Construction of an optical sensor for molybdenum determination based on a new ionophore immobilized on a polymer membrane

2.1 Instrumentation

An Orion research model 601 A/digital analyzer pH meter was employed to investigate he pH of solutions. A Hamilton syringe (10 μL) was used to deliver small volumes of reagent metal into the cell. UV–vis spectrophoto-meter model V 53 from JASCO (Tokyo, Japan) was achieved for assessing the spectra and the absorbance assessments. A Perkin-Elmer model 5300 DV; ICP-AES (Waltham, MA, USA) was applied for all ICP-AES measurements. The operating factors were set as received by the manufacturer. The absorbance assessments were set by mounting the optical membrane sensors samples inside a quartz cuvette. The absorbance of the optical membrane sensor samples was detected in respect to air and a blank optode sample.

2.2 Reagents

Except for vacuum drying, all reagents from Merck (Dermasdat, Germany) were analytical reagent quality and used as purchased, with no additional purification. In the spectrophotometric solution investigations, sodium tetraphenylborate (NaTPB) was applied as an ionic strength stabilizer and an additive (in the membranes). High molecular weight poly vinyl chloride (PVC), dibutyl phthalate (DBP), and newly distilled tetrahydrofuran (THF) were utilized to prepare the membranes. The metal ions that were evaluated were the nitrate salts of the metal ions. All the solutions were prepared using reagent grade substances and doubly distilled water. Standard stock solution of Mo⁶⁺ (100 µg/mL) was prepared by dissolving of 0.0184 g of (NH₄)₆Mo₂₄·4H₂O (Merck) in bi-distilled water and diluted to 100 mL in a standard flask and subsequent standardization by EDTA titration. Analar grade or comparable grade water-soluble salts were used to make a large number of inorganic ions. Special dissolve procedures were used for insoluble compounds. Throughout the experiment, double-distilled water was supplied.

1-(2-Benzothiazolylazo)-2-hydroxy-3-naphthoic acid (BTAHN) used in the present paper was synthesized according to the procedure ascribed previously (Amin, 1999; Amin, 2000). Acetate, borate, thiel, phosphate and universal buffer solutions of various pH values 2.0–12 were prepared. Interfering ion stock solutions of 20000 µg/mL were made by dissolving specified quantities of relevant salts in double distilled water.

2.3 Preparation of the membrane sensor

The sensor membranes were prepared by mixing 62 mg plasticizer (DBP), 31 mg PVC, 2.5 mg of BTAHN and 4.5 mg NaTPB. These components were fully dissolved in 2.0 mL THF. The THF cocktail was poured and put onto the glass plate at a volume of 200 µ L. All glass plates were washed with pure THF to eliminate dust and organic contaminants subsequent to coating. The membrane was left to dry in air for 15 min after 3.0 min of spinning (by a spin-on instrument) at 600 rpm. The thickness of the membrane was 5.0–7.0 µm. The reference membrane was identical to the test membrane except that it was devoid of reagent.

2.4 Procedure

The sensor membrane was inserted in a spectrophotometer cell with 2.5 mL of pH 7.25 acetate buffer. Then adding calculated amount of Mo⁶⁺ ion solution, the sample's absorption spectra was evaluated at 1.0 cm intervals against a reference blank membrane in the wavelength range 400–750 nm.

2.5 Analysis of water samples

A tap water sample was gathered from Benha city line, a commercial natural drinking water and mineral water collected from local market in Benha, Egypt. These samples were analyzed to its Mo⁶⁺ ion content. The water samples were filtered through Whatman filter paper (No. 40). Then the previous technique was used after applying 2.5 mL of pH 7.25 buffer solution.

A seawater sample was filtered to eliminate suspended particles. A 100-mL portion of the sample was taken into a 250-mL flask and diluted with water to the mark (dilution was required to reduce a high ionic strength of the solution). The Alexandria seawater was employed as a matrix for the verification of the procedure. Molybdenum that existent initially in the sample was precipitated with a 0.1 % solution of α-benzoinoxime at pH 1.0–7.0. The precipitate was extracted with chloroform quantitatively. The analysis was achieved by the added–found method 5.0–60 ng/mL Mo⁶⁺ was added.

