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Voltammetric Determination of Selected Nitro Compounds at a Polished Silver Solid Amalgam Composite Electrode
kvorov,a Jana Tvrdkov,a il,*a Toms Navrtil,b Ales Dan hel,a Jan De dk,a Zuzana Krejc ov,a Lucie S Vlastimil Vyskoc a Barek Jir
Charles University in Prague, Faculty of Science, Department of Analytical Chemistry, UNESCO Laboratory of Environmental Electrochemistry, Hlavova 2030/8, 12843 Prague 2, Czech Republic b J. Heyrovsky kova 3, 18223 Prague 8, Czech Republic Institute of Physical Chemistry of the AS CR, v.v.i., Dolejs *e-mail: vyskoci1@natur.cuni.cz Received: July 14, 2010; & Accepted: October 17, 2010 Abstract Voltammetric behavior of selected biologically active organic nitro compounds, namely 2-nitrofluorene, picric acid (2,4,6-trinitrophenol) and metronidazole, has been investigated using direct current voltammetry (DCV) and differential pulse voltammetry (DPV) at a polished silver solid amalgam composite electrode (p-AgSA-CE). The optimum conditions have been found for their determination in a 1 : 1 mixture of methanol and aqueous BrittonRobinson buffer of pH 5.0 for 2-nitrofluorene and in the aqueous BrittonRobinson buffer solutions of pH 2.0 and 4.0 for picric acid and metronidazole, respectively, with the limits of quantification (LQs) 4, 0.1 and 2 mmol L1 (DCV at p-AgSA-CE) and 3, 1 and 4 mmol L1 (DPV at p-AgSA-CE) for 2-nitrofluorene, picric acid and metronidazole, respectively. An attempt to increase the sensitivity using adsorptive stripping voltammetry was not successful for all three test substances. For comparison, the UV-vis spectrophotometric determinations of studied compounds have also been carried out in methanol for 2-nitrofluorene (LQ % 1 mmol L1) and in deionized water for picric acid (LQ % 3 mmol L1) and metronidazole (LQ % 2 mmol L1). Practical applicability of the newly developed voltammetric methods was verified on direct determination of the studied compounds in drinking and river waters with LQs around 106 mol L1 for all the studied compounds. Keywords: Voltammetry, Silver solid amalgam composite electrode, Drinking water, River water, Nitro compounds, 2-Nitrofluorene, Picric acid, Metronidazole
a

DOI: 10.1002/elan.201000428

Dedicated to the Memory of Dr. Miloslav Kopanica One of the Pioneers of Composite Electrodes on the Occasion of His Unattained 80th Birthday Presented at the 13th International Conference on Electroanalysis, ESEAC 2010, Gijn, Spain

1 Introduction
The bonding of nitro group imparts a variety of chemical, physical and/or biological characteristics to unsubstituted organic compounds. Among them, nitrated cyclic and heterocyclic aromatic compounds (nitro aromatics or nitro arenes) represent structural units of known or newly synthesized explosives, pesticides, herbicides, dyes, pharmaceuticals, etc. In natural environment, nitro aromatic pollutants are produced by incomplete combustion of fossil fuels and the indiscriminate use of nitro aromatics in the past, due to wide applications, has also resulted in inexorable environmental pollution [1]. The majority of nitro aromatic compounds in the biosphere are at least genotoxic, some of them act as mutagens and/or carcinogens [2]. On the other hand, nitro aromatic compounds are widely used in medicine, often as anticancer drugs [3].
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There is an ever increasing demand for sensitive and selective methods for determination of various nitro aromatic compounds of biological and ecological interest [4]. The easy electrochemical reduction of nitro groups at the systems with conjugated double bonds, whose mechanism is discussed in [5], permits very sensitive determinations of a number of biologically active organic nitro compounds using modern voltammetric techniques such as differential pulse voltammetry (DPV) at a hanging mercury drop electrode (HMDE) [6] and adsorptive stripping voltammetry (AdSV) at HMDE [7]. Nevertheless, due to increasing fears of liquid mercury toxicity during the last decades [8], which has resulted in the mercury phobia [9], attention has been paid to the development of solid electrodes (e.g., solid amalgam electrodes [1014], solid composite electrodes [1520], carbon paste electrodes [21] or boron-doped diamond film electrodes [22]), which can also be used for determination of various nitro com129

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Scheme 1. Structural formulas of tested compounds.

