Extraction-Spectrophotometric Studies on the Ion-Pairing Between Some 2 , 3 , 5-Substituted Monotetrazolium Cations and Anions Deriving from 4-( 2-Thiazolylazo ) resorcinol or 4-( 2-Pyridylazo ) resorcinol

The ion-pairing between some 2,3,5-substituted monotetrazolium cations (T + ) and anions deriving from 4-(2pyridylazo)resorcinol (PAR) or 4-(2-thiazolylazo)resorcinol (TAR) was studied by water-chloroform extraction and spectrophotometry. The following tetrazolium salts (TS) were used as a source of T + : i) 2,3,5-triphenyl-2H-tetrazolium chloride (TTC); ii) 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT); iii) 3-(2-naphtyl)-2,5-diphenyl-2Htetrazolium chloride (Tetrazolium violet, TV); and iv) 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT). The spectral characteristics of the extracted species were established at different pH and TS concentration. The composition and stability of the ion-pairs were determined at pH 9, where the extraction of neutral PAR (H2PAR) and TAR (H2TAR) species was negligible. The results showed that the ion-pairs can be expressed by the following formulae (T + )(HTAR – ) (where T + = TT + , MTT + , TV + or INT + ), (T + )(HPAR – ) (where T+ = TT + , MTT + or TV + ) and [(INT + )(HPAR – )]2. Relationships involving the molecular masses of the ion-pairs (MIP) or T + (MT+) and the values of the constants of association () or conditional molar absorptivities (’) were examined, namely Log  = f(Log MIP) and ’ = f(Log MT+). Some practical aspects concerning the investigation of metal complexes with TS-PAR/TS-TAR were discussed.


Reagents and apparatus
Azo derivatives of resorcinol (ADR) PAR (96%) and TAR (97%) were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). 210 -3 mol L −1 aqueous solutions of these ADRs were prepared by dissolving weighted amounts of PAR or TAR in water alkalised with KOH. The final pH of the obtained solutions was 7-8. More dilute ADR solutions (510 -4 mol L −1 ) were prepared by appropriate dilution with distilled water. MTT, TV, INT and TTC were purchased from Alfa Aesar (Karlsrue, Germany), Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany), AppliChem GmbH (Darmstad, Germany) and Loba Feinchemie GmbH (Fischamend, Austria), respectively. Aqueous solutions of these reagents were prepared. The concentrations were 210 -3 mol L −1 (for TV and INT), 310 -3 mol L −1 (for MTT), and 410 -3 mol L −1 (for TTC). Redistilled chloroform was used. The acidity of the aqueous medium was set by the addition of buffer solution prepared by mixing 2 mol L −1 aqueous solutions of CH3COOH and NH4OH. The resulting pH was checked by HI-83140 pH meter. A Camspec M508 spectrophotometer (United Kingdom), equipped with 10 mm path-length cells, was employed for reading the absorbance.

Procedure
1 mL of buffer solution and aliquots of ADR solution (PAR or TAR) and TS solution (TTC, MTT, TV or INT) were pipetted into 100-mL separatory funnels. The volumes were made up to 5 mL with distilled water. Then 5 mL of chloroform were added. The funnels were closed with stoppers and shaken for 2 minutes. After separation of the layers, portions of the chloroform extracts were transferred through filter papers into cells. The absorbance was read against chloroform or an appropriate blank sample (containing buffer-ADR or buffer-MTT).

Preliminary data and observations
Several optically different PAR and TAR species exist in aqueous solution in dependence on the acidity: H4PAR 2+ , H3ADR + , H2ADR, HADR and ADR 2- [16,[33][34][35] (Table 2). Studying the chloroform extraction of these reagents from buffered aqueous medium (pH 1-10) Marić and Široki [16] established maximum and practically constant extraction of PAR and TAR under the pH ranges of 3.5-5 and 1.5-5, respectively, where their neutral H2ADR species predominate ( Table 2). On the other hand, the concentration of H2ADR is negligible at pH 9. As a result, PAR and TAR cannot be extracted with chloroform alone. However, they can be extracted as ion-pairs of the type (Ph4X + )(HADR -), where Ph4X + is a cation deriving from tetraphenylphosphonium chloride and tetraphenylarsonium chloride [16]. Our preliminary investigations on the TS-ADR extraction systems at pH 9.0 suggested the formation of similar compounds. The recorded wavelengths of maximum absorption (max) ( Table 3) were quite close to these reported by Marić and Široki: max=400 nm for (Ph4X + )(HPAR -) and max=498 nm for (Ph4X + )(HTAR -) [16]. However, different conditional molar absorptivity (') rows were observed in both series (ADR=PAR and ADR=TAR) at equal conditions (Table 3) Table 3 and Fig. 1). This anomaly showed that there must be some big difference between both extracted INT-containing compounds.

