Analytica Chimica Acta 448 (2001) 239–250

Application of modified Gran functions and derivative methods to

potentiometric acid titration studies of the distribution of inorganic
carbon species in cultivation medium of marine microalgae
Marilda Rigobello-Masini a , Jorge C. Masini b,∗
a Instituto de Biociências, Universidade de São Paulo, C.P. 11461, 05422-970 São Paulo, Brazil
b Instituto de Quı́mica, Universidade de São Paulo, C.P. 26077, 05513-970 São Paulo, Brazil
Received 1 March 2001; received in revised form 13 July 2001; accepted 30 July 2001

Abstract
This paper presents an evaluation of the modified Gran functions (MGFs) and derivative methods for assessing the concen-
trations of inorganic carbon species, CO2 , HCO3 − and CO3 2− , in the cultivation medium of the marine microalgae Tetraselmis
gracilis. Both methods were applied to simulated and experimental potentiometric titration data and were able to detect small
variations of inorganic carbon species during 10 days of batch cultivation. The MGF were able to determine the concentration
of an additional titratable species with pKb values between 9.5 and 9.9 that appears in the medium after the 4th day of cultiva-
tion. This species may be assigned to carboxylate groups of organic carbon compounds excreted by the algae. Treatment of
the titration data with the derivative method did not permit the quantitative determination of the concentration of the additional
titratable species, but no interference was observed in the results obtained for the inorganic carbon species. © 2001 Elsevier
Science B.V. All rights reserved.
Keywords: Titrimetry; Alkalinity; Carbon assimilation; Algae cultivation; Speciation

1. Introduction of communities in marine ecosystem. Accurate mea-

Due to the greenhouse effect, nowadays there is a
great interest in studying the carbon biogeochemical
cycle on Earth and how it is influenced by the anthro-
pogenic emissions of CO2 . About 40% of the carbon
fixed from the atmosphere is taken by the biologi-
cal ocean pump, through microalgal photosynthetic
processes [1]. Different phytoplanktonic species may
behave in a diverse way through variations in in-
organic carbon levels, leading to different growth
rates; this can bring consequences for the structure
∗ Corresponding author. Fax: +55-11-3818-5579.
E-mail address: jcmasini@quim.iq.usp.br (J.C. Masini).
surements of the concentrations of inorganic carbon
species in marine or other aquatic environments can
give support for a better understanding of these pro-
cesses, especially at the enzymologycal level. Rubisco
is the enzyme responsible for the carboxylation re-
action in the dark part of the photosynthesis, using
CO2 as substrate, although at the pH of seawater,
bicarbonate is the major carbon species present. Car-
bonic anhydrase is an enzyme that, when present
at the exofacial cell wall in the algae, promotes the
acquisition of inorganic carbon as bicarbonate ions.
Internally, in the cytoplasm or in the organelles,
especially the chloroplast, Carbonic anhydrase can
catalyze the bicarbonate dehydration, releasing it as

