Flow-injection system with enzyme reactor for differential amperometric determination of hydrogen peroxide in rainwater

Analytica Chimica Acta 441 (2001) 73–79

Flow-injection system with enzyme reactor for differential amperometric determination of hydrogen peroxide in rainwater Renato Camargo Matos a , Jairo José Pedrotti b , Lúcio Angnes a,∗ a

Instituto de Qu´ımica, Universidade de São Paulo, Av. Prof. Lineu Prestes 748, 05508-900 São Paulo, SP, Brazil b Departamento de Qu´ımica, Universidade Presbiteriana Mackenzie, São Paulo, Brazil Received 7 February 2001; received in revised form 4 May 2001; accepted 4 May 2001

Abstract Differential determinations of hydrogen peroxide (H2 O2 ) have been performed by amperometry, combining flow-injection analysis (FIA) and a tubular reactor containing immobilized enzymes. A gold microelectrode modified by electrochemical deposition of platinum was employed as working electrode. Hydrogen peroxide was quantified in rainwater using amperometric differential measurements at +0.60 V versus Ag/AgCl(sat) . For the enzymatic consumption of H2 O2 , a tubular reactor containing immobilized catalase was constructed, using a novel way for immobilization of enzymes on Amberlite IRA-743 resin. The linear dynamic range in H2 O2 extends from 1 to 100 × 10−6 mol/l, at pH 7.0. At flow rate of 2.0 ml/min and injecting 150 ␮l sample volumes, the sampling frequency of the 90 determinations/h is afforded. The reproducibility of the current peaks for hydrogen peroxide in 10−5 mol/l range concentration shown a R.S.D. better than 1%. The detection limit of the method is 2.9 × 10−7 mol/l (1.5 ng of H2 O2 in a 150 ␮l sample). The rainwater samples analyses were compared with the parallel amperometric determinations using static mercury electrode and by spectrophotometry, showing very good correlation between the three methods. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Hydrogen peroxide; Rainwater; Catalase; Modified microelectrode; Platinum

1. Introduction Hydrogen peroxide, H2 O2 , performs an important role in atmospheric and biochemical processes. Atmospheric hydrogen peroxide is formed from the interactions of hydroperoxyl (HO2 • ) and hydrated hydroperoxy (H2 O• HO2 ) radicals, which are produced by the photochemical reactions of atmospheric trace gases, such as ozone and volatile organic compounds [1]. Among the multiple reactions in the troposphere, hydrogen peroxide was characterized as the most efficient oxidant for the conversion of dissolved sulfur ∗ Corresponding author. Fax: +55-11-3815-5579. E-mail address: [email protected] (L. Angnes).

dioxide (SO2 ) to sulfuric acid (H2 SO4 ), which is the main contributor to the acidification of rainwater [2–7]. The reaction between SO2 and H2 O2 is particularly important because, it is relatively rapid even at pH values below five, whereas the oxidation of SO2 by other oxidants, such as O3 and O2 in the presence of Fe and Mn as catalysts is retarded in acidic atmospheric waters [2,8]. During the last two decades, a number of investigations have been carried out concerning the measurement of H2 O2 in the atmospheric gas and liquid phases [1,4–6,9–11]. The results of these investigations have shown that the ambient concentration of H2 O2 varies from 0.1 to 2 ppb in the gas phase and from <10−7 to 10−4 mol/l in fog, cloud, and rainwater with higher concentrations observed in the summer

0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 1 ) 0 1 0 9 4 - 7

74

R.C. Matos et al. / Analytica Chimica Acta 441 (2001) 73–79

and lower concentrations in the winter. Different research groups have also studied the effects of several meteorological and chemical factors on the concentration of H2 O2 in cloud and rainwater [1,4–6,9–12]. Actually, chemical sensors with the ability of continuously sensing analytes attract considerable attention and many applications have appeared in the literature. In the last two decades, many analytical methods have been reported for such measurements, including spectrophotometry [13,14], fluorometry [15,16], chemiluminescence [17,18], amperometry [12,19] and voltammetry [20]. Electrochemical determinations of hydrogen peroxide are generally performed by oxidation on a platinum electrode [21]. Depending upon the pH of the solution, a quite high positive potential must be applied for the oxidation of hydrogen peroxide, typical applied potentials are in the range of +0.7 to +0.9 V versus SCE [22]. As a result, many substances can interfere with the measurements. The use of biosensors with immobilized enzymes, such as peroxidase and catalase has been extensively investigated for hydrogen peroxide analysis, based on spectrophotometry [23], fluorometry [24,25], chemiluminescence [26,27] and electrochemical [28,29] techniques. In the present work, we describe a versatile method for differential amperometric determination of hydrogen peroxide in rainwater, using a gold microelectrode modified by electrodeposition of platinum, combined with an on-line tubular reactor containing catalase immobilized on resin (Amberlite IRA-743). In the next sections we present a new method of enzyme immobilization and the advantages of the differential amperometric method for applications to real samples. Comparison with spectrophotometric and amperometric methods (using mercury electrodes) are also included. 2. Experimental

