Extractive Spectrophotometric Determination and Theoretical Investigations of Two New Vanadium(V) Complexes

Two new vanadium (V) complexes involving 6-hexyl-4-(2-thiazolylazo)resorcinol (HTAR) and tetrazolium cation were studied. The following commercially available tetrazolium salts were used as the cation source: tetrazolium red (2,3,5-triphenyltetrazol-2-ium;chloride, TTC) and neotetrazolium chloride (2-[4-[4-(3,5-diphenyltetrazol-2-ium-2-yl)phenyl]phenyl]-3,5-diphenyltetrazol-2-ium;dichloride, NTC). The cations (abbreviated as TT+ and NTC+) impart high hydrophobicity to the ternary complexes, allowing vanadium to be easily extracted and preconcentrated in one step. The complexes have different stoichiometry. The V(V)–HTAR–TTC complex dimerizes in the organic phase (chloroform) and can be represented by the formula [(TT+)[VO2(HTAR)]]2. The other complex is monomeric (NTC+)[VO2(HTAR)]. The cation has a +1 charge because one of the two chloride ions remains undissociated: NTC+ = (NT2+Cl−)+. The ground-state equilibrium geometries of the constituent cations and final complexes were optimized at the B3LYP and HF levels of theory. The dimer [(TT+)[VO2(HTAR)]]2 is more suitable for practical applications due to its better extraction characteristics and wider pH interval of formation and extraction. It was used for cheap and reliable extraction–spectrophotometric determination of V(V) traces in real samples. The absorption maximum, molar absorptivity coefficient, limit of detection, and linear working range were 549 nm, 5.2 × 104 L mol−1 cm−1, 4.6 ng mL−1, and 0.015–2.0 μg mL−1, respectively.


Introduction
Vanadium can be classified as a dispersed trace element with an average content in the upper continental crust of 97 mg kg −1 [1].It enters the biosphere through natural phenomena (mechanical and chemical rock weathering, volcanism, forest fires, and aeolian processes) and human activity.The main sources of anthropogenic vanadium are ore mining and processing, fossil fuel combustion, agricultural chemicalization, and the production of glass, ceramics, pigments, rubber, redox batteries, plastics, and sulfuric acid [2][3][4][5][6][7].Recently, its anthropogenic enrichment factors (AEFs) have increased: vanadium ranks first among the trace elements in the atmosphere and fourth among the trace elements in the world's rivers [2,5,8].The problem is global [5] and should not be underestimated, as the most common anthropogenic vanadium form-V(V)-is almost as toxic as mercury, arsenic, lead, and cadmium [9].This form resembles phosphorous (V) in chemical behavior [10] and can interfere with important biochemical processes by inhibiting the activity of key enzymes such as phosphatases and kinases [11,12].
To study the LLE of the V(V)-HTAR species in the presence of tetrazolium salt; 2.
To find the ground-state equilibrium geometries of the extracted species using quantum chemical calculations at the B3LYP and HF levels of theory; 3.
To develop a competitive LLE-spectrophotometric method for determining V(V) in real samples.

Absorption Spectra
The spectra of the extracted species are shown in Figure 2. The absorbance of the two complexes (1 and 2) at their absorption maxima (λmax) is practically the same.However, there is a small difference in the position of these maxima: λmax (1) = 549 nm and λmax (2) = 556 nm.The spectral band of the V(V)-HTAR-TTC complex (1) is slightly broader, which may be due to the aggregation of the extracted species [34].
The absorbance of the blank samples (1′ and 2′) at the absorption maxima of the corresponding complexes is low.Although the concentrations of the reagents under the optimal extraction conditions (see below) are higher for the HTAR-TTC system, the absorbance of the blank is lower.This is a prerequisite for achieving better repeatability with this TS.

Absorption Spectra
The spectra of the extracted species are shown in Figure 2. The absorbance of the two complexes (1 and 2) at their absorption maxima (λ max ) is practically the same.However, there is a small difference in the position of these maxima: λ max (1) = 549 nm and λ max (2) = 556 nm.The spectral band of the V(V)-HTAR-TTC complex (1) is slightly broader, which may be due to the aggregation of the extracted species [34].
The absorbance of the blank samples (1 and 2 ) at the absorption maxima of the corresponding complexes is low.Although the concentrations of the reagents under the optimal extraction conditions (see below) are higher for the HTAR-TTC system, the absorbance of the blank is lower.This is a prerequisite for achieving better repeatability with this TS.

