Mechanochemical Synthesis of a Fluorescein-Based Sensor for the Selective Detection and Removal of Hg2+ Ions in Industrial Effluents.

Environmentally benign mechanochemistry-assisted high-yielding synthesis of fluorescein-phenylalaninol (FPA) conjugates as a Schiff base receptor is reported herein. This newly synthesized fluorescent probe is found to be most exciting and efficient because of its simultaneous detection and removal of mercury ions (Hg2+) in aqueous medium and industrial effluents through precipitate formation. The receptor successfully worked as a chemosensor in selectively sensing the Hg2+ ion through the rapid transition from yellow to pink in the colorimetric as well as quenching of fluorescence intensity in the fluorometric assay. The removal of mercury ions was confirmed by the inductively coupled plasma analysis of the supernatant. The lower detection limit of Hg2+ ions for the receptor FPA is 1.65 and 0.34 μM as determined through absorption and fluorescence spectroscopic methods, respectively. The high removal efficiency (∼98%) of the mercury ions is promising and could be achieved via the formation of the complex in a 1:1 stoichiometric ratio of receptor to Hg2+ ions. Furthermore, this probe may be a practical alternative for use in a paper-based portable device for achieving on-site detection of mercury ions in solid, solution, and vapor phases.

Introduction

Efficient synthesis of an organic compound is crucial for environmental, economical, and academic benefit. From an ecological perspective, mechanochemistry is one of the emerging methods[1−3] not only because it is an energy- and time-efficient, high yielding, and atom-economic protocol but also because it avoids the excessive use of volatile organic compounds, reagents, and harsh conditions. The direct release of the untreated wastage material after large scale production from industries became a threat to the health of humans and other animals. In this context, the residues of heavy metals released with industrial effluents (IEs) are an ever-growing challenge to a safe and clean aquatic environment. Water pollution by heavy metals and IEs has profoundly influenced the critical water supply and quality of drinking water in recent times; this contaminated drinking water has become one of the leading causes of death every year.[4] Amongst them, mercury is unhealthy and harmful to human beings and ecosystems[5,6] as it shows the bioaccumulation in most forms of life on Earth from the environment.[7−10]

The Earth’s crust, combustion of fossil fuels, incineration of mercury-containing products, random and uncontrolled use of mercury-containing pesticides, bleach production, metal mining and ore processing,[11a] and various industrial and medical activities are the primary sources of mercury in the ecosystem.[11b,12] It is also responsible for Pink disease, Hunter-Russell syndrome, Minamata disease, and so on.

A wide variety of methods, based on organic fluorophores[13] or chromophores,[14] semiconductor nanocrystalline materials,[15] cyclic voltammetry,[16] polymeric materials,[17] proteins[18] and so on[19,20] have been established for the detection of Hg2+. However, most of the above-mentioned materials are incompatible for the detection of mercury ions in an aqueous medium because of their poor water solubility or less sensitivity. Moreover, the simultaneous detection and removal of Hg2+ in an aqueous medium are still inadequate and also a portable device for on-site detection is limited. Therefore, the search for efficient sensing systems in an aqueous environment to simultaneously detect and remove Hg2+ ions is highly necessary. The colorimetric and fluorometric sensing are high throughput detection techniques for the on-site detection of heavy metal ions in biological and environmental samples.[21] In recent years, our research group has already attempted to develop a sensitive and efficient fluorometric and colorimetric chemosensor for selective Cu(II) detection. Herein, we report the mechanochemical synthesis of a sensitive sensor for the simultaneous detection and removal of Hg2+ from IEs and the construction of a paper-based portable device.

Results and Discussion

In the course of our continuous exploration of fluorescent probes/chemosensors for ion recognition, we have synthesized a potential Hg2+ ion sensor, based on a fluorescein-phenylalaninol (FPA) conjugate as the Schiff base. The introduction of hydroxy and imine functionalities into the framework of the probe makes the parent molecule hydrophilic, which facilitates to bind Hg2+ in an aqueous medium. The sensor FPA was synthesized by an economical and environmentally benign mechanochemical method, as described in Scheme from a commercially inexpensive fluorescein dye. In our previous work, we have produced[22] fluorescein monoaldehyde applying Reimer–Tiemann’s method in 28% yield upon refluxing a mixture of a fluorescein dye (1), aqueous NaOH solution, CHCl3, and the catalyst 15-crown-5 in methanol for 12 h. This solution-based (SB) method was replaced by a mechanochemical (MC) approach using a mortar-pestle set for grinding them in a solid phase at room temperature for 30 min, providing fluorescein monoaldehyde 2 in 30% yield and allowing us to scale up the product using 1.0 g (3.0 mmol) of fluorescein. The structure of the compound was characterized by 1H and 13C{1H} NMR spectroscopy (Figures S1 and S2).

