Square-Wave Adsorptive Cathodic Stripping Voltammeteric Determination of Manganese ( II ) Using a Carbon Paste Electrode Modified with Montmorillonite Clay

Manganese is an essential micronutrient for all organisms; however at high concentrations it has a toxic effect. Manganese toxicity is a serious constraint to crop cultivation since it is taken-up by plants and can easily be passed into the food chain again causing symptoms of Parkinson’s disease. A fully validated square-wave adsorptive cathodic stripping voltammetry method has been developed for determination of Mn (II) as a complex with 2-(5’-bromo-2’-pyridylazo) 5-diethylaminophenol in aqueous solutions using a carbon paste electrode (CPE) modified with montmorillonite-Na clay. The results showed that the modified CPE (90% (w/w) graphite powder and 10% (w/w) montmorillonite-Na clay) exhibited excellent electrochemical activity towards the investigated Mn (II) complex in acetate buffer of pH = 5.0. Factors affecting the performance of the modified carbon paste electrode and the sensitivity of the described squarewave stripping voltammetry method, including the electrode composition, concentration of ligand, pulse parameters and preconcentration conditions were examined. A detection limit (S/N = 3) of 0.015 μg·L (2.73 × 10 mol·L) Mn (II) was achieved when a preconcentration time of 240 s was applied. Insignificant interferences from various inorganic and organic species were estimated. The described square-wave adsorptive cathodic stripping voltammetry method coupled with the modified carbon paste electrode has been successfully applied to Mn (II) analysis in different water samples.


Introduction
Manganese is an essential micronutrient for all organisms [1] but at high concentrations it has toxic effect [2] contributing for example to the early development of Parkinson's disease symptoms in susceptible people [3].Manganese toxicity is also a serious constraint to crop cultivation since it is taken-up by plants and can easily be passed into the food chain again causing symptoms of Parkinson's disease [4].
Stripping voltammetry has shown numerous advantages including speed of analysis, good selectivity, sensitivity and inexpensive for determination of various metal ions [24].However, anodic stripping voltammetric determination of Mn (II) at the hanging mercury drop electrode [25,26] or mercury film electrode [27] suffers from the low solubility of manganese in mercury, the closeness of its reduction potential to that of hydrogen ion (-1.7 V vs. SCE) and the formation of inter-metallic compounds at the mercury electrode.
Cathodic stripping voltammetry technique was suc-cessfully used for the determination of manganese at the hanging mercury drop electrode [28][29][30], glassy carbon electrode [31][32][33] and carbon paste electrode [34,35].However, the toxicity of mercury limits the usage of the mercury electrodes in the analytical practice and excludes them from the out-of-laboratory applications.Moreover, the sensitivity of glassy carbon and carbon paste electrodes is relatively poor in the determination of metal ions.In order to improve this, a fascinating and effective way is to modify it with a unique substance.Clay minerals have become attractive electrode modifier since the first example of the use of clay as modifier was reported [36].Montmorillonite-Na clay has well-layered lattice structure, high chemical and mechanical stability, high cationic exchange capacity and strong adsorptive properties attributed to the expandability of its internal layers.

Apparatus
A computerized Electrochemical Trace Analyzer Model 394-PAR (Princeton Applied Research, Oak Ridge, TN, USA) controlled via 270/250 PAR software was used for the voltammetric measurements.A micro-voltammetric cell consisting of a C-2 stand (BAS model MF-2063) with a carbon paste electrode body (BAS model MF-2010), an Ag/AgCl/3 M KCl reference electrode (BAS model MF-2079) and a platinum wire counter electrode (BAS model MW-4130) was used.The body of the carbon paste electrode was a Teflon rod with end cavity of 3 mm diameter and 1 mm deep bored at one end for paste filling.Contact was made with a copper wire through the center of the Teflon rod.A magnetic stirrer (PAR-305) with a Tefloncoated magnet was used to provide the convective transport during the preconcentration step.The whole measurements were automated and controlled through the programming capacity of the apparatus.
A Shimadzu Flame Atomic Absorption Spectrometer (FAAS) Model AA-670 interfaced with a data processor was used for determination of the examined metal ion.A Mettler balance (Toledo-AB104, Greifensee, Switzerland) was used for weighing the solid materials.A pH-meter (Crison, Barcelona, Spain) was used for measuring the pH of solutions.A micopipetter (Eppendorf-Multipette ® plus) was used for transferring the solutions throughout the present experimental work.

