Electrochemical Oxidation of 2,5-Dimercapto-1,3,4-thiadiazole on Carbon Electrodes Modified with Ru(III) Schiff Base Complex

The thiol compound 2,5-dimercapto-1,3,4-thiadiazole is a potential cathode material. The redox reactions of the mentioned thiol compound are slow at room temperature but can be enhanced using electron transfer mediators. The electrochemical oxidation of 2,5-dimercapto-1,3,4-thiadiazole on the surface of carbon electrodes modified with Ruthenium(III) Schiff base complex was studied by voltammetric methods and amperometric flow injection analysis. The electrocatalytic properties of Ruthenium(III) Schiff base complex on glassy carbon and screen printed carbon electrodes are enhanced by the addition of multi-walled carbon nanotubes and Nafion. Voltammetric studies showed that anodic oxidation of DMcT on a modified glassy carbon electrode occurs at a potential of +0.28 V vs. Ag/AgCl in Britton-Robinson buffer (pH 6.50). Flow injection amperometric measurements were performed at +0.20 V vs. Ag/AgCl in Britton-Robinson buffer solutions pH 6.50 at a 0.40 cm3 min−1 flow rate. The results of amperometric measurements for modified screen printed and glassy carbon electrodes showed that the screen printed electrode had a lower value of detection limit (0.38 mg dm−3) and quantification (1.28 mg dm−3), and a linear dynamic range from 1 to 500 mg dm−3 of 2,5-dimercapto-1,3,4-thiadiazole. Modified glassy carbon electrode provided a linear dynamic range up to 750 mg dm−3 of 2,5-dimercapto-1,3,4-thiadiazole with a detection limit of 3.90 mg dm−3 and quantification of 13.20 mg dm−3.


Introduction
The thiol compound 2,5-dimercapto-1,3,4-thiadiazole (DMcT) has been the most studied thiol compound in the last decade as a potential cathode material due to its high theoretical capacity. 1 Except as a cathode material, DMcT is also used as a corrosion inhibitor, 2,3 biocide, 4 as an intermediate or starting material for pharmaceuticals and dyes, and metal chelating agent. 5,6 Due to its wide range of uses, it is important to study its oxidation and detection. There is a need to develop sensitive, simple, fast, and easily accessible methods for determining DMcT. The most commonly used analytical techniques for the determination and quantification of DMcT are capillary zone electrophoresis (CZE) and high performance liquid chromatography (HPLC) with UV detection. 7,8 These methods require expensive and non-portable instrumentation, trained personnel, sophisticated sample preparation, and suffer from numerous chemical interferences. The electrochemical techniques have some advantages like simplicity, fast response, wide linear dynamic range, ease of miniaturization, high sensitivity and low cost compared to other methods. 9 The oxidation of thiol compounds at the solid electrodes (such as Au, Pt, carbon and graphite), at room temperature, is very slow and requires a potential of at least +1.00 V. 10 Therefore, finding appropriate electron transfer mediators for low potential and fast determination of DMcT is of great importance. Organic compound poly(3,4-ethylenedioxythiophene) has been used as a mediator for DMcT oxidation at different electrodes. 11,12 Ru complexes have been the subject of numerous studies because of their catalytic, anti-tumour, and electron transfer mediator activity. 13,14 Ruthenium(III) complexes were studies as chemical modifiers for carbon electrodes due to their electrochemical behaviour, they are efficient mediators and catalyse reactions of organic substances. To solve problems with overpotential for the quantification of L-cysteine and ascorbic acid with carbon electrodes, in our previous research we studied electron transfer mediator properties of Ru(III) complex compound. 15,16 Immobilized on the surface of the carbon electrode, the Ru complexes transfer electrons between the analyte solution and the electrode substrate, resulting in a decrease in activation overpotential.