Effect of pH and the Amount of Buffer
The effect of pH was studied using a series of ammonium acetate buffer solutions with a known pH (Figure 3).The V(V)-HTAR-TTC complex is maximally extracted in a pH range of 3.9-5.0.The maximum extraction range of the V(V)-HTAR-NTC complex is shorter: 4.4-5.0.In this range, the absorbance of both complexes is the same.
To elucidate the effect of the amount of buffer, a series of experiments were carried out at pH 4.7 (one of the two pH values for which the buffering capacity is maximal [35]).It was found that there was no difference in absorbance in the presence of 1, 2, or 3 mL of the buffer solution.For reasons of economy, all subsequent experiments were performed with 1 mL of the buffer.

Effect of Extraction Time
The effect of extraction time is shown in Figure 4. Two minutes are needed to reach extraction equilibrium in the V(V)-HTAR-TTC system.Equilibrium in the other system is established a little slower (2.5 min).Since the shaking rate is a parameter that is difficult to control and depends on the experimenter, in order to avoid random and systematic errors, it is recommended that the extraction time be extended to 2.5 min and 3.0 min, respectively.

Effect of pH and the Amount of Buffer
The effect of pH was studied using a series of ammonium acetate buffer solutions with a known pH (Figure 3).The V(V)-HTAR-TTC complex is maximally extracted in a pH range of 3.9-5.0.The maximum extraction range of the V(V)-HTAR-NTC complex is shorter: 4.4-5.0.In this range, the absorbance of both complexes is the same.

Effect of pH and the Amount of Buffer
The effect of pH was studied using a series of ammonium acetate buffer solutions with a known pH (Figure 3).The V(V)-HTAR-TTC complex is maximally extracted in a pH range of 3.9-5.0.The maximum extraction range of the V(V)-HTAR-NTC complex is shorter: 4.4-5.0.In this range, the absorbance of both complexes is the same.
To elucidate the effect of the amount of buffer, a series of experiments were carried out at pH 4.7 (one of the two pH values for which the buffering capacity is maximal [35]).It was found that there was no difference in absorbance in the presence of 1, 2, or 3 mL of the buffer solution.For reasons of economy, all subsequent experiments were performed with 1 mL of the buffer.

Effect of Extraction Time
The effect of extraction time is shown in Figure 4. Two minutes are needed to reach extraction equilibrium in the V(V)-HTAR-TTC system.Equilibrium in the other system is established a little slower (2.5 min).Since the shaking rate is a parameter that is difficult to control and depends on the experimenter, in order to avoid random and systematic errors, it is recommended that the extraction time be extended to 2.5 min and 3.0 min, respectively.To elucidate the effect of the amount of buffer, a series of experiments were carried out at pH 4.7 (one of the two pH values for which the buffering capacity is maximal [35]).It was found that there was no difference in absorbance in the presence of 1, 2, or 3 mL of the buffer solution.For reasons of economy, all subsequent experiments were performed with 1 mL of the buffer.

Effect of Extraction Time
The effect of extraction time is shown in Figure 4. Two minutes are needed to reach extraction equilibrium in the V(V)-HTAR-TTC system.Equilibrium in the other system is established a little slower (2.5 min).Since the shaking rate is a parameter that is difficult to control and depends on the experimenter, in order to avoid random and systematic errors, it is recommended that the extraction time be extended to 2.5 min and 3.0 min, respectively.

Effect of HTAR and TS Concentrations
The effect of the HTAR and TS (TTC or NTC) concentrations is shown in Figures 5  and 6, respectively.Saturation is more easily reached in the V(V)-HTAR-NTC system.The optimal reagent concentrations for the two systems, among other optimized parameters, are shown in Table 1.

Effect of HTAR and TS Concentrations
The effect of the HTAR and TS (TTC or NTC) concentrations is shown in Figures 5 and 6, respectively.Saturation is more easily reached in the V(V)-HTAR-NTC system.The optimal reagent concentrations for the two systems, among other optimized parameters, are shown in Table 1.

