Extraction-Chromogenic Systems Containing Iron ( III ) , 4-( 2-Thiazolylazo ) Resorcinol and Ditetrazolium Salts

Liquid-liquid extraction systems containing FeIII, 4-(2-thiazolylazo)resorcinol (TAR), and a ditetrazolium salt (DTS: Neotetrazolium chlortide, Blue Tetrazolium chloride, or Nitro Blue Tetrazolium chloride), water and chloroform were investigated. The optimum conditions for iron extraction, some equilibrium constants (association constants, distribution constants, and extraction constants), and characteristics (molar absorptivities, Sandell’s sensitivities, limits of detection and quantification) were found. The extracted species are ternary complexes with a general formula of (DT2+)[FeII(TAR) 2 ]. In this formula the metal ion is in oxidation state II, the azo dye is in doubly deprotonated form (TAR2"), and the ditetrazolium cation (DT) has a charge of 2+.


MATERIALS AND METHODS
A stock iron(III) solution (1 mg mL -1 ; 1 L) was prepared by dissolving 8.6350 g of FeNH 4 (SO 4 ) 2 .12H 2 O (99.1%; Reanal, Hungry)in water containing 5 mL of conc.H 2 SO 4 .Working solutions (50 µg mL -1 ) were prepared every day by suitable dilution of the stock solution with 0.01 mol L -1 H 2 SO 4 (27, 28).TAR (97%), NTC ("for microbiology" grade), BTC ("formicrobiology" grade), and NBT (98%) were purchased from Sigma-Aldrich Chemie GmbH.Aqueous solutions of the mentioned reagents were prepared: C TAR =3×10 -3 mol L -1 , and C DTS =2×10 -3 mol L - 1 .The chloroform was additionally distilled before use.The acidity of the aqueous medium was set by the addition of buffer solution, prepared by mixing 2 mol L - 1 aqueous solutions of CH 3 COOH and NH 4 OH.The pH was checked by a Hanna HI 83140 pH meter.A Camspec M507 spectrophotometer (United Kingdom), equipped with 0.5cm path-length cells, was used for reading the absorbance of the extracts.A Microwave Plasma -Atomic Emission Spectrometer Agilent 4200 MP-AES was employed for determining the iron content in residual aqueous phase obtained after extraction.

Procedure for establishing the optimum conditions
Aliquots of Fe(III) solution, TAR solution (up to 2.0 mL), DTS solution (up to 2.5 mL) and buffer solution (3 mL; pH ranging from 4.4 to 8.5) were introduced into 125-mL separatory funnels.The resulting mixtures were diluted with distilled water to a total volume of 10 mL. 10 mL of chloroform were added and the funnels were shaken for a fixed time (up to 5.0 min).A portion of each organic extract was transferred through a filter paper into a cell and the absorbance was read against a blank by the UV-VIS spectrophotometer.

Procedure for determining the fractions extracted and constants of distribution
The fractions extracted (E%) and constants of distribution (K D ) were calculated by comparison of the iron content (determined by MP-AES at wavelengths 371.993, 373.486,and 302.064 nm) in the aqueous phase before and after the extraction.The initial concentration of Fe III in the aqueous phase was 2.24×10 -5 mol L -1 and the extraction was performed under the optimum conditions (Table 1; pH=6.3).