2.6 Procedure of soil sample

A sample (3.0–4.0 g) of air-dried soil was pre-calcined at 450 °C and placed in a glassy-carbon crucible. Concentrated H₂SO₄ (0.6 mL per 1.0 g of soil) was added to the sample, the mixture was gently heated until the dissolution was accomplished. The residue accomplished was dissolved in 2.0 mol/L NaOH to the complete precipitation of Fe(OH)₃. The Fe(OH)₃ precipitate was separated carefully by filtration through a white-ribbon filter. The filtrate was acidified with HCl added drop wise to pH ∼ 2.0, 30 mL of water was added. The solution was moved to a 250-mL flask and diluted with water to the mark. To verify the procedure, the total molybdenum was assessed in soil sample by the added–found method. Several replicates, was prepared approximately 0.08 or 0.16 wt% molybdenum was added, and the initial amount of Mo⁶⁺ in the sample was neglected. The achieved solution was light yellow; and the previous procedure was applied for the assessment of molybdenum.

2.7 Determination of molybdenum in human urine samples

The recommended procedure has been applied to assess Mo⁶⁺ level in urine samples “normal” individuals human urine samples between 25 and 50 years old. The samples were acidified with 1.0 % HNO₃ to the pH of the standard Mo⁶⁺ solution and evaporated if necessary to known volume. The above method was used after adjustment of pH to 7.25 for the assessment of Mo⁶⁺ in urine samples. Some of the samples containing high concentration of Mo⁶⁺, and thus it were essential to dilute them to known volume prior to analysis.

2.8 Procedure for food and beverage samples

For the assessment of Mo in food samples, approximately 1.0 g of the each sample (apple, banana, cucumber, peas, rice, soybeans, tomato, and tea) was first ashed at 500 °C for 6.0 h in a crucible. The ash was slightly moistened after cooling with 2.0 mL of 1:1 (v/ v) HNO₃. Then, the content was heated to proximate dryness using a hotplate. In 5.0 mL 1.0 mol/L HNO₃, the residue was dissolved. The solution was gathered in a 25 mL measuring flask after filtration and diluted with water to the mark. Then, 1.0 mL of the resulting clear solution was poured into a 50 mL measuring flask, made up to volume with water and analyzed by means of both the proposed and independent ICP-AES procedures under the optimum conditions. Externally spiked three point standard addition calibration curves at various concentration levels were used to characterize the amounts of Mo⁶⁺, depending on the type and composition of the sample.

For the beverage samples analysis, a wet digestion method was applied. A 3.0 mL of concentrated HNO₃ and 5.0 mL of 30 % (w/w) H₂O₂ were added to 30 mL of beverage sample. Then, to eliminate excess H₂O₂, minimize the matrix influence, and analyte loss due to volatilization, the samples were evaporated near dryness and diluted to 50 mL with 0.1 mol/L HNO₃ solution. Then, the pH of samples was adjusted to approximately 7.25 by using the NH₄OH solution of 2.0 mol/L, and aliquot of 10 of these diluted solutions was analyzed. In order to suppress the interference effect of WO₄²⁻ ions, masking agents like sodium–potassium tartrate was added whenever necessary. The solution was filtered and then examined using the above-mentioned technique.

2.9 Procedure for leaves of plants samples

The plants leaves (mint, pepper black and fenugreek) were gathered from growing lands in a city vicinity of Moshtohor, Egypt. The samples were positioned in plastic bags. To remove any debris or soil, the leaves of the plants were carefully rinsed with tap water before being cleansed with distilled water. At 25 ± 2.0 °C, the samples were dried on a sheet of paper to avoid any excess moisture. The leaves were dried in an oven at 80 °C before being pulverized in a mortar and sieved to achieve a consistent particle size. The samples were then kept in a clean, dry glass container with a stopper.

