Applied Surface Science 370 (2016) 486–495

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Applied Surface Science

journal homepage: www.elsevier.com/locate/apsusc

Characterization of acid functional groups of carbon dots by nonlinear

regression data fitting of potentiometric titration curves
Larissa A. Alves a , Arthur H. de Castro a , Fernanda G. de Mendonça b , João P. de Mesquita a,∗

a
Department of Chemistry, Federal University of the Jequitinhonha and Mucuri Valleys, Rodovia MGT 367 – Km 583, n◦ 5000, Alto da Jacuba,
CEP 39100-000 Diamantina, MG, Brazil
b
Department of Chemistry, Federal University of Minas Gerais, Av. Antônio Carlos, 6627 – Pampulha, Belo Horizonte, MG CEP 31270-901, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The oxygenated functional groups present on the surface of carbon dots with an average size of
Received 30 November 2015 2.7 ± 0.5 nm were characterized by a variety of techniques. In particular, we discussed the fit data of
Received in revised form 9 February 2016 potentiometric titration curves using a nonlinear regression method based on the Levenberg–Marquardt
Accepted 13 February 2016
algorithm. The results obtained by statistical treatment of the titration curve data showed that the best
Available online 17 February 2016
fit was obtained considering the presence of five Brønsted–Lowry acids on the surface of the carbon
dots with constant ionization characteristics of carboxylic acids, cyclic ester, phenolic and pyrone-like
Keywords:
groups. The total number of oxygenated acid groups obtained was 5 mmol g−1 , with approximately 65%
Nanostructures
Oxidized carbon dots
(∼2.9 mmol g−1 ) originating from groups with pKa < 6. The methodology showed good reproducibility
Acids functional groups and stability with standard deviations below 5%. The nature of the groups was independent of small
Potentiometric titration variations in experimental conditions, i.e. the mass of carbon dots titrated and initial concentration of
Electrochemical techniques HCl solution. Finally, we believe that the methodology used here, together with other characterization
Nonlinear regression techniques, is a simple, fast and powerful tool to characterize the complex acid–base properties of these
so interesting and intriguing nanoparticles.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
Carbon dots (CD) or carbon quantum dots (CQD) are the newest
starlets of carbon materials. These carbon nanostructures were
discovered in 2004 during the electrophoretic purification of
single-wall carbon nanotubes [1]. In addition to the traditional
properties of carbon materials (chemical inertness, thermal sta-
bility, high specific surface area and others), CD exhibit distinct
electronic and optical properties [2]. Extensive investigations on
photoinduced charge separation and electron transfer processes
have shown that CD have an excellent ability to receive and donate
electrons from their band structure [3–5]. Due to these charac-
teristics, the potential application of these nanostructures in the
development of new photocatalysts, sensors and light-energy con-
version devices and related areas has been considered. However,
CD optical properties and their fluorescence emissions in particular
∗ Corresponding author at: Department of Chemistry – FACET, Federal University
of the Jequitinhonha and Mucuri Valleys, Diamantina, MG 39100-000, Brazil.
E-mail addresses: joaopm2000@yahoo.com.br, joao.mesquita@ufvjm.edu.br
(J.P. de Mesquita).
have attracted increasing interest in recent years with their great
potential for fluorescent bioimaging in vitro and in vivo. All this
interest arises from the multiple advantages of CD over semicon-
ductor quantum dots, including biocompatibility, low biotoxicity,
environmental friendliness with comparable optical properties and
good chemical and photochemical stability [6]. Although a widely
accepted mechanism for electron transfer and optical properties of
CD remains unavailable there is a consensus that both mechanisms
exhibit dependence on the size of the nanoparticles [7] and surface
properties [8,9].
The majority of CD are rich in oxygenated functional groups.
Depending on the synthetic approaches used, the oxygen content
of CD ranges up to 50 wt% [10]. The presence of such groups on the
surface of the CD, especially at the edges of the graphene layers,
besides interfering directly with the electronic properties of the
CD, contributes to their excellent water solubility and utility as a
suitable chemical platform for further functionalization and surface
passivation with different chemical compounds.
There are different ways to characterize the functional
groups either qualitatively or quantitatively. For qualitative
characterization, spectroscopic techniques such as Fourier trans-
form infrared (FTIR) and X-ray photoelectron (XPS) are typically