0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 0 3 - 2 6 7 0 ( 0 1 ) 0 1 3 1 2 - 5
240 M. Rigobello-Masini, J.C. Masini / Analytica Chimica Acta 448 (2001) 239–250
CO2 that can be concentrated around Rubisco’s active
site [2].
The determination of inorganic carbon species con-
centrations in sea, or estuary waters, as well as in cell
cultivation media, is possible with the measurement of
the pH and the total CO2 concentration [3–7]. These
two parameters allow the computation of the distri-
bution of inorganic carbon species from the thermo-
dynamic dissociation constants of carbonic acid. The
total CO2 concentration is usually determined by acid
titration using a variety of approaches to detect the sto-
ichiometric points [5]. Many data have been obtained
by potentiometric Gran titrations [8–10], which allow
the graphic determination of the end points due to
the carbonate and bicarbonate species, permitting the
computation of the total CO2 concentration. The accu-
racy and precision of these methodologies are limited
by the presence of titratable species other than those
involved in the carbonate system, such as borate and
silicate ions, or carboxylic and phenolic groups from
natural organic matter [4,6,7]. The use of the classical
Gran methodology to determine the total CO2 concen-
tration in seawaters has lead to systematically positive
errors, mainly in coastal waters, a fact that has been
assigned to the presence of naturally occurring organic
compounds (fulvic and humic acids) [4,7]. The devel-
opment of accurate methods for treatment of data ob-
tained in potentiometric titration of mixtures of acids
has been described in the literature [11–23], so that it is
possible to determine the concentration of acidic com-
ponents with pKa values differing by only one, or even
less [23]. In addition, simultaneous determinations of
concentration and ionization constants of the mixture
components have also been described [11–23]. These
methods are based on linearization by modified Gran
plots [11,15,21,22], or other linear equations including
Scatchard, Scott, Hofstee and Lineweaver-Burk plots
[18,19], and on methods based on multiple sample
addition [16,17]. Nonlinear regression methods have
also been described for this purpose [20–23].
Recently, Hernández-Ayón et al. [4] described a
simple and fast potentiometric titration method with a
difference derivative readout for determination of total
CO2 concentration in sea and coastal waters, over-
coming the systematic positive error observed with
the classical Gran methodology. This paper presents
the application and evaluation of the modified Gran
functions (MGFs) and the derivative method for
determination of the daily distribution of carbon
species during 10 days of batch cultivation of the
marine algae Tetraselmis gracilis.
2. Experimental
2.1. Cultivation and sample preparation
Unialgal cultivation of Tetraselmis gracilis was per-
formed in a cultivation room kept at 20.0 ± 0.5◦ C at
the Oceanographic Institute of the University of São
Paulo. Large volume batch culture (4 l) were grown
in f/2 medium [24] with composition shown in Ta-
ble 1, prepared in sterilized seawater, under a photon
flux density of 300 ␮E m2 s−1 provided by white fluo-
rescent tubes with 12/12 light–dark cycle. Filtered air
was continuously bubbled through the algae cultures.
Cultivation growth rate was evaluated daily by cell
counting using a hemocytometer-type chamber at an
optical microscope.
Samples of the culture were collected daily at 3:00
P.M., filtered using 0.45 ␮m cellulose acetate mem-
branes, and the cell free medium immediately trans-
ferred to the titration flask.
2.2. Apparatus and reagents
All reagents were of analytical grade from Merck.
Standard 0.1 mol l−1 NaOH solutions were prepared
by diluting 7 ml of previously filtered 50% (m/v)
NaOH stock solution with CO2 -free deionized wa-
ter in a 1 l volumetric flask containing 35 g of NaCl
and 4 g of Na2 SO4 . This solution was stored in a
high-density polyethylene flask and standardized with
hydrogen phthalate previously dried at 115◦ C for at
least 1 h. Aliquots of this solution were withdrawn
Table 1
Initial composition of the cultivation medium for the algae
Tetraselmis gracilis
Additive Concentration
NaNO3 (mmol l−1 ) 0.88
NaH2 PO4 (␮mol l−1 ) 36.3
Na2 SiO3 (mmol l−1 ) 0.054–0.107
Cyanocobalamin (␮g l−1 ) 0.5
Biotin (␮g l−1 ) 0.5
Thiamine HCl (␮g l−1 ) 100
 M. Rigobello-Masini, J.C. Masini / Analytica Chimica Acta 448 (2001) 239–250
with a siphon system and the air entering the flask
passed through a tube containing soda lime to remove
the atmospheric CO2 .
Standard 0.1 mol l−1 HCl solutions were prepared
in the presence of 35 g l−1 NaCl and 4 g l−1 Na2 SO4
by diluting 8.3 ml of the concentrated acid in a 1 l
volumetric flask. The solution was standardized with
Na2 CO3 previously dried at 270◦ C using bromocresol
green as indicator.
The measurements of potential were performed us-
ing a Metrohm 654 pH-meter (precision of 0.1 mV, or
0.001 units of pH) coupled to a Metrohm combina-
tion glass electrode with Ag/AgCl internal reference
electrode, using saturated KCl/AgCl internal solution.
Additions of titrant were performed with a Gilmont
GS 4200 micro burette with capacity of 2.5 ml and
precision of 0.1 ␮l.
2.3. Procedure for titration of samples
Aliquots of filtered samples were rapidly transferred
to a titration cell with capacity of 25 ml, which was
immediately closed with the cover containing the elec-
trodes and burette tip [5,25]. After equilibration at
temperature of 20.0 ± 0.5◦ C, the titration was started
by addition of small increments of acid (10–20 ␮l).
These increments were changed during the titration to
avoid potential jumps greater than 10 mV. To maintain
this criterion for all samples, titration of medium ob-
tained after 5th day of cultivation (increased dilution)
were performed with a 0.05 mol l−1 standard acid. Af-
ter the 2nd end point, the increments were increased
to 100 and 200 ␮l, and the titration continued until
the potential reached values between 270 and 280 mV
(pH close to 2.5). A total of 50–70 points (volume
and potential) were taken per titration. The potential
at each point was read only after a drift smaller than
0.555 mV min−1 , monitored in the control panel of the
pH-meter used.
2.4. Measurement of hydrogen and hydroxyl ion
concentrations
The electrode calibration was performed in terms
of hydrogen ion concentrations instead activities to
avoid errors that arise from the lack of knowledge
of ionic activity coefficients in concentrated ionic
medium as the seawater. For the calibration, an aliquot
241
of 25 ml of a solution containing 35 g l−1 NaCl and
4 g l−1 Na2 SO4 plus 1.5 ml of the standard HCl solu-
tion (in the same ionic medium of NaCl and Na2 SO4 )
was titrated potentiometrically with a standard NaOH
solution, also containing 35 g l−1 NaCl and 4 g l−1
Na2 SO4 , using increments of 100 ␮l of base. The
titration was performed in a vessel that permits a tight
fit of the glass electrode and burette tip and circula-
tion of N2 to avoid contamination of the atmospheric
CO2 . Before starting the titration, the solutions were
equilibrated at the laboratory temperature, which
was kept constant and monitored at the interval of
20.0 ± 0.5◦ C. Calibrations were performed daily just
before the titration of samples.
The measured potential, E, at each experimental
point is related to the hydrogen ion concentration,
[H+ ], by the Eq. (1) [26], which is valid at tempera-
ture of 20◦ C
E = Ea0 + 58.14 log[H+ ] + Ej (1)
where Ea0 is a constant that includes the standard po-
tential electrode and the ion activity coefficient of hy-
drogen ion at the ionic medium and temperature of the
experiment. Ej the liquid junction potential, which is
given by Eq. (2)
Kw
Ej = JH [H+ ] + JOH (2)
[H+ ]
where JH and JOH are the numerical adjustable param-
eters characteristic of the ionic medium in acidic and
basic solutions, respectively. Kw is the ionic product of
water. The terms Ea0 and JH are obtained with data of
the acid branch of the calibration titration, where the
concentration of hydroxyl ions is negligible. A plot of
E − 58.141 log[H+ ] versus [H+ ] gives Ea0 and JH as
the intercept and slope, respectively, according Eq. (3)
E − 58.14 log[H+ ] = Ea0 + JH [H+ ] (3)
Similarly, JOH is obtained with data from the alkaline
branch of the calibration titration, where [H+ ] is neg-
ligible. In addition to JOH , a term Eb0 , which includes
Ea0 plus the ionic product of water, Kw , is obtained
from the plot of E + 58.141 log[OH− ] versus [OH− ],
which provides Eb0 and JOH as intercept and slope re-
spectively, according to Eq. (4)
E + 58.14 log[OH− ] = Eb0 + JOH [OH− ] (4)
242 M. Rigobello-Masini, J.C. Masini / Analytica Chimica Acta 448 (2001) 239–250

