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Electrochemical study of a glassy carbon electrode modified by poly-4-

nitroaniline murexide and its sensitivity for metal ions

Article in Analytical Biochemistry · August 2018

DOI: 10.1016/j.ab.2018.08.023

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Accepted Manuscript

Electrochemical study of a glassy carbon electrode modified by poly-4-nitroaniline

murexide and its sensitivity for metal ions

C. Ben Ali Hassine, H. Barhoumi

PII: S0003-2697(18)30325-7
DOI: 10.1016/j.ab.2018.08.023
Reference: YABIO 13118

To appear in: Analytical Biochemistry

Received Date: 27 March 2018

Revised Date: 22 August 2018
Accepted Date: 25 August 2018

Please cite this article as: C. Ben Ali Hassine, H. Barhoumi, Electrochemical study of a glassy
carbon electrode modified by poly-4-nitroaniline murexide and its sensitivity for metal ions, Analytical
Biochemistry (2018), doi: 10.1016/j.ab.2018.08.023.

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 ACCEPTED MANUSCRIPT
Electrochemical study of a glassy carbon electrode modified by
poly-4-nitroaniline murexide and its sensitivity for metal ions

C. Ben Ali Hassine1*, H. Barhoumi1

1 Laboratory of Interfaces and Advanced Materials (LIMA) FSM Monastir 5000, Tunisia

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*Corresponding author: Ben Ali Hassine Chedia, Laboratory of Interfaces and Advanced Materials
(LIMA), FSM Monastir, Tunisia, Tel: +21620874578, Email: bechedia@yahoo.fr

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Abstract

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The electrochemical modification of a glassy carbon electrode using reduced poly-
4nitroaniline (P-4NA) and it’s applicability for determination of metallic ions was performed

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in this study. The electrode modification was performed by cyclic voltammetry in the
potential range between 0,9V and 1,4V vs Ag/Ag+ (in 10mM AgNO3) at the scan rate of
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100mV/s by 50 cycles in non-aqueous media. The reduction of nitro groups on the P-4NA
modified glassy carbon electrode surface was performed in the potential range between -0,1V
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and -0,8V vs Ag/AgCl(Sat. KCl) at a scan rate of 100mV/s in 100mM aqueous HCl solution .
The reduced P-4NA glassy carbon surface was modified with the murexide. The affinity of
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the modified glassy carbon electrode with some metallic ions was investigated by
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electrochemical impedance spectroscopy method in phosphate buffer solution (pH=5).

Keywords: Glassy carbon electrode; 4-nitroanoline; murexide; electrochemical

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measurements; metal ions complexation.

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1. Introduction
Metallochromic indicator can be used for sensor application by spectrophotometric method or
other methods like zincon [1], Alizarin yellow R [2], 1-(2-Pyridylazo)-2-naphthol [3],
dithizone [4],erichrom black [5] and murexide [6]. Murexide has been used in many studies
as a metallochromic indicator for its complexometric properties[7]. Murexide is the
ammonium salt of purpuric acid named as ammonium 2,6-dioxo-5-(2,4,6-trioxo-tetrahydro-

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pyrimidine-5-ylidene amino)-1,2,3,6-tetrahydro-pyrimidine-4-olate, which in its dry state has
the appearance of a reddish purple powder slightly soluble in water and is used in analytical

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chemistry as a complexometric indicator. Murexide is commonly used in conventional EDTA
titrations, most often of calcium ions, but also of Cu, Ni, Co and rare earth metals. In addition

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to its classical use as a metallochromic indicator, murexide has been employed as an efficient
scavenger for superoxide and hydroxyl radicals and recently as a chromogenic agent for
conventional spectrophotometric determination of some metals [8,9]. Heavy metals are