http://dx.doi.org/10.1016/j.apsusc.2016.02.128
0169-4332/© 2016 Elsevier B.V. All rights reserved.
 L.A. Alves et al. / Applied Surface Science 370 (2016) 486–495
used. On the other hand, temperature programmed desorption
(TPD) and potentiometric titration are the two most commonly
used methods to quantify the oxygen functional groups present
on the surface of carbon materials [11]. Potentiometric titration is
a robust method that presents the best cost–benefit ratio to quan-
tify the acidic groups typically found in different carbon materials.
The classic way to quantify protic functional groups is based on
neutralizing the acidic groups with an alkali, usually NaOH, fol-
lowed by back titration of the excess of base. In this methodology
and even in Boehm’s titration, which use bases of various basici-
ties (e.g., NaHCO3 , Na2 CO3 , NaOH, NaOC2 H5 ), the results obtained
for various materials represent a simplified description of the true
surface chemical heterogeneity since equivalent functional groups
(e.g., carboxyl) may exhibit significant differences in acidity due
to the influence of neighboring groups anchored on the surface of
the graphene layers [12]. In addition, Boehm’s titration is time con-
suming and quite difficult to carry out as it requires considerable
manipulation (e.g., filtration and back titrations). The manipulation
of basic solutions in open systems is also highly susceptible to con-
tamination with CO2 from the air, directly influencing the results
obtained. On the other hand, because of the variety of structures,
acid–base strength and small number of functional groups on the
solid surface, the potentiometric titration curve shows no point
of inflection [13]. Therefore, traditional methods of the first and
second derivatives do not provide reliable results.
The application of different mathematical methods for the
determination of equivalence points started with Gran, more than
half a century ago, in order to improve accuracy in identifying the
equivalence volume for titration of a weak acid in which use of
the first and second derivatives did not provide accurate results
[14]. Nowadays methods of nonlinear regression [15,16], proton
affinity distributions obtained by solving the integral adsorption
of the proton equation [17–19] and principle component analysis
(PCA) [20] have been used to calculate the pKa distribution and acid
site concentrations of different materials. The non-linear regres-
sion method has been used for the multiparameter adjustment of
potentiometric titration curves of clay [21], collagen [22], activated
carbon [13,23], graphite [24], graphene [24] and multi-walled car-
bon nanotube suspensions [11], presenting excellent reproducibil-
ity and results consistent with other characterization techniques.
Here we will report the characterization of oxygenated func-
tional groups, mainly on acid–base potentiometric titrations in
order to determine the number and nature of accessible acidic
sites in carbon dots prepared from dehydration/oxidation of cot-
ton cellulose, and in particular on using a nonlinear regression
method based on the Levenberg–Marquardt algorithm to fitting
the experimental data from the potentiometric titration curves. The
Levenberg–Marquardt algorithm is used to optimize the parame-
ters of an implicit function describing the titration of a mixture
of a strong acid with n weak acids (surface acid sites) that allows
quantification and determination of the nature of functional groups
from their strength acid (pKa). With the help of other charac-
terization techniques, e.g. FTIR, the contributions of the different
oxygenated functional groups to the total acidity of the carbon
dots will be discussed as well as the reproducibility and robust-
ness of the methodology. In addition, the thermal stability of the
functional groups present on the surface of the carbon dots is also
briefly discussed.
2. Experimental
2.1. Preparation of oxidized carbon dots (OCD)
OCD preparation was performed as described in the literature
with some modifications [25]. First, 5 g of cotton were dispersed
487
in 20 mL of H2 SO4 at 80 ◦ C. After 40 min, the reaction was inter-
rupted by adding 100 mL of water. The carbon material obtained
was washed and redispersed in HNO3 (2 M). The mixture was then
refluxed for 12 h and then neutralized with Na2 CO3 and dialyzed
until constant conductivity was reached. Finally an aqueous solu-
tion was obtained with a concentration of 1.22 g L−1 of OCD. This
methodology has been described elsewhere [26].
2.2. Characterization
Transmission electron microscopy (TEM) images of the OCD
were taken using a FEI Tecnai G2-Spirit with 200 kV acceler-
ation voltage. The OCD suspensions were deposited from an
aqueous dispersion on electron microscopic carbon film grids.
FTIR spectra were obtained in a Nicolet 6700 FT-IR spectrome-
ter using the attenuated total reflectance (ATR) accessory (Smart
iTRTM ). Simultaneous thermal analysis was carried out using a
PerkinElmer Simultaneous Thermal analyzer model STA8000. The
curves were obtained with a heating rate of 5 ◦ C min−1 . Thermo-
gravimetry/coupled mass (TG–MS) analysis was performed in a
NETZSCH TGA equipment (STA 449 F3), coupled to a NETZSCH
Aëolos mass spectrometer (QMS 403C) using a heating rate of
5 ◦ C min−1 under argon atmosphere.
2.3. Potentiometric titration
All potentiometric titration curves were performed at 25 ◦ C
using a SCHOTT automatic titrator (Titroline 7000) with a combined
pH electrode model N6280 (SCHOTT) under nitrogen atmosphere.
A CO2 -free solution of NaOH (0.0134 mol L−1 ) was used as a titrant.
This aqueous solution was prepared by dilution from a stock solu-
tion (15 mol L−1 ) in previously boiled water for 30 min. To maintain
the activity of H+ ions constant throughout the experiment, the
ionic strengths of the HCl and NaOH solutions were adjusted to
0.10 mol L−1 by dissolution of sodium chloride.
2.3.1. Calibration of the electrode
Prior to titration of the carbon dot samples, a potentiomet-
ric titration with standardized solutions of an acid and a strong
base was performed to calibrate the electrode and determine the
ionization constant of water (Kw) under the experimental condi-
tions (ionic strength, temperature). Thus, it is possible to relate the
potential (E) measured by the electrode and the concentration of H+
ions by Eq. (1), since the concentrations of H+ and OH− are known
at each point on the titration curve.
 Kw 