The ionic product of water under the conditions of
temperature and ionic strength used in the experiment
is computed from Eq. (5)
Eb0 − Ea0
log Kw =
58.14
The parameters Ea0 , JH , JOH and Kw used in Eq. (1)
permit the computation of [H+ ], and consequently the
pc H (pH expressed in terms of hydrogen ion concen-
tration) from the potential measurements.
before the start of the titration (the internal volume of
the closed titration cell mounted with electrode, bu-
rette tip and piston), VBn the volume of HCl at the
stoichiometric point of the titration of the base Bn ,
(5) and CHCl is the concentration of HCl in mol l−1 . [H+ ]
and [OH− ] are the concentrations of hydrogen and hy-
droxyl ions, respectively. The term Aj is defined by
KBj
Aj = (8)
KBj + [OH− ]

2.5. Computation and the terms VBj , KBj and Aj have same meaning as
the terms VBn , KBn and An .
2.5.1. MGF According to Eqs. (6) and (7), the slope of the plot
The MGF methodology is described elsewhere of the MGF versus volume of HCl gives the negative
[11,13], and its application to acid titration of the sys- of the hydrolysis constant of Bn . The volume of HCl at
tem HCO3 − /H2 CO3 in presence humic acid was pre- the stoichiometric point is given at the intercept of the
sented by Masini [14] for speciation of components MGF with the ordinate axis (FBn = 0). This program
of the acid neutralizing capacity (ANC) of simulated was compiled in Quick Basic 4.5 software using PC
fresh waters. For a mixture of N weak bases, the MGF, 486 DX-2 computer.
FBn , for the strongest base, B1 , is given by Eq. (6)
2.5.2. Derivative method [4]
FB1 = VB1 KB1 − vKB1