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considered as highly toxic components since they are non-biodegradable and lead to greater
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risks to the human health and the environment. Increasing industrial activities are causing the
release of heavy metals into the environment. Heavy metals such as Hg, As, Pb, and Cu are
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considered highly toxic and carcinogenic at a trace level. They are accumulated in the
biosphere and penetrate living organisms through the alimentary chain [10]. Heavy metal
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pollution has become a concern for global sustainability. However, it is essential to keep track
of the level of heavy metals in the environment, drinking water, food, and biological fluids.
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Several analytical techniques such as inductively coupled plasma-mass spectroscopy, atomic

absorption spectroscopy and atomic fluorescence spectroscopy are being used to detect these
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dangerous toxic metal ions [11-14]. However, these methods are expensive, time consuming
and bulky. Electrochemical sensors can be employed to overcome the limitations of other
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methods. In deed, they provide an easier portability, a rapid response, a high sensitivity and a
low detection limit [15,16]. The murexide being a complexing agent against various metals
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can be used to detect metal ions. Recently, there has been increasing interest in electrode
modification with some polymers such as electrochemically synthesized polyaniline [17,18]
to have better analytical performances. The electrochemical determination of Cu(II) ions by
differently modified electrodes has been realized by some other research groups [19]. A
modified electrode with 4-Formylphenylboronic Acid used for detection of copper ions have
been reported [20]. In this work, a glassy carbon electrode was modified by poly-4-
nitroaniline reduced using cyclic voltammetry in HCl (0,1M). The modification of a glassy

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carbon electrode by poly-4-nitroaniline was studied [21]. After the electrochemical
modification of the glassy carbon electrode, the murexide was deposited on the poly-4-
nitroaniline-red modified glassy carbon electrode. The modified glassy carbon electrode
sensitivity was tested towards Hg2+, Cu2+, Ni2+ and Co2+ cations by impedance spectroscopy
in a redox reagent.

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2. Material and Methods
2.1. Chemicals

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Ferricyanide (K4Fe(CN)6) and ferrocyanide (K3Fe(CN)6), Potassium monohydrogen
phosphate (K2HPO4) and Potassium dihydrogen phosphate were purchased from Fluka. 4-

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nitroaniline, Tetra-n-butylammonium tetrafluoroborate, acetonitrile and murexide were
purchased from Merck. KCl (potassium chloride) was purchased from PROLABO. Aqueous
solutions were prepared using distilled-deionised water.

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2.2. Apparatus
A traditional three-electrode cell system was used in all electrochemical experiments. A
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platinium wire was used as a counter electrode and GC electrodes were used as working
electrodes with a geometric area of 0.07cm2. The reference electrode was used as a Ag/AgCl
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in saturated KCl (Ag/AgCl/(sat.KCl)), which was used in aqueous media and a Ag/Ag+ (in
10mM AgNO3), which was used in non-aqueous media. The electrochemical experiments
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were carried out using a potentiostat (DY200 POT Eco-chemie) for cyclic voltammetry (CV)
and an Autolab PGSTAT 320N potentiostat for impedance spectroscopy spectra
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measurements controlled by computer with software (NOVA 1.5) for data analysis. For
impedance measurements, all experiments were carried out inside a Faraday cage. UV-visible
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absorbance spectra were recorded with a 67 series spectrophotometer Model 6705 UV/Vis
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JENWAY. FTIR spectra were recorded on a Perkin Elmer 1600 FTIR spectrometer from 400
to 4000 cm-1 after redisollution of the deposited layers in dichloromethane.

2.3. Electrode modification procedure

The surface of the GC electrode was hand-polished with a 10µm alumina–water slurry using a
polishing cloth. The polished electrode was washed and sonicated in acetone for 5 min in
order to clean the electrode’s surface. An electrochemical pretreatment step was used under
cyclic voltammetry in 0.5 M NaOH at 50 mV/s from 0.6 to 1.4 V versus Ag/AgCl followed

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by rinsing with deionised water [22]. Then, the electrode was sonicated in a mixture of 1:1
(v/v) CH3CN with isopropylalcohol for10 min. The electrode was modified employing 50
reversible potential cycles between +0.9V and +1.4V in CH3CN containing 100mM TBATFB
at the scan rate of 100mV.s-1(Figure2). The electrochemical reduction of nitro groups on the
P-4NA/GC surface to amino groups was performed by 45 potential cycles in 100mM aqueous
HCl solution at the potential range between −0.1V and −0.8V applying a scan rate of

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100mV/s (Figure3). The reduced P-4NA glassy carbon surface was dipped into a solution
containing 1mM murexide for 19 h in order to covalently bind the –C=O groups of murexide

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and the –NH2 groups of P-4NA-red/GC surface. Then, the modified glassy carbon electrode
was characterized using electrochemical methods (CV, EIS…).

