Hydroxyl Radicals quantification by UV spectrophotometry

Article abstract of DOI:10.1016/j.electacta.2014.02.047

Hydroxilation of aromatic compounds is an oxidative process that has been employed as indirect method to quantify the produced hydroxyl radicals during an advanced oxidation process (^a-OH). This is usually complicated by the radical high reactivity and short life time and therefore a scavenging agent such as salicylic acid is commonly employed. This usually implies a rather sophisticated analytical method such as chromatography. In this work, however, a relatively simple and low cost method is proposed to achieve the aforementioned objective. This method is based on UV-Vis spectrophotometry and was employed to quantify the hydroxyl radicals produced during the electrochemical oxidation of water when employing a platinum anode in a galvanostatic type process. Salicylic acid was employed as ^a-OH scavenger. The concentration of the hydroxilated resulting products, 2,3-dihidroxybenzoic Acid and 2,5-dihydroxibenzoic Acid, was determined by UV-Vis Spectroscopy and utilized to quantify the hydroxyl radicals. In addition, mineralization was followed by Chemical Oxygen Demmand measurements.

Full text of DOI:10.1016/j.electacta.2014.02.047

Electrochimica Acta 129 (2014) 137–141
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Electrochimica Acta
journal homepage: www.elsevier.com/locate/electacta
Hydroxyl Radicals quantification by UV spectrophotometry
E. Peraltaa,b, G. Roab,∗, J.A. Hernandez-Servinc, R. Romerob, P. Balderasb, R. Natividadb
a Universidad del Mar, Ciudad Universitaria s/n, Puerto Ángel, Oaxaca, México,70902
b Centro Conjunto de Investigación en Química Sustentable UAEMex-UNAM Carretera Toluca Atlacomulco km 14.5, Toluca, Estado de México, México, 50200
c Faculty of Engineering, Universidad Autonoma del Estado de México, Cerro de Coatepec S/N, Toluca, Edo. De México, México, 50130
a r t i c l e i n f o
a b s t r a c t
Article history:
Hydroxilation of aromatic compounds is an oxidative process that has been employed as indirect method
to quantify the produced hydroxyl radicals during an advanced oxidation process (᭹OH). This is usually
complicated by the radical high reactivity and short life time and therefore a scavenging agent such
as salicylic acid is commonly employed. This usually implies a rather sophisticated analytical method
such as chromatography. In this work, however, a relatively simple and low cost method is proposed
to achieve the aforementioned objective. This method is based on UV-Vis spectrophotometry and was
employed to quantify the hydroxyl radicals produced during the electrochemical oxidation of water
when employing a platinum anode in a galvanostatic type process. Salicylic acid was employed as ᭹OH
scavenger. The concentration of the hydroxilated resulting products, 2,3-dihidroxybenzoic Acid and 2,5-
dihydroxibenzoic Acid, was determined by UV-Vis Spectroscopy and utilized to quantify the hydroxyl
radicals. In addition, mineralization was followed by Chemical Oxygen Demmand measurements.
© 2014 Elsevier Ltd. All rights reserved.
Received 15 October 2013
Received in revised form 2 February 2014
Accepted 13 February 2014
Available online 24 February 2014
Keywords:
Advanced oxidation process
Salicylic Acid
2 ;3-dihidroxybenzoic Acid
2 ;5-dihydroxibenzoic Acid
1. Introduction
Phenylalanine, benzoic acid, salicylic acid (SA) and the esterificated
form of aspirin have been reported for this purpose. Among these
Recently, Advanced Oxidation Processes (AOP) have been rec-
ognized and employed as efficient methods to remove dangerous
and recalcitrant environmental pollutants. These processes have
in common the production of hydroxyl radicals (᭹OH) to conduct
oxidation reactions [1]. Albeit other oxidants, hydroxyl radicals are
attractive for effluents purification treatment since their short life
time minimizes the undesirable side effects observed with chlo-
rine and ozone, for example [2]. Therefore, it is not surprising that
in the last decades the research on organic compounds mineraliza-
tion has been strongly addressed to producing hydroxyl radicals by
several forms, i.e. photocatalysis, electrochemistry, ozonation and
photolysis of H2O2. At this point, it becomes desirable to quantify
the produced hydroxyl radicals as an attempt to understand the
difference among each AOP. Regarding this matter, some meth-
ods have been developed such as aromatic hydroxylation and this
has become one of the most sensitive methods to quantify ᭹OH.
