**Simultaneous Underway Measurements of pH, *f*CO~2~, and TCO~2~ during
CLIVAR P16N**

R/V Thomas G. Thompson, 3250TT191

13 February -- 30 March 2006

Papeete, Tahiti -- Honolulu, HI - Kodiak, AK, USA

![](media/image1.jpeg){width="4.476388888888889in"
height="6.675694444444445in"}

+------------+--------+-----------------------------------------------+
| *          |        | Dr. Robert H. Byrne                           |
| *Principal |        |                                               |
| Inves      |        | Email: <byrne@marine.usf.edu>                 |
| tigator:** |        |                                               |
+------------+--------+-----------------------------------------------+
| **Parti    | **Leg  | Dr. Xuewu Sherwood Liu, Dr. Renate Bernstein  |
| cipants:** | 1:**   |                                               |
+------------+--------+-----------------------------------------------+
|            | **Leg  | Dr. Robert Byrne, Dr. Zhaohui 'Aleck' Wang,   |
|            | 2:**   | Dr. Johan Schijf, Mr. Ryan Bell               |
+------------+--------+-----------------------------------------------+
| **Inst     |        | College of Marine Science, University of      |
| itution:** |        | South Florida                                 |
|            |        |                                               |
|            |        | 140 7th Avenue South, St. Petersburg, FL      |
|            |        | 33701.                                        |
+------------+--------+-----------------------------------------------+
| **Data     |        | Dr. Zhaohui 'Aleck' Wang                      |
| Pro        |        |                                               |
| cessing:** |        | Email: awang@marine.usf.edu                   |
+------------+--------+-----------------------------------------------+

**Summary**

The automated Multi-parameter Inorganic Carbon Analyzer (MICA) developed
by the College of Marine Science at the University of South Florida was
used to simultaneously measure underway surface seawater pH, *f*CO~2~,
and TCO~2~ during the CLIVAR P16N cruise. The MICA data can be compared
to measurements from the PMEL underway LiCOR *p*CO~2~ system (surface
seawater *f*CO~2~) and discrete surface sampling (surface seawater pH
and TCO~2~) to evaluate performance.

Technical details and performance evaluation of the MICA system can be
found in Wang et al. (2007). The system consists of three seawater
channels (surface seawater *f*CO~2~, TCO~2~, and pH). All measurements
(three channels) are made at constant temperature (25 ˚C) and are based
on similar spectrophotometric principles. The system operates
autonomously with a sampling frequency of \~7 per hour. For each sample,
all three parameters are measured and recorded simultaneously.

The MICA system performed well during both legs of the P16N cruise. Its
measurements agree well with conventional state-of-the-art measurements
of carbon system parameters. The data are internally consistent based on
thermodynamic calculations.

**Principle of Measurements**

**1. pH**

Spectrophotometric pH measurements are based on the method described in
Clayton and Byrne (1993), but utilize thymol blue as the pH indicator
(Zhang and Byrne, 1996; Wang et al., 2007). Thymol blue is directly
injected into a stream of underway seawater and absorbances are
monitored spectrophotometrically. Sample pH is linked to the absorbance
ratio (R), dissociation constant (K~I~), and molar absorbance ratios
(e~1~, e~2~, and e~3~) of the indicator with the following equation:

+--------------------------------------------------------------+-------+
| > $\text{pH}T = - \text{log}                                 | \(1\) |
| _{T}^{}K_{I} + \text{log}\frac{R - e_{1}}{e_{2} - \Re_{3}}$, |       |
+--------------------------------------------------------------+-------+

where the subscript T denotes parameters expressed on the total hydrogen
ion scale. The calibrated constants of thymol blue are given by Zhang
and Byrne (1996) as:

