Amperometric detection of nitrite and nitrate at tetraruthenated porphyrin-modified electrodes in a continuous-flow assembly

Analytica Chimica Acta 452 (2002) 23–28

Amperometric detection of nitrite and nitrate at tetraruthenated porphyrin-modified electrodes in a continuous-flow assembly José Roberto Caetano da Rocha, Lúcio Angnes, Mauro Bertotti∗ , Koiti Araki, Henrique Eisi Toma Instituto de Qu´ımica, Universidade de São Paulo, 05508-900 São Paulo, Brazil Received 4 May 2001; received in revised form 28 August 2001; accepted 2 October 2001

Abstract The modification of a glassy carbon surface by coating with an electrostatically assembled film of tetraruthenated cobalt porphyrin/(meso-tetra(4-sulphonatephenyl)porphyrinate zinc(II) yields an indicator electrode that allows the determination of nitrite to be performed with a limit of detection of 0.1 ␮M in a flow injection configuration. The dynamic range extends up to 1000 ␮M and the repeatability of the measurements was evaluated to be 1.5% with a throughput of 50 samples per hour. The efficiency of the bilayered film to mediate the electron transfer allows the determinations to be performed at a less positive potential (+0.75 V) with enhanced sensitivity. The coating also prevents the surface poisoning and its stability is maintained over several weeks. The same detector was used for determination of nitrate after reduction to nitrite in a reductor column containing copperised cadmium. This method was used for the determination of nitrate and nitrite in mineral water, saliva and cured meats, the results being in agreement with certified values and those obtained by using recommended procedures. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Nitrite; Nitrate; Porphyrins; Electrocatalysis; Modified electrodes

1. Introduction The use of nitrite for colour enhancement and preservation of several industrialised foods is usually controlled because this substance may react with haemoglobin and if sufficient haemoglobin is oxidised, then blood oxygen-carrying capacity may be markedly reduced [1]. Nitrite ions can also interact with amines to form nitrosamines, which are well-known carcinogenic substances [2]. Besides nitrite, determination of nitrate is also of relevance for environmental, food and agricultural fields. For instance, in the environment, nitrate is found at mod∗ Corresponding author. Tel.: +55-113-815-5579. E-mail address: [email protected] (M. Bertotti).

erate concentrations but owing to human activities, such as the use of fertilisers, contamination may reach dangerous levels and become a subject of concern, especially because nitrate may be reduced to nitrite by the intestinal flora [3]. Because of the relevance of the problem, various analytical methods have been described in the literature to determine both analytes. Most of these are based on the formation of a coloured compound with a high absorptivity in diazotisation reactions [4]. In this case, nitrate is determined after its previous quantitative reduction to nitrite and one of the most frequently employed flow injection methods uses a copper-coated cadmium column. Based on this typical configuration, the simultaneous determination of both analytes have been extensively performed, samples including

0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 1 ) 0 1 4 4 0 - 4

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wastewater [5], river water and soil extracts [6] and biological matrices [7]. In spite of the good detection limits of spectrophotometric procedures based on the Griess reaction, some important inconveniences should be mentioned, particularly the complex sample manipulation and the toxicity of reagents utilised. Electrochemical methods offer useful alternatives since they allow faster, cheaper and safer analyses. Therefore, methods involving the indirect determination of nitrite by measuring the amount of triiodide formed in chemical oxidation of iodide have been proposed, the detection being performed by amperometry at bare platinum disc microelectrodes [8,9] or at two platinum wire electrodes in a biamperometric mode [10,11]. A more convenient approach involves direct anodic oxidation of the analyte at a polarised electrode. Accordingly, it is well known that nitrite is oxidised at platinum [12–14], glassy carbon [15], gold [16,17], diamond [18,19] and transition metal oxides electrodes [20]. However, the application of these sensors is limited because several species may poison the electrode surface and decrease the electrode’s sensitivity and accuracy [21]. Therefore, modification of electrode surfaces provides a means of extending the dynamic range in analytical determinations, particularly if coupled with flow injection procedures. Two main advantages are envisaged: some problems related to the stability of the electrode surface are alleviated and the kinetics of electrode reactions may be accelerated, thereby increasing the sensitivity of the determinations. Oxidation of nitrite at modified electrodes has been largely described. Methods include use of thin films of mixed-valence CuPtCl6 [22], silicotungstic heteropolyanions [23], enzymes [24], osmium polymers [25], ruthenium porphyrins [26] and poly(4-vinylpyridine) containing IrCl6 2− [27]. Reduction of nitrite has also been studied at Cu–Ni (30%) alloys and the significant catalytic activity of the surface was used in nitrite/nitrate speciation [28]. Sensors for nitrate have exploited the catalytic reduction of the analyte at copper surfaces [29] and its selective reduction by enzymes immobilised at the electrode surface [30–32]. Metalloporphyrin-coated electrodes have demonstrated beneficial features as analytical sensors because they can lower substantially the overvoltage for redox processes of several species with slow electron-transfer reactions [33,34]. As a result, lower