 This method is based on differentiating the titra-
([OH− ] − [H+ ])(V0 + v)(KB1 + [OH− ]) tion curve, so that the almost imperceptible inflection
=
CHCl points due to titration of the CO3 2− and HCO3 −

N species in diluted samples are converted into two
+v[OH− ] − (VBn − VBn−1 )(An )(KB1 well-defined peaks. This computation was performed
n=2 with the measured potential or pH values using the
+[OH− ]) (6) Microcal Origin 4.3 software.

For the subsequent weak bases (n > 1), the MGF are 2.5.3. Simulation of acidimetric titration curves for
given by Eq. (7) model studies
Simulated titration data for a mixture of N weak
FBn = VBn KBn − vKBn

 bases with HCl was based on the general equation
([OH− ] − [H+ ])(V0 + v)(KBn + [OH− ]
=
CHCl f (V , [OH− ])
+v[OH− ]+VBn−1 KBn −VB1 A1 (KBn +[OH− ])

 Kw−
= VCHCl + [OH ] − (V0 + v)
  n−1 [OH− ]
− (VBj − VBj −1 )Aj 
N

j =1, n>1 − (VBn − VBn−1 )An CHCl (9)


N  n=1
+ (VBj − VBj −1 )Aj (KBj + [OH− ])
 All terms in Eq. (9) have the same meaning as the
j =n+1
terms of Eqs. (6)–(8). The pOH values were calcu-
(7)
lated at pre-set volumes of hydrochloric acid using
In these equations, v is the volume of HCl added the Newton–Raphson method, as described in previ-
to the titration cell, V0 the initial volume of sample ous papers [21,22].
 M. Rigobello-Masini, J.C. Masini / Analytica Chimica Acta 448 (2001) 239–250 243

2.5.4. Computation of the distribution of inorganic Table 2

carbon species Hypothetical compositions of cultivation media used to generate
simulated acidimetric titration curvesa
With the total concentration of CO2 determined by
the MGF, or by the derivative method, and the hydro- Species pKb Concentrations
studied (mmol l−1 )
gen ion concentration of the sample before the start of
the titration, the distribution of the inorganic carbon Phosphate 1.300; 6.800 0 or 0.06450
and 11.900
species was determined by the following equations: Borate 4.800 0.3900
Silicate 4.300 0.2848
T[CO2 ] [H+ ]2
[H2 CO3 ] = (10) Carbonate 3.700 and 7.704 1.975; 1.000
[H+ ]2 + K1 [H+ ] + K1 K2 and 0.5000
Sulfate 12.00 29.57
− T[CO2 ] [H+ ]K1 Salt of organic acid 8.500 0.2000
[HCO3 ]= + 2 (11)
[H ] + K1 [H+ ] + K1 K2 Salt of organic acid 9.500 0.2000
Salt of organic acid 10.500 0.2000
T[CO2 ] K1 K2
[CO3 2− ] = (12) a Ionization product of water = 2 × 10−14 .
[H+ ]2 + K1 [H+ ] + K1 K2
where T[CO2 ] is the total concentration of CO2 species were generated for mixtures that simulated the typ-
expressed in mol l−1 , while K1 and K2 are the condi- ical composition of ionizable species found in the
tional dissociation constants of the carbonic acid in the media of batch cultivation of marine microalgae.
ionic medium of 35 g l−1 NaCl and 4 g l−1 Na2 SO4 , Table 2 shows the components, concentrations and
at the temperature of 20◦ C. These constants were de- hydrolysis constants for the mixtures of weak bases
termined in a separate set of titrations of a standard that are involved in the composition of the cultiva-
0.1038 mmol l−1 carbonate solution (prepared from tion medium and were used to simulate the titration
Na2 CO3 ) using the modified Gran method for data curves.
treatment. Table 3 shows the results obtained with simu-
lated titration data generated assuming that T[CO2 ] is
1.975 mmol l−1 , but with the initial pH varying from
3. Results and discussion 8.2 to 9.6. In this simulation, no salt of organic acid
was included in the model and the phosphate con-
3.1. Model studies centration was assumed to be 64.5 ␮mol l−1 . Results
show that the T[CO2 ] was systematically higher than
To verify the accuracy of the results obtained for the expected 1.975 mmol l−1 , with errors between 1.5
T[CO2 ] and alkalinity (ALK) by the MGF and by the and 2.0% for the MGF, and between 3.0 and 3.2%
derivative method, model acidimetric titration data for the derivative method. When, the titration curve

