THE AGRICULTURAL SCIENTIST Lead PHILIPPINE Detection Using a Pineapple Bioelectrode Vol. 89 No. 2, 134-140 June 2006

E-R.E. ISSNMojica 0031-7454 et al.

Lead Detection Using a Pineapple Bioelectrode Elmer-Rico E. Mojica1,2*, Socrates P. Gomez1, Jose Rene L. Micor1 and Custer C. Deocaris3,4,5 1Institute

of Chemistry, College of Arts and Sciences, University of the Philippines Los Baños, Laguna, Philippines

2Department of Chemistry, Natural Sciences Complex, University at Buffalo, The State University of New York, Buffalo,

New York 14260-3000, USA 3Department of Chemistry and Biotechnology, University of Tokyo, 7-3-1, Hongo, Tokyo, Japan 4Gene Function Research Center, 1-1-1 Higashi, AIST Central 4, Tsukuba 305-8562, Japan 5 Institute of Chemistry, College of Science, University of the Philippines Diliman, Quezon City, Philippines *Author for correspondence; e-mail: [email protected]

Freeze dried pineapple fruit peelings were powderized and used as a modifier of a carbon paste electrode. The pineapple bioelectrode was used to determine trace levels of lead in aqueous solution. Accumulation was followed by medium-exchange to an electrolyte solution where surface bound lead ions were measured. The response of the modified electrodes was assessed in terms of the supporting electrolyte used, proportion of the pineapple in the bioelectrode, pH, accumulation time, deposition time, deposition potential, regeneration and lead concentration. The surface was successfully renewed by soaking the electrode in an EDTA solution. The relative standard deviation for ten accumulation/measurement/renewal cycles with a 1 mg L-1 lead ion solution was 4.2%. The peak current was directly proportional to the concentration of lead ions in the range of 1–10 mgL-1 (R=0.991). The presence of other metals such as Cr(III), Cd(II), Zn(II), Co(II), Ni(II), Ca(II), Mg(II), Mn(II), Al(II) and Fe(III) did not interfere with the determination of Pb(II) although Hg(II), Ag(I) and Cu(II) reduced the peak of Pb(II) by 11.82–34.61%. The pineapple bioelectrode was successfully used to measure the lead content of spiked water and a laboratory waste sample with values very similar to those obtained by atomic absorption spectrometer.

Key words: lead ions, bioelectrode, pineapple peelings Abbreviations: Ag/AgCl – silver/silver chloride, CME – chemically modified electrodes, CMCPEs – chemically modified carbon paste electrodes, CPE – carbon paste electrode, CV – cyclic voltammetry, DPASV – differential pulse anodic stripping voltammetry INTRODUCTION The field of chemically modified electrodes (CMEs) has evolved into a widely researched area since the 1970s (Lane and Hubbard 1973; Moses et al 1975). CMEs have been studied from the fundamental aspects (e.g., electron transfer and mass transport) to diverse applications particularly in biosensing and electroanalysis (Murray 1984; Arrigan 1994). In electroanalysis, the chemically modified carbon paste electrodes (CMCPEs) are among the most popular due to several advantages: ease and speed of preparing and obtaining a reproducible surface, ability to vary the modifier and low residual current. CMCPEs can be fabricated even in ordinary laboratories as it entails

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homogenizing carbon powder, modifier and paraffin oil into a paste and loading this paste into a convenient holder (Kalcher 1990; Kalcher et al. 1995). In this experiment, pineapple fruit peelings were chosen as modifier of a carbon paste electrode based on the precept that they are a rich source of cysteine proteases like bromelain, ananain and comosain, which contain amino acids or functional groups that can serve as potential binding sites for complexing with lead ions. The primary metalbinding amino acid side chains are imidazole, carboxylate, thiol, thioester, hydroxyl, indole, quanidinium and amide. A study utilizing keratin as modifier in a carbon paste electrode for silver (I) determination proved that proteins could be used on the basis of interaction of metals with thiol and

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Lead Detection Using a Pineapple Bioelectrode

disulfide groups of keratin (Sugawara et al. 1998). It is also possible to take advantage of the presence of cellulose, hemicellulose and lignin in the pineapple peel (Hartley 1978; Rani and Nand 2004). The fabricated electrode or bioelectrode is used for the voltammetric analysis of lead ions in aqueous samples. This study assessed the response of pineapple fruit peelings-modified electrode in terms of the supporting electrolyte used, the proportion of the pineapple peelings in the bioelectrode, pH, accumulation time, deposition time, deposition potential, regeneration and lead concentration.

