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Talanta 75 (2008) 301306

Differential amperometric determination of hydrogen peroxide in honeys using ow-injection analysis with enzymatic reactor
R omulo Augusto de Abreu Franchini, Maria Auxiliadora Costa Matos, Rosana Colombara, Renato Camargo Matos
NUPIS (N ucleo de Pesquisa em Instrumenta ca o e Separa co es Anal ticas), Departamento de Qu mica, Instituto de Ci encias Exatas, Universidade Federal de Juiz de Fora, 36036-330 Juiz de Fora, MG, Brazil Received 30 August 2007; received in revised form 30 October 2007; accepted 7 November 2007 Available online 17 November 2007

Abstract Hydrogen peroxide (H2 O2 ) present in honey was rapidly determined by the differential amperometric method in association with ow-injection analysis (FIA) and a tubular reactor containing immobilized enzymes. A gold electrode modied by electrochemical deposition of platinum was employed as working electrode. Hydrogen peroxide was quantied in 14 samples of Brazilian commercial honeys using amperometric differential measurements at +0.60 V vs. Ag/AgCl(sat) . For the enzymatic consumption of H2 O2 , a tubular reactor containing immobilized peroxidase was constructed using an immobilization of enzymes on Amberlite IRA-743 resin. The linear dynamic range in H2 O2 extends from 1 to 100 106 mol L1 , at pH 7.0. At ow rate of 2.0 mL min1 and injecting 150 L sample volumes, the sampling frequency of the 90 determinations per hour is afforded. This method is based on three steps involving the ow-injection of: (1) the sample spiked with a standard solution, (2) the pure sample and (3) the enzymatically treated sample with peroxidase immobilized. The reproducibility of the current peaks for hydrogen peroxide in 105 mol L1 range concentration showed a relative standard deviation (R.S.D.) better than 1%. The detection limit of this method is 2.9 107 mol L1 . The honey samples analyses were compared with the parallel spectrophotometric determination, and showed an excellent correlation between the methods. 2007 Elsevier B.V. All rights reserved.
Keywords: Amperometry; Honey; Hydrogen peroxide; Peroxidase immobilized

1. Introduction Honey contains a complex matrix of components, which presents a considerable analytical challenge. It is a liquid (or semiliquid) product made up of about 80% solids. It is produced by bees from the nectar of plants, as well as from honeydew. Bees and plants are the primary sources of components such as: carbohydrates (fructose, glucose, maltose and sucrose with traces of many other sugars depending on the oral origin), water, traces of organic acids, enzymes, aminoacids, pigment, and other components like pollen and wax which arise during honey maturation [1]. The chemical analysis of honey has three main purposes: (1) to determine the geographical and botanical origin, (2) verication of adulteration and (3) identication of pharmacological active compounds. The rst and second points assist with cer-

Corresponding author. Fax: +55 32 3229 3314. E-mail address: renato.matos@ufjf.edu.br (R.C. Matos).

tication of quality of the product which is commonly used as a food product; and the third area allows the examination of content for the use of honey in medicinal purposes. Hydrogen peroxide is a product of many biological reactions catalyzed by several oxidase enzymes. All honeys contain peroxide, which imbues them with antibacterial properties. It has been shown that the antibacterial activity of honey occurs due to hydrogen peroxide generation [25]. Therefore, the determination of hydrogen peroxide is important in the characterization and selection of honey samples for its use as an antimicrobial agent. Hydrogen peroxide is generated by the enzyme glucose oxidase when honey is diluted and maximum levels of hydrogen peroxide encountered in the diluted honeys are in the range of 12 mmol L1 [6]. Dilution is needed to decrease the acidity of the medium and for adjusting the pH for proper action of glucose oxidase. Weston [5] stated that the level of hydrogen peroxide in honey is essentially determined by the amount of catalase, which originates from ower pollen, and glucose oxidase, which originates from the hypopharyngeal glands of

