Electrochemical Reduction of Isatin-monohydrazone on Mercury Electrode

Electrochemical behaviour of isatin monohydrazone (IM) on a hanging mercury drop electrode in the Britton-Robinson (B-R) buffer solution of pH = 2.00 – 9.00 has been investigated using square-wave voltammetry (SWV) and cyclic voltammetry (CV) techniques. In the pH range of 2.00 – 5.00, the voltammogram of IM exhibited a single cathodic irreversible peak. When the pH value exceeds 5.00, a new cathodic irreversible peak was also seen. According to the voltammetric data, a plausible electrode reaction mechanism of IM was proposed. The first reduction peak of IM is resulted from the reduction of =N–NH– group with consumption of 2e–/2H+. Also, its second cathodic peak is formed by the participation of 2e–/2H+ for the reduction of –N=N– group on its tautomeric form.


INTRODUCTION
The chemistry of isatin (indole-2,3-dione) and its derivatives is particularly interesting owing to their wide application for analytical and synthetic purposes.It has important biological activities such as anti-inflammatory, antibacterial, anticonvulsant and antioxidant properties. 1,2Many new compounds of isatin were synthesized and reported for antibacterial, 3 antifungal, 4 anti-HIV 5 and anticonvulsant. 6,7The monohydrazone 8 and bishydrazone compounds 9 from isatin have been prepared and widely used in medicine.For example, isatin hydrazone was used as a detecting reagent for 3-ketosteroids. 10][13][14] The electrochemical behaviors of isatin and some of its derivatives 9,[15][16][17][18][19][20][21] were examined.The knowledge of the redox properties of hydrazone compounds is important for a better understanding of their behaviors in both chemical and biological processes.3][24][25][26][27][28][29] The hydrazones are electroactive compounds.1][32][33][34][35][36][37][38][39] Isatin-3-hydra-zone (IM) was detected during the electroreduction of 3-diazo-oxindole at pH > 8.5. 40,41In a recent paper, the redox behavior of isatin-3-hydrazone at a glassy carbon electrode has been studied by Olivera et al. 21evertheless, the comprehensive research of the electrochemical behavior of IM on the hanging mercury drop electrode (HMDE) will be also useful in the clarification of the electron transfer pathways of biological reactions of the hydrazones which possess some pharmaceutical activities. 42n the present research, the redox properties of IM on the mercury electrode in different pH media have been examined by using square-wave and cyclic voltammetry techniques.This study has reported the results together with a discussion of the electrode reaction mechanism of IM.

Materials and Reagents
Isatin and hydrazine hydrate (reagent grade 98.0 %) were obtained from Sigma-Aldrich.Methanol was of analytical grade and purchased from Merck.All solutions were prepared using triple distilled and deinonized water.B-R buffers were made from boric acid, acetic acid, and phosphoric acid, all 0.04 M, and titrated to the desired value with 0.2 M NaOH.B-R buffers were prepared using analytical grade reagents (Merck or Sigma) and were used as supporting electrolyte.

Apparatus
An EG&G PARC Model 303A HMDE three electrode system (mercury working electrode, platinum counter electrode and Ag|AgCl |KClsat.reference electrode) along with EG&G PAR 394B polarographic analyzer used for voltammetric measurements at room temperature.

Method
Before each measurement, the solutions were deaerated by a stream of pure nitrogen.A known volume of 1 × 10 −3 M IM solution was added to the electrochemical cell, which was closed, deaerated, and blanketed with oxygen-free nitrogen.Then, the voltammograms were recorded.The square-wave voltammograms were obtained using the following experimental parameters: pulse height, 20 mV; scan increment, 2 mV; frequency, 50 Hz; scan rate, 100 mVs -1 ; drop size medium and equilibrium time 5 s.Cyclic voltammograms were obtained for drop size medium and equilibrium time 5 s in the range of scan rate of 200 -1000 mVs -1 .Each measurement was carried out on a fresh mercury drop.

General Procedure for Synthesis of IM
According to the procedure, reported by Murukan and Mohanan, 43 IM was prepared by refluxing together equimolar solutions of isatin and hydrazine hydrate in methanol for 3 h (Scheme 1).On cooling, the monohydrazone crystallized from the reaction mixture.Recrystallization from methanol afforded beautiful yellow crystals of the monohydrazone, m.p. 226 °C.The melting point of IM is different from those of isatin (m.p. = 203 °C) 44 and hydrazine hydrate (m.p. = -51 °C). 45The m.p. of IM is almost same with that (225 °C) of the synthesized by Kulkarni et al. 46