compounds, however, SA has been preferred due to (i) high reac-
tion rate (5 × 109 M−1
s
−1), (ii) its high competitiveness against
some other scavengers and (iii) the stability of its reaction prod-
ucts which facilitates the samples treatment and analytical process
[3].
Some analytical methods like High Performance Liquid Chro-
matography (HPLC) have been employed to detect ᭹OH by using
SA as scavenger in a Fenton process [4]. Other employed analytical
methods for the same purpose are capillary zone electrophoresis
with amperometric detection (CZE-AD) [5], Electronic Spin Reso-
nance (ESR), employing phenyl-ter-butilnitrone (PBN) as scavenger
in a Fenton process [2]. Also dimethyl sulfoxide (DMSO) has been
employed as scavenger in a Fenton reaction, photolysis of H2O2
and ␥ irradiation of H2O [6]. Other employed ᭹OH scavengers are
salicilate and 4-hydroxybenzoate [7]. In all the aforementioned
studies the use of a scavenger is a constant and the employment of
a sophisticated analytical technique becomes compulsory in order
to identify the reaction products of the scavenger. These methods,
although highly accurate, also offer some inherent disadvantages
like complexity and cost. This encouraged this work to be dedicated
to develop a rather simple and less costly method based on UV-Vis
Spectrophotometry to establish the production of hydroxyl radicals
in an AOP. In this case the employed AOP was electrochemical and
∗
Corresponding author. Tel.: +5201 722 766610Ext7716;
fax: +5201 722 2 175109.
E-mail addresses: e pere70@hotmail.com, groam@uaemex.mx (G. Roa),
xoseahernandez@gmail.com (J.A. Hernandez-Servin), rromeror@uaemex.mx
(R. Romero), patbh2003@yahoo.com.mx (P. Balderas), reynanr@gmail.com
(R. Natividad).
http://dx.doi.org/10.1016/j.electacta.2014.02.047
0013-4686/© 2014 Elsevier Ltd. All rights reserved.

138
E. Peralta et al. / Electrochimica Acta 129 (2014) 137–141
a Pt electrode was used since it has been reported that generates
᭹OH without reaching mineralization [8].
2. Experimental
2.1. Reagents
Analytical grade Salicylic Acid (SA), 2,3-dihidroxybenzoic Acid
(2,3-DHBA) and 2,5-dihydroxibenzoic Acid (2,5-DHBA) were pur-
chased from Sigma Aldrich. Reagent grade Sulfuric acid was
purchased from ACS and deionized water was obtained from a
Millipore Direct Q3 UV with resistivity >18 Mꢀ at room tempera-
ture. Standard solutions of SA (1 × 10−3 M), 2,3-DHBA (1 × 10−3 M),
2,5-DHBA (8 × 10−4 M) and H2SO4 (0.5 M) were prepared with
deionized water.
2.2. Production and scavenging of ᭹OH
The production and scavenging of ᭹OH radicals was conducted
in a 5 ml typical electrochemical cell (not shown). For this purpose,
3 mL of a 1 × 10−3 M SA solution were mixed with 1 mL of 0.5 M
H2SO4 solution. After mixing the SA concentration was 8 × 10−4 M.
The experiments were carried out at 0.2 A and 4 V, 0.4 A and 5 V with
a platinum electrode as anode and a graphite electrode as cathode.
Both experiments were performed at room temperature, pH of 1.4
and a reaction time of 105 min. The power source was GWINSTEK
GPR-1820HD. pH was monitored with a HI 9811 HANNA instru-
ments potenciometer. Chemical oxygen demand was established
with a HACH DR/4000U and an Orion COD 165 thermoreactor. Sam-
ples were taken every 15 minutes and analyzed in a UV-Vis Perking
Elmer Precisely Lambda 25 spectrophotemeter.