MATERIALS AND METHODS Reagents Deionized distilled water was used all throughout the experiment. The 1000 mg L-1 stock standard solutions of Pb, an AAS-standard purchased from AJAX chemicals, was used to prepare the solutions. Deactivated carbon powder, mineral oil and sodium acetate were obtained from Sigma Aldrich Co. (St. Louis, Mo., USA). Analytical grade hydrochloric acid, sodium chloride and sodium hydroxide, sodium phosphate and sodium biphosphate were from Ajax Chemicals. Acetic acid was purchased from J.T. Baker. Preparation of Pineapple Bioelectrode Rind of the pineapple fruit, smooth Cayenne variety from a local market in Los Baños, Laguna, Philippines, was rinsed with deionized water and homogenized. The mixture was then lyophilized and the resulting powder was stored at 20 oC. The bioelectrode was prepared by making a homogenous paste with various amounts of pineapple powder and carbon powder (100 mg). Twenty µL of mineral oil was added as binder. The paste mixture was firmly packed inside a 3-mm polyethylene tube, which is in contact with a copper wire.

was immersed in the stirred lead ion solution for a given time at open circuit. The electrode was then placed in the three-electrode system containing supporting electrolyte for voltammetric measurement. Cyclic voltammetry (CV) was performed with both bare carbon paste electrode (CPE) and the pineapple bioelectrode in a 0.01 M HCl solution. A potential range of –1500 to 1500 mV was applied and reversed. Scan rate of 100 mV s-1 was used in the analysis. Optimization of parameters like supporting electrolyte, proportion of pineapple peeling in the bioelectrode, pH, accumulation time, deposition time and deposition potential was done using differential pulse anodic stripping voltammetry (DPASV) analysis. During DPASV, the modified electrode was pre-concentrated under open circuit, rinsed with deionized distilled water and then connected to Metrohm 693VA processor for voltammetric measurements. The parameters used for the DPASV on the different heavy metals are as follows: U. amplitude: 50 mV t. step: 0.20 s U. meas: -1000 mV U. start: -1000 mV U. end: 1000 mV

Rot Speed: 0 min-1 t. meas: 16.7 ms t. pulse: 33.3 ms U step: 10 mV Sweep rate: 50 mV s-1

RESULTS AND DISCUSSION The evaluation of the pineapple bioelectrode was conducted and compared with the unmodified carbon paste electrode (Fig. 1). It can be seen that both bare CPE and pineapple bioelectrode produced similar voltammograms. However, the cyclic voltammogram of the pineapple

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Current,I (uA)

Voltammetry All voltammetric measurements were done on the Metrohm 693 VA Processor. The Metrohm is connected to a threeelectrode system which was made up of the working electrode (pineapple bioelectrode), the auxiliary electrode (platinum wire) and the reference electrode (silver/silver chloride [Ag/AgCl] electrode). All potentials were reported relative to the reference electrode. The voltammetric data processed on the Metrohm are displayed on the computer via the RS232 interface. The computer has 693 VA back up software compatible with the Metrohm output data. Experiments were done using the accumulation/medium exchange/ voltammetry/ regeneration scheme. In the accumulation step, the modified carbon paste electrode

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bare CPE bioelectrode

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-500 -1.5

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Fig. 1. Cyclic voltammograms of bare CPE and pineapple bioelectrode. Working conditions: scan rate of 100 mv s -1 and 0.01M HCl supporting electrolyte (CPE - carbon paste electrode)

