An extraction-chromogenic system for vanadium(IV,V) based on 2,3-dihydroxynaphtahlene

Abstract A liquid-liquid extraction-chromogenic system for vanadium(IV, V) containing 2,3-dihydroxynaphtahlene (DN), 2,3,5-triphenyl-2H-tetrazolium chloride (TTC), water and chloroform was studied in detail. When the vanadium is in the oxidation state of IV, the extracted species are aggregates containing three 1:2:1 (V:DN:TTC) ion-pair units composed of triphenyltetrazolium cations (TT+) and chelate anions {[VIVO(DN)(DNH)]− (I) and/or [VIV(OH)(DN)2]− (II)}. When the initial oxidation state of vanadium is V and the DN concentration is high, vanadium(V) is reduced by DN to a lower oxidation state, V(IV). However, at low DN concentration, vanadium(V) can enter the organic phase as a part of an ion-pair consisting of TT+ and [VVO2(DN)]− (III). The ground-state equilibrium geometries of the anions I, II, and III were optimized by quantum chemical calculations using BLYP/6-31++G⋆. The following characteristics were determined under the optimum conditions for VIV extraction: absorption maximum λmax = 333 nm, molar absorptivity ε333= 2.1x104 dm3 mol−1 cm−1, Sandell’s sensitivity SS = 2.4 ng cm−2, and fraction extracted E = 98%. The conditional extraction constant was calculated by two independent methods. The calibration graph was linear in the range 0.1-3.1 μg cm−3 (R2=0.9994) and the limit of detection was 0.03 μg cm−3.


Introduction
Vanadium is a biologically important trace element with many industrial applications. Making up about 0.014% of the Earth's crust, it is the fifth-most abundant transition metal [1]. Key factors that determine vanadium's roles in physiological systems are its oxidation-reduction properties (e.g. ability to switch easily between oxidation states V and IV) and flexible stereochemistry [2,3].
Many methods have been proposed for vanadium determination and speciation [4][5][6]. Among the most sensitive and cost effective are the extractionspectrophotometric methods based on ternary complexes involving catecholic ligands [6][7][8][9]. However, the mechanism of color development in such methods is disputable since it is not always clear whether the main spectral bands are due to the formation of coordination compounds or products of oxidation and polymerization of the reagent(s) [10].
Several papers [11][12][13][14] describe liquid-liquid extraction of V V with 2,3-dihydroxynaphthalene (DN) -a catechol type ligand of analytical importance [15]. However, the possibility of V V → V IV reduction caused by DN, a phenomenon well documented by Adediran and Pratt [16], is underestimated in these papers. For example, the time dependent absorbance at pH values higher than 5.25 in the V V -DN -iodonitrotetrazolium chloride (INT) system [11] has been attributed solely to a redox reaction between DN and INT, assuming that the tetrazolium salt INT is able to inhibit the reduction of V V to V IV caused by ortho-diphenolic compounds [17]. A convenient way to throw additional light on the mentioned assumption is to compare results obtained with V IV and V V . To the best of our knowledge, such a comparison has not been performed so far and V IV -DN ternary complexes have never been studied. Here, we report our results for extraction-chromogenic systems containing V IV or V V , DN, 2,3,5-triphenyl-2Htetrazolium chloride (TTC), water and chloroform. We selected TTC for the present study since it is the simplest commercially available tetrazolium salt. Moreover, TTC has many applications based on ion-association [17][18][19][20][21][22] and oxidation-reduction [23][24][25]. Molecular parameters of TTC and some compounds containing its cation TT + are given in the literature [26,27].

Reagents and Apparatus
Stock V IV aqueous solution (5×10 -2 mol dm -3 ) was prepared from VOSO 4 ⋅5H 2 O (Fluka, purum) and standardized by potassium permanganate titration. Working solutions at a concentration of 2×10 -4 mol dm -3 were prepared daily as described previously [28]. V V solution (2×10 -4 mol dm -3 ) was prepared by dissolving NH 4 VO 3 (VEB Laborchemie Apolda, puriss. p.a.) in distilled water. DN solution (2×10 -3 mol dm −3 ) was prepared daily by dissolving an appropriate amount of the solid reagent (Fluka, purum) in chloroform. The concentration of TTC (from Loba Feinchemie GMBH, p.a.) was 5×10 -3 mol dm −3 (aqueous solution). The chloroform was additionally distilled. The acidity of the aqueous medium was set by the addition of buffer solution, prepared by mixing 2.0 mol dm -3 aqueous solutions of CH 3 COOH and NH 4 OH. The resulting pH was checked by a Hanna HI 83141 pH meter (manufactured in Romania) calibrated according to manufacturer's instructions. A Camspec M508 spectrophotometer (United Kingdom), equipped with 10 mm path-length glass cells, was used for the absorbance measurements. All experiments were performed at room temperature ∼23 °C (air-conditioner).

