pH and temperature responsive redox behavior of biologically important aniline derivatives

Article abstract of DOI:10.1039/c5ra10822e

The redox behavior of two biologically important aniline derivatives, 2-(4-aminophenyl) ethanol and 4-aminobenzoic acid was scrutinized in a wide pH range of 2.0-12.0 using modern electrochemical techniques. The results of cyclic voltammetric experiments conducted at different temperatures were used for the evaluation of kinetic and thermodynamic parameters such as diffusion coefficient, heterogeneous electron transfer rate constant, Gibbs free energy, enthalpy and entropy changes. Differential pulse voltammetry was employed for the determination of the acid-base dissociation constant (pK_a) and evaluation of the number of electrons and protons involved in redox reactions. The comparatively more rapid and sensitive voltammetric technique, square wave voltammetry allowed the determination of quite a low limit of detection and quantification of the analytes. Computational studies were performed for the identification of the most favorable oxidizable electropores of the compounds and for developing structure-activity relationships in conjunction with the experimental results. On the basis of electrochemical and computational findings a comprehensive redox mechanism was proposed which may provide useful insights about the role by which this class of compounds exert their biological action. The proposed mechanism was found to be in line with the analysis of the product scratched from the electrode surface.

Full text of DOI:10.1039/c5ra10822e

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Lashin, U. A. Rana, N. Al-Arifi, K. Ahmad, H. Hussain, R. Qureshi, S. Ali, M. A. Zia, H. Kraatz and A. Shah,
RSC Adv., 2015, DOI: 10.1039/C5RA10822E.
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1
0
0
0
0
0
.0
.8
.6
.4
.2
0
.15
A
0.10
B
0.05
0.00
3
00 400 500 600 700 800
Wavelength/nm
B.L
Cmpd
15 dpv
25 dpv
50 dpv
75 dpv
.0
2
00
300
400
500
600
700
800
Wavelength / nm
The electrochemical oxidation of anilines resulted in azodye formation which was evidenced
by the appearance of a new peak in the UV-visible spectra (A) and colour change of
solution (B).

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pH and Temperature Responsive Redox Behavior of Biologically
Important Aniline Derivatives
a
Hanif Subhan , Aref Lashin , Usman Ali Rana , Nassir Al-Arifi , Khurshid Ahmad ,
b,c
d
e
a
f
Hidayat Hussain , Rumana Qureshi , Saqib Ali , Muhammad Abid Zia , Heinz-Bernhard
a
a
g
h
Kraatz and Afzal Shah
a,h*
a
Department of Chemistry, Quaid-i-Azam University, 45320, Islamabad, Pakistan
b
King Saud University, College of Engineering - Petroleum and Gas Engineering Department,
P.O. Box 800, Riyadh 11421 - Saudi Arabia.
c
Benha University, Faculty of Science - Geology Department, P.O. Box 13518, Benha-
Egypt.
d
Sustainable Energy Technologies Center, PO-box-800, King Saud University, Riyadh
1
1421, Saudi Arabia
King Saud University, College of Science - Geology and Geophysics Department, P.O. Box
455, Riyadh 11451 - Saudi Arabia.
e
2
f
UoN Chair of Oman's Medicinal Plants and Marine Natural Products, University of Nizwa,
Birkat Al-Mauz, Nizwa 616, Oman
g
University of Education, Attock campus Attock
h
Department of Physical and Environmental Sciences, University of Toronto
Scarborough, 1265 Military Trail, Toronto, M1C 1A4, Canada
*To whom correspondence should be addressed
Dr Afzal Shah
Tel: +92-5190642110
E-mail: afzals_qau@yahoo.com and afzal.shah@utoronto.ca
1

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Abstract
The redox behavior of two biologically important aniline derivatives, 2-(4-aminophenyl)
ethanol and 4-aminobenzoic acid was scrutinized in a wide pH range of 2.0-12.0 using
modern electrochemical techniques. The results of cyclic voltammetric experiments
conducted at different temperatures were used for the evaluation of kinetic and
thermodynamic parameters such as diffusion coefficient, heterogeneous electron transfer rate
constant, Gibbs free energy, enthalpy and entropy changes. Differential pulse voltammetry
was employed for the determination of acid-base dissociation constant (pKa) and evaluation
of number of electrons and protons involved in redox reactions. The comparatively more
rapid and sensitive voltammetric technique, square wave voltammetry allowed the
determination of quite lower limit of detection and quantification of the analytes.
Computational studies were performed for the identification of the most favorable oxidizable
electropores of the compounds and for developing structure-activity relationships in
conjunction with the experimental results. On the basis of electrochemical and computational
findings a comprehensive redox mechanism was proposed which may provide useful insights
about the role by which this class of compounds exert their biological action. The proposed
mechanism was found in line with the analysis of the product scratched from the electrode
surface.
Key words: Redox behavior; pH responsiveness; Kinetics; Thermodynamics; Computational
studies.
2