Effect of pH
The electrochemical behavior of IM was studied by using SWV and CV techniques in B-R buffer solutions (pH = 2.00 -9.00).For the pH range of 2 -5, the squarewave voltammogram of IM exhibited one well-defined reduction peak (peak I) in the potential range of 0.0 to -1.2 V vs. Ag|AgCl |KClsat.(Figure 1).When the pH value exceeded 5.0, a new reduction peak (peak II) was observed (Figure 1).In addition, the reduction potential of peak II is more negative than that of peak I.The peak II appears clearly only at at pH range of 6.00-8.00(Figure 1) for high IM concentrations (  1 × 10 -5 M).This peak (peak II) was not observed at glassy carbon electrode by Olivera et al. 21In the literature, 47 it was reported that the multiplicity waves of various hydrazone derivatives were ascribed to equilibrium among hydrazone, azo, and ene-hydrazine forms.Therefore, these peaks on the voltammograms of IM can be sourced from the ketohydrazo and enol-azo tautomeric forms of IM (Scheme 2).Especially, the appearance of new peak may be attributed to the reduction of the enol-azo form.
Keto-enol tautomerism is catalyzed by either acid or base.Usually the enol is the minority tautomer, but it is more reactive. 48[51] Scheme 2. Keto-hydrazo and enol-azo tautomeric forms exhibited by IM In the range 2  pH  5, the current of peak I considerably increased with increasing pH and then reached a maximum value, which confirms that IM is more electrochemically active at pH = 5.The azo tautomeric form of IM may be present in solution but in very small quantities that does not exhibit a voltammetric signal in the pH range of 2 -5.However, in the range 6  pH  9, the current of peak II continues to increase up to pH = 8 whereas that of peak I decreases with increasing pH (Figure 2).Increase in the alkalinity of the medium leads to a shift in the equilibrium towards to the enolazo form. 52So, in the range 6  pH  9, the appearance of the peak II may be caused from the reduction of the azo tautomeric form of IM, present in effective amount under these conditions.Moreover, the potentials (Ep) of both reduction peaks (I and II) shifted to more negative values with increasing pH of the electrolyte (Figure 3), indicating that hydrogen ions are involved in the reduction process.The hydrogen ion concentration decreases by increasing pH.In this case, the required energy for the reduction of IM increases.Therefore, the peak potentials of IM shifted towards to more cathodic values.
For the peak I, the graph of Ep vs. pH exhibits two linear relationships in the pH range 2.00-9.00.These two linear parts graphically break at pH = 7.41 (Figure 3).This pH value may be attributed to a pKa, corresponding to the deprotonation of enolic form of IM (Scheme 3).The determined pKa value (7.41) of IM in the present study is different from the value (~ 9) reported by Olivera et al. 21This case might arise from the difference in the experimental conditions (e.g., temperature, ionic strength and the solvent dielectric constant).Because it is well known that pKa depends on the experimental conditions. 53 addition, the potential of peak II is also dependent on pH (Figure 3).By the effect of pH, in the range 7  pH  9, the magnitude of shift in the potential of first peak (I) is greater than that of the shift of new peak (II) (Figure 3).So, the new peak II overlaps with the first peak I to produce a shoulder peak at pH = 9 (Figure 4).
Examination of cyclic voltammograms obtained in the pH range 2.00 -9.00 reveals that no anodic counter parts of the peaks I and II are seen on the reverse sweep (Figure 5).This result indicated that the electrode process of IM is irreversible under the experimental conditions examined in this study.However, cathodic peak II is only observed at pH values higher than 5 (Figure 5).As can be seen from Figure 5, the peak II is slightly observed at pH = 6.Consequently, cyclic voltammetric results of IM are nearly in agreement with those   obtained by square-wave voltammetry technique on the formation and characteristic values (cathodic currents, peak potentials, etc.) of the peaks I and II.

Effect of the Scan Rate
The influence of the scan rate (ν) on the peak currents (Ip) of IM was investigated by cyclic voltammetry.The peak current changes linearly with scan rate according to the equation Ip = Aν x . 54The x values 1.0 and 0.5 are expected for adsorption-controlled and diffusion-controlled reactions, respectively. 55,56For cathodic peaks I and II, the linear relationships between log Ip and log ν are observed at different pH values (Figure 6).The experimental results are listed in Table 1.As can be seen in Table 1, the slope values of peaks I and II at pH = 6.0 are very close to the theoretically expected value of 0.5 for a purely diffusion-controlled current.At more acidic or basic solutions, the slope values are bigger than 0.5.This result indicates that the electrode process of IM is a diffusion-controlled mass transfer, involving a slightly contribution of adsorption.
The half-peak-width, W1/2 (expressed in V), at the peak half height can be used to calculate αn at each pH value: [57][58][59] W1/2 = 3.52 R T/ α n F where F is the Faraday constant (96 485 C mol -1 ), R is the standard gas constant (8.314J mol -1 K -1 ), and T is the temperature (expressed in K).The calculated αn values were given in Tables 2 and 3.
If an electrode process includes a protonation step, the currents become pH dependent.The number of protons, mH + , involved per molecule of IM in the ratedetermining step (RDS) are calculated from the slopes (= 0.059 mH + /αn) of the graph of Ep vs. pH (Figure 3) and αn values which is determined by means of the halfpeak-width (Tables 2 and 3).The determined proton numbers in the RDS of the electrode reaction are also given in Tables 2 and 3.As can be seen in Table 2, at the RDS of the electrode reaction of the first reduction peak of IM, the values of mH + calculated lie in the range of 1.55-1.69,indicating that two protons are consumed.In addition, for the peak II, the values of mH + calculated lie in the range of 1.90-2.00,showing the involvement of two protons in the RDS in the pH range 6.009.00(Table 3).The data presented in Table 2 are also different from the results of IM at RDS on glassy carbon electrode. 21heme 5. Tentative reduction mechanism for peak II (in the pH range 6-9).

Figure 1 .
Scheme 1.The synthesis of IM

Figure 3 .
Figure 3. Dependence of the potentials of peak I ( ) and peak II ( ) on pH for 1 ×10 -5 M IM.

Table 1 .
The cyclic voltammetric data from plots of log Ip νs.log ν for the first reduction peak (I) and the second reduction peak (II) of 1×10 -4 M IM at pH values of 2, 3, 5, 6, 8 and 9 (for the scan rate range: 200 -1000 mVs -1 )

Table 2 .
The square-wave voltammetric data obtained for peak I

Table 3 .
The square-wave voltammetric data obtained for peak II Tentative reduction mechanism for peak I (in the pH range 2-8).