Absorption spectra and effect of pH
Absorption spectra of the chloroform extracted ternary complexes are shown in Fig. 1.All three Fe-TAR-DTS complexes have maxima at ca. 618-620 nm (curves 1-3).Additional maxima can be observed in the spectra when the extraction is performed at relatively low pH: e.g.pH 4.1 for the systems with NTC and BTC (curves 1', 2'), or pH 5.7 for the system with NBT (curve 3').These maxima are the most pronounced for the NBT complex.This fact can be explained with the lowest stability and molar absorptivity of (NBT 2+ )(TAR -) 2 21 -the salt-like compound which determines the absorption of the blank: Log  NBT-TAR = 8.3,  max =1.95×10 4 L mol -1 cm -1 .The corresponding values for (NTC + ) 2 (TAR -) 2 and (BT 2+ )(TAR -) 2 are higher 21 .Hence, the resultant absorption A l = A Fe-TAR-DTS -A TAR-DTS is strongly affected by the A TAR-DTS , especially when TAR is predominantly in anionic form (pH>pK TAR ;pK TAR =6.23 38 ) and < 550 nm.As a result, the peaks of the ternary Fe-TAR-DTS complexes in this spectral region disappear (curves 1-3).At pH>pK TAR , the maxima of the ternary Fe-TAR-DTS complexes appears at 618 or 620 nm (Table 1).The absorbance of the blank is negligible in this spectral region (Figure 2, curves 1'-3') and the obtained spectrophotometric results are stable in time and repeatable.Hence, these maxima were used for our further spectrophotometric measurements.
The effect of pH on the absorbance is shown in Fig. 2. It can be assumed that the shape of the pH- curves is governed by two factors: (i) doubly protonated TAR speciespredominate at low pH values; and (ii) Iron hydrolysis 39 exert noticeable effects on the complex formation at pH higher than pH opt .

Effect of reagents concentration
The effect of TAR and DTS concentrations on the absorbance is shown in Fig. 3 and Fig. 4, respectively.The optimum reagents concentrations deduced from these figures are shown in Table 1.

Effect of shaking time
The extraction equilibria in the Fe-TAR-DTS systems are reached for ca.90 seconds.We shook the funnels for 2 min in our further experiments.

Composition, molecular formulae and equilibrium constants
The molar TAR-to-Fe and DTS-to-Fe ratios were determined from the experimental results presented in Fig. 3 and Fig. 4, respectively.Two different methods were used: the mobile equilibrium method 40 (Table 3) and the straight-line method of Asmus 41 .The results show that the molar ratio between the components of the ternary complex -Fe, TAR and DTS -is 1:2:1.This molar ratio, along with the spectral characteristics (which are similar to these for the Fe-TAR-MTS complexes 27 ), give us grounds to assume that the iron ion has an oxidation state of II,TAR is in doubly deprotonated form (TAR 2-), and DT z+ has a charge of 2 (DT 2+ ).
If we assume that the Fe III reduction takes place in the aqueous phase, we can write the following equations of ion-association (1)  The constants of association () describing eq.1were determined by several methods: the Holme-Langmyhr method 42 , the Harvey-Manning method 43 , and the mobile equilibrium method 40 .The constants of distribution (K D ) describing eq. 2 were calculated by the formula K D =E/(1-E), where E is determined by MP-AES at the optimum extraction conditions (as described above).The attempts to determine these constants spectrophotometrically by comparison of the absorbance values obtained after single (A 1 ) and triple (A 3 ) extractions in equal final volumes [26][27][28] were unsuccessful, since A 1 was higher than A 3 .This anomaly was greater than that observed in 27 for the Fe III -TAR-TTC system (TTC=2,3,5-triphenyl-2Htetrazolium chloride).It can be attributed to the formation of substances with different colour properties during the oxidation-reduction process involving iron or during the dilution of the organic extracts with chloroform.
The extraction constants (K ex ) were calculated by the formula K ex =K D × 44 .All experiments were performed atroom temperature of ca.22°C.The results are given in Table 2.

Beer's law and analytical characteristics
The adherence to Beer's law for each Fe III -TAR-DTS-water-chloroform system was examined under the optimum extraction-spectrophotometric conditions.Then the molar absorptivities, Sandell's sensitivities, limits of detection (LOD) and limits of quantification (LOQ) were calculated.The results are given in Table 4.

CONCLUSION
Cations deriving from DTSs were studied for the first time as components of ion-association systems containingiron.These cations form chloroform-extractable ion-pairs with the anionic Fe-TAR chelate, which can be represented with the formula [Fe II (TAR 2-) 2 ] 2-, independently of the fact that the initial oxidation state of iron was III.The calculated equilibrium constants and characteristics suggest that the DTS-TAR couples canbe applied for iron extraction.However, the greatest potential in this field has NBT which form the most stable and extractable complex.