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1
. Introduction
In spite of the fact that aniline is toxic, a plethora of its derivatives possess potent
1
-3
anticancer anti-HIV, antioxidant,
4
5-7
8
9
antifungal and antimicrobial activities. Aniline
mustards have been reported to exhibit significant antitumor properties and two
representatives (chlorambucil and melphalan) of this class have achieved clinical status for
1
cancer treatment . Likewise, aniline containing sorafenib and its derivatives are promising
1
0
11
antitumor agents. Homoallylamines possess good antifungal activities. Similarly, the
12
antifungal properties of hetaryl groups bearing substituted anilines have been reported.
Thiourea derivatives containing aniline moiety have also been documented as potent
13
antibacterial agents . A survey of recent literature reveals that electron transfer reactions are
14
responsible for the biological activities of aniline derivatives. Consequently, detailed
knowledge about the redox behavior of this class seems conceivable for comprehending the
mechanism of their biological action. Literature survey reveals that extensive information is
15-18
available about the use of anilines as redox probes and electron transfer mediators.
However, meager information about the pH dependent electron transfer reactions of anilines
and no information about the thermodynamics of their proton coupled electron transfer
processes are available in literature. Thus, the current work is an effort for the partial
fulfilment of this gap in literature.
In agriculture, a range of aniline based compounds are used as chemical weapons for
eradicating the undesirable plants. Sulphonylureas, phenylureas and chloroacetanilides
represent important families of aniline based herbicides which have registered their potency
19,20
on commercial scale.
These herbicides exert their action either by diverting the flow of
electron in photosynthesis from its normal pathway or inhibit acetolactose-synthatase
1
9,21
enzyme.
Therefore, our current investigations about the redox mechanistic pathways of
3

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aniline derivatives are expected to provide some useful guidelines for facilitating prediction
of herbicidal role of this class of compounds.
Polyaniline is the most extensively studied ionic conducting polymers due to its high
conductivity, robust electrochemistry and electrochromic effects. Polyanilines find extensive
22
23
applications in rechargeable batteries, microelectronics and electrochromic display
24
devices. Moreover, polyanilines of high water solubility are developed for lithography and
25
mass inspection in scanning electron microscopy. Changes in conditions such as
temperature and pH of the medium can have a remarkable influence on the course of electron
transfer reactions. Thus, spurred on by the broad range applications of aniline derivatives, we
probed the pH and temperature responsive redox mechanism of two unexplored
representatives of this class by electrochemical techniques and computational studies. The
previous investigators have reported that oxidation of anilines leads to the formation of azo
2
6
dyes which can act as electro-optical sensors. Interestingly, the results of our experiments
also show that the oxidation product of anilines is both electroactive and photoactive. We
have experimentally confirmed the final product of the proposed redox mechanism and thus,
the current work is expected to be a valuable addition to the literature of aniline derivatives.
2
.
Experimental
2.1. Chemicals and reagents
Analytical grade 4-aminophenyl alcohol (4-APA) and 4-aminobenzoic acid (4-ABA) were
obtained from sigma Aldrich and used as received. Stock solutions of 2.0 mM concentration
o
were prepared in ethanol and stored at 4 C. Fresh solutions in buffer and ethanol (1:1)
mixture were prepared from stock solutions for experimental work. Britton–Robinson (BR)
buffer of 0.2 M ionic strength in the pH rang 2.0-12.0 was employed as supporting
4

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electrolyte. Bidistilled water of ≤0.1 ꢀS /cm conductance was used for the preparation of BR-
buffer solutions.
2.2. Equipments and measurements
All voltammetric experiments were performed using µAutolab running with GPES 4.9
2
software Eco-Chemie, The Netherlands. Glassy carbon (GC) having surface area of 0.071cm
was employed as working electrode while Pt-wire was used as counter electrode. Besides
this, Ag/AgCl electrode consisting of 3 M KCL solution was used as reference electrode.
Before recording any voltammetric scan, the dissolved oxygen was flushed out by purging
argon for 10 minutes and the surface of GCE was polished with diamond spray (having
particle size of 1 µm) followed by thorough rising with a jet of doubly distilled water. The
reproducibility of experimental results was ensured by placing the polished electrode in the
buffer electrolyte of desired pH followed by recording various voltammograms up to the
attainment of a steady state base line. For differential pulse voltammetry, pulse amplitude of
50 mV and pulse width of 70 ms were set as experimental conditions. Similarly for square
wave voltammetry (SWV), the experimental conditions were 2 mV potential increments and
-
1
5
0 Hz frequency with an effective sweep rate of 100 mVs . Temperature was kept constant
by immersing the measurement cell in a water circulating bath (IRMECO I-
400 GmbH Germany). Gaussian 03 package was used for computational studies. The
2
geometric structures were first optimized at hybrid Becke three- parameter Lee–Yang–Parr
exchange correlation functional (B3LYP) level of 631-G basis set and then the most stable
conformations were proceeded for charge distribution calculation.
5

