LIMNOLOGY

and Limnol. Oceanogr.: Methods 14, 2016, 864–873

OCEANOGRAPHY: METHODS VC 2016 The Authors Limnology and Oceanography: Methods published by Wiley

Periodicals, Inc. on behalf of Association for the Sciences of Limnology and Oceanography
doi: 10.1002/lom3.10137

Spectrophotometric measurement of freshwater pH with purified

meta-cresol purple and phenol red
Chun-Ze Lai,1 Michael D. DeGrandpre,*1 Brandon D. Wasser,1 Taymee A. Brandon,1 Daniel S. Clucas,1
Emma J. Jaqueth,1 Zachary D. Benson,1 Cory M. Beatty,1 Reggie S. Spaulding2
1
Department of Chemistry and Biochemistry, University of Montana, Missoula, Montana
2
Sunburst Sensors, Missoula, Montana

Abstract
Impurities in indicator salts can significantly bias spectrophotometric pH determinations. In this work,
two purified sulfonephthalein indicators, meta-cresol purple (mCP) and phenol red (PR), were tested for anal-
ysis of freshwater pH on the free hydrogen ion concentration scale. These two purified indicators were char-
acterized for the first time under low ionic strength conditions, providing their molar absorption coefficients
and dissociation constants along with their temperature dependence from 8 8C to 30 8C. At 25 8C, the infinite
dilution constants (pKI o ) were determined to be 8.6606 and 8.0642 for mCP and PR, respectively. The accura-
cy and precision of the method, evaluated with a variety of buffers with known pH, were found to be
10.0014 pH units and 60.0022 pH units, respectively (n 5 30). The pH values of different freshwater samples
were also determined using both indicators. The mCP and PR results were all within 6 0.01 pH units of each
other with three out of seven pH differences within 6 0.001 pH units, indicating the high consistency
between these two indicator methods. The work presented here is the first parallel comparison with two puri-
fied indicators used to determine pH of the same freshwater samples.

Spectrophotometric pH has long been the preferred meth- other errors (Davison and Woof 1985; Stauffer 1990), and
od for pH analysis of seawater because of its innate reproduc- the inherent drift and the need to frequently calibrate pH
ibility and excellent precision (Byrne and Breland 1989; electrodes, limit the usefulness of pH electrode data for dis-
Clayton and Byrne 1993; DeGrandpre et al. 2014). Wide- cerning long-term trends and computing inorganic carbon
spread acceptance of the method was preceded by careful and other chemical speciation in freshwater systems (French
characterization of the optical and thermodynamic proper- et al. 2002; Martz et al. 2003; Abril et al. 2015).
ties of sulfonephthalein indicators in a seawater matrix The freshwater spectrophotometric pH method can enhance
(Robert-Baldo et al. 1985). There have been similar efforts to the utility of pH data for many different applications but fur-
characterize these indicators for freshwater analysis (Yao and ther validation is needed. In Yao and Byrne (2001), the pKI o s
Byrne 2001; French et al. 2002; Yuan and DeGrandpre 2008) for bromocresol purple and phenol red (PR) were determined at
and for other low ionic strength solutions (Yamazaki et al. infinite dilution and excellent precision was obtained for river
1992; Raghuraman et al. 2006). However, most freshwater water (60.001 pH units) but there was no assessment of accura-
studies and monitoring programs continue to rely on glass cy. French et al. (2002) measured the pH of dilute phosphate
pH electrodes. For example, the U.S. Geological Survey still buffers and river water with cresol red after determining the
uses glass electrodes for their extensive water quality moni- equilibrium constant. The measurements compared to within
toring program (Butman and Raymond 2011) and many lim- 0.003 6 0.008 pH units of the buffer pH. Yuan and DeGrandpre
nological research programs use pH electrode measurements (2008) evaluated the dependence of buffer intensity on the pH
(Talling 2010; Wallin et al. 2010; Nimick et al. 2011). While perturbation of freshwater using cresol red and bromothymol
these measurements provide long-term methodological con- blue. None of these studies have compared different indicators
sistency, the uncertainty in pH due to liquid junction and for analysis of the same sample. This comparison could provide
valuable insights into the accuracy of the method. Additional-
*Correspondence: [email protected] ly, none of the previous freshwater work has utilized purified
sulfonephthalein indicators. Indicator impurities have been
This is an open access article under the terms of the Creative Commons
Attribution License, which permits use, distribution and reproduction in found to cause significant seawater pH errors (Yao et al. 2007;
any medium, provided the original work is properly cited. Liu et al. 2011; Patsavas et al. 2013), and have led to use of