detection limits and improved selectivity are to be expected and in some cases the need for elaborate sample pre-treatment of the electrode surface is obviated. Accordingly, anodic oxidation of nitrite has been investigated at electrostatically assembled films deposited layer-by-layer, by alternating cationic meso-tetra(4-pyridyl) porphyrins (MTRP) coordinated to four [Ru(bipy)2 Cl]+ groups) and anionic Zn-TPPS (TPPS = meso-tetra (4-sulphonatephenyl) porphyrinate anion) coatings [35]. As shown previously [36], this second porphyrin minimises losses of the immobilised material by solubilisation. Preliminary FIA experiments and rotating disk data have demonstrated the effectiveness of this film in the catalysis of the anodic oxidation of nitrite [35]. A current enhancement has been detected at around 0.75 V, proportionally to the concentration of nitrite ions. Studies on the influence of the film thickness on the current signal were performed and it has been concluded that the rate of the electron transfer is relatively high when compared with the rate of the cross-exchange reaction. Hence, in this paper some results on the use of this modified electrode as a sensor for analysis of nitrite in a FIA configuration are discussed. By incorporating a copper–cadmium column in the FIA manifold, nitrate analysis was also achievable, both analytes being determined in several samples.

2. Experimental 2.1. Chemicals The [Co-TRP](TFMS)5 ·4H2 O and Na4 [Zn-TPPS] 10H2 O were synthesized as described previously [36]. The corresponding solutions were prepared by dissolution of the compounds in methanol (1 mg/5 ml, Co-TRP) and water (1 mg/5 ml, Zn-TPPS). Sulphanilamide and N-(1-naphtyl)-ethylenediamine solutions were prepared as described in literature [4]. Aqueous solutions were prepared by dissolving the reagents in deionised water treated with a nanopure infinity water purification system. 2.2. Electrochemical experiments Voltammetric experiments were carried out by using an EG&G Princeton Applied Research potentio-

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stat, model 273A. A glassy carbon disk (r = 1.5 mm) embedded in a Teflon cylinder was used as working electrode. A platinum wire and a Ag/AgCl (3 M KCl) electrode were used as counter and reference, respectively. Rotating disk voltammetry was carried out using a Pine Instrument Co. analytical rotator. Modification of the electrode surface. Porphyrins were deposited on a glassy carbon surface previously polished with alumina slurry (1 ␮m) and rinsed with water. Electrostatically assembled films were prepared on the electrode surface by transferring 1 ␮l of Co-TRP solution and allowing it to dry in air. Then, 1 ␮l of the Zn-TPPS solution was placed on the top and after a minute, the bilayer coat was rinsed with water. By measuring the charge in a voltammogram recorded with the modified electrode it was evaluated that this procedure yielded a coat containing approximately 50 monolayers of Co-TRP. Thicker films were prepared by repeating the procedure described above. 2.3. FIA apparatus The flow injection system comprised of a Ismatec peristaltic pump (model 78016-30, Cole Parmer), a home-made rotary injection valve with a 130 ␮l sample loop, a cadmium-copper pellet column, carrier solution reservoir and an EG&G 273A potentiostat. The flow-through electrochemical cell was built-up using two acrylic blocks separated by a Teflon spacer. The glassy carbon modified electrode was adapted in cell centre, counter (stainless steel tube) and reference (Ag/AgCl) electrodes being positioned downstream. The system manifold used (Fig. 1) was composed of PTFE tubings (0.8 mm internal diameter) and has two