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3
.
Results and Discussion
3.1. Cyclic Voltammetry (CV)
CV was used for getting a general portrait of the redox behavior of the compounds. CVs of 4-
APA and 4-ABA shown in Fig. 1 depict a prominent and well defined anodic wave, 1a, at
0.79 and 0.82 V respectively. The appearance of a very small cathodic peak corresponding to
the reduction of the oxidized product of 1a in the reverse scan demonstrates the quasi-
reversible nature of the inclusive oxidation process. The different current intensities (Ia/Ic ≠
1) of the anodic and cathodic signals point to follow up reactions of the oxidized product. The
broad reduction signals of 4-APA and 4-ABA at -0.64 and -0.62 V disappear after purging
with argon and hence these emerge due to reduction of interrupting dissolved oxygen.
Besides the above mentioned signals, 4-ABA also shows a reduction wave, 1c*, at -0.10 V
with corresponding oxidation signal (2a) at -0.03 V in the next successive scan. The peak
separation of 49 mV demonstrates the reversible nature of the redox couple labeled as 2a and
1c. Moreover, the comparatively lower oxidation potential of 4-APA suggests this compound
as a comparatively stronger reducing agent than 4-ABA. The rationale behind the lower
oxidation potential of 4-APA can be related to the electron donating effect (via hyper
conjugation) of the alkyl group to encourage the oxidation of amino group as compared to 4-
ABA possessing COOH as electron withdrawing group at the aromatic ring.
The effect of sweep rate variation on the CV response of the analytes was examined
in a medium of pH 7.4 for the determination of diffusion coefficient (D) at different
temperatures. The CVs shown in Fig. S1 demonstrate the intensification of the peak current
with rise in temperature due to possible decrease in the viscosity of solution. Moreover, the
peak exhibits a slight shift with rise in temperature and sweep rate, thus, suggesting the quasi-
2
reversible nature of the electrochemical oxidation. The D values of the analytes were
7
6

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evaluated at different temperatures from the slopes of the linear plots (Fig. 2) of peak
1
/2
28
current (I ) vs square root of scan rate (υ ) using Randles-Sevcik equation.
p
ꢍ
ꢍ
ꢍ
ꢏ
ꢇ
ꢏ
ꢐꢑꢒ ꢏ
ꢃ.ꢄꢅꢀ×ꢄꢆ ꢈꢉꢊ ꢈꢌ
ꢎ
ꢎ
ꢓ
ꢎ
ꢋ
ꢀ
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢁ =
3.1
ꢂ
ꢍ
ꢏ
ꢔ
ꢎ
a
Where n denotes the number of electrons involved in oxidation reaction, α the anodic charge
2
transfer coefficient, A the area of electrode (cm ), D the diffusivity constant (cm/s) and C the
3
bulk concentration (mol/cm ) of analyte. The α
a
n values listed in Table 1 were evaluated
from the difference of E and E of the cyclic voltammetric signals shown in Fig. S1 using
p p/2
ꢆ.ꢆꢗꢘꢘ
the equation, ꢕ − ꢕ
ꢂ
=
. Inspection of table 1 reveals that the value of D
ꢂ
ꢏ
ꢖ
ꢊ ꢈ
ꢋ
progressively increases with rise in temperature because of lower solution resistance due to
increase in kinetic energy of the species at higher temperature. The D value of 4-APA is
greater than 4-ABA due to comparatively lower molecular mass and less polar nature (thus
experiencing lesser attraction with the solvent) as evidenced by its computationally
determined smaller dipole moment (vide infra in table 5). The linear log plots of peak current
versus sweep rate shown in Fig. S2 having slope values quite close to the theoretical value of
0.5 point to the diffusion controlled nature of the oxidation processes of 4-APA and 4-ABA
29-31
according to the criteria reported by the previous investigators.
The value of
heterogeneous electron transfer rate constant (k), an imperative parameter for predicting the
nature of redox process was calculated on the basis of peak 1a at different temperatures using
3
2
the equation reported in literature. An examination of table 1 and Fig. S1 reveals that the
oxidation potential drifts towards lower potentials and the values of k increase with rise in
temperature thus, signifying the facile electron abstraction at higher temperature.
Ipa/nFAC =k
3.2
7