Reagents and Solutions
Britton-Robinson (B-R) universal buffer (pH 2.0 -11.0), acetate buffer (pH 4.0 -6.0), and phosphate buffer (pH 2.0 -7.5) were prepared in de-ionized water and were tested as supporting electrolytes.A solution of 1 × 10 −3 mol•L −1 2-(5'-bromo-2'-pyridylazo) 5-diethylaminophenol was prepared by dissolving an appropriate amount of the compound (Sigma) in spec-pure methanol.Desired standard solutions of K (I), Na (I), Mg (II), Ca (II), Al (III), Cu (II), Cd (II), Pb (II), Sb (III), Bi (III), Se (IV), Zn (II), Mn (II), Ni (II), Co (II) and Fe (III) were prepared by accurate dilution of their standard stock solutions (1000 mg•L −1 dissolved in aqueous 0.1 mol•L −1 HCl, supplied from Cica, Japan) by de-ionized water.Standard solutions of Cl − , 3 , obtaining a low background current.After that, aliquot of the Mn (II) standard solution was introduced into the electrolysis cell and a selected preconcentration potential was then applied to the modified CPE for a selected preconcentration time, while the solution was stirred at 400 rpm.At the end of the precocentration time, the stirring was stopped and a 5 s rest period was allowed for the solution to become quiescent.The voltammogram was then recorded by scanning the potential towards less positive direction using the square-wave potential waveform.
For regeneration of the electrode surface after recording each voltammogram, the modified CPE was transferred to a blank electrolyte solution in the voltammetric cell and series of cyclic scans were continued (between 1.2 to -0.2 V versus Ag/AgCl/3 M KCl) until a voltammogram corresponding to the residual current (lower background current) was obtained.The electrode was then ready for use in the next measurement.

Analyzed Environmental Water Samples
Various water samples were subjected to analysis by means of the described square-wave cathodic stripping voltammetry method utilizing the modified CPE.These include groundwater, bottled natural water (available in the Egyptian market) and coastal seawater samples (collected from the sea coast at Alexandria City, Egypt).The sea water sample was taken a few meters from the coast where the water was 3 -4 m deep.Then, the seawater sample was UV-digested (3 hs) after acidification with HCl to pH 1.0 with a 1 KW high-pressure mercury-vapor lamp to avoid possible interferences caused by natural organic compounds and to breakdown organic-metal complexes.ing preconcentration at open circuit conditions (Figure 1, curve c).This behavior suggests the formation of electro-active Mn (II)-5-Br-PADAP complex species in solution.However, following preconcentration at the bare CPE by adsorptive accumulation at +1.2 V for 60 s the cathodic peak of Mn (II)-5-Br-PADAP complex was better enhanced (Figure 1, curve d) indicating the interfacial adsorption nature of the formed Mn (II)-complex at the bare CPE.Addition of 0.01% Triton X-100 to solution of the examined Mn (II) complex leads to strong suppression of the SW-AdCS voltammetric peak current of the investigated Mn (II)-complex.This behavior indicated that the Mn (II)-complex was adsorbed at the surface of the bare CPE.As reported in the literature, the molar ratio of Mn (II) to 5-Br-PADAP ligand in the complex was found to be 1:2 [47,48], therefore, the form of the present complex is Mn (II)-(5-Br-PADAP) 2 .

