A Simple and Highly Structured Procaine Hydrochloride as Fluorescent Quenching Chemosensor for Trace Determination of Mercury Species in Water A Simple and Highly Structured Procaine Hydrochloride as Fluorescent Quenching Chemosensor for Trace Determination of Mercury Species in Water

An ultrasensitive, simple and highly selective spectrofluorometric strategy for quantifying traces of mercury(II) in environmental water has been established using the fluorescent probe procaine hydrochloride (PQ + .Cl − ). The procedure was based upon the formation of the ternary ion associate complex [(PQ + ) 2 .(HgI 4 ) 2− ] between PQ + .Cl − and mercury(II) in iodide media at pH 9.0–10.0 with its subsequent extraction onto dichloromethane accom- panied by a change in fluorescence intensity at λ ex/em = 268/333 nm. The developed strategy exhibited a linear range of 1–114 μg L −1 with lower limit of detection (LOD) and quanti-fication (LOQ) of mercury(II) 1.3 and 3.98 nM, respectively. Intra and inter-day labora - tory accuracy and precision for trace analysis of mercury(II) in water were performed. Complexed mercury(II) in environmental water, chemical speciation and successful lit- erature comparison was performed. The proposed system offered excellent selectivity towards mercury(II) ions examined in the presence of competent ions in excess, relevant to real water samples. The method was applied for analysis of mercury(II) in tap water samples. Statistical comparison (Student’s t and F tests) of the proposed method with the refer-ence ICP-OES method revealed no significant differences in the accuracy and precision. water samples by ICP-OES data and statistical treatment of data in terms of significant tests, e.g. F and Student’s t tests. The method could be expanded for ultra-trace analysis of mercury(II) ions in water after on-line enrichment on nanosized solid phase extractor, e.g. polyurethane foam packed column followed by elution with selective reagent [83] and/or its proposed coupling with the advanced microextraction techniques [84]. Therefore, the present work suggested the suitability of the proposed method for use in routine analysis and appli-cable strategy for analysis of mercury(II) in complex matrices.


Introduction
Heavy metal pollution is global level environmental concern, which poses serious implications towards human health [1,2]. Heavy metal pollution has attained considerable interest in the recent past [3]. Among heavy metals, mercury is considered among the most toxic and dangerous ion due to its wide existence as an ore cinnabar in nature and its applications as pigment vermilion, detoxification/anticorrosive medicines and mercury fulminate detonator in explosives [3,4]. Due to its wide presence in environment, it enters the biological membranes through respiratory and gastrointestinal tissues [4,5]. It can also cause permanent harm to the endocrine and central nervous systems if accumulated in the food chain and ultimately in human body [5][6][7][8][9][10][11][12][13]. Moreover, a low-level exposure of mercury will affect the endocrine and nervous systems, brain and kidneys [14,15].
Several forms of mercury including elemental, organic and inorganic or elemental display different levels of toxicity and contamination in natural water resources and drinking water [2]. The most stable and prevalent form of mercury that contributes to wider contamination is the solvated divalent mercuric ion (Hg 2+ ), due to its high solubility in water [16]. The US Environmental Protection Agency (US-EPA) and World Health Organization (WHO) have set the permissible level (MPL) for mercury to 10 nM [1,17]. Traces of mercury ions have shown significant toxicity, and therefore developing highly sensitive methodologies are considered essential [18]. Thus, recent trends have been oriented towards developing highly sensitive and selective procedures for monitoring and/or enrichment of mercury in various water samples [20][21][22].