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3. Results and discussion
3.1. Modification of GC electrode

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Cyclic voltammograms of poly-4-nitroaniline were performed in CH3CN and 100mM
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TBATFB at the scan rate of 100mV.s-1. Figure 1 shows the cyclic voltammograms of the GC
electrode surface electrochemically modified with P4NA. From Figure 1, it can be seen that
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polymerization of 4NA is indicated by changes in cyclic voltammograms registered during

modification of the GC electrode. At the first potential scan an irreversible peak is registered
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at approximately +1150 mV, which could be addressed to 4NA molecules adsorbed on the
GC surface. Subsequently, a decrease in the current density was noticed in the cyclic
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voltammograms spectrum. This decrease is caused by the formation of conjugated P4NA.

This is explained by the specific effects of the presence of an electron withdrawing nitro
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group and an electron donating amino group in para position in the aromatic system of the
P4NA [21]. The electrochemical reduction of nitro groups into amino groups was performed
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by cyclic voltammetry in HCl (0.1M) at a potential range between −0.1V and −0.8V and a
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scan rate of 100 mV/s. Figure 2 shows the cyclic voltammograms for the reduction of P4NA
at the electrode surface. An irreversible peak is observed at around -0.4V in the first cycle
corresponding to the reduction of the 4-nitrophenyl group to 4-aminophenyl (Ar–NH2) [25].
The peak current gradually disappeared during scans, indicating the reduction of the amino
groups. Afterwards, the electrode was washed carefully with deionised water and then dipped
into a solution of 1V:1V water:ethanol containing 1mM murexide for 19 hours.

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3.2. Determination of pKa of the deposited layer

The substitutional groups with a strong electron withdrawing substituents such as nitro group
can significantly affect the polymerization process of aniline. From literature, when the pH is
high (pH> pKa) nitro groups of P-4NA dissociates and become negatively charged. When the

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pH is low (pH <pKa) the nitro group on the layer should be protonated. Therefore, acidity
constants are varied [21]. That is why, different cyclic voltammograms were registered at

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3-/4-
different pH in the presence of redox couple [Fe(CN)6] . The nitro group of this polymer
exhibits significant pH dependence as shown in Figure 3 and Figure 4. In order to estimate the

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pKa value of the deposited polymer, we have plotted the characteristic curve dIpa/dpH=f(pH).
From Figure 4(A), the estimated pKa is equal to 2.54. This value is similar to literature for the

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poly-4-nitroaniline (pKa '= 2.59) [21]. The Figure 4(B) indicates that after reduction of P-
4NA and deposition of murexide, three pKa were found pKa '= 2.557, pKa '= 4.556 and pKa'
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= 6.7. The first two acidity constants are corresponding respectively to the acid constants of
the groups -NO2 and -NH2. The third acidity constant can be attributed to murexide molecules
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covalently bound to the surface of the modified electrode.

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3.3 Electrochemical characterization of bare GC, P4NA/GC, Reduced-P4NA/GC and

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Mu/Reduced-P4NA/GC electrodes

The modified electrode was characterized using cyclic voltammetry and impedance
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spectroscopy. In order to characterize the formation of the P4NA layer on the GC electrode
surface, the cyclic voltammograms were recorded in the presence of the redox active
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compounds: Fe(CN)63−/Fe(CN)64− in 0.1M PBS pH=5, and ferrocene in CH3CN containing

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100mM of TBATFB (Figure 5,6). Ferrocene is a metallocene (MCP2) (Cp: cyclopentadienyl

anion) and belongs to the most important class of organometallic compounds. Ferrocene
(FeCp2) has reversible redox properties according to the reaction [23]:
→ +
Figure 5 and 6 shows the cyclic voltammograms before and after modification of the GC
electrode. The voltammograms of ferrocyanide redox system present a reversible behavior at
the bare and modified electrodes with a small peak separation for the bare electrode
∆E=78mV and a larger peak separation for the modified electrode ∆E=284mV for the P-

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4NA/GC electrode, ∆E=142mV Reduced-P-4NA/GC electrode, ∆E=161mV Mu/Reduced-P-
4NA/GC electrode. As it is shown in Figure 5, after the modification of the GC electrode by
P-4NA a decrease in the peak current is observed due to the negative charges of the polymer
(-NO2) and the negative charges of ferrocyanide. After reduction and murexide incorporation
an increase in the peak current appeared. The voltamograms of ferrocene redox system
present a reversible behavior at the bare and modified electrodes (Figure 6). After the

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modification of the GC electrode an increase in the peak current of ferrocene in non-aqueous
solution is observed and demonstrate the formation of P-4NA layer on the GC surface.