Voltammetric Response of Mn (II)-Complex at the Modified CPE
Voltammograms of 10 μg•L −1 Mn (II) in the presence of 50 μmol•L −1 5-Br-PADAP in 0.1 mol•L −1 acetate buffer (pH = 5.0) at the CPE modified with MMT-Na clay under various experimental conditions (Figure 2) demonstrated that the modified CPE remarkably improved the SW-AdCS voltammetric peak current magnitude of the investigated Mn (II) complex in comparison to that recorded at the bare CPE (Figure 2, curve a).This behavior Such enhancement of stripping peak current magnitude is expected due to the superior adsorptive ability of MMT-Na clay.However, the conductivity of the modified CPE dropped with increasing the ratio of the non-conducting MMT-Na clay (curves d and e); hindering the electron transfer process and increasing the background current.Therefore, a CPE modified with 10% (w/w) of MMT-Na clay was used over the rest of the present analytical studies.indicated that modification of the CPE with MMT-Na clay significantly enhances the stripping peak current magnitude of Mn (II) complex under the experimental conditions which may be attributed to the superior adsorptive ability of MMT-Na clay.Following preconcentration of Mn (II)-complex by adsorptive accumulation at the modified CPE surface, it stripped out from the electrode surface by scanning the potential towards less positive direction resulting in a cathodic voltammetric peak (Figure 2).The overall electrode process may be expressed in two steps as: 1) Preconcentration (by adsorptive accumulation) step: 2) Cathodic stripping step: {Mn 2+ − (5-Br-PADAP) 2 } (surface) + 2e - ⎯→ (Mn 0 ) + 2 (5-Br-PADAP) As shown in Figure 2 (curves b-e), the ratio (w/w) of MMT-Na clay in the graphite paste remarkably influences the square-wave cathodic stripping voltammetry peak current magnitude of the Mn (II) complex compared to that at the bare CPE (Figure 2, curve a).The stripping peak current magnitude firstly increased upon the increase of the MMT-Na clay ratio up to 10% (w/w) in the carbon paste (Figure 2, curve c) and then decreased at higher clay ratios (Figure 2, curves d and e).
For optimization of a square-wave adsorptive cathodic stripping voltammetry (SW-AdCSV) method for sensitive determination of Mn (II) as a complex with 5-Br-PADAP utilizing the modified CPE, the following were carried out.

Effect of Supporting Electrolyte and Its pH
SW-AdCS voltammograms of 10 μg•L −1 Mn (II) in the presence of 50 μmol•L −1 5-Br-PADAP were recorded in Britton-Robinson (pH 2.0 -11.0), acetate (pH 4.0 -6.0) and phosphate (pH 2.0 -7.5) buffers following preconcentration of the Mn (II) complex at the modified CPE by adsorptive accumulation at +1.2 V (vs.Ag/AgCl/3 M KCl) for 60 s.The results showed that, better enhanced SW-AdCSV peak current was achieved in the acetate buffer of pH values 4.0 -5.0 (Figure 3) in comparison to that obtained in the other two media.
The dramatic decrease in the SW-AdCS voltammetric peak current magnitude at higher pH values (Figure 3) may be attributed to the precipitation of Mn (II) as Mn(OH) 2 in alkaline media.In addition, the influence of ionic strength of the acetate buffer (0.05 -0.20 mol•L −1 ) was also studied while keeping the pH value at 5.0.Better enhanced peak current was achieved in 0.10 mol•L −1 acetate buffer of pH = 5.0, therefore, it was chosen as a supporting electrolyte in the rest of the present analytical study.

Effect of Concentration of 5-Br-PADAP as a Ligand
SW-AdCS voltammograms of 100 μg•L −1 Mn (II) in the presence of increased amount of 5-Br-PADAP (10 to 50 μmol•L −1 ) were recorded in acetate buffer of pH = 5.0 following preconcentration of the Mn (II) complex by adsorptive accumulation at the modified CPE at +1.2 V for 60 s.The voltammograms showed that the peak current (i p ) magnitude of Mn (II) complex increased with concentration of 5-Br-PADAP up to ∼ 20 μmol•L −1 and then leveled off.Therefore, 20 μmol•L −1 of the tested ligand was used in the rest of analytical study since it is sufficient enough for the formation of complex with Mn (II) ions in concentrations up to 100 μg•L −1 .Reaction kinetics of Mn (II) with 5-Br-PADAP were identified from its voltammograms recorded after different times of mixing the reactants.The SW-AdCS voltammetric peak current (i p ) magnitude of the examined Mn (II) complex was practically constant with the reaction time, indicating the immediate formation of Mn (II)-(5-Br-PADAP) 2 complex at the time of mixing the reactants in the electrochemical cell.Therefore, heating of the reactants solution was not required in the present work.