Reagents and materials
Glasswares were pre-cleaned with HNO 3 (20% m/v), acetone and deionized water. Analytical reagent grade chemicals and solvents were used as received. Low-density polyethylene (LDPE) bottles, Nalgene were used for storage of the stock solutions. A stock solution (1.0 mg mL −1 ) of standard mercury(II) solution was prepared in ultra-pure water. Stock solution (1000 μM) of mercury(II) was prepared from HgCl 2 (BDH Chemicals, Poole, England) in ultra-pure water. More dilute solutions of mercury(II) ions were prepared by series dilution in deionized water. Potassium iodide solution (10% w/v) was prepared in ultra-pure water. Stock solutions of other cations were synthesized either from their chloride or nitrate salts in deionized water. Britton-Robinson (B-R) (pH 2-11) buffers were prepared from BDH purchased H 3 BO 3 , H 3 PO 4 , CH 3 COOH and NaOH in deionized water as reported [65]. A stock solution (1000 μM) of procaine hydrochloride (Sigma-Aldrich) was prepared by dissolving the required weight in aqueous solution.

Apparatus
Fluorescent measurements were recorded on a Perkin-Elmer LS55 spectrofluorometer, USA equipped with quartz cuvettes of path length 10 × 10 mm. UV-visible (190-1100 nm) spectra were recorded on a Perkin-Elmer spectrophotometer (Lambda 25, Shelton, CT, USA). For method validation, a Perkin-Elmer inductively coupled plasma-optical emission spectrometry (ICP-OES) (California, CT, USA) was utilized for mercury determination at the optimum operational parameters ( Table 1). Deionized water was obtained from Milli-Q Plus system (Millipore, Bedford, MA, USA). A digital micropipette (Plus-Sed) and pH meter (inoLap pH/ ion level 2) were used for preparation of stock and more diluted solutions of reagent and mercury(II) and pH measurements, respectively.  After equilibrium, the organic phase was separated out, shaken with anhydrous Na 2 SO 4 to remove trace of water and finally the absorbance of the organic extract was finally measured at λ max 310 nm versus the reagent blank at room temperature.

Spectrofluorometric procedure
In a series of glass test tubes (10.0 mL), appropriate concentrations (20-140 nM) of mercury(II) and 100 μL of 10% KI (w/v) were added. The test solutions were completed to the mark with B-R buffer of pH 10.0 after adding 1.0 mL (20 μM) of PQ + .Cl − and shaken well for 3.0 min with dichloromethane (5.0 mL). The organic phase was separated after equilibrium, shaken with anhydrous Na 2 SO 4 and analyzed at λ ex/em = 268/333 nm versus a reagent blank at room temperature. The quenched fluorescence in signal intensity (∆F) of PQ + .Cl − by mercury(II) was computed employing the following equation: where F 0 and F are the fluorescence intensities of the reagent before and after addition of mercury(II) under the optimized analytical parameters, respectively. The fluorescence signal intensity (F) of the formed ion associate complex was measured versus a reagent blank as described above. The quenched fluorescence (∆F) was finally determined. Following the recommended procedures, the selectivity of the proposed method was examined in the presence of a series of the concurrent diverse ions, e.g. Ca

Calibration curve of mercury(II)
To construct the linear calibration plot, a series of solutions (10.0 mL) containing various known (5.0 mL, 20-140 nM) concentrations of mercury(II) and PQ + .Cl − (1.0 mL, 20 μM) were transferred to measuring flask (25 mL) prepared. The test solutions were completed to the mark with B-R buffer of pH 10 and shaken well with dichloromethane (5.0 mL) for 3.0 min. After equilibrium, the organic phase was separated out, shaken with anhydrous Na 2 SO 4 and analyzed at λ ex/em = 268/333 nm versus a reagent blank at room temperature. The quenched fluorescence signal intensity (∆F) of the formed colorless complex species was finally measured versus a reagent blank as described above.

Analytical applications
In LDPE sample bottles, pre-cleaned as described in Section 3.

Electronic and fluorescence spectra of reagent and its mercury(1I) ion associate complex
The absorption spectrum of the ion pairing reagent PQ + .Cl − is demonstrated in Figure 2.

Analytical parameters
The effect of various experimental conditions on the fluorescence characteristics of both ion associate complex was studied, including type of solvents, pH, reagent concentration and equilibrium time.