Fig. 1. Schematic diagram showing the immobilization of enzymes. IRA: resin Amberlite IRA-743; GLU: glutaraldehyde; ENZ: enzyme (catalase or peroxidase).

ing boron extraction. The immobilization process is presented schematically in Fig. 1. The enzyme immobilization process is begun with the addition of 100 ␮l glutaraldehyde 0.1% on 250 mg of resin and this mixture is maintained under stirring for 5 min. Following this, 200 units of enzymes are introduced into the mixture and stirred for a further 10 min. The next step consists in filling the reactor with the resin and washing out the excess of reagents. Reactors so prepared have presented high stability for at last 15 days under intense use in flowing solutions. After this period, a decrease on the order of 18–23% on the enzyme activity has been observed. When not in use, the reactors were stored in a freezer at −20◦ C.

2.1. Enzymes immobilization 2.2. Reagents and chemicals The procedure adopted to immobilize peroxidase and catalase enzymes is simple and fast. Amberlite IRA-743 resin was chosen as support, which has active amine groups in its chemical structure. This resin was originally manufactured for applications involv-

All solutions used were of analytical grade. Hydrogen peroxide, mono- and di-hydrogen phosphate were obtained from Merck (Darmstadt, Germany). Solutions were prepared with distilled, deionized

R.C. Matos et al. / Analytica Chimica Acta 441 (2001) 73–79

water obtained from Nanopure system (Barnstead Thermolyne, Dubuque, IA). Commercial catalase (EC 1.11.1.6–1300 kU/ml) and peroxidase (EC 1.11.1.7–115 U/mg) were obtained from Sigma (St. Louis, MO). The amberlite IRA-743 ion-exchange resin and glutaraldehyde were obtained from Aldrich (Milwaukee, USA). All solutions were prepared just before their use. 2.3. Sample collection The sample collection site is located on the balcony (10 m above ground) of the Chemistry Institute of São Paulo University (São Paulo, Brazil). The rainwater samples were collected in an automatic rain collector [30], build in the lab. The rain samples were analyzed rapidly after collection or preserved by freezing and stored at −20◦ C. 2.4. Electrodes and Instrumentation The electrochemical cell comprised an array of platinum-modified gold microelectrodes [31], similar to that used with success in the differential amper-

75

ometric analysis of ascorbic and uric acids in urine [32] and ascorbic acid in soda, natural juices, beers and commercial Vitamin C tablets [33]. The array of gold microelectrodes composed of 24 microwires was obtained by removing the polymeric material from the top of a conventional electronic integrated circuit chip [31]. The resulting microelectrodes present an elliptical geometry, usually with 25 ␮m for the smaller diameter and 30–80 ␮m for the larger diameter. Modification was done by electrochemical deposition of Pt (K2 PtCl6 2 × 10−3 mol/l, pH 4.8, at −1.00 V for 15 min). Microscopic observation of the electrodes after electrodeposition showed uniform platinum deposit, with a very rough surface. Electrodes so modified were stable for at least 1 week under intense use. The reference electrode was a miniaturized Ag/AgCl(sat) electrode constructed in our laboratory [34] and a stainless steel tube (1.2 mm i.d.) was used as auxiliary electrode. In this work, a double channel flow system was employed. The solutions were propelled by pressurization, utilizing an aquarium air pump to avoid the undesirable pulsation observed when peristaltic pumps are employed [35]. Control of the flow rate was done

Fig. 2. FIA-amperometric manifold for the determination of hydrogen peroxide: (A) aquarium air pump; (B) electrolyte; (C) control valve; (D) sampling loop; (E) tubular reactor; (F and G) channels with and without reactor, respectively; (H) electrochemical cell; (I) waste; (J) potentiostat and (L) microcomputer.