pounds. From this point of view, working electrodes based on silver amalgam powder mixed with nonconducting binder, such as polished silver solid amalgam composite electrode (p-AgSA-CE) [18], represent a suitable nontoxic alternative to the traditional mercury electrodes; the novel types of electrode materials based on silver amalgam containing no liquid mercury produce negligible amount of mercury vapors in comparison with liquid mercury and materials containing liquid mercury [23]. A good mechanical stability, simple handling and regeneration including an electrochemical pretreatment of the electrode surface are among the main advantages of the p-AgSA-CE. The wide field of analytical applications of these electrodes has been reviewed [13, 24]. This work aims at developing direct current voltammetric (DCV), DPV and adsorptive stripping voltammetric (AdSV) methods for the determination of trace amounts of selected structurally different biologically active organic nitro compounds 2-nitrofluorene, picric acid and metronidazole (see Scheme 1) using p-AgSA-CE and comparing the results obtained with UV-vis spectrophotometric determinations of these substances. Furthermore, practical applicability of the newly developed voltammetric methods was verified on the direct determinations of the studied compounds in drinking and river waters. 2-Nitrofluorene was selected as a representative and model marker of nitrated polycyclic aromatic hydrocarbons (NPAHs) well-known class of genotoxic environmental pollutants [25]; 2-nitrofluorene is a proven carcinogen to rats and a possible carcinogen to humans (IARC group 2B) [26], intermediates of its nitro reduction interact in vitro with DNA [27]. Picric acid (2,4,6-trinitrophenol) represented a member of the group of nitro aromatic explosives [1] and metronidazole (IARC group 2B) [28, 29], an antibiotic and antiprotozoal drug derived from heteroaromatic imidazole [30], represented an abundant group of nitro aromatic drugs.

public, was prepared by dissolving the substance in 100.0 mL of methanol (99 %, p.a. purity; Lachema, Brno, Czech Republic). The stock solutions of picric acid (CAS Number 88-89-1, CAS Name Phenol, 2,4,6-trinitro-; p.a. purity; Lachema) and metronidazole (CAS Number 44348-1, CAS Name 1H-Imidazole-1-ethanol, 2-methyl-5nitro-; crystalline, p.a. purity; Sigma-Aldrich), both of concentration 1 103 mol L1, were prepared by dissolving the appropriate pure substances in 100.0 mL of deionized water. For 2-nitrofluorene, methanol was used as a solvent due to the limited solubility of 2-nitrofluorene in water. UV-vis spectrophotometric studies have demonstrated that the stock solutions are stable for at least one year [3133]. More dilute solutions were prepared by diluting the stock solutions with methanol or deionized water. The BrittonRobinson (BR) buffers were prepared in a usual way, i.e., by mixing a solution of 0.04 mol L1 in phosphoric acid, 0.04 mol L1 in acetic acid and 0.04 mol L1 in boric acid with the appropriate amount of 0.2 mol L1 sodium hydroxide solution (all p.a. purity; Lachema). Potassium chloride, p.a. purity grade, was supplied by Lachema. Deionized water was produced by Milli-Qplus system (Millipore, Billerica, USA). All the chemicals were used without further purification and all the solutions were stored in glass vessels in dark at laboratory temperature. 2.2 Apparatus The voltammetric measurements were performed using an Eco-Tribo Polarograph driven by PolarPro 5.1 software (both Polaro-Sensors, Prague, Czech Republic). The software worked under the operational system Microsoft Windows XP Professional (Microsoft Corporation, Redmond, USA). The voltammetric measurements were carried out in a three-electrode system, a platinum auxiliary electrode PPE, a RAE 113 (1 mol L1 KCl) Ag/AgCl reference electrode (both Monokrystaly, Turnov, Czech Republic) and a p-AgSA-CE (the disc diameter, 2.9 mm, Institute of and the disc area, 6.6 mm2 ; J. Heyrovsky Physical Chemistry of the AS CR, v.v.i., Prague, Czech Republic) as working electrode. The scan rate, 20 mV s1, was used for both DCV and DPV, the pulse amplitude, 50 mV, and pulse width, 100 ms, were used in DPV, with current sampling for the
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2 Experimental
2.1 Chemicals and Reagents The stock solution of 2-nitrofluorene (c = 1 103 mol L1; CAS Number 607-57-8, CAS Name 9H-Fluorene, 2-nitro-; 98 %), obtained from Sigma-Aldrich, Prague, Czech Re130
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Fig. 1. Scheme of p-AgSA-CE (A) and the detailed photo of polished electrode surface (B); 1) composite mixture of silver amalgam and epoxy resin, 2) copper wire, 3) glass tube, 4) plastic fixture to the analyzer, 5) electric contact.