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Lai et al. Freshwater pH with purified indicators

purified indicators for seawater pH analysis (Liu et al. 2011; A50:50921ðT2298:15Þ38:531024 (6)
DeGrandpre et al. 2014). It is unknown to what extent the
impurities could affect freshwater pH accuracy, but it is worth- where T is the temperature in Kelvin. Equation 5 can be
while to obtain accurate “impurity-free” thermodynamic and used for ionic strengths  0.05 M.
optical properties of the indicators for future applications.
In this work, we have purified meta-cresol purple (mCP) Materials and procedures
and PR and have quantified their molar absorption coeffi- Materials
cients and pKI o at low ionic strengths. The indicator mCP Indicators mCP (Lot No. 11517KCV, 90 wt%) and PR (Lot
was selected because we have purified mCP available for our No. 13912PS, 95 wt%) were purchased from Sigma-Aldrich
seawater applications; whereas, PR was selected because its (St. Louis, MO) and purified before use (see below). The buf-
pKI o is optimal for freshwater analysis and is close enough to fers used in this work (CH3COOH, Na2HPO4, KH2PO4) were
the pKI o of mCP that comparisons can be made. Here, the analytical grade and obtained from Fluka (Buchs, Switzer-
two indicators are used to analyze a wide range of low ionic land) and Mallinckrodt Baker (Paris, KY). Certified standard
strength samples, including weak buffers and a variety of solutions of 0.1 N HCl and 0.1 N NaOH were purchased
natural water samples. from Fisher Scientific (Pittsburgh, PA). Acetonitrile (HPLC
grade) was obtained from Fisher Scientific and trifluoroacetic
Theory acid was obtained from Sigma-Aldrich. Nanopure water (17.9
The spectrophotometric pH method, which is used in this MXcm specific resistance), obtained with a Barnstead water
work, is based on the equilibrium of a weak diprotic acid purification system (Thermo Scientific, Waltham, MA), was
indicator: used for all solutions.
The mCP and PR stock solutions used for pH analyses had
HI2 $ H1 1I22 (1) concentrations of 9.25 3 1024 molkg soln21 and 1.25 3
1023 molkg soln21, respectively. To minimize the perturba-
where HI2 is the protonated (acid) form and I22 is the tion of the sample pH, each of the indicator solutions was
deprotonated (base) form. The second dissociation constant adjusted to a pH of 7.5 with a 0.1 N HCl or 0.1 N NaOH
of the indicator is solution while being monitored with an Orion combination
½H1 ½I22  cH1 cI22 pH electrode connected to a dual channel pH/ion meter
KI 5  (2) (AccumetTM, model AR25, Fisher Scientific).
½HI2  cHI2
Phosphate buffer salts Na2HPO4 and KH2PO4 were dried
where c denotes activity coefficient. The pH, determined on at 105 8C for 2 h before weighing. The solutions were pre-
the free hydrogen ion concentration scale, is expressed as: pared using Nanopure water that was degassed by boiling
  under vacuum. The acid component of the buffer (KH2PO4)
R2e1 c 1 c 22 was first added to water with approximately half total sol-
pH52log ½H1 5pKIo 1log 1log H I (3)
e2 2Re3 cHI2 vent volume to prevent CO2 contamination at dissolution.
The base component (Na2HPO4) was then added and the
where R is the ratio of indicator absorbances at the absor- flask was filled to the final volume. Buffer solutions were
bance maxima of I22 and HI2 . For mCP, k1 5 434 nm and purged with N2 gas in the head-space when transferred to a
k2 5 578 nm, and for PR, k1 5 433 nm and k2 5 558 nm for Nalgene bottle for storage.
HI2 and I22 , respectively. pKI o is the negative logarithm of
the second dissociation constant of the indicator at zero ion- Purification of mCP and PR
ic strength. The term ðR2e1 Þ=ðe2 2Re3 Þ is equal to the ratio Indicators mCP and PR were purified with a flash chroma-
R