76

R.C. Matos et al. / Analytica Chimica Acta 441 (2001) 73–79

by adaptation of the aquarium valve and used to pinch a tygon tube inserted in the line. Teflon tubing of 0.5 mm i.d. was used throughout the flow system. A potentiostat constructed in the authors’ laboratory, operating in the amperometric mode, was employed for FIA measurement [36]. A schematic representation of the flow system is depicted in Fig. 2. The system is constituted of an aquarium air pump, a pinch valve, sampling loop, a tubular reactor (∅ = 0.26 and 2.2 cm of length) with catalase chemically immobilized in Amberlite IRA-743 resin (immobilization process depicted in Fig. 1), an electrochemical cell and the potentiostat.

experiments involving consecutive injections of hydrogen peroxide solution were performed. Response of a gold microelectrode modified by electrodeposition of platinum for injections of 150 ␮l 1×10−5 mol/l hydrogen peroxide for a channel without enzyme, with immobilized catalase and with immobilized peroxidase were obtained. For a channel without enzyme a current of 2.5 nA was measured, while for the reactors with immobilized catalase and peroxidase currents of 0 and 1.25 nA were respectively found. The reactor with immobilized catalase was chosen as the most effective, once it is able to eliminate completely the H2 O2 , a fundamental condition for applications in differential measurements.

2.5. Procedure For amperometric detection of hydrogen peroxide, +0.60 V (versus Ag/AgClsat ) was found the most favorable potential to be applied to the gold electrode modified with platinum. The differential determination of this analyte requires at last two measurements, one involving the sample passing in the channel without the reactor (Fig. 2(G)) and a second measurement in which a similar sample passes through the enzyme reactor. In the first case a signal, corresponding to hydrogen peroxide plus the interfering components is registered. In the second case, the signal corresponds to the sample without H2 O2 (i.e. only to the interfering species). The calculated difference is compared with a calibration plot for H2 O2 . 3. Results and discussion Preliminary tests employing platinum-modified electrodes showed a very interesting behavior in the presence of hydrogen peroxide. The current enhancement was remarkable and in addition a decrease in the oxidation potential of hydrogen peroxide occurs when the electrodes are modified. Part of the increase in current can probably be attributed to the increase in the effective area of the electrodes. Observations with a microscope showed the formation of a very porous surface after platinum deposition.

3.2. Optimization of the flow system The influence of parameters, such as flow rate and sample volume was studied. Amperometric responses of a gold microelectrode modified with platinum for injections of 150 ␮l of 1×10−5 mol/l−1 hydrogen peroxide, as a function of the flow rate, varied from 0.5 to 5.0 ml/min. The signal remains virtually constant when the flow rate is varied from 0.5 to 3.0 ml/min. For high flow rates, the catalase immobilized in the tubular reactor was unable to eliminate completely the hydrogen peroxide. A flow rate of 2.0 ml/min was chosen as the most favorable, since it combines good reproducibility, high throughput (90 samples/h), lower consumption of carrier solution and complete elimination of the hydrogen peroxide. The influence of the sample volume on the analytical signal was also evaluated. Loops with internal volumes varying from 50 to 250 ␮l were tested. When the volume of the sample is increased, the amperometric signal increases, but the time required for each analysis also increases, since the cell washout process also requires a longer time. A volume of 150 ␮l was chosen as the working volume in subsequent experiments. For all the volumes studied the catalase immobilized in the tubular reactor totally eliminated the hydrogen peroxide in the samples.

3.1. Catalase and immobilized peroxidase

3.3. Calibration plot

To examine the efficiency of the tubular reactor containing immobilized catalase and peroxidase in a resin,

Fig. 3 shows the amperometric response of the modified gold microelectrode for successive injections of

R.C. Matos et al. / Analytica Chimica Acta 441 (2001) 73–79

77

Fig. 3. FIA-amperometric measurements involving injections of 150 ␮l solution containing (1–10)×10−6 mol/l of hydrogen peroxide. The inset shows the respective calibration plot. Conditions — sample volume: 150 ␮l; flow rate: 2.0 ml/min; applied potential: +0.60 V vs. Ag/AgCl(sat) .