last 20 ms. The p-AgSA-CE (see Figure 1) consisted of a drawn-out glass tube, whose tip was filled with a composite mixture of fine silver amalgam powder and epoxy resin (80 : 20, w/w), connected to an electric contact, polished with emery papers of various granulity and, afterwards, polished to glassy glance with wet velvet [18, 20]. The p-AgSA-CE could be used for several weeks, only its polishing was repeated every week. UV-vis spectra were measured in quarz cuvettes (optical path length 1.0 cm; Hellma, Heiloo, Netherlands) vs. methanol or deionized water using Agilent 8453 UV-visible Spectrophotometer driven by UV-visible ChemStation 9.01 software (both Agilent Technologies, Santa Clara, USA). The solution pH was measured by Conductivity & pH meter 4330 (Jenway, Chelmsford, UK) with a combined glass electrode (type 924 005). 2.3 Procedures Before starting the work, as well as after electrode passivation or every break in the voltammetric measurements longer than 1 hour, the electrochemical activation of p-AgSA-CE was carried out in 0.2 mol L1 KCl at 2200 mV under stirring for 300 s followed by rinsing with deionized water. Unless stated otherwise, the general voltammetric procedure was as follows: An appropriate volume of the stock solution of a test compound in methanol or deionized water was measured into a 10.0 mL volumetric flask (methanol was added to a total volume of 5.0 mL in the case of 2-nitrofluorene), the flask was filled up to 10.0 mL with a BR buffer of the appropriate pH and transferred into the voltammetric cell. Oxygen was removed by bubbling with nitrogen (purity class 4.0; Linde, Prague, Czech Republic) for 5 min and the voltammogram at p-AgSA-CE was recorded. Regeneration of p-AgSA-CE lasting about 30 s preceded each measurement (except for the investigation of p-AgSA-CE passivation during measurements without regeneration). It was based on application of 300 polarizing
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cycles (switching the electrode potential from E1,reg to E2,reg for 50 ms). E1,reg was about 50 to 100 mV more negative than the potential of the anodic dissolution of the electrode, E2,reg was about 50 to 100 mV more positive than the potential of the hydrogen evolution in the given supporting electrolyte. Under these conditions, eventual oxides of mercury or silver are reduced and adsorbed molecules are desorbed [18, 34]. The appropriate values of the potential and the time of regeneration were inset in the software of the computer-controlled instrument used and the regeneration of p-AgSA-CE was thus carried out automatically. All the curves were measured 3 times and all the measurements were carried out at laboratory temperature. The DCV peak height (Ip) was evaluated from the extrapolated linear portion of the voltammogram before the onset of the peak. DPV peaks were evaluated from the straight line connecting the minima before and after the peak. The calibration curves parameters (e.g., slope, intercept) were calculated using the OriginPro 8.0 software (OriginLab Corporation, Northampton, USA) [35]. The limit of quantification (LQ) was calculated as the analyte concentration corresponding to tenfold the standard deviation of the ten parallel measurements of the lowest measurable concentration [35, 36]. 2.4 Model Water Samples Drinking water from the public water pipeline in the building of Faculty of Science of the Charles University in Prague and the river water from the Vltava river in Prague (filtered through a glass frit funnel of porosity S4), spiked with appropriate amounts of stock solutions of test compounds, were used as the model samples. The procedure for the DC or DP voltammetric determination of 2-nitrofluorene, picric acid and metronidazole in the model samples was as follows: 9.0 mL of a model water sample spiked with appropriate amounts of the studied compound were filled up to 10.0 mL with a BR buffer of the appropriate pH and, after deaeration with nitrogen, DC or DP voltammograms were recorded.