Square-Wave Pulse Parameters
In order to obtain a well developed and better enhanced SW-AdCS voltammetric peak for 10 μg•L −1 Mn (II) in the presence of 20 μmol•L −1 5-Br-PADAP using the acetate buffer (0.1 mol•L −1 ) of pH = 5.0 as a supporting electrolyte following preconcentration at the modified CPE by adsorptive accumulation at +1.2 V for 60 s, the square-wave pulse parameters (frequency f, scan increment ΔE i and pulse-amplitude a) were optimized.The peak current magnitude of the examined Mn (II)-(5-Br-PADAP) 2 complex was increased linearly with frequency within the range 20 -120 Hz; its corresponding regression equation was: i p (μA) = 0.0425 f (Hz) -0.019 (r = 0.997) On increasing the scan increment within the range 2 -10 mV, the peak current magnitude of the examined Mn (II) complex was also increased linearly; its corresponding regression equation was: i p (μA) = 0.3434 ΔE i (mV) + 1.859 (r = 0.998)Although the peak current (i p ) magnitude was increa-sed linearly with the pulse-amplitude within the range 20 -60 mV, the best peak morphology with the lower baseline was obtained at pulse-amplitude of 25 mV.Accordingly, the optimal square-wave pulse-parameters used over the rest of the present analytical study were: frequency f = 120 Hz, scan increment ΔE i = 10 mV and pulse-amplitude a = 25 mV.

Preconcentration Conditions
Effect of varying the preconcentration (accumulation) potential E acc from +1.3 to +1.0 V (vs.Ag/AgCl/3M KCl) on the peak current magnitude of the SW-AdCS voltammograms for 10 μg•L −1 Mn (II) in acetate buffer (0.1 mol•L −1 ) of pH = 5.0 in the presence of 20 μmol•L −1 5-Br-PADAP was evaluated following its preconcentration at the modified CPE by adsorptive accumulation for 60 s (Figure 4).The results showed that better enhanced peak current magnitudes were achieved over the potential range +1.2 to +1.1 V. Therefore, a preconcentration potential of +1.1 V (vs.Ag/AgCl/3 M KCl) was used throughout the present analytical study.
On the other hand, SW-AdCS voltammograms of 5 and 10 μg•L −1 Mn (II) in the presence of 20 μmol•L −1 5-Br-PADAP were recorded under the optimum operational conditions following increased preconcentration time from 0.0 to 300 s by adsorptive accumulation at +1.1 V (Figure 5).The peak current magnitude of the Mn (II) complex was increased linearly with preconcentration time over the examined time period.At a higher concentration of Mn (II) (10 μg•L −1 ), adsorptive saturation of the electrode surface (adsorption equilibrium) was reached at 240 s and hence the peak current leveled off.Accordingly, the preconcentration time should be chosen according to the concentration level of Mn (II) in the examined solution.

Linearity Range and Limit of Detection
SW-AdCS voltammograms of various concentrations of Mn (II) in the presence of 20 μmol•L −1 5-Br-PADAP were recorded under the optimum operational conditions using the modified CPE (Figure 6).Rectilinear relations between the peak current magnitude (i p ) and concentrations (C) of Mn (II) following preconcentration for various times (60, 120 and 240 s) at the modified CPE were obtained over the ranges shown in Table 1.