Fig. 1. Absorption spectra of SA, 2,3-DHBA and 2,5-DHBA.
and its energy decreases proportionally to the strength of the
formed hydrogen bridge bond. The electronic excitation occurs
in such a short time that the system is not able to adapt to that
new electronic distribution. Thus the mobility of an electron from
the n to the ␲* orbital eliminates the possibility of stabilization
of the excited state and the global result is the aforementioned
hypsochromical displacement.
3.2. Calibration curve
Different calibration curves were built for the three analytes in
the concentration range given in Table 1. These calibration curves
were fitted to linear models (column 3, Table 1). The determination
coefficient of such models and their concentration range of linearity
are also presented in Table 1.
2.3. Chemical Oxygen Demand (COD)
Chemical Oxygen Demand measurements were carried out
according to the Standard Methods for the Examination of Water
and Wastewater [9]. The closed reflux with calorimetric mea-
surements was the followed standard method. This analysis was
employed to verify the effectiveness of the conducted electrolysis.
In this case, however, the aim was not to decrease the COD but
to establish the operating conditions to achieve only the partial
oxidation of SA towards 2,3-DHBA and 2,5-DHBA.
Once the calibration curves for the three analytes were estab-
lished, the quantification of ᭹OH was carried out.
3.3. Identification of species
In previous studies, when employing SA as free ᭹OH scavenger,
the following reaction products were identified, 2,3-DHBA, 2,5-
DHBA and catechol [5,7,10–12]. However, 2,5-DHBA and catechol
have been reported to occur in very small quantities [10] or to do
not appear at all [11]. It has been reported that 2,3-DHBA is pro-
duced in higher quantity than 2,5-DHBA when ᭹OH radicals are
chemically produced while 2,5-DHBA is favored over 2,3-DHBA
when ᭹OH radicals are electrochemically produced [13]. The pro-
duced hydroxylated compounds at 0.2 A, 4 V and 0.4 A, 5 V, were
analyzed in a UV-Vis Spectrophotometer and the obtained spectra
are presented in Figs. 2 and 3. In these Figures, it can be observed
that the maximum absorbance gradually diminishes and presents a
hypsochromic displacement due to the reaction between Salicylic
Acid and ᭹OH radicals. The maximum absorbances were found at
303 nm for SA, 310 nm for 2,3-DHBA and 329 nm for 2,5-DHBA (see
Fig. 1). As can be observed in Figs. 2 and 3, the maximum absorbance
hypsochromic displacement tends to the maximum absorbance of
the corresponding hydroxilated products. It is worth noticing that
these products are due to the reaction between SA and ᭹OH radi-
cals. These radicals are produced during the water oxidation that is
expected to occur in the platinum anode according to Ec. (1) [12].
3. Results and discussion
3.1. Analysis of salicylic acid and hydroxylated derivatives
standards
Standard solutions of SA, 2,3-DHBA and 2,5-DHBA with 1 × 10−3
,
1 × 10−3 y 8 × 10−4 M concentrations, respectively, were prepared
and analyzed in order to establish the wavelength at which the
maximum absorption is observed for every analyte. Fig. 1 depicts
the resulting absorption spectra. The aforementioned compounds
*
are carboxylic acids and therefore present n␲ transitions. Since
these compounds can be classified as chromophores, their maxi-
mum absorbance is expected to occur at a wavelength higher than
230 nm. Indeed, it can be observed in Fig. 1 that the maximum
absorbance of SA, 2,3-DHBA and 2,5-DHBA is at 303 nm, 310 nm
and 329 nm, respectively. This hypsochromical displacement of
the n␲* bands is expected and is due to the mobility of an electron
from a n to a ␲* orbital. When a carbonyl group, like the one in the
SA, becomes in contact with ᭹OH radicals, the free electrons pair
of the carbonyl oxygen can bond with ᭹OH groups by hydrogen
bridges. The molecules configuration will be the one that favors
such interaction. The basic energy of the system is then stabilized
H2O→•OH + H+ + e−
(1)

E. Peralta et al. / Electrochimica Acta 129 (2014) 137–141
139
Table 1
Regression equation and statistics.