Saturation curves at pH 9
The dependences between the absorbance of extracted ion-pairs and concentration of TS were studied at pH 9 ( Figure 2). The concentration of ADR (PAR or TAR) was kept constant in all series (C=5.0×10 -5 mol L -1 ) in order to ensure identical experimental conditions. The absorbances of the extracted species were recorded against blank samples containing ADR (curves 1, 3, 4, 1', 2', 3' and 4') or MTT (curve 2). The absorbance of the MTT-PAR species was found by a different manner due to the noticeable absorption of MTT at max(MTT-PAR). In contrast to the other studied TSs (which exhibit maxima in the UV region) MTT has an additional maximum at about 370-380 nm [1,3,36]. The results presented in Figure  2 show that the following TS n-fold excesses (in parenthesis) are necessary for maximum ADR extraction: i) ARD=PAR; TTC (48), MTT (18), TV (6.4), and INT (5); ii) ARD=TAR; TTC (48), MTT (30), TV (9.6), and INT (24). It should be mentioned that at TS excess conditions the recorded max were slightly different from these reported in Table 3. The specific max and max values for each ADR-TS system are shown in the caption of

Composition of the ion-pairs
One can expect the following ion-pair reaction for the system of ADR and TS: Job's isomolar curves [37] (Figure 3) and the saturation curves presented in Figure 2 were used to check the validity of Equation 1. The data shown in Figure 2 were processed by the straight-line method of Asmus [38], Bent-French method [39] and mobile equilibrium method [40]. The results confirmed the formation of (T + )(ADR -) species in all systems except for the INT-PAR system. The mobile equilibrium method [40] (Figure 4 and Figure 5) and the dilution method [41] (Figure  6), which are appropriate to distinguish between 1:1 and 2:2 species, showed that the INT-PAR compound had a composition of 2:2. Hence, the ion-pair formation in this case can be expressed with Equation 2: The established composition of 2:2 explains well the highest conditional molar absorptivity of PAR-INT in the PAR-TS series (Table 3): the dimerization leads to enhanced bulkiness and hydrophobicity of the compound and this improves its extraction behavior.

Constants of association 
The equilibrium constants  characterizing the processes of ion-pairing described above (Equations 1 and 2) were calculated by several independent methods, namely the mobile equilibrium method (Figures 4 and 5) [40], the Harvey-Manning method [42], the Holme-Langmyhr method [43] and the dilution method [41] (Figure 6). The results are presented in Table 4.

Relationships between Log  and Log M
At least two factors noticeably influence the stability of tetrazolium ion-associates. The first factor was formulated by Alexandrov et al [1,44]: the higher the molecular mass (M), the higher the association constant (). The second factor is bound up with the presence of nitrophenyl dubstituent(s) in the tetrazolium ring [3,45]: The value of  is significantly lower than the expected one if the tetrazolium cation contains NO2-group(s).
The results shown in Table 4 for Log (TT+)(ADR-), Log (MTT+)(ADR-) and Log (TV+)(ADR-) and calculated logarithms of the molecular masses of the mentioned ion-pairs (Log MIP) were used to check the validity of the first factor. The obtained squared correlation coefficients (R 2 ) were R 2 =1.0000 (for ADR=PAR) and R 2 =0.9524 for (ADR=TAR).

Absorption spectra at pH 6.
At pH-values lower than 8 the spectral curves of the extracted ion-pairs were affected in some extent by the simultaneous extraction of neutral PAR and TAR species [16]. Having in mind the different stability of the extracted TS-ADR ion-pairs and the nature of the used blank samples (buffer-PAR or buffer-TAR) one can expect more remarkable spectral differences for the systems containing species with closer max values. H2PAR and H2TAR have absorption maxima at 382 nm ( Figure 8, curve 5) and 413 nm (Figure 9, curve 5), respectively. HPAR and HTAR -, in their turn, have absorption maxima at approximately 400 nm and 495-500 nm. Hence, the PAR-TS systems contain species with the closer spectral characteristics (18 nm); that is why the recorded max values for PAR-TTC, PAR-MTT, PAR-TV and PAR-INT, appeared in relatively wide interval (see Figure 8). Contrariwise, the big difference between the maxima of H2TAR and HTAR -(84 nm) resulted in a relative stability of the established max values: max=491 nm (TAR-MTT and TAR-INT systems), 492 nm (TAR-TV system) or 493 nm (TTC-TAR system). However, an additional effect can arise in these TAR-TS systems, namely negative absorbance values (A = ATAR+TS -ATAR; ATAR > ATAR+TS) recorded near the wavelength region of maximum H2TAR absorption (Figure 9). This effect is expected to be stronger when CTAR and  are higher. Figure 9 shows that the minimum in the spectral curve for the TAR-TV system is the deepest (min405-410 nm, A=-0.130); in fact, this system produces the most stable ion pair in the TAR-TS series. It should be mentioned that we observed similar minima in the spectra of some ternary complexes containing a metal ion (V, Co), TAR and TS [5,30]. These minima caused noticeable distortion of the main spectral band of the complexes, especially when TV or TTC were used [5].
One can notice from Figure 8 that in contrast to the spectral curves (1) and (3), the PAR-INT spectral curve (4) is not symmetrical. The shoulder observed on the left side of the peak can be regarded as a sign of dimerization [46]. As a result of the overlapping of two bands, the half-width of the recorded complex band is wider and max appears at shorter wavelengths (405 nm). Such an explanation fits well with the observed facts. , and between the conditional molar absorptivity ' (pH=9, CADR=CTS) and Log MT+ (or Log MIP). The relatively big differences between the studied ion-pairs can be attributed to the different nature of the substituents in tetrazolium ring. In contrast to the ion-pairs studied by Marić and M. Široki [16] (Ph4X + )(HADR -), in which X is situated in the core of the cation (X=P or As), the different substituents in our case are located in the outside part of the tetrazolium cation T + . Hence, they can contribute more dramatically to the overall performance of T + and the ion-pairs itself.