Recognition of Heavy Metal Ions by Using E-5-((5-Isopropyl-3,8-Dimethylazulen-1-Yl) Dyazenyl)-1H-Tetrazole Modified Electrodes

S S symmetry

Article Recognition of Heavy Metal Ions by Using E-5-((5-Isopropyl-3,8-Dimethylazulen-1-yl) Dyazenyl)-1H-Tetrazole Modiﬁed Electrodes

Adina-Maria Păun, Ovidiu-Teodor Matica, Veronica Anăstăsoaie, Laura-Bianca Enache, Elena Diacu * and Eleonora-Mihaela Ungureanu

Faculty of Applied Chemistry and Materials Science, University “Politehnica” of Bucharest, Gheorghe Polizu 1-7, Sector 1, 011061 Bucharest, Romania; [email protected] (A.-M.P.); [email protected] (O.-T.M.); [email protected] (V.A.); [email protected] (L.-B.E.); [email protected] (E.-M.U.) * Correspondence: [email protected]; Tel.: +40-722366378

Abstract: Chemically modified electrodes (CMEs) based on polymeric films of E-5-((5-isopropyl- 3,8-dimethylazulen-1-yl) diazenyl)-1H-tetrazole (L) deposited on the surface of the glassy carbon electrode have been used for the recognition of heavy metal (Me) ions. The electrochemical study of L was done by three methods: differential pulse voltammetry (DPV), cyclic voltammetry (CV), and rotating disk electrode voltammetry (RDE). The CV, DPV, and RDE studies for L were performed at different concentrations in 0.1 M tetrabutylammonium perchlorate solutions in acetonitrile. The polymeric films were formed by successive cycling or by controlled potential electrolysis (CPE). Citation: P˘aun,A.-M.; Matica, O.-T.; The film formation was proven by recording the CV curves of the CMEs in ferrocene solution. An˘ast˘asoaie,V.; Enache, L.-B.; Diacu, The CMEs prepared at different charges or potentials were used for detection of heavy metal ions. E.; Ungureanu, E.-M. Recognition of Synthetic samples of heavy metal ions (Cd(II), Pb(II), Cu(II), Hg(II)) of concentrations between 10−8 Heavy Metal Ions by Using E-5-((5- and 10−4 M were analyzed. The most intense signal was obtained for Pb(II) ion (detection limit of Isopropyl-3,8-Dimethylazulen-1-yl) about 10−8 M). Pb(II) ion can be detected by these CMEs in waters at such concentrations. The ability Dyazenyl)-1H-Tetrazole Modified of the ligand L to form complexes with Pb(II) and Hg(II) ions was also tested by UV-Vis spectrometry. Electrodes. Symmetry 2021, 13, 644. The obtained results showed the formation of Me(II)L complexes. https://doi.org/10.3390/ 2 sym13040644 Keywords: azulene derivative; chemically modified electrodes; voltammetric techniques; complexing

Academic Editors: polymeric ﬁlms; UV-Vis spectrometry; heavy metal ions Christophe Humbert and Enrico Bodo

Received: 10 March 2021 1. Introduction Accepted: 9 April 2021 Chemically modified electrodes (CMEs) with complexing properties are alternative Published: 11 April 2021 tools for the recognition of heavy metal ions which can be done by using very sensitive methods such as atomic absorption spectroscopy [1], emission spectroscopy [2], cold vapor Publisher’s Note: MDPI stays neutral atomic fluorescence spectrometry [3] and inductively coupled mass spectrometry [4]. with regard to jurisdictional claims in The last techniques require laborious sample preparation and well-controlled experimental published maps and institutional affil- conditions being also expensive tools. That is why electrochemical detection, which uses iations. methods such as anodic stripping voltammetry, is a promising answer, its main advantage being the fact that it is a portable method at a low cost. Regarding to toxicity concerns, solid electrodes which can be further modified to increase the selectivity and sensitivity can replace the toxic mercury electrodes usually used for stripping [5]. The CMEs can Copyright: © 2021 by the authors. be prepared by physical absorption of several compounds or by electropolymerization Licensee MDPI, Basel, Switzerland. of specific complexing monomers on solid electrodes. For instance, Zhou [6] developed This article is an open access article a sensor for the specific detection of Cd(II) and Pb(II) by using amino acids that have distributed under the terms and cysteine as a functional side chain. The use of graphene oxide nanoparticles led to the conditions of the Creative Commons increase the electrochemical signal. Complexing CMEs can be obtained by covering the Attribution (CC BY) license (https:// electrode surface with polymer complexing films. The most efficient preparation is the creativecommons.org/licenses/by/ direct electropolymerization of a complexing monomer [7]. This allows for the obtaining of 4.0/).

Symmetry 2021, 13, 644. https://doi.org/10.3390/sym13040644 https://www.mdpi.com/journal/symmetry Symmetry 2021, 13, 644 2 of 12

Symmetry 2021, 13, 644 the electrode surface with polymer complexing films. The most efficient preparation 2is of the 11 direct electropolymerization of a complexing monomer [7]. This allows for the obtaining of a film with a thickness that is easy to control [8] in a single step; the method is generally areliable film with and a reproducible. thickness that Different is easy to monomers control [8] have in a singlebeen used step; for the modification method is generally of elec- reliabletrode surfaces and reproducible. to detect heavy Different metals monomers (pyrrole, have thiophene). been used Compared for modification to them, of electrode azulene surfaceshas special to properties, detect heavy such metals as an (pyrrole,easy polyme thiophene).rization due Compared to its polar to them,character. azulene Its push- has specialpull structure properties, with suchseparate as an loads easy on polymerization the two cycles due (a cyclopentadien to its polar character.yl anion Its joined push-pull with structurea cycloheptatrienyl with separate cation loads having on the6 π twoelectrons cycles in (a each cyclopentadienyl ring [9]) allows anion for the joined use of with az- aulene cycloheptatrienyl derivatives in oxidation cation having and reduction 6 π electrons processes. in each Due ring to [their9]) allows particular for thechemistry, use of azulenesuch azulene derivatives derivatives in oxidation have been and reductionlittle used processes. in applications Due to such their as particular the electroanalysis chemistry, suchof heavy azulene metal derivatives ions. However, have beenthe CMEs little usedwith inpolymeric applications azulene such derivative as the electroanalysis films can be ofused heavy as sensing metal ions. tools However,for monitoring the CMEs the concen with polymerictrations of azulenedifferent derivative targets, such films as canheavy be usedmetals as in sensing wastewater tools forsamples monitoring [10]. the concentrations of different targets, such as heavy metalsThe in monomer wastewater used samples to prepare [10]. the complexing films in this paper is a tetrazole az- uleneThe (Figure monomer 1). Our used research to prepare led to the obtainin complexingg new films CMEs in based this paper on the is aazulene tetrazole derivative azulene (FigureL (L-CMEs)1). Our and research testing led their to obtainingability to new complex CMEs heavy based metals. on the azuleneThe electrooxidative derivative L (L-CMEs)polymerization and testing of L was their used ability to cover to complex the electrode heavy metals. surfaces The with electrooxidative complexing polymeric polymer- izationfilms by of whichL was the used recognition to cover the of electrodeheavy metal surfaces cations with (Hg(II), complexing Cu(II) polymeric, Pb(II), Cd(II)) films be- by whichcame possible. the recognition of heavy metal cations (Hg(II), Cu(II), Pb(II), Cd(II)) became possible.