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possible routes. One of them directs the flow through the cadmium column reducing quantitatively nitrate to nitrite, then the nitrate +nitrite content is measured. The other directs the flow directly to the detector in order to measure solely the nitrite content. The determination of nitrate is performed by subtracting the concentration of nitrite. A 0.1 M sodium acetate + 0.2 M sodium perchlorate solution (pH = 8.0) was used as a carrier electrolyte since at this pH condition the reduction of nitrate to nitrite is quantitative. In all FIA experiments the flow rate was 2.4 ml min−1 . 2.4. Reduction column The reduction column was a glass tube (10 cm length, 2 mm internal diameter) filled with cadmium pellets (20–40 mesh) previously copperised by reaction of cadmium (5 g) with 125 ml CuSO4 (20 g l−1 ) for 15 min. An evaluation of the column efficiency for reduction of nitrate was verified by comparing FIA signals obtained after independent injections of 10 ␮M solutions of nitrite and nitrate in the FIA system. The reduction efficiency was found to be better than 96%. 2.5. Spectrophotometric analysis The results obtained by the proposed amperometric method were validated by using the Griess method. Briefly, sulphanilamide and N-(1-naphtyl)-ethylenediamine solutions were added to the samples. Absorbance signals of the resulting solutions were measured after 15 min at 540 nm in a Beckman DU-70 equipment using a 1 cm path length glass cell. 2.6. Sample treatment

Fig. 1. Flow injection diagram for amperometric determination of nitrate and nitrite. C: carrier solution (0.1 M NaAc + 0.2 M NaClO4 , pH = 8); P: peristaltic pump; S: sample; RC: copper–cadmium reduction column; D: detector; W: waste.

Unstimulated whole saliva samples were collected directly into glass test tubes. Immediately upon collection, aliquots were diluted with 0.1 M NaOH solution in order to prevent microbial reduction of nitrate and nitrite. These samples were centrifuged at 1000 rpm for 5 min to separate solid materials. Nitrite and nitrate were extracted from cured meat by leaving crushed samples in 0.1 M sodium acetate solution (pH = 8) under stirring for 30 min [37]. After 30 min the liquid was filtered with filter paper and the remaining solution was diluted with acetate solution prior to

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injections in the FIA system. In another procedure, meat samples were conveniently treated to remove proteins [38]. This was accomplished by adding to meat samples (10 g) 5 ml of sodium borate solution (50 g l−1 ) and 40 ml of deionised water. The mixture was heated at 70 ◦ C for 15 min under manual stirring and after cooling to room temperature, 2 ml of potassium hexacyanoferrate(II) solution (106 g l−1 ) and 2 ml of zinc acetate solution (220 g l−1 ) were added. The remaining solution was shaken for 30 min with a magnetic stirrer and then filtered using filter paper.

3. Results and discussion The electrochemical behaviour of the Co-TRP modified electrode is shown in Fig. 2A, an anodic and a cathodic peak being observed at 0.75 and 0.66 V, respectively. The appearance of the cathodic peak in the RDE voltammogram confirms that the material is immobilised at the electrode surface. The separation of both peaks is an indication that electron motion involving the Ru(III)/(II) moieties is not sufficiently fast to maintain both forms in thermodynamic equilibrium with the electrode potential. However, with relatively thin films charge transport is fast, peak currents being proportional to potential scan rate. The addition of nitrite to the supporting electrolyte solution causes an increase in anodic current with

Fig. 2. Rotating disk voltammetric curves of the modified electrode immersed in 0.2 M sodium perchlorate before (A) and after addition of nitrite to give the following final concentrations: 1 mM (B) 3 mM (C) and 5 mM (D). Rotation rate = 100 rpm, scan rate = 20 mV s−1 .

a concurrent decrease in the cathodic peak signal Fig. 2B. This latter observation accounts for consumption by the substrate of the Ru(III) electrogenerated in the positive scan. Previous RDE experiments have shown that at relatively high nitrite concentrations and strong convection situations, anodic oxidation of nitrite leads to non-linear IL versus ω1/2 plots, demonstrating that the film cannot handle with such high flux of substrate due to some kinetic limitation [35]. Notwithstanding, at sub-mM levels and for stationary solutions the enhancement in the anodic current is proportional to the concentration of nitrite. After subtracting the signal for the immobilised material the resulting current is significantly higher than the one obtained with a bare electrode, demonstrating the advantages of this modification. Amperometric sensors for nitrite usually operate at potentials near to 1.0 V at bare electrode surfaces [13,14]. However, the anodic process is anticipated when the electrode surface is coated with ruthenated porphyrin. For instance, working at 0.75 V, a 15-fold current enhancement is observed with respect to results obtained with the bare electrode. The effect of film thickness on the amperometric signal was also evaluated. Fiagrams were recorded at 0.75 V by using modified electrodes containing 1, 3, 5, 7, 10 and 15 bilayer coats. The catalytic current decreased rapidly as the thickness was increased, probably because of increased resistivity associated with the counter ion diffusion [35]. Nevertheless, in thin films the cross-exchange reaction is the rate-limiting step of the nitrite electrocatalytic oxidation in such way that the substrate may penetrate deeply into the film. However, the relatively small peak width in the fiagrams, comparable with those at bare electrodes, showed that they are relatively impermeable and excluding with respect to nitrite ions. The implications of this characteristic in terms of analytical applications are a favorable frequency of analysis and the absence of memory effects due to penetration of the substrate into the modifier film. The stability of the coated electrode was investigated by recording voltammograms over a large period of time. Fig. 3 shows these results and a small decrease (around 15%) in the current is observed after 25 days. In this period the modified electrode was used for approximately 90 h and when not in use it was stored in air. The excellent stability of this porphyrin coated