[I22 ]/[HI2 ], where e1, e2, and e3 refer to molar absorption tography system (CombiFlashV Rf, Teledyne ISCO, Lincoln,
coefficient ratios corresponding to HI2 or I22 at k1 or k2: NE) (Liu et al. 2011; Patsavas et al. 2013). The reversed phase
C-18 column (C18Aq, Teledyne ISCO) was loaded with 1 g
eHI;k2 eI;k2 eI;k1
e1 5 e2 5 e3 5 (4) mCP or PR dissolved in a 100 mL of solvent containing
eHI;k1 eHI;k1 eHI;k1
water, acetonitrile and trifluoroacetic acid (95.0 : 5.0 : 0.05,
v : v : v). The purified indicators were eluted during a gradi-
After the determination of eis and pKI o (described below),
ent that increased the acetonitrile from 5% to 40% for mCP
the pH on the free hydrogen ion concentration scale can be
or to 45% for PR. The eluent was captured in sequential test
obtained using:
tubes and then combined and dried at room temperature in
   pffiffiffi 
R2e1 l a stoppered flask with ultrapure air blown onto the solution.
pH5pKIo 1log 24A pffiffiffi 20:3l (5)
e2 2Re3 11 l The air-dried sample was then dried at 35 8C and 15 mm Hg
vacuum for 2 h. The typical yield was 50–60% for both mCP
where l is the ionic strength and and PR. After purification, based on the change in the ratio

865
Lai et al. Freshwater pH with purified indicators

We typically add 40 lL of stock indicator solution for each

of three additions, corresponding to an indicator concentra-
tion range from 1.2 3 1026 molkg soln21 to 3.7 3 1026
molkg soln21 for mCP and 1.6 3 1026 molkg soln21 to 5.0
3 1026 molkg soln21 for PR and an absorbance range from
0.5 to 1.5 a.u. The total concentration of indicator in the
sample solution is calculated and plotted along with the cal-
culated pH (Fig. 1). The y-intercept, where no indicator is
present, is assumed to be the true pH, or perturbation-free
pH, for the sample (French et al. 2002). The SAMI-pH sensor
technology uses an automated form of this methodology
(Martz et al. 2003; Seidel et al. 2008).

Fig. 1. Effect of total indicator concentration (䉬 for mCP and 䉱 for PR) Indicator characterization
on the measured pH. Data shown in the figure were collected by For mCP, an acetic acid solution with pH 4.4 was used to
sequential addition of indicator to a Clark Fork River sample. The y-inter- determine the molar absorption coefficient of HI2 at 434 nm
cept is assumed to be the true sample pH where no indicator is present.
and 578 nm. At this pH, the presence of the other two forms
(H2I) and (I22 ) is minimized (their concentrations are about
of peak areas of the contaminants to the peak area of the 104 times lower than ½HI2 ). Higher or lower pH would
indicator, 96.4% of contaminants were removed from mCP, increase the absorbance of the other two forms, causing inac-
resulting in a purity of 99.7%, and 86.0% of contaminants curate determination of molar absorption coefficient of ½HI2 :
were removed from PR, resulting in a purity of 99.3%. For PR, similarly, an acetic acid solution with pH 4.4 was used
Spectrophotometric measurements to determine the molar absorption coefficient of HI2 at
All spectrophotometric measurements were carried out on 433 nm and 558 nm. A 0.01 M NaOH solution with pH 12 was
a benchtop spectrophotometer (Cary 300, Varian) with a used to determine the molar absorption coefficients of I22 for
10 cm path length, capped, quartz cuvette. The performance mCP and PR at 434 nm and 578 nm, and 433 nm and 558 nm,
of the spectrophotometer was routinely checked with wave- respectively. The molar absorption coefficients were measured
length and absorbance standards (DeGrandpre et al. 2014). at four temperatures, 8, 17, 22 and 30 8C. The molar absorp-
The solution temperature was controlled with a water- tion coefficients at these ionic strengths (0.010 M) are not
jacketed spectrophotometric cell holder. The actual tempera- expected to differ significantly from ionic strengths over the
ture of the indicator solution in the cuvette was measured freshwater range because others have shown the salinity
by a high accuracy temperature probe (Eutechnics, 15-060- dependence is weak (DeGrandpre et al. 2014). It should also
381, Fisher Scientific), immersed in the sample via a hole be noted that, while being stored in the dark, the absorbance
drilled in the cell stopper. Before measurement, sample bot- intensity of mCP at pH 12 degraded by 0.0005 a.u./d for a
tles were placed in a water bath set to the approximate mea- 10 cm cell. Therefore, we prepared fresh pH 12 indicator solu-
surement temperature. After 20 min temperature tions to minimize the error in measurements if the experi-
equilibration, the cuvette, containing a stir bar, was filled ments spanned more than 1 d.
with sample, capped, wiped dry, and placed in the spectro- As shown in Eq. 3, the pH measurement accuracy directly
photometer. A magnetic stirring wand was mounted to the depends on the accuracy of pKI o . However, pKI o values for
upper wall of the cell holder to mix the indicator without purified mCP and PR under low ionic strength conditions
removing the cell. Light absorption was measured at the have not been reported. Therefore, pKI o and its temperature
absorbance maxima and at 780 nm (to account for offsets dependence were determined in this work by adding indica-
between the blank and sample). Absorbance offsets at tor to phosphate buffers with known pH. The pKI o was then
calculated using the following equation (Yao and Byrne,
780 nm were typically < 0.002 absorbance units (a.u.).
2001):
As stated in the introduction section, indicators them-
 