3 Results and Discussion

3.1 Voltammetric Behavior of the Test Substances The influence of the pH on the DCV and DPV responses (see Figure 2) of 1 104 mol L1 2-nitrofluorene at p-AgSA-CE was investigated first in mixtures of methanol and the BR buffer with pH values of 2.0 to 12.0 (1 : 1); BR buffer was replaced by 0.1 mol L1 NaOH for preparation of strong alkaline medium of pH 13.0. Voltammetric behavior of picric acid and metronidazole (see also Figure 2) was then investigated in aqueous solutions of BR buffer with pH values of 2.0 to 12.0 (1 : 1). 2-Nitrofluorene yielded one to three DC or DP voltammetric peaks over the whole pH range (see Figures 2A and 2B). Although some DCV responses, not only for 2www.electroanalysis.wiley-vch.de

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Full Paper nitrofluorene, had a sigmoidal shape similar to DC polarographic waves, all DCV responses of the test compounds were evaluated conventionally as DCV peaks (see Section 2.3.). 2-Nitrofluorene gave rise only to one well-developed irreversible voltammetric peak (pI) in acidic, neutral and weak alkaline media, corresponding to the four-electron reduction of the nitro group to the hydroxyamino group [37]. At strong alkaline media (pH 10.0 to 13.0), the height of this peak is decreasing (pII is formed) and peak pIII is observable at more negative potentials, as the mechanism of the nitro group reduction is changing to two-step mechanism [5]. The first step includes a fast reversible uptake of one electron under formation of a nitro radical anion (Ar-NO2C); the second step is a slow three-electron irreversible reduction forming the hydroxyamino group [37]. At p-AgSA-CE, the voltammetric peak pIV was newly observed for 2-nitrofluorene; this peak has not been observed using HMDE [38, 39] or mercury meniscus modified silver solid amalgam electrode (m-AgSAE) [14]. It was proved that its appearance is connected with application of the regeneration potentials and peak pIV corresponds to the reversible two-electron reduction of nitroso group (running predominantly in neutral and alkaline media [5]) formed beforehand. By application of more negative regeneration potential (E2,reg), 2-hydroxyaminofluorene is formed at the electrode surface during such electrochemical regeneration prior to DCV or DPV scan. This is subsequently oxidized at more positive regeneration potential (E1,reg) to 2-nitrosofluorene [37]. After the last regeneration cycle (of 300 polarizing cycles) ending at E2,reg, the residue of previously formed 2-hydroxyaminofluorene can be adsorbed at the p-AgSA-CE surface, even if the solution is stirred. At the beginning of following DCV or DPV scan, the adsorbed 2-hydroxyaminofluorene is immediately oxidized to 2-nitrosofluorene which is subsequently reduced back (peak pIV) to 2-hydroxyaminofluorene during this voltammetric scan. While the regeneration potentials are applied in opposite order (the more negative regeneration potential at first), peak pIV disappears in the following voltammetric scan. Picric acid and metronidazole also yielded only one well-developed cathodic peak over the whole pH range (see Figures 2C and 2D). Although picric acid includes three nitro groups which give rise to three well-separated peaks at HMDE [40], only one voltammetric peak (pV) probably corresponds to the simultaneous reduction of all three nitro groups to hydroxyamino groups at p-AgSACE; this peak includes the current contributions from the reductions of three nitro groups and is thus more suitable, because of its height, for analytical purposes. Voltammetric behavior of metronidazole at p-AgSA-CE is then similar to that observed at mercury electrodes [41]; peak pVI corresponds also to the formation of hydroxyamino group from the nitro group by the uptake of four electrons and four protons. The peak potential values of all the observed peaks are shifted toward more negative potentials with increasing 132
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pH. This shifts can be explained by preliminary protonation of the tested substances (or intermediates of their electrochemical reduction) leading to a decrease in the electron density at nitrogen atom and resulting in easier electron acceptance at low pH values. 3.2 Voltammetric Determination of the Test Substances An irreversible reduction step, corresponding to the formation of hydroxyamino group from nitro group, provided the highest voltammetric responses (both in the DCV and DPV modes) of all the test compounds. These best developed and highest voltammetric peaks were obtained under the conditions summarized in Table 1. Repeated measurements can cause pronounced passivation of the electrode, probably by the adsorption of the electrode reaction products, resulting in decreasing peak heights and a shift toward more negative potentials. The optimum regeneration potentials (E1,reg and E2,reg) thus had to be found for each analyte (see Table 1). The regeneration was carried out in the same supporting electrolyte as that used for the analyte determination. The procedure was based on potential switching (300 polarizing cycles) between two limiting potentials. These potentials were kept at each of these regeneration potential values for 50 ms. The repeatability of the measurements (expressed in terms of the relative standard deviation (RSD) of the highest peak Ip values, evaluated from twenty subsequent voltammetric measurements at the p-AgSA-CE) varied from 0.4 to 17 %, in dependence on the analyte concentration (always measured for the highest and the lowest measurable concentrations of the given analyte). As it results from Figure 3, the regeneration step applied prior to each voltammetric measurement led not only to improvement of the determination repeatability (e.g., the RSD of DCV determinations of metronidazole, at concentration 1 104 mol L1, decreased from 6.7 % to 2.4 % and of DPV determinations of picric acid, at the same concentration, from 2.1 % to 1.4 %), but also to the stabilization and enhancement of both DC and DP voltammetric responses of the studied analytes. Under the optimum conditions (see Tables 1 and 2), the DCV and DPV calibration curves were measured over concentration ranges from zero to 1 105 (see Figure 4) and 1 104 mol L1 for all the test substances and, moreover, in concentration range from zero to 1 106 mol L1 for picric acid using DCV at p-AgSA-CE. The calibration curves parameters are summarized in Table 2. The sensitivity slightly differs between particular concentration orders. This behavior is typical for voltammetry at solid electrodes. The course of the calibration curve in wider concentration range corresponds to the shape of an adsorption isotherm. Therefore, it can be supposed that the tested analytes are adsorbed at the electrode surface. Nevertheless, within the particular concentration orders, the concentration dependences can be approximated by straight lines. The sensitivities of voltammetric determinations of 2-nitrofluorene and metronidaElectroanalysis 2011, 23, No. 1, 129 139