0039-9140/$ see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2007.11.011

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bees. Manzoori et al. [7] have proposed the spectrouorometric determination of hydrogen peroxide in several honey samples using crude extract of kohlrabi (Brassica oleracea gongylodes), which is a rich source of peroxidase. Franchini et al. [8] have described a versatile method for spectrophotometric determination of micromolar hydrogen peroxide in commercial Brazilian honey samples using a peroxidase immobilized on resin and the determination of trace metals using capillary zone electrophoresis without any treatment of honey samples. Electrochemical determinations of hydrogen peroxide are generally performed by oxidation on a platinum electrode [9]. Depending upon the pH of the solution, a very high positive potential must be applied for the oxidation of hydrogen peroxide. The typical applied potentials are in the range of +0.7 to +0.9 V vs. SCE [10]. 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 [8,11], uorometry [7,12], chemiluminescence [13,14] and electrochemical [15] techniques. Strategies have been investigated to adapt quantication methods to the range of sample concentrations with low cost. In the analytical methods using enzymes, the reduction in cost of the determination is generally associated with reduced enzyme consumption. Recently, various ion-exchange resins have gained considerable attention not only for separation purposes but also as carriers of catalytic active substances. Considerable thought has been paid to their application for immobilization of enzymes [11,14,1619]. The resins should meet several requirements. Their porous structure must be strong enough to withstand the enhanced pressure usually applied in forced ow bioreactors. Furthermore, the membrane material must be chemically and physically resistant. These requirements can be met by various aromatic and aliphatic polyamides. Therefore, resin prepared from these polymers is a suitable substrate for the immobilization of enzymes [20]. The covalent binding of the enzyme to the polymer matrix is one of the most prospective methods for immobilization. In the present work, we describe a versatile method for differential amperometric determination of hydrogen peroxide in honey, using a gold microelectrode modied by electrodeposition of platinum, combined with an on-line tubular reactor containing peroxidase immobilized on resin (Amberlite IRA743) without any treatment of samples. The concentration of the hydrogen peroxide in each sample was calculated based on the difference between the current measurement before and after the enzymatic treatment. 2. Experimental 2.1. Enzymes immobilization The procedure adopted to immobilize the peroxidase enzyme was quick and very simple [15]. Amberlite IRA-173 resin was selected as support, because it has active amine groups in its chemical structure. The enzyme immobilization process begins with the addition of 100 L of glutaraldehyde 0.1% to 250 mg

of resin, and this mixture was stirred for 5 min. Subsequently, 200 units of enzymes were introduced into the mixture and stirred for an additional 10 min. In the next step, the resin was transferred to a length of tygon tubing (2.5 mm of i.d. and 25 mm long) with one of its extremities closed with a thin layer of glass wool to assemble the reactor. At this point, the other extremity of the tubing was then closed with glass wool. To adapt the enzymatic reactor to a FIA (ow-injection analysis) system, the tubing (0.8 mm of i.d) was attached at in each of its extremities with the aid of a small piece of silicone tubing (1.3 mm i.d. and 5 mm long). Finally, the reactor was washed with 10 mmol L1 phosphate buffer solution (pH 7.0) to remove the excess of peroxidase. 2.2. Reagents and chemicals All solutions used were of analytical grade. Hydrogen peroxide, mono- and di-hydrogen phosphates were obtained from Merck (Darmstadt, Germany). Solutions were prepared by dissolving the solids in distilled water that was also treated with a nanopure system. Commercial peroxidase (EC 1.11.1.7115 U mg1 ) was obtained from Sigma (St. Louis, MO, USA). The Amberlite IRA-743 ion-exchange resin and glutaraldehyde were obtained from Aldrich (Milwaukee, WI, USA). Diluted solutions of hydrogen peroxide were prepared daily using deionized water. 2.3. Sample collection This work was carried out on 14 samples in Brazil. The samples were stored in the dark at room temperature prior to analysis. For determination of hydrogen peroxide, 1 g of honey was dissolved in 10 mL of puried water and injected in the ow-injection system. Each sample was injected in triplicate. 2.4. Electrodes and instrumentation The electrochemical cell comprised a platinum-modied gold electrode (3.0 mm diameter). Modication was done by electrochemical deposition of Pt (K2 PtCl6 2 103 mol L1 , 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 modied were stable for at least 1 week under intense use. The reference electrode was a miniaturized Ag/AgCl(sat) electrode constructed in our laboratory [21] and a stainless steel tube (1.2 mm i.d.) was used as auxiliary electrode. In this work, a double channel ow 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 [22]. Control of the ow rate was done by adaptation of the aquarium valve outlet with a pinched tygon tube inserted in the line. Teon tubing of 0.5 mm i.d. was used throughout the ow system. The ow system used during the development of this work consisted of two lines, in rst the sample was added in the detection system, in the second the sample was inserted in the line that