150 ␮l hydrogen peroxide from (a) 1 to (e) 10 ␮mol/l. The proportionality between the amperometric current and the hydrogen peroxide concentrations was confirmed from the calibration plot shown in the inset (i (nA) = 8.21 × 10−12 + 8.38 × 10−5 [H2 O2 ] (␮mol/l), correlation coefficient, 0.999). Notice the very favorable signal-to-noise ratio, demonstrated by the very stable base line obtained for these low micromolar concentrations. The detection limit for the conditions adopted in present study was found as 2.9×10−7 mol/l (three times the standard deviation of the blank) [37].

Fig. 4. Comparison of the results obtained by differential amperometric analysis for 10 different samples of rainwater using a gold microelectrode modified by the deposition of platinum and (A) differential amperometric analysis using a mercury microelectrode and (B) spectrophotometric methods for the analysis of hydrogen peroxide.

Table 1 Results obtained in analysis of hydrogen peroxide in rainwater (n = 3) Samples of rainwater

[H2 O2 ] (␮mol/l) Au/Pt microelectrode

1 2 3 4 5 6 7 8 9 10

1.68 1.77 4.78 4.23 3.86 4.05 2.83 29.5 16.9 22.4

± ± ± ± ± ± ± ± ± ±

0.04 0.03 0.04 0.08 0.04 0.05 0.05 0.4 0.4 0.1

Hg microelectrode 1.87 1.80 4.78 3.59 3.53 3.95 3.17 31.6 16.4 21.2

± ± ± ± ± ± ± ± ± ±

0.02 0.01 0.04 0.07 0.07 0.06 0.09 0.3 0.2 0.5

Spectrophotometric method 1.73 1.79 4.79 4.15 3.68 3.99 2.96 30.5 15.9 22.0

± ± ± ± ± ± ± ± ± ±

0.04 0.04 0.03 0.05 0.03 0.02 0.02 0.2 0.2 0.5

78

R.C. Matos et al. / Analytica Chimica Acta 441 (2001) 73–79

3.4. Determination of hydrogen peroxide in rainwater by flow-injection analysis (FIA) The samples to be analyzed were mixed on-line with buffer solution, used as the carrier solution. Table 1 and Fig. 4 compare the results of the analyses performed by amperometry developed in this work and using the amperometric method with a mercury electrode [38] and spectrophotometric detection [39] for 10 different samples (in triplicates). Linear regression between the amperometric method with a gold microelectrode modified by electrodeposition of platinum and with a mercury microelectrode gives a slope and intercept very close to unity and zero, respectively. The confidence interval for the slope and intercept are (1.03 ± 0.03) and (−0.26 ± 0.38)/␮mol, respectively, for a 95% confidence level. Comparing the amperometry with gold/platinum microelectrode and spectrophotometry gives a slope and intercept very close to unity and zero, respectively. The confidence interval for the slope and intercept are (1.01 ± 0.02) and (−0.12 ± 0.23)/␮mol, respectively, for a 95% confidence level. Taking into account these results, no significant differences between the three methods were observed, which strongly indicates the absence of systematic errors.

4. Conclusions This work demonstrated the potentiality of the amperometric method using gold microelectrodes modified with platinum coupled with FIA techniques, for the detection of hydrogen peroxide in rainwater using catalase immobilized in a tubular reactor. The very high sensitivity provided by amperometry, combined with the low volume of the flow cell, allows us to work with small sample volumes and at low concentrations. The association of amperometric detection with FIA and the possibility of avoiding cumbersome processes, such as separation, extraction and filtration substantially increase the speed of analysis. These advantages offer a very favorable way for the rapid analysis of hydrogen peroxide in rainwater samples (throughput of 90 samples/h). Others compounds can be studied with the same amperometric-FIA system associated with different enzymes.

Acknowledgements The authors acknowledge Professor Christopher A.M. Brett (University of Coimbra) for revision on the text and to Dr. Claudimir L. Lago (University of São Paulo) for suggestion and for provide the resin used in this study. This work was supported with grants and fellowships from FAPESP (proc. 97/04268-3), CNPq (proc. 304031/85-2) and MACKPESQUISA (proc. 18/99).