E = E 0 + 0.0592 log [H+ ] + JH [H+ ] + JOH (1)
H+
In Eq. (1), first proposed by Pehrsson et al. [27,28], E is the poten-

tial measured in the electrode, and E0 is a term that includes
the standard electrode potential and the activity of H+ under
the conditions of ionic strength in the solution. JH and JOH are
junction potentials in acidic and alkaline medium, respectively.

Under acidic conditions, the values for E0 and JH can be deter-
mined from the plot E − 0.05916 log [H ] versus [H+ ]. On the other
+
hand, under alkaline conditions, the Kw is determined through
the plot E + 0.05916 log [OH− ] versus [OH− ] [15]. The calibra-

tion parameters E0 , JH , JOH and Kw can be used to convert the
measured potential, E, obtained for the carbon dot titration in
hydrogen ion concentration. In this titration a linear titration
method (available equipment) was used with constant additions
of 0.01 mL of titrant. As a criterion for the equilibrium of the
acid–base reaction a potential variation of less than 1 mV/min was
adopted. The methodology used here has been described elsewhere
[15,16].
488 L.A. Alves et al. / Applied Surface Science 370 (2016) 486–495
2.3.2. Potentiometric titration curves of carbon dots
To obtain the potentiometric titration curves of the carbon dots,
a volume of OCD solution was added to a solution of HCl to a total
volume of 20 mL. Typically, 5 mL of OCD solution (1.22 g L−1 ) was
added to 15 mL HCl (0.0036 mol L−1 ) titrated with CO2 -free NaOH
(0.0134 mol L−1 ). The titrations were conducted with a dynamic
titration method (available equipment) where titrant injection vol-
umes ranged from 0.010 to 0.500 mL. As the calibration titration, a
potential variation of less than 1 mV/min was adopted the criterion
to the equilibrium of acid–base reaction. Through of the titration
calibration it was established that approximately 1 mV corresponds
to 0.015 pH units. The data on pH and volume of titrant used was
then adjusted by a nonlinear regression program briefly described
in the following.
2.3.3. Computational algorithm used to fit the experimental data
of potentiometric titration
The titration of a mixture of a strong acid with n weak acids by
a strong base may be described by Eq. (2) [15,16]. This equation is
obtained through a rigorous balance of mass and charge of all the
chemical species present in the solution.
 
+ + Kw
f (Vi , [H ]i ) = (Vi − VHA0 )Cb + [H ]i − + (V0 + Vi )
[H ]i

N
KHAn
− (VHAn − VHAn−1 )Cb
KHAn + [H+ ]i
j=1
Eq. (2) is an implicit function in which the independent variable
is added titrant volume (Vi ) and the dependent variable is the
hydrogen ion concentration ([H+ ] = 10−pHi ). V0 and Cb are the initial
volume present in the titration cell and the concentration of alka-
line titrant, respectively. Eq. (2) can be used to fit the experimental
data of titration curves of solutions containing a strong acid and
several weak acids, with constant acidity and unknown concen-
trations, by determining the parameters KHAn (ionization constant
of weak acid), VHAn (volume equivalence of weak acid) and VHA0
(equivalent volume of strong acid). To determine these parameters
a non-linear regression program was used. This is program has been
Fig. 1. The flowchart of the computational algorithm used in adjusting the potentiometric titration curves.
described elsewhere [11,13]. Following is a brief description of the
calculation procedures used in this computational routine.
First the experimental data are read: pH, Vi , V0 , Cb and ini-
tial estimates of the number of functional acidic groups present
on the surface of the nanomaterial (n), and also of the ionization
constants of the weak acids (KHAn ), equivalent volumes of weak
acids (VHAn ) and volume equivalence of the strong acid (VHA0 ).
Since Eq. (2) is an implicit function, the use of an iterative method
to determine the dependent variable ([H+ ]i (calc) = 10−pHi (calc) ) is
required. Although we tested the traditional Newton–Raphson
iterative method, it presented convergences highly dependent on
initial estimates and most often the method found negative roots
for the concentration of ions H+ ions; here, we used the Pegasus
method [29]. The method uses Eq. (3) to determine the depend-
ent variable [H+ ]i (calc) by performing various iterations until the
condition [(pHi,k+1 − pHi,k )/pHi,k ] < 0.00001 is obtained [11].
f (pHi,k )(pHi,k − pHi,k−1 )
pHi,k+1 = pHi,k − (3)
f (pHi,k ) − f (pHi,k−1 )
The following was used to calculate the sum of the squares of the
errors, S (Eq. (4)).