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contain the enzymatic reactor before of the detection system. A potentiostat (-AUTOLAB) operating in the amperometric mode was employed for FIA measurement. Temperature control was achieved using a THERMOMIX 18 BU B thermostatic bath Braun Biotech. International. The system contains an aquarium air pump, a pinch valve, sampling loop, a tubular reactor ( = 0.25 cm and 2.5 cm of length) with peroxidase chemically immobilized in Amberlite IRA-743 resin, an electrochemical cell and the potentiostat. 2.5. Procedure For amperometric detection of hydrogen peroxide, +0.60 V was found the most favorable potential to be applied to the gold electrode modied with platinum. The differential determination of this analyte requires at last three measurements, one involving the sample containing a standard addition in the channel without the reactor, a second containing just the sample in the channel without the reactor, and the third measurement involves a sample passes through the enzyme reactor. In the rst case a signal, corresponding to hydrogen peroxide standard, hydrogen peroxide of the sample and plus the interfering components is registered. In the second case, the signal corresponds to the sample without hydrogen peroxide standard. In the third case, the signal corresponds to the sample without H2 O2 (i.e., only to the interfering species). The calculated difference is compared with a H2 O2 standard. 3. Results and discussion Preliminary tests employing platinum-modied 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 modied. 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. 3.1. Immobilized peroxidase and optimization of the ow system To examine the efciency of the tubular reactor containing immobilized peroxidase in a resin, experiments involving consecutive injections of hydrogen peroxide solution were performed. Responses of a gold electrode modied by electrodeposition of platinum for injections of 150 L of hydrogen peroxide 1 105 mol L1 for a channel without enzyme and with immobilized peroxidase were obtained. For a channel without enzyme a current of 0.17 A was measured, while for the reactors with immobilized peroxidase currents of 0 A was found. The reactor with immobilized peroxidase was effective, once it was able to eliminate completely the H2 O2 , a fundamental condition for applications in differential measurements. The inuence of parameters such as ow rate and sample volume was studied. Fig. 1A shows the amperometric
Fig. 1. Repetitive injections of hydrogen peroxide 1 105 mol L1 to nd the most suitable working conditions. (A) Flow rate from 0.5 to 5.0 mL min1 and (B) samples volume injected from 50 to 250 L. Measurements made with a gold electrode modied by electrodeposition of platinum. Applied potential, +600 mV vs. Ag/AgCl(sat) .

responses of a gold electrode modied with platinum for injections of 150 L of hydrogen peroxide 1 105 mol L1 , as a function of the ow rate, varied from 0.5 to 5.0 mL min1 with and without reactor. The signal remains virtually constant when the ow rate is changed from 1.5 to 3.0 mL min1 . For high ow rates, the peroxidase immobilized in the tubular reactor was unable to eliminate completely the hydrogen peroxide. The elimination reaction rate has to decrease when the ow rate decreases. A ow rate of 2.0 mL min1 was chosen as the most favorable, since it combines good reproducibility, high throughput (90 samples h1 ), lower consumption of carrier solution and complete elimination of the hydrogen peroxide. Fig. 1B shows the inuence of the sample volume on the analytical signal which 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 grew but the analysis time also increased, once the cell wash-out process also requires a longer time. The volume of 150 L was selected as the working volume in the following experiments.

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Fig. 2. FIA-amperometric measurements involving injections of 150 L solution containing 1 10 106 mol L1 of hydrogen peroxide. The inset shows the respective calibration plot. Conditions: sample volume, 150 L; ow rate, 2.0 mL min1 ; applied potential, +600 mV vs. Ag/AgCl(sat) .