Scheme 1

Synthesis of a FPA Hybrid as a Schiff Base

The synthesis of the FPA (4) hybrid was also carried out by exploring both the SB and MC methods. In the SB method, fluorescein monoaldehyde (2), and l-phenylalaninol (3) in methanol were stirred at room temperature for 12 h at pH 6.5 in the presence of a catalytic amount of AcOH to produce the Schiff base FPA (4) in 68% yield after chromatographic purification. As per the MC method, the synthesis of FPA (4) was also achieved by grinding fluorescein monoaldehyde (2), phenylalaninol (3), and methanol (few drops for proper mixing) together in a mortar/pestle for 20 min to complete the reaction. It was noted that over time, the product formation was also observed with the naked eye by the transition of the color from yellow to orange (Figure ). On completion, the reaction mixture was purified through the removal of unreacted starting materials by washing with a MeOH/DCM (1:40 v/v) mixture to produce 4 in 76% yields.

Figure 1

Change of color during the mechanochemical reaction.

In the 1H NMR spectrum, the disappearance of the signal of the aldehydic proton at δ 10.63 ppm and the appearance of a new signal at δ 8.85 ppm of the imine group along with additional signals at δ 5.11 (s, 1H), and in between δ 3.89 and 2.92 ppm confirmed the attachment of phenylalaninol with the fluorescein dye. In the 1H NMR spectrum in DMSO-d6, the peak appeared at δ 14.92 ppm for FPA (4) indicates the presence of a strong hydrogen bond between the phenolic −OH of fluorescein and the N-atom of the imine group (Figures S3 and S4). In the Fourier transform infrared (FTIR) spectrum, the peaks at 3053 (O–H stretching), 2956, 2925, 2876, 2841 (C–H stretching of l-phenylalaninol), and 1646 (C=N stretching of imine) cm–1 can be assigned to the functionalities present in FPA. The structure of the FPA was also corroborated from the mass peak that appeared at m/z [HRMS (ESI-TOF)]: calcd for C30H24NO6 ([M + H]+), 494.1603; found, 494.1580 (Figure S5).

The fundamental physicochemical properties, that is, the solubility in protic and aprotic solvents, optical behavior, and influence of pH on the absorbance of the receptor, were studied by absorption spectroscopy. It is noteworthy to mention that at a fixed concentration, FPA exhibits the highest absorbance at λmax 490 nm in water; this might be due to the hydrogen bonding effect of water with FPA (Figure S6), while significant absorbance intensity of the receptor was not observed in toluene presumably due to increased aggregation of the insoluble probe in the above-mentioned solvent. Thus, overall a yellowish fluorescence of the receptor in protic solvents is observed due to the hydrogen bonding interaction with the solvent while a light pink or colorless fluorescence in aprotic nonpolar organic solvents is found due to insolubility and natural aggregation of the probe (Table S1).

Several factors, such as high quantum yield, absorption and emission maxima of fluorescein in the visible region, and the participation of a Schiff base as a recognition unit for coordination to metal cations as a Lewis acid were considered in the design of a fluorescein-derived Schiff base. In addition to the above-mentioned parameters, the pH as an exogenous factor affected the chromogenic behaviour (or selectivity and sensitivity) of the receptor. Generally, fluorescein exists in equilibrium between a fluorescent, ring-opened carboxylic acid form, and a non-fluorescent closed lactone form, which is very sensitive to the pH of the medium.[23] Thus, the receptor, which contains a fluorescein core as a signalling unit, is highly sensitive to pH alterations in sensing metal ions in aqueous environments. The absorption spectra of the free probe under different pH conditions were recorded (pH 2.0, 4.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 12.0) (Figure S7A). The absorption peak at 490 nm of the metal-free receptor in water was affected by strongly acidic or alkaline solutions of Tris-buffer because of the existence of an equilibrium between the open and closed form of the fluorescein core. The results indicated that a neutral to basic medium might be the suitable condition for the effective photophysical interaction (Figure S7B).