Analyte
Linear Range (M)
Regression Equation
R2
SA
2,3-DHBA
2,5-DHBA
6 × 10−5–1 × 10−3
6 × 10−5 - 1 × 10−3
6 × 10−5 - 8 × 10−4
A303nm= 3047[SA] 90 + 0.06 0.05
0.995
0.999
0.999
A310nm = 2720[2,3-DHBA] 32.36 + 0.08 0.018
A329nm = 3335[2,5-DHBA] 36.8 + 0.06 0.016
analyte from this type of spectra a special method should be fol-
lowed. In such a method, the total absorbance of a mixture is equal
to the sum of the absorbances of each individual compound (Eq. 2),
AT = Ax + Ay + Az
(2)
By employing the Beer-Lambert Law, Eq. (2) can be written in
terms of molar absorptivity and concentration of every analyte.
Thus, if the unknown variable is the concentration of every analyte,
this can be known by establishing an equations system with many
equations as unknown analytes concentrations. To do so, a number
of pure standards equal to the number of analytes is prepared and
the wavelength at which they present the maximum absorbance
of energy is recorded. In this case there are three analytes so that
three wavelengths for maximum absorbance were determined. At
these wavelengths, when analyzing a mixture, three absorbances
can also be determined. Thus a system with three equations was
established and was solved in a matricial way at every reaction
time. At this point it is worth noticing that this method is limited
to the feasibility of establishing linearly independent equations.
This is plausible in cases where experimentation is conducted
under careful control. In addition, special attention should be
given to the significance of the selected absorbance values so that
they are not strongly influenced by signal noise or measurement
errors.
Fig. 2. Effect of reaction time on mixture UV-Vis spectrum. Reaction conditions: 0.2
A and 4 V, pH = 1.4, SA initial concentration (t = 0 min) = 8 × 10−4 M.
3.4. Hydroxyl radicals quantification
Formally, let us define ε*i = X, Y or Z and the indice i = 1,2,3 cor-
responds to wavelengths ꢁ1, ꢁ2 and ꢁ3, respectively. Therefore we
have,
A method to quantify hydroxyl radicals has been previously
reported [14] and is based on employing SA as ᭹OH scavenger to
produce 2,3-DHBA and 2,5-DHBA. According to this method and to
the reaction stoichiometry, the sum of the hydroxilated compounds
concentration is equal to the hydroxyl radicals concentration.
Thus, the problem of quantifying ᭹OH radicals is solved when the
hydroxilated compounds concentration is established. To do so by
UV-Vis, however, it should be taken into account that the mixture
of the three analytes absorbs energy of only one wavelength (see
Figs. 2 and 3). According to quantitative analytical chemistry basic
literature [15], in order to establish the concentration of every
⎛
⎞
⎛
⎞
⎛
⎞
A1(t)
εX1 εY1 εZ1
[X](t)
[Y](t)
[Z](t)
⎜
⎝
⎟
⎠
⎜
⎟
⎠
⎝
⎠
A (t)
2
= b εX
εY2 εZ2
(3)
(4)
⎝
2
A3(t)
εX3 εY3 εZ3
which can be solved as,
⎛
⎞
⎛
⎞
−1
⎛
⎞
εX1 εY1 εZ1
A1(t)
[X](t)
[Y](t)
[Z](t)
⎜
⎝
⎟
⎠
⎜
⎝
⎟
⎠
−1
⎝
⎠
= b
εX
2
εY2 εZ2
A (t)
2
εX3 εY3 εZ3
A3(t)
where, A = Absorbance of mixture
t = reaction time
ε = Molar Absorptivity, M−1cm−1
b= Cell optical length = 1 cm
[X]= Molar salicilic acid concentration in the mixture
[Y]= Molar 2,3-DHBA concentration in the mixture
[Z]= Molar 2,5-DHBA concentration in the mixture
The absortivities for every compound were determined by a
commonly employed procedure [15] at the wavelengths of 290,
310 and 330 nm (where the pure standards show a maximum of
absorbance). Table 2 shows the value of the calculated absortivities.