selves are weak acids or bases. A pH change of < 0.005 pH ½H2 PO4 2  R2e1
pKIo 5pK2o 2log 2log (7)
units due to addition of the indicator is typical for seawater ½HPO4 22  e2 2Re3
samples (Chierici et al. 1999). Because freshwaters usually
have lower buffer intensity, the effect of the indicator on the where pK2o is the negative logarithm of second dissociation
pH can be more pronounced (Yuan and DeGrandpre 2008). constant of the phosphate buffer (Bates and Acree 1943).
Consequently, for freshwater analyses, the pH perturbation The actual concentrations of H2 PO4 2 and HPO4 22 for use in
should be determined for every sample to obtain the best Eq. 7 were calculated using an in-house program written in
accuracy. To determine the perturbation-free pH value, a Visual Basic in Excel. Since the typical freshwater ionic
solution of mCP or PR is sequentially added to the sample. strength is between 0 and 0.005 M (Domenico and Schwartz

866
Lai et al. Freshwater pH with purified indicators

Fig. 2. Temperature dependence of the molar absorption coefficients measured using purified (a) mCP and (b) PR at the wavelengths of maximum
absorption for each chemical species.

1990; Pankow 1991), the ionic strengths of diluted phos- avoid the change of pH due to CO2 exchange in the proce-
phate buffer used for the pKI o determination were dure. Freshwater samples were tested back to back, e.g., one
all  0.010 M. sample with mCP followed by one sample with PR, or in the
opposite order. Measurements were carried out on
pH accuracy evaluation temperature-equilibrated samples with three sequential addi-
The accuracy of the pH determined with mCP and PR was
tions of 40 lL of indicator stock solution as described above
evaluated by measuring the pH of a series of phosphate buf-
(see Fig. 1). All freshwater samples were measured at 25 8C.
fers. The actual pH values of these buffers were known
through the calculation using the in-house equilibrium pro-
Assessment
gram that includes the temperature dependence of pKw (the
negative logarithm of the water ion product) (Millero 2007) Molar absorption coefficients and ratios
With the successful purification of indicators in this work,
and pK2 o of the phosphate buffer. The pH values were mea-
the molar absorption coefficients for mCP and PR could be
sured over a wide temperature range (8–30 8C). Ionic
accurately determined at low ionic strength for the first
strengths for all phosphate buffers were approximately
time. Figure 2 shows the temperature dependence of the
0.010 M. An independent accuracy evaluation was also car-
molar absorption coefficients at the wavelengths of maxi-
ried out by determining pH values of a borate buffer (ionic
mum absorption for both mCP and PR. The molar absorp-
strength was 0.010 M) repeatedly with both mCP and PR.
tion coefficients decrease with increasing temperature at the
pH of freshwater samples absorbance maxima for acid and base forms for both mCP
Different freshwater samples, including tap water, and and PR. Conversely, for the molar absorption coefficients
samples collected during several field visits to local streams that are not at the absorbance maxima (i.e., eHI2 ;578 and
including Rattlesnake Creek, Blackfoot River, Clark Fork eI22 ;434 for mCP, and eHI2 ;558 and eI22 ;433 for PR), the molar
River near Gold Creek (labeled as Gold Creek) and near Mis- absorption coefficients increase with increasing temperature
soula (labeled as Missoula), were also analyzed with both for both mCP and PR. These trends have been observed
indicators. Samples were kept in the dark to limit any possi- before and are due to both a slight spectral shift to longer
ble alteration of the sample and were typically analyzed wavelengths in the I22 peaks and to shorter wavelengths for
within 4 h. Samples were used directly without filtering to the HI2 peaks (Harris 2013).

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Lai et al. Freshwater pH with purified indicators

Table 1. Molar absorption coefficients for mCP and PR and their temperature dependence at the analytical wavelengths.