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Fig. 2. DC (A) and DP (B) voltammograms of 2-nitrofluorene (c = 1 104 mol L1) at p-AgSA-CE in the BR buffermethanol (1 : 1) medium; the BR buffer pH: 3.0 (1), 5.0 (2), 7.0 (3), 9.0 (4) and 11.0 (5); voltammogram (6) obtained in the 0.1 mol L1 NaOH of pH 13.0 methanol (1 : 1) medium. DC voltammograms of picric acid (C) and metronidazole (D) (both for c = 1 104 mol L1) at p-AgSA-CE in the BR buffer medium; the BR buffer pH: 2.0 (1), 4.0 (2), 6.0 (3), 8.0 (4), 10.0 (5) and 12.0 (6). Polarization rate, 20 mV s1; bold voltammograms represent the optimum conditions selected for the determination of the test compounds; for peaks description, see text.

zole are comparable to each other, whereas, the slopes of calibration curves for picric acid are about ten times higher. This can be explained by the presence of three nitro groups bonded on the phenolic skeleton in picric acid, the simultaneous reduction of three nitro groups (see Section 3.1. for details) can give higher voltammetric response in contrast to the reduction of only one nitro group in 2-nitrofluorene or in metronidazole. As it can be seen in Figure 4D, the curve of the supporting electrolyte includes the response of the residue of 2-nitrofluorene. This electrode memory is probably caused by the corrosion of epoxy resin at the surface of p-AgSA-CE caused by methanol. This corrosion resulted in adsorption of 2-nitrofluorene at electrode surface, which resulted in its peak observable even in pure supporting electrolyte. Corrected values of Ip were thus used
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for the construction of calibration straight lines in the case of 2-nitrofluorene. Picric acid and metronidazole, measured in purely aqueous media, have not shown this type of adsorption. The above described and discussed adsorption of 2-hydroxyaminofluorene (see Section 3.1.) can thus be causally connected also with this corrosion of epoxy resin. A further increase in the sensitivity of the determination could be achieved by adsorptive accumulation of the test substance on the p-AgSA-CE surface [7]. Optimum conditions, found for the DPV determination of 2-nitrofluorene and metronidazole at p-AgSA-CE and for the DCV determination of picric acid at p-AgSA-CE, have been used for investigating the possible accumulations. Suitable potentials of accumulation (Eacc) were tested with time of accumulation (tacc) varied from zero to
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Fig. 3. DC voltammograms of metronidazole (c = 1 104 mol L1) at p-AgSA-CE in the BR buffer of pH 4.0 without applying (A) and with applying the regeneration cycles (E1,reg = + 150 mV, E2,reg = 1100 mV) (B) prior to each measurement (n = 20). DP voltammograms of picric acid (c = 1 104 mol L1) at p-AgSA-CE in the BR buffer of pH 2.0 without applying (C) and with applying the regeneration cycles (E1,reg = + 200 mV, E2,reg = 950 mV) (D) prior to each measurement (n = 20). Polarization rate, 20 mV s1; the dashed line represents the first measurement; the corresponding peak heights are depicted in the insets.

