
7.2 The cause of the pHT perturbation by the addition of HgCl2 ······················································· 18-19
7.3 Internal consistency of CO2 system parameters ··········································································· 19-20
8 Summary ··········································································································································· 20
Acknowledgements ····························································································································· 21
Appendices:
Appendix A: Calculation of change in pHT through CO2 gas exchange
between headspace and seawater sample in a bottle ··························································· 22
Appendix B: Calculation of the pHT change derived from the addition ofsaturated HgCl2 solution ····································································································· 23-25
References ··········································································································································· 26-28

List of Tables and Figures
TablesTable 1 Repeatability of onboard pHT measurements of near-surface waters along the equator during cruiseMR02-K06.
Table 2 pHT of CRMs.
Table 3 pHT of working standards.
Table B.1 Stability constants for various Hg2+ complexes.

FiguresFig. 1

Schematic diagram of pHT measurement system.

Fig. 2

Example of pHT perturbation caused by the addition of dye solution.

Fig. 3

Effect of sample temperature on the measured pHT.

Fig. 4

Horizontal distributions of a) pHT, b) temperature and c) salinity in near-surface water along theequator observed in January 2003 during the R/V Mirai’s MR02-K06 cruise.

Fig. 5Differences in pHT between duplicate measurements, 'pHT, of bottle samples without HgCl2
addition.

Fig. 6Comparison of pHT between bottle and onboard measurements without HgCl2 addition.

Fig. 7

Effect on pHT of headspace of 2 cm3 in a 250 cm3 sample bottle.

Fig. 8Observed pHT change caused by the addition of 0.2 cm3 of saturated HgCl2 solution to 250 cm3
seawater.

Fig. 9Time course of pHT of surface water samples stored in glass bottles with HgCl2.

Fig. 10 Difference between the pHT values measured on board as soon as possible after water samples
arrived on deck, pHT(asap), and after 50-day storage, pHT(stored).
Fig. 11 Comparison of pCO2 values observed and calculated from measured pHT and TCO2 using
dissociation constants for carbonic acid given by Mehrbach et al. (1973; diamond), Hansson(1973; bar), Dickson and Millero (1987; plus), Goyet and Poisson (1989; circle), Roy et al. (1973;
triangle), Lueker et al. (2000; box), and Mojica Prieto and Millero (2002; cross)Technical Reports of the MRI, No. 57

1.

2008

Introduction
The concentration of carbon dioxide (CO2), a major greenhouse gas, has been increasing in theatmosphere because of anthropogenic activities such as fossil fuel burning (IPCC, 2007b; WDCGG, 2007).
The current concentration of CO2 in the atmosphere is approximately 100 ppm higher than that during thepreindustrial era, i.e. early 18th century (280 ppm; IPCC, 2007a). To predict future atmospheric CO2
concentrations on the basis of emission scenarios and the potential alteration of carbon cycles caused byclimate change, the processes that control the flux of CO2 and its temporal variability among global carbon
reservoirs, including the atmosphere, the terrestrial biosphere and the oceans, must be understood.
The oceans are the important sinks for anthropogenic CO2. The latest estimate of ocean CO2
uptake is 2.2 ± 0.5 GtC yr–1 (IPCC, 2007b). Recent model simulation revealed that uptake of anthropogenic
CO2 by the oceans will lower the average pH of surface seawater by approximately 0.1 compared with the
pH in 1750 (Caldeira and Wickett, 2003). The increase in CO2 concentration in the ocean’s surface layer
affects the chemical equilibrium in seawater. As CO2 concentrations increase, surface waters become more
acidic, and the concentration of carbonate ion consequently decreases. This change in chemical equilibrium
reduces the capacity of the ocean to take up additional CO2 (Sabine et al., 2004). Moreover, model
simulations have indicated that anthropogenic ocean acidification might impact calcifying organisms at
high latitudes, particularly in the Southern Ocean, within the next few decades, not centuries as suggested
previously (Orr et al., 2005). Therefore, monitoring ocean acidification and its influence on biogeochemical
processes by means of field observations is highly important.
At the hydrogen ion concentration range of seawater observed in the oceans, CO2 predominantly
exists as hydrogencarbonate (HCO3–) or carbonate (CO32–) ions. The concentrations of these ions are
determined by mass balance, charge balance and the acid–base equilibrium within the carbonate system inseawater. This carbonate system can be described by four measurable parameters: CO2 partial pressure(pCO2), total inorganic carbon (TCO2), hydrogen ion concentration ([H+]) and total alkalinity (TA). The
hydrogen ion concentration, usually reported as pH (= –log [H+]), determines the ratio of carbonate to TCO2
and the ratio of hydrogencarbonate to TCO2. Given two of these four parameters, one can calculate theremaining two from the thermodynamic relationships between the parameters (e.g., Lee and Millero, 1995).
We have been observing pCO2 in the atmosphere and in near-surface seawater (i.e., air in which
CO2 gas exchange between a great excess of near-surface water has attained the equilibrium) for more than
two decades and TCO2 in seawater for more than a decade, mainly in the western Pacific Ocean and theequatorial Pacific Ocean (Inoue, 1995; Inoue, 2000; Ishii et al., 2001; Midorikawa et al., 2005). We aimed

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to describe the temporal and spatial variations of these parameters and to understand the factors that control
them. In addition, we started to measure pH in seawater with high comparability and precision in the late
1990s to describe the variability in the carbonate system not only in surface waters but also at greater
depths. We continually improve our methods to measure these parameters precisely.
Midorikawa et al. (2005) reported that pCO2 values in the atmosphere and in surface waters in thewestern subtropical North Pacific Ocean increased at rates of 1.60 ± 0.03 ȝatm yr–1 and 1.6 ± 0.2 ȝatm yr–1,
respectively between 1984 and 2003. This rate of increase in oceanic pCO2 corresponds to an annualchange in pH of –0.002 yr–1 at a constant TA of 2300 ȝmol kg–1. Therefore, a pH measurement precision of
0.002 or better is required to monitor long-term oceanic acidification (DOE, 1994).
Seawater pH was formerly measured by a potentiometric method using a pair of glass and
Ag/AgCl reference electrodes (e.g., Grasshoff, 1976) based on the operational definition of pH in theabsence of seawater-based reference material. However, because of experimental problems such as thefluctuation in electrode sensitivity and residual liquid junction potential, a precision better than ±0.01 wasrarely achieved, even with very careful measurements (Dickson, 1993). For more precise measurements, aspectrophotometric method has been developed (Byrne et al., 1988; Dickson, 1993). In this study, weconstructed an automated apparatus with a flow-type spectrophotometric cell in a closed circuit. Using thisapparatus, we achieved the precision of ±0.0002 for on board measurements. The reproducibility ofmeasurements and the effects of sample storage were also examined.

2. Methods and materials
2.1 Principles
In the spectrophotometric method, pH is determined by measuring the visible absorption spectraof seawater samples containing indicator. Our method was based on that described by Clayton and Byrne(1993), who used m-cresol purple as an indicator dye.
At the pH range of seawater (7.4–8.2), the predominant pair of acid (HI–)1 and base (I2–)1 formsof m-cresol purple are in equilibrium with each other (Eq. (1)):

HI–

ĺ
ĸ

H+ + I2–.