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Where n, A, F and k represent number of electrons, active surface of working electrode,
Faraday’s constant and heterogeneous electron transfer rate constant.
For getting insights about the thermodynamics of the electrode processes, some
valuable thermodynamic parameters such as Gibbs free energy (∆G), enthalpy (∆H) and
entropy changes (∆S) were determined. The values of k calculated at five different
temperature values were subsequently employed for ∆G determination by using the following
33
equation.
∆
G = 5778.8(5.096- log k)
3.3
The slope of the Arrhenius plots shown in Fig. 3 were used for the determination of
activation energy (E
a
). Enthalpy and entropy changes were then determined according to the
33
following equations.
ꢁ
H = E
a
– RT
3.4
3.5
ꢁG = ꢁH – TꢁS
Thermodynamic parameters listed in Table 2 show positive values of ∆G and ∆H thus,
demonstrating the non-spontaneous and endothermic nature of the oxidation process while
negative ∆S points to entropy opposed process. Negative ∆S means that the product has
lower entropy than the reactant, hence, the ordered nature of the oxidized product can be
related to its adsorption over the electrode surface. Furthermore, the gradual decrease in ∆G
with temperature rise shows the inclination of redox reaction towards reversibility. In
contrast, the increase in ∆S values with elevation of temperature indicate increase of
randomness in the system as expected.
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3.2. Differential pulse voltammetry (DPV)
The redox behaviour of aniline derivatives was also probed by DPV, as this technique has the
ability of minimizing the charging current and extracting Faradaic current which leads to
34
enhanced sensitivity and greater accuracy. The DPVs of 4-APA depicted in Fig. 4A shows
two small peaks 2a and 3a besides the main oxidation signal in acidic media while no such
signals can be seen in the DPVs of 4-ABA (Fig. 5A). The peak potentials of both compounds
shift cathodically with increasing pH of the medium indicating facile electron abstraction
under higher pH conditions. The half peak width (W1/2) values calculated according to
35
equation 3.6, show one electron transfer during oxidation of the selected aniline derivatives.
The number of protons accompanying the electron transfer reactions was determined from
p
equation 3.7. Below pH 6, the slope of E -pH plot corresponding to peak 1a is 21 mV per pH
unit which indicates the loss of 0.14 proton and one electron from a single molecule of 4-
APA. This suggests the loss of one proton and seven electrons from seven molecules of 4-
APA in acidic conditions of pH ≤ 6. The slope of 37 mV per pH unit in neutral and alkaline
conditions reveals the oxidation by the involvement of 0.25 protons and one electron per
single molecule of 4-APA. Thus, the oxidation of 4 molecules of 4-APA occurs by the
+
abstraction of 1H and 4e. The E
p
versus pH plot of 4-APA (Fig. 4B) shows the switching of
mechanism at pH around 6.0 and this behavior is in good agreement with the reported results
36,37
of structurally related compounds.
Similar calculations for peaks 2a and 3a of 4-APA
indicate the participation of protons during electron transfer reactions (Fig. 4C). In case of 4-
ABA, the involvement of three electrons and one proton per three molecules is suggested in
the light of DPV results obtained in media of pH less than 7.9. In media of pH higher than
this value, protons involvement is not evidenced during electron transfer reactions (Fig. 5B).
The parameters obtained from DPVs can be seen in Table 3.
W
1/2 = 90.4 / αn
a
3.6
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No. of protons= (dE/dpH) (αn
a
)/ 0.059
3.7
3.3. Square wave voltammetry (SWV)
Due to the associated advantages of detecting lower concentration of the analytes, higher
38
sensitivity and rapid pace of analysis, SWV was employed for the determination of the limit
of detection (LOD) and the limit of quantification (LOQ) of aniline derivatives. The peak
current of the calibration standards regularly increase with rise in concentration of the
substrate as expected (Fig. 6). Nicely linear calibration plots in the concentration range of 30
µM to 0.4 mM and 7.5 µM to 0.11mM were obtained for 4- APA and 4-ABA (see inset of
Fig.6). The slope values of the linear range of the calibration curves were used for the
39
calculation of LOD and LOQ using equations 3.8 and 3.9.
LOD = 3SD / m
LOQ = 10SD / m
3.8
3.9
Where SD stands for the standard deviation of intercept and m represents slope of the linear
segment of I plotted against concentration.
p
The LOD and LOQ of both compounds and the regression parameters of the
calibration curve are listed in Table 4. The obtained detection values clearly indicate that
very low concentrations of the selected aniline derivatives can be detected using square wave
voltammetric method. The detection limits are comparable with the reported values of
structurally related aniline derivatives.
3.4. Computational studies
Computational studies were carried for the identification of the most favorable oxidizable
moiety in the compounds. First, the geometry of the compounds was optimized followed by
Mullikan charge densities calculation using standard 6-31-G basis set in Gaussian-3 package.
1
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Both aniline derivatives exhibited the highest negative charge on nitrogen atom of NH
group, suggesting it as the most favorable centre for electrochemical oxidation as shown in
Fig. 7. Moreover, the less negative energy of the highest occupied molecular orbital (EHOMO
2
)
of 4-APA than 4-ABA demonstrate its easier oxidation thus, supporting cyclic voltammetric
results. We used computational chemistry for the determination of ELUMO, ionization energy,
electron affinity and dipole moment (Table 5) of the compounds. These parameters helped in
offering explanation to the voltammetric observations; for instance, the smaller D of 4-ABA
than 4-APA was explained on the basis of its higher dipole moment. So, the more polar 4-
ABA will experience greater friction thus, hindering its mobility towards the electrode
surface.
3.5. Redox mechanism of aniline derivatives
In the light of computational findings and experimental results obtained from the three
electrochemical techniques, pH dependent redox mechanisms were suggested. Both the
compounds displayed a prominent and stable oxidation peak, 1a, in all the studied pH
conditions (Fig. 1A). Based on recent literature survey and computational investigations, it is
4
0
proposed that signal 1a emerges due to oxidation of -NH
width at half peak height of peak 1a represents the loss of one electron from this moiety. The
slope of E -pH plot corresponding to peak 1a indicates the accompaniment of only 0.14
proton during an electron transfer reaction of 4-APA. Thus, based upon the quasi-reversible
2
group of the compounds. The
p
41
a c
oxidation as evidenced by cyclic voltammetric result of I /I ≠ 1 and literature report, the
molecule of 4-APA is suggested to form a cationic radical followed by the formation of a free
radical through chemical deprotonation as shown in Scheme 1. In the next step two free
radicals may combine thus leading to the formation of a stable dimer. The two small anodic
signals, 2a and 3a, in 4-APA are ascribed to successive one electron and one proton loss from
the dimer resulting in the formation of a stable azodye. The formation of such dyes has also
1
1