Methods and electrode preparation
Voltammetric and amperometric measurements were performed using an Autolab Potentiostat /Galvanostat (PG-STAT 12) coupled to PC. The Autolab software GPS Version 4.8 was used to control the instrument. The working electrodes were a bare and modified glassy carbon electrode (GC electrode, Metrohm, 0.28 cm 2 surface area), and a screen printed carbon electrode (SPC electrode, Coors Ceramic GmbH, USA, 0.28 active area: 35 × 4 mm). All obtained potentials are given versus the Ag/AgCl reference electrode at room temperature. Cyclic voltammograms (n = 3) of DMcT were recorded in the potential range between −0.25 V and +0.35 V (scan rate of 100 mV s −1 ) in BR buffer solutions pH 6.50, using bare and modified GC electrodes as working electrode, Ag/AgCl electrode (Metrohm 6.0733.100 LL) as a reference, and a platinum wire as a counter electrode. Differential pulse voltammograms of DMcT were recorded in potential range from 0.00 to +0.60 V applying scan rate of 20 mV s −1 in BR buffer solutions pH 6.50, a pulse time of 0.05 s, pulse amplitude +0.025 V, using bare and modified GC electrodes as working, a platinum wire as counter, and Ag/AgCl as a reference electrode. Square wave voltammetry was measured in the potential range from 0 to +0.40 V, pulse interval 5 s, frequency 25 Hz, scan rate 40 mV s −1 in BR buffer solutions pH 6.50, amplitude +0.020 V. SWV measurements as well as other voltammetric measurements were performed in a cell with a volume of 5 cm 3 . Amperometric measurement was carried out using flow injection system that involved of a high performance liquid chromatographic pump ( . After the addition of MWCNTs and Nafion to the modification mixture, the oxidation peak is more intense (∆I pa = 12.80 µA) and appears at potential +0.29 V (Fig. 3B- 24 introduced oxidation of DMcT at bare disposable screen printed graphite electrodes at the potential of +1.30 V, which is a significantly higher value related to the potential of +0.28. The mechanism of oxidation of DMcT involves the transfer of two electrons and two protons forming a disulphide dimer in the acidic medium. 25 Similar to those for other thiol compounds, the oxidation process of DMcT on the surface of solid electrodes formed thiyl radical (reaction 1), which further dimerizes to disulphide (reaction 2). 26,27 R -SH → R -S ads 2R -S ads Since Nafion has a strong capacity to dissolve MWCNTs and Na[RuCl 2 (SB) 2 ], it has been used for dissolving and binding of those substances to the surface of carbon electrodes during the electrochemical measurement. 15,27 The mediating properties of the Ru(III)/Ru(II) pair for the oxidation of DMcT on the glassy carbon electrode were demonstrated also using differential pulse voltammetry and square wave voltammetry. The well-defined anodic peak obtained using a modified GC electrode at +0.28 V corresponding to DMcT oxidation, clearly indicates excellent mediating properties of Na[RuCl 2 (SB) 2 ] (Fig. 4).
Furthermore, square wave voltammetry was used to investigate electrochemical oxidation of DMcT at a modified GC electrode. As shown in Fig. 5A, an increase in DMcT concentration causes an increase of anodic peak current (+0.28 V), suggesting a good electrochemical response of the modified electrode. Fig. 5B shows the dependence of the DMcT concentration versus anodic current response.
The modified electrode demonstrates a linear dependence, with correlation factor R 2 = 0.9882.
At the surface of the Na[RuCl 2 (SB) 2 ]/MWCNTs/Nafion GC electrode, the DMcT is oxidised to 5,5'-disulfanediylbis (1,3,4-thiadiazol-2-amine), while the Ru(III) is reduced to a Ru(II) complex compound. The resulting Ru(II) complex is electrochemically re-oxidised to the Ru(III) complex, producing an oxidation current proportional to the DMcT concentration (Fig. 2). The oxidation of DMcT at the Na[RuCl 2 (SB) 2 ]/MWCNTs/Nafion GC electrode was recorded at a potential of +0.28 V for all three voltammetric methods. The potential of +0.28 V is a significantly lower value than those reported in the literature. 24

Optimisation of working conditions
Since Na[RuCl 2 (SB) 2 ] and MWCNTs showed remarkable mediating properties in DMcT oxidation, the operating parameters for the analytical determination of DMcT were optimised using flow injection amperometry. Fig. 6A shows the dependence of the (a) background current, and (b) amperometric response for 200 mg dm −3 DMcT at an applied potential from −0.20 V to +0.40 V in BR buffer solution (pH = 6.50), and flow rate 0.40 cm 3 min −1 for Na[RuCl 2 (SB) 2 ]/MWCNTs/Nafion SPC electrode. For negative potential values, the DMcT current response has low values, as well as an unstable background current. Increasing the potential value also increases the current response. However, high values of positive potentials are not recommended and electrodes are modified with mediators to prevent overpotential of the analyte. The previously reported results showed that the oxidation of DMcT takes place at positive values of the potential and its reduction at negative ones, which is in accordance with the results of this study. 30 At a potential of −0.20 V to 0 V, no anode peaks were observed. Working potential of +0.20 V was selected for subsequent amperometric detection in the FIA experiments because: (i) the background current was stable and had values near to zero, (ii) it was low enough to reduce any interferences, (iii) the amperometric response was satisfactory and very reproducible.
The electrochemical behaviour of DMcT was studied in phosphate and BR buffer solution in the pH range from 3.50 to 9 (Fig. 6B). The amperometric response of oxidation DMcT on the modified GC electrode was found to have a higher value in BR buffer compared to phosphate buffer. The highest value of the current response was obtained at a pH value of 6.50. Therefore, all further meas-urements were performed in BR buffer at a pH value of 6.50. Next, the flow rate was evaluated in order to obtain the highest signal for DMcT oxidation.

Calibration curves, detection and quantification limits
Linearity, the limit of detection and quantification were investigated for three Na[RuCl 2 (SB) 2 ] modified carbon electrodes. One GC electrode and two SPC electrodes with and without MWCNTs addition. The amperogram for the modified GC electrode is shown in Fig. 8. For the same electrode, the linear relation between the amperometric peak current and different concentrations of DMcT was tested by injecting (n = 3) standard solutions into the FIA system under optimal parameters. The experiments revealed a good linear response for DMcT concentrations ranging from 5 to 750 mg dm −3 , as shown in Fig. 9B, in agreement with the following equation i(μA) = 0.0114 [DMcT] (mg dm −3 ) + 0.0570, R 2 = 0.9949.
After amperometric studies of DMcT oxidation at the modified GC electrode, measurements were also performed for the SPC electrodes. Figs and MCNTs is significantly higher than the SPC electrode modified with the mixture without MW-CNTs. Furthermore, for an MWCNTs-modified electrode, the lowest detectable concentration value is 1 mg dm −3 , whereas, for an unmodified electrode it is 5 mg dm −3 . The widest linearity was achieved at the GC electrode modified with Ru(III) complex and MWCNTs (Fig. 9). Additionally, the lowest detection limit, among the used electrodes, was obtained at the SPC electrode modified with Ru(III) Schiff base complex and MWCNTs.  demonstrated better sensitivity (LOD = 1.55 mg dm −3 ), while a wide linear range was obtained by the GC electrode (5-750 mg dm −3 ). In conclusion, findings from the present study emphasize the application of these modified electrodes for the determination of DMcT in real samples.