1(1)

Though the net charges of the acid form and the base form of m-cresol purple are 0 and –1, respectively,
we express these ions as HI– and I2– for the sake of convenience, as such notation is conventional fordibasic acids.

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The dissociation constant of HI– (K2) is expressed as

K2[H  ] T [I 2  ][HI  ],

(2)

where [H+]T denotes the concentration of hydrogen ions (mol kg–1 seawater) expressed by the totalhydrogen ion concentration scale: [H+]T = [H+]F(1 + [SO4]T/KHSO4), where [H+]F is the concentration of freehydrogen ions, [SO4]T is the total concentration of sulphate ions and KHSO4 is the acid dissociation constantof hydrogensulphate ion (Dickson, 1990). Seawater pHT is then expressed as Eq. (3),pH T

 log[H  ] T

pK 2  log[ I 2- ][HI  ].

(3)

The dye absorption spectrum in the visible wavelength region is used to estimate [I2–]/[HI–]. The
maximum absorbance is observed at 578 nm for I2– and at 434 nm for HI–. The [I2–]/[HI–] ratio is calculated
from the ratio R = A578/A434 and the ratios of the molar extinction coefficients e1, e2 and e3, which are
expressed as İ578(HI–)/İ434(HI–), İ578(I2–)/İ434(HI–) and İ434(I2–)/İ434(HI–), respectively (İȜ(X) denotes themolar extinction coefficient of ion X at wavelength Ȝ). Then, pHT of seawater is calculated from Eq. (4):pH T

pK 2  log[ I 2- ][HI ]§ R  e1 ·¸.
pK 2  log¨¨¸© e 2  Re 3 ¹(4)

The ratios of extinction coefficients e1, e2 and e3 have been determined by Clayton and Byrne (1993). The
value of pK2 (= –log K2/k0, k0 = 1 mol kg–1) has also been expressed as a function of temperature T (inKelvin) and salinity S (in practical salinity unit, psu) by the same authors, but the calculated value has beensubsequently corrected by 0.0047 on the basis of a revised pHT value accounting for “tris” buffer (DelValls
and Dickson, 1998):

pK2 = pK2 (Clayton & Byrne, 1993) + 0.0047= 1245.69/T + 3.8322 + 0.00211 (35 – S).
293 K  T  303 K; 30  S  37
e1 = 0.00691; e2 = 2.2220; e3 = 0.1331.

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For practical calculation of R, measured absorbances are corrected for shifts in baseline absorbance and forthe absorbance of the sample itself (background absorbance) by means of Eq. (6):R

A578 (SW  Dye)  A578 (SW)  A730 (SW  Dye)  A730 (SW),A434 (SW  Dye)  A434 (SW)  A730 (SW  Dye)  A730 (SW)(6)

where AȜ(SW) is absorbance of seawater and AȜ(SW+Dye) is absorbance of seawater that contains dye atwavelength Ȝ.

2.2

Apparatus
We constructed an automated pHT measurement system (Fig. 1) that consists of an

ultraviolet/visible (UV/VIS) spectrophotometer (Varian Instruments, Cary 50), a sample and dye solution
circuit with an optical flow cell, a temperature-controlled water bath (ADVANTEC®, LP-3110) and anauto-sampling unit. This apparatus was used for onboard pHT analysis of surface seawater and bottle
samples. Details of sample collection are described in Section 2.6. All tubing was made of fluorinatedethylene-perfluoroalkoxyethylene copolymer (Teflon® PFA) except the peristaltic pump (Pharmed® tube)
and the inlet for the dye solution (polyether-etherketone, PEEK). All commands and sample temperature
data were sent to a personal computer (PC) through an I/O controller. Absorbance data were acquired
through the PC board, which was directly connected to spectrophotometer.
To achieve precise analysis, a constant dye/sample mixing ratio, precise temperature control andprecise absorbance measurement are critical. To keep the dye/sample mixing ratio constant, fixed volume
loops (shown as bold lines in Fig. 1) for dye solution and for samples connected to a 6-port switching valve(2 positions) were introduced to the apparatus. First, the sample loop that included the optical cell of thespectrophotometer was filled with a portion of sample (13.16 cm3), and absorbances of sample atwavelength 730 nm, 578 nm, 488 nm and 434 nm (A730(SW), A578(SW), A488(SW) and A434(SW),respectively) were measured. Then, a small amount of dye solution (0.0512 cm3) was introduced intoanother loop. After switching the 6-port valve to its second position, the sample and dye solutions weremixed together by circulation at a flow rate of ca. 75 cm3/min. To accelerate mixing, a capillary made of
PFA tubing (0.5 mm inner diameter) was placed at the inlet of the cell. Reproducibility and completeness of
mixing were confirmed by monitoring the absorbance at the isosbestic point of m-cresol purple (488 nm).

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Approximately 1.5 minutes (ca. 9 cycles) was required to homogenize the dye concentration by circulation
through the combined loop. The final concentration of dye in the sample solutions was 6.2 ȝmol/kg andvaried within ±1%.
The optical cell is a specially designed water-jacketed cylindrical quartz cell with a path length of
8 cm. The inner tube of the cell was tilted to facilitate ejection of bubbles in the sample. The temperaturesin the water jacket (outer tube) and cell holder were regulated within 25.00 ± 0.05 °C by circulating
temperature-controlled water, and the temperatures were monitored just outside the cell with a Pt-100ȍ
resistance thermometer calibrated with a standard resistance thermometer (Pt-25ȍ). Using a flow cell in aclosed circuit, seawater sample was not drained from the optical cell before the dye solution was injected.
As the result, we reduced the shift in baseline absorbance (±0.0005) as well as the sample volume required
for analysis. To regulate the sample temperature after the seawater sample and dye solutions were mixed,
2.5 minutes was required. This time was also reduced by circulating seawater sample through thetemperature controlled closed circuit for approximately 9 cycles. Each complete sample analysis procedure
took 10 minutes.

Switching valve unit
Sample selector unit

N2 gas
Com

Waste

NO NC

Pure water

STD
BottleSeawater
supplyValve NCc)
Switchingvalve

Spectrophotometerd) Optical cell
& cell compartment

Waste
c)
Waste

Sample
pump

Dye
pump

c)

a) Sample loopb) Dye loop

Bottle samples, STD & Dyee) Water bath25.0 0 ±0.05℃
Sample tubings
Thermostat tubings

Fig. 1 Schematic diagram of pHT measurement system. a) Sample loop (13.16 cm3), b) dye loop (0.051 cm3), c)
automated switching valve, d) optical cell and e) water bath. NO, NC and Com indicate the valve position to benormal open (when switched off), normal close, and common (open at all time), respectively.

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Technical Reports of the MRI, No. 57

2.3

2008

Preparation of dye solution
We prepared the indicator dye solution at a concentration of 1.6 mmol kg–1: approximately 1.3 g of

m-cresol purple sodium salt (C21H17O5NaS) (Acros Organics, water soluble) was dissolved in 2.0 kg of purified
water obtained with a Milli-Q SP TOC system (Millipore). Approximately 0.4 cm3 of ca. 1.0 M NaOH solution
was added to this dye solution while monitoring the absorbance of approximately 0.5 cm3 aliquot in a 0.5-mm
path length quartz cell until the absorbance ratio of the dye solution, Rdye = A578/A434, reached 1.6 ± 0.1.