Table 3
Influence of the initial pH on results of T[CO2 ] and ALK obtained by the MGF and derivative method in model studiesa

Initial pH Expected ALK MGF Derivative method

(mmol l−1 )
T[CO2 ] (mmol l−1 ) ALK (mmol l−1 ) pKB1 pKB2 T[CO2 ] (mmol l−1 ) ALK (mmol l−1 )

8.249 2.156 2.005 (+1.5%) 2.156 (0%) 5.906 7.693 2.034 (+3.0%) 2.155 (−0.05%)
8.729 2.356 2.006 (+1.6%) 2.354 (−0.08%) 5.389 7.691 2.035 (+3.0%) 2.353 (−0.1%)
9.249 2.756 2.011 (+1.8%) 2.755 (−0.04%) 4.886 7.691 2.034 (3.0%) 2.761 (+0.2%)
9.578 3.156 2.015 (+2.0%) 3.156 (0%) 4.601 7.691 2.038 (3.2%) 3.160 (+0.1%)
9.177b 2.627 1.970 (−0.25%) 2.625 (−0.08%) 4.879 7.700 1.986 (+0.55%) 2.635 (+0.3%)
a Simulated acidimetric titration data were generated assuming: T −1 −1
[CO2 ] = 1.975 mmol l ; C HCl = 0.1000 mol l ; initial volume in the
titration cell (V0 ) = 25.00 ml. The concentrations of other components are the same as described in Table 2, but in absence of salt of
organic acid.
b Results obtained in absence of phosphate. The values in parenthesis are the relative error in comparison to expected values.
244 M. Rigobello-Masini, J.C. Masini / Analytica Chimica Acta 448 (2001) 239–250
is generated assuming a phosphate free medium,
the errors observed for T[CO2 ] with the computation
methods are around of 0.3–0.5%, indicating that the
species HCO3 − and HPO4 2− are determined as only
one species. This may be explained by the prox-
imity of both hydrolysis constants of these species,
in agreement with previous studies using the MGF,
which verified that the difference between the pK
values should be at least one to permit accurate indi-
vidual determination of each titratable species [21].
Subtracting the phosphate concentration, the results
obtained by the MGF are systematically negative for
the T[CO2 ] , with errors between 1.2 and 1.7%, while
the derivative method provides more accurate results,
with negative errors between 0.05 and 0.25%.
The ALK was estimated as the sum of titratable
species with pK b < 10, so that the concentrations of
H2 PO4 − and SO4 2− were not included in the expected
value presented in Table 3, as well as, the fraction
of the concentrations of species already protonated at
the initial pH. With this assumption, the accuracy of
the ALK obtained by the computation methods was
excellent, as is shown in Table 3.
The pKb values determined by the MGF are also
shown in Table 3. Two classes of titratable species
were characterized, B1 and B2 , where B1 resulted from
the numerical fitting that reflects the stoichiometric
point of the titration of PO4 3− , CO3 2− , borate and sil-
icate species. The pKb depends on the initial pH of
the medium, reflecting the contribution of the major
species in solution at the pH values used for computa-
tion, so that the greater the initial pH, the greater the
pKB1 .
The pKB2 values presented in Table 3 are fitting
parameters obtained for the pH range where HCO3 −
and HPO4 2− species are titrated. As a consequence,
the pKB2 values are systematically lower than the
pKb of HCO3 − (7.704), and higher than the pKb of
HPO4 2− (6.800). The proximity of pKB2 to the pKb
of HCO3 − is explained by the greater concentration
of this species in comparison to the concentration of
HPO4 2− . As can be observed in Table 3, and also
in Tables 4 and 5, when the titration curve is gener-
ated in absence of phosphate, the pKB2 determined
is very close to the expected value of 7.704 for the
HCO3 − , indicating that this is the only species being
titrated in the studied pH range, allowing an accurate
determination of the T[CO2 ] .
Table 4 presents the results obtained with acidimet-
ric titration curves generated with different concentra-
tions of T[CO2 ] in presence and absence of phosphate,
at initial pH around 9.0, and in absence of salts of or-
ganic acids. These results confirm that HPO4 2− is de-
termined together with the titration of HCO3 − , being
a serious interference for determination of the T[CO2 ]
and distribution of inorganic carbon species in cultiva-
tion media. The ALK, on the other hand, is determined
in very good agreement with the expected value.
Table 5 presents the results obtained from titration
curves generated assuming the T[CO2 ] of 1.0 mmol l−1 ,
absence of phosphate, initial pH 9.0 and the presence
of a salt of organic acid with pKb values 8.50, 9.50
or 10.50. The results obtained with the MGF show
that the T[CO2 ] can be determined with errors between
+0.5 and −0.3%. The organic salt is represented by
B3 in Table 5 and its concentration was determined
by the MGF with errors of −3.6 and +1.3% when the
original pKb values were 8.50 and 9.50, respectively.
The fitted pKB3 values were 8.517 and 9.520. When,
the titration curve was generated assuming the pKb of
the salt of organic acid as 10.5, this species was not
determined by the MGF, indicating that it was treated
by the computational program as a strong electrolyte
that cannot be distinguished from other species like
H2 PO4 − and SO4 2− . In this case, the determined
ALK had a significant error, since the species with
pKb 10.5 was not accounted in the sum of species
contributing to acid neutralizing properties of the
mixture.
Studying the influence of the salt of organic acid by
the derivative method, the results of Table 5 show that
the presence of a titratable species with pKb 8.5 leads
to a significant positive error of 16.3% in the calcu-
lated T[CO2 ] . This fact can be explained by a partial
overlapping between the equilibriums involving the
hypothetical organic species and the HCO3 − , moving
the 2nd peak position toward larger volumes of titrant.
For pKb values 9.5 and 10.5, this overlap is minimized
and the T[CO2 ] is determined with very good accu-
racy, as shown in Table 5. The ALK determined by
the derivative method in presence of the organic acid
salt was underestimated in all the three pKb cases.
The salt of organic acid depresses the peak height of
the 2nd stoichiometric point in the derivative method.
Hernández-Ayón et al. [4] verified that the peak de-
pression can be used for detection of the organic acid,
M. Rigobello-Masini, J.C. Masini / Analytica Chimica Acta 448 (2001) 239–250 245
246 M. Rigobello-Masini, J.C. Masini / Analytica Chimica Acta 448 (2001) 239–250