About 1.0–2.0 g of each oven-dried plant sample was put in a silica crucible, which was mineralized by heating it to 550 °C for 2.0 h and then maintaining it at this temperature for another 2.0 h. After moistening the ash with 1.0–2.0 mL of water, 1.0 mL of concentrated HCl was added. On a hot plate, the mixture was heated to near-dryness. The residue was dissolved in 10 mL of bi-distilled water, filtered and the insoluble part was washed again with 5.0 mL of hot water. The filtrate and washings were placed in a 25 mL measuring flask, which was then filled with bi-distilled water to bring the total volume to 25 mL. The above method was applied assessment of Mo⁶⁺.

3.1 Effect of reagent

The influence of reagent amount within range of 0.25–3.25 mg at fixed concentration of Mo⁶⁺ (2.5 × 10⁻⁵ mol/L) was demonstrated. In the absence of reagent and because of low complexation and low sensitivity is illustrated. By adding BTAHN up to 2.5 mg, the amount of Mo⁶⁺ ion migrates to membrane increased expressively and complex amount increased significantly. More reagent addition does not have a significant effect on the sensitivity sensor and response. So, 2.5 mg of BTAHN is described for successive investigations.

3.2 Effect of membrane compositions

It is well recognized that the sensitivity and selectivity accomplished for a specified ionophore established on composition of the membrane and the solvent mediator and additives nature expressively (Oehme et al., 1998). As a result, the influence of the plasticizer kind, ionophore concentration, and NaTPB as a lipophilic additive (anionic site) on the response behaviour of the provided sensor was investigated. As revealed in Fig. 2, an response improvement of the applied sensor and further addition of NaTPB did not effect significantly on its response as a result in increasing of NaTPB amount level up to 4.5 mg. So, 4.5 mg of NaTPB is favorable for consequent works.

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Fig. 2

Effect of amount of Na-TPB on theresponse of the sensor of BTAHN with 2.5 × 10^–5 mol/L of Mo⁶⁺ at the optimum conditions.

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Addition of plasticizer (solvent mediator) compatible with PVC as a common polymer is needed to have a homogenous organic phase. DBP, DES, and DMS were accurate as possible plasticizers with varying polarity in the suggested investigation. Addition of DMS to membrane causes improper physical properties. Membranes that rely on the other two plasticizers have comparable qualities and sensitivity, while the inclusion of DBP improves the preferred sensor's feature and signal characteristics. In 2.5 × 10⁻⁵ mol/L of Mo⁶⁺ for BTAHN, absorbance measurements for the sensor membrane with several kinds of plasticizer were accomplished against a reference membrane. Furthermore, addition of 62 mg of DBP is the optimum amount to achieve the satisfactory results.

3.3 Effect of pH

View glance to the ionophore structure presents the existence of hydroxyl group and nitrogen atoms as more reactive atom for binding Mo⁶⁺ ions with high pH dependency of their complexation tendency. Thus, the sensor response depends on incorporation of BTAHN ionophore expressively influenced by the buffer pH. In the pH range of 2.5–12, the influence of pH on the sensor response (extent of complexation) in buffer and non-buffer solutions was investigated, and the findings are seen in Fig. 3. The sensor response reached its best value at pH 7.25, as ascribed. This pH was ideal for future research. At lower pH possibly owing to reactive sites protonation and proton competition with Mo⁶⁺ ions the sensitivity decreased considerably. At higher pH because of hydrolysis of Mo⁶⁺ ion and/or probably because of carriers deprotonation and charged species creation in membrane and their possible bleeding from membrane to aqueous solution, the sensitivity is reduced. On the other hand, at higher pH, ionophores' bleeding to bulk solution increased significantly, possibly owing to the negative charge appearance, resulting in a decrease in optode response and sensitivity.

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Fig. 3

Effect of pH on the response of the proposed optodes of BTAHN with 2.5 × 10^–5 mol/L of Mo⁶⁺ at the optimum conditions.