Effect of HTAR and TS Concentrations
The effect of the HTAR and TS (TTC or NTC) concentrations is shown in Figures 5  and 6, respectively.Saturation is more easily reached in the V(V)-HTAR-NTC system.The optimal reagent concentrations for the two systems, among other optimized parameters, are shown in Table 1.

Effect of HTAR and TS Concentrations
The effect of the HTAR and TS (TTC or NTC) concentrations is shown in Figures 5  and 6, respectively.Saturation is more easily reached in the V(V)-HTAR-NTC system.The optimal reagent concentrations for the two systems, among other optimized parameters, are shown in Table 1.3.0 a Optimization studies conducted at room temperature (22 ± 1 • C), V aq = 10 mL, and V chloroform = 5 mL.b Ammonium acetate buffer (1 mL).

Stoichiometry, Formulas, and Equations
The small differences in the spectra (Figure 2) and optimal extraction conditions (Table 1) indicate possible differences in stoichiometry due to aggregation of the extracted species.Therefore, a variety of methods were used to elucidate the formulas of the complexes (Table 2), including those applicable to compounds of the type A n B m (where n = m > 1) [36,37].
Other methods with more limited capabilities.
Figures 7a and 8a present the results of determining the V(V):HTAR molar ratio in the ternary complexes via the mobile equilibrium method [36].They show that the molar ratios in the two complexes are not the same: 2:2 (for the complex deriving from TTC) and 1:1 (for the complex deriving from NTC).This can be seen from the slopes (n) of the obtained straight lines, which match well with the correct value of m.

Stoichiometry, Formulas, and Equations
The small differences in the spectra (Figure 2) and optimal extraction conditions (Table 1) indicate possible differences in stoichiometry due to aggregation of the extracted species.Therefore, a variety of methods were used to elucidate the formulas of the complexes (Table 2), including those applicable to compounds of the type AnBm (where n = m > 1) [36,37].
a Methods applicable to complexes of the type A2B2.b A method capable of distinguishing A1B1 complexes from AnBn (n > 1) complexes.c Other methods with more limited capabilities.
Figures 7a and 8a present the results of determining the V(V):HTAR molar ratio in the ternary complexes via the mobile equilibrium method [36].They show that the molar ratios in the two complexes are not the same: 2:2 (for the complex deriving from TTC) and 1:1 (for the complex deriving from NTC).This can be seen from the slopes (n) of the obtained straight lines, which match well with the correct value of m.A similar conclusion is reached when studying the TS:V(V) molar ratio.When TS = TTC, the molar ratio is 2:2 (Figure 7b), and when TS = NTC, the molar ratio is 1:1 (Figure 8b).A similar conclusion is reached when studying the TS:V(V) molar ratio.When TS = TTC, the molar ratio is 2:2 (Figure 7b), and when TS = NTC, the molar ratio is 1:1 (Figure 8b).
An additional independent method [37] was used to confirm the stoichiometry in the TTC complex (Figure 9).As can be seen, a straight line is obtained for a complex of type A2B2.It is known from the literature [39,42,43] that Job's method [38] can be used to distinguish 1:1-complexes from AnBn (n > 1) complexes (e.g., A2B2, A3B3, A4B4, etc.).The criterion for distinction is the presence or absence of concavities at the ends of the isomolar curve.Although such a distinction is not always reliable, it can be judged from Figure 10 that the NTC complex is most probably of the A1B1 type (no concavities), and the TTC complex is of the AnBn type (n > 1).If this conclusion is compared with the results presented above obtained via the mobile equilibrium method [36] and the dilution method [37], it can be deduced that nTTC:nV = 2:2.An additional independent method [37] was used to confirm the stoichiometry in the TTC complex (Figure 9).As can be seen, a straight line is obtained for a complex of type A 2 B 2 .A similar conclusion is reached when studying the TS:V(V) molar ratio.When TS = TTC, the molar ratio is 2:2 (Figure 7b), and when TS = NTC, the molar ratio is 1:1 (Figure 8b).
An additional independent method [37] was used to confirm the stoichiometry in the TTC complex (Figure 9).As can be seen, a straight line is obtained for a complex of type A2B2.It is known from the literature [39,42,43] that Job's method [38] can be used to distinguish 1:1-complexes from AnBn (n > 1) complexes (e.g., A2B2, A3B3, A4B4, etc.).The criterion for distinction is the presence or absence of concavities at the ends of the isomolar curve.Although such a distinction is not always reliable, it can be judged from Figure 10 that the NTC complex is most probably of the A1B1 type (no concavities), and the TTC complex is of the AnBn type (n > 1).If this conclusion is compared with the results presented above obtained via the mobile equilibrium method [36] and the dilution method [37], it can be deduced that nTTC:nV = 2:2.It is known from the literature [39,42,43] that Job's method [38] can be used to distinguish 1:1-complexes from A n B n (n > 1) complexes (e.g., A 2 B 2 , A 3 B 3 , A 4 B 4 , etc.).The criterion for distinction is the presence or absence of concavities at the ends of the isomolar curve.Although such a distinction is not always reliable, it can be judged from Figure 10 that the NTC complex is most probably of the A 1 B 1 type (no concavities), and the TTC complex is of the A n B n type (n > 1).If this conclusion is compared with the results presented above obtained via the mobile equilibrium method [36] and the dilution method [37], it can be deduced that n TTC :n V = 2:2.
The performed experiments give reason to assume that the complex obtained from TTC is an aggregate obtained in the organic phase via the dimerization of two 1:1:1 complexes represented by the formula (TT + )[VO 2 (HTAR)].Such dimerization was reported for similar extraction systems containing V(V) and 5-methyl-4-(2-thiazolylazo)resorcinol [44,45].
The obtained values, along with the values of distribution ratios (D) and fractions extracted (E), are given in Table 3. Equations of complex formation and extraction, based on information for the state of V(V) [13] and HTAR (H 2 L) [47,48] at the optimum pH value, are as follows:
The obtained values, along with the values of distribution ratios (D) and fractions extracted (E), are given in Table 3.

