Cyclic voltammetry, Copper (II), HMDE, 1,2,4-triazole, Schiff base, Water samples, Human fluids
Heavy metals found in a trace concentration in natural biological systems and exert a beneficial or harmful effect on human metabolism and creature life. Pollution sources may be domestic, agriculture or industrial. Heavy metals such as copper, lead, nickel and its compounds are listed as pollutants of priority interest by the environmental protection agency of the United States and the Council of the European Communities [1,2]. The introduction of trace metal pollutants from human activities in many parts of the world has seriously degraded aquatic environment [1,3,4]. Heavy metal distribution varies among aquatic species depends on physiological factors. Tuna and shrimp being able to concentrate high amounts of heavy metals which recently using for bio monitoring of environmental contamination.
Recently, the feeding of aquatic mammals has increased in importance because the protein, saturated fat, and omega fatty acids become as known to affect human health. However, the health risk related with consumption of aquatic species and food chain polluted by heavy metals is an important worldwide concern [5,6]. Environmental measurements of heavy metal levels are generally approved by conventional analytical techniques, including atomic absorption spectroscopy (AAS) , and inductively coupled plasma-mass spectrometry (ICP/MS) [7,8]. These methods are well recognized, but they are affected by quit a few drawbacks, such as time consumer, expensive, and deficiency of multi elemental analysis. Moreover, most of the analytical instruments for separation and detection of heavy metal species are based on the line coupling of high-performance liquid chromatography (HPLC) to element-specific detectors, such as a hydride generation atomic absorption spectrometer (HG-AAS) [1-4] flam atomic absorption spectrometry (FAAS) [9,10], hydride generation-atomic fluorescence spectrometer (HG-AFS) , inductively coupled plasmaoptical emission spectrometer (ICP-OES) [12,13].
Copper is recognized as an essential micronutrient for biological systems, though the wellbeing boundary between its nutrient and toxic dosages is very slight. Spectrophotometric methods for speciation analysis of copper have been presented in the recent years. Furthermore, the previous work interested and presented the copper speciation, and focused on hyphenated instrumental technique, as well as the problems encountered . These techniques are unusable for on-site screening or for quantification as part of a conclusion instrument owing to their size and high work and analytical expenses. Hence, there is a need for moveable analytical systems that can be met by using electrochemical techniques for the analysis of copper and other [15-17]. Cyclic voltammetry (CV) has been recognized as a dependable trace metal analysis method appropriate to diverse samples.
A recent literature survey on the analytical application of the triazole–Schiff base has revealed no study on the use of this compound for the voltammetric determination of copper (II) specie. Consequently, the overall goals of the work presented in this paper, a CV method was applied to the determination of Cu(II) in the acetate buffer media. The method is based on the Cu (II)-TSB chelate formation at HMDE surface. The Cu(II)- TSB complex was determined in various samples, including fresh and marine water, and human fluids (urine and plasma) using hanging mercury dropping electrode (HMDE). In general, the copper metal should be included the human fluids a result of eating many foods which contained this metal. On the other hand, copper metal as a toxic metal may be found in underground water and sea water as a result of throwing industrial waste in these places. According to the previous information, the copper (II) metal has been electrochemically determined at these samples as in the present study.
A 1, 2, 4-triazole–Schiff base (TSB) was synthesized through the condensation reaction and chemical characterized as discussed by Alghamdi and Rezki in 2016 . The chemical structure of TSB and the nature of the electrode reaction and the most possible reduction mechanism of copper (II) with TSB chelate by use hanging mercury working electrode were shown in Scheme 1.
The electrochemical determination of copper (II)-TSB was carried out by cyclic voltammetric technique onto 797 VA Metrohm (Switzerland). A three-section borosilicate which included a hanging mercury dropping electrode (HMDE, 0.6 mm2 drop surface area) as a working electrode, double-junction Ag/AgCl as a reference electrode and platinum wire as a counter electrode in the electrochemical cell (10 mL). Digital pH-meter (model pH 211, Hanna instrument) was used for pH measurements. De-ionized water was attained from Milli-Q Plus system water purification system (Milford, MA, USA) which used throughout the work. A labofuge 200 (Heraeus Sepatech, Germany) was used for centrifuging the biological samples.
Reagents and materials
For the determination of copper (II)-TSB, the chemicals were used accordantly to analytical reagent grade. Stock solution of the 1,2,4-triazole–Schiff base was prepared by dissolving an precise weight of the synthesized compound in a smallest volume of solvent and completed to the mark with ethanol at 50 mL volumetric flask. In addition, the stock solution of CuSO4.2H2O (1 × 10-2 mol L-1) was prepared in doubly deionized water at 100 mL volumetric flask. The diluted solutions of copper (II) were freshly prepared in deionized water according to a nature of electrochemical work. A sequence of acetate, Britton-Robinson (B-R), carbonate, and phosphate buffers of pH 1.5 to 10 were prepared [19,20], to obtain the high CV current for the determination of copper(II)-TSB.