m
2
S= [pHi (Exp) − pHi (calc)] (4)
i=1
Wherein m is the total number of experimental data (Vi , pHi (Exp)).
(2) The parameters VHA0 , VHAn and KHAn were adjusted through the
Levenberg–Marquardt algorithm by calculating the vector d (Eq.
(5)).
−1
{d} = {[D]T [D] + [I]} [D]T {E} (5)
where [I] is the identity matrix,  is a positive scalar which starts
with a high value and decreases with the number of iterations and
E is a vector defined as the difference between the experimental
and the calculated pH. [D] is the Jacobian matrix [30]. Since the
value of d is calculated, the values of the adjustable parameters
VHAn , KHAn and VHA0 are refreshed and the value of S (Eq. (4)) is
recalculated. The whole procedure described above is repeated sev-
eral times until the value of S remains approximately constant [11].
Fig. 1 shows the flowchart of the computational algorithm used to
 L.A. Alves et al. / Applied Surface Science 370 (2016) 486–495 489

Fig. 2. TEM (a and b) and HRTEM (c) images of OCD prepared from acid dehydration/oxidation of cellulose.

Fig. 3. (a) Thermogravimetric (TG) and differential thermal (DTA) analysis. (b) Thermogravimetric (TG) and differential thermogravimetric (DTG) analysis obtained for the
OCD.

adjust the potentiometric titration curves obtained for the carbon

dots.

3. Results and discussion

The presence of carbon nanostructures was confirmed with TEM

and HRTEM images (Fig. 2). The OCD obtained exhibited particles
with an average diameter of 2.7 ± 0.5 nm (detail in Fig. 2). The CD
typically comprised amorphous to crystalline cores with predomi-
nantly graphitic or graphene sheets modified by diamond-like sp3
hybridized carbon insertions [6]. The carbon dots obtained here
have a quasi-spherical morphology in which can be observed the
presence of 002 planes of the graphitic structure (Fig. 2c).
The optical properties of carbon dots obtained here have been
previously reported [26]. They presented optical absorption in the
ultraviolet region that extends to ∼500 nm. On the other hand, the
emissions are broad and excitation wavelength-dependent [26].