Figure 1. Structure of the azuleneazulene derivativederivative LL usedused asas monomer.monomer. 2. Materials and Methods 2. Materials and Methods The synthesis of azulene derivative L shown in Figure1 was performed accord- The synthesis of azulene derivative L shown in Figure 1 was performed according to ing to the described methodology [11]. Tetrabutylammonium perchlorate (TBAP, Fluka, the described methodology [11]. Tetrabutylammonium perchlorate (TBAP, Fluka, Mu- Munich, Germany, analytical purity ≥ 99.0%) and acetonitrile (CH3CN, Sigma Aldrich, nich, Germany, analytical purity ≥ 99.0%) and acetonitrile (CH3CN, Sigma Aldrich, elec- electronic grade 99.999% trace metals) were used as supporting electrolyte and solvent, tronic grade 99.999% trace metals) were used as supporting electrolyte and solvent, re- respectively. 0.1 M acetate buffer solution (pH = 4.5) was prepared from 0.2 M acetic acid spectively. 0.1 M acetate buffer solution (pH = 4.5) was prepared from 0.2 M acetic acid solutions (Fluka, Munich, Germany, >99.0%, trace select), 0.2 M sodium acetate (Riedel de solutions (Fluka, Munich, Germany, >99.0%, trace select), 0.2 M sodium acetate (Riedel de Haën, Seelze, Germany), and ultrapure water. Stock solutions of heavy metal salts were Haën, Seelze, Germany), and ultrapure water. Stock solutions of heavy metal salts were prepared by dissolving salts of mercury (II) acetate, cadmium acetate dihydrate (Fluka, prepared by dissolving salts of mercury (II) acetate, cadmium acetate dihydrate (Fluka, Munich, Germany, ≥98%), lead (II) acetate trihydrate (Fluka, Munich, Germany, ≥99.5%) Munich, Germany, ≥98%), lead (II) acetate trihydrate (Fluka, Munich, Germany, ≥99.5%) and copper (II) acetate monohydrate (Fluka, Munich, Germany, ≥98%) in ultrapure water. and copperFor the (II) electrochemical acetate monohydrate experiments (Fluka, cells Munich, with three Germany, electrodes ≥98%) were in ultrapure connected water. to an AUTOLABFor the potentiostat. electrochemical For experiments the preparation cells of with CMEs, three the electrodes working electrodewere connected were glassy to an carbonAUTOLAB disks potentiostat. (d = 3 mm, For Metrohm, the preparation Herisau, of Switzerland), CMEs, the working the reference electrode electrode were glassy was carbon disks (d = 3 mm, Metrohm, Herisau, Switzerland), the reference electrode was Ag/10 mM AgNO3, 0.1 M TBAP/CH3CN, and the counter electrode a Pt wire. The po- tentialsAg/10 mM were AgNO ﬁnally3, 0.1 referred M TBAP/CH to the3CN, reversible and the system counter ferrocene/ferricenium electrode a Pt wire. The (Fc/Fc poten-+). + Beforetials were each finally experiment, referred the to glassy the revers carbonible electrode system wasferrocene/ferricenium polished with diamond (Fc/Fc paste). Before and rinsedeach experiment, with acetonitrile. the glassy CV carbon curves electrode were recorded was polished at a scan with rate diamond of 0.1 V/s paste or at and different rinsed potentialwith acetonitrile. scan rates CV (0.1–1curves V/s) were when recorded the at inﬂuence a scan rate of thisof 0.1 parameter V/s or at different was particularly potential studied.scan rates DPV (0.1–1 curves V/s) werewhen obtained the influence at the of scan this rate parameter of 0.01 V/s. was RDE particularly curves were studied. recorded DPV atcurves0.01 V/swereby obtained using different at the scan electrode rate of rotation0.01 V/s. rates RDE (500, curves 1000, were 1500 recorded rpm). Forat 0.01 the V/s recog- by nitionusing different experiments electrode using rotation CMEs, therates reference (500, 1000, electrode 1500 rpm). was Ag/AgClFor the recognition and the counter experi- electrodements using was CMEs, a Pt wire. the reference electrode was Ag/AgCl and the counter electrode was a Pt wire.UV-Vis absorption spectra were recorded between 800 and 200 nm on JASCO V-670 spectrophotometerUV-Vis absorption at room spectra temperature, were recorded using acetonitrilebetween 800 as and solvent. 200 nm on JASCO V-670 spectrophotometerL-modified electrodes at room were temperatur preparede, in using solutions acetonitrile of the ligand as solvent.L in 0.1 M TBAP/CH3CN. After preparation, each CME was taken out from the monomer solution, equilibrated and overoxidized in 0.1 M acetate buffer solution (pH = 4.5), according to previously described procedure [12,13]. Then, this L-CME was immersed for 15 min under stirring in a solution of heavy metal ions. After that the modified electrode having the accumulated ions was Symmetry 2021, 13, 644 3 of 12