The absorbances were measured at the same wavelengths but at
various reaction times. At these reaction times the concentrations
of the analytes were also determined by solving (4) and Figs. 4 and 5
were generated. The evolution of the reagent and products concen-
tration when applying 0.2 A and 4 V can be observed in Fig. 4. It
is observed that the production of ᭹OH radicals is instantaneous
since the SA concentration decays as soon as an electrical current
Fig. 3. Effect of reaction time on mixture UV-Vis spectrum. Reaction conditions: 0.4
A and 5 V, pH = 1.4, SA initial concentration (t = 0 min) = 8 × 10−4 M.

140
E. Peralta et al. / Electrochimica Acta 129 (2014) 137–141
Table 2
Molar absortivities for every analyte.
Analyte
␭ (nm)
Absortivity, ␧
(M−1 cm−1
)
S A
290
290
290
310
310
310
330
330
330
2434.70
1316.13
635.14
2591.04
2706.30
2402.49
216.97
2,3–DHBA
2,5–DHBA
S A
2,3–DHBA
2,5–DHBA
S A
2,3–DHBA
2,5–DHBA
1694.40
3238.56
Fig. 4. Concentration profiles of Salicylic Acid, 2,3-DHBA and 2,5-DHBA. Operating
conditions: 0.2 A and 4 V.
Fig. 7. Effect of time on chemical oxygen demand (COD).
thought that this observed 2,3-DHBA concentration diminishment
is due to the appearance of 2,5-DHBA. This would suggest that the
occurrence of these products is consecutive rather than in parallel.
However, by analyzing Fig. 7, it can be concluded that the dimin-
ishment of 2,3-DHBA concentration is due to mineralization (COD
decreases) and therefore the production of both acids occurs in
parallel rather than in series and this is in concordance with pre-
vious reports, [4,13,15], the mechanism is illustrated in Fig. 6. In
concordance to the stoichiometry of this reaction scheme, in the
first stage of the reaction when only 2,3-DHBA is formed (before
15 minutes), ᭹OH radical concentration is equal to 2,3-DHBA con-
centration. According to previous reports [14], in the second stage
of the reaction, when 2,5-DHBA appears, ᭹OH radical concentration
Fig. 5. Concentration profiles of Salicylic Acid, 2,3-DHBA and 2,5-DHBA. Operating
conditions: 0.4 A and 5 V.
is applied. In the first 15 minutes of reaction, this decay can be
ascribed to the oxidation of SA by ᭹OH radicals and the evidence
of this reaction is the appearance of its main products 2,3-DHBA
and 2,5-DHBA. 2,3-DHBA concentration reaches a maximum after
15 minutes of reaction. At this point this concentration starts to
decay and practically extinguishes after 45 minutes. It could be
COOH
OH
COOH
OH
2,3-Dihidroxybenzoic acid
OH
COOH
OH
+
OH
Salicylic acid
2,5-Dihidroxybenzoic acid
HO
Fig. 6. Reaction of Salicylic Acid and hydroxyl radical.

E. Peralta et al. / Electrochimica Acta 129 (2014) 137–141
141
would be equal to the sum of 2,3-DHBA and 2,5-DHBA concentra-
Acknowledgments
tions. In this case, however, COD results (Fig. 7) shows that ᭹OH
radicals are not only consumed to produce the hydroxilated prod-
ucts but also are being consumed in their mineralization. It can also
be inferred from Fig. 4, that reaction 1 is more rapid than reaction 2.
When 0.4 A and 5 V are applied to the system, both reactions may be
accelerated and this may explain why only 2,5-DHBA was detected
(see Fig. 5). The concentration of this compound is observed to
reach a maximum around 30 minutes and after this time a slight
diminishment is observed.
The authors acknowledge CONACYT for financial support
through Project 168305 and E. Peralta thanks CONACYT for schol-
arship 87037 to conduct Posgraduate studies.
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UV-Vis spectrophotometry can be used to indirectly quantify
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is 2 × 10-5 mol·L−1·min−1
.