For all the volumes studied the peroxidase immobilized in the tubular reactor completely eliminated the hydrogen peroxide in the samples when used ow rate of 2.0 mL min1 . An important characteristic observed for the immobilized enzyme was a storage stability of at last 2 week under intense use with hydrogen peroxide standard. After this period, a decrease on the order of 3045% of the enzyme activity was observed. When applied in the determination of hydrogen peroxide in honey, the enzymatic reactor showed a loss in the enzyme activity after 50 injections, requiring construction of a new reactor. When not in use, the reactors were stored in a freezer at 20 C [8]. 3.2. Calibration plot Fig. 2 shows the amperometric response of the modied gold electrode for successive injections of 150 L of hydrogen peroxide from (a) 1 mol L1 to (e) 10 mol L1 . The proportionality between the amperometric current and the hydrogen peroxide concentrations was conrmed from the calibration plot shown in the inset (i (A) = 3.77 103 + 1.67 102 [H2 O2 ](mol L1 ); correlation coefcient, 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 2.9 107 mol L1 (3 times the standard deviation of the blank) [23]. 3.3. Determination of hydrogen peroxide in honey by ow-injection analysis The samples to be analyzed were mixed on-line with buffer solution, used as the carrier solution. Fig. 3(AC) shows three sequences of hydrogen peroxide analysis for three samples. Each group of three peaks corresponds to 150 L injections of the honey sample containing (I) a standard addition (2 106 mol L1 of the standard hydrogen peroxide), (II) the sample without treatment, and (III) the sample after enzymatic treatment with peroxidase immobilized. The standard addition
Fig. 3. Differential FIA-amperometric measurements of hydrogen peroxide in three different samples of honey: (A) sample 3; (B) sample 4; and (C) sample 7. The triplicate injection correspond to: (I) 150 L of sample +2 106 mol L1 of the standard hydrogen peroxide solution, (II) 150 L of the sample and (III) 150 L of the sample after enzymatic treatment with peroxidase immobilized. Other conditions as in Fig. 2.

R.A.d.A. Franchini et al. / Talanta 75 (2008) 301306 Table 1 Results obtained in analysis of hydrogen peroxide (mg kg1 ) in honey samples Number of sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Mean Vmin Vmax Geographical origin Vic osa Teres opolis Teres opolis Teres opolis Teres opolis Teres opolis Teres opolis Esp rito Santo Belo Horizonte Tabuleiro Coronel Pacheco Coronel Pacheco Volta Redonda Juiz de Fora H2 O2 (mg kg1 ) (amperometry) 150 3 139 3 56 2 130 3 30 1 80 205 2 157 3 140 2 148 4 47 1 80 2 155 4 37 0 103 8 205

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H2 O2 (mg kg1 ) (spectrophotometry) 155 2 135 4 71 6 131 6 28 1 90 214 4 158 5 139 3 142 4 62 3 91 3 147 6 47 1 109 9 214

method was carried out with the peaks (I) and (II) after subtracting of the height of the peak (III). Table 1 and Fig. 4 compare the results of the analyses performed by amperometry developed in this work and using the spectrophotometric detection [8] for 14 different samples (in triplicates). Comparing the amperometry with gold/platinum electrode and spectrophotometry gives a slope and intercept very close to unity and zero, respectively. The condence interval for the slope and intercept are (0.95 0.03) and (8.14 4.04) mg kg1 , respectively, for a 95% condence level. Taking into account these results, no signicant 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 electrodes modied with platinum coupled with ow-injection analysis techniques, for the detection of hydrogen peroxide in honey using peroxidase immobilized in a tubular reactor. The very high sensitivity provided by amperometry, combined with the low volume of the ow cell, allows us to work with small sample volumes and at low concentrations. The association of amperometric detection with ow-injection analysis and the possibility of avoiding cumbersome processes such as separation, extraction and ltration substantially increase the speed of analysis. These advantages offer a very favorable way for the rapid analysis of hydrogen peroxide in honey samples (throughput of 90-samples h1 ). Acknowledgements o de Authors would like to thank FAPEMIG (Fundac a ` Pesquisa do Estado de Minas Gerais), CNPq Amparo a (Conselho Nacional de Desenvolvimento Cient co e Tecnol ogico) and PROPESQ/UFJF (Pr o-Reitoria de Pesquisa e o da Universidade Federal de Juiz de Fora) for P os-Graduac a nancial support and grants. References
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Fig. 4. Comparison of the results obtained by differential amperometric analysis for 10 different samples of rainwater using a gold microelectrode modied by the deposition of platinum and (A) differential amperometric analysis using a mercury microelectrode and (B) spectrophotometric methods for the analysis of hydrogen peroxide.

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