L-modified electrodes were prepared in solutions of the ligand L in 0.1 M TBAP/CH3CN. After preparation, each CME was taken out from the monomer solution, equilibrated and overoxidized in 0.1 M acetate buffer solution (pH = 4.5), according to Symmetry 2021, 13, 644 3 of 11 previously described procedure [12,13]. Then, this L-CME was immersed for 15 min under stirring in a solution of heavy metal ions. After that the modified electrode having the accumulated ions was taken out, cleaned with distilled water and immersed in 0.1 M ac- etatetaken buffer out, cleanedsolution with(pH = distilled 4.5), where water it was and polarized immersed for in 3 0.1 min M to acetate −1.2 V bufferand the solution stripping(pH =curves 4.5), where were itrecorded was polarized between for − 31.2 min V to and−1.2 +0.8 V andV. The the strippingmethod used curves for were recognition recorded wasbetween based− on1.2 the V andstripping +0.8 V. analysis The method on L-CME used complexing for recognition modified was based electrodes on the using stripping the DPVanalysis technique on L-CME available complexing in the potentiostat modified electrodes software. using the DPV technique available in the potentiostat software. 3. Results 3. Results 3.1. Electrochemical Study of L 3.1. Electrochemical Study of L Three methods have been used to study the electrochemical behavior of L: DPV, CV Three methods have been used to study the electrochemical behavior of L: DPV, and RDE. Figure 2 shows the DPV and CV curves registered at oxidation and reduction CV and RDE. Figure2 shows the DPV and CV curves registered at oxidation and reduction for different concentrations of L in the supporting electrolyte (0.1 M TBAP/CH3CN). The for different concentrations of L in the supporting electrolyte (0.1 M TBAP/CH CN). curves at the bottom (A) are the DPV curves, and present 2 main anodic peaks (a1, a2)3 for The curves at the bottom (A) are the DPV curves, and present 2 main anodic peaks (a1, a2) L and 3 secondary peaks (a3–a5) which overlap the oxidation domain of the solvent. When for L and 3 secondary peaks (a3–a5) which overlap the oxidation domain of the solvent. scanning in the cathodic direction, 5 DPV cathodic peaks (c1–c5) are noticed, of which the When scanning in the cathodic direction, 5 DPV cathodic peaks (c1–c5) are noticed, of which highest (in absolute value) are c1, c2, and c3. The CV curves represented at the top of the highest (in absolute value) are c1, c2, and c3. The CV curves represented at the top of Figure 2B show two oxidation peaks corresponding to a1 and a2 DPV peaks and a large Figure2B show two oxidation peaks corresponding to a1 and a2 DPV peaks and a large peak on the potential domain covering a3–a5 DPV peaks. In the cathodic scans in the CV, peak on the potential domain covering a3–a5 DPV peaks. In the cathodic scans in the CV, five peaks are noticed in good agreement with c1–c5 DPV peaks. The anodic and cathodic ﬁve peaks are noticed in good agreement with c1–c5 DPV peaks. The anodic and cathodic peakpeak currents currents in in the the CV CV and and DPV DPV curves curves increase increase with with the the concentration concentration of of LL. .The The other other smallsmall signals signals at at negative negative potentials potentials (−1.2 (− 1.2V–− V–1.4− V)1.4 are V) attributed are attributed to the to reduction the reduction of oxy- of gen traces (secondary process) from residual water (O2/O2−). − oxygen traces (secondary process) from residual water (O2/O2 ).

Figure 2. (A)—DPV (with currents in absolute value) and (B)—CV (0.1 V/s) curves on glassy carbon Figure 2. (A)—DPV (with currents in absolute value) and (B)—CV (0.1 V/s) curves on glassy car- bonfor forL in L 0.1 in 0.1 M TBAP/CH3CNM TBAP/CH3CN at at different different concentrations concentrations (mM): (mM): 0 (dotted0 (dotted blue blue line), line), 1 1 (green (green line), line),2 (red 2 line).(red line). Figure3A shows CV curves at different scan rates (V s −1) in L solution (1 mM) that Figure 3A shows CV curves at different scan rates (V s−1) in L solution (1 mM) that tested, the appearance of a cathodic peak c1’ corresponding to the return scan was noticed, tested, the appearance of a cathodic peak c1’ corresponding to the return scan was noticed, attesting to the fact that c1 is a quasi-reversible process. The peak-to-peak separation attesting to the fact that c1 is a quasi-reversible process. The peak-to-peak separation be- between c1 and c1’ is about 200 mV, while for ferrocene/ferrocenium reversible couple this tween c1 and c1’ is about 200 mV, while for ferrocene/ferrocenium reversible couple this value is in the same conditions (without IR compensation, in the same cell) of about 90 mV value is in the same conditions (without IR compensation, in the same cell) of about 90 (see further). That is why the process c1 has been considered as quasireversible (Table1).

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mV (see further). That is why the process c1 has been considered as quasireversible (Table 1). In order to check the diffusion control, the plots of the peak current (ip) as a function of the square root of the scan rate (v1/2) has been done for the anodic (a1) and cathodic (c1) peaks (Figure 3B). The linear dependency of the peak currents on v1/2 can be noticed: ipa1 (µA) = 1.00 + 60·v1/2 (R2 = 0.997); ipc1 (µA) = 8.05–95·v1/2 (R2 = 0.994). The slope for c1 is almost double compared to that for a1, which indicates a different number of electrons involved in these processes. From the slope of the a1 line vs. v1/2 the diffusion coefficient (of about 2.5·10−4 cm2·s−1) for L was calculated using the Randles-Sevcik equation for a three electrons transfer. The value of 3 for the number of electrons involved in the first Symmetry 2021, 13, 644 peak a1 agrees with the mechanism for oxidation and reduction proposed for an azulene4 of 11 derivative [13] with similar structure.

Figure 3. CV curves on glassy carbon at different scan rates (A); the dependence of current (ip) on Figure 3. CV curves on glassy carbon at different scan rates (A); the dependence of current (ip) on the square root of the scan rate for a1 and c1 peaks in L solution (1 mM) in 0.1 M TBAP/CH3CN (B). the square root of the scan rate for a1 and c1 peaks in L solution (1 mM) in 0.1 M TBAP/CH3CN (B). Table 1. Values (in V) of anodic (a) and cathodic (c) peak potentials from CV and DPV curves