Sensing Studies

For assessing the efficacy of an effective cation probe, the key factor is the ability to detect a specific metal ion in the presence of other metal ions. Sustainable scanning studies were performed with a series of metal ions (125.0 μM) to find out the change in adsorption behavior of the receptor FPA (4) (25.0 μM) in an aqueous medium by absorption spectroscopy. The scanning study (Figure ) revealed that out of a large number of environmentally and physiologically relevant cations (Li+, Na+, K+, Mg2+, Ca2+, Fe2+, Co2+, Cu2+, Ni2+, Pb2+, Hg2+, Cd2+, As3+, Er3+, Gd3+, Pr3+, and Sm3+), mercury ions caused an instant color change of the receptor from fluorescent green to light pink (Figure S8). This was confirmed by a photophysical study of the FPA–Hg2+ complex providing the hypsochromic shift of 30 nm in comparison to the λmax value of FPA (Figure , inset). An absorption band at around 460 nm is observed due to intramolecular charge transfer where the Hg2+ strongly binds with phenolic −OH of the fluorescein core, and −OH/–NH of the phenylalaninol segment of the chemosensor FPA.

Figure 2

Absorbance spectra of the receptor FPA (4) (25.0 μM) upon addition of various cations (125.0 μM) in water; (inset) color transition of the FPA in water upon addition of Hg2+ ions.

With a firm understanding of the spectroscopic properties and responses of FPA towards Hg2+ in hand, the fluorescein-based probe was applied to provide a rapid estimation of the total mercury content and its removal from IEs. The metal-free receptor FPA (4) produced a strong fluorescence emission at λemi 521 nm in water, and excitation at 495 nm where the slit width is 3.0 nm (Figures , S9, and S10). The fluorometric response was recorded before and after the addition of cationic salts, and the results of the fluorescence titration experiment indicated that Hg2+ quenched the fluorescence intensity of FPA by 8 fold with a red shift of 3.0 nm showing λemi maxima at 524 nm while Cu2+ provided a quenching signal with a blue shift of 4.0 nm with λemi maxima at 517 nm.

Figure 3

Emission spectra of FPA (4) (5.0 μM) upon addition of various cations (50.0 μM) in water with excitation at 495 nm, emission maxima at 521 nm, slit width 3 nm, and medium sensitivity. (Inset) The appearance of receptor 4 upon addition of Hg2+ and Cu2+.

In order to validate the high selectivity of the sensor FPA towards divalent metal ions, an interference study with other metal ions was performed by absorbance spectroscopy. The absorption spectra indicated no considerable interference from other metal ions (Li+, Na+, K+, Mg2+ Ca2+, Fe2+, Co2+, Cu2+ Ni2+, Pb2+, Cd2+, As3+, Er3+, Gd3+, Pr3+, and Sm3+) during Hg2+ ion detection (Figure S11A) and the intensity of absorbance was almost similar to FPA–Hg2+ complex absorbance intensity (Figure S11B).

The sensitivity and selectivity of the receptor FPA for the detection of Hg2+ at various pH values from acidic to basic in 10.0 mM Tris-buffer was recorded by the absorption spectroscopy study of FPA (4) upon addition of Hg2+ salt (Figure S12A). The difference of the absorbance intensity (A0 – A) of the receptor before and after the addition of Hg2+ ions against different pH values was plotted in Figure S12B, which indicates that the receptor is capable of detecting Hg2+ in the range of pH 2–8 and the highest difference of the absorbance intensities is detected at pH 4.0.

