Electrochemical andspectrastudies of some sulfa drug azodyes and their metal complexes in aqueous solution

The electrochemical behavior of some azo compounds derived from sulfa drugs derivatives in B.R. buffer solutions of different pH containing 20 (v/v) ethanol was investigated at the mercury electrode using different techniques (DC,DPP,CV and Coulometry) to investigate the effect of medium on the electro reduction process and suggestion the electrode reaction mechanism. The obtained results denoted that these compounds were reduced undergo a single irreversible 4electron polarographic wave in acid and neutral solutions which represent the cleavage of the N=N center to the amine stage, whereas in alkaline solution, two wave are obtained the second is 2-electron irreversible wave corresponding to the reduction ofCHO group to CH2OH. The DPP and CV data showed a single peak in solutions of pH< 8, whereas three peaks are in alkaline solutions. The dissociation constants of the investigated compounds were determined by using spectrophotometric and potentiometric methods. Also the metal – ligand formation constants were determined potentiometrically and found in the order Cu˃ Co˃ Ni ˃Zn.


Instrumentation 2.2.1. Voltammetric measurements
DC-polarograms were recorded on an ink recording SARGENT WELCHPolarograph model 4001. The capillary used as a dropping mercury electrode has the following characteristics in 0.1 M KCl (open circuit): m = 1.7 mg/s, t = 3 sec., at mercury height (h) = 60 cm. The mercury used was purified according to the recommended method [32]. DP polarograms and cyclic voltammograms of the studied compounds were recorded with a Polarographic Analyzer Model 264A -PARC (from EG&G) and the electrode assembly Model 303 , with a hanging mercury drop electrode (HMDE) as a working electrode, Ag/AgCl as a reference electrode and Pt wire as a counter electrode. The constituents of the electrolysis cell were the same as in the DC-polarography. X-Y recorder Model RE 0091, Houston Instrument Division (from EG&G) was used for recording the DPpolarograms and voltammograms. A digital coulometer (Model 179) from EG&G was used for the coulometry measurements.

UV-visible spectra
Electronic absorption spectra were recorded at room temperature within the wavelength range 200-600 nm using a Shimadzu UV-visible recording spectrophotometer Model1600 A.

Potentiometric measurements
Potentiometric measurements were performed using a Digital ORION pH-meter Model 201 accurate to =0.02 pH units having combined glass and calomel electrode, with a magnetic stirrer and a semi micro burette with divisions of 0.01 ml. The electrode was standardized before andafter titration with buffer solutions produced by FISHER (New Jersey, USA). These solutions were titrated potentiometrically with 0.02 M NaOH solution. The NaOH solution was titrated against a standard solution of sodium carbonate.

Electrochemical studies 3.1.1. DC Polarography
The polarograms of 1x10 -4 M of salicyladehydeazosulfa-methazine (I), salicyldehydeazo-sulfamerazine (II),salicyldehydeazo-sulfadiazine (III) and sulfamethazineazo-phenol (IV) in Britton-Robinson buffer solutions of pH 2-11 containing 20% (v/v) ethanol were recorded. The addition of ethanol is due the low solubility of these compounds in pure aqueous media. The polarograms of the azo compounds (I-III) consist of two reduction waves of unequal heights. The second wave in acidic solutions (pH < 7) is ill-defined and coaleases with the hydrogen evolution. The first wave is an irreversible 4-electron step represents the reduction of the azo group, whereas the second one is an irreversible 2-electron wave and due to the reduction of the aldehyde group. Fig. 1Arepresents the polarograms of azo compound (I) as atypical example of this series. However, in solutions of pH > 8.0, the first wave of azo compounds (I-III) splits into two waves of unequal heights. On increasing the pH of the solution, the limiting current ( il ) of the second splitting wave increases at the expense of the first splitting one whereas the total limiting current is remained constant.The il-pH curves of the single wave and the two splitting waves recalled a dissociation curve (Z-shaped) and an association curve (S-shaped) (Fig. 1B), thebehaviour which may be attributed to an acid-base equilibrium in that pH range [33].  The half-wave potential (E1/2) of the polarographic wave shifted to more negative values on increasing the pH of the solution, denoting the consumption of protons in the reduction process and the proton uptake precedes the electron transfer [33]. The effect of mercury height (h) on the limiting current (il) of the polarographic waves using the equation [34]: il = k h x , revealed that the reduction process for all azo compounds is controlled mainly by diffusion with some adsorption contribution.
The value of the exponent (x) at different pH values amounts to 0.5-0.75 (Table 1). Analysis of the polarographic waves at different pH values using the basic equation for reversible polarographic wave [32]showed that the electrode reaction proceeds irreversibly. The value of the transfer coefficient (α) and the number of electrons participating in the rate-determining step (na) were determined from the reciprocal slopes (S1) of the logarithmic analysis plots and given in Table 1. It was found that, the most probable (α) values were obtained at na equals to one or two depending on the pH of the medium, whichrevealing that the rate-determining step of the reduction process may involve two electronsin acidic solution and neutral solutions, and one electron in alkaline ones ( Table 1).
The E1/2 -pH plots of all azo compounds gave straight lines of three segments and the breaks occur at pH 4-5 and 8-9. From the slope values (S2) of these plots and the slopes of logarithmic analysis (S1), the number of protons (ZH + ) participating in the rate-determining step was determined using the following relation [34]: The value of ZH + for all compounds were calculated at different pH values and found approximately equal to unity (