and half-wave potential (E1/2) from RDE for L (1 mM), and the associated processes characteristics obtainedThe CV from curves the CVfor curvesthe azulene on different derivative scanning L in domains 0.1 M andTBAP/CH rates. 3CN solution (1 mM) on different scan domains are presented in Figure 4. Based on the shape of the CV peaks in Figures 3 and 4 obtained from the CVMethod curves on different scanning domains and rates, and thePeak peak-to-peak separation for the direct and reverse processes (suchProcess as Characteristics c1 and c1’), CV DPV RDE (E1/2) it can be appreciated that a1, c2, c5 correspond to irreversible processes, and a2, c1, c3, and a1 0.57 0.55 Irreversible c4 to quasi-reversiblea2 0.93 processes (Table 0.90 1). Quasireversible The values of the peak potentials from the CV−1.867 and (500 DPV rpm) curves measured for L solution (1 mM)c1 are also− presente1.95 d in Table−1.91 1. The potential−1.888 (1000 of the rpm) first oxidationQuasireversible peak for L is +0.55 V. This value is clearly lower than that obtained−1.906 (1500for a rpm)similar unsubstituted derivative [13],C’1 which has− the1.70 potential of a1 peak at the potential of +0.91 V. It means - that L − − oxidizesc2 much easier.2.26 The comparison2.23 of potentials of the first cathodic peak Irreversible of the same −2.288 (500 rpm) derivativesc3 shows that−2.34 L is reduced−2.32 more hardly− 2.292(−1.91 (1000 V). rpm)The differenceQuasireversible between their oxidation potentials (0.36 V) is higher than that for−2.330 reduction (1500 rpm) potentials (0.15 V in absolute value), thec4 first reduction−2.57 peak for− th2.55is unsubstituted derivative being at − Quasireversible1.76 V [13]. The values obtainedc5 for −L3.11 are explained− by3.05 the effects of alkyl groups that increase Irreversible the electron density on azulene nucleus, making the oxidation easier and the reduction harder. In order to check the diffusion control, the plots of the peak current (ip) as a function of the square root of the scan rate (v1/2) has been done for the anodic (a1) and cathodic (c1) peaks (Figure3B). The linear dependency of the peak currents on v 1/2 can be noticed: ipa1 (µA) = 1.00 + 60·v1/2 (R2 = 0.997); ipc1 (µA) = 8.05–95·v1/2 (R2 = 0.994). The slope for c1 is almost double compared to that for a1, which indicates a different number of electrons involved in these processes. From the slope of the a1 line vs. v1/2 the diffusion coefﬁcient (of about 2.5 ×10−4 cm2·s−1) for L was calculated using the Randles-Sevcik equation for a three electrons transfer. The value of 3 for the number of electrons involved in the ﬁrst peak a1 agrees with the mechanism for oxidation and reduction proposed for an azulene derivative [13] with similar structure. The CV curves for the azulene derivative L in 0.1 M TBAP/CH3CN solution (1 mM) on different scan domains are presented in Figure4. Based on the shape of the CV peaks in Figures3 and4 obtained from the CV curves on different scanning domains and rates, and the peak-to-peak separation for the direct and reverse processes (such as c1 and c1’), it can be appreciated that a1, c2, c5 correspond to irreversible processes, and a2, c1, c3, and c4 to quasi-reversible processes (Table1). Symmetry 2021, 13, 644 5 of 12

Symmetry 2021, 13, 644 5 of 11

Figure 4. CV curves (0.1 V s−1) on glassy carbon at different potential domains for L (1 mM) in 0.1 M FigureTBAP/CH 4. CV3CN. curves (0.1 V s−1) on glassy carbon at different potential domains for L (1 mM) in 0.1 M TBAP/CHThe values3CN. of the peak potentials from the CV and DPV curves measured for L solution (1 mM) are also presented in Table1. The potential of the first oxidation peak for L is +0.55 V. This value is clearly lower than that obtained for a similar unsubstituted derivative [13], Tablewhich 1. Values has the potential(in V) of a1anodic peak at the(a) potentialand cathodic of +0.91 V. (c) It meanspeak that potentialsL oxidizes from CV and DPV curves and much easier. The comparison of potentials of the first cathodic peak of the same derivatives 1/2 half-waveshows thatpotentialL is reduced (E more) from hardly RDE (−1.91 for V). L The (1 difference mM), and between the their associated oxidation processes characteristics ob- tainedpotentials from the (0.36 CV V) is curv higheres than on that different for reduction scanni potentialsng domains (0.15 V in absolute and value),rates. the first reduction peak for this unsubstituted derivative being at −1.76 V [13]. The values obtained for L are explained by the effects of alkylMethod groups that increase the electron density Peakon azulene nucleus, making the oxidation easier and the reduction harder. Process Characteristics Figure5 shows theCV RDE (A) curves at differentDPV rotation rates for RDEL at concentration (E1/2) of 1 mM compared to the corresponding anodic and cathodic DPV curves plotted on the top (B).a1 The anodic RDE curves0.57 show only one0.55 wave corresponding as potential to a1 peak in Irreversible DPV. The cathodic curves show two clear waves (w1, w2) which correspond to the DPV peaksa2 c1, and c2–c3 (which0.93 are situated to0.90 very close potentials to be separated through Quasireversible RDE, but they can be distinguished by DPV), respectively. The increase of the rotation rate of the electrode leads to the increase of the limiting currents−1.867 for the cathodic(500 rpm) waves. Thec1 anodic RDE wave−1.95 does not increase with−1.91 the rotation rate− of1.888 the electrode. (1000 At rpm) more Quasireversible positive potentials than that for a2 DPV peak, the current decreases suddenly. The same behavior has been noticed in other cases of azulene derivatives−1.906 when the (1500 electrode rpm) was covered with a film. The values of the RDE half-wave potential for the cathodic processes presentedC’1 in Table1 agree−1.70 with the peak potentials obtained through DPV and CV methods. - c2 −2.26 −2.23 Irreversible −2.288 (500 rpm) c3 −2.34 −2.32 −2.292 (1000 rpm) Quasireversible −2.330 (1500 rpm) c4 −2.57 −2.55 Quasireversible c5 −3.11 −3.05 Irreversible

Figure 5 shows the RDE (A) curves at different rotation rates for L at concentration of 1 mM compared to the corresponding anodic and cathodic DPV curves plotted on the top (B). The anodic RDE curves show only one wave corresponding as potential to a1 peak in DPV. The cathodic curves show two clear waves (w1, w2) which correspond to the DPV peaks c1, and c2–c3 (which are situated to very close potentials to be separated through RDE, but they can be distinguished by DPV), respectively. The increase of the rotation rate of the electrode leads to the increase of the limiting currents for the cathodic waves. The anodic RDE wave does not increase with the rotation rate of the electrode. At more positive potentials than that for a2 DPV peak, the current decreases suddenly. The same behavior has been noticed in other cases of azulene derivatives when the electrode was covered with a film. The values of the RDE half-wave potential for the cathodic processes presented in Table 1 agree with the peak potentials obtained through DPV and CV methods.

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FigureFigureFigure 5. 5. ( (AA 5.)—RDE)—RDE(A)—RDE and and ( (B andB)—DPV(with)—DPV(with (B)—DPV(with currents currents currents in in absolute absolute in absolute value) value) curves value)curves on curveson glassy glassy on carbon carbon glassy at carbonat at different rotation rates (rpm) of RDE for L (1 mM) in 0.1 M TBAP/CH3CN. differentdifferent rotation rotation rates rates (rpm) (rpm) of RDE of RDE for forL (1L mM)(1 mM) in 0.1 in 0.1M TBAP/CH M TBAP/CH3CN.3CN.