Table 1. Parameters of the p-AgSA-CE regeneration and the repeatability of Ip measurements under optimum conditions for the highest and the lowest measurable concentrations of the tested compounds. Analyte 2-Nitrofluorene Technique DCV DPV Picric acid DCV DPV Metronidazole DCV DPV Medium BR buffer pH 5.0 methanol (1 : 1) BR buffer pH 5.0 methanol (1 : 1) BR buffer pH 2.0 BR buffer pH 2.0 BR buffer pH 4.0 BR buffer pH 4.0 c (mol L1) 1 104 2 106 1 104 2 106 1 104 1 107 1 104 2 106 1 104 2 106 1 104 2 106 E1,reg (mV) 350 350 350 350 + 200 + 200 + 200 + 200 + 150 + 150 + 150 + 150 E2,reg (mV) 1100 1100 1100 1100 950 950 950 950 1100 1100 1100 1100 Repeatability (%) [a] 0.37 6.7 0.48 7.4 2.0 1.7 1.4 6.7 2.4 17 0.59 13

[a] Repeatability of measurements expressed in terms of the relative standard deviation of the Ip values for twenty subsequent voltammetric measurements at the p-AgSA-CE with the regeneration step (potential switching between E1,reg and E2,reg) prior to each measurement.

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Fig. 4. DC voltammograms of picric acid (A) and metronidazole (B) and DP voltammograms of picric acid (C) and 2-nitrofluorene (D) at the p-AgSA-CE; for experimental conditions, see Table 1; concentrations measured: 0 (1), 2 (2), 4 (3), 6 (4), 8 (5) and 10 (6) mmol L1. Polarization rate, 20 mV s1; the corresponding calibration straight lines are given in the insets.

Fig. 5. Influence of accumulation time on the peak current of 2-nitrofluorene (c = 1 106 mol L1) (A), picric acid (c = 1 107 mol L1) (B) and metronidazole (c = 1 106 mol L1) (C) at the p-AgSA-CE in the BR buffer of pH 5.0 (A) (AdSDPV with Eacc = 100 mV and E1,reg = 350 mV, E2,reg = 1100 mV), pH 2.0 (B) (AdSDCV with Eacc = 100 mV and E1,reg = + 200 mV, E2,reg = 950 mV) and pH 4.0 (C) (AdSDPV with Eacc = 300 mV and E1,reg = + 150 mV, E2,reg = 1100 mV), respectively (n = 3). Polarization rate, 20 mV s1.
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Table 2. Parameters of the calibration straight lines for the determination of the tested compounds at the p-AgSA-CE. Analyte Technique Medium BR buffer pH 5.0 methanol (1 : 1) BR buffer pH 5.0 methanol (1 : 1) BR buffer pH 2.0 c (mol L1) Slope [a] (mA mol1 L) Intercept [a] (nA) r [b] 3.82 0.18 1.88 0.15 2.59 0.15 2.47 0.12 17.23 0.58 23.96 0.92 25.51 0.95 10.88 0.18 12.89 0.74 5.97 0.20 4.05 0.16 3.26 0.10 3.16 0.13 + 36 15 6.3 1.3 9.5 3.3 6.2 1.1 52 38 12.9 8.1 2.3 2.2 42 12 + 2.6 4.5 + 3 13 1.0 1.1 4.6 4.3 + 4.0 1.1 0.997 0.995 0.997 0.994 0.998 0.995 0.954 0.999 0.992 0.998 0.999 0.999 0.997 LQ [c] (mol L1) 4 106 3 106 1 107 1 106 2 106 4 106 (210) 105 (210) 106 (210) 105 (210) 106 (210) 105 (210) 106 (110) 107 BR buffer pH 2.0 (210) 105 (210) 106 BR buffer pH 4.0 (210) 105 (210) 106 BR buffer pH 4.0 (210) 105 (210) 106

2-Nitrofluorene DCV DPV Picric acid DCV

DPV Metronidazole DCV DPV

[a] Intervals represent the lower and upper confidence limits (a = 0.05); [b] correlation coefficient; [c] limit of quantification (10s ; a = 0.05).