References [1] B.A. Watkins, D.D. Parrish, M. Trainer, R.B. Norton, J.E. Yee, J. Geophys. Res. 100 (1995) 22831. [2] C. Brandt, R. van Eldik, Chem. Rev. 95 (1995) 119. [3] Y. Zuo, J. Hoigné, Science 260 (1993) 71. [4] H. Sakugawa, I.R. Kaplan, W. Tsai, Y. Cohan, Environ. Sci. Technol. 24 (1990) 1452. [5] R.M. Peña, S. Garc´ıa, C. Herrero, T. Lucas, Atmos. Environ. 35 (2001) 209. [6] D.W. Gunz, M.R. Hoffmann, Atmos. Environ. 24A (1990) 1601. [7] Y. Deng, Y. Zuo, Atmos. Environ. 33 (1999) 1469. [8] C.S. Fung, P.K. Mistra, R. Bloxam, S. Wong, Atmos. Environ. 25A (1990) 1601. [9] J. Willey, R.J. Kieber, R.D. Lancaster, J. Atmos. Chem. 25 (1996) 149. [10] P.A. Tanner, A.Y.S. Wong, Anal. Chim. Acta 370 (1998) 279. [11] J. Yuan, A.M. Shiller, Atmos. Environ. 34 (2000) 3973. [12] I.G.R. Gutz, D. Klockow, Fresenius Z. Anal. Chem. 335 (1989) 919. [13] B.C. Madsen, M.S. Kromis, Anal. Chem. 56 (1984) 2849. [14] Y. Fujita, I. Mori, M. Toyada, T. Matsuo, Anal. Sci. 10 (1994) 827. [15] H. Hwang, P.K. Dasgupta, Anal. Chim. Acta 170 (1985) 347. [16] Y. Ci, L. Chen, Fenxi Huaxue 17 (1989) 404. [17] W. Qin, Z. Zhang, B. Li, S. Liu, Anal. Chim. Acta 372 (1998) 357. [18] A.N. Diaz, F.G. Sanchez, J.A.G. Garcia, Anal. Chim. Acta 327 (1996) 125. [19] M. Somasundrum, K. Kirtikara, M. Tanticharoen, Anal. Chim. Acta 319 (1996) 59. [20] M. Somasundrum, M. Tanticharoen, K. Kirtikara, J. Electroanal. Chem. 407 (1996) 247. [21] J.E. Harrar, Anal. Chem. 35 (1963) 893. [22] G.G. Guibault, G.L. Lubrano, Anal. Chim. Acta 64 (1973) 439. [23] J.M. Fernandez-Romero, M.D. Luque de Castro, Anal. Chem. 65 (1993) 3046. [24] F. Schubert, F. Wang, H. Rinnerberg, Mikrochim. Acta 121 (1995) 237. [25] H.E. Posh, O.S. Wolfbeis, Mikrochim. Acta 1 (1989) 41.

R.C. Matos et al. / Analytica Chimica Acta 441 (2001) 73–79 [26] R.W. Marshall, T.D. Gibson, Anal. Chim. Acta 266 (1992) 309. [27] U. Spohn, F. Preuschoff, G. Blankenstein, D. Janasek, M.R. Kula, A. Hacker, Anal. Chim. Acta 303 (1995) 109. [28] P.D. Wentzell, S.J. Vanslyke, K.P. Bateman, Anal. Chim. Acta 246 (1991) 43. [29] J.H. Qian, Y.C. liu, H.Y. Liu, T.Y. Yu, J.Q. Deng, Anal. Biochem. 236 (1996) 208. [30] A. Fonaro, P.C. Isolani, I.G.R. Gutz, Atmos. Environ. 27 (1993) 307. [31] M.A. Augelli, V.B. Nascimento, J.J. Pedrotti, I.G.R. Gutz, L. Angnes, Analyst 122 (1997) 843. [32] R.C. Matos, M.A. Augelli, C.L. Lago, L. Angnes, Anal. Chim. Acta 404 (2000) 151.

79

[33] R.C. Matos, M.A. Augelli, J.J. Pedrotti, C.L. Lago, L. Angnes, Electroanalysis 10 (13) (1998) 887. [34] J.J. Pedrotti, L. Angnes, I.G.R. Gutz, Electroanalysis 8 (1996) 673. [35] R.C. Matos, R.S. Fontenele, I.G.R. Gutz, L. Angnes, J.J. Pedrotti, Qu´ımica Nova, accepted for publication. [36] R.C. Matos, L. Angnes, C.L. Lago, Instrum. Sci. Technol. 26 (1998) 451. [37] J.C. Miller, J.N. Miller, Statistics for Analytical Chemistry, Harwood, Chichester, 1992. [38] F.R., Rocha, I.G.R. Gutz, 2001, in press. [39] H.U. Bergmeyer, Methods of Enzymatic Analysis, 2nd Edition, Academic Press, New York, 1974.