3.2.3.2. 3.2.Preparation Preparation Preparation of of L L-CMEs of-CMEsL-CMEs TheTheThe glassyglassy glassy carboncarbon carbon electrodeselectrodes electrodes werewere were modifiedmodified modified withwith with polypoly polyLL filmsfilmsL films inin 1 1 in mM mM 1 mM L L solutionsolutionL solution inin in 0.1 M0.1 TBAP/CH M TBAP/CH3CN byCN successive by successive cycling cycling of the ofpotential the potential in the anodic in the anodicdomain domain with dif- with 0.1 M TBAP/CH3CN3 by successive cycling of the potential in the anodic domain with dif- ferentferentdifferent potentialpotential potential limitslimits limits(0.75(0.75 V,V, (0.75 11 V)V) V, oror 1 V)byby orCPECPE by usingusing CPE using differentdifferent different chargescharges charges andand potentialspotentials and potentials (1(1 mC,mC,( 1 0.750.75 mC, V/1V/10.75 V;V; V/1 22 mC,mC, V; 2 11 mC, V).V). The 1The V). numbernumber The number ofof cyclescycles of cycles hashas not hasnot been notbeen been optimized,optimized, optimized, andand and 2020 cyclescycles 20 cycles werewerewere usuallyusually usually used, used, used, asas shownshown as shown inin FigureFigure in Figure 6A. 6A.6 A. TheThe The CMEsCMEs CMEs werewere were transferredtransferred transferred after after after preparationpreparation preparation to ato cell a cellcontaining containing ferrocene ferrocene solution solution (1 mM) (1 mM) in 0.1 in 0.1M MTBAP/CH TBAP/CH3CN3 CNto record to record the theCV CV to a cell containing ferrocene solution (1 mM) in 0.1 M TBAP/CH3CN to record the CV curves and compare them with those on bare electrode (Figure6A,B). It can be seen that curvescurves andand comparecompare themthem withwith thosethose onon barebare elelectrodeectrode (Figure(Figure 6A,B).6A,B). ItIt cancan bebe seenseen thatthat the ferrocene signal for the polyL film on CME prepared by successive cycling or CPE thethe ferroceneferrocene signalsignal forfor thethe polypolyLL filmfilm onon CMECME preparedprepared byby successivesuccessive cyclingcycling oror CPECPE isis is slightly shifted as anodic and cathodic peak potential, and the peak currents remain slightlyslightly shifted shifted as as anodic anodic and and cathodic cathodic peak peak potential, potential, and and the the peak peak currents currents remain remain prac- prac- practically the same. The ferrocene/ferrocenium system has a peak-to-peak separation ticallytically the the same. same. The The ferrocene/ferrocenium ferrocene/ferrocenium system system has has a a peak-to-peak peak-to-peak separation separation of of about about of about 100 mV on both CMEs and on bare electrode This indicates the formation of 100100 mV mV on on both both CMEs CMEs and and on on bare bare electrode electrode This This indicates indicates the the formation formation of of thin thin films. films. thin films.

−1 Figure 6. CV curves (0.1− V1 s ) recorded after the transfer of the L-CMEs in ferrocene solution (1 mM) FigureFigure 6. 6. CV CV curves curves (0.1 (0.1 V V s s−)1) recorded recorded after after the the transfer transfer of of the the L-CMEs L-CMEs in in ferrocene ferrocene solution solution (1 (1 mM)in in 0.1 0.1 M M TBAP/CH TBAP/CH3CN3CN compared compared to to those those recorded recorded on on bare bare electrode electrode (dashed (dashed lines); lines);L-CMEs L- were mM) in 0.1 M TBAP/CH3CN compared to those recorded on bare electrode (dashed lines); L- A CMEsCMEsprepared were were prepared prepared by ( ) 20 by by cycles ( (AA)) 20 20 in cycles thecycles domains in in the the domains domains of a1 peak of of (whena1a1 peak peak scanning (when (when scanning scanning from 0 V from from to 0.75 0 0 V V V) to to and0.75 0.75in the V)V) and anddomain in in the the of domain domain a2 peak of of (when a2 a2 peak peak scanning (when (when scanning scanning from 0 V from from to 1 V,0 0 V andV to to 1 by1 V, V, CPE and and (by Bby) CPE at:CPE 0.75 ( (BB)) Vat: at: and 0.75 0.75 1 V V mC, and and 1 1 1 V and mC,mC,2 1 1 mC,V V and and 1 V 2 2 andmC, mC, 1 1 1 mC V V and and (solid 1 1 mC mC lines). (solid (solid lines). lines).

TheTheThe filmfilm ﬁlm formationformation formation onon L onL-CME-CMEL-CME waswas was evidencedevidenced evidenced byby byelectrochemicalelectrochemical electrochemical experimentsexperiments experiments (see(see (see FiguresFiguresFigures S1–S7) S1–S7) S1–S7) by by CV byCV CVin in the the in supporting thesupporting supporting electrolyte electrolyte electrolyte (S1) (S1) (S1)and and andferrocene ferrocene ferrocene solution solution solution in in support- support- in support- inging ingelectrolyte electrolyte electrolyte (S2). (S2). (S2). The The peaks Thepeaks peaks corresponding corresponding corresponding to to the the topolymer polymer the polymer oxidation oxidation oxidation and and reduction reduction and reduction are are are situated at 0.052 and 0.038 V (S3). However, much more systematic studies and sta- tistical analysis on several sets of electrodes for different ﬁlm thicknesses are needed.

Symmetry 2021, 13, 644 7 of 11

Supplementary Figure S4 shows the chronoamperograms of films of different thicknesses. Supplementary Figure S5 shows the CV curves by which the electrode is equilibrated, and Supplementary Figure S6 the CV curves by which the film is overoxidized on the electrode. However, there is no expected concordance between the charge used to prepare the film and the peak current heights obtained for different film thicknesses (S7). Supplementary Figure S8 shows the CV curves during the formation of L-CMEs by successive potential scanning (20 cycles) with anodic limit in the potential domain of a1 and a2 processes. As shown in Supplementary Figure S8, the peak current for a2 decreases in successive cycles especially when the potential is scanned in the domain of a2 process. This is a proof of a covering of the electrode with the polymeric film. The transfer in ferrocene solution of the modified electrodes obtained by cycling in the domain of a1 and a2 processes leaded to the cyclic voltammograms showed in Figure6. All these studies will be the subject of systematic investigations related to the optimization of the conditions for the preparation and use of future sensors based on chemically modified electrodes with L. The research is in progress.