2.4

Perturbation caused by addition of dye solution
The injection of m-cresol purple solution affects the pHT of seawater samples because theacid-base equilibrium of the seawater is disrupted by the addition of the dye acid-base pair (DOE, 1994).
There are two approaches to correct for the perturbation caused by the dye solution: a) an empirical method
in which a second aliquot of dye solution is added to the seawater sample (DOE, 1994; Clayton and Byrne,1993) and b) a theoretical method in which the acid–base equilibrium is calculated (Hunter and Macaskill,1999). We used the empirical method. In this method, the absorbance ratio R extrapolated at zero dye
addition was calculated by subtracting the change in absorbance ratio (ǻR) after the addition of the first andsecond aliquots of dye solution (ǻR2-1 = R2 – R1) assuming ǻR2-1 to be identical to the change effected by
the addition of first aliquot (ǻR1 = R1 – R) (Eq. (7)):ǻR = R2 – R1 = R1 – R (Assumption),(7)

R = R1 – ǻR.

The value of R obtained by subtracting ǻR from R1 was used to calculate pHT by means of Eq. (4).
We measured ǻR over a range of seawater pHT values and determined a calibration curve for ǻR
as a function of R1 for each batch of the dye solution (Fig. 2a), since ǻR is also a function of the differencein pH between the sample solution and the dye solution. As described above, Rdye was adjusted to 1.6 ± 0.1.
The pH value corresponding to Rdye was 7.9 ± 0.1. A quadratic regression (Eq. (8)) reproduced themeasured values well with a correlation coefficient of 0.9946. The standard error of ǻR was ±0.0019:

ǻR = – 0.0185 R12 + 0.0221 R1 + 0.0029(Rdye = 1.60)(8)

The change in pHT caused by the addition of the indicator dye ranged from –0.013 to +0.008 (Fig.

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Technical Reports of the MRI, No. 57

20082b) when 0.051 cm3 of the dye solution (1.6 mmol kg–1) was added to 13 cm3 of sample (dye concentrationin the sample corresponding to this mixing ratio was 6.2 Pmol kg–1).

C

D




R*6R*6

44































4







R*6

Fig. 2 Example of pHT perturbation caused by the addition of dye solution. The difference between the additionof the second and first aliquots of dye solution for batch 784C was expressed as a) absorbance ratio R and b) pHT.
Such plots were constructed for each batch of the dye solution used in the measurements.

2.5

Correction for temperature deviations
As described in Section 2.2, the temperature of seawater samples was maintained at 25.00 ± 0.05

ºC and monitored in our pHT measurement system. However, a change in sample temperature of 0.05 ºC
could give rise to the change in pHT of ca. 0.001 (Fig. 3a). Therefore, the pHT determined at a temperature t(pHT(t), with t in °C) was corrected to the pHT at 25.00 ºC (pHT(25)) with Eq.(9):(pHT(t) – pHT(25)) / (t – 25.00)= (2.00170 – 0.735594 pHT(25) + 0.0896112 pHT(25)2 – 0.00364656 pHT(25)3).

(9)This equation was determined as follows. Values of pHT(t), TCO2 and salinity were obtained for all bottle
samples collected during cruise MR02-K06 (described below). TA was then calculated from each set ofmeasured pHT(t), TCO2 and salinity values by means of the procedure described in DOE (1994). The
equilibrium constants given by Lueker et al. (2000) at temperature t (25.00 ± 0.05 ºC) were used for
calculation. Finally, pHT at temperature t’ in the range from 24.7 to 25.3 ºC was calculated from TCO2,
salinity and calculated TA values. The slope of the linear regression of pHT(t’) – pHT(25) versus t’ – 25 was
expressed as a cubic function of pHT(25) with a correlation coefficient of 0.99993 (p < 10–8, Fig 3b).

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Technical Reports of the MRI, No. 57

2008

D

R*6 








R*6 V R*6 

R*6 V R*6  VQ%

C
















[Z  Z
Z 
4






VQ%

R*6 



Fig. 3 Effect of sample temperature on the measured pHT at temperatures from 24.7 °C to 25.3 °C. The difference
between pHT(t) and pHT(25) was proportional to the sample temperature (a). The slope of the plot pHT(t) – pHT(25) vs. t –25 was expressed by a cubic function of pHT(25) with a correlation coefficient 0.99993 (b).

2.6

Seawater samples
Seawater samples were collected during cruises MR00-K08 (January 2001) and MR02-K06(January 2003) of the research vessel Mirai, which belongs to the Japan Agency for Marine-Earth Scienceand Technology. Samples consisted of near-surface waters, which were pumped continuously from thesea-chest at about 4.5 m below the surface and collected onboard, and of water samples at depth collectedwith a CTD/carousel sampler.
Onboard measurements for near-surface waters were made along the equator from 160° E to 160°
W in the western Pacific warm pool and in the equatorial divergence zone of the Pacific. A portion of water(ca. 0.5 dm3 min–1), which was a branch of seawater pumped up continuously from the sea-chest (ca. 20
dm3 min–1), was used for pHT measurements. Each sample was introduced into the measurement systemthrough the Teflon® PFA tube. Samples were collected twice every 1.5 hours, concurrently with onboard
TCO2 and pCO2 measurements.
Bottle samples were collected with Niskin bottles (30 dm3 polyvinyl chloride) on the
CTD/carousel sampler for deep water and with a bucket for surface water. The samples were transferred toborosilicate glass bottles (Shibata, 250 cm3). A small portion (2 cm3) of sample was removed to allow
thermal expansion of the seawater, and 0.2 cm3 of saturated HgCl2 solution (0.25 mol kg–1) was injected
into the larger portion as a bactericide. The bottles were sealed with greased ground-glass stoppers(Apiezon® grease, type L). The addition of the bactericide and the sealing of the bottles prevented anincrease in CO2 caused by biological activity in the bottle and prevented CO2 exchange with ambient air.
The effects of bottling and addition of HgCl2 solution are examined in Section 4.

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Technical Reports of the MRI, No. 57

2.7

2008

Certified reference materials (CRMs) and working standards
We examined the repeatability of the pHT measurements for a variety of time ranges from a day

to a few years using batches of certified reference materials (CRMs; provided by Andrew Dickson;
http://andrew.ucsd.edu/co2qc/) used as standards for TCO2 and TA analyses in seawater. We analysed theCRMs at the beginning and the end of each run of pHT measurements. Since TCO2 and TA of a CRM arecertified, its pHT is expected to be stable at constant temperature, although its pHT value has not beencertified yet. In addition, we prepared several batches of reference seawaters as working standards, by amethod similar to that used to create the CRMs (Dickson, 1991). These reference seawaters were sterilizedwith HgCl2, sealed as described above, and stored in 250 cm3 borosilicate glass bottles.

3.