Table 5
Influence of the pKb of a salt of organic acid (B3 ) present at concentration of 0.2000 mmol l−1 on the results for T[CO2 ] and ALK obtained
by the MGF and derivative method in model studiesa
pKb MGF Derivative method

T[CO2 ] (mmol l−1 ) ALK (mmol l−1 ) [B3 ] (mmol l−1 ) pKB1 pKB2 pKB3 T[CO2 ] (mmol l−1 ) ALK (mmol l−1 )

8.500 1.005 (+0.5%) 1.615 (0%) 1.928 (−3.6%) 5.007 7.706 8.517 1.163 (+16%) 1.579 (−2.2%)
9.500 0.9968 (−0.3%) 1.619 (+0.2%) 2.03 (+1.3%) 5.030 7.705 9.520 1.005 (+0.5%) 1.429 (−11.5%)
10.500 0.9980 (−0.2%) 1.418 (−12%) N.D.b 5.036 7.707 N.D. 0.9998 (−0.02%) 1.424 (−11.8%)
a Simulated acidimetric titration data were generated assuming: T −1 −1
[CO2 ] = 1.000 mmol l ; C HCl = 0.1000 mol l ; initial volume in the
titration cell (V0 ) = 25.00 ml; initial pH = 9.010; expected ALK = 1.615 mmol l−1 . The concentrations of other components are the same
as described in Table 2.
b N.D.: not determined; values in parenthesis are the relative error in comparison to expected values.

as well as a rough estimation of its concentration if
this is larger than 1 × 10−5 mmol l−1 , once the mag-
nitude of peak depression presented a quasi-linear re-
lationship with the logarithm of the concentration of
the organic acid. The slope of the graph of peak de-
pression versus the logarithm of the organic acid con-
centration depends on the pKb . Increasing the pKb
(stronger acid), a decrease in the slope is observed,
reflecting the less important overlapping involving the
equilibrium between HCO3 − and the salt of organic
acid. This suggests that both, concentration and ion-
ization constants, could be estimated exploiting the
depression of the peak due to the 2nd stoichiomet-
ric point in acidimetric titrations. This point, however,
was not investigated further in the present work, since
other factors including the T[CO2 ] and the concentra-
tion of titrant solution can also have some influence
on the peak height.