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The characterization of the thin layer on the GC electrode was also performed by EIS
measurements at an applied potential of 215mV in the presence of redox compounds
Fe(CN)63−/Fe(CN)64 in 0.1M KCl as shown in Figure 7. Impedance spectra were fitted to

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equivalent circuits using NOVA 1.5 and the obtained results are given in Table 1. The nyquist
plots of the bare GC electrode represents at low frequency a straight line with a small semi-

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circle at high frequency region. On the other hand the nyquist plots of P-4NA/GC electrode
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represent a semi-circle at high frequency which is related to an increase in the charge transfer
resistance (Table 1). This increase can be explained by a repulsion between the nitro groups
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of P-4NA on the surface of the electrode negatively charged and the redox couple used.

3.4 Caracterization using FTIR and UV-visble spectroscopy

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FTIR spectra of P-4NA, P-4NA–RED and P-4NA–RED/Mu were registered on a Perkin

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Elmer 1600 FTIR spectrometer from 400 to 4000 cm-1. FTIR spectra of were depicted in
Figure 8. Characteristic FTIR band of P-4NA are observed [24]. After addition of murexide a
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band appeared at 3300 cm-1 it’s due to amide stretching vibration, the same band is observed
for murexide spectra. The UV–visible absorption spectra of P-4NA, P-4NA–RED and P-
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4NA–RED/Mu recorded at room temperature are given in Figure 9. Figure 9(a) shows that 2
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distinctive peaks of P-4NA appear at about 230 nm and 278 nm. From Figure 9(b) it can be
noted that the peak at around 278nm desapeared and the peak at about 230 nm has been
shifted to 240nm becoming larger. From Figure 9(c) relative to the P4NA-red/Mu, a large
peak appear at around 300 nm and can be explained by the incorporation of murexide to the
layer.

3.5 Complexation-ability behaviour of the Mu/Reduced-P4NA/GC electrode with some

metal ions

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In order to check the complexation-ability of the Mu/Reduced-P4NA/GC electrode surface,
EIS measurements were employed in 0.1M of PBS buffer solution containing 1.0mM
equivalent molar ratio of Fe(CN)63−/Fe(CN)64−, pH =5.0. The complexation reactions studied
in this work were investigated between Mu/Reduced-P4NA/GC and Co2+, Ni2+, Cu2+, Co2+
and Hg2+. Results are shown in Figure 10. Impedance spectra were registered with different
concentrations of ions solution. Standard Randles equivalent model circuit used to estimate

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the analytical parameters is presented in Figure 10. Equivalent circuit includes the solution
resistance (Rs), the charge transfer resistance (RCt), the Warburg resistance (Wo) and the

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constant phase element (CPE). The Nyquist plots represent a semi-circle at high frequency
region and a straight line at low frequency. An increase of the impedance spectrum is noticed

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for the electrode after ions complexation. This is explained by an increase in the charge
transfer resistance. In order to evaluate the modified electrode selectivity, we have plotted the
variation of (log Z) as a function of the (-log([X(mol/L)]) at frequency value of 3.0 Hz (Figure

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11). The features of the Mu/Reduced-P-4NA/GC electrode as well as linear ranges and
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detection limits are gathered in Table 2. According to the results, the sensitivity of the
Mu/Reduced-P-4NA/GC electrode with the transition metal ions varies in this order Cu2+ ›
Hg2+›Ni2+› Co2+. In previous studies the complexation of murexide and some metallic ions
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were investigated by spectrophotometric method [26, 27]. The order of stability constants for
the obtained metal ion–murexide complexes was investigated in DMSO and found that Cu2+
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ions have the best stability of complexation which is in conformity with our results [27]. The
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stability constants of metallic ions and murexide complexes was studied and determinated in
previous works [26-28].
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4. Conclusion
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According to this work, electrochemical polymerization of 4NA in a non-aqueous media was

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performed. The electrochemical reduction of nitro groups to amino groups was done by cyclic
voltammetry and it was followed by attachment of Murexide ligand. The formed
Mu/Reduced-P4NA/GC electrode exhibited an attractive ability to accumulate efficiently
trace of metal ions from aqueous solutions. Results show that the sensitivity of the
Mu/Reduced-P4NA/GC electrode with the transition metal ions varies in this order Cu2+ ›
Hg2+›Ni2+› Co2+.