+--------------------------------------------------------------+-------+
| > $- \text{log}_{T}^{}K_{I} =                                | \(2\) |
|  \frac{4\text{.}\text{706S}}{T} + \text{26}\text{.}\text{330 |       |
| 0} - 7\text{.}\text{17218  logT} - 0\text{.}\text{017316S}$, |       |
+--------------------------------------------------------------+-------+
| > *e*~1~ = --0.00132 + 1.6×10^-5^T,                          | \(3\) |
| >                                                            |       |
| > *e*~2~ = 7.2326 -- 0.0299717T + 4.6×10^-5^T^2^, and\       |       |
| > *e*~3~ = 0.0223 + 0.0003917T.                              |       |
+--------------------------------------------------------------+-------+

where T is absolute temperature in K, and S is salinity.

**2.** *f*CO~2~

For seawater *f*CO~2~ measurements, Teflon AF 2400 (DuPont) is used as
both a CO~2~ permeable membrane and a liquid-core waveguide (LCW) (Wang
et al., 2007). Phenol red is used as the indicator (Yao and Byrne,
2001). During each CO~2~ measurement, the indicator solution, composed
of Na~2~CO~3~ with constant total alkalinity (TA), is motionless inside
the LCW. The seawater samples are directed to flow outside the LCW.
After CO~2~ molecules equilibrate by diffusion with the LCW's internal
solution, the equilibrium pH is determined by measurements of absorbance
ratios. *f*CO~2~ is then derived from the equilibrium pH with the
following equation:

+--------------------------------------------------------------+-------+
| > $f\text{CO}_{2} = \frac{\t                                 | \(4\) |
| ext{TA}}{2K_{0}K_{1}^{'}K_{2}^{'}\lbrack H^{+}\rbrack^{- 2}  |       |
| + K_{0}K'_{1}\lbrack H^{+}\rbrack^{- 1}} = \frac{a}{L} + b$, |       |
+--------------------------------------------------------------+-------+

where K~0~ is the Henry's Law constant, K'~1~ and K'~2~ are carbonic
acid dissociation constants, and L
=$2K_{0}K_{1}^{'}K_{2}^{'}\lbrack H^{+}\rbrack^{- 2} + K_{0}K'_{1}\lbrack H^{+}\rbrack^{- 1}$.
Parameters "a" and "b" are derived through a calibration procedure using
CO~2~ gases at known concentrations. The pH of the indicator solution is
determined spectrophotometrically, and the L term in Equation 4 is then
calculated. Since the TA of the internal solution is constant, sample
*f*CO~2~ has a linear dependence on 1/L with a slope of "a" and an
intercept of "b". The calibration constants, "a" and "b", account for
all uncertainties in Equation 4 including the absolute value of TA.

**3. TCO~2~**

Spectrophotometric measurements of DIC using Teflon 2400 AF liquid core
waveguides have been described by Byrne et al. (2002). Water samples are
acidified before measurements (pH 2.7), whereupon the total
$\text{CO}_{2}^{}$concentration equals the TCO~2~ concentration. After
the internal Na~2~CO~3~ -indicator (bromcresol purple) solution attains
CO~2~ equilibrium with the acidified water samples across the LCW
CO~2~-permeable walls, the TCO~2~ of the outer solution can be written
as:

+------------------------------------------------------------------+---+
| > $$\text{log}\text{DIC} = \text{log}\left( \frac{(              | \ |
| K_{0})_{\text{ex}}}{(K_{0})_{i}} \right) + B - \text{log}\left(  | ( |
| \frac{R - e_{1}}{1 - \frac{\Re_{3}}{e_{2}}} \right),\text{and}$$ | 5 |
|                                                                  | \ |
|                                                                  | ) |
+------------------------------------------------------------------+---+
| > $$\text{log}\left( \frac{(K_{0                                 | \ |
| })_{\text{ex}}}{(K_{0})_{i}} \right) = \left( \frac{\text{50}\te | ( |
| xt{.}\text{20}\left( \mu_{\text{ex}} - \mu_{i} \right)}{2\text{. | 6 |
| }\text{303}} \right)\left( 0\text{.}\text{023517} - 0\text{.}\te | \ |
| xt{023655}\left( \frac{T}{\text{100}} \right) + \text{ 0}\text{. | ) |
| }\text{0047036}\left( \frac{T}{\text{100}} \right)^{2} \right)$$ |   |
+------------------------------------------------------------------+---+

where B is an experimentally derived constant determined via calibration
of the TCO~2~ channel against CRM. The subscript "ex" refers to the
acidified outer solution, "i" indicates the internal solution, and µ is
ionic strength.