Procedure
Aliquots of V IV or V V solution, buffer solution and TTC solution were transferred into separatory funnels. The resulting solutions were diluted with distilled water to a total volume of 10 cm 3 . Aliquots of DN chloroform solutions were added and the organic phase was made up to 10 cm 3 with chloroform. The funnels were shaken for extraction for a fixed time (10 -300 s). After separation of the layers, portions of the chloroform extracts were transferred through filter papers into cells. The absorbances were measured against simultaneously prepared blank solutions (containing all of the reagents with the exception of vanadium) or chloroform.

Theoretical details
The structures of the anionic chelates were optimized at the BLYP/6-31++G* level of theory. For structure I the charge and the multiplicity were set to -1 and singlet, while for the structures II and III they were -1 and doublet. Subsequent frequency calculations were performed to prove that the found structures lie in minima on the PESs.
The initial structures of the anionic chelates for energy gradient optimizations were constructed relying on experimental data for the molar ratios V-DN and V-TTC. Technically the initial molecular complexes were constructed keeping in mind the positions and the orientations of the electron-accepting and electrondonating centrums as well as our intuition for producing a stable complex. All calculations were performed with the GAUSSIAN 03 program package [29].

Statistical calculations
All statistical calculations were performed with a spreadsheet program (Microsoft Office Excel 2010) at a probability of 95%. Figure 1 shows the absorption spectra of the chloroformextracted complexes and their corresponding blanks. Figure 1a represents the results obtained with high DN concentration (8.0×10 −4 mol dm −3 ). Spectrum 1 (with V IV ) and 2 (with V V ) are practically identical and this is an indication that under the given conditions V V is reduced to V IV . The small differences at wavelengths where the blank (spectrum 3) absorbs intensively, could be regarded as a result of an oxidation-reduction interaction between V V and DN, converting a portion of DN to its oxidation product [16,30,31] with different spectral and distribution properties. Another explanation could be the different degree of aggregation of the complexes derived from V IV and V V . It is supported by the observation (Figs. 1 and S1) that the main absorption band at 310 -350 nm for the species obtained with V IV is more clearly split into two components (see the blue circle in Fig. 1b). This evidence of aggregation [32] must be related in some way to another type of aggregation, characteristic for the blank samples, whose visible manifestation (spectra 3 in Figs. 1 and S1) is a very narrow peak (at ca. 325 nm) which depends on the DN concentration (spectra 3 and 3' in Fig. 1b).