Onboard measurements
We first evaluated the repeatability of onboard pHT measurements in the laboratory on land byanalysing seawater stored in a large-volume polyethylene container (20 dm3). This seawater was a mixture
collected at stations along the equator from 145° E to 160° W during cruise MR00-K08 and kept in the dark
at room temperature after the addition of HgCl2. The average pHT (with standard deviation) of this test
seawater was 7.8598 ±0.0002 (n = 32). The standard deviation was attributable to the baseline fluctuationof the spectrophotometer.
We then examined the repeatability of onboard pHT measurements at sea from the standard
deviations of pHT data measured when the ship was stopped at several stations during cruise MR02-K06.
Although the number of measurements was limited to 5 at most, the standard deviations of pHT data ranged
from 0.0001 to 0.0011 (Table 1). As evident from fluctuations in salinity, seawater samples taken at eachstation were not as homogeneous as the seawater mixed in a polyethylene container in the laboratory.
However, the standard deviations of the pHT data in onboard measurements suggest that measurement
repeatability was comparable to that obtained in the laboratory on land.
Table 1. Repeatability of onboard pHT measurements of near-surface waters along the equator during cruise MR02-K06.

Date
2003/1/17
2003/1/18
2003/1/21
2003/1/22
2003/1/24
2003/1/25
2003/1/28

Longitude
164°29.4’E – 164°33.6’E
169°59.1’E – 169°59.3’E
179°17.0’E – 179°18.8’E
175°38.3’W – 175°37.5’W
169°59.5’W – 169°57.2’W
164°48.2’W – 164°47.8’W
159°58.9’W – 159°58.4’W

pHT
8.1345 ± 0.0006 (n = 5)
8.1396 ± 0.0001 (n = 4)
8.1472 ± 0.0002 (n = 5)
8.1557 ± 0.0003 (n = 4)8.1001 ± 0.0011 (n = 3)
8.0934 ± 0.0002 (n = 4)
8.0689 ± 0.0006 (n = 3)

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−9−

Salinity (psu)34.434 ± 0.010
34.337 ± 0.005
34.101 ± 0.005
34.143 ± 0.012
34.170 ± 0.009
35.321 ± 0.003
35.265 ± 0.003

Technical Reports of the MRI, No. 57

2008

Such onboard measurements of pHT withgood repeatability, taken every 45 minutes at aninterval of about 12 nautical miles, can be used to findfine structures of pHT distribution in near-surface
seawater. During cruise MR02-K06, a clear boundary
was observed near 172° W at the east end of thewestern Pacific warm pool (Fig. 4). In the warm pool,
pHT was higher than 8.120. It exceeded 8.140 in theregion from the international dateline to 172° W. In thedivergence zone to the east of 160° W, pHT decreased
towards the east (minimum 8.069 at 160° W). The
results of these observations will be described in a
future report.

Fig. 4 Horizontal distributions of a) pHT, b) temperatureand c) salinity in near-surface water along the equator
observed in January 2003 during the R/V Mirai’s
MR02-K06 cruise.

4.

Measurements of bottle samples taken at depth
For pHT analysis of bottle samples collected at depths with the CTD/carousel sampler, it has been

recommended that the seawater samples be directly withdrawn from Niskin bottles into the optical cells(DOE, 1994). However, it is not convenient to prepare and handle many optical cells during fieldwork.
Therefore, we examined the use of sampling bottles and the effect of sterilization with HgCl2.
In our study, ca. 500 cm3 of water sample was drawn with a silicone rubber tube, which wasconnected to the outlet cock of a Niskin bottle, into a 250 cm3 borosilicate glass bottle (250 cm3 wasoverflowed). After the temperature of the sample was adjusted to 25.00 ± 0.05 °C in a water bath, thesample was introduced into the sample loop of the measurement system with a peristaltic pump (see Fig. 1).
During cruise MR02-K06, bottle samples were taken in duplicate from a Niskin bottle. The repeatability forthe measurement of bottle samples as inferred from duplicate measurements (Fig. 5) was 0.0011 (1ı, n =32). Problems associated with the comparability and precision of pHT measurements of bottle samples- 10 -− 10 −

Technical Reports of the MRI, No. 57

2008

include a) CO2 exchange between the sample and ambient air during bottling and during withdrawal of thesample from a rigid glass bottle, b) hydrolysis of HgCl2 and dilution caused by addition of saturated HgCl2
solution and c) pHT change during storage owing to imperfect sterilization or sealing.







$QVVNGR*6

'R*6




































1PDQCTFR*6

5GSWGPEGPWODGT

Fig. 5 Differences in pHT between duplicate
measurements, ΔpHT, of bottle samples without HgCl2
addition. The short-term standard deviation (repeatability)of the measurements was 0.0011, calculated with Equation
3 in SOP 23 described in DOE (1994).

4.1



Fig. 6 Comparison of pHT between bottle and onboard
measurements without HgCl2 addition. Error bars indicate
the standard deviation of measurements. The solid line
indicates a slope of 1. Broken lines indicate the error limits(±2σ).

Effects of bottling and headspace
The effects of CO2 exchange with ambient air during bottling and exchange with the headspace ina bottle, which arose from withdrawing a seawater sample from a rigid glass bottle, were examined bycomparing the analytical results of onboard pHT measurements with those acquired from bottle sample
measurements.
Bottle samples were taken from near-surface water (see Section 2.6) immediately after onboard
measurement. No HgCl2 solution was added to these samples. The difference in pHT (bottle minus onboard)
was –0.0019 ± 0.0010 (1ı, n = 14; Fig. 6) and was significant, considering the repeatability of onboard
measurements (0.0002) and that of bottle sample measurements (0.0011). However, the uncertainty due tobottling and short-term storage of seawater samples in bottles was as small as the required precision
described in the introduction (±0.002).

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Technical Reports of the MRI, No. 57

2008

Using a method similar to that used to correct for pCO2 perturbation in bottle samples, we
calculated the change in pHT caused by CO2 exchange between a sample and headspace in a bottle (DOE,
1994; see Appendix A for details). For surface water (initial pHT of ~8.12), the estimated effect of theheadspace on pHT ranged from –0.0003 to +0.0000, depending on the initial headspace pCO2 value (Fig. 7).
These changes were sufficiently smaller than the above-mentioned repeatability of pHT measurement forbottle samples. Therefore, the effect of headspace, which is less than 1% of the sample volume, on thechange in pHT was negligible for surface water. For deep waters (initial pHT of 7.48), the estimated effectof the headspace on pHT reached a maximum of +0.0012 for an initial headspace pCO2 value of 350 ȝatm(Fig. 7). This change was also comparable to the repeatability of pHT measurements and was hardly
detectable. With regard to bottling and headspace, we concluded that pHT values for bottle samples should
reasonably agree with onboard measurements within ±2ı (0.002) of the repeatability in bottle sample
























Fig. 7 Effect on pH T of headspace of 2 cm 3 in a
250 cm 3 sample bottle. a) Quantity of CO2 (μmol)
exchanged between sample and headspace and b) pHTchange caused by CO2 exchange between a seawater
sample and air in a bottle.




R*6






Fig. 8 Observed pHT change caused by the additionof 0.2 cm 3 of saturated HgCl 2 solution to 250 cm 3seawater (closed circles). The broken line shows
the linear regression of observed values, pHT(0.4) –pHT(0.2) = (–0.00094 ± 0.00059) pHT(0.2) + (0.00616± 0.00465), γ 2= 0.17, p = 0.14. Calculated values (solidline) were obtained from TCO2 and TA using stability
constants β of Hg 2 + complexes at ionic strength
0.7. Dotted lines indicate the uncertainty (±2σ) of
calculation derived from the variability of inputted
TCO2 and TA.

