Anion Recognition : Fabrication of a Highly Selective and Sensitive HPO 4 2-PVC Sensor Based on a Oxo-Molybdenum Methyl-Salen

Um sensor de monohidrogenofosfato (MHP) altamente seletivo e sensível foi obtido, através da preparação de uma membrana de PVC, contendo oxo-molibdênio com ligantes derivados do salen (MS) como carregador neutro, brometo de hexadeciltrimetil amônio (HTAB) como aditivo catiônico e benzilacetato (BA) como solvente plastificante mediador. A membrana sensora exibe uma resposta linear no intervalo de concentração de 1,0 × 10 a 4,0 × 10 mol L de MHP. O eletrodo mostra uma inclinação Nernstiana de –28,6±0,3 mV década em pH 8,5. O sensor proposto também exibe um tempo de resposta menor que 15s. O limite de detecção do sensor é 2,0 × 10 mol L (~20 ng mL), e pode ser usado por um período superior a oito semanas, sem alterações significantes na resposta. A seletividade do sensor, com relação a outros ânions comuns, orgânicos e inorgânicos (por exemplo, cloreto, brometo, iodeto, nitrato, sulfito, sulfato, carbonato, perclorato, cianeto, tiocianato, diidrogenofosfato, pirofosfato de tetrasódio e íons tripolifosfato de sódio) é excelente. A utilidade prática do sensor foi demonstrada pelo uso como eletrodo indicador na titulação potenciométrica de MHP com íons Ba e pela determinação direta de MHP em amostras de fertilizantes granular e líquido e em amostras de água de descarte.


Introduction
Phosphate levels in freshwaters have increased in the past 50 years, which may have a negative effect on aquatic ecology and water quality. 1 Recent studies show that leaching of PO 4 2-as small as 10 μg L -1 from agricultural land can contribute to eutrophication. 2 Thus, due to the increasing use of phosphate fertilizers in agricultural industries, monitoring of phosphate ions is very important.
The main methods for determination of phosphate ions in real samples are ion chromatography and spectrophotometric methods such as molybdenum blue, complex of molybdophosphate with basic dye compounds and indirect lanthanum chloranilate methods.
The development of novel sensors for fast, accurate, sensitive and selective determination of monohydrogen phosphate (MHP) ion in various species is of great interest due to its existence in clinical, environmental, agricultural and medical systems.][18][19][20][21][22][23][24][25][26] In this report we introduce the synthesis of a oxomolybdenum methyl-salen and its application in fabrication of a highly selective and sensitive MHP membrane sensors for fast monitoring of MHP in various granular fertilizers as well as waste water of fertilizer industry.

Reagents
Reagent grade benzyl acetate (BA), dibutyl phthalate (DBP), nitrobenzene (NB), acetophenone (AP), hexadecyltrimethylammonium bromide (HTAB), tetrahydrofuran (THF), and high relative molecular weight PVC were purchased from Merck chemical company and used as received.The sodium or potassium salts of the anions, NH 4 VO 3 , (NH 4 ) 6 Mo 7 O 24 .4H 2 O, KH 2 PO 4 used (all from Merck or Aldrich) were of the highest purity available, and used without any further purification except for vacuum drying over P 2 O 5 .Doubly distilled de-ionized water was used throughout.

Synthesis of MS
The Schiff-base, bis(2-hydroxy-acetophenone)ethylendimiimine, was prepared according to the literature through the well known, as follows: 1,2-diaminoethane (0.3 g, 5 mmol) was dissolved in 40 mL ethanol and then transferred into a 250 mL three-necked flask.Under reflux, 1.36g (10 mmol) of 2-hydroxy-acetophenone in 40 mL ethanol was added dropwise to the flask.The mixture was stirred for 50 min, under reflux, and the cooled to room temperature.The solid product was filtered, and the product was recrystallized from chloroform.

Preparation of electrodes
Membrane solutions were prepared by thoroughly dissolving 7.0 mg of the oxo-molybdenum methyl-salen (MS), 58.0 mg of BA, 33.0 mg of PVC and 2.0 mg of HTAB in 3 mL of fresh THF.The resulting solution was evaporated slowly until an oily mixture was obtained.A Pyrex tube (5 mm o.d) was dipped into the mixture for about 10 s so that a nontransparent membrane of 0.3 mm thickness was formed.The tube was then pulled out from the mixture, and kept at room temperature for 10 h.The tube was then filled with an internal solution (1.0 × 10 -3 mol L -1 MHP).The electrode was finally conditioned for about 12 h by soaking in a 1.0 × 10 -2 mol L -1 MHP solution.A silver/silver-chloride electrode was used as the internal reference electrode.