Absorption spectra, composition, formulae and equations
Spectra 1 and 2 in Figs. 1 and S1 are characterized by a main absorption maximum at λ = 333 nm. Another maximum of interest is that at λ = 630 nm. At high or moderate concentration of DN the position of these maxima is not affected by changes in pH and the absorption ratio A 333 /A 630 changes only slightly. However, when the concentration of DN is low (e.g. 8.0×10 −5 mol dm −3 ) the main maximum for the sample with V V shifts to ca. 340 nm and the absorbance at the second maximum (at 630 nm) becomes very low (Fig. 1b, spectrum 2'); as a result, the A 333 /A 630 ratio increases significantly. The spectral changes accompanying the decrease of the DN concentration could be attributed to retention of the initial fifth oxidation state of vanadium and related differences in the composition of the extracted species. Fig. 2 illustrates the dependence of the absorbance at 333 nm and 630 nm on the concentration of DN. Note that the shape of the curve 1 (obtained with V IV at λ = 333 nm) is normal, while that of curve 2 (obtained with V V at the same conditions) is complicated due to oxidationreduction events.
The dependence of the absorbance on the concentration of TTC, when the DN concentration is 4×10 −4 mol dm −3 , is shown in Fig. 3. There are no anomalies in the shape of the curves obtained with V V . One can conclude comparing curves 1 and 2 that saturation is reached more easily when the initial oxidation state of vanadium is V (curve 2). In this case, there is a welldefined plateau (with a constant maximum absorbance in a wide concentration range). When the initial oxidation state of vanadium is IV (curve 1), a gradual increase in the absorbance is observed up to c TTC close to 1.2×10 −3 mol dm −3 . This is typical for both wavelengths (333 nm and 630 nm) and shows that there might be some difference in the mechanisms of complex formation for V IV and V V .
To determine the molar ratios in the ternary complexes, we used the mobile equilibrium method [33], the straight-line method of Asmus [34], and the Job's method of continuous variations [35]. The results based on the saturation curves shown in Figs. 2 and 3 are given in Figs. 4 -6 (mobile equilibrium method) and Figs. S2 -S5 (straight-line method of Asmus). Fig. 4 shows that the vanadium:DN molar ratio is 1:2 when the initial oxidation state of vanadium is IV (straight lines 1 and 1'; slopes close to 2). When the initial oxidation state is V (straight lines 2 and 2'), the vanadium:DN molar ratios are different for 333 nm and 630 nm (1:1 and 1:2 respectively). The same result was obtained by the method of Asmus (Fig. S3). It should be noted that the result 1:1 was obtained for relatively low DN concentrations (up to 1×10 -4 mol dm −3 ).
When the DN concentration is low, the chance of a fast oxidation-reduction process between the diphenolic compound and V V is lower [36]. Therefore, V V can be partially extracted in the organic phase under the form of a ternary complex according to Eq. 1.
Eq. 1 is constructed in accordance with the established V V :TTC molar ratio of 1:1 (Fig. 5, line 2; Fig. S5a) and the state of V V [6] and the tetrazolium salt [17] in the aqueous phase. This reaction involves the formation of an anionic complex [V V O 2 (DN)] − whose ground-state equilibrium geometry is shown in Fig. 7, structure I. Studies with other catecholic compounds [36] have shown that the formation of the 1:1 (V V :DN) complex is a fast process favoured by increasing the pH [31]. The fact that there is a serious difference in the shape of the pH curves for λ = 333 nm (Fig. S6, curves 1 and 2) for V IV (maximum at pH 5.5) and V V (maximum at pH 7) shows that eq. 1 is really an important stage in the overall extraction-chromogenic process for the system with V V (especially for DN concentrations lower than ca 5×10 −4 mol dm −3 ). If we assume that the absorption maximum at 630 nm is due to the formation of a 1:2 V IV -to-DN ternary complex (Fig. 4, lines 1' and 2'; Figs. S2b and S3b), two formulae are of importance considering that the V:TTC molar ratio is 1:1 at this wavelength (  (Fig. 7, structure III). These isomeric structures differ by the position of the hydrogen atom H13. In the structure II, H13 is bonded to the oxygen atom O3 from DN and in the structure III -to the oxygen atom of the VO group.
Line 1 in Fig. 5, obtained for the complex derived from V IV at 333 nm, has a slope which is statistically different from unity: 1.22 ± 0.03. There are two explanations of this result: Simultaneous extraction [37] of complexes with 1.
The mobile equilibrium method [33] is a useful tool for determining the composition of complex species with m = n [19,38,39]. The results shown in Fig. 6 (slope of 3 for n = m = 3) demonstrate that the extracted species can be regarded as aggregates made of three subunits with a V IV :TT molar ratio of 1:1. If we assume that these three subunits are identical, e.g. (TT + )[V IV O(HDN − )(DN 2− )], one can write the following equation under the optimum conditions for V IV extraction (Table 1) A parallel process, which is the main process that occurs in the blank, is the interaction between TTC and ND. Based on our experience with similar systems containing tetrazolium salts and diprotic chromogenic reagents H 2 L [42], and the above mentioned indication of aggregation in the blank (a very narrow peak at λ = 325 nm), we can propose the following equation for this process.
It should be mentioned that in contrast to the previously reported system V V -DN-INT [11] the spectral characteristics of the blank are stable in time (Fig. S9). This indicates that (i) x can be regarded as a constant for the given reaction conditions and (ii) there is no oxidation-reduction reaction between DN and TTC. Having in mind eq. 3, we can rewrite eq. 2 in the following way: As noted above, there is a difference in the extraction mechanism when the initial oxidation state of vanadium is V (and the concentration of the DN is not very high). In this case, the complex formation passes through the formation of an extractable V V complex (TT + )[V V O 2 (DN)] (step 1). The next step, described by Kustin et al [30] for similar catecholic compounds in aqueous solutions, is the destruction of the complex due to changes in DN which accompany its oxidation by V V (step 2; rate determining step). The last step 3 is the formation of the final V IV -DN-TT complex. In our conditions, the slow step 2 occurs in the organic phase. Consequently, it can affect the processes of aggregation therein and the formula of the final structure should be [(TT + )[V IV O(HDN − )(DN 2− )]] y with y less than 3.

Constant of extraction and fraction extracted
We calculated the conditional constant of extraction K ex by the Likussar-Boltz method [43] (Fig. 8) extended by us with equation 5 which is valid for M m L n species with m = n = 3 (where M and L are the cation and anion, respectively).
The fraction extracted was calculated by the formula E% = 100 × A 1 /A 3 , where A 1 and A 3 are the absorbances measured for single and triple extractions at the optimum conditions (Table 1) in equal final volumes of 25 cm 3 as described previously [11]. The value obtained was E = (98 ± 2) % (four replicate experiments).

Conclusions
Vanadium forms well chloroform-extractable species with DN and TTC. They are stable and intensively colored.
When the concentration of DN is high, the oxidation state of vanadium in the final complex is IV independently of the initial oxidation state of vanadium -IV or V. In this case the extracted species are aggregates of three 1:1:2 (TT-V IV -DN) complexes. There is a difference in the extraction mechanism for V V and V IV when the DN concentration is low, since V V is quickly extracted under the form of a 1:1:1 complex, (TT + )[V V O 2 (DN)]. As a result, the oxidationreduction process between V V and DN (which is followed