- 12 -− 12 −

R*6 R*6

R*6  R*6 

measurements.

Technical Reports of the MRI, No. 57

4.2

2008

Perturbation of pHT caused by addition of saturated HgCl2 solution
The pHT measurements of 27 bottle samples collected from various depths at a given station took

about 8 hours. If a sample is not sterilized, its pHT and its TCO2 could change owing to biological activity
occurring prior to measurement. Therefore, bottle samples must be sterilized with saturated HgCl2 solution.
However, the addition of saturated HgCl2 solution may affect pHT because of hydrolysis of HgCl2 anddilution of the sample. To evaluate the perturbation of pHT caused by the addition of HgCl2 solution and tocorrect for this perturbation empirically, we compared the pHT values of 14 pairs of samples taken fromseveral ocean layers ranging from the surface to a depth of 794 m in the equatorial Pacific Ocean (from170° W to 165° W) during the cruise MR02-K06. Duplicate samples were collected at each depth, anddifferent volumes of saturated HgCl2 solution (0.2 and 0.4 cm3) were added to each of a given pair ofsamples.
The injection of additional 0.2 cm3 saturated HgCl2 solution to each sample already containing
0.2 cm3 saturated HgCl2 caused pHT changes (pHT(0.4) – pHT(0.2)) ranging from –0.0024 to –0.0003 (Fig.
8, closed circles). These changes appeared larger for samples with higher pHT values, but the correlation
was insignificant (Ȗ2 = 0.17, p > 0.1). Therefore, we corrected for perturbation caused by HgCl2 addition byadding a constant value of 0.00123 (average of pHT(0.4) – pHT(0.2)) to measured pHT values. The cause ofthis perturbation is discussed later in Section 7.2.

4.3

Storage of samples
We investigated whether it was possible to store seawater samples for pHT analysis for a periodof a few months. To monitor the change in pHT of seawater samples stored in borosilicate glass bottles, wecollected approximately 20 litters of surface water at 4° 10’ N, 156° 40’ E during the cruise MR02-K06.
This large sample was subsampled into 30 borosilicate glass bottles 250 cm3 each and then sterilized with
0.2 cm3 of saturated HgCl2 solution before being plugged with a greased ground-glass stopper. The sealed
samples were stored at room temperature. The mean pHT for samples stored for 50 days was 8.1252 ±0.0011 (1ı, n = 21; Fig. 9). There was no significant temporal change (Ȗ2 = 0.06, p > 0.2). This result
suggests that surface water samples can be stored for pHT analysis with a precision that is comparable to
the repeatability of pHT measurements of samples without HgCl2 addition (±0.0011).
For deep-water samples, 42 pairs of duplicate samples were collected at layers ranging from thesurface to a depth of 5104 m at 0°, 160° W during the cruise MR02-K06. One of the duplicate samples was- 13 -− 13 −

Technical Reports of the MRI, No. 57

2008



“asap”), and another was analysed 50 days later at alaboratory on land (referred to as “stored”). Thechanges in pHT ranged from –0.001 to +0.007 after 50

R*6analysed within 15 hours after bottling (referred to as

days of storage (Fig. 10). The change in pHT was largerat 400–1500 m, where pHT was lower than 7.5. Thenegative correlation between the change in pHT (=

pHT(stored) – pHT(asap)) and pHT(asap) (Fig. 10b) andthe positive correlation between the change in pHT andpCO2 (Fig. 10c) suggest the possibility of CO2 gas









5VQTKPIVKOGFC[UFig. 9 Time course of pHT of surface water samples
stored in glass bottles with HgCl2. The mean (solid line)
and the standard deviation (broken line) were 8.1252
and 0.0011, respectively.

exchange between ambient air and samples taken at
depth, where pHT was lower than 7.5. A change in pHTof +0.005 corresponds to a change in TCO2 of –4 ȝmolkg–1 at constant TA. However, TCO2 values previously
have been observed to remain unchanged whensamples are stored by the method described here (Ishii
et al., 2000). Since pCO2 is high in deep-water samples
with lower pHT and higher TCO2 than those innear-surface

seawater,

CO2

could

escapefromdeep-water samples during sampling and measuring
processes. Further studies are needed regarding thestorage of seawater for pHT analysis.

Fig. 10 Difference between the pHT values measured on
board as soon as possible after water samples arrived on
deck, pHT(asap), and after 50-day storage, pHT(stored),versus a) depth, b) pHT(asap) and c) pCO2 calculated
from TCO2 and pHT(asap). 0.2 cm3 of saturated HgCl2
solution was injected into each sample.

- 14 -− 14 −

Technical Reports of the MRI, No. 57

4.4

2008

Uncertainty in pHT measurements of bottle samples
The pHT values of bottle samples agreed reasonably with the values measured onboard. The

precision of bottle sample measurements as estimated from duplicate sample measurements was 0.0011(Fig. 5 in Section 4). Taking into account the perturbation due to storage, the uncertainty of bottle sample
measurements was 0.001 for surface waters (Fig. 9 in Section 4.3) and a maximum of 0.006 for deep waters(Fig. 10 in Section 4.3) over a period of 50 days.

5. Consistency of measured and calculated pHT values for CRMs andcomparability of pHT data among several cruises
We measured pHT values of several batches of CRMs used as standards for TCO2 and TA
analyses to investigate the consistency of pHT measurements obtained by means of our system with thecalculated pHT values of CRMs certified for TCO2 and TA. Although the CRMs were not certified for pHT,
their pHT values could be calculated from the certified TCO2 and TA values because these values are stable.
We compared our measured pHT values with the values calculated from the certified TCO2 and TA valuesof the CRMs using the dissociation constants of carbonic acid in seawater reported by Lueker et al. (2000).
We adopted the dissociation constants of Lueker et al. (2000) for the following reasons:a) The fugacity of CO2 (fCO2) calculated from TCO2 and TA using these dissociation constants agreed
well with measured values in the range up to 500 ȝatm (Lueker et al., 2000).
b) The dissociation constants of Lueker et al. (2000) are based on the data of Mehrbach et al. (1973). Lee
et al. (2000) reported that fCO2 calculated from pHT and TCO2 using dissociation constants based on
the data of Mehrbach et al. (1973) agreed well with measured fCO2 values.
The pHT values of CRMs measured during several cruises agreed well with pHT values calculated
from the certified values of TCO2 and TA (Table 2). Although the difference between the measured andcalculated pHT values for batch 65 was larger than for any other batch, this difference was within the rangeof experimental error (mean ±2ı). We concluded that the pHT values obtained by our measurement systemwere consistent with certified TCO2 and TA values and that the pHT measurements we obtained during thedifferent cruises (in which different dye solutions and working standards were used) were comparable.