Determination of orthophosphate by colorimetric method
The molybdovanadophosphoric acid method was used as colorimetric method in this work.According to this method, addition of molybdate to an acidic solution containing orthophosphate and vanadate, results in the formation of yellow-orange molybdovanadophosphoric acid having the P:V:Mo ratio of 1:1:11.Ammonium metavanadate, 0.25% solution.Dissolve 1.25 g of NH 4 VO 3 in 250 mL of hot water.Cool the solution and add 10 mL of conc.HNO 3 .Allow the solution to stand overnight, filter (if necessary), and dilute with water to 500 mL.Store solution in a polyethylene container.Ammonium molybdate, 5% solution.Dissolve in 250 mL of water (at about 50 °C) 25 g of (NH 4 ) 6 Mo 7 O 24 .4H 2 O. Allow the solution to stand overnight, filter (if necessary), dilute with water to 500 mL, and store in a polyethylene container.Standard phosphorus (V) solution (KH 2 PO 4 ): 1 mg mL -1 .Procedure 10 mL of HCl 10% (m/m), is added to the 100-500 mg of each sample.Then the solution is filtered after 10 min shaking.The resulted solution was diluted to 250 mL.To the slightly acidic sample solution containing not more than 0.3 mg of P, add successively 2.5 mL of nitric acid (1+1), 2.5 mL of the vanadate solution, and 2.5 mL of the molybdate solution, mixing the solution after the addition of each reagent.Dilute the solution to volume with water in a 25 mL standard flask.After 30 min, measure the absorbance at 400 nm against a reagent blank solution. 28

UV-Vis study
The preferential response towards MHP is believed to be associated with the coordination of MHP with the central metal of the ion-carrier MS.With UV/Vis spectra, as illustrated in Figure 2, it was possible to distinguish the interactions between the central metal and MHP.The substantial increases in the absorbance at 292.5 nm after the contact of the carried solution with a monohydrate phosphate-containing phase suggested that the absorbing species had increased in size and axial coordination was thought to take place.At the same time, the effects of other anions such as chloride, bromide, sulfate, perclorate on the spectrum of the carrier were investigated and no detectable changes in the UV/Vis spectra were noted.These results revealed that the oxo-molybdenum methylsalen has especial tendency to the MHP ions respect with other common anions.The obtained results denoted that the mechanism of the response of the membrane sensor to MHP is based on coordination of MHP ion to oxomolebdenum and its rapid exchanges with the solution containing MHP ions.
Thus, in the next experiments MS was used as a suitable active component in the fabrication of a number of PVC-membrane sensors for a wide variety of common inorganic anions including chloride, bromide, iodide, nitrate, sulfite, sulfate, carbonate, perclorate, cyanide, thiocyanate, dihydrogen phosphate, tetrasodium pyrophosphate, and sodium tripoly phosphate ions, the potential responses of which are shown in Figure 3.As can be seen from Figure 3, while the slopes of the linear parts of the potential responses of the sensors for all other anions tested, are much lower than those expected by the Nernst equation, the resulting MHP potential response shows a near-Nernstian behavior.

Membrane characteristics
The properties of the membranes, namely sensitivity, detection limit, response time, lifetime, selectivity, and chemical stability depend on the ratio of electro-active phase, and the binder materials.Membranes with different compositions were prepared and preliminary studies revealed that a membrane having a composition of 7.0: 2.0: 33.0: 58.0 of MS: HTAB: PVC: BA gave the best response characteristics (Table 1, membrane F).
The influences of the solvent mediators (AP, DBP, BA and NB), ionic additive (HTAB), and amount of  the active component on the performance of the membranes were also considered, and the results are summarized in Table 1.As seen, these data reveal that four different plasticizers used, have almost the same results if the optimum composition is used.However, because BA is a low polar solvent (in comparison with NB with high polarity and BA, and DBP with low polarity), leading to the extraction of MHP and interfering ions within the relatively lower lipophile anions such as Cl -, Br -, and NO 3 -, which may have positive effects on the selectivity behavior of the sensor, BA was chosen as the solvent mediator.This may seem to lead to the poorer extraction of MHP ion, having a relatively high charge density, but this seemed to be compensated by the selective complexation of MS with the MHP ions.This way, not only was the slope of the sensor response maintained, but its selectivity pattern was also improved.
The optimization of permselectivity of membrane sensors is known to be highly dependent on the incorporation of additional membrane component.In fact, it has been demonstrated that, the presence of lipophilic positively charged additives improves the potentiometric behavior of certain selective sensors not only by reducing the ohmic resistance, 23,24 improving the response behavior, and selectivity, 25,26 but also, in cases where the extraction capability of the ionophore is poor, by enhancing the sensitivity of the membrane electrodes.Moreover, the additives may catalyze the exchange kinetics at the sample-membrane interface. 29able 1 shows that if 2% of HTAB is present as a suitable additive, the slope of the proposed sensor increases from -15.3 (membrane C) to -28.6 mV decade -1 (membrane F).

Working concentration range and slope
The potential response of the membranes as a function of MHP activity is shown in Figure 4.It is seen that membrane F shows a wide working concentration range (1.0 × l0 -1 -4.0 × 10 -7 mol L -1 ) with a detection limit of 2 × 10 -7 mol L -1 (~20 ng mL -1 ), and a Nernstian slope of -28.6±0.3 mV per decade of activity.