Binding Constant and Limit of Detection

To get a further insight into the sensing properties of FPA, a quantitative investigation of the binding affinity of the sensor to Hg2+ ions was carried out by absorption and fluorometric titration. The absorption spectrum of FPA (4) (25.0 μM) in water was recorded during the titration with various concentrations of [Hg2+] (0–40.0 μM) (Figure A). The linearity of detection and lower limit of detection (LOD) for Hg2+ are calculated by plotting a graph of A0 – A at 460 nm against different concentrations of Hg2+ ions in Figures B and S13. A linear response of the absorbance (A460) intensity as a function of [Hg2+] was detected from 1.75 to 20.0 μM (R2 = 0.9869) at 460 nm and the LOD of FPA toward Hg2+ is 1.65 μM. The binding constant (Kb) was calculated from the intercept and slope of the straight line of the plot of 1/(A0 – A) against 1/[Hg2+] at 460 nm following the Benesi–Hildebrand equation; it exhibits linearity in the range 2.7 × 104 to 5.7 × 104 M–1 (R2 = 0.9971) (Figure S14) assuming a 1:1 binding stoichiometry. The estimated value was found to be Kb = 1.7 × 104 M–1 for the FPA–Hg2+ complex, indicating the strong binding of Hg2+ with FPA in water.

Figure 4

(A) Absorption titration spectra of FPA (4) (25.0 μM) upon the addition of different amounts of Hg2+ ions (0.01–5.0 equiv) in water. (B) A plot of the difference of intensity of absorbance (A0 – A) upon addition of different amounts of Hg2+ ions (0–40.0 μM) in FPA at 460 nm.

Further studies to strengthen the strong interaction of mercury ions with FPA, the LOD was measured through fluorometric titration with different concentrations of Hg2+ ions (0.01–10.0 equiv) against the receptor 10.0 μM FPA in water (Figure A). The LOD of FPA for the Hg2+ was determined to be 0.34 μM by plotting the change of intensity F0 – F at 517 nm against [Hg2+]. Reasonable linearity of the curve was found in the range of 0.10–4.0 μM Hg2+ ion concentration in water (Figure B,C). The LOD of FPA for Hg2+ in fluorometric titration is 3.7 times lower than the value determined by the absorption titration method. On a serious note, a LOD of this sensor in fluorometric titration is found to be superior compared to that of many reported receptors, which are not even able to detect Hg2+ in the pure aqueous medium (Table S2). In this context, the receptor FPA (4) is highly proficient for the colourimetric and fluorometric detection of Hg2+ in an aqueous medium.

Figure 5

(A) Emission spectra for titration of FPA (4) (5.0 μM) with Hg2+ (0.1–60.0 μM) in water with a slit width = 3 nm, λex = 495 nm, and medium sensitivity. (B) Difference of fluorescence intensities (F0 – F) at 517 nm upon addition of different amounts of Hg2+ ions. (C) Linear curve for LOD determination.

The detection ability for Hg2+ was assessed by the fluorescence quenching of FPA with mercury ions at 517 nm upon addition of various concentrations of Hg2+ ions in water, and the quenching constant Ksv was calculated using the Stern–Volmer equation.F0 and F represent the steady-state fluorescence intensities of the fluorophore of FPA in the absence and presence of the quencher Hg2+, respectively. [Q] is the concentration of the fluorescence quencher and Ksv is the quenching constant. The rate of fluorescence quenching increased with an increase in the Hg2+ ion concentration. Figure S15 shows a linear relationship between the rate of fluorescence quenching upon increasing the concentration of Hg2+. In the range from 10 to 60.0 μM, it follows the regression equation Y = 0.2206X + 0.4873 with a correlation coefficient R2 = 0.9995 (Figure S15) where X is the concentration of Hg2+, and Y is the rate of fluorescence quenching. The quenching constant calculated for Hg2+ is Ksv= 2.0 × 105 M–1. This study suggested that the quenching of the fluorescence intensity of FPA by Hg2+ follows a static quenching pathway.

Binding Stoichiometry and the Binding Site

The complexation mode between FPA and Hg2+ ions in water was determined by Job’s titration method using absorption spectroscopy at 460 nm with a varying mole fraction of FPA (4) and Hg2+ ions. The graph in Figure indicates that the binding stoichiometry of mercury ions to the receptor is in the ratio of 1:1. This FPA–Hg2+ complexation mode was further strengthened using the HRMS (ESI-TOF) spectrum (Figure S16a) wherein a characteristic peak at 496.1751 m/z corresponding to [FPA + 3H]+ and a peak at 714.3174 m/z upon addition of Hg2+ appeared for [FPA + Hg2+ + H2O + H]+. This study again confirms the 1:1 mercury ion to FPA binding in FPA–Hg2+ complexation.

Figure 6

Job’s plot of FPA–Hg2+ at 460 nm in an aq. medium.