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3.4 Response time

The recommended sensor response time significantly indicates the needed time for the analyte diffusion from the bulk of solution into the membrane interface followed by association of ionophore for 2.5 × 10⁻⁵ mol/L of Mo⁶⁺ ions. The sensor dynamic response time (distinguished as the time required to reach 95 % of the last equilibrium) that was established to be 3.0–5.0 min for BTAHN based sensor in solution with various levels of Mo⁶⁺ ion was checked as a time function. The concentration of the Mo⁶⁺ ion, which governs its passage into the membrane, moreover the ionophore loading procedure, influence response time (Absalan et al., 2004) and membranes thickness. The response time of other methods (Pelle et al., 2019; Nakano et al., 2010) is more higher than 15 min, demonstrating that the response time of the proposed sensor method is quick.

3.5 Life time

The sensor membrane lifetime was assessed by the addition of buffer solution of pH 7.25 in cuvette containing membrane. The absorbance was detected at λ^max of 619 cross a period of time (about 10 h). During this time, there is no significant loss of carriers. A stable absorbance versus time was achieved by radiation exposure to membrane, which might be attributable to the constant membrane composition (absence of significant carrier bleeding from membrane to the bulk aqueous solution). No drift in absorbance was occurred. However, prepared membrane was preserved under water when not in use to avoid them from drying out.

3.6 Sensor selectivity

The coexisting ions effects in various water samples on the molybdenum recovery were also demonstrated. In these experiments, 2.5 mL solutions were containing 2.5 × 10⁻⁵ mol/L of Mo⁶⁺. The tolerated levels of each ion were taken as those, which caused less than a ± 5.0 % alteration in the absorbance. Under the optimum conditions for the proposed sensor especially the effect of plasticizers and type and amount of lipophilic additive; a selectivity coeffcient higher than 12,500 was achieved for NH₄⁺, Na⁺, K⁺, Li⁺, Tl⁺, Mg²⁺, Ca²⁺, F⁻, Cl⁻, Br⁻, I⁻, ClO₄⁻, NO₃⁻, SO₄²⁻, PO₄³⁻, and thiourea; and higher than 7500 for Fe²⁺, Ni²⁺, Mn²⁺, Pd²⁺, La³⁺, Y³⁺, Sc³⁺, Cd²⁺, Ga³⁺, Al³⁺, Cr³⁺, Cr⁶⁺, citrate and tartrate; and higher than 1000 for oxalate, Fe³⁺, Cu²⁺, Sm³⁺, Yb³⁺, Nb⁵⁺, Co²⁺, La³⁺, Lu³⁺, V⁵⁺ and Zn²⁺. The interfering effect of W⁶⁺ could be eliminated by adding of sodium–potassium tartrate.

In biological samples, abundant elements include Na, K, Ca, Mg, Fe, O, C, N, and P and major microelements like Cu, Mn, Zn, Ni, Co, Cr and Al, other elements being current in only trace amounts. It was establish that the tolerance limits for these elements were mostly in excess of 1.0 mg, especially K, Na, Ca, Mg, and Fe with tolerance limits > 5.0 mg. This property can be developed to create a favorable procedure for the assessment of trace molybdenum without any separation in plants, foods and seeds. An ultra trace level of molybdenum was assessed in biological samples simply by increasing the sample mass owing to better selectivity of the suggested method.