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40, 42
been reported by the previous researchers for compounds structurally related to 4-APA.
In solutions of pH higher than 6.0, only the main oxidation signal 1a was registered in the
voltammograms. From the W1/2 values and slope of the plot between E and pH, the redox
p
mechanism shown in Scheme 2 was suggested. The proposed oxidation mechanism of 4-
ABA is presented in Scheme 3.
Confirmation of the product (Azo dye): Electronic absorption spectroscopy and color
response of the solution provided clues about the formation of azo dye. The UV-Vis spectrum
of 4-APA showed no signal at 485 nm but the spectra of the product scratched from the
electrode surface after recording multiple differential pulse voltammograms indicated an
increase in absorbance that finally developed into a noticeable band around this wavelength
region as shown in Fig. 8A. The color response of the solution also offered evidence about
the formation of azo-dye. The solution of the starting compound is colorless but the solution
of the product scratched from the electrode surface after 75 DPV scans appears light red (Fig.
8B). The azo dye possesses -N=N-chromophore which gives a well-defined signal in the
43,44
visible region as documented by some investigators.
4
.
Conclusion
Both the aniline derivatives 4-aminophenyl alcohol (4-APA) and 4-aminobenzoic acid (4-
ABA) exhibited pH dependent and diffusion controlled quasi-reversible mode of oxidation at
GC-electrode. The appearance of corresponding small cathodic signals and values of
heterogeneous electron transfer rate constant suggested the quasi-reversible nature of the
redox processes. Temperature effect monitored for thermodynamic assessment of the electron
transfer reactions indicated non-spontaneous, endothermic and entropy opposed nature of the
oxidation processes. The increase in diffusion coefficient with rising temperature can be
1
2

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related to the decrease in viscosity and concomitant increase in kinetic energy of the
molecules. Likewise, the value of heterogeneous rate constant increased with elevation in
temperature leading the redox process to incline towards reversibility. Moreover, the
#
#
lowering of ∆G and increasing of ∆S values with rise in temperature reveals the drift of
oxidation process towards spontaneity and increase in degree of disorder of the resulting
product. Unlike the analytes, their oxidized products were found to show reversible redox
behavior as evidenced by the appearance of signals corresponding to a reversible redox
couple. The redox mechanistic pathways of the selected anilines were proposed on the basis
of experimental and computational findings. Interestingly the analysis of the product formed
at the electrode surface supported the suggested mechanism.
Acknowledgments
The authors are thankful to Higher Education Commission and Quaid-i-Azam University,
Islamabad, Pakistan. The authors also extend their sincere appreciations to the Deanship of
Scientific Research at the King Saud University for its funding this Prolific Research Group
(PRG-1436-08).
1
3