Ground-State Equilibrium Geometries of the Cations
The optimized ground-state equilibrium geometries of the cations (TT + and NTC + ) are shown in Figure 11a .The lack of significant differences is consistent with the conclusion that the internal dimensions "are largely insensitive to the local environment" [30,52].As seen in Figure 11b, the chloride ion is located near the right-hand tetrazolium ring (closest to N24).It is pincered by two hydrogen atoms (H(64) and H( 61)) of the substituent groups.

Ground-State Equilibrium Geometries of the Cations
The optimized ground-state equilibrium geometries of the cations (TT + and NTC + ) are shown in Figure 11а .The lack of significant differences is consistent with the conclusion that the internal dimensions "are largely insensitive to the local environment" [30,52].As seen in Figure 11b, the chloride ion is located near the right-hand tetrazolium ring (closest to N24).It is pincered by two hydrogen atoms (H( 64) and H( 61)) of the substituent groups.

Ground-State Equilibrium Geometries of the Complexes
The next step was to correctly pair each of the two cationic structures (Figure 11a,b) with the anion [VO 2 (HTAR)] − optimized in a previous paper [23] (Figure 11c).Two different NTC + -[VO 2 (HTAR)] − structures were constructed and fully optimized at the HF/3-21G theoretical level (Figure 12a,b).Better stacking between the counterions was observed in Structure 1 (Figure 12a).Its energy is 15 kJ mol −1 lower than that of Structure 2.
The structure of [(TT + )[VO 2 (HTAR)]] 2 was optimized in two steps.In the first step, the parent ions were assembled in two different ways, as shown in Figure 13a,b.In structure M1, the O(23) of the VO 2 group is located near the tetrazolium ring.In the more stable structure M2, both oxygen atoms from the VO 2 group are involved in additional interactions.Oxygen( 24) is near the tetrazolium ring, and O (23) participates in a hydrogen bond C(50)-H(65)•••O (23).This structure has 20 kJ mol −1 lower energy.
Figure 14 shows structures of dimers obtained in the second step-pairing of monomers.The dimeric structure D1 (a) is derived from two structures, M1, the dimeric structure D2 (b) is derived from two structures, M2, and the dimeric structure D3 (c), is derived from one structure, M1, and one M2.
The structure of [(TT + )[VO2(HTAR)]]2 was optimized in two steps.In the first step, the parent ions were assembled in two different ways, as shown in Figure 13a,b.In structure М1, the O( 23) of the VO2 group is located near the tetrazolium ring.In the more stable structure М2, both oxygen atoms from the VO2 group are involved in additional interactions.Oxygen( 24) is near the tetrazolium ring, and O( 23    The structure D2 (Figure 14b) has 46 kJ mol −1 lower energy than the energy of D1.The change of ΔG°298 for the formation of the dimer from two M2 fragments is 18 kJ mol −1, and the heat effect is ΔH°298 = −44 kJ mol −1 .