3.1. Thermal analysis and FTIR of oxidized carbon dots

Characterization of the surface groups with thermogravimetric

analysis and FTIR were carried out for comparison purposes and
to aid in interpreting the results of potentiometric titration of the
acidic oxygenated functional groups introduced after the dehydra-
tion and oxidation of the cotton cellulose.
The thermal gravimetric analysis and differential thermal anal- Fig. 4. FTIR spectra of the cellulose (I), dehydrated cellulose with H2 SO4 (II) and OCD
(III). Detail: region between 1900 and 1450 cm−1 of OCD FTIR spectrum.
ysis, besides helping to identify and quantify the functional groups,
provides important information on the thermal stability of these
groups, contributing to future reaction protocols to control the four well-defined thermal events at around 270, 500, 680, and
electronic properties of carbon dots. The thermogravimetric (TG) 830 ◦ C. The mass losses which occurred at these temperatures
analyses (Fig. 3a) profile clearly showed the presence of oxygenated coincided with the decomposition temperatures of different oxy-
functional groups on the carbon dots [23]. The DTA (Fig. 3a) showed genated functional groups [31]. The peak at around 270 ◦ C (weight
490 L.A. Alves et al. / Applied Surface Science 370 (2016) 486–495
Fig. 5. Titration of 20.00 mL of a standard solution of HCl (0.0027 mol L−1 ) with 0.0134 mol L−1 NaOH (a). The values plotted in (b) and (c) were calculated from data shown
in (a).
loss ∼23%) can be attributed to the decomposition of carboxylic
groups and NO2 groups (Fig. 3b) (see Fig. S1 in supplemen-
tary information). Moreover, the results obtained for other carbon
nanostructures with temperature programmed desorption (TPD)
showed the water eliminated from 150 to 450 ◦ C attributed to the
formation of carboxylic anhydrides from the dehydration of adja-
cent carboxyl groups [32]. Another possible source of this water
peak could be the dehydration of phenolic hydroxyl groups [32].
In Fig. 3b, the peaks at around 500 ◦ C and 680 ◦ C were found to be
associated with the decomposition of carboxylic anhydrides and
lactones, which are more stable than carboxyl groups (Fig. S1, CO2
profile). The weight loss of approximately ∼5% at around 830 ◦ C
is associated with the decomposition of phenol, quinone, ether
groups, and also the decomposition of pyrones [33]. Finally, the
results indicate a material with complex thermal properties due to
its surface heterogeneity.
FTIR is the most popular technique for qualitative evaluation of
the chemical structures of carbon materials. Despite the difficulties
in obtaining good spectra and their very complex interpretation,
the technique is useful for identifying different functional groups
[34]. Thus, the results obtained with FTIR contribute effectively
to the identification of the acidic groups characterized by poten-
tiometric titration. FTIR spectra of the OCD are shown in Fig. 4.
Before dehydration, the absorptions typically observed in cellulose
type I are O H, C H, ␦CH2 and saccharide structure centered
at 3340, 2899, 1431 and 1200–1000 cm−1 , respectively [35]. After
acid dehydratation there is a significant change in the structure
of the cellulose, with destruction of the saccharide structure and
intense absorptions at 1029, 1188, 1620, 1722 and 3412 cm−1
mainly assigned to the stretching of the functional groups S O,
C O, C C, C O and O H, respectively. OCD is obtained from the
oxidation of this dehydrated material. Due to the strong oxidative
attack of the HNO3 on the carbon structure, significant changes are
observed in the FTIR spectra. The bands at 1120 and 1040 cm−1
are attributed mainly to the C O and S O of different func-
tional groups. Moreover, the investigation of sulfur groups with
TG/MS suggesting the elimination these functional groups after
oxidation (Fig. S1, SO2 profile). On the other hand, there is an
increase in the relative intensity of the band centered at 1374 cm−1 .
These bands are attributed to N O, particularly NO2 [36–39].
The presence of nitrogen functional groups was investigated by
TG/MS analysis (Fig. S1). The presence of intense m/z signals 30
related to NO was observed which suggests the presence of large
amounts of these functional groups. It is observed also an intense
and broad absorption are observed between 1450 and 1900 cm−1 .
These absorption bands arise from the C C of aromatic struc-
tures, C O of quinone and C O from various functional groups
such as carboxyl and anhydride (∼1770 and 1700 cm−1 ) [34].
According to the results it is clear that OCD prepared by dehy-
dratation/carbonization with H2 SO4 and HNO3 present significant
numbers of oxygenated acidic groups, i.e. OH and COOH, on the
surface.
 L.A. Alves et al. / Applied Surface Science 370 (2016) 486–495
Fig. 6. Experimental data V, pH obtained for the titration of 6.1 mg of OCD in the presence of 20.00 mL of a standard solution of HCl (0.0027 mol L−1 ) with 0.0134 mol L−1
NaOH. The best fit found considering the absence of any acidic group on the surface of the OCD, n = 0 (a). The best fit obtained by resolution of Eq. (2) given the existence of 1
(b), 2 (c), 3 (d), 4 (e) and 5 (f) acidic groups on the surface of the OCD.
3.2. Potentiometric titration of oxidized carbon dots
Because of the variety of structures, acid–base strengths and
particularly the small numbers of functional groups on the carbon
surface dots, experiments must be conducted carefully and under
inert atmosphere to avoid contamination by dissolved atmospheric
CO2 (see supplementary information, Fig. S2). Although the concen-
trations of carbonates and bicarbonates produced by the reaction
of CO2 and H2 O were very low, the values of these concentrations
were of the same order of magnitude as the surface groups found
on the carbon dots.
3.2.1. Titration of electrode calibration
Prior to titrating samples it is necessary to perform a titration of
electrode calibration to determine the parameters, e.g. the junc-
tion potential and auto-ionization constant of water under the
actual experimental conditions, i.e., ionic strength and tempera-
ture [15,16]. These parameters will be used in the conversion of
electrical potential measurements (mV) obtained from titration
of solutions containing carbon nanostructures into pH. In Fig. 5a,
a typical titration curve obtained for 20 mL of a standard solu-
tion of HCl (0.0027 mol L−1 ) is shown. Fig. 5b and c shows plots
used to determine the calibration parameters (Eq. (1)). From the
491
linear regression of the data shown in these figures, the results
 
obtained for E0 , E0 , JH , JOH and Kw were 383.5 mV, −421.4 mV,
39.5 mV L mol , 518.9 mV L mol−1 and 2.51 × 10−14 , respectively.
−1
3.2.2. Experimental and fitted potentiometric titration curves of
oxidized carbon dots
Once calibrated, the electrode parameters were used to con-
vert the measured potentials (mV) into pH by solving Eq. (1). The
Levenberg–Marquardt algorithm (LM) was used to optimize equiv-
alent volumes (VHA0 and VHAn ) and ionization constants (KHAn ).
The LM method is also known as the modified Gauss–Newton
method. The Gauss–Newton method has very good convergence
when the initial estimates are close to true values. However, it
may oscillate around the minimum error or diverge if initial esti-
mates are poor, which most often occurs. Thus, modifications have
been proposed for the method, such as the Levenberg–Marquardt
method, which uses the method of gradient descent ( → ∞ in
Eq. (5)) when the parameter values are far from optimal, and
the Gauss–Newton method ( → 0 in Eq. (5)) when the values of
the parameters are close to the optimum [30]. According to the
theoretical model represented by Eq. (3), it is necessary to esti-
mate the nature of the functional acidic groups present on the
surface of the carbon dots (n) as well as the values of equivalent
492 L.A. Alves et al. / Applied Surface Science 370 (2016) 486–495

Table 1
Results obtained by fitting experimental titration curves of OCD with four and five surface acidic groups to the model.