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Table captions
Table 1: Electrochemical parameters of modified GC electrode from Nyquist plot. Equivalent
circuit used is [(R(Q[RW]].

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Table 2: Analytical performances of the Mu/Reduced-P4NA/GC electrode toward metal ions.

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Figure captions

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Graphical abstract: Illustration of the modification steps of the GC electrode
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Figure 1: Cyclic voltammetry based modification of GC electrode by electrochemical
polymerization of 4NA dissolved in CH3CN containing 100mM TBATFB by 50 reversible
potential cycles. The scan rate is 100 mV/s vs. Ag/Ag+ (in 10mM AgNO3).
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Figure 2: Electrochemical reduction of nitro groups in HCl (0.1M) by cyclic voltammetry.

Figure 3: The cyclic voltammograms of 1mM of Fe(CN)63−/Fe(CN)64− in 0.1M PBS buffer
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solution at various pHs. The scan rate is 100 mV/s vs Ag/AgCl/(sat. KCl).
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Figure 4: Relationship between the anodic peak current and pH values and differential curve
versus pH.
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Figure 5: The cyclic voltammograms of 1.0mM of Fe(CN)63−/Fe(CN)64− in 0,1M PBS of (a)

bare GC, (b) P4NA/GC, (c) Reduced-P4NA/GC and (d) Mu/Reduced-P4NA/GC electrodes.
At potential sweep rate of 100 mV/s vs. Ag/AgCl/(sat. KCl).
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Figure 6: The cyclic voltammograms of 1.0mM ferrocene in CH3CN containing 0.1M of

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TBATFB of (a) bare GC, (b) P4NA/GC, (c) Reduced-P4NA/GC and (d) Mu/Reduced-
P4NA/GC electrodes. At potential sweep rate of 100 mV/s vs. Ag/Ag+ (in 10mM AgNO3).
Figure 7: Nyquist plots of 1.0mM Fe(CN)63−/Fe(CN)64− solution in 0.1M of KCl of (a) bare
GC, (b) P4NA/GC, (c) Reduced-P4NA/GC electrode and (d) Mu/Reduced-P4NA/GC
electrode. Frequency range is from 0.05 to 75,000 Hz, the modulation amplitude is 10 mV,
working electrode potential is 215mV and the Equivalent circuit applied for calculations
[(R(Q[RW]].
Figure 8: FTIR spectra P4NA, P4NA-red, P4NA-red/Mu and murexide.

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Figure 9: Absorbance of the (a) P4NA, (b) P4NA-red and (c) P4NA-red/Mu deposited and
dissolved in dichloromethane.
Figure 10: Nyquist plots of 1.0mM Fe(CN)63−/Fe(CN)64− solution in 0.1M of PBS of
Mu/Reduced-P4NA/GC electrode after immersion in different concentration of copper ions
and the Equivalent circuit applied for calculations Working electrode potential is +0.250 V.
Figure 11: Variation of log(Z) as a function of p[X]. At frequency f=3 Hz and polarization

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potential E=250 mV/s vs Ag/AgCl(Sat KCl).

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Graphical abstract

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Figure 1

U
AN
100

Cycle 1
80
M
Current (µA)

60
D

40

Cycle 50
TE

20

0
EP

0,9 1,0 1,1 1,2 1,3 1,4

Potential (V)
C
AC

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Figure 2

SC
5
Cycle 45

U
0
AN
-5
Current (µA)

-10

-15
M

-20 Cycle 1
-25
D

-30
TE

-0,9 -0,8 -0,7 -0,6 -0,5 -0,4 -0,3 -0,2 -0,1 0,0

Potential (V)
C EP
AC

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Figure 3

SC
40

U
20
Current (µA)