**Materials and Measurements**

**1. Reagents**

For each of the three indicators used, three wavelengths are chosen for
measurement of absorbances. Two wavelengths assess the absorbance peaks
of acid and base forms of the indicator, while a third wavelength serves
as a reference wavelength. Absorbances vary at the acid and base
wavelengths in response to pH changes, but not at the reference
wavelength. Absorbance measurements at acid and base indicator maxima
are used for determination of all CO~2~ system parameters. The
wavelengths chosen for the three channels are listed in Table 1.

  -------------------- --------------- -------------- -------------- --------------
  **Table 1.                                                         
  Wavelengths used for                                               
  spectrophotometric                                                 
  determination of                                                   
  inorganic carbon                                                   
  species.**                                                         

  **Channel**          **Indicator**   **Acid         **Base         **Reference
                                       Wavelength**   Wavelength**   Wavelength**

  Seawater *f*CO~2~    Phenol red      434 nm         558 nm         700 nm

  TCO~2~               Bromcresol      432 nm         589 nm         700 nm
                       purple                                        

  pH                   Thymol blue     435 nm         596 nm         730 nm
  -------------------- --------------- -------------- -------------- --------------

The indicator solution for CO~2~ measurements consists of a solution of
2 µM phenol red, 225 µmol kg^-1^ total alkalinity (Na~2~CO~3~), and 0.2
µM sodium lauryl sulfate. For TCO~2~ measurements, the indicator
solution is made of 2 µM bromcresol purple in 1000 µmol kg^-1^ total
alkalinity (Na~2~CO~3~) and 0.2 µM sodium lauryl sulfate. The reference
solutions of the CO~2~ and TCO~2~ measurements are similar solutions
that contain no indicator. For pH measurements, the thymol blue stock
solution is made in Milli-Q water with a concentration of 1.5 mM. The R
ratio of thymol blue solution is adjusted (R\~0.77) to minimize the
magnitude of indicator-induced pH perturbations. All indicator and
reference solutions are stored in gas-impermeable laminated bags. 3N HCl
in Milli-Q water is stored in a 250 ml glass bottle and used to acidify
TCO~2~ samples.

**2. MICA system**

Three Ocean Optic 2000 spectrophotometers are used to determine
indicator absorbances for each of the three measurement channels. The
light assemblies, spectrophotometers, and optical cells are connected
with optic fibers. The light assembly of each channel consists of a
high-temperature tungsten lamp with blue and short-pass filters in order
to achieve an improved balance of spectral intensity between 430 and 700
nm.

The optical cells of the CO~2~ and DIC channels are custom-machined from
PEEK rods. The PEEK optical cell has a 27 mm O.D. and a 2 mm I.D. with a
length of 15 cm. A Teflon AF 2400 LCW is located inside this optical
cell. The sample inlet and outlet, and two optical fibers that connect
the optical cell with the light source and spectrophotometer, are
inserted into the ends of the LCW with two custom-made PEEK connectors.
The ends of the LCW are sealed with two O-rings that are housed inside
the connectors. The PEEK connectors allow both reagent and light to pass
through the LCW. The pH optical cell is machined from a PEEK rod, but
does not require special connectors since no LCW is used.