Recommended cyclic voltammetry procedure for copper (II)-TSB determination
The voltammetric cell was pre-cleaned by soaking in nitric acid (10% v/v) and washed with deionized water. The general method used to get cyclic voltammetry (CV) was as followed: a precise volume (10 mL) of an aqueous solution containing acetate, Britton-Robinson, carbonate and phosphate buffers as supporting electrolyte at the required pH value (1.5-10) and volume (300 μL) of the TSB (3.0 × 10-6 mol L-1) were transferred into the electrochemical cell. The solution was stirred and purged with nitrogen gas for 180 seconds. Then the stirrer was stopped, the background voltammogram of the supporting electrolyte and the TSB were recorded by applying a negative going potential scan from 0.0 to -1.0 V vs. Ag/AgCl electrode at accumulation potential -0.2 V, accumulation time of 60 s, and scan rate of 100 mVs-1. After recording the voltammogram of the TSB solution, an accurate volume (50 μL) of CuSO4.2H2O solution (5 × 10-6 mol L-1) was added. The CV scan was repeated with a new mercury drop under the same experimental conditions of the Schiff base (TSB) analysis. After 30 s latency time, the voltammogram of the copper(II)-TSB complex was totally observed at potential of -550 mV, by applying a negative going potential scan from 0.0 to -1.0 V vs. Ag/AgCl reference electrode.
Preparation of Schiff base and biological samples
A stock solution of TSB (1 × 10-3 mole L-1) was prepared and diluted by ethanol solvent at 50 mL of volumetric flask . Human urine and plasma were prepared by addition 1.0 mL of 5.0% ZnSO4.7H2O, 0.1 mL of NaOH and 1.0 mL of methanol to 0.5 mL of human urine and plasma in a centrifuge tube  under 5000 rpm at 8.0 min. The various concentrations of TSB and Cu (II) were added to the prepared biological samples. The CV procedure was applied for the determination Cu (II) by recovery.
Recommended procedure for the analysis of free copper (II) complex in fresh and marine samples
Alternatively, the spiking (addition) method was used 2.0 mL of the test water samples adjusted to pH 2 into electrochemical cell at the optimum experimental conditions. The concentration of copper (II) ion, in the presence of TSB, was finally determined from the calibration curve of the standard addition. Fresh or Red sea water samples collected from Badur, Almundasah, Hamra Alasad, and Umluj area (Red Sea) were filtered through 0.45 μm cellulose membrane filter and stored in LDPE sample bottles. The recommended electrochemical procedures that used for the standard curve of Cu(II) determination were followed for all samples in the presence of TSB.
Study of analytical parameters
The results of CV approved high degree of adsorption and sensitivity for the reduction peak (-0.55 V) of Copper (II)- TSB complex. While the 1,2,4-triazole–Schiff base used for the cyclic voltammetric determination of copper (II), the influence of diverse parameters that affected the reduction peak currents of the cathodic wave of copper (II)-TSB complex was critically should be studied using HMDE vs. Ag/AgCl reference electrode. The effect of the various buffer solutions on the peak current height at the reduction peak was studied over a wide range of pH 3-10 at 30 s pre-concentration time. Maximum height of the reduction peak current for the determination of Cu (II)-TSB complex, was observed by use acetate buffer pH3. Acetate buffer was also used over the range 1.5 to 6 of pH, resulting pH 2 was given a high current and chosen a best one for the future studies (Figure 1).
At meanwhile, the peak potential of Cu(II)-TSB complex was found to be depended on the pH values. A gradual shift to a more negative potential was recorded from -450 to -700 mV when increasing pH value over the range 1.5-6.0, shown consumption of electrons (hydrogen ions) in the electrode reaction . The linear relation was found between the reduction peak potential versus pH values as plotted in Figure 2.
The accumulation time (tacc) was included the very important parameters that controlled the current height of complex cathodic peak. This parameter was examined in range of 0-180 s as shown in Figure 3. Maximum peak current and well defined peak was observed at 60 s. At longer time; the cathodic peak current began to be constant because of the HMDE surface was saturated with free TSB. Hence, in the subsequent work, accumulation time of 60 s was selected for the quantitative determination of copper (II) ion.