Cyclic voltammetry measurements
The cyclic voltammetry of azo compounds (I-IV)was measured in buffer solutions of different pH valuescontaining 20% (v/v) ethanol. The voltammograms were recorded at different scan rates (20 -500 mV/s). As shown in Fig. 2A (as a typical example), a single cathodic peak was observed for azo compounds (I-III) in solutions of pH < 8, whereas in alkaline solutions (pH > 8) three cathodic peaks were observed. Thisbehavior agreed with thoseof DCpolarography. However, for azo compound IV, a single cathodic peak is observed within the entire pH range. The absence of any peaks in the reverse scan (anodic direction) for all the voltammograms confirmed the irreversibility of the reduction process of these compounds.
f,h is the heterogeneous formal rate constant (cm/s), Dois the diffusion coefficient (cm 2 /s), R is the gas constant, T is the absolute temperature, F is the faraday constant, αna has its usual significant Thisequation relates the values of Ep and αna. The plots of Ep versus lnѵ (logarithm of sweep rate) were straight lines, from their slopes; α values were calculated and given in Table 2. It was found that the most probable values of (α) were obtained at na = 1.0 in all pH solutions. This may be due to the high resolution of cyclic voltammetry compared to DCpolarography.
On plotting the peak current (ip) as a functionof thesquare root of sweep rate (ѵ)according to the following equation [36]: In which n is the total number of electrons, A is the electrode surface area (cm 2 ), Co is the concentration of the depolarizer and the remainder terms have their usual significance Linear correlations were obtained which deviated from the origin, confirming that the reduction process is mainly controlled by diffusion with some adsorption contribution[37].

DP-Polarography
The DP-polarograms of all azo compounds (I-IV) showed a single dp-polarographic peak in acidic solutions (pH < 8), whereas in alkaline ones three peaks were obtained. On the other hand, the DP-polarograms of azo compound (IV) showed a single peakin allpH solutions. Fig. 1C represents the DP-polarograms of azo compound (I) as a typical example of this series in solution of pH 2.2. The results of the DPP measurements are in agreement with those obtained from DC and CV measurements.

Controlled potential electrolysis measurements (Coulometry)
The total number of electrons involved in the electrode reaction was determined using controlled potential electrolysis technique (coulometry) using a mercury pool cathode. The accumulated charge (Q) was taken from the digital coulometer at a potential corresponding to the limiting current of the polarographic wave. Applying the equation: where w is the weight of the sample (in grams) and M is the gram molecular weight, the values of (n) along the first waveof azo compounds (I-III) and the single wave of azo compound (IV) was found to be 4 electrons which represent the reduction of azo group whereas for azo compounds (I-III) along the second wave (n) equals to two electrons representing the reduction of aldehyde group attached to the phenyl ring.The data of coulometry are given in Table (3).