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References
1
2
3
4
5
6
7
8
9
1
1
A. Prakash, W.A. Denny, T.A. Gourdie, K.K. Valu, P.D. Woodgate and L.P. Wakelin,
Biochemistry, 1990, 29, 9799-9807.
X.-F. Huang, X. Lu, Y. Zhang, G. Q. Song, Q. L. He, Q. S. Li, X. H. Yang, Y. Wei
and H. L. Zhu, Bioorg. Med. Chem., 2012, 20, 4895-4900.
N. Kapuriya, K. Kapuriya, X. Zhang, T.C. Chou, R. Kakadiya, Y. T. Wu, T. H. Tsai,
Y. T. Chen, T. C. Lee, and A. Shah, Bioorg. Med. Chem., 2008, 16, 5413-5423.
L. Zhang, P. Zhan, X. Chen, Z. Li, Z. Xie, T. Zhao, H. Liu, E. De Clercq, and C.
Pannecouque, J. Balzarini, Bioorg. Med. Chem., 2014, 22, 633-642.
H. Nawaz, Z. Akhter, S. Yameen, H.M. Siddiqi, B. Mirza and A. Rifat, J. Organomet.
Chem., 2009, 694, 2198-2203.
M. Gizdavic-Nikolaidis, J. Travas-Sejdic, P. Kilmartin, G. Bowmaker and R. Cooney,
Curr. Appl. Phys., 2004, 4, 343-346.
J.P. Silva, V.A. Machado, R.C. Calhelha, M.-J.R. Queiroz and O.P. Coutinho, Drug
Discov Ther., 2010, 4, 246-256.
F.D. Suvire, M. Sortino, V.V. Kouznetsov, L.Y. Vargas M, S.A. Zacchino, U.M. Cruz
and R.D. Enriz, Bioorg. Med. Chem., 2006, 14, 1851-1862.
I. Yilmaz, C. Kazak, S. Gümüꢂ, E. Ağar and Y. Ardali, Spectrochim Acta., 2012, 97,
4
23-428.
0 J. Yao, Z. He, J. Chen, W. Sun, H. Fang and W. Xu, Bioorg. Med. Chem. Lett., 2012,
2, 6549-6553.
2
1 L.Y. Vargas M, M.a.V. Castelli, V.V. Kouznetsov, J.M. Urbina G, S.N. López, M.
Sortino, R.D. Enriz, J.C. Ribas and S. Zacchino, Bioorg. Med. Chem., 2003, 11, 1531-
1550.
1
4

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1
1
1
2 V.V. Kouznetsov, L.Y. Vargas Méndez, M. Sortino, Y. Vásquez, M.P. Gupta, M.
Freile, R.D. Enriz and S.A. Zacchino, Bioorg. Med. Chem., 2008, 16, 794-809.
3 S.Y. Abbas, M.A. El-Sharief, W.M. Basyouni, I.M. Fakhr and E.W. El-Gammal, Eur.
J. Med. Chem., 2013, 64, 111-120.
4 E. Bendary, R. Francis, H. Ali, M. Sarwat and S. El Hady, Annals. Agric. Sci., 2013,
58, 173-181.
15 L. Zhang, Q. Lang and Z. Shi, Am. J. Anal. Chem., 2010, 1, 102-112.
16 J. Yano and S. Yamasaki, Synthetic Metals, 1999, 102, 1157-1157.
17 X. H. Cai, K. Kalcher, C. Neuhold and B. Ogorevc, Talanta, 1994, 41, 407-413.
18 S. Mu, Biosensors and Bioelectronics, 2006, 21, 1237–1243.
19 A. Can, I. Yildiz and G. Guvendik, Environ Toxicol Pharmacol., 2013, 35, 369-379.
20 A.B. Hill, P.R. Jefferies, G.B. Quistad and J.E. Casida, Mutat Res Genet Toxicol
Environ Mutagen., 1997, 395, 159-171.
2
1 F.E. Dayan and M.L.D.M. Zaccaro, Pest Biochem Physiol., 2012, 102,189-197.
2 K.S. Ryu, K.M. Kim, Y.-S. Hong, Y.J. Park and S.H. Chang, Bull. Korean Chem.
Soc., 2002, 23, 1144-1148.
2
2
2
2
3 M. Angelopoulos, IBM J Res Dev., 2001, 45, 57-75.
4 P.R. Somani and S. Radhakrishnan, Mater Chem Phys., 2003, 77, 117-133.
5 M. Angelopoulos, N. Patel, J.M. Shaw, N.C. Labianca and S.A. Rishton, J. Vac. Sci.
Technol B., 1993, 11, 2794-2797.
2
6 K.R. Raghavendra and K. Ajay Kumar, Int. J. ChemTech Res., 2015, 5, 1756-1760.
7 A. Shah, A. Ullah, A. Rauf, Z.U. Rehman, S. Shujah, S.M. Shah and A. Waseem, J.
Electrochem. Soc., 2013, 160, H597-H603.
2
2
8 I. Ullah, A. Naveed, A. Shah, A. Badshah, G.S. Khan and A. Nadeem, J Surfactants
Deterg., 2014, 17, 243-251.
1
5