5 min. Because methanol is also usually adsorbed on the electrode surface, it was not contained in the supporting electrolyte in the case of 2-nitrofluorene. However, it has been proven that none of the test substances significantly increased voltammetric response in dependence on the tacc. As it follows from the Figure 5, the peak height of 2-nitrofluorene (at concentration 1 106 mol L1) practically does not vary with increasing taac, whereas the peak heights of picric acid (at concentration 1 107 mol L1) and metronidazole (at concentration 1 106 mol L1) increase negligibly. Nevertheless, this increase in voltammetric responses is too insignificant than that to be successfully usable for increasing the sensitivity of previously described DC and DP voltammetric methods.

the case of using DCV technique for the picric acid determination, LQ reached was even one order of magnitude lower than that reached using UV-vis spectrophotometry. 2-Nitrofluorene, picric acid and metronidazole have also been determined in the past using the voltammetry at HMDE. The lowest reported LQs were 3 109 mol L1 (AdSV at HMDE) [14, 38], 3 109 mol L1 (square wave voltammetry at HMDE) [41] and 3 107 mol L1 (square wave voltammetry at HMDE) [42] for 2-nitrofluorene, picric acid and metronidazole, respectively.

3.4 Voltammetric Determination of the Test Substances in Drinking and River Waters In order to verify practical applicability of the DCV or DPV methods developed, the determination of 2-nitrofluorene, picric acid and metronidazole was carried out in model samples of drinking and river waters in micromolar or submicromolar concentration ranges under optimum conditions. Calibration curves were measured using a mixture of 9.0 mL of a spiked model water sample and 1.0 mL of a BR buffer of appropriate pH. For 2-nitrofluorene and metronidazole, DPV at p-AgSA-CE was chosen as suitable for their determination in model water samples, whereas for picric acid, DCV at p-AgSA-CE was chosen, mainly because of its higher sensitivity and lower LQ attained (see Table 2).

3.3 UV-vis Spectrophotometric Determination of the Test Substances UV-vis spectrophotometric determination of 2-nitrofluorene, picric acid and metronidazole has been used for comparison with the newly developed voltammetric methods of determination of these compounds. The calibration straight lines were constructed from absorbance values evaluated at wavelengths of absorption maxima of the test substances (see Table 3). The LQs attained using DC and/or DP voltammetry at p-AgSA-CE are comparable with those attained using UV-vis spectrophotometry. In

Table 3. Parameters of the calibration straight lines for UV-vis spectrophotometric determination of the tested compounds. Analyte 2-Nitrofluorene Picric acid Metronidazole Medium/wavelength Methanol/ 330 nm Deionized water/ 357 nm Deionized water/ 320 nm c (mol L1) (210) 105 (210) 106 (210) 105 (210) 106 (210) 105 (210) 106 Slope [a] (mmol1 L) 20.65 0.21 21.68 0.17 15.79 0.15 15.67 0.80 10.07 0.10 10.48 0.11 Intercept [a] 0.009 0.014 0.015 0.011 0.024 0.010 0.018 0.015 0.004 0.012 0.005 0.011 r [b] 0.999 0.999 0.999 0.996 0.999 0.999 LQ [c] (mol L1) 1 106 3 106 2 106

[a] Intervals represent the lower and upper confidence limits (a = 0.05); [b] correlation coefficient; [c] limit of quantification (10s ; a = 0.05).

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Fig. 6. A) DP voltammograms of 2-nitrofluorene at p-AgSA-CE in spiked drinking waterBR buffer of pH 5.0 (9 : 1); c(2-nitrofluorene) in drinking water: 0 (1), 0.4 (2), 0.6 (3), 0.8 (4), 1.0 (5), 2.0 (6) and 4.0 (7) mmol L1 (6); E1,reg = 350 mV, E2,reg = 1100 mV. B) DC voltammograms of picric acid at p-AgSA-CE in spiked river waterBR buffer of pH 2.0 (9 : 1); c(picric acid) in river water: 0 (1), 1 (2), 2 (3), 4 (4), 6 (5), 8 (6) and 10 (7) mmol L1; E1,reg = + 200 mV, E2,reg = 950 mV. Polarization rate, 20 mV s1; the corresponding calibration straight lines are given in the insets.