Indicator Fitted equation* r2 Residual e at 25 8C (M21 cm21)†

mCP eHI2 ;434 5 24.6960 3 1023T2 2 3.0214 3 101T 1 2.7092 3 104 0.99904 0.00 6 9.3 17,666
eHI2 ;578 5 21.1158 3 1024T2 1 4.1248 3 1021T 2 6.7050 0.99986 0.0 6 3.6 3 1022 106
eI22 ;434 5 26.7456 3 1022T2 1 4.6026 3 101T 2 5.5793 3 103 1.0000 1.7 3 102168.7 3 1022 2147.0
eI22 ;578 5 1.5877T2 2 1.0392 3 103T 1 2.0937 3 105 0.99484 1.4 3 10167.4 3 101 40,669
PR eHI2 ;433 5 3.4018 3 1021T2 2 2.3446 3 102T 1 6.2560 3 104 0.99762 0.0 6 1.6 3 101 22,896
23 2 2
eHI2 ;558 5 4.1299 3 10 T 2 1.2731T 1 1.0929 3 10 0.99999 1.1 3 102263.4 3 1022 96.836
eI22 ;433 5 23.5570 3 1022T2 1 2.7201 3 101T 2 2.6943 3 103 0.99996 8.9 3 102263.5 3 1021 2253.7
eI22 ;558 5 2.3897T2 2 1.5575 3 103T 1 3.1796 3 105 0.99997 21.3 3 10167.4 66,020
*The temperature is in Kelvin.
†
M21 cm21 5 100 M21 m21.

The fitted equations for data shown in Fig. 2 are listed in method over a range of temperatures. As shown in Fig. 4,
Table 1 along with the r2, mean residual and standard devia- the pH values with the purified indicators match well with
tion. In addition, molar absorption coefficients for both the calculated pH of the phosphate buffers in the tempera-
mCP and PR at 25 8C are listed as examples. The molar ture range 8–30 8C. The pH values only slightly deviate from
absorption coefficient ratios, eis, for mCP and PR were then the 1 : 1 line. For mCP, the average absolute residual is
calculated according to Eq. 4 using the actual data. Because 0.0016 6 0.0009 pH units (n 5 12) and the maximum residual
it is critical to account for the ei temperature dependence is 0.0026 pH units, and for PR, the average absolute residual
(DeGrandpre et al. 2014), the ei temperature dependence is 0.0016 6 0.0013 pH units (n 5 9) and the maximum residu-
along with the r2 and mean residuals are shown in Table 2. al is 0.0040 pH units. These small pH errors confirm the
The ei values for mCP and PR at 25 8C are also listed as high accuracy of the spectrophotometric method with puri-
examples. fied mCP and PR under low ionic strength conditions. Nota-
bly, mCP performed as well as PR even though its pKI o is
Indicator pKI o
considered “out of range,” i.e., the pH of some of the sam-
Figure 3 shows the results of the pKI o determination using
ples were not within the pKI o 6 1 range. In this case, the
the phosphate buffers and includes the best fit curves and
molar absorption coefficient of the base form of mCP is
residuals. The temperature dependence of pKI o for both mCP
about twice of that of acid form (Table 1); therefore, there is
and PR exhibits the same trend of decreasing pKI o with
still sufficient absorbance to obtain good accuracy and preci-
increasing temperature. This relationship is due to endother-
sion at the low pHs (Fig. 4). It is also evident from Fig. 4
mic acid dissociation reactions that are more favorable at
that most pH values with both mCP and PR are slightly
higher temperature. Both mCP and PR data are accurately fit-
higher than the actual pH value. There are also systematic
ted with an equation of the form pKIo 5A1BT1 TC 1Dln T.
deviations at the extreme ranges of temperature and pH for
The mean residuals are indiscernible from zero (Table 3)
the PR analyses (Fig. 4 middle and right panels). The reasons
with no clear trend with temperature (Fig. 3). The pKI o s for
for these small positive errors are not clear.
unpurified PR calculated using the equations in Yao and
These tests also show that the spectrophotometric pH
Byrne (2001) follow the same trend but are lower than the
method has very high precision. The average standard devia-
corresponding pKI o s for purified PR. Table 3 lists the pKI o
tion for all pH values at different temperatures is 6 0.0006
equations for purified mCP and PR between 8 8C and 30 8C.
for both mCP and PR (n 5 21). Furthermore, five additional
With these equations, pKI o at 25 8C is 8.6606 for mCP and
buffer measurements with mCP were carried out at 25.19 6
8.0642 for PR, respectively. For comparison, pKI o at 25 8C for
0.08 8C and the standard deviation is 6 0.0008.
unpurified PR at zero ionic strength is 8.0341 (Yao and Byrne
The accuracy of pH measurements with purified mCP and
2001). The lower pKI o for unpurified PR could be the result
PR was more directly assessed by measuring phosphate buf-
of several different factors; for example, the impurities in PR
fers with both pH (ranging from 7.520 to 8.131) and ionic
could deprotonate at a lower pH or they could bias the
strength (ranging from 0.0050 M to 0.0100 M) that differed
determination of the ei values and therefore the pKI o deter-
from that used to determine pKI o (Table 4). The larger uncer-
mined using Eq. 7.
tainties were not due to the spectrophotometric pH measure-
Accuracy evaluations ment because mCP and PR matched to within
After determining the pKI o s, we used a phosphate buffer 20.0009 6 0.0082 pH units for these samples. These results
with pH different from that used to determine the pKI o but support the findings of Yuan and DeGrandpre (2008) that
with 0.010 M ionic strength to evaluate the accuracy of the accuracy and precision degrade with lower buffer intensity;