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Fig. 3. Voltammetric curves recorded with the modified electrode in 0.2 M sodium perchlorate solution at different days after its preparation: 1 (A), 8 (B), 10 (C) and 25 (D). During this period of time the modified electrode was used in the FIA configuration for analysis of nitrite and nitrate for approximately 90 h. When not in use the electrode was stored in air. Scan rate = 50 mV s−1 .

electrode is a result of the use of a high concentration of electrolyte in the carrier solution as well as formation of the electrostatically assembled porphyrin film by deposition of the anionic Zn-TPPS complex, both procedures minimising leaching of the immobilised material from the electrode surface. FIA responses for nitrite injections in the range 1–9 ␮M yielded linear least square with a slope of 0.90 nA ␮M−1 and a correlation coefficient of 0.9994. The calibration plot for nitrite was extended upto 1 mM and the relative standard deviation (n = 20) for signals obtained after injecting 5 ␮M nitrite solutions was 1.5%. The analytical frequency was estimated to be 50 samples per hour. The detection limit using the criterion of the nitrite concentration that yields a

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current signal of three times the background uncertainty was 0.1 ␮M. The application of the above procedure for the determination of nitrate requires preliminary stoichiometric reduction of the analyte in flow conditions. Analytical characteristics for pure nitrate samples determinations were found to be as follows: linear dynamic range: 5–100 ␮M, repeatability of determinations: 1.5% (n = 20), limit of detection: 1 ␮M. The dynamic range was relatively short due to the incompleteness of the nitrate reduction for concentrations higher than 100 ␮M. This problem could be worked out by increasing the reduction column length; notwithstanding, the dispersion pattern of the proposed FIA manifold would be significantly influenced. The use of the ruthenated porphyrin allows analytical determinations to be performed with some more selectivity since at the modified surface the anodic oxidation of nitrite may be monitored at relatively less positive potentials. The major interferents with this nitrite modified electrode sensor are anions capable to be oxidised at the working potential and this group includes bromide and iodide. Since nitrate and oxygen are non-electroactive under the conditions of operation, both species do not cause interference in nitrite analysis. In order to evaluate the practical utility of the method, nitrite and nitrate were determined in several samples, results being compared with those obtained by using the recommended spectrophotometric method. Tables 1 and 2 show the results and, in all cases, differences did not exceed 3%. For mineral water samples data are also in good agreement with certified values, demonstrating the reliability of the proposed amperometric method and the absence of interfering species.

Table 1 Determination of nitrite and nitrate in saliva and mineral water samples Samples

Saliva Sample1a Sample 2a Sample 3a Sample 4a Sample 5a a

Mineral water.

Nitrite (␮mol l−1 )

Nitrate (␮mol l−1 )

Amperometry

Spectrophotometry

Amperometry

Spectrophotometry

Label value

18.9 – – – – –

19.4 – – – – –

45.5 51.5 139.1 253.5 117.0 318.9

46.6 53.6 138.3 250.4 113.1 316.8

– 50 140 240 100 330

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Table 2 Determination of nitrite and nitrate in cured meat samples Samples

Extraction Extraction + deproteinisation

Nitrite (mmol kg−1 )

Nitrate (mmol kg−1 )