Identification of the electrolysis products
The completely electrolysed solutions of azo compounds (I) at pH 2and 10 were chosen as an example for the compounds under consideration. These solutions were concentrated and the buffer ingredients were removed by extracting the slurry with ether. Thin layer chromatography (TLC) was applied for the etheral extract in chloroformethanol mixture (90:10); two products (two spots) were observed. On comparing with authentic sulfamethazine sample, it is concluded that the later is one of the two electrolysis products which indicated the cleavage of N=N centre. On the other hand, the UV-spectra of the continuously electrolysed solution of azo compounds (I) at pH 2 and different time intervals showed that the band appeared at max = 350 nm which characteristic to  - * transition of N=N group decreases gradually and disappeared completely for the completely electrolysed solution which confirmed also the cleavage of the N=N bond, (Fig.2C).

Mechanism of the electrode reaction
The data obtained from coulometric measurements along the first wave for azo compounds (I-III) and the single wave for azo compound (IV) revealed that the electro-reduction process of these compounds involved 4 electrons and 4 protons leading to the cleavage of the N=N center within the entire pH range and the corresponding amine is obtained. On the other hand, coulometry measurements along the second wave of the azo compounds (I-III) revealed that 2 electrons are involved in the reduction processwhich represents, the reduction of the electroactive aldehyde group (CHO) to (CH2OH) The reduction mechanism can be represented as follows (Scheme 2):

Analytical micro-determination
Analytical microdetermination of azo compound (I) as an example of this series was investigated in buffer solution of pH 2. The micro-determination was done using the more sensitive dp-polarography. The total volume of the electrolysis solution in the polrographic cell was kept to be 10 ml containing the same percent of ethanol. The concentration of the depolarizers was increased from1.43 x10 -4 to5.26x10 -7 mol/L. The dp-polarorams were recorded and represented in

Electronic absorption spectra of the sulfa drug azo compounds
The electronic absorption spectra of 1 × 10 -4 Mof azo compounds (I-IV) investigated in buffer solutions of varying pH (2)(3)(4)(5)(6)(7)(8)(9)(10)(11) were recorded within the range 200-700 nm. Fig.4A, represents the spectra of azo compound (I) as a typical example of this series. All the investigated compounds exhibited three bands; the first band (not shown) appeared at 230-250 nm is due to the local excitation of the π-π* transition of the aromatic moiety, while the second and third bands appeared within the range 380-450 nm are attributed to the charge transfer (C.T) interaction within the whole molecule. Also, a clear one or two isobestic points were observed indicating more than one species are present in equilibrium.

Determination of the dissociation constant values
To determine the dissociation constants of the azo compounds (I-IV); the limiting absorbance method (LAM)  Table ( 4). The results indicated that two pka values for each azo compound (I-IV) were obtained; the first is due to the dissociation of proton of amide group whereas the second is attributed to the dissociation of the hydroxyl group in the phenyl ring.

3.3.1.Proton-ligand dissociation constants
The acid dissociation constants of the azo compounds (I-IV) were calculated from the titration curves of the hydrochloric acid with sodium hydroxide solution in absence as well as in presence of azo compounds. However, potentiometric titration of the ligands with sodium hydroxide in the presence of (10 -3 M) hydrochloric acid was carried out at ionic strength (μ=0. where: Y is the total number of dissociable protons attached to the ligand, is the molarity of the sodium hydroxide solution, is the molarity of the hydrochloric acid solution, V1 and V2 are the volumes of sodium hydroxide required to reach a definite pH value for the free acid titration curve and for that acid in presence of a ligand, respectively, is the total concentration of the ligand in molar scale and is the initial volume of the titrated solution (50 ml).
On plotting n A vs. pH gives the proton-ligand formation curves (Figure omitted for brevity) of azo compound (I) as a typical example of this series. The values of proton-ligand dissociation constants of the investigated compounds were calculated by interpolation at half n A values, at n A = 0.5, 1.5 and 2.5 and given in Table (4). These values indicated that the azo compounds (I-IV) have two dissociable protons (two pka values). The second is due to dissociation of proton of phenolic OH group in the phenyl ring whereas the first is attributed to dissociation of amide group (NH) within the sulfa drug moiety. It is important to noted that, the data obtained from spectrophotometric measurements are in agreement with those calculated by potentiometric ones.