Accuracy and Precision
Accuracy and precision of the described SW-AdCSV method for determination of Mn (II) complex utilizing the modified CPE were estimated as recovery (% R) and standard deviation (% SD) by analyzing three reference standard concentration levels of Mn (II) for three times (Table 2) under the optimized operational conditions.Mean percentage recoveries and standard deviations obtained by applying the calibration curve and standard addition methods (Table 2) indicated the accuracy and precision of the described SW-AdCSV method coupled with the modified CPE for determination of Mn (II) as 5-Br-PADAP complex.Moreover, the results obtained by the described stripping voltammetry method, applying the calibration curve, were statistically compared with those obtained by flame atomic absorption spectrometry (FAAS).The results showed that the described voltammetric method utilizing the modified CPE is much more sensitive than the FAAS method.Since the calculated value of F-statistic does not exceed the theoretical one (Table 2), there was no significant difference between the described SW-AdCSV and FAAS methods with respect to reproducibility [48].Also, no significant differences were noticed between the two methods regarding accuracy and precision as revealed by t-values [50] (Table 2).

Interferences
Interferences from some foreign cations (K + , Na + , Mg 2+ ,   as non-ionic surfactant to the assay of the investigated metal ion was insignificant up to 0.001%.Higher concentrations of the surfactant cause deformation and great suppression of the voltammetric peak current magnitude of Mn (II) complex.Influence of surfactants, if present in the analyzed water samples, can be completely eliminated by thorough mineralization of water sample prior to the analysis.

Application
The described SW-AdCSV method coupled with the modified CPE was successfully utilized for determination of Mn (II) in ground water, bottled natural water and sea water samples.The same water samples were also analyzed by FAAS.Comparison of the results obtained by the described SW-AdCSV method with those obtained by FAAS (Table 4) revealed the capability of the modified CPE for determination of Mn (II) at trace and ultratrace concentrations.

Conclusion
A simple, precise and accurate fully validated squarewave adsorptive cathodic stripping voltammetry method has been developed for determination of Mn (II) as a complex with 2-(5'-bromo-2'-pyridylazo) 5-diethylami-nophenol in aqueous solutions using a carbon paste electrode modified with 10% (w/w) montmorillonite-Na clay.The developed voltammetric method coupled with the modified carbon paste electrode has been successfully applied for analysis of Mn (II) in different water samples without interferences from various cations and anions.The modified carbon paste electrode is much more sensitive than most of the previously applied electrodes for determination of manganese in various matrices due to the superior adsorption property of montmorillonite-Na clay as a modifier.

Figure 3 .
Figure 3. SW-AdCS voltammetry peak currents (i p ) as a function of pH of acetate buffer (0.1 mol•L −1 ) for 10 μg•L −1 Mn (II) in presence of 50 μmol•L −1 5-Br-PADAP following preconcentration by adsorptive accumulation for 60 s at the CPE modified with 10% (w/w) MMT-Na clay.other parameters are as those given in Figure 1.

Figure 5 .
Figure 5. SW-AdCS voltammetric peak currents (i p ) as a function of the preconcentration time (t acc ) in acetate buffer (0.1 mol•L −1 ) of pH = 5.0 for a solution containing; (a) 5 and (b) 10 μg•L −1 Mn (II) in presence of 20 μM 5-Br-PADAP following preconcentration at the CPE modified with 10% (w/w) MMT-Na clay by adsorptive accumulation at +1.1 V. other parameters are as those given in Figure 4.

Table 1 . Characteristics of the calibration curves for the determination of Mn (II) as 5-Br-PADAP complex following pre- concentration by adsorptive accumulation at the modified CPE with 10% (w/w) MMT-Na, E acc. = +1.1 V.
*Average of three determinations.

Table 2 . Accuracy and precision of the described SW-AdCS voltammetry method as recovery (%R) and standard devia- tion (%SD) for determination of Mn (II) as 5-Br-PADAP complex following preconcentration by adsorptive accumu- lation at the modified CPE with 10% (w/w) MMT-Na clay.Table 4 . Determination of Mn(II) as 5-Br-PADAP complex in ground, bottled and sea water samples by the described SW-AdCS voltammetry method and flame atomic absorp- tion spectrometry (FAAS).
*F-static = 5.24 and t-test = 1.63 (A) Calibration curve method and (B) Standard addition method.* Under limit of detection.Theoretical F-statistic = 6.39 and t-test = 2.3 at 95% confidence limit for n 1 = n 2 = 5.