Page 17 of 28
RSC Advances
View Article Online
DOI: 10.1039/C5RA10822E
2
9 A.J. Bard and L.R. Faulkner, Electrochemical Methods: Fundamentals and
Applications, Wiley, 2001.
30 J. Wang, Analytical electrochemistry, John Wiley & Sons, 2006.
31 S.A. Ali and M. Sami, Pak. J. Pharm. Sci., 2005, 18, 6-17.
32 S. Munir, A. Shah, A. Rauf, A. Badshah, S.K. Lunsford, H. Hussain and G.S. Khan,
Electrochim. Acta, 2013, 88, 858-864.
3
3 S.A. Yasin, Portugaliae Electrochim Acta, 2006, 24, 23-36.
4 A.C. Michael and L. Borland, Electrochemical methods for neuroscience, CRC Press,
3
2007.
3
5 T.A. Enache and A.M. Oliveira-Brett, J. Electroanal. Chem., 2011, 655, 9-16.
6 O. Thomas and C. Burgess, UV-visible spectrophotometry of water and wastewater,
Elsevier Science, 2007.
3
3
7 C.A. Hollingsworth, P.G. Seybold and C.M. Hadad, Int J Quantum Chem., 2002, 90,
1396-1403.
3
8 S. Altınöz and B. Uyar, Analytical Methods, 2013, 5, 5709-5716.
3
9 K. Ahmad, A.H. Shah, B. Adhikari, U.A. Rana, C. Vijayaratnam, N. Muhammad, S.
Shujah, A. Rauf, H. Hussain and A. Badshah, RSC Adv., 2014, 4, 31657-31665.
0 R.M. Kotkar and A.K. Srivastava, Sensor Actuat B Chem., 2006, 119, 524-530.
1 J. Liu, L. Cheng, B. Liu and S. Dong, Langmuir, 2000, 16, 7471-7476.
2 A. Zaouak, F. Matoussi and M. Dachraoui, Int. J. Electrochem., 2011, 2011, ID
4
4
4
9
04570, 1-6.
3 M. S. Zakerhamidi, A. ghanadzadeh and M. Moghadam, Chem. Sci. Trans., 2012, 1,
-8.
4 M. S. Masoud, A. E. Ali, M. A. Shaker and M. A. Ghani, Spectrochim. Acta, 2004,
0, 3155-3159.
4
4
1
6
1
6

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Schemes
7
H N
2
H N
2
HN
6
OH
OH
1 H+
OH
-
follow up chemical reaction (deprotonation)
6
HN
OH
HO
H
N
N
2
HN
H
OH
OH
2e, 2H
HO
N
N
OH
Scheme 1.
Proposed oxidation mechanism of 4-APA in solutions of pH ≤ 6.
1
7

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4
H N
2
H N
2
HN
3
OH
OH
1 H+
OH
-
follow up chemical reaction (deprotonation)
3
HN
OH
HO
H
N
N
2
HN
H
OH
OH
2
e, 2H
HO
N
N
OH
Scheme 2.
Proposed oxidation mechanism of 4-APA in media of pH ˃ 6.
O
O
O
OH
OH
OH
H N
2
H N
2
H N
2
-
1H
HO
H
N
N
O
O
O
HN
H
OH
OH
Scheme 3.
Proposed oxidation mechanism of 4-ABA corresponding to peak 1a.
1
8

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Tables
Table 1.
Diffusion coefficient and heterogeneous rate constant values for 4-APA and 4-
ABA calculated at different temperatures.
-
5
/10 cm s
2
-1
-3
/10 cm s
-1
Compounds
Temperature/K
α
a
n
D
k
3
3
318
3
3
08
13
0.40
0.45
0.51
0.55
0.62
5.20
5.39
5.63
5.88
6.35
3.33
3.43
3.76
4.12
4.52
4
-APA
23
28
3
3
313
3
3
3
03
08
0.43
0.49
0.54
0.57
0.61
0.63
4.75
4.97
5.31
5.52
6.07
6.56
3.24
3.44
3.83
4.26
4.48
4.83
4
-ABA
18
23
28
1
9

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Table 2.
Thermodynamic parameters of aniline derivatives as determined from CV
#
G /(kJ/mol) Ea/(kJ/mol)
#
H /(kJ/mol)
#
S /(J/K.mol)
Comp. Temperature/K
∆
∆
∆
3
3
3
3
3
08
13
18
23
28
43.77
43.69
11.13
11.09
11.05
11.00
10.97
-12.75
-12.53
-12.26
-12.00
-11.75
43.46
13.69
43.24
4-APA
43.00
3
3
3
3
3
3
03
08
13
18
23
28
43.84
43.69
11.26
11.22
11.17
11.13
11.09
11.05
-12.93
-12.68
-12.39
-12.11
-11.89
-11.66
43.42
13.78
43.15
4-ABA
43.02
42.83
Table 3. Number of electrons, protons and pKa of aniline derivatives calculated from DPV
No. of
electrons plot (V/pH)
Slope
E
p
-pH
No. of
protons
PKa
Compounds Peaks
1
a
1
-0.020
-0.035
-0.083
-0.090
0.14
0.25
1
4
-APA
6.0
2
3
a
a
1
1
1
1
2a
a
1
1
1
-0.043
---
---
0.30
1
1
7.9
4
-ABA
1c
2
0