3.7 Repeatability and regeneration

The sensor response repeatability at λ^max 619 nm using a single membrane was measured by performing various replicate measurements for 2.5 × 10⁻⁵ mol/L. For these assessments, the relative standard deviation (RSD) was estimated to be 2.1 %. The regeneration of the membrane response was tested by multiple usages of each sensor for Mo⁶⁺ ion detection in test solutions of 2.5 × 10⁻⁵ mol/L Mo⁶⁺ solution. After each absorbance reading, the membrane was regenerated by 0.1 mol/L EDTA solution, rinsed with deionized water and soaked in thiel buffer solution of pH 7.25 for a few minutes. Good regeneration was developed for Mo⁶⁺ ion of 2.5 × 10⁻⁵ mol/L. The corresponding RSD value was ± 2.22 %. The sensor's short-term stability was evaluated by assessments of absorbance variance in contact with 2.5 × 10⁻⁵ mol/L of Mo⁶⁺ ion solution at pH 7.25, after a period of 8.0 h. From the absorbance taken every 15 min (n = 32), after 8.0 h of checking, it was revealed that the reaction is complete, with just a 2.65 % increment in absorbance. The membrane lifetime was assessed across a 50-day period, during which four produced membranes were stored at 4.0 °C in 5.0 % (v/v) ethanol. The mean absorbance variances of the membranes were found to be 0.022 (±0.002) and 0.028 (±0.003), before and after this period, respectively.

3.8 Dynamic range

Under the optimal circumstances, the dynamic range of Mo⁶⁺ ions concentration assessed was linear from 8.5 × 10⁻⁸ to 4.4 × 10⁻⁵ mol/L (Table 1). Both of detection and quantification limits were established to be 2.5 × 10⁻⁸ and 8.4 × 10⁻⁵ mol/L. It was seen that the LOD gained is much lower than earlier demonstrated procedures (Comitre and Reis, 2003; Shrivas et al., 2009; Varghese et al., 2006; Lopez-Garcıa et al., 2007; Sung et al., 1997; Filik et al., 2009; Carvalho et al., 2020; Rojas-Romo et al., 2020; Pelit et al., 2013; Soylak et al., 1997; Madrakian and Ghazizadeh, 2008; Ahmed et al., 2014; Santos et al., 2001) [Table 2]. In this case, it founds that the DPB increased the more Mo⁶⁺ ions extraction from the aqueous phase and consequently enhanced the sensitivity of sensor. In addition, exhibits higher sensitivity as compared to other reported procedures for Mo⁶⁺ ions. Although BTAHN is a photometric reagents for niobium (Amin, 2000), cadmium (Amin, 2001), gallium (Amin and Moalla, 2016) and palladium (Hassan and Amin, 2017) ions, its selectivity can be achieved and increased by using the proposed sensor which is more selective compared to the above previously methods [Table 2].

3.9 Analytical applications

The validity of the achieved sensor was further proven by analyzing spiked Mo⁶⁺ ion (water, soil and urine) samples. For this purpose, molybdenum solutions with the concentration between 5.0 ng/mL and 70 ng/mL were spiked to sample prepared by using 100 mL of water sample. Molybdenum was detected by direct calibration method after homogenizing the samples and using the method. In calculations, analytical factors derived from standard studies assumed in the achieved sensor were used. Table 3 listed the results of spiked molybdenum samples. RSD were less than 2.15 %. Calculated recoveries were found between 98.0 and 102.2 % for waters. Accuracy was measured by comparing results with these achieved using ICP-AES. Applying the paired t-test and F-value (Miller and Miller, 2005) no significant variance at 95 % confidence level was detected.

Moreover, the suggested procedure accuracy was certified by applying the method to assess molybdenum in some food and plant leaves samples collected from supermarkets in Benha, Egypt. The recommended method was applied to many categories of samples containing, peas, native banana, rice, tomato, cucumber, apple, soybeans, green tea, pepper black leaves, fenugreek leaves, and mint leaves with diverse matrix cation and anion concentration. The results are listed in Table 4–6. It can be described that the molybdenum content varies from 34.7 to 110.8 ng/g. Also, recovery values accomplished by the standard addition method proved that the advanced procedure is not effected by matrix interferences and can be applied reasonably for natural food and plant leaves analysis. Reliability was tested either by spiking the sample or/and comparing the achieved results with data assessed by ICP-AES. The spiked samples recovery is improved, and there is reasonable agreement between the results and data obtained by ICP-AES analysis, indicating that the recommended technique is reliable for the sample type tested, according to the results.