Voltammograms at the p-AgSA-CE representing direct determinations of 2-nitrofluorene in spiked drinking water (over the whole measurable concentration range of 0.44 mmol L1) and of picric acid in spiked river water (within a concentration range of 110 mmol L1) are depicted in Figure 6. It can be seen from the Figure 6A that the response of the residue of 2-nitrofluorene in the supporting electrolyte, observable in Figure 4D and discussed in Section 3.2, disappeared. This is probably because of the methanol was not present in the model water samples. Moreover, the sensitivity of DPV determination of 2-nitrofluorene increased about four times as compared with the determination in methanol containing medium. This behavior is well-known from the polarography/voltammetry of nitro compounds with low solubility in water, the polarographic/voltammetric peaks are narrower and higher with decreasing content of alcohol in the supporting electrolyte [43]. However, this limited solubility of 2-

nitrofluorene also caused that the samples of drinking and river water spiked by 2-nitrofluorene could be prepared only at concentrations equal or lower than 4 mmol L1. Therefore, in the case of 2-nitrofluorene, only the measurable concentration range from zero to 4 mmol L1 was used for proving the applicability of developed method. The parameters of the calibration curves obtained are summarized in Table 4. These results confirm the possible application of the proposed methods for both drinking and river water.

4 Conclusions
It has been demonstrated that polished silver solid amalgam composite electrode, combined with direct current voltammetry or differential pulse voltammetry, is a suita-

Table 4. Parameters of the calibration straight lines for the determination of the test compounds at the p-AgSA-CE in various matrices. DW: drinking water; RW: river water. Analyte 2-Nitrofluorene Picric acid Technique DPV DPV DCV DCV Metronidazole DPV DPV Matrix Spiked DWBR buffer pH 5.0 (9 : 1) Spiked RWBR buffer pH 5.0 (9 : 1) Spiked DW BR buffer pH 2.0 (9 : 1) Spiked RW BR buffer pH 2.0 (9 : 1) Spiked DW BR buffer pH 4.0 (9 : 1) Spiked RW BR buffer pH 4.0 (9 : 1) c (mol L1) (0.44) 106 (0.64) 106 (210) 105 (0.610) 106 (210) 105 (110) 106 (210) 105 (210) 106 (210) 105 (210) 106 Slope [a] (mA mol1 L) 9.85 0.97 8.56 0.45 14.97 0.84 15.25 0.33 12.05 0.55 11.46 0.30 6.52 0.48 6.93 0.54 5.46 0.45 5.52 0.54 Intercept [a] (nA) 0.58 0.19 0.91 0.93 5.4 1.5 1.1 1.0 1.8 2.3 + 0.5 1.8 2.5 1.7 1.1 1.2 3.8 1.9 2.5 1.5 r [b] 0.999 0.993 0.996 0.995 0.996 0.998 0.997 0.996 0.992 0.988 LQ [c] (mol L1) 6 107 8 107 6 107 2 106 2 106 4 106

[a] Intervals represent the lower and upper confidence limits (a = 0.05); [b] correlation coefficient; [c] limit of quantification (10s ; a = 0.05).
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Full Paper ble sensor for the determination of micromolar or submicromolar concentrations of model nitro aromatic compounds (namely 2-nitrofluorene, picric acid and metronidazole) and can thus, to some extent, substitute mercury electrodes for this purpose. It provides stable and reproducible responses during long-time measurements; the preparation of this electrode and its pretreatment is simple, the electrochemical activation of its surface is relatively easy. However, reached limits of quantification are roughly one to three orders of magnitude higher than those obtained at hanging mercury drop electrode. Although the attempts at increasing the sensitivity using adsorptive stripping voltammetry were not successful for all three test substances, the sensitivity can certainly be increased by suitable preconcentration technique such as liquid-liquid or solid phase extraction. Using direct current voltammetry or differential pulse voltammetry at the polished silver solid amalgam composite electrode, the limits of quantification for all the three tested compounds are similar to those attained using UVvis spectrophotometric determination. Moreover, this electrode, when connected to a portable computer controlled potentiostat, can be used under field conditions, e.g., for the on-site analysis of contaminated waters or soils. The applicability of polished silver solid amalgam composite electrode to voltammetric determination of the test nitro aromatic compounds in model samples of drinking and river water has been verified. It is possible to suppose that the tested electrode is a suitable sensor not only for voltammetric determination of nitro aromatics, but also for their amperometric detection. The possible utilization of this electrode for determinations of the above or structurally similar analytes in flowing systems (flow injection analysis and high performance liquid chromatography with amperometric detection) is under investigation.

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Acknowledgements
Financial support of this work, provided by The Ministry of Education, Youth and Sports of the Czech Republic (Projects LC 06035, MSM 0021620857 and RP 14/63), by the Grant Agency of Charles University in Prague (Projects 89710/2010/B-Ch/PrF and SVV 261204) and by the Grant Agency of the Academy of Sciences of the Czech Republic (Project No. IAA400400806), is gratefully acknowledged.
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