3.3. Recognition of Heavy Metal Ions Using Poly L For the recognition of heavy metal ions, the L-CME were prepared by CPE in 1 mM L solution in 0.1 M TBAP/CH3CN at a potential of 1 V and using a charge of 1 mC. The used procedure was the same as in the case of other azulene derivatives [13]; after that, the L-CME was introduced in the conditioning cell containing 0.1 M acetate buffer solution (pH 4.5) and was equilibrated (15 CV cycles at 0.1 V s−1 between −0.9 V and +0.6 V) and overoxidized (15 CV cycles at 0.1 V s−1 between −0.2 V and +1.5 V). Afterwards, the L-CME was cleaned with distilled water and introduced into the accumulation solution containing a mixture of heavy metal ions (Cd(II), Pb(II), Hg(II) and Cu(II)) at a given concentration under magnetic stirring for 15 min. Different concentrations of accumulation solutions were tested in this order: 10−8, 10−7, 10−6, 10−5, 10−4 (mol L−1). After accumulation, the CME complexed with metal ions was rinsed with distilled water and introduced into the analysis cell ﬁlled with 0.1 M acetate buffer, pH 4.5, where it was polarized for 3 min Symmetry 2021, 13, 644 8 of 12 at −1.2 V to reduce the accumulated cations. Afterwards, the anodic redissolution curves were recorded between −1.2 V and 0.8 V using DPV (Figure7).

Figure 7. DPV curves (0.01 V s−1) recorded on L-CME for different concentrations (M) of mixed Figure 7. DPV curves (0.01 V s−1) recorded on L-CME for different concentrations (M) of mixed metal ion accumulation solutions. metal ion accumulation solutions. The recorded DPV curves show peaks for all metal ions in solution (Cd(II), Pb(II), Cu(II), Hg(II) that occur at about −0.76 V, −0.55 V, −0.10 V and +0.25 V, respectively, vs. Ag/AgCl) (Figure7). The dependence of the DPV peak currents on the concentration of the cations in the accumulation solutions is presented in Figure8. It can be observed

Figure 8. Dependence of the stripping currents from DPV on the metallic ions’ concentration.

3.4. UV-Vis Study of Hg(II) and Pb(II) Metal ion Complexation Using L In order to follow the interaction of L with the metal ions of Pb(II) and Hg(II), the spectrophotometric titration was performed in acetonitrile solutions of L (from 10−6 M concentration solution). Changes in the spectra were found following the addition of increasing concentrations of Pb(II) and Hg(II) solutions (several µL of metal salts 10−3 M in water) to 3 mL solution of L in 7 µM CH3CN. The hypo-, batho- and hypsochromic shifts in respect to the initial spectrum of L (Figures 9A and 10A) represent evidence of the formation of complexes between Me and L. A significant hypochromic shift of the ligand L band from λmax ~ 487.7 nm is observed in the UV-Vis spectra recorded when increasing [Me(II)]/[L] ratio. The calibration curves (Figures 9B and 10B) for the formation of complexes with Hg(II), and Pb(II), respectively, were obtained from the decrease of the absorbance peak at 487.7 nm when the metal concentration increases. The increase in the [Me(II)]/[L] molar ratio results initially in a linear decrease in absorbance, followed by a saturation stage (with plateau) in which the ligand was fully complexed with Pb(II) or Hg(II) metal ions. The absorbances are practically constant after the addition of 0.6 equivalents of Pb(II), but decrease slowly (descending plateau) for Hg(II).

Symmetry 2021, 13, 644 8 of 12

Symmetry 2021, 13, 644 8 of 11

that the most intense signals were obtained for the Pb(II) ion in solutions of 10−8 to Figure−4 7. DPV curves (0.01 V s−1) recorded on L-CME for different concentrations (M) of mixed2+ 10 M concentrations. From the linearity of the points in the graph ipeak = f (cPb ) for Pb metal ion accumulation solutions. (Figure8) in the interval 10−8–10−5 M, it can be concluded that the L-CMEs can be used for the analysis of the Pb (II) ion in waters, even at low concentrations.

Figure 8. Dependence of the stripping currents from DPV on the metallic ions’ concentration. Figure 8. Dependence of the stripping currents from DPV on the metallic ions’ concentration. 3.4. UV-Vis Study of Hg(II) and Pb(II) Metal ion Complexation Using L 3.4. UV-VisIn order Study to follow of Hg(II) the and interaction Pb(II) Metal of L ionwith Complexation the metal ions Using of Pb(II) L and Hg(II), the spec- trophotometricIn order to titrationfollow the was interaction performed of in L acetonitrile with the metal solutions ions ofofL Pb(II)(from and 10− 6Hg(II),M concen- the spectrophotometrictration solution). Changes titration in was the spectraperformed were in found acetonitrile following solutions the addition of L (from of increasing 10−6 M concentrationconcentrations solution). of Pb(II) Changes and Hg(II) in solutions the spectra (several were µfoundL of metal following salts 10the− 3additionM in water) of in- to −3 creasing3 mL solution concentrations of L in 7 µofM Pb(II) CH3CN. and TheHg(II) hypo-, solutions batho- (several and hypsochromic µL of metal shiftssalts 10 in respect M in water)to the to initial 3 mL spectrum solution of L in(Figures 7 µM 9CHA and3CN. 10 TheA) hypo-, represent batho- evidence and hypsochromic of the formation shifts of incomplexes respect to between the initial Me spectrum and L. A significantof L (Figures hypochromic 9A and 10A) shift represent of the ligand evidenceL band of from the λfor-max mation~ 487.7 of nm complexes is observed between in the UV-Vis Me and spectra L. A si recordedgnificant when hypochromic increasing shift [Me(II)]/[L] of the ligand ratio. L band Thefrom calibration λmax ~ 487.7 curves nm is (Figuresobserved9B in and the 10 UV-VisB) for spectra the formation recorded of when complexes increasing with [Me(II)]/[L]Hg(II), and ratio. Pb(II), respectively, were obtained from the decrease of the absorbance peak at 487.7The nm calibration when the metalcurves concentration (Figures 9B increases.and 10B) for The the increase formation in the of [Me(II)]/[L] complexes molarwith Hg(II),ratio results and Pb(II), initially respectively, in a linear were decrease obtained in absorbance, from the decrease followed of bythe aabsorbance saturation peak stage Symmetry 2021, 13, 644 at(with 487.7 plateau) nm when in the which metal the concentration ligand was fully increases. complexed The increase with Pb(II) in the or [Me(II)]/[L] Hg(II) metal molar9 of ions. 12

ratioThe absorbancesresults initially are in practically a linear decrease constant in after absorbance, the addition followed of 0.6 by equivalents a saturation of Pb(II),stage (withbut decrease plateau) slowly in which (descending the ligand plateau) was fully for complexed Hg(II). with Pb(II) or Hg(II) metal ions. The absorbances are practically constant after the addition of 0.6 equivalents of Pb(II), but decrease slowly (descending plateau) for Hg(II).