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Optimization Supporting electrolyte. Different stripping solutions were used and resulted in different current peak heights. The supporting electrolyte at which the adsorption of lead (II) ions is best on the surface of the modified electrode and gave highest pulse was 0.01 M HCl (Fig. 4). This could be due to the fact that HCl has the ability to enhance the current by complexation reaction between Pb (II) and chloride in giving an extra driving force for the oxidation. In contrast, the use of NaOH and NaCl, where the positive inert species like Na+ competes in carrying the current (can also be oxidized or stripped), resulted in interferences. This was observed in a study involving the use of chloride media such as NH4Cl and HCl in determining the trace amount of heavy metals because of the presence of species capable of complexing the metal ion formed during the anodic scan which favors the oxidation reaction (Bartlett et al, 2000). Furthermore, the characteristic of the chloride ion as a better leaving group enhances and provides a well-defined peak current signal and higher sensitivity. Therefore, 0.01 M HCl was used as the supporting electrolyte for stripping analysis in the entire study. Proportion of the modifier. The proportion of the pineapple peeling to the carbon powder is an important factor on the current peak signal, wherein the current signal was

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bare CPE bioelectrode

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Fig. 2. Cyclic voltammogram of pineapple bioelectrode and bare CPE immersed in lead (II) solution. Same working conditions as Fig. 1. (CPE - carbon paste electrode) 70

- - - -unmodified modified

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Current (uA)

bioelectrode has slightly higher background current compared to the bare CPE. It is noted that this is the potential window where the analyte such as lead ions is reduced. The roughness of the resulting current response within the potential window indicates the presence of interfering ions that may be bound to the modifier. Figure 2 shows a cyclic voltammogram of the pineapple electrode pre-concentrated in a 100 mg L-1 lead (II) solution. The cyclic voltammogram produced a distinct peak at voltage range of –600 to –400 mV, which is the voltage range for the reduction of lead (II) ions. Moreover, the peak current produced was at the anodic peak, which means that the lead (II) ions were favorably oxidized rather than reduced. From here, the anodic peak (anodic stripping voltammetry) was chosen for the peak current measurement of lead (II) ions. The quantification of lead (II) ions (differential pulse voltammetry) is coupled with anodic stripping voltammetry where the resulting peak (Fig. 3) would be known to be due to the Pb (II) ions and not the pineapple peelings or modifier. The modifier only functions to adsorb or complex the analyte species and must not be oxidized, otherwise, it would be an interference making it unsuitable for electroanalytical analysis. It can be concluded that the utilization of pineapple peelings as modifier for the bioelectrode proved successful in analyzing trace metals like lead (II) which has peak current within the potential window of the modified electrode.

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Potential, E (mV)

Fig. 3. Differential pulse voltammogram of the pineapple biolectrode and bare CPE immersed in lead (II) solution. Working conditions: scan rate of 40 mV s -1 and 0.01 M HCl supporting electrolyte (CPE - carbon paste electrode)

observed to decrease as the amount of the modifier was increased (Fig. 5). The optimum peak current signal was obtained using 5% ratio of pineapple modifier to graphite powder. This could be due to the physical form of the pineapple modifier which is a powdered solid. By increasing the amount of the modifier, the amount of carbon powder decreases, disabling the transfer of electrons in the bioelectrode. Even though there would be more active sites at higher proportions of the modifier in binding metals, the conductivity at the surface of the electrode was greatly affected. In effect, less of the analyte species (lead) is deposited and oxidized as the ratio is increased.

The Philippine Agricultural Scientist Vol. 89 No. 2 (June 2006)

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Lead Detection Using a Pineapple Bioelectrode

60 55 50

0.01 M NaCl 0.01 M NaOH 0.01 M HCl

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Fig. 4. Differential pulse voltammogram of pineapple bioelectrode at different supporting electrolytes. Working conditions: 5% modifier, 3 min accumulation time and 60 sec deposition time

Fig. 6. Effect of pH on the peak current of the pineapple bioelectrode. Working conditions: same as in Fig. 4 and 5.