To find out the binding sites of FPA in complexation with Hg2+ ions, NMR analysis was set out in the presence and absence of Hg2+ ions (Figure S17). The host–guest interaction between FPA and Hg2+ ions was studied through 1H NMR titrations of FPA in DMSO-d6 with the addition of mercury ions as the perchlorate salt in different (0.5–3.0 equiv) concentrations to FPA. It has been observed that the signals corresponding to phenolic and aliphatic −OH of the fluorescein core and the l-phenylalaninol segment of FPA at δ 14.92 and 5.11 ppm, respectively, disappeared upon the addition of Hg2+ salt, while the signals of the remaining phenolic protons and the imine functionality (CH=N−) shifted toward downfield at δ 12.78 and 10.21 ppm, respectively, with reduced intensity and broadening of the NMR peaks. Hence, the significant changes observed at the position of phenolic −OH of the fluorescein ring and the −OH associated with l-phenylalaninol are assumed to engage in the formation of the complex with Hg2+ ions (Scheme ).

Scheme 2

Proposed Binding of Hg2+ Ions to FPA (4)

Real-Time Sensing of Mercury Ions

FPA-Containing Paper-Strip Sensor

One of the expeditious and economical methods of sensing metal ions is the construction of a paper-strip sensor. The paper-strip sensor of FPA was prepared to enhance the sensing ability for real-time sensing of mercury ions. This approach might be beneficial for the effective detection of Hg2+ under various experimental conditions. Herein, the prepared strip was coated with FPA, dried and exposed to Hg2+ ions in the solid, solution, and vapor states and a color change of the paper-strip sensor was observed (Figure A). Upon exposure to mercury ions, the paper strip rapidly changes the color from light yellow to yellow in visible light; from green to dark green in absorbance, and greenish-yellow to dark blue under fluorescence light (Figure B,C). The constructed paper strip sensor provides rapid and straightforward detection of mercury ions in all three states.

Figure 7

Images of the paper-strip in visible (A), UV (B), and fluorescent light (C) upon exposure to mercury ions in solid, solution, and vapor phases. (D) Images of the color transition of FPA-containing paper-strip in water at different concentrations of mercury ions under fluorescence light.

Such a paper-based sensor was further applied to find the LOD for Hg2+ ion detection in aqueous solution. The images obtained upon dipping the paper strip in different concentrations of Hg2+ ion solutions from 10–2 to 10–6 M are illustrated in Figure D. The FPA is found to be effective for the detection of Hg2+ ions in water up to 10–5 M concentration. A comparison of the paper-based sensing studies is provided (Table S3).

Detection of Hg2+ Ions in IEs

The receptor was applied to detect Hg2+ in IEs to find its broad applicability in real-time sensing. The absorption spectrum of FPA at pH 5.0 in the IEs was recorded before and after the addition of a known amount of Hg2+, and the outcome is demonstrated in Figure . The FPA selectively detects the spiked mercury ions in IEs, and the LOD was calculated for Hg2+ ion detection in IEs by plotting the difference of absorbance intensities A0 – A at 460 nm (Figure S18), and it is found to be 41.8 μM at pH 5.0.

Figure 8

Absorbance spectra of FPA (4) upon addition of IE and spiking of Hg2+ ions into IEs.

Removal Study of the Metal Ions by ICP

Capacity for the removal of mercury ions by FPA was confirmed by inductively coupled plasma (ICP) analysis in water.[24,25] In order to assess the ability of the sensor for sensing Hg2+ ions in water, 0.01–10.0 ppm (or 2.5–250.0 μM) solution of Hg(ClO4)2 salt was gradually added to the 25.0 μM FPA solution and allowed to settle or precipitate out as the FPA–Hg2+ complex for 24 h (Figure ). The supernatant of the mixture (5.0 mL) was used for ICP analysis. From the concentration of the Hg2+ ions in the stock solution and the concentration of Hg2+ ions obtained after precipitation (Table ), it is demonstrated that the mercury ion concentration is reduced in stoichiometric ratio as compared to the stock solution. Thus, this probe finds an excellent application for the removal of Hg2+ ions from water and IEs.

Figure 9

FPA–Hg2+ complex precipitation in water after 24 h at different mercury ion concentrations.