Acid functional groups Four surface acidic groups model (n = 4) Five surface acidic groups model (n = 5)

pKa Surface acidic groups/mmol g−1 pKa Surface acidic groups/mmol g−1

Carboxyl 3.42 1.91 3.29 1.71

Carboxyl 4.89 1.19 4.62 1.23
Lactone 6.03 0.59
Lactone 6.47 0.50 7.64 0.20
Phenol 8.99 0.81 9.22 0.81

Total acidic functional groups – 4.42 – 4.55

Residual sum of squares (S) 0.08821 0.02981

volumes (VHA0 and VHAn ) and ionization constants (KHAn ). Although

all are a priori arbitrary, the origin of the sample, the experimental
procedure used in its preparation and physical–chemical charac-
terization performed with other techniques, e.g. FTIR, indicates the
possibility of certain functional groups being present. Therefore,
these initial estimates cannot be considered totally arbitrary. Typi-
cally, data fitting is started by assuming the presence of traditional
functional groups (carboxylic acid, lactone, and phenol). After pre-
liminary results are obtained, more sites are added to the model
until no statistically significant improvement is observed. However,
in order to determine the number of acidic species that best repre-
sent the experimental data and demonstrate the reliability of the
model, first, we fit the experimental data from potentiometric titra-
tion of the OCD considering the presence of different numbers of
acidic functional groups anchored on the surface of the carbon dots
prepared herein. The results are shown in Fig. 6. An overview of
the results presented in Fig. 6 shows clearly that when additional
species are added to the model, an improvement in the fitting is
observed. Fig. 7. Potentiometric titration curves obtained for carbon dot samples in triplicate.
In Fig. 6a–c, optimizations were conducted considering the pres-
ence of no (n = 0), 1 (n = 1) or 2 (n = 2) acidic functional groups, may return results without any physical meaning, for example, a
respectively, on the surface of the OCD. The results obtained with negative volume of equivalence (VHAn ).
these models clearly show an inadequate number of acidic groups Since the fit of the experimental data considering the presence
present on the surface of the nanomaterial. In addition, these results of six different acidic groups on the surface of the OCD did not gen-
suggest the need to consider the presence of at least three distinct erate an improvement in the value of S (S6 = 0.0445), a test was
acidic groups (n = 3). In Fig. 6d, the results of this estimation are applied to evaluate the significance of additional parameters (vol-
shown. Visually this model appears to represent well the acidic ume of equivalence and constant of ionization of the fifth acidic
groups present on the surface of the carbon dots. However, the rel- functional group) in the model that considers the presence of four
ative error values are high and with a poor dispersion (Fig. S3d). surface acidic groups. The testing involved calculating the F ratio
Moreover, the resolution of Eq. (2) considering the presence of four (Eq. (6)) [40].
(n = 4) and five (n = 5) distinct functional acidic groups on the sur-
(S4 − S5 )/(m − p5 )
face of the OCD (Fig. 6e and f) represent an excellent fit of the F(p5 − p4 , m − p5 ) = (6)
experimental data with a high dispersion and uniform distribu- S5 /(m − p5 )
tion of relative errors (Fig. S3e and f). The fits of these data using Here, m is the number of fitted data (V, pH), S5 and S4 are the
four and five surface acidic groups in the model are summarized in residues of the sums of squares and p4 and p5 are the number of
Table 1. The increase in the adjustable parameters, VHAn and KHAn , adjustable parameters considering the presence of five and four
has generated a decrease in the residual sum of squares, S, defined functional acidic groups, respectively. This test has been used to
as the difference between the calculated and experimental pH. This determine the ionizable groups on proteins [15] and characterize
fact may be due to the real presence of titratable species, but it ligand-binding systems [40] as well as carboxylic and pheno-
may also be due to flexibility in the mathematical model, which lic groups in humic acids [41]. According to the experimental

Table 2
Result of nonlinear regression fitting for potentiometric titration curves of OCD.

Functional group content and pKa interval

1st titration 2nd titration 3rd titration Mean and standard deviation

pKa Acid groups/mmol g−1 pKa Acid groups/mmol g−1 pKa Acid groups/mmol g−1 pKa Acid groups/mmol g−1

3.29 1.71 3.02 1.92 3.16 1.92 3.16 ± 0.13 1.85 ± 0.12
4.62 1.23 4.46 1.36 4.58 1.30 4.55 ± 0.09 1.30 ± 0.07
6.03 0.59 5.73 0.61 5.95 0.55 5.91 ± 0.15 0.58 ± 0.03
7.64 0.20 7.09 0.31 7.43 0.23 7.39 ± 0.28 0.24 ± 0.06
9.22 0.81 9.22 0.90 9.19 0.84 9.21 ± 0.02 0.85 ± 0.05