Effect of solvent
The influence of extraction solvent, e.g. n-hexane, chloroform, dichloromethane, toluene and cyclohexane, on the fluorescence quenching of the formed mercury(II) complex was studied. The results are demonstrated in Figure 4. Stable and maximum change in fluorescence  quenching was observed in dichloromethane. Therefore, a detailed study on the influence of dichloromethane volume on the fluorescence quenching of PQ + .Cl − reagent by mercury(II) was critically studied (Figure 5). Thus, dichloromethane was adopted at 2.0 mL volume in the subsequent work pertaining to its higher performance compared to other volume fractions.

Effect of pH
The effect of pH on the fluorescence quenching of the formed mercury(II)-PQ + complex was studied in B-R (pH 3-12) buffer solutions. Maximum quenching by mercury(II) quencher was achieved at pH ≈ 9-10. The extraction rate at pH < 10.0 for ternary ion associate complex [(PQ + ) 2 .(HgI 4 ) 2− ] was lower. Several factors including hydrolysis, instability and/or incomplete extraction, and the slow dissociation of the complex K 2 [HgI 4 ] 2− account for the decrease in the amount of mercury extracted at pH values other than pH ≈ 9-10 [62,63]. Thus, in the next work, the solution media were adopted at pH ≈ 10 due to the ease in formation of the ternary ion associate complex [(PQ + ) 2 .(HgI 4 ) 2− ].

Effect of extraction time
The stability and the fluorescence signal intensity of the emission spectrum of the formed ternary ion associate complex [(PQ + ) 2 .(HgI 4 ) 2− ] considerably depend on the reaction time. Therefore, the fluorescence intensity of the emission spectrum of the ternary ion associate complex [(PQ + ) 2 .(HgI 4 ) 2− ] was measured at various time intervals (0.5-14 min) at λ ex/em = 268/333 nm at the optimum conditions. The results are demonstrated in Figure 6. Maximum stability in signal intensity was achieved after 2.0 min and remained constant for longer time up to 14.0 min. Therefore, a standing time of 2.0 min was adopted in the subsequent work.

Effect of PQ + .Cl − concentration
The influence of PQ + .Cl − concentration on the fluorescence quenching of mercury(II) at concentration of 80 nM was studied in KI (0.1% w/v). Thus, various fractions of PQ + .Cl − solution were added to [HgI 4 ] 2− . The fluorescence quenching (∆F) increased on increasing PQ + .Cl − concentration up to 5.0 μM and leveled off at higher concentration. This possible self-absorption and aggregation of the reagent at high concentration contribute to this enhanced behavior [77]. Thus, in the subsequent work, a concentration of 5.0 μM of PQ + .Cl − was selected, capable of successfully quantifying the target up to trace level proportions.

Selectivity
Microenvironment resembling real sample matrix containing competent interfering ion was designed to critically examine the applicability of the proposed method. Thus, the quenching in the fluorescence intensity of the ternary ion associate complex [(PQ + ) 2 .(HgI 4 ) 2− ] before and after adding the interfering ions in the presence of relatively high concentration (50-500 μgL −1 ) of other metal ions (K + , Ca 2+ , Ba 2+ , Cu 2+ , Pb 2+ , Fe 3+ , As 3+ , Ag + , Al 3+ Sn 2+ , Cd 2+ , Bi 3+ , WO 4 2− , MnO 4 − , F − , CO 3 2− , SO 4 2− ) under the same condition was studied. The results are shown in Figure 7, where no noticeable quenching changes were observed. The data indicated that PQ + .Cl − possess good selectivity in probing mercury(II). As shown in Figure 7, the signal intensity in the presence of 2.0 μg L −1 mercury(II) mixed with 500 μg L −1 of other metal ions in KI (0.1% w/v) was found similar to mercury(II) alone at 2.0 μg L −1 in KI (0.1% w/v). Thus, it can be concluded that the probe is selective towards mercury(II) in the presence of potent interfering ions in higher concentration.