The influence of accumulation potential (Eacc) on the CV reduction peak current was evaluated over a wide range of potential -0.8 to + 0.2 V (Figure 4) versus Ag/AgCl reference electrode after 60 s accumulation time. The cathodic peak current was reached a maximum value at -0.2 V (Eacc) for Cu(II)-TSB reduction peak versus Ag/AgCl reference electrode. Thus, an accumulation potential of -0.2 V was selected for the subsequent studies. The effect of the hanging mercury drop size on the electrochemical CV reduction peak of the Cu(II)-TSB was discovered over a wide range (0.15-0.6 mm2) as given in Figure 5, under the previous optimal conditions. The data revealed excellent, symmetric reduction peak and maximum peak currents of the proposed complex were achieved on mercury drop size 0.6 mm2. Therefore, in the subsequent experiments, drop size of 0.6 mm2 was selected as an optimum value. Similarly, the peak current can be maximized by choosing the faster stirring rate, yet, to reach a largest possible amount of the studied complex to a working electrode surface, so a 3000 rpm stirring rate was recorded a high current and it was selected as the best rpm value for the future work (Figure 6).
The effect and importance of scan rate
In this study, the scan rate was monitored over the range 10-200 mVs-1 for the studied solution which contained TSB (1 × 10-5 mol L-1) in the existence of copper (II) ion (5 × 10-6 molL-1) onto the surface of HMDE under the optimal accumulation time, accumulation potential and an acidity of supporting electrolyte. The CV reduction peak of the Cu (II)-TSB complex steadily on raising the scan rate value from 10 to 200 mVs-1. Through this analysis, the potential was gradually increased in the negative direction from 410 to 550 mV, so the potential shift around -140 mV was confirmed an irreversibility nature of the cyclic voltammetric procedure for the determination of copper (II)-TSB. Otherwise, the effect of the scan rate (log ʋ) on the cathodic peaks potential at pH 2 was studied on a freshly drop of the HMDE. The Cu (II)-TSB reduction peak potential was shifted cathodically with increasing the scan rates approving the irreversible nature of the recorded reduction process of Cu (II)-TSB complex. Furthermore, as shown in Figure 7, the plot of log (ipcCu-TSB) vs. log(ʋ) was been linear with slope of 0.71 that closed to 1.0, which confirmed an adsorption process for the analysed complex on the HMDE surface controlled by adsorption .
Analytical performance of electrochemical CV procedure
The validation of the proposed CV procedures for copper (II)-TSB determination under the previously optimized parameters was performed via the calibration curve, limit of detection, limit of quantification, reproducibility and recovery. The CV voltammograms of copper (II)-TSB were recorded as in the calibration curve. The plot of the reduction peak currents (Ered= -0.55 V) measured by the electrochemical CV procedure versus copper (II) concentrations was created over the concentration range 2 × 10-6-1 × 10-5 mol L-1 (Figure 8). After a 1 × 10-5 mol L-1 concentration, the calibration curve has a tendency to level off because of the adsorption saturation on the HMDE. The calibration plot was shown a linear relation between cathodic current and complex concentration with a good regression (r2=0.99). According to IUPAC, the LOD and LOQ under the parameters established for copper (II) was estimated, where the values of LOD and LOQ calculated to become 2.76 ppb and 9.21 ppb, respectively.
Figure 8: The left figure shows the CV voltammograms of the effect of copper (II) concentration on the reduction peak current of Cu(II)-TSB complex at concentrations A: acetate buffer; B: TSB (1 × 10-4 mole L-1) while C: 2 × 10-6; D: 4 × 10-6; E: 6 × 10-6; F: 8 × 10-6 mole L-1 and G: 1 × 10-5 mole L-1 of Cu(II) at acetate buffer pH 2, Eacc= -0.2 V; and scan rate=200 mVs-1. The plot shows the linear relation at various concentration of Cu(II) vs. cathodic peak of Cu(II)-TSB.
Other analytical characteristics were investigated such as recovery, reproducibility and stability. In the present work, the accuracy and the precision of the selected electrochemical method was evaluated by the study of the recovery of 2 х 10-6 mol L-1 and yielded in average 104% ± 1.87 (Table 1). Furthermore, reproducibility of 5 × 10-6 mol L-1 of Cu(II) was reported for ten CV measurements and yielded almost stable cathodic peak current with 0.58% relative standard deviation (RSD %) (Table 2). The stability of the cyclic voltammetric signal for Cu(II)-TSB was studied at period 120 min to obtain the stable current values.
|Recovered Cu Conc. 2.0 × 10-6 mol L-1||Found ( mol L-1)||Recovery %|
|2.1 × 10-6||105|
|2.1 × 10-6||105|
|2.12 × 10-6||106|
|2.04 × 10-6||102|
|2.04 × 10-6||102|
|Standard Deviation||± 1.87|
Table 1: Cyclic voltammetric accuracy for Cu(II), recovery; 5 × 10-5 mol L-1 of ligand.
|Complex concentration (mol L-1)||Current (nA)||Current Average ± SD||RSD %|
|3 × 10-5 TSB + 5 × 10-6 Cu (II)||141||141.3±0.82||0.58|
Table 2: Cyclic voltammetric precision for complex (Reproducibility).