Total 4.55 Total 5.10 Total 4.84 Total 4.83 ± 0.27

 L.A. Alves et al. / Applied Surface Science 370 (2016) 486–495 493

Fig. 9. Concentration of the acidic functional groups originating from different

Fig. 8. (Symbols) Experimental data V, pH obtained for the titration of OCD with
0.0134 mol L−1 NaOH. Experimental conditions of the titrations: (I) 3.7 mg of OCD in
the presence of 20.00 mL of a standard solution of HCl (0.0030 mol L−1 ); (II) 6.1 mg
of OCD in the presence of 20.00 mL of a standard solution of HCl (0.0027 mol L−1 );
(III) 12.2 mg of OCD in the presence of 20.00 mL of a standard solution of HCl
(0.0018 mol L−1 ). The solid lines correspond to the best fit obtained by resolution
of Eq. (2) with the five surface acidic group’s model.
conditions employed in this work, m = 139, p4 = 9, p5 = 11, and S4
and S5 are shown in Table 1. From Eq. (6) an F(2,128) = 125 is
obtained. This result is greater than the critical value F(2,∞) = 5.5
with a confidence level of 99.5%. It is therefore concluded that at the
99.5% confidence level, the five acidic groups model is more appro-
priate to represent the experimental data of OCD potentiometric
titration.
Given the number of distinct acidic surfaces it is necessary
to identify the corresponding functional groups of each of the
Brønsted–Lowry acids from pKas obtained with the fitting (Table 1).
The surface acidic groups of the carbon dots exhibited acidic
strength similar to that on corresponding organic acids with sim-
ilar structures. The pKa of an acidic functional group is influenced
mainly by substituents on the ␣-carbon, largely through induc-
tive effects. Electron-withdrawing groups increase the acidity and
electron-donating groups decrease the acidity. Thus, assigning
acidic functional groups based on pKas may be challenging since
closely similar acidic functional groups mutually interfere in their
acid–base properties. In addition, because of the use of HNO3 in
the preparation of the OCD, the presence of large amounts of the
NO2 group is expected [42]. These chemical species are strong
electron-withdrawing groups. In carbon materials pKa’s between
2.0 and 5.0 are typically attributed to carboxylic acids [43]. pKas
between 6 and 8 are assigned to acidic groups formed by the
hydrolysis of cyclic esters (lactone groups) in medium acid or base,
and phenolic groups are assigned pKas greater than 9 [44]. More-
over, the presence of ether functional groups is highly expected in
preparation batches of oxidized carbon dots.
cellulose dehydration reactions [45,46]. Some studies have shown
that pyrone-like functionalities at the edges of graphene layers are
predicted to exhibit a broad range of pKa values (4–13) depending
on the relative position of the ketone and etheric rings [47]. Because
of the broad range of pKa values observed for pyrone-like function-
alities, we speculate there is a large probability these groups had
acid–base properties in the range of ionization constants obtained
with fitting of experimental data. According to Table 1, 65% of
the acidic functional groups present on the surface of the OCD
presented pKa < 5, typically attributed to carboxylic acids, 17%
are lactones and the remaining 18% are attributed to phenolic
groups.
Having identified the model that best represents the number
of distinct surface acid groups present on the surface of the OCD,
we evaluated the repeatability of the titration curves under the
same experimental conditions and the robustness of the method-
ology. The repeatability of the experimental methodology was
evaluated and the titration curves obtained under the same exper-
imental conditions are shown in Fig. 7. It is clear in Fig. 7 that
the experimental procedure is stable and reproducible. The level
of agreement between the results of fits using these successive
measurements carried out under the same measuring conditions
is shown in Table 2. In general, the potentiometric titration of car-
bon dots showed high reproducibility and stability, with standard
deviations below 5%.
The concept of the robustness of an analytical method is con-
nected to its ability to remain unchanged under small variations
in the parameters of the method [48]. To evaluate the robustness
of the methodology used here, we realized titrations with differ-
ent masses of carbon dots and changed the initial concentration
of the HCl solution. The titration curves obtained under different

Table 3
Results of fitting potentiometric titration curves of OCD obtained with different masses of titrated sample and initial concentration of the HCl solution.

Functional group content and pKa intervala Mean and standard deviation

Experimental conditions I Experimental conditions II Experimental conditions III

pKa Acid groups/mmol g−1 pKa Acid groups/mmol g−1 pKa Acid groups/mmol g−1 pKa Acid groups/mmol g−1

3.80 2.12 3.29 1.71 3.68 1.81 3.59 ± 0.26 1.88 ± 0.21
5.44 1.05 4.62 1.23 4.96 1.13 5.01 ± 0.41 1.13 ± 0.09
6.04 0.17 6.03 0.59 6.51 0.42 6.19 ± 0.27 0.39 ± 0.21
6.97 0.34 7.64 0.20 8.09 0.19 7.57 ± 0.56 0.24 ± 0.08
9.23 1.45 9.22 0.81 9.45 0.61 9.30 ± 0.13 0.96 ± 0.43