Analytical Characteristics and Application
The relationships between the measured absorbance and the V(V) concentration in the aqueous phase were investigated under optimal conditions (see Table 1).The linear regression equations and some associated parameters related to the application of these The dimer complex has 53 kJ mol −1 higher energy than D2 and only 7 kJ mol −1 than D1.In other words, the energy analysis of the three V(V)-HTAR-TT complexes led to the following stability series: D2 > D1 > D3.

Analytical Characteristics and Application
The relationships between the measured absorbance and the V(V) concentration in the aqueous phase were investigated under optimal conditions (see Table 1).The linear regression equations and some associated parameters related to the application of these systems for the determination of V(V) are included in Table 4. Advantages of the TTC-HTAR system are a wider linear range, a lower LOD, a lower cost of TTC (in comparison to NTC), and the tolerance of higher amounts of side ions such as Al(III), Ba(II), Br − , Ca(II), Cl − , Cr(VI), I − , Mo(VI), NO 3 − , and Re(VII) (Table 5).Advantages of the NTC-HTAR system are the lower reagents concentrations (Table 1) and higher tolerable levels of Zn(II), Cd(II), Hg(II), Mg(II), Pb(II), F − , and HPO 4 2− (Table 5).Table 5.Effect of foreign ions on determining 5 µg vanadium(V).

Foreign Ion (FI) Added
Added Salt Formula HTAR-TTC System HTAR-NTC System The HTAR-TTC system was used to determine V(V) in real samples such as vanadiumdepleted catalysts from sulfuric acid production and pharmaceuticals.The results of the catalyst analysis are shown in Table 6.They are statistically indistinguishable from those obtained via an alternative spectrophotometric method [53].The results of V(V) determination in spiked pharmaceutical samples are displayed in Table 7.The recoveries were in the range of 97.2-105%, with relative standard deviations from 2.0% to 4.6%.

Instrumentation
An Ultrospec 3300 pro scanning spectrophotometer (Garforth, UK), equipped with 10 mm path-length quartz (or glass) cuvettes, was used during the work.The pH measurements were made with a WTW InoLab 7110 pH meter (Weilheim, Germany) with a glass electrode.

Samples
Pharmaceuticals were purchased from a local pharmacy: (i) Marimer inhalation (2.2% hypertonic seawater); (ii) Sterimar for nasal hygiene (a 100% natural, purified seawaterbased nasal spray); and (iii) a solution of 0.9% NaCl for intravenous infusion.Aliquots of 5 mL of these solutions were subjected to the extraction procedure.
Samples of spent silica-supported catalysts (from the sulfuric acid production) were provided by KCM SA, Plovdiv (Bulgaria).They were ground in a mortar and prepared for analysis according to a known procedure [59].Aliquots of 0.5 mL of the obtained solutions were used for the analysis.

Procedures for Optimization and Determination of Extraction Constants
The following solutions were mixed in a 125 mL separatory funnel: V(V), ammonium acetate buffer, HTAR, and TS (TTC or NTC).Water was added to a total volume of 10 mL.The volume of chloroform was 5 mL (in the optimization experiments) or 10 mL (in the determination of the extraction constant; Figures 7-10).After pouring the organic solvent, the funnel was stoppered and shaken for a fixed time.Part of the resulting chloroform extract was filtered through filter paper and transferred to the cuvette.Absorbance was measured against a simultaneously prepared blank containing no V(V).