Fig. 1. Titration curve of 25 ml of the cultivation medium segmented and linearized by the MGF, FBn . (A) Fifth day of cultivation,
performed with a 0.1006 mol l−1 HCl solution, and (B) 10th day of cultivation, performed with a 0.05003 mol l−1 HCl solution.
 M. Rigobello-Masini, J.C. Masini / Analytica Chimica Acta 448 (2001) 239–250 247

3.2. Experimental studies

The pKb values of the carbonate system deter-

mined by the MGF, at 20◦ C and ionic medium of
35 g l−1 NaCl and 4 g l−1 Na2 SO4 , were 7.74 ± 0.03
and 4.32 ± 0.02 (n = 3). These values were further
used in the computation of inorganic carbon species
with Eqs. (10)–(12), after conversion to pKa values
(5.96 and 9.38, respectively). The ionic product of
water under the above mentioned conditions was
(2.0 ± 0.1) × 10−14 .
Fig. 1 shows the titration curves of the medium
sampled in the 5th and 10th days of cultivation. The
titration curves appear segmented and linearized by
three MGF, FB1 , FB2 and FB3 , which correspond to
three classes of titratable species characterized.
The function FB1 can be assigned to the titration of
a mixture of species that include the carbonate, sili-
cate and borate anions, as previously observed in the
model studies. The characterization of the individual
contribution of each one of these species was not pos- Fig. 2. pc H of the cultivation medium and pKb fitted for titratable
sible due to the proximity of the hydrolyses constants, species B1 , B2 and B3 determined by the MGF in the samples
so that this range of the titration curve is fitted by taken daily from the cultivation. The results correspond to an
only one function and the pKb value determined average of three experiments of cultivation.

Fig. 3. Titration curve of 25 ml of the cultivation medium and the derivative function for: (A) 5th day of cultivation, performed with a
0.1006 mol l−1 HCl solution; and (B) 10th day of cultivation, performed with a 0.05003 mol l−1 HCl solution.
248 M. Rigobello-Masini, J.C. Masini / Analytica Chimica Acta 448 (2001) 239–250

is an average value of the species titrated in that nutrients by the cells and release of metabolites. Fig. 2
range. shows the pKb values of titratable species character-
The MGF FB2 corresponds to the titration of the ized in the daily samples of 10 days of cultivation, as
HCO3 − to H2 CO3 , giving the total concentration well as the pH (in terms hydrogen ion concentration).
of the CO2 system T[CO2 ] in Eqs. (10)–(12)), which One can observe that the pKb of species B1 decreases
together with the pH of the medium, permits the significantly from the 1st to the 4th day of cultivation,
computation of the distribution of inorganic carbon approaching toward the 2nd hydrolysis constant of the
species. As verified in the model studies, at this pH carbonate. This fact can be explained by the contri-
range of the titration curve, the protonation of hy- butions of mixed species titrated in this pH range, as
drogenophosphate ion interferes due to the overlap- observed in the model studies (Table 3). The pKb of
ping of its hydrolysis constant with that of the HCO3 − species B2 remains constant, within the experimental
ion, so that its contribution has been included in the error at the 95% confidence level, reflecting the lit-
definition of ALK [27–29]. Since, the individual con- tle influence of other titratable species than hydrogen
tribution of each anion cannot be discriminated by the carbonate in this pH range.
MGF, the phosphate concentration was determined Fig. 3 shows the derivative graphs of titrations pre-
by an independent methodology and discounted from viously shown in Fig. 1. The 1st peak, which leads
the concentration of species B2 . The total phosphate to the 1st stoichiometric point, corresponds to species
concentration in the original medium (before start- B1 characterized by the MGF, while the 2nd peak
ing the cultivation) was around of 3.2 ␮mol l−1 , but corresponds to species B2 , so that the difference be-
dropped down to below 3.2 ␮mol l−1 at the 4th day tween the stoichiometric points of B1 and B2 permits
as the phosphate was depleted from the medium by
the growing cell population. After the 4th day, the
phosphate was not detected by the spectrophotometric
molybdenum blue method [30].
Species B3 , characterized by the function FB3 , is
only detected after the 4th day of cultivation and may
be assigned to carboxylate groups (pKb values in the
range of 9.5–9.9, see Fig. 2) of organic compounds
excreted to the medium by the cells. The presence of
this species leads to an overestimation of the T[CO2 ]
when one applies the classical Gran function (F =
(V0 + v)10E/58.14 ) [8,9] to the points corresponding
the excess of strong acid to determine the end point
of the titration.
In the MGF used in this work, the dissociation or
hydrolysis constant, are adjustable parameters in the
program, as well as the stoichiometric points. This is
advantageous in comparison to the MGF proposed by
Hansson and Jagner [10], where the dissociation con-
stants for the carbonic acid, as well as the dissociation
constants and concentrations of boric acid, hydrogen
sulfate and hydrofluoric acid have to be entered in the
equations, which requires the knowledge of the con-
stants at the temperature and ionic medium (salinity) Fig. 4. Concentration of titratable species determined in daily
samples of a cultivation of the algae Tetraselmis gracilis by the
of the sample. Despite these constants and concen-
MGF and derivative methods. The sum of the concentrations gives
trations are well documented for seawater, the condi- the ALK. For comparison, the ALK determined by the classical
tions of cultivation media may be quite different and Gran function [8] applied to the points corresponding to the excess
change during the cultivation, owing to the uptake of of strong acid (ALK-Gran) is also presented.
 M. Rigobello-Masini, J.C. Masini / Analytica Chimica Acta 448 (2001) 239–250 249