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Technical Reports of the MRI, No. 57

2008

Table 2 pHT of CRMs.
Mean ± standard deviation (bottles, measurements) during individual cruises
Experimentperiods(ddmmyy)140103-190303

Apparatus

Dye soln.
batch

WS
batch

Batch 58

Batch 62

Batch 65

Batch 72

1

784C

V

7.9118 ± 0.0007(2, 4)

301003-111103

1

826C

W

7.8760 ± 0.0011(3, 8)
7.8740 ± 0.0007(3, 6)
7.8747 ± 0.0007 a(1, 2)
7.8709 ± 0.0014(3, 6)
7.8747 ± 0.0005 a(1, 2)
7.8712 ± 0.0016(3, 6)
7.8690 ± 0.0001 a(1, 2)

170304-210404

1

826C

W

270804-100904

2

837C

W,X

241004-271204

1

837C

X

180805-180805

2

957C

X,Y

091105-290306

2

957C

Y

7.9155 ± 0.0006(2, 4)
7.9079 ± 0.0001 a(1, 2)

Calculated from TCO2 and TA b

7.9118 ± 0.0007(2, 4)
7.9119 ± 0.0010

7.8731 ± 0.0025(16, 34)
7.8735 ± 0.0014

7.9129 ± 0.0039(3, 6)
7.9189 ± 0.0015

7.9009 ± 0.0001 a(1, 2)
7.8962 ± 0.0006 a(1, 2)
7.8949 ± 0.0022(3, 7)
7.8956 ± 0.0027(7, 15)7.8986 ± 0.0017

130706-140806

2

957C

Y,Z

131206-200107

2

957C,
1058C

27

Measured – Calculated–0.0001–0.0005–0.0059–0.0029

Whole period

Mean ± standard deviation for the difference in analytical results between duplicate measurements.
Means ± standard deviations of pHT calculated from certified values of TCO2 and TA using dissociation
constants for carbonic acid in seawater reported by Lueker et al. (2000).
a

b

6.

Procedure to measure pHT with high comparability and precision
On the basis of our experimental results, we recommend the following experimental procedure

for pHT measurements at sea and on land. CRMs should be measured at the beginning and the end of a
cruise or a series of experiments. Working standards should be measured at the beginning and the end ofeach day of pHT measurements during a cruise. Prior to data collection during a cruise, we recommend
making a property control chart of measured pHT values (SOP 22 in DOE (1994)) and calculating the meanand standard deviation from at least 12 data points obtained from working standards measurements. If a
newly measured value is out of the range of mean ± 2ı, an additional bottle of working standard should beanalysed. If a couple of measured values are out of the range of mean ± 3ı, another batch of working
standards or CRMs should be analysed. If the results are out of the range of each mean ± 3ı, the apparatus
or reagents should be checked to determine the reason for discrepancy. The mean and standard deviation
are updated by adding newly accepted data.
The following data processing method is recommended:
1) pHT is calculated from spectrophotometric data by means of Eqs. (4) and (5).
2) pHT should be corrected for the perturbation induced by the addition of dye and saturated HgCl2
solutions to the sample. Dye correction is expressed by the term 'R and expressed as a quadratic
function of R1 (Eqs. (7) and (8)). The coefficients in equation (8) should be determined for each batch- 16 -− 16 −

Technical Reports of the MRI, No. 57

2008

of indicator solution. To correct for the perturbation caused by the addition of saturated HgCl2 solution,
the constant value of 0.00123 is added to the pHT data of a sample that has been sterilized with HgCl2.
3) pHT measured at temperature t (°C) is normalized at temperature of 25.00 ºC by means of Eq. (9).

7. Discussion
7.1 Standard deviation of pHT in reference materials
The averages and standard deviations of the pHT values of CRMs and working standards for asingle cruise and for several cruises are listed in Tables 2 and 3, respectively. The standard deviations forworking standards (0.0004–0.0059) during each cruise were on the whole larger than those for CRMs
(0.0006–0.0022), except the values from duplicate measurements. The standard deviations were somewhat
larger than the repeatability of bottle sample measurements described in Section 4.3 (0.0011). This result
indicates that the CRMs and, in particular, working standards were somewhat inhomogeneous. Working
standard batch W is likely to be more inhomogeneous than others, because the standard deviation of pHT
for working standard W was larger than that for working standard V and X, which was measured in thesame periods of experiments with same apparatus (Table 3). If we exclude the results of working standard
batch W, we can conclude that the repeatability of our measurements within a cruise or a series ofexperiments was better than 0.002.
Table 3 pHT of working standards.
Mean ± standard deviation (bottles, measurements) during individual cruises
Experimentperiods(ddmmyy)140103 –190303

Apparatus

Dye soln.
batch

Batch V

1,

784C

171003 –221003

1

826C

301003 –111103

1

826C

7.7776 ± 0.0011(43, 43)7.7756 ± 0.0015(7, 9)
7.7810 ± 0.0025(4, 4)

170304 –210404

1

826C

270804 –100904

2

837C

300904 –011004

1

837C

041004 –081004

2

837C

241004 –271204

1

837C

180805 –180805

2

957C

091105 –290306

2

957C

130706 –140806

2

957C

131206 –200107

2

957C,
1058C

Whole perioda7.7776 ± 0.0017(54, 56)

Batch W

7.8613 ± 0.0059(7, 9)
7.8704 ± 0.0057(40, 41)7.8640 ± 0.0038(45, 45)7.8718 ± 0.0040(3, 5)

7.8667 ± 0.0061(95, 100)

Batch X

Batch Y

Batch Z

7.9177 ± 0.0021(93, 101)
7.9187 ± 0.0010(5, 5)
7.9169 ± 0.0001 a(2, 2)
7.9177 ± 0.0021(101, 109)

7.8356 ± 0.0013(7, 7)
7.8348 ± 0.0021(40, 40)
7.8358 ± 0.0021(6, 6)
7.8330 ± 0.0024(54, 54)7.9576 ± 0.0020(23, 27)
7.9494 ± 0.0013(6, 6)
7.9550 ± 0.0020(8, 11)7.9504 ± 0.0015(31, 32)
7.9550 ± 0.0004(7, 7)

7.9537 ± 0.0037(75, 83)

Mean ± standard deviation for the difference in analytical results between duplicate measurements.

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In contrast, cruise-to-cruise differences were observed in the pHT measurements. The standard
deviations in pHT measurements of CRMs for the whole period (0.0007–0.0039) were larger than those foreach cruise (0.0006–0.0022) (Table 2). For example, the standard deviations of the pHT values in CRM
batch 62 and in working standard batch X measured during the same cruises were 0.0025 and 0.0037,
respectively. When we analysed the CRMs and working standards at the same time, both pHT values tended
to change similarly. This result suggests that the variations of pHT were caused not by changes in thequality of the CRMs or working standards but by changes in the conditions of apparatus or indicatorsolutions. If we corrected the pHT values to reflect the amount of change we commonly observed for the
CRM and working standard measurements, the standard deviation of the measured pHT values of working
standard batch X decreased from 0.0037 to 0.0023; therefore we can expect to obtain more precise
measurements than present measurements. However, there is no guarantee that we can apply this correction
to the whole pHT range in seawater, because the pHT range of the CRMs or working standards was limited
between 7.8 and 7.9. We would be able to confirm the possibility to correct for the cruise-to-cruise
difference by measuring several sets of working standards with different pHT values.
For precise pHT measurements, it is important to satisfy the requirements for analyses ofreference materials before measuring a series of samples, as described in Section 6. Our experimental
results suggest that the procedure described here for analysing reference materials effectively enables us toobtain pHT measurements with an uncertainty of less than 0.003 during different cruises.