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4 5 6 Pe rcent Modifier

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Fig. 5. Effect of amount of pineapple on the peak current of the bioelectrode. Working conditions: 0.01 M HCl supporting electrolyte, 3 min accumulation time, 60 sec deposition time

Fig. 7. Effect of accumulation time on current peak of the pineapple bioelectrode. Working conditions same as in Fig. 6.

pH of accumulating solution. The effect of pH (adjusted using different buffers) was examined and pH 7.0 showed the highest peak current (Fig. 6). This is in agreement with the optimum pH of bromelain, which is at pH 6.8 and close to neutral pH in catalyzing a variety of substrates (Rowan et al. 1990). However, Heinicke and Gortner (1957) emphasized that bromelain is a mixture of four proteases: bromelain pH 4.5, bromelain pH 5.5, bromelain pH 7.0 and bromelain pH 8.5. Bromelain pH 4.5 and 5.5 show many similarities while bromelain pH 7.0 and 8.5 show similar characteristics (Rowan et al. 1990). Since the fruit enzyme is an acidic protein (Heinicke and Gortner 1957), a higher peak response was observed at pH 4–5 (Fig. 6). Lead ions compete with protons for the negative charges of the car-

boxylic groups. However, the activity of these two bromelains decreases after hydrolysis (pH 6.0). At pH 7.0, the optimum pH characterized by very high peak current signal was obtained. This is due to the interaction between the reactive sulfhydryl groups and lead ions. The amino groups also play a role. However, the thiol groups predominate in binding the Pb (II) ions. Cysteine, with a pKa of 8.35, which is the optimum pH, favors the dissociation of the proton of the thiol group and the formation of a complex with Pb (II) ions. Accumulation time. It can be observed that the peak current increases linearly with increasing accumulation time (Fig. 7). The current response signal is appreciable since more lead ions are oxidized because of greater amount of

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the analyte deposited on the surface of the electrode at longer accumulation times. However, after 180 s, there was a decrease in the peak current and the size of the stripping peak attained constant value. This is due to the saturation of the binding sites of the modifier as time progresses or the attainment of equilibrium between the bound Pb2+ and the ions in solution. Therefore, it is assumed that there is a fixed number of binding sites exposed to the Pb (II) ions and the constant reading indicates that the maximum number of Pb2+ has been accumulated at these sites. Deposition time. An increasing trend was observed for the current signal as the deposition time was increased (Fig. 8). Increasing the deposition time would mean more time for the deposition of Pb (II) ions on the surface of the electrode. As a result, there would be an increase in the peak height indicating the higher concentration of the analyte species being stripped into the solution until it reaches a near equilibrium. Deposition potential. There was an increase in the peak current as the deposition potential is changed to a more negative potential (Fig. 9) since more Pb2+ are converted to its reduced form Pb (0) during electrolysis. The conversion of the metal cation to its metallic form takes longer to scan towards the more negative potential, hence, higher concentrations of lead will bind to the electrode resulting in a more distinct and higher peak current response. On the contrary, as the deposition potential was increased to –1500 mV, the peak height suddenly decreased (Fig. 9). The deposition potential of –1,500 mV deviates from the trend as it is observed to decrease suddenly.

it from the remaining analyte, by use of regenerating solutions such as ethylenediammine tetraacetic acid EDTA or acids, or even through electrochemical means (Zen and Lee 1993). The use of 0.01 M EDTA proved to be successful in regenerating the electrode surface by soaking the electrode for at least 10 min (Fig. 10). However, the performance of the regenerated electrode started to decrease after the second regeneration.

Regeneration of the Electrode Surface Regeneration of the electrode surface is done to make an electrode usable again. This is carried out by “cleansing”

Fig. 9. The effect of various deposition potential in the determination of Pb (II). Working conditions: same as in Fig. 6.

Fig. 8. The effect of various deposition time in the determination of Pb (II). Working conditions: same as in Fig. 6.

Fig. 10. Regeneration of the pineapple bioelectrode.

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Electrode Response to Pb2+ The relationship between the peak current and the concentration was studied. The peak current was directly proportional to the concentration of lead ions in the range 1–10 mg L-1 (R = 0.991). The relative standard deviation in 1 mg L-1 lead solution was calculated to be 4.2% (n=10).