Amperometry

Spectrophotometry

Amperometry

Spectrophotometry

1.1 1.3

1.2 1.4

11.0 12.8

11.8 12.2

Acknowledgements The authors wish to thank FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo and CNPq (Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnológico) for the financial support. References [1] Y.-G. Huang, J.-D. Ji, Q.N. Hou, Mutat. Res. 358 (1986) 7. [2] I.A. Wolff, E.A. Wasserman, Science 177 (1972) 4043. [3] A.E. Williams, L.J. Lund, J.A. Johnson, Z.J. Kubala, Environ. Sci. Technol. 32 (1998) 32. [4] A.I. Vogel, A Textbook of Quantitative Inorganic Analysis, 4th Edition, Longman, London, 1978, p. 755. [5] A. Cerdà, M.T. Oms, R. Forteza, V. Cerdà, Analyst 121 (1996) 13. [6] M.F. Giné, H. Bergamin, E.A.G. Zagatto, B.F. Reis, Anal. Chim. Acta 114 (1980) 191. [7] P.F. Pratt, K. Nithipatikom, W.B. Campbell, Anal. Biochem. 231 (1995) 383. [8] M. Bertotti, D. Pletcher, Anal. Chim. Acta 337 (1997) 49. [9] V. Mori, M. Bertotti, Anal. Lett. 32 (1999) 25. [10] A. Hulanicki, W. Matuszewski, M. Trojanowicz, Anal. Chim. Acta 194 (1987) 119. [11] J. Lichtig, M.O.O. Rezende, Electroanalysis 5 (1993) 251. [12] R. Guidelli, F. Pergolo, G. Raspi, Anal. Chem. 44 (1972) 745. [13] S.M. Silva, C.R. Alves, S.A.S. Machado, L.H. Mazo, L.A. Avaca, Electroanalysis 8 (1996) 1055. [14] D. Pletcher, M. Bertotti, J. Braz. Chem. Soc. 8 (1997) 391. [15] A.Y. Chamsi, A.G. Fogg, Analyst 113 (1988) 1723. [16] X. Xing, D.A. Scherson, Anal. Chem. 60 (1988) 1468. [17] S.M. Silva, L.H. Mazo, Electroanalysis 10 (1998) 1200.

[18] N. Spataru, T.N. Rao, D.A. Tryk, A. Fujishima, J. Electrochem. Soc. 148 (2001) E112. [19] M.C. Granger, J.S. Xu, J.W. Strojek, G.M. Swain, Anal. Chim. Acta 397 (1999) 145. [20] S. Sunohara, K. Nishimura, K. Yahikozawa, M. Ueno, M. Enyo, Y. Takasu, J. Electroanal. Chem. 354 (1993) 161. [21] J.N. Barisci, G.G. Wallace, Anal. Lett. 24 (1991) 2059. [22] J.H. Pei, X.Y. Li, Talanta 51 (2000) 1107. [23] L. Cheng, S. Dong, J. Electrochem. Soc. 147 (2000) 606. [24] B. Strehlitz, B. Grundig, W. Schumacher, B.M.H. Kroneck, K.D. Vorlop, H. Kotte, Anal. Chem. 68 (1996) 807. [25] M.M. Malone, A.P. Doherty, M.R. Smith, J.G. Vos, Analyst 117 (1992) 1259. [26] K. Araki, L. Angnes, C.M.N. Azevedo, H.E. Toma, J. Electroanal. Chem. 397 (1995) 205. [27] J.A. Cox, P.J. Kulesza, J. Electroanal. Chem. 175 (1984) 105. [28] M.J. Moorcroft, L. Nei, J. Davis, R.G. Compton, Anal. Lett. 33 (2000) 3127. [29] A.G. Fogg, S.P. Scullion, T.E. Edmonds, B.J. Birch, Analyst 116 (1991) 573. [30] S. Cornier, B. Galland, C. Innocent, J. Electroanal. Chem. 433 (1997) 113. [31] S.A. Glazier, E.R. Campbell, W.H. Campbell, Anal. Chem. 70 (1998) 1511. [32] L.M. Moretto, P. Ugo, M. Zanata, P. Guerriero, C.R. Martin, Anal. Chem. 70 (1998) 2163. [33] F. Bedioui, J. Devynck, Acc. Chem. Res. 28 (1996) 30. [34] M. Biesaga, K. Pyrzynska, M. Trojanowicz, Talanta 51 (2000) 209. [35] J.R.C. Rocha, M. Bertotti, K. Araki, H.E. Toma, J. Electroanal. Chem., submitted to publication. [36] C.M.N. Azevedo, K. Araki, L. Angnes, H.E. Toma, Electroanalysis 10 (1998) 467. [37] C.D. Usher, G.M. Telling, J. Sci. Food Agric. 2 (1975) 1793. [38] W.H. Lara, M.Y. Takahashi, N. Silveira, Rev. Inst. Adolfo Lutz 38 (1978) 167.