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Table 4.
LOD and LOQ of aniline derivatives and other linear fit parameters calculated
from calibration curves.
-
7
-5
Compounds
SD /10
A)
Slope/10
3
LOD/
LOQ/
(Adm (µmol) ) (µmolL ) (µmolL )
Linearity
range ( M)
-1
-1
-1
(
µ
4
4
-APA
-ABA
2.469
6.128
6.127
12.09
12.08
40.29
40.29
7.5-80
30-479
2.468
Table 5. Computationally determined parameters of aniline derivatives
Parameters
4-APA
4-ABA
EHOMO / (e V)
-5.974
-5.269
E
LUMO / (e V)
0.0591
-
1.083
I.E / (e V)
E.A / (e V)
5.269
0.059
5.974
0.040
Oxidation potential / (V)
Dipole moment / (Debye)
0.725
3.788
0.863
8.028
2
1

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Figures
4
0
0
0
4
0
0
0
1a
A
B
1a
2
2
2a
-20
-40
-60
Without purging
After 10 min N purging
I
2
Scan1
Scan2
Scan3
Scan4
Scan5
-
20
40
1c*
-
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5
E / V vs. Ag / AgCl
E / V vs. Ag / AgCl
Fig. 1. CVs of 1mM (A) 4-APA and (B) 4-ABA obtained in a medium of pH 7.4 at a scan rate of 100
-1
mVs .
Temp.
/
K
Slope
Temp/ K Slope
303 9.72E-5
308 1.03E-4
4
3
3
2
2
1
0
5
0
5
0
5
A
40
B
3
3
08 9.52E-5
13 1.02E-5
35
318 1.1E-4
3
13 1.11E-4
3
3
23 1.16E-4
28 1.26E-4
318 1.13E-4
323 1.24E-4
30
25
20
15
10
0
.12 0.15 0.18 0.21 0.24 0.27 0.30 0.33
0.12
0.16
0.20
0.24
0.28
0.32
1
/2
-1 1/2
)
1/2
-1 1/2
υ
(Vs
υ
(
Vs
)
Fig. 2. Plots of current intensity of peak 1a versus square root of scan rate using 1mM
solution of (A) 4-APA and (B) 4-ABA.

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-5.32
-5.39
-5.46
-5.53
-5.60
-5.67
-5.74
4-ABA Slope = -1671.40
4
-APA
Slope = -1647.05
0
.00305 0.00310 0.00315 0.00320 0.00325
-1
1/T(K)
o
Fig. 3. Plots of lnk as a function of 1/T.

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3
.6
.8
1
2
A
B
0
0
0
0
.77
.70
.63
.56
1
0
.0
pKa = 6
3
.6
11
9
.8
I
7
5
2
0
.0
.5
.0
4
0.49
4.4
2.2
0.0
2
4
6
8
10
12
3
1a
2
1
0
pH
2
a
3a
0
.0
0.5
1.0
1.5
E / V vs. Ag/AgCl
1.19
1.12
1.05
0.98
0.91
C
Peak 3
Slope = 0.0904
Peak 2
Slope = 0.083
2.4 2.7 3.0 3.3 3.6 3.9 4.2
pH
-1
p
Fig. 4. (A) DPVs of 4-APA recorded in in the pH range 3 - 11at 5 mVs . E - pH plots of 4-
APA corresponding to peak (B) 1a (C) 2a and 3a.

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-1
Fig. 5. (A) DPVs of 4-ABA obtained in the pH range 3 - 11at 5 mVs and (B) Plot of peak
potential as a function of pH.
Fig. 6. SWVs of different concentrations of (A) 4-APA and (B) 4-ABA obtained at 100 mV
-1
s in a medium of pH 7.4. Insets show current intensity vs. concentration plots.

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Fig. 7. Mullikan charge distribution on atoms of 4-APA.

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1
0
0
0
0
0
.0
.8
.6
.4
.2
0
.15
A
0.10
B
0
.05
.00
0
3
00 400 500 600 700 800
Wavelength/nm
B.L
Cmpd
15 dpv
25 dpv
50 dpv
75 dpv
.0
2
00
300
400
500
600
700
800
Wavelength / nm
Fig. 8. (A) UV-Vis spectra of 4-APA recorded after different number of DPV scans, inset
spectra is a demonstration of better visualization of signal around 485 nm (B) Colorless
solution of 4-APA and light red colored solution of its oxidized product obtained from the
electrode surface after 75 scans.