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Lai et al. Freshwater pH with purified indicators

Table 2. Molar absorptivity ratios (eis) (Eq. 4) and their temperature dependence.

Fitted equation* r2 Residual e at 25 8C

mCP
e1 e1 5 1.5036 3 1027T2 2 5.8331 3 1025T 1 1.0044 3 1022 0.99968 0.0 6 0.0 0.006
e2 e2 5 3.1400 3 1025T2 2 2.0527 3 1022T 1 5.6349 1.0000 23.2 3 102366.7 3 1023 2.306
e3 e3 5 22.8764 3 1026T2 1 2.2697 3 1023T 2 2.9948 3 1021 0.99982 21.4 3 102567.2 3 1025 0.1215
PR
e1 e1 5 2.6445 3 1027T2 2 9.9420 3 1025T 1 1.0361 3 1022 1.0000 0.0 6 0.0 0.004
e2 e2 5 6.2236 3 1025T2 2 3.8762 3 1022T 1 8.9077 0.99225 0.0 6 1.9 3 1023 2.883
e3 e3 5 23.0956 3 1026T2 1 2.2264 3 1023T 2 2.9019 3 1021 0.99992 26.2 3 102663.4 3 1025 0.0984
*The temperature is in Kelvin.

Fig. 3. Temperature dependence of the dissociation constants for purified mCP (top) and PR (bottom) along with residuals (fitted pKI o – measured
pKI o ) of the fit (right). For comparison, the pKI o s for unpurified PR predicted with the equation by Yao and Byrne (2001) are also shown (䉱).

Table 3. Temperature dependence of pKI o :

Indicator Fitted equation for pKI o * r2 Residual pKI o at 25 8C

mCP pKI o 5 25:02423104
T 2 3:54333102 lnT 1 6:140931021 T 1 2:01293103 0.99993 0.0 6 5.4 3 1024 8.6606
o 2 21 3
PR pKI 5 23:53263104
T 2 2:5305310 lnT 1 4:4364310 T 1 1:4360310 0.99997 0.0 6 2.7 3 1024 8.0642
*The temperature is in Kelvin.

however, the results were also not as good as previously error of 0.0019 pH units, and 8.3181 6 0.0054 (n 5 4) at an
obtained with the 0.010 M phosphate buffer. This larger average temperature of 25.02 8C (actual pH 5 8.3170) by PR
uncertainty might be due to techniques used by different with an error of 0.0011 pH units. These two pH values only
analysts and reflect the challenge in reproducible prepara- differ by 0.0008 pH units after correction for temperature,
tion of the weak buffers. indicating the high consistency of measurements between
Finally, as an additional accuracy assessment, a borate purified mCP and PR.
buffer with known pH (ionic strength 5 0.010 M) (Bower and
Bates 1955) was repeatedly analyzed. The borate pH was Determination of pH for different freshwater samples
determined to be 8.3182 6 0.0019 (n 5 5) at an average tem- The purified mCP and PR were used to determine the pH
perature of 25.10 8C (actual pH 5 8.3163) by mCP with an for different freshwater samples. The comparison of pH

869
Lai et al. Freshwater pH with purified indicators

Fig. 4. pH of 0.010 M ionic strength phosphate buffers measured with purified mCP and PR (left panels, the dashed lines are the 1 : 1 relationships)
and the calculated pH errors (measured pH – actual pH) for phosphate buffers at different temperatures (middle panels), and different buffer pH (right
panels). The error bars indicate the standard deviation for the measured pH obtained from at least three replicates at the same temperature.