The effect of some possible metals may be presented with copper ion at the studied solution was also evaluated in this research. Zinc (Zn2+), Nickel (Ni2+) and Aluminum (Al3+) were studied in terms of their effects on the reduction current of Cu(II)-TSB(2 × 10-6 mol L-1 of Cu (II) and 2 × 10-5 mol L-1 TSB) solution. These metal ions were added at various concentrations such as 1, 5 and 10 times than copper ion concentrations at acetate buffer pH 2. Additions of Zn2+ and Al3+ ions were positively affected on the copper ion signal by 5-8% and 44-48%, respectively. While addition of Ni2+ was negatively affected on the reduction peak by 15-42%.
Cyclic voltammetric behavior of copper (II)-TSB complex
According to the previously optimum parameters; acetate buffer, pH 2, 60 s accumulation time, -0.2 V accumulation potential and 100 mVs-1 scan rate; the sensitive and good sharp of cyclic voltammogram was obtained as illustrated in Figure 9. As it shown as in this figure; the CV procedure was confirmed an irreversible reduction process for Cu(II)- TSB. For more detail, the CV of copper (II)-TSB (Figure 10), displayed three well-defined reduction peaks, the first one was recorded at -0.12 V, so in the recorded potential, the investigated cathodic peak is most likely assigned to the reduction peak for Cu(II) only. The second peak was appeared for TSB ligand only at -0.35 V; while the third peak was observed for Cu(II)-TSB complex at -0.55 V versus Ag/AgCl reference electrode. Therefore, it can be decided that, the mechanism of electrochemical irreversible reduction process for Cu (II)-TSB complex has been suggested as in Scheme 1.
Analysis of copper (II) in fresh and marine water
The validation of the presented electrochemical CV method was evaluated by determining Cu (II) in various water samples such as Red Sea water, Mundasa, Badur and Hamra Alasad areas. These samples were collected and filtered as described in experimental section. Then, the test water samples were analyzed by the selected CV method. The results of the analysis of the total amount of Cu (II) after spiking of water samples are summarized in Table 3. The calculated results indicated the accuracy and the suitability of the selected CV method for determination Cu (II) using the reagent TSB.
|Recovered Cu(II) (2.0 × 10-6 mol L-1)
3 × 10-5mol L-1of TSB
|Badur||Red Sea||Mundasa||Hamra Alasad|
|Standard Deviation||± 1.0||± 0.71||± 1.41||± 1.58|
Table 3: Levels of the tested metal Cu(II) in Badur, Red Sea, Mundasa, and Hamra Alasad water samples which determined by the proposed CV method.
Analysis of copper (II) in human fluids
For more validation of the proposed method, the selected Cu(II)-TSB chelate was determined, using CV method, in human fluids (urine and plasma) by recovery of 2 × 10-6 mol L-1 of Cu(II) after preparation the samples as previously described in experimental section. The accuracy of the analytical procedure was indicated recovery values at averages of 95 ± 0.71% and 96 ± 2.12% for urine and plasma, respectively as listed in Table 4.
|Recovered Cu(II) (2.0 × 10-6 mol L-1)
3 × 10-5mol L-1of TSB
|Standard Deviation||± 0.71||± 2.12|
Table 4: Levels of athe tested metal Cu(II) in human fluids (urine and plasma) which determined by electrochemical CV method.
The proposed CV method was used for the determination of Cu (II)-TSB complex using acetate buffer as supporting electrolyte at pH 2. The electrochemical method provides an excellent alternative method for the determination of copper because of its satisfactory precision, applicability and high sensitivity determination for Cu (II) with TSB chelate. The LOD and LOQ were investigated with good values. The accuracy and precision of the adapted method were indicated by recovery and relative standard deviation, respectively. The sensitivity and selectivity of the electrochemical proposed method could be enhanced to detect lower level of copper (II) by prior accumulation from large sample volumes. However, the work is still continuing for voltammetric analysis of selected copper (I and II) in various environmental samples.
The authors would like to thank Dr. Nadjet Rezki, from department of chemistry, faculty of science, Taibah University for her assistance in doing our research.
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