7.2

Origin of the pHT perturbation caused by the addition of HgCl2
To determine the reasons for the pHT decrease of –0.0024 to –0.0003 caused by the addition of

HgCl2 solution (see Section 4.2), we calculated pHT from TCO2 and TA before and after addition of the
HgCl2 solution. In this calculation, we took into account the effect of dilution and the effect ofcomplexation of Hg2+ with OH– and with CO32– ions (see Appendix B for details).
The result of this calculation supports the experimental results that the addition of HgCl2 solution
reduced the pHT in seawater. The change in pHT calculated for the addition of 0.2 cm3 of saturated HgCl2
solution to 250 cm3 of seawater ranged from –0.0039 to –0.0015, depending on the combination of thevalues of TCO2 and TA of seawater (Fig. 8). These calculated results of the negative differences and thenegative correlation to pHT were consistent with experimental results. Although the calculated results
differed significantly from the experimental results at a 99% confidence level (p = 6 × 10–9), the magnitudeof the calculated change in pHT was within the same order as that of the experimental data. A change in TA- 18 -− 18 −

Technical Reports of the MRI, No. 57

2008(–3.8 ȝmol/kg) due to hydrolysis of Hg2+ and HgCO3 formation corresponded to a change in pHT of–0.0025. Most of the experimentally observed changes in pHT caused by HgCl2 addition could be
accounted for by the change in TA and dilution. The difference between experimental and calculated values
might be derived from uncertainty in the stability constants of Hg2+ complexes.

7.3

Internal consistency of CO2 system parameters
When we measure three of four parameters controlling the CO2 system (TCO2, TA, pHT andpCO2), we can examine each data point by comparing measured values and the one parameter calculated
from the other two parameters (e.g., Lee and Millero, 1995; McElligott et al., 1998; Lee et al., 2000). We
calculated pCO2 from measured pHT and TCO2 and compared the results with measured pCO2 using the
calculation procedure described in DOE (1994). Various dissociation constants for carbonic acid, including
those reported by Lueker et al. (2000), Mojica Prieto and Millero (2002), Roy et al. (1993), Dickson andMillero (1987), Goyet and Poisson (1989), Mehrbach et al. (1973) and Hansson (1973), were used for thesecalculations and were compared on the scale of pHT.
The pHT, TCO2 and pCO2 values insurface water were measured simultaneously
thecruiseMR02-K06.

samples for onboard measurements of thesethree parameters were obtained from the sameonboardpumpingdetermined

by

the

system.

TCO2

coulometricSeawaterwasmethoddescribed by Ishii et al. (1998) and calibrated
with the same CRMs described above. pCO2

R%1OGCUR%1ECNE PCVO

duringwas measured by the method described by









Inoue (2000) and was calibrated by the MRI87





R%1

scale that is traceable to the WMO scale.
The difference between measured

OGCU







PCVO

Fig. 11 Comparison of pCO 2 values observed andcalculated from measured pHT and TCO2 using dissociation
constants for carbonic acid given by Mehrbach et al.
(1973; diamond), Hansson (1973; bar), Dickson andMillero (1987; plus), Goyet and Poisson (1989; circle),
Roy et al. (1973; triangle), Lueker et al. (2000; box) andMojica Prieto and Millero (2002; cross).

and calculated pCO2, ǻpCO2 = pCO2meas –pCO2calc, ranged from –6 to +18 ȝatm (< 4%),depending on the dissociation constants used
(Fig. 11). The ǻpCO 2 value calculated with- 19 -− 19 −

Technical Reports of the MRI, No. 57

2008

dissociation constants reported by Lueker et al. (2000) was in the middle of these differences. Taking into
account the uncertainties of ±2 ȝatm for pCO2meas and ±2 ȝatm for pCO2calc derived from onboard
measurements of pHT (±0.0002) and TCO2 (±2 ȝmol kg–1), pCO2calc calculated with dissociation constants
reported by Lueker et al. (2000) was approximately equivalent to pCO2meas.
Lee et al. (2000) reported that pCO2 and TA calculated from measured TCO2 and pHT showed thebest agreement with measured values when the dissociation constants of Mehrbach (1973), which were
given as functions of temperature and salinity by Dickson and Millero (1987), were used. In the study of
Lee et al. (2000), the difference in fugacity, 'fCO2 = fCO2meas – fCO2calc, was +1.4 ± 1.4 (1ı) % (+5.2 ± 5.2
ȝatm at 370 ȝatm) for seawater collected at the 170° W meridian. For our pHT results, the calculation with
dissociation constants of Mehrbach (1973) gave 'pCO2 of +2.3 ± 0.4 (1ı) % (+8.7 ± 1.4 ȝatm). This valueof 'pCO2 differed insignificantly from the value described by Lee et al. (2000).
Although further consideration may be required to the comparability of pHT measurements and tothe variability of calculation caused by the uncertainty of dissociation constants of carbonic acid, the pCO2
values measured in the equatorial Pacific Ocean were reproduced within the range of ±11 ȝatm bycalculation from our measured pHT and TCO2 values.

8.

Summary
We developed an automated, precise pHT measurement system based on a spectrophotometrictechnique with the indicator dye m-cresol purple. We proposed a procedure for precise pHT measurement
involving the analysis of reference materials, and we evaluated the uncertainty of the pHT values obtainedwith our analytical procedure. We also examined the effects of bottling and storage, as well as the effect of
the addition of HgCl2 solution, on the pHT measurements obtained from bottle samples.
The standard deviations of pHT measurements obtained with our system were 0.0002 for onboard
measurements, 0.002 for bottle samples of surface waters and 0.006 for deep waters after 50 days ofstorage. Our measured pHT values for CRMs were consistent with those calculated from certified TCO2 andTA values with the dissociation constants of carbonic acid reported by Lueker et al. (2000). The onboard
pHT measurements were consistent with those derived from TCO2 and pCO2 measurements in the fieldstudy.

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Acknowledgements
We thank Mr. A. Kudo of Nippon ANS Co. for his helpful suggestions on the design andspecifications of the apparatus. We thank the scientists and crew of the research vessel Mirai for theirsupport during cruises MR02-K06 and MR00-K08. We also thank the staff of the Geochemical Research
Department, Meteorological Research Institute, Japan Meteorological Agency, for their support of thisstudy. This study was funded by the research programme “Global Carbon Cycle and Related Mapping
based on Satellite Imaginary Programme” (FY1998-2002) by the Research Promotion Fund from the
Ministry of Education, Culture, Sports, Science and Technology, Japan, and by the research programmes“Observational Study on the Seasonal and Interannual Variability and its Mechanisms of the Carbon
Dioxide System at the Air-Sea Interface and in the Upper Layer of the Ocean (FY2003)”, “Observational
Study on the Variability of Carbon Cycle in the Ocean, I” (FY2004-2006) and “Observational Study on the
Variability of Carbon Cycle in the Ocean, II” (FY2007-2008) of the Meteorological Research Institute,Japan Meteorological Agency’s research fund.