Table 1

Removal Capacity of Mercury Ions by FPA

stock (equiv of Hg2+ to FPA)	stock (ppm)	measure stock concn (ppm)	supernatant after 24 h	mercury ion removal (ppm)	mercury ion removal (%)
0.1	0.05	0.077 ± 1.7 × 10–3	–195 ± 1.7 × 10–3	ND	98
0.5	0.25	0.284 ± 1.5 × 10–3	–0.284 ± 2.7 × 10–3	ND	98
1.0	0.5	0.488 ± 1.1 × 10–3	0.105 ± 1.5 × 10–3	0.383 ± 5.8 × 10–4	78.4 ± 5.9 × 10–2
2.0	1.0	0.858 ± 1.4 × 10–3	0.506 ± 1.4 × 10–3	0.352 ± 3.3 × 10–4	41.0 ± 6.4 × 10–2
10.0	5.0	4.005 ± 2.0 × 10–3	3.557 ± 2.0 × 10–3	0.448 ± 3.3 × 10–4	11.8 ± 0.7 × 10–2
1.0 equiv spiking	0.25	0.302 ± 1.2 × 10–3	0.056 ± 2.0 × 10–3	0.246 ± 8.8 × 10–4	81.5 ± 55 × 10–2

A bar graph of Hg2+ ion removal in percentage is plotted against the different amounts of Hg2+ ions to receptor concentration in the equivalent ratio shown in Figure . The removal efficiency (∼98%) of the mercury ions was achieved in the presence of 2.0 equiv of FPA respective to the mercury ion concentration. FPA also successfully eliminates the mercury ions present in IEs efficiently and at a 0.5 equiv concentration of Hg2+ to the receptor and 81.5% removal of the mercury ions were observed. A comparison of the detection of mercury ions with literature data has been provided (Table S2).

Figure 10

Bar graph of Hg removal in percentage from water and IEs at different concentrations of Hg2+ ions.

Conclusions

A mechanochemical high-yielding synthesis of an FPA receptor with ease of working and purification has been developed successfully. The fundamental physicochemical behavior, that is, the solubility and pH effect, was studied by absorption spectroscopy. The synthesized receptor acted as a colorimetric and fluorometric sensor for the simultaneous detection and removal of toxic mercury ions in aqueous solution. In colourimetry, the FPA detected mercury ions by changing the color from fluorescent green to light pink, while it quenched the fluorescence intensity of FPA in the fluorometric study. The LODs of FPA for Hg2+ ions are 1.65 μM and 0.34 μM calculated by absorption and fluorescence spectroscopy, respectively. The binding constant is calculated by an absorption study and found to be Kb = 1.7 × 104 M–1. The receptor FPA is capable of removing ∼98% mercury ions in the presence of 2.0 equiv of the receptor to mercury ions from IEs applying the ICP technique. The sensor is also advantageous to detect mercury ions using the simple paper-strip movable technique. Therefore, the receptor FPA may constitute a simple and inexpensive chemodosimeter, which could demonstrate a highly viable and useful application for the detection and effective removal of mercury ions from an aqueous environment and IEs.

Experimental Section

Materials and Reagents

All the salts were purchased from Sigma-Aldrich, SRL, and Alfa Aesar. All solvents used were of spectroscopic grade and used without further purification unless mentioned. Ultrapure Millipore water was used throughout the experiments. IEs were collected from the Vapi IE plant, Gujarat, India.

Instrumentation

The progress of the reactions was monitored by thin layer chromatography (TLC) analysis on aluminium plates precoated with silica gel 60 F254. Purifications are carried out by column chromatography using silica gel (200–400 mesh size). Absorption spectra were recorded by using analytical an UV SPECTRO 2060+ UV–vis spectrophotometer, and fluorescence spectra were obtained from a Jasco FP 6500 spectrophotometer. Infrared spectra were studied on a PerkinElmer’s Spectrum 65 FT-IR spectrometer using KBr pellets as a reference. 1H NMR spectra of the samples were analysed on a Bruker AVANCE 500 MHz NMR spectrometer. Multiplicities are abbreviated as s = singlet, d = doublet, t = triplet, quart = quartet, quint = quintet, sext = sextet, and m = multiplet. Metal removal data were examined using inductively coupled plasma ICP–OES 7300 DV, PerkinElmer.