AN
0

pH=1,5
M

-20 pH=2,45
pH=3,5
pH=4,5
pH=5,5
-40 pH=6,5
D

-0,3 0,0 0,3 0,6 0,9 1,2

Potential (V)
TE
C EP
AC

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Figure 4

46 60 10
I (µA) 10 I (µA)
dIpa/dpH dIpa/dpH

U
44 8
8
50
6 6
42
AN
4 4
40 40
I (µA)
dipa/dpH
I (µA)

dipa/dpH
2
2
38 0
30
0
-2
36
M

-4 -2
20
34
-6 -4

32 -8
10 -6
1 2 3 4 5 6 7
1 2 3 4 5 6 7 8
D

pH pH
TE

(A) (B)
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Figure 5

SC
40

U
30

20
AN
10
Current (µA)

0
M

-10

-20
(a)
-30 (b)
D

(c)
-40 (d)
TE

-0,4 -0,2 0,0 0,2 0,4 0,6 0,8 1,0 1,2

Potential (V)
C EP
AC

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Figure 6

50

U
(a)
40
(b)
AN
(c)
30
(d)
20
Current (µA)

10
M

-10
D

-20
TE

-30

-40
-0,2 -0,1 0,0 0,1 0,2 0,3 0,4
Potential (V)
C EP
AC

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Figure 7

U
2500 AN
2000

1500
Z'(Ω)

1000

(a)
D

500
(b)
(c)
(d)
TE

0 500 1000 1500 2000 2500 3000 3500

-Z"(Ω)
C EP
AC

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Figure 8

105 105

U
100
100
-1

95
3354 cm

-1
-1

3365 cm
720 cm

90
95

-1
-1

-1
1069 cm
-1

AN
1706 cm
1069 cm

1746 cm

T%

85 90
-1
-1
1379 cm
1450 cm

80
85
T%

75

70 80

65
-1

75
2922 cm

60
70
55
(P-4NA) (P-4NA_RED/Mu)
50 65
0 500 1000 1500 2000 2500 3000 3500 4000 4500 0 500 1000 1500 2000 2500 3000 3500 4000 4500
-1
D

Wavenumber (cm ) -1
Wavenumber (cm )
TE

105

100
100

90
95
-1
EP
-1

1450 cm
-1

1728 cm
-1

1180 cm
740 cm

90 80
-1
1650 cm
-1
T%

85
-1
1370 cm

70
1464 cm

-1
880 cm
T%

80
60
-1
3337 cm
C

75
-1
1050 cm

50
-1
2900 cm

70
(P-4NA_RED) 40 Murexide
AC

65
0 500 1000 1500 2000 2500 3000 3500 4000 4500 0 500 1000 1500 2000 2500 3000 3500 4000 4500
-1
Wavenumber (cm ) -1
Wavenumber (cm )

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Figure 9

1,4

U
(a)
1,2
230 nm

242 nm

(b)
235 nm

(c)
1,0
AN
Absorbance (a.u)

0,8
265 nm

350 nm

0,6
280 nm
0,4
M

285 nm
0,2

0,0

300 400
D

Wavelength (nm)
TE
C EP
AC

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Figure 10

U
3000
0M
-9
AN
10 M
-9
2500 5.10 M
-8
10 M
-8
5.10 M
2000 -7
10 M
-7
M
-Z"(Ω)

5.10 M
-6
1500 10 M
-5
10 M
-4
10 M
1000 -3
D

10 M

500
TE

500 1000 1500 2000 2500 3000 3500 4000 4500

Z'(Ω)
C EP
AC

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Figure 11

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3,30

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2+
Ni
2+
Co
3,25 2+
Hg
AN
2+
Cu
3,20
log(Z(Ω))

3,15
D

3,10
TE

3,05

3 4 5 6 7 8 9

-log [X(mol/L)]
C EP
AC

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Table 1

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Type de surface RS (Ω) CPE (µF) Rtc (Ω) W (µF) X2

Bare GCE 240 5.35 339 386 0.06295

P4NA/GCE 352
U 6.89 1110 376 0.04590
AN
Reduced-P4NA/GCE 393 4.84 422 413 0.05064

Mu/Reduced-P4NA/GCE 348 6 651 381 0.0195

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Table 2
D

-1
Metal-ions Sensibility (Ω/mol.L ) Domaine of linearity
TE

(mol/L)
2+ -9 -3
Hg 0.01705 10 -10
EP

2+ -9 -5
Co 0.01497 5x10 -10
C

2+ -9 -3
Ni 0.01515 10 -10
AC

2+ -8 -3
Cu 0.0266 10 -10

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Highlights
• Poly-4-nitroaniline/Murexide composite was deposited on the surface of glassy carbon
electrode.
• The modified electrode was characterized using electrochemical and spectroscopic
methods.
• The affinity of the modified electrode for some metallic ions was tested using

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impedance spectroscopy.

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