Fig. 5. Concentrations of inorganic carbon species and cells of Tetraselmis gracilis in samples collected during 10 days of batch cultivation.
Results correspond to the average of three cultivations. (A) HCO3 − and CO3 2− ; (B) H2 CO3 plus CO2 .

the computation of T[CO2 ] . No evidence of systematic
differences was observed at the 95% confidence level
between the results obtained for T[CO2 ] by the deriva-
tive and MGF methods, as is evidenced in Fig. 4,
where both results are superposed for comparison. An
interesting feature of the derivative method is that de-
spite the small inflection of the titration curves during
the last days of cultivation, which is explained by the
increased dilution caused by the uptake of inorganic
carbon, the two peaks were clearly defined in all sam-
ples, permitting an easy location of the stoichiometric
points, as can be verified in Fig. 3.
The characterization of species B3 by the deriva-
tive method could be possible as proposed by
Hernández-Ayón [4], since a clear peak depression
is observed in the graphs of Fig. 3. This point, how-
ever, was not exploited further in this work owing
to the variation of the media composition during the
cultivation, including T[CO2 ] , ALK, phosphate con-
centration, along with the liberation of the B3 species
by the cells. All these variations can modify the
peak height, leading to ambiguous interpretation of
the results.
Fig. 5 shows the distribution of inorganic carbon
species during 10 days of cultivation of the marine al-
gae Tetraselmis gracilis together with the cell concen-
tration. The inorganic carbon species were computed
using Eqs. (10)–(12) from T[CO2 ] values determined
by the MGF. No systematic differences were observed
when the distribution of inorganic carbon species was
computed from T[CO2 ] values obtained by the deriva-
tive method. During the 1st day of cultivation an in-
crease in the total CO2 concentration is observed as a
consequence of the constant aeration of the cultivation
medium and the small number of cells. The concentra-
tion of species CO2 plus H2 CO3 in the medium before
starting the cultivation is 9 ± 1 ␮mol l−1 , reaching its
maximum value at the end of the 1st day of cultiva-
tion (33.8 ± 0.8) ␮mol l−1 , and decaying significantly
the next 2 days, remaining at the level between 2 and
4 ␮mol l−1 the rest of the experiment. This behavior
may be explained by the combination of factors in-
volving uptake of CO2 by the cells and increase of
the pH of medium. As the exponential growth occurs,
from 4th to 6th day, the HCO3 − concentration drops
abruptly, which can be assigned to the uptake of
250 M. Rigobello-Masini, J.C. Masini / Analytica Chimica Acta 448 (2001) 239–250
inorganic carbon by the cells, once the pH of medium
remains approximately constant during this phase.
4. Conclusion
Both MGF and derivative computation methods
were adequate to assess the distribution of inorganic
carbon species in cultivation medium of the algae
Tetraselmis gracilis. Since the composition of the
medium changes as the cell population grows, the
advantage of the MGF and derivative methods to
this kind of sample is that no dissociation constant
and species concentration must be known a priori to
locate the stoichiometric end points of the titration.
Phosphate at elevated concentrations is a serious in-
terference in the determination of T[CO2 ] , but in the
case described in this paper, the phosphate concentra-
tions were low. The derivative method is very simple
to apply, giving results similar to those obtained by
the more complex MGF for the inorganic carbonic
species.
Acknowledgements
Authors are grateful to CAPES, CNPq and FAPESP
for financial support.
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