Total 5.12 Total 4.55 Total 4.15 Total 4.61 ± 0.48

a
The experimental conditions are the same as described in the legend of Fig. 8.
494 L.A. Alves et al. / Applied Surface Science 370 (2016) 486–495
Fig. 10. Schematic representation of the surface chemistry of carbon dots prepared from cotton cellulose with the number of oxygenated acid groups obtained from fitting
the data in the potentiometric titration curve.
conditions are shown in Fig. 8 and the results obtained with the fits
of these experimental data are summarized in Table 3.
In general, the results obtained under the three experimen-
tal conditions evaluated are very close, i.e., the pKa distributions
and numbers of acidic functional groups obtained by fitting are
independent of the mass and HCl concentration used in these pro-
cedures. Moreover, in Table 3, a slight increase can be noted in the
concentration of acidic groups with decreasing amount of sample
used in the titration. This behavior has been observed in the char-
acterization of other samples of carbon and has been attributed to
the close conditions of chemical equilibrium in experiments with
smaller amounts of samples [13]. A good example of these consid-
erations is the hydrolysis of the lactol and lactone groups, which
depend on the concentration and acid–base properties of the solu-
tion [12].
In addition, we carried out the titration three different batches
of OCDs samples (Fig. S4). The results obtained with the fits of
these experimental data are summarized in Table S1. The pKa val-
ues are very similar indicating that the OCDs preparation method
always form functional groups with the same acid–base properties.
In Fig. 9 are shown the concentrations of the acid functional groups
obtained these three different samples. In general, the results are
close. The exception was the concentration of the functional group
with pKa = 9.57 (3rd synthesis). Moreover, the relative concentra-
tions of the functional groups in three batches of preparation are
very similar. Given the complexity of the surface of these nanoparti-
cles and the experimental procedures carried out, i.e., dehydration,
oxidation (difficult to control reaction) and dialysis; the results sug-
gest that the method is suitable for the preparation of carbon dots
with a well-defined surface properties.
Cellulose is a high molecular weight homopolymer formed by
ˇ(1 → 4) linked d-glucose units that contains 40 wt% carbon. It is
the most abundant organic compound in the biosphere and an
interesting candidate for the preparation of new carbon materi-
als, e.g. carbon dots. The carbon quantum dots soluble in water
prepared here are obtained by a dehydration reaction which pro-
ceeds until aromatization/carbonization of the carbohydrate. Then,
the obtained material is oxidized with HNO3 , a strong oxidant that
attacks the carbon surface, eroding its structure and introducing
oxygen functional groups by different mechanisms [49]. Fig. 10
shows a schematic representation of the dehydration and oxidation
process of cotton cellulose forming carbon dots with a proportional
number of oxygenated functional groups obtained from the fit of
the potentiometric titration data.
4. Conclusions
The acidic oxygenated functional groups present on the surface
of highly oxidized carbon dots were characterized by a simple and
fast procedure based on fit data of potentiometric titration curves
using a nonlinear regression method. The Pegasus iterative method
was used together with the Levenberg–Marquardt algorithm, and
proved highly efficient and robust. From the fit it was found that the
carbon dots prepared from cotton cellulose have a surface consist-
ing of Brønsted–Lowry acids with five different ionization constants
with a density of oxygenated acids (phenol, lactone, carboxyl and
pyrone-like) of ∼5 mmol g−1 . Of this total, 2.9 mmol g−1 are groups
with pKa between 3 and 6, typically attributed to carboxylic func-
tional groups. Under the experimental conditions carried out in this
work, the potentiometric titration of the carbon dots showed high
reproducibility and stability with standard deviations below 5%.
The robustness of the methodology was evaluated from the amount
of titrated sample and the initial concentration of the acid solu-
tion. The results obtained within the pH range analyzed showed
the identity of the groups is independent of the mass of the carbon
dots titrated and the initial concentration of the HCl solution. More-
over, a slight decrease of the number of acidic functional groups
was found with the increase of sample mass titrated. The results
of TG/DTA suggest that oxygenated functional groups exhibit ther-
mal behavior similar to that observed for other carbon materials.
Since the electronic structure and optical properties of the CD are
highly dependent on surface chemistry, and the surface chemistry
of carbon materials in general is practically indistinguishable from
that of the oxygen acidic groups, the methodology presented here
is extremely utilities and if performed in parallel with other char-
acterization techniques, e.g. FTIR, TPD or XPS, the interpretation of
the results is more accurate and realistic. In general, the method
is useful for planning experimental procedures and an excellent
alternative for monitoring chemical surface reactions that use
these functional groups as a platform for specific functionalization
reactions.
Acknowledgements
The authors wish to thank CAPES, FAPEMIG (Process APQ-
00614-13), RQ-MG (Process CEX – RED-00010-14) and CNPq
(grants # 478228/2013-9) for the financial support and to the Grupo
de Tecnologias Ambientais (GruTAm – UFMG).
 L.A. Alves et al. / Applied Surface Science 370 (2016) 486–495
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.apsusc.2016.02.
128.
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