Analytical performance
Under the optimized experimental conditions, the influence of various known mercury(II) concentrations (20-140 nM) on the fluorescence spectra of mercury(II)-PQ + was recorded. The plot of mercury(II) concentrations (20-140 nM) versus ∆F was linear (Figure 8) with the following regression equation: where C and ∆F are the concentration of mercury(II) ions and quenched fluorescence, respectively.
The precision of the method was checked by means of ANOVA by performing five successive replicates per day for 5 days for a typical sample containing 100 nM mercury(II) ( Table 2).
The precision of the proposed method was evaluated. The calculated value of F (F = 1.64) ( Table 3) was found lower than the tabulated F value (F = 2.87) at 95% confidence level [78].

Fluorescence quenching mechanism
The fluorescence quenching process of PQ + .Cl − by mercury(II) as a quencher was critically investigated to evaluate the fluorescence mechanism for the formed ternary ion associate    upon introduction of varying concentrations (0.3-1.0 μg L −1 ) of quencher (mercury(II) ions) are shown in Figure 9. The fluorescence intensity of PQ + .Cl − decreases regularly with increasing quencher concentration. The Stern-Volmer (K SV ) constant was calculated by employing the equation:

Sum of squares (SS) Degrees of freedom (df) Mean square (MS) F value S 1 /S 2
where F ∞ represents the emission intensity of the ternary ion associate complex [(PQ + ) 2 .

Analytical applications
Applications of the proposed method were tested for analysis of mercury(II) in tap water samples (King Abdulaziz University, Jeddah, KSA). Samples were spiked with known concentrations (20-100 nM) of mercury(II) ion and analyzed by the developed method. In each sample, the fluorescence quenching of PQ + .Cl − was immediately measured after spiking of mercury(II) onto the water samples. For method validation, mercury(II) concentrations in the spiked samples were also determined by the standard ICP-OES method. The results are summarized in Table 5. The recovery percentage of the measured mercury(II) added to the real samples by the developed and the standard ICP-OES methods was in the range from 97.74 to 104.7% and 98.05 to 102.7%, respectively. The calculated values of the Student's t and F tests were found lower than the tabulated Student's t and F tests at 0.05 probability [78] revealing no significant differences between both methods. Thus, it can be concluded that the proposed fluorescence probe can be used as a potential assay for sensing of mercury(II) in complex matrices.

Conclusion
A new and facile extractive spectrofluorometric method for cost effective, precise, accurate and selective determination of trace levels of mercury(II) in water. The proposed method was based upon formation of ternary ion associate complex [(PQ + ) 2 .(HgI 4 ) 2− ] between [HgI 4 ] 2− and the fluorescence probe PQ + .Cl − . The proposed system offered excellent selectivity towards mercury(II) ions over most anions and metal cations. The detection process could be performed quickly at room temperature without any catalyst or oxidizer. The proposed method provides LOD (1.3 nM) lower than the value set by WHO (10 nM) and USEPA for drinkable water [1,17]. The developed method is easy to operate as it does not require sophisticated experimental techniques, and the proposed assay is useful for point-of care applications. Moreover, the method opens capable ways for developing fluorescence assay strategies. The proposed approach was validated successfully by analysis of mercury(II) in environmental  water samples by ICP-OES data and statistical treatment of data in terms of significant tests, e.g. F and Student's t tests. The method could be expanded for ultra-trace analysis of mercury(II) ions in water after on-line enrichment on nanosized solid phase extractor, e.g. polyurethane foam packed column followed by elution with selective reagent [83] and/or its proposed coupling with the advanced microextraction techniques [84]. Therefore, the present work suggested the suitability of the proposed method for use in routine analysis and applicable strategy for analysis of mercury(II) in complex matrices.