Table 4. Accuracy and precision for measurements of phos- indicates that there is no systematic deviation with pH. The
phate buffers with varied ionic strengths and pHs. match between mCP and PR also implies that the two indi-
cators have very similar ionic interactions in different ionic
Measured Measured media, i.e., the activity coefficients are the same for both
with mCP* with PR* indicators. To our knowledge, this is the first reported paral-
Ionic strength
(M) pH error SD pH error SD lel comparison with two purified indicators of different types
for the same freshwater samples.
0.0100 (n 5 16) 20.0038 0.0074 20.0101 0.0067
It also should be noted that the pH values for the fresh-
0.0076 (n 5 16) 20.0243 0.0073 20.0235 0.0075
water samples do not include the activity coefficient contri-
0.0050 (n 5 12) 20.0173 0.0280 20.0132 0.0268
bution (see Eq. 5) due to the unknown sample ionic
*Data shown are based on four different pHs for 0.0100 M and strength. We can, however, estimate the uncertainty due to
0.0076 M ionic strengths, and three different pHs for 0.0050 M ionic
this missing information. As reported previously (Yuan and
strength.
DeGrandpre 2008), the mean ionic strength for Rattlesnake
Creek was approximately 9 3 1024 M and therefore would
values for the two indicators is shown in Fig. 5 (top). Clearly, shift the pH by 20.0588 pH units. The ionic strength for the
the determinations with purified mCP and PR provide highly Clark Fork River was estimated to be 5.3 3 1023 to 8.8 3
comparable pH values for a wide range of sample types and 1023 M and would result in a shift of pH by 20.1350 to
pHs. To better visualize this, the pH differences are also 20.1693 pH units (Lynch 2007). Because this correction is
shown in Fig. 5 (bottom). Their pH differences are all the same for mCP and PR, the resultant pH is the same. A 6
within 6 0.01 pH units. For three out of seven of the fresh- 20% error in determination of ionic strength could result in
water samples, the pH differences are within 6 0.001 pH 20.0056 to 0.0063 pH unit error for a low ionic strength
units. The precision for the measurement in freshwater sam- environment (0.001 M) and 20.0113 to 0.0127 for a high
ples ranges from 6 0.0023 to 6 0.0162 pH units and 6 0.0033 ionic strength environment (0.005 M). To accurately deter-
to 6 0.0129 pH units for mCP and PR, respectively, and the mine the pH value of a freshwater sample, ionic strength
mean standard deviation is 6 0.0082 pH units. Figure 5 also should be quantified. The measurement of conductivity, e.g.,

870
Lai et al. Freshwater pH with purified indicators

in Fig. 1 and explained above, it is important to determine

the perturbation-free pH for freshwater samples. For exam-
ple, the pH for the Clark Fork River sample as determined by
using mCP and PR is 8.1887 and 8.1858, respectively (Fig. 1).
Under this specific circumstance, the fitting of the data finds
that the perturbation of pH due to the addition of mCP or
PR is 21.58 3 1023 pH/(lmolkg21) and 21.06 3 1023 pH/
(lmolkg21), respectively. Therefore, theoretically, with the
addition of 2.0 lmolkg21 mCP or PR to this freshwater sam-
ple, the perturbations would be 20.0032 and 20.0021 pH
units, respectively, for a 10 cm cell. When the experimental
condition changes, including the pH of the sample, the buff-
er intensity of the sample, the pH of the indicator and the
concentration of the indicator solution (e.g., 10X increase if
using an optical pathlength of 1 cm), the extent of perturba-
tion will also be changed. Consequently, the perturbation
should be quantified for each analysis if accuracy better than
0.01 pH units is desired.
Future studies should include further validation of the
equations in Tables 1, 2 with standard reference materials
(e.g., those available from the U.S. National Institute of
Standards and Technology). Spectrophotometric pH should
also be combined with dissolved inorganic carbon or total
alkalinity measurements on freshwater samples to verify that
the partial pressure of CO2 (pCO2) can be accurately pre-
dicted using spectrophotometric pH. These studies are
important because pCO2 is commonly calculated using pH
(from glass electrodes) and alkalinity (by titration) (Lynch
et al. 2010; Butman and Raymond 2011; Finlay et al. 2015).
These studies will establish the internal consistency of the
spectrophotometric pH method like those completed for sea-
water pH (e.g., Clayton et al. 1995). Extension of the charac-
terization of the purified indicators to estuarine conditions
Fig. 5. Comparison of pH measured with both mCP and PR (top) and should also be conducted.
calculated pH differences for these data (bottom) for different samples.
It is also important to note that, being an optical mea-
surement, the optical transparency of the sample is impor-
using a conductivity meter such as Orion STAR Conductivity tant. The freshwater samples tested in this work were not
Meter (Thermo Scientific), is a rapid and inexpensive way of filtered to avoid the change of pH due to CO2 exchange. If
determining the ionic strength of a freshwater sample necessary, samples could be filtered using a syringe with
(Richards 1954) using the linear relationship between ionic zero head space. The effect of suspended particulates or col-
strength and electrical conductivity (Griffin and Jurinak ored dissolved organic matter on measurements needs to
1973). be more systematically studied in the future. In addition,
the spectrophotometric method with purified indicators,
Discussions, implications, and recommendations such as mCP and PR in the present work, can be used for
The characterization of the purified indicators, mCP and direct calibration of glass pH electrodes in freshwater and
PR, are reported under low ionic strength conditions for the evaluation of the electrode performance (Easley and Byrne
first time. The spectrophotometric pH measurements with 2012).
purified mCP and PR have high accuracy and precision based While freshwater pH is still primarily measured with pH
on analysis of phosphate and borate buffers with known pH. electrodes due to their ease of use and low cost, the inaccu-
Furthermore, purified mCP and PR have been successfully racy of these measurements reduces the pH’s usefulness for
used for measurement of different freshwater samples. The geochemical modeling, especially for predicting inorganic
molar absorption coefficients and pKI o s can therefore be carbon speciation. This work lays the foundation for future
used in future studies to accurately measure pH if purified studies using spectrophotometric pH for long-term measure-
indicator is available and ionic strength is known. As shown ments of freshwater systems and combining the pH with