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Appendix A: Calculation of change in pHT due to CO2 gas exchange
between headspace and seawater sample in a bottle.
Initial pHT and TCO2 values (pHT,i and TCO2,i) in the seawater samples without CO2 gas
exchange were chosen arbitrarily so as to reproduce the variety of seawater conditions that were observed
during cruise MR02-K06. Then, initial pCO2 and TA values in the seawater (pCO2,iSW and TAi) were
calculated from pHT,i and TCO2,i using the equilibrium relations for carbonates (DOE, 1994). The initial
pCO2 in the headspace of the sealed bottle (pCO2,iHS) was chosen from the values between 350 ȝatm(uncontaminated atmosphere) and 1000 ȝatm (room air). The final pCO2 in the headspace (pCO2,fHS) was
assumed to be close to pCO2,iSW. From the initial and final pCO2 values in the headspace, the change in thenumber of micromoles of CO2 (ǻnCO2) in the headspace with a volume of VHS was calculated with thefollowing equation of state (Eq. A.1), assuming that CO2 behaved as an ideal gas:'n CO 2HS
HS
pCO HSfV
2, f  pCO 2,i f p  V

RT.

(A.1)җ

Variables in Eq. A.1 that have not been defined are as follows:
fp: Factor to convert pressure unit (101325 Pa atm–1).

VHS: Volume of the headspace in a bottle (2 cm3).

fV: Factor to convert volume unit (10–6 m3 cm–3).

R: Gas constant (8.31451 J mol–1 K–1).

T: Temperature in K.

The change in TCO2 in the seawater sample (in ȝmol kg–1) was calculated with the following equation:'TCO 2

TCO 2, f  TCO 2,i'n CO 2

UV SW

,

(A.2)

where ȡ and VSW are the sample density (in kg dm–3) and volume (0.250 dm3), respectively. Then, theperturbed pHT was calculated from the equilibrium relationship for carbonates from perturbed TCO2,f andinitial TAi, which does not change during CO2 gas exchange.

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Appendix B: Calculation of the pHT change derived from the addition ofsaturated HgCl2 solution.
The Hg2+ ion reacts with various inorganic ligands to form metal complexes. The stability
constants of major Hg2+ complexes are given in Table B.1 (Stumm and Morgan, 1996). The formation ofcomplexes with hydroxide and carbonate affects TA (Eq. B.1) and TCO2 (Eq. B.2) in seawater and hence
perturbs pHT.

Table B.1 Stability constants for various Hg2+ complexesa.

Ligands
Complexes
HgL
HgL2
HgL3
HgL4
HgOHL

OH–

Cl–

CO32–

SO42–

10.6
21.8

7.2
14.0
15.1
15.4
18.1

16.1

2.5
3.6

Constants are given by the logarithms of the overall formation constants, β, at 25 °C and an ionic strength ofzero. The stability constants were corrected to an ionic strength of 0.7 mol kg–1 with activity coefficients (–log γ)
of 0.146 and 0.584 for charges of ±1 and ±2, respectively.
aTAmix = [HCO3–] + 2[CO32–] + [B(OH)4–] + [OH–] – [H+]F – [HSO4–]+ [Hg(OH)+] + 2[Hg(OH)2] + [Hg(OH)Cl] + 2[Hg(CO3)]
TCO2mix = [CO2*] + [HCO3–] + [CO32–] + [Hg(CO3)]

(B.1)

(B.2)

The basic algorithm for calculating the pHT change caused by the addition of saturated HgCl2 solution
involves correcting for the consumption of hydroxide and carbonate ions caused by the formation of Hg
complexes in the calculation of pHT from TCO2 and TA.
To use the stability constants in Table B.1, which are given at an ionic strength I = 0 mol kg–1, we
corrected for the activity coefficient to obtain the constants at the ionic strength of seawater (0.7 mol kg–1).
The thermodynamic stability constant ȕ of complex HgLx is expressed by Eq. B.3:

E HgL xaHgL xaHg aLx[HgL2x-xQ ](m0 ) x J HgL x
[Hg 2  ][LQ  ]x J Hg J Lx .

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2008

Variables in Eq. B.3 are as follows:aM: Activity of an ion M (dimensionless).

[M]: Concentration of an ion M in mol kg–1

m0

: Standard value of concentration (1 mol kg–1).

ȖM: Activity coefficient of ion M (dimensionless).

Eq. B.3 can be rearranged to Eq. B.4 to give the stability constant XHgLx:

log X HgL x§ [HgL2x- xȞ ](m 0 ) x ·¸log¨¨ [Hg 2  ][LȞ  ] x ¸
©
¹
(B.4)

log E HgL x  log J HgL x  log J Hg  x  log J L

The values of log ȕHgLx in Eq. B.4 are given in Table B.1. The values of –log Ȗ in Eq. B.4 are substituted by
0.584 or 0.146 depending on the charge of the ionic species (Stumm and Morgan, 1996). For example, log
XHgLx for HgCl42– is calculated as follows: log ȕHgCl4 = 15.4; – log ȖHgCl4 = 0.584; + log ȖHg = – 0.584; + log
ȖCl = – 0.146. Then, log XHgCl4 = 15.4 + 0.584 – 0.584 – 4 × 0.146 = 14.8.
We used the following procedure to calculate the pHT in seawater that contained HgCl2:a) First, TA was calculated from pHT, TCO2, temperature and salinity measured for seawater obtained in
the equatorial Pacific Ocean during the cruise MR02-K06 with the dissociation constants for carbonicacid reported by Lueker et al. (2000). Because the samples were diluted with saturated HgCl2 solution
by the ratio of volume VSW:VHg, the diluted values of TCO2 (TCO2mix), TA (TAmix) and salinity (Smix)
were calculated with the following equations:

TCO2mix = TCO2 · VSW / (VSW + VHg) ,TAmix = TA · VSW / (VSW + VHg) ,Smix = S · VSW / (VSW + VHg) .

b) The effect of dilution on pHT was then calculated by iterative methods from TCO2mix and TAmix to give
pHTmix after the addition of HgCl2 solution without considering the effect of Hg2+ complexes.
c) Concentrations of hydroxide ion [OH–] and carbonate ion [CO32–] were calculated from pHTmix and
TCO2mix. The concentrations of chloride and sulphate ions were calculated from salinity Smix (DOE,
1994).

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Technical Reports of the MRI, No. 57

2008d) The concentration of each Hg2+ complex was calculated from Eqs. B.5 and B.6, using the result ofprocedure c):e)[HgLx2–xȞ] = XHgLx [Hg2+] [LȞ–]x ,(B.5)

CHg = [Hg2+] + 6 [HgLx2–xȞ] = [Hg2+] (1 + 6X HgLx [LȞ–]x) .

(B.6)

Perturbations of TA by HgOH+, Hg(OH)2, Hg(OH)Cl and HgCO3 from TAmix in Eq. B.1 were
calculated to give a corrected TA value, TA’. This procedure corresponds to the correction for the
consumption of OH– and CO32– caused by HgCl2 addition.

f) The concentration of HgCO3 was subtracted from TCO2mix in Eq. B.2 to give a corrected TCO2 value,
TCO2’. This procedure corresponds to the correction for the consumption of CO32–.
g) A corrected pHT value, pHT’, was calculated from TCO2’ and TA’ by the same method described inprocedure b).
h) If the difference between pHT’ and pHTmix was larger than 0.00001, pHTmix was substituted by pHT’.
Then procedures c) through h) were repeated. The value of pHT’ converged after two or three
iterations.

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Technical Reports of the MRI, No. 57

2008

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