Procedure for Determination of Distribution Ratios and Fractions Extracted
The liquid-liquid distribution ratios for each of the systems were calculated after considering the absorbance in single extraction (A 1 ) and triple extraction (A 3 ) experiments: D = A 1 /(A 3 − A 1 ).The single extraction and the first step of the triple extraction were performed with 10 mL of chloroform under the optimal extraction conditions (Table 1).The organic layers were transferred into 25 mL volumetric flasks.The flask for the single extraction was brought to the mark with chloroform.The second and third steps of the triple extraction were carried out by adding 7 mL of chloroform to the aqueous phase of the previous step.The organic extracts were combined in the second 25 mL volumetric flask, and chloroform was added to the mark.Absorbances were measured against corresponding blanks.

Procedure for Determination of Vanadium(V) with HTAR and TTC
An aliquot of the analyzed solution (containing 0.015-2.0µg mL −1 of V) was transferred in a 125 mL separatory funnel.Solutions of the buffer (1 mL, pH 4.7), HTAR (0.4 mL, 2 × 10 −3 mol L −1 ), and TTC (0.8 mL, 3 × 10 −3 mol L −1 ) were added, and the sample was diluted with water until a total volume of 10 mL.Then, 5 mL of chloroform was added, and the mixture was shaken for 2.5 min.After the phase separation, a portion of the chloroform extract was poured into the cuvette, and the absorbance was measured at 549 nm against a blank.The unknown V(V) concentration was calculated from a calibration plot prepared using the same procedure.

Procedure for Determination of Vanadium(V) with HTAR and NTC
An aliquot of the analyzed solution (containing 0.023-1.1 µg mL −1 of V) was transferred in a 125 mL separatory funnel.Solutions of the buffer (1 mL, pH 4.7), HTAR (0.2 mL, 2 × 10 −3 mol L −1 ), and NTC (0.7 mL, 2 × 10 −3 mol L −1 ) were added, and the sample was diluted with water until a total volume of 10 mL.Then, 5 mL of chloroform was added, and the mixture was shaken for 3.0 min.After the phase separation, a portion of the chloroform extract was poured into the cuvette, and the absorbance was measured at 556 nm against a blank.The unknown V(V) concentration was calculated from a calibration plot prepared using the same procedure.

Theoretical
The ground-state equilibrium geometries of the cations, TT + and NTC + , were optimized at the B3LYP/6-311++G** and B3LYP/6-31+G* levels of theory, respectively.Each of these cations was then paired in different ways with the anionic complex [VO 2 (HTAR)] − studied in a previous work [23] as described above.
The NTC + and [VO 2 (HTAR)] − counterions were assembled in two ways and optimized at the HF/3-21G level of theory.Furthermore, the thermodynamic characteristics of the obtained structures were calculated and compared.
The TT + and [VO 2 (HTAR)] − counterions were also assembled in two different ways and optimized at the HF/3-21G level of theory to obtain monomeric structures (M1 and M2) of the type (TT + )[VO 2 (HTAR)].These structures were combined into three dimers (M1-M1, M2-M2, and M1-M2).Optimization was performed at the HF/3-21G level of theory, and the stability of the obtained species was evaluated.
All calculations were performed for the gas phase using the GAUSSIAN 16 program package [63].The ChemCraft program (https://chemcraftprog.com (accessed on 13 September 2023)) was used for the visualization of the computed results [64].

Figure 14
Figure 14 shows structures of dimers obtained in the second step-pairing of monomers.The dimeric structure D1 (a) is derived from two structures, M1, the dimeric structure D2 (b) is derived from two structures, M2, and the dimeric structure D3 (c), is derived from one structure, M1, and one M2.The formation of D1 from two M1 structures is attended with а change of the Gibbs

Table 1 .
Optimum conditions a .

Table 2 .
Molar ratios in the ternary V(V)-HTAR-TS complexes obtained via different methods.

Table 1 .
Optimum conditions a .

Table 2 .
Molar ratios in the ternary V(V)-HTAR-TS complexes obtained via different methods.

Table 4 .
Characteristics concerning the application of the two extraction-chromogenic systems for determining V(V).

Table 5 .
Cont.Higher FI:V(V) mass ratio not studied.b Masked by 10 mg F − . a

Table 7 .
Determination a of vanadium(V) in pharmaceutical samples.
a Three replicate analyses (mean ± standard deviation).

Table 8 .
Comparison with other spectrophotometric procedures.