871
Lai et al. Freshwater pH with purified indicators

dissolved inorganic carbon or total alkalinity measurements DeGrandpre, M. D., R. S. Spaulding, J. O. Newton, E. J.
on freshwater samples to accurately predict the inorganic Jaqueth, S. E. Hamblock, A. A. Umansky, and K. E. Harris.
carbon speciation. Although the spectrophotometric method 2014. Consideration for the measurement of spectropho-
may be more time-consuming and less portable, it is more tometric pH for ocean acidification and other studies.
reliable because the measurement is based on well-defined Limnol. Oceanogr.: Methods 12: 830–839. doi:10.4319/
thermodynamic principles. Moreover, the challenge in sam- lom.2014.12.830
ple handling, i.e., the sample needs to be put into a small Domenico, P. A., and F. W. Schwartz. 1990. Physical and
cuvette where CO2 exchange is more possible and tempera- chemical hydrogeology. Wiley.
ture is difficult to measure and control, can be minimized if Easley, R. A., and R. H. Byrne. 2012. Spectrophotometric cali-
it is done carefully as demonstrated in this work. In addi- bration of pH electrodes in seawater using purified m-
tion, the spectrophotometric method can be made autono- cresol purple. Environ. Sci. Technol. 46: 5018–5024. doi:
mous, as shown with the SAMI-pH (Martz et al. 2003; 10.1021/es300491s
Spaulding et al. 2014), providing drift free, accurate, long- Finlay, K., R. J. Vogt, M. J. Bogard, B. Wissel, B. M. Tutolo,
term monitoring for freshwater systems. G. L. Simpson, and P. R. Leavitt. 2015. Reduction in CO2
efflux from northern hardwater lakes with increasing
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Acknowledgments
2007.11.013
This manuscript is presented in memory of Zachary Benson, a B.S.
Spaulding, R. S., M. D. DeGrandpre, J. C. Beck, R. D. Hart, B. chemistry graduate of UM, who worked on this project as an undergrad-
Peterson, E. H. De Carlo, P. S. Drupp, and T. R. Hammar. uate in its early stages. Zach passed away from complications due to
2014. Autonomous in situ measurements of seawater alka- leukemia in September 2008. Financial support was provided by the
linity. Environ. Sci. Technol. 48: 9573–9581. doi:10.1021/ Montana University System Research Initiative (contract 51030-
MUSRI2015-02), the M.J. Murdock Charitable Trust and the Montana
es501615x Board of Research and Commercialization Technology. Undergraduate
Stauffer, R. E. 1990. Electrode pH error, seasonal epilimnetic researchers (BW, TB, DC and EJ) were supported by the U.S. National
pCO2, and the recent acidification of the Maine lakes. Water Science Foundation under grants ARC-1107346 and OCE-1459255.
Air Soil Pollut. 50: 123–148. doi:10.1007/BF00284788
Talling, J. F. 2010. pH, the CO2 system and freshwater sci- Conflict of Interest
None declared.
ence. Freshw. Rev. 3: 133–146. doi:10.1608/FRJ-3.2.156
€
Wallin, M., I. Buffam, M. Oquist, H. Laudon, and K. Bishop.
Submitted 14 June 2016
2010. Temporal and spatial variability of dissolved inor-
Revised 9 August 2016
ganic carbon in a boreal stream network: Concentrations
Accepted 10 August 2016
and downstream fluxes. J. Geophys. Res. 115: G02014.
doi:10.1029/2009jg001100 Associate editor: John Smol

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