Figure 9. Cont.

Figure 9. (A) UV-Vis titration spectra for L (1 mM) with 0–3 equivalents of Hg(II) ions in solution; the black arrows indicate the isosbestic points, the red arrows indicate the direction in which the absorbance values move with the increase of Hg(II) concentration; (B) Maximum visible peak absorbance vs. [Hg]/[L], [L] ≈ constant.

Using the method of absorbance variation on the molar ratio [Me(II)]/[L] [14], from Figure 11A the probable formula of the complexes formed with Pb(II) and Hg(II) were obtained. Since [Pb(II)]/[L] = 0.53 ≈ 1/2 and [Hg(II)]/[L] = 0.61 ≈ 1/2, it follows that the probable formula of the formed complexes is Me(II)L2. The same stoichiometric formula of the complex was obtained using the method of continuous variations (Job) of the absorbance with the molar fraction X = [Me(II)]/([Me(II)] + [L]) of the metal [14] from a series of solutions (Figure 11B), in which X varied between 0 and 0.75. For both Pb(II) and Hg(II): [Me(II)]/([Me(II)] + [L]) = 0.472 ≈ 1/2 → the probable formula of the formed complexes is Me(II)L2.

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Symmetry 2021, 13, 644 9 of 11

Figure 9. (A) UV-Vis titration spectra for L (1 mM) with 0–3 equivalents of Hg(II) ions in solution; Figurethe black 9. (A) arrows UV-Vis indicate titration the spectra isosbestic for L points,(1 mM) thewith red 0–3 arrows equivalents indicate of theHg(II) direction ions in insolution; which the Symmetry 2021, 13, 644 10 of 12 theabsorbance black arrows values indicate move the withisosbestic the increase points, th ofe Hg(II)red arrows concentration; indicate the ( Bdirection) Maximum in which visible the peak absorbanceabsorbance values vs. [Hg]/[L], move with [L] the≈ constant.increase of Hg(II) concentration; (B) Maximum visible peak absorbance vs. [Hg]/[L], [L] ≈ constant.

Figure 10. (A) UV-Vis titration spectra for L (1 mM) with 0–3 equivalents of Pb(II) ions in solution; Figurethe black 10. (A arrows) UV-Vis indicate titration the spectra isosbestic for L (1 points, mM) with the red 0–3 arrowsequivalents indicate of Pb(II) the directionions in solution; in which thethe black absorbance arrows indicate values shiftthe isosbestic with the points, increase the of red Pb(II) arrows concentration; indicate the ( Bdirection) Maximum in which visible the peak absorbanceabsorbance values vs. [Pb]/[L], shift with [L] the≈ inconstant.crease of Pb(II) concentration; (B) Maximum visible peak absorbance vs. [Pb]/[L], [L] ≈ constant. Using the method of absorbance variation on the molar ratio [Me(II)]/[L] [14], from Figure 11A the probable formula of the complexes formed with Pb(II) and Hg(II) were obtained. Since [Pb(II)]/[L] = 0.53 ≈ 1/2 and [Hg(II)]/[L] = 0.61 ≈ 1/2, it follows that the probable formula of the formed complexes is Me(II)L2. The same stoichiometric formula of the complex was obtained using the method of continuous variations (Job) of the absorbance with the molar fraction X = [Me(II)]/([Me(II)] + [L]) of the metal [14] from a series of solutions (Figure 11B), in which X varied between 0 and 0.75. For both Pb(II)

Figure 11. Absorbance vs. [Me(II)]/[L] (A) and absorbance vs. [Me(II)]/([L] + [Me(II)]) (B) for the visible peak of L during the complexations with Pb(II) (blue stars) and Hg(II) (red squares).

Symmetry 2021, 13, 644 10 of 12

Symmetry 2021, 13, 644 10 of 11 Figure 10. (A) UV-Vis titration spectra for L (1 mM) with 0–3 equivalents of Pb(II) ions in solution; the black arrows indicate the isosbestic points, the red arrows indicate the direction in which the absorbance values shift with the increase of Pb(II) concentration; (B) Maximum visible peak ab- sorbanceand Hg(II): vs. [Pb]/[L], [Me(II)]/([Me(II)] [L] ≈ constant. + [L]) = 0.472 ≈ 1/2 → the probable formula of the formed complexes is Me(II)L2.

FigureFigure 11. 11. AbsorbanceAbsorbance vs. vs. [Me(II)]/[L] [Me(II)]/[L] (A) (andA) and absorbance absorbance vs. [Me(II)]/([L] vs. [Me(II)]/([L] + [Me(II)]) + [Me(II)]) (B) for (B the) for the visiblevisible peak peak of ofL duringL during the the complexations complexations wi withth Pb(II) Pb(II) (blue (blue stars) stars) an andd Hg(II) Hg(II) (red (red squares). squares). 4. Conclusions The electrochemical study of compound E-5-((5-isopropyl-3,8-dimethylazulen-1-

yl)diazenyl)-1H-tetrazole (L) in 0.1 M TBAP/CH3CN showed that a polymeric film which can complex the heavy metal cations can be obtained through the oxidative polymerization of L on a glassy carbon electrode. By recording the CV curves of the ferrocene signal for the CME with polyL prepared by CPE or successive cycling, it has been shown that the electrode was covered with a thin insulating film. The recognition of heavy metal ions using L-CME was performed by chemical preconcentration and anodic stripping from aqueous solutions of metal cations (Cd(II), Pb(II), Cu(II), and Hg(II)) with concentrations between 10−8 and 10−4 M. Pb(II) and Hg(II) ions have shown the best signals. The detection limit was estimated at 10−8 M for Pb(II) ion. The UV-Vis absorption spectra of L solutions in presence of heavy metal ions confirmed the formation of Me(II)L2 complexes with Pb(II) and Hg(II). It was shown through voltammetric techniques and UV-Vis spectroscopy that L can be used to detect Pb(II) and Hg(II) ions, with these methods being of interest for the analysis of these ions in different water samples.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/sym13040644/s1, Figure S1: CV curves for L-CME in 0.1 M TBAP, CH3CN at different scan rates (V/s): 0.01—green; 0.05—red; 0.1—blue; 0.2—magenta; 0.3—navy; 0.5—cyan; 1—olive; L-CME was prepared in 1 mM solution of L in 0.1 M TBAP, CH3CN by CPE at 1 V, 1 mC. Figure S2. CV curves for L-CME in 1 mM ferrocene in 0.1 M TBAP, CH3CN at different scan rates (V/s): 0.01—blue; 0.02—red; 0.025—green; 0.03—wine; 0.05—cyan; 0.1—magenta, 0.2—black; L-CME was prepared in 1 mM solution of L in 0.1 M TBAP, CH3CN by CPE at 1 V, 1 mC. Figure S3. CV curves (0.05 V/s) for L-CME (red line) and GC (blue dashed line) in 0.1 M TBAP, CH3CN; L-CMEs was prepared in 1 mM solution of L in 0.1 M TBAP, CH3CN by CPE at 1 V, 1 Mc. Figure S4. Chronoamperograms during the preparation of L-CMEs in 1 mM solution of L in 0.1 M TBAP, CH3CN by CPE at 1V and using different charges: 0.5 (blue short line—electrode E1), 1 (blue longer line—electrode E2), Symmetry 2021, 13, 644 11 of 11