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Interferences The selectivity of the bioelectrode was also determined. Several metal ions were used for their possible interference in lead (II) determination. The determination of a 10 mg L-1 Pb(II) using the optimized conditions was not affected by the presence of 100 mg L-1 of Cr(III), Cd(II), Zn(II), Co(II), Ni(II) Ca(II), Mg(II), Mn(II), Al(II) and Fe(III). However, the presence of Hg(II), Ag(I) and Cu(II) at 50 mg L-1 reduced the Pb(II) peak by 34.61%, 33.05% and 11.82%, respectively. No Pb (II) peak was observed when 500 mg L-1 of Hg(II) and Ag(II) was used. However, the use of masking reagents like KSCN and KCN removed the interferences caused by these metal ions (Molina-Holgado et al. 1995). Application to Lead Ion Determination The lead content of water samples from a river and laboratory waste samples was determined by the use of the pineapple bioelectrode using the optimized parameters. No lead ions were found in river samples and this was confirmed by graphite furnace AAS. Addition of lead ions to the river water sample showed a linear increase of peak current (data not shown). For the laboratory waste sample, the lead content was found to be 14.07 ± 0.32 mg L-1 using standard addition while AAS analysis gave 14.10 ± 0.03 (Table 1). To further test the effectiveness of the biolectrode, a deionized water sample was used and spiked with known amount of lead ions. No lead was detected in the deionized water sample and spiking it with 5.0 mg L-1 lead ions gave a mean value of 4.95 ± 0.10 or a percent recovery of 99%. This was compared with flameless AAS which gave a reading of 5.01 ± 0.05 on the spiked samples or a percent recovery of 100%. Mechanism The ability of the pineapple bioelectrode to detect lead is probably due to the affinity of the lead ions with the functional groups present in pineapple peelings. Pineapple peelings are a rich source of cysteine proteases like bromelain,

ananain and comosain (Rowan et al. 1990). These enzymes belong to the group of thiol or cysteine proteases just like papain and ficin that contains different amino acids whose functional groups can serve as potential binding sites for complexation with metals like lead ions (Ota et al. 1964). These amino acids or peptides can serve as a very effective and specific ligand for a variety of metal ions since they contain a great number of potential donor atoms through the peptide backbone and amino acid side chains (Gooding et al. 2001). The complexes formed exist in a variety of conformation that is sensitive to the pH environment of the complex (Sigel and Martin 1982; Kozlowski et al. 1999). Another possible site of complexations may be the lignocellulosic materials present in the pineapple peelings. These materials are responsible for the binding of lead ions in kapok fiber (Mojica et al. 2002). Lignins are a group of phenolic polymers (Lignin Institute 2001) with derivatives well known for their ability to bind heavy metal ions (Varma et al. 1990). They contain an abundant amount of oxygen-containing functional groups such as phenolic, alcoholic and carboxylic structures that could possibly form lignin-metal macromolecular complexes with high stability through ionic, hydrogen and coordinate covalent bonding. Pineapple contained phenolic acids (ferulic, p-coumaric, and diferulic) that are bound to polysaccharide components (Hartley 1978). These polysaccharides components (usually cellulose) can also bind with metal by different mechanisms. Alginic acid which is a polymeric carbohydrate was successfully used as modifier of carbon paste electrode to detect lead by ion exchange mechanism (Wang et al 1991). Although the active sites have yet to be determined, it is possible that the hydroxyl group and the carbonyl groups are the main binding sites. Other mechanisms for the lead affinity of pineapple peelings are by adsorption and chelation in which the functional groups in pineapple peelings play a very important part.

Table 1. Determination of lead in lead-spiked water and laboratory waste sample using five pineapple bioelectrodes and atomic absorption spectrometer. Lead (mgL -1) Using bioelectrodes Spiked lead in deionized water Lab waste samples Using AAS Spiked lead in deionized water Lab waste samples

E1 4.9623

E2 4.8553

E3 5.0973

E4 4.9712

E5 4.8678

Mean 4.95078

SD 0.097493

13.7612

14.3142

14.1492

14.4334

13.6909

14.06978

0.330545

R1 5.01

R2 5.07

R3 5.06

R4 4.95

R5 4.97

Mean 5.012

SD 0.053104

14.13

14.1

14.05

14.11

14.09

14.096

0.029665

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