Indicator and reference solutions are pumped through separate lines into
their respective channels by digital peristaltic pumps. Surface seawater
is obtained continuously with a shipboard pumping system. It first flows
through a SBE 49 CTD that records salinity and temperature, and is then
pumped through three seawater channels (*f*CO~2~, TCO~2~, and pH).
Before entering the TCO~2~ channel, seawater samples are acidified with
\~3 N HCl using a digital peristaltic pump. The mixing ratio is
approximately \~700 (seawater to HCl). An in-line coil is used to
facilitate mixing. For pH measurement, thymol blue is mixed with
seawater sample at a mixing ratio of \~700 (seawater to thymol blue),
whereupon the final thymol blue concentration in the mixed sample is \~
2 µM. This low indicator concentration results in very small
indicator-induced pH perturbations (\< 0.001 pH units). An in-line
mixing coil is also used in this case.

All channels are thermostated in a Lauda E100 water bath that is set to
25 ± 0.1°C. All samples, reference and indicator solutions are
temperature pre-equilibrated to 25 ˚C in the water bath using PEEK and
glass coils. All measurements, as well as calibrations, are taken at
this temperature.

All components of the system are connected to a custom-made electronic
motherboard that is controlled by a PC. The interface program cycles to
operate the MICA autonomously. The time required for each measurement
cycle depends on the equilibration time (7 minutes for the seawater
*f*CO~2~ and TCO~2~ channels) and flushing time selected for the
indicator/reference solution and samples (\~2 minutes). The
equilibration time required for pH measurements is very short. The
following sequence is used during a measurement cycle:

1.  Flush pH reference (seawater samples without indicator solution).

2.  Flush reference for seawater *f*CO~2~ and TCO~2~.

3.  Read and store reference readings.

4.  Flush indicator solutions for seawater *f*CO~2~ and TCO~2~; mix
    thymol blue with seawater samples (pH measurements); acidify TCO~2~
    samples.

5.  Seawater *f*CO~2~ and TCO~2~ equilibration (7 minutes).

6.  Read and store measurements.

7.  Repeat Steps 4-6 five times.

8.  End of one measurement cycle and repeat from the beginning.

**3. Standards and Calibration**

Prior to the cruise, the CO~2~ channel was calibrated against five
standard CO~2~ gases ranging from 280 to 550 ppm (XCO~2~). TCO~2~ was
also calibrated before the cruise using a Certified Reference Material
(CRM). Thymol blue has been calibrated previously for seawater pH
measurements (Zhang and Byrne, 1996). During the cruise, CO~2~ gas
standards and CRM were used periodically to evaluate calibration
consistency for CO~2~ and TCO~2~ measurements. Re-calibration was
performed if necessary.

**Results**

**1. Precision**

The precisions of all measured parameters, estimated by replicate
measurements at the concentration ranges encountered, are given as
follows:

  ------------------------------------------ ----------------------------
  pH                                         ± 0.001

  Seawater *f*CO~2~                          ± 1 µatm

  TCO~2~                                     ± 1-3 µmol/kg
  ------------------------------------------ ----------------------------

**2. Corrections**

The calibration for the CO~2~ channel is related to XCO~2~ readings of
the standard CO~2~ gas tanks, even though it is *f*CO~2~ that drives
equilibrium across the LCW membrane. Based on DOE (1994), XCO~2~,
*p*CO~2~ and *f*CO~2~ can be calculated from each other at various
measurement conditions (temperature, salinity, and pressure). The MICA
seawater *f*CO~2~ measurements reflect *f*CO~2~ at 25°C with 100% water
vapor content. The *f*CO~2~ at 25 ˚C was corrected to in-situ
temperature to compare with PMEL LiCOR underway *p*CO~2~ measurements.
The temperature correction used in this case is given by (Millero,
2007):

  ------------------------------------------------------------------------------------------------------------------------ ------------
  $$d(\text{ln}f\text{CO}_{2}\frac{)}{\text{dT}}(\text{deg}^{- 1}) = 0\text{.}\text{044} - (8 \times \text{10}^{- 5})S$$   \(7\)

  ------------------------------------------------------------------------------------------------------------------------ ------------

No correction was required for the MICA TCO~2~ measurements since TCO~2~
is expressed in µmol kg^-1^. The MICA pH measurements give pH values on
the total scale at 25 ˚C.