and 6 (red line—electrode E3). Figure S5. CV curves (0.1 V/s) during the equilibration of the electrode E3, prepared as shown in Figure S4 in 0.1 M acetate buffer solution (pH = 4.5), according to previously described procedure [12,13] giving the electrode E3e. Figure S6. CV curves (0.1 V/s) during the overoxidation in 0.1 M acetate buffer solution (pH = 4.5), according to previously described procedure [12,13] giving the electrode L-CME (E3eo). Figure S7. DPV curves (0.01 V/s) recorded for L-CMEs prepared as in Figure S4 (electrodes E1, E2, E3), equilibrated and overoxidized in 0.1 M acetate buffer solution (pH = 4.5), according to the previously described procedure [12,13], after their immersion in a mixed metal ion accumulation solution of 10−5 M concentration (for each cation); L-CMEs were prepared in 1mM solution of L in 0.1 M TBAP, CH3CN by CPE at 1V and using different charges: 0.5 (green line), 1 (blue line), and 6 (red line). Figure S8. CV curves (0.1 V/s) during the formation of L-CMEs by successive potential scanning (20 cycles) with anodic limit in the potential domain of a1 and a2 processes; L-CME 1 mM solution of L in 0.1 M TBAP/CH3CN. Author Contributions: Conceptualization, E.D. and E.-M.U.; writing—original draft preparation, A.-M.P.; writing—review and editing, V.A. and L.-B.E.; validation, E.-M.U. and E.D.; investigation, A.-M.P.; L.-B.E.; V.A., and O.-T.M.; supervision, E.-M.U. and E.D. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Data Availability Statement: Available data are presented in the supplementary materials. Acknowledgments: The authors are grateful for the financial support from: PN-III-P3-3.1-PM- RO-FR-2019-0309. Conflicts of Interest: The authors declare no conflict of interest.

References 1. Welz, B.; Sperling, M. Atomic Absorption Spectrometry, 3rd ed.; Wiley-VCH: Weinheim, Germany, 1999; pp. 335–475. [CrossRef] 2. Evans, E.H.; Day, J.A.; Palmer, C.D.; Price, W.J.; Smith, C.M.M.; Tyson, J.F. Atomic spectrometry update. Advances in atomic emission, absorption and fluorescence spectrometry, and related techniques. J. Anal. At. Spectrom. 2005, 20, 562–590. [CrossRef] 3. Zhang, Y.; Adeloju, S.B. Coupling of non-selective adsorption with selective elution for novel in-line separation and detection of cadmium by vapour generation atomic absorption spectrometry. Talanta 2015, 137, 148–155. [CrossRef][PubMed] 4. Montes-Bayón, M.; DeNicola, K.; Caruso, J.A. Liquid chromatography–inductively coupled plasma mass spectrometry. J. Chromatogr. A 2003, 1000, 457–476. [CrossRef] 5. Philips, M.F.; Gopalan, A.I.; Lee, K.P. Development of a novel cyano group containing electrochemically deposited polymer film for ultrasensitive simultaneous detection of trace level cadmium and lead. J. Hazard. Mater. 2012, 237, 46–54. [CrossRef][PubMed] 6. Zhou, W.; Li, C.; Sun, C.; Yang, X. Simultaneously determination of trace Cd2+ and Pb2+ based on L-cysteine/graphene modified glassy carbon electrode. Food Chem. 2016, 192, 351–357. [CrossRef][PubMed] 7. March, G.; Nguyen, T.D.; Piro, B. Modified Electrodes Used for Electrochemical Detection of Metal Ions in Environmental Analysis. Biosensors 2015, 5, 241–275. [CrossRef][PubMed] 8. Buică, G.O.; Bucher, C.; Moutet, J.C.; Royal, G.; Saint-Aman, E.; Ungureanu, E.M. Voltammetric Sensing of Mercury and Copper Cations at Poly(EDTA-like) Film Modified Electrode. Electroanalysis 2009, 21, 77–86. [CrossRef] 9. Arsene, P.; Marinescu, C. Organic Chemistry; E.D.P.: Bucharest, Romania, 2016; pp. 253–254, ISBN 9786063103285. 10. Buică, G.O.; Ungureanu, E.M.; Birzan, L.; Răzus, A.C.; Popescu, L.R. Voltammetric sensing of lead and cadmium using poly(4- azulen-1-yl-2,6-bis(2-thienyl)pyridine) complexing films. J. Electroanal. Chem. 2013, 693, 67–72. [CrossRef] 11. Birzan, L.; Cristea, M.; Tecuceanu, V.; Hanganu, A.; Ungureanu, E.M.; Răzus, A.C. 5-(Azulen-1-yldiazenyl)tetrazoles; Syntheses and Properties. Rev. Chim. 2020, 71, 251–264. [CrossRef] 12. Buica, G.O.; Lazar, I.G.; Birzan, L.; Lete, C.; Prodana, M.; Enachescu, M.; Tecuceanu, V.; Stoian, A.B.; Ungureanu, E.-M. Azulene- ethylenediaminetetraacetic acid: A versatile molecule for colorimetric and electrochemical sensors for metal ions. Electrochim. Acta 2018, 263, 382–390. [CrossRef] 13. Enache, L.B.; Anăstăsoaie, V.; Birzan, L.; Ungureanu, E.M.; Diao, P.; Enăchescu, M. Polyazulene-Based Materials for Heavy Metal Ion Detection. 2. (E)-5-(azulen-1-yldiazenyl)-1H-Tetrazole-Modified Electrodes for Heavy Metal Sensing. Coatings 2020, 10, 869. [CrossRef]

14. Cordos, E.; Frentiu, T.; Ponta, M.; Rusu, A.; Darvasi, E. Analiza Prin Spectroscopie de Absorbt, ie Moleculară in Ultraviolet-Vizibil;

Institutul National de Optoelectronică: Bucures, ti, Romania, 2001; pp. 199–200.