Lifetime and reproducibility of the sensor
The membranes were used over a period of eight weeks without showing any significant changes in the slope and detection limit of the sensor.During usage, the membranes were stored in 0.01 mol L -1 MHP solution and were reequilibrated with 0.1 mol L -1 MHP solution whenever any drift in potentials was observed (Table 2).Repeated monitoring of potentials (20 measurements) at 1.0 × 10 -3 mol L -1 concentration gave a standard deviation of 0.4 mV.

Dynamic response time
Dynamic response time is one of the important factors for any ion-selective electrode.In this study, the practical response time was recorded by changing the MHP concentration in solution; over a concentration range 1.0 × 10 -1 mol L -1 to 1.0 × 10 -6 mol L -1 .The actual potential versus time traces is shown in Figure 5.As can be seen from Figure  5, in the whole concentration range, the electrode reaches its equilibrium response in a very short time (<15 s).This is most probably, due to the fast exchange kinetics of complexation-decomplexation of MHP ion with the MS at the test solution-membrane interface.

Effect of pH
The pH dependence of the MHP membrane sensor was tested over a pH range of 4.0-10.0 in a 1.0 × 10 "4 mol L -1 MHP solution, and the results are illustrated in Figure 6.As it can be seen, the potential remains fairly constant in the pH range of 8.5-9.2.Beyond this range, a gradual change in the potential was detected.The observed potential drift at the higher and lower pH values could be due to the decreasing of the concentration of MHP (formation of H 2 PO 4-and PO 4 3-at lower and higher pH, respectively).

Potentiometric selectivity
The influence of interfering ions on the response behavior of ion-selective membrane electrodes is usually described in terms of selectivity coefficients.The potentiometric selectivity coefficients of the MHP membrane sensor were evaluated by the matched potential method (MPM) and separation solution method (SSM). 30he resulting values of the selectivity coefficients are given in Table 3.As shown in Table 3, for all common anions tested, the selectivity coefficients are in the order of 1.0 × 10 -4 or smaller, which seems to indicate negligible interferences in the performance of the electrode assembly.Such remarkable selectivity of the MHP membrane sensor over other common anions, reflects the high affinity of the MS used as ion carrier toward the MHP ions.
Table 4, compares the response time, detection limit, linearity concentration range and major interfering ions (log K < -4) of the proposed sensor with those reported for previously reported MHP membrane sensors.As it is seen, the sensor in the case of the response time and detection limit is superior to the best previously reported MHP ion selective membrane electrodes. 12,16
To assess the practical applicability of the sensor in real samples an attempt was made to determine MHP in the three phosphate fertilizer samples, and the results are given in Tables 5, 6 and 7 (for mixed nitrogen phosphate potassium fertilizer (NPK), triple super phosphate (TSP), and single super phosphate (SSP), respectively).As it is seen, the MHP contents obtained from triplicate measurements were found to be in satisfactory agreement with those obtained by standard method (colorimetry). 28he monohydrogen phosphate sensor was used for the direct monitoring of phosphate ions in the waste water samples of monocalcium phosphate industry (Faridshimi Co. Hashtgerd, Iran).2.0 mL of each sample was taken and dissolved in distilled water in a 100 mL volumetric flask.To this solution 5.0 mL of 0.1 mol L -1 EDTA (pH of 10.0) was added to the flask and diluted to the mark with distilled water.The concentration of each sample was measured by the monohydrogen phosphate sensor, using the calibration method.The results obtained by the direct potentiometry and spectrophotometry are depicted in Table 8.As can be seen, there is a good correlation between the both methods.

Figure 3 .
Figure 3. Potential responses of different ion-selective electrodes based on MS.

Figure 4 .
Figure 4. Calibration curves of phosphate electrode based on MS.

5 Figure 5 .
Figure 5. Dynamic response time of the potentiometric sensor for different concentration of phosphate.

Figure 6 .
Figure 6.The effect of the pH of the test solution (1.0 × 10 -4 mol L -1 ) on the potential response of the membrane sensor.

Table 1 .
Composition of membrane ingredients

Table 2 .
The lifetime behavior of the monohydrogen phosphate

Table 3 .
Selectivity coefficients of various interfering ions

Table 4 .
The characteristics of the proposed sensor were compared with those of the best previously reported

Table 6 .
28termination of P 2 O 5 in three granular TSP fertilizers using colorimetry28and proposed monohydrogen phosphate sensor based on MS

Table 5 .
28termination of P 2 O 5 in three samples of NPK fertilizers using colorimetry28and proposed monohydrogen phosphate sensor based on MS a Results are based on five measurements.

Table 8 .
Determination of phosphate content in waste water samples using colorimetry and proposed monohydrogen phosphate sensor based on MS a Results are based on five measurements.

Table 7 .
28termination of P 2 O 5 in three granular SSP fertilizer samples using colorimetry28and proposed monohydrogen phosphate sensor based on MS a Results are based on five measurements.