The auxiliary salinity, temperature and pressure data used for these
corrections and calculations were obtained from underway measurements
using shipboard instruments.

**3. Quality control**

Quality control analysis was performed in three steps. First, suspect
data were removed, as necessary, following reported cruise log
malfunctions (e.g. possible sample contamination and observed
malfunction of the thermostat) and spectrophotometric anomalies (e.g.,
sudden decrease of baseline light signal indicating entrapment of air
bubbles). Secondly, data points with deviations \> 3 standard deviations
(p \< 0.01) from the running average of the data were removed. Finally,
two of the three measured carbonate parameters (*f*CO~2~, TCO~2~, pH)
were used to calculate the third parameter, which was compared with
direct MICA measurements of the parameter. Suspect data were eliminated
based on this internal consistency check.

**4. Data Display**

Figures 1 and 2 show the longitudinal distribution of surface water
*f*CO~2~, TCO~2~, and pH obtained with the MICA underway system. For
comparison, pH~T~ data calculated from *f*CO~2~ and TCO~2~ measurements
(using the K~1~ and K~2~ constants of Mehrbach et al. (1973) refit by
Dickson and Millero (1987)) were also plotted.

![](media/image2.wmf){width="6.497222222222222in"
height="8.231944444444444in"}

Figure 1. USF underway measurements of *f*CO~2~ (a), TCO~2~ (b), and pH
(c) during the P16N Leg 1.

![](media/image3.wmf){width="6.497222222222222in"
height="8.231944444444444in"}

Figure 2. USF underway measurements of seawater *f*CO~2~ (a), TCO~2~
(b), and pH (c) during the P16N Leg 2.

References

Byrne, R. H.; Liu, X.; Kaltenbacher, E.; Sell, K. 2002.
Spectrophotometric measurement of total inorganic carbon in aqueous
solutions using a liquid core waveguide. Analytica Chimica Acta, 451,
221-229.

Clayton, T.D., and R.H. Byrne. 1993. Spectrophotometric seawater pH
measurements: Total hydrogen ion concentration scale calibration of
m-cresol purple and at-sea results. Deep-Sea Res., 40: 2315-2329.

Dickson, A.G. and F. Millero. 1987. A comparison of the equilibrium
constants for the dissolution of carbonic acid in seawater media.
Deep‑Sea Research, 34, 1733‑1743.

DOE Handbook. 1994. Handbook of Methods for the Analysis of the Various
Parameters of the Carbon Dioxide System in Sea Water. Version 2, A. G.
Dickson & C. Goyet, eds. ORNL/CDIAC-74

Mehrbach, C. Cuberson, C.H., Hawley, J.E. and Pytkowicz, R.M. 1973.
Measurement of the apparent dissociation constants of carbonic acid in
seawater at atmospheric pressure. Limnol. Oceanogr., 18, 897-907.

Wang, Z. A., Liu, X., Byrne, R. H., Wanninkhof, R. H., Bernstein, R. E.,
Kaltenbacher, E. A., Patten, J. 2007. Simultaneous spectrophotometric
flow-through measurements of pH, carbon dioxide fugacity, and total
inorganic carbon in seawater. Analytica Chimica Acta, 596: 23-36.

Yao, W.; Byrne, R. H. 2001. Spectrophotometric determination of
freshwater pH using bromocresol purple and phenol red. Environmental
Science and Technology, 35, 1197-1201.

Zhang, H.; Byrne, R. H. 1996. Spectrophotometric pH measurements of
surface seawater at in-situ conditions: absorbance and protonation
behavior of thymol blue. Mar. Chem., 52, 17-25.