Synthesis and Characterization of Fluorescein Monoaldehyde (2)

It was already synthesized through the SB method and reported in our previous work.[22]

Mechanochemical Method

Fluorescein (1) (1.0 g, 3.0 mmol) was mixed with solid NaOH (700.0 mg) in a mortar with a pestle adding a few drops of MeOH. The mixture was ground for 10 min, and CHCl3 (1.0 mL) was added dropwise and ground for another 20 min. The crude was purified through column chromatography to obtain compound 2.

Characterization Data[22]

Rf = 0.65 (DCM/MeOH = 20:1); 1H NMR (500 MHz, DMSO-d6): δ 11.87 (s, 1H), 10.63 (s, 1H), 10.26 (s, 1H), 8.01 (d, 1H, J = 6.6 Hz), 7.80 (t, 1H, J = 1.6 Hz), 7.72 (t, 1H, J = 7.6 Hz), 7.33 (d, 1H, J = 7.7 Hz), 6.96 (d, 1H, J = 8.9 Hz), 6.86 (s, 1H), 6.70 (d, 1H, J = 9.1 Hz), 6.62 (s, 2H) ppm; 13C{1H} NMR (125 MHz, DMSO-d6): δ 192.9, 168.6, 163.0, 159.6, 152.2, 152.3, 150.8, 136.5, 135.8, 130.3, 129.0, 125.9, 124.8, 124.0, 113.6, 113.5, 109.8, 109.3, 109.3, 102.7, 81.8 ppm.

Synthesis and Characterization Data of FPA (4)

A few drops of methanol was added to a mixture of fluorescein monoaldehyde (2) (200.0 mg, 0.55 mmol), and l-phenylalaninol 3(26) (80.2 mg, 0.53 mmol) in a mortar and ground well with a pestle for 20 min. The progress of the reaction was monitor by TLC techniques. The crude residue was purified by washing with dichloromethane (DCM) and DCM/MeOH in 50:1 ratio. The yield of FPA is found to be 206.3 mg, 76%.

SB Method

To a solution of fluorescein monoaldehyde (2) (100.0 mg, 0.28 mmol) in methanol (20.0 mL), in the presence of catalytic amount of acetic acid at pH 6.5 and l-phenylalaninol (40.1 mg, 0.265 mmol) was added. The method for monitoring and purification was applied as similar to the MC method. The yield of FPA = (93.96 mg, 68%). Rf = 0.4 [DCM/MeOH (20:1)]; 1H NMR (500 MHz, DMSO-d6): δ 14.92 (s, 1H), 10.21 (s, 1H), 8.85 (s, 1H), 7.98 (d, 1H, J = 7.2 Hz), 7.77 (t, 1H, J = 7.6 Hz), 7.70 (t, 1H, J = 7.6 Hz), 7.26–7.17 (m, 6 H), 7.19 (t, 1H, J = 7.2 Hz), 6.75 (s, 1 H), 6.55 (d, 2 H, J = 8.2 Hz), 6.42 (s, 1H), 5.11 (s, 1H), 3.89 (s, 1H), 3.68 (m, 1H), 3.53 (m, 1H), 3.05 (dd, 1H, J = 4.8, 13.1 Hz), 2.92 (dd, 1H, J = 7.9, 13.1 Hz); 13C{1H} NMR (125 MHz, DMSO-d6): 168.7, 159.9, 159.4, 152.3, 150.6, 138.3, 138.0, 135.7, 132.9, 130.2, 129.4, 129.3, 129.0 (2C), 128.6 (2C), 128.4, 126.4, 126.2, 124.7, 124.0, 116.7, 113.2, 109.5, 104.4, 104.1, 102.4, 68.4, 63.5, 40.1 ppm; FTIR (KBr): νmax/cm–1 3053.7 (O–H stretching), 2956.4, 2925.8, 2876, 2841.6 (C–H stretching of l-phenylalaninol) 1646.7 (C=N stretching of amine); HRMS (ESI-TOF)] m/z: calcd for C30H24NO6 ([M + H]+), 494.1603; found, 494.1580.

Solution Preparation for Solubility, Solvent, and pH Effect

A stock solution of 1 × 10–2 M concentration of FPA (4) (4.9 mg, 10.0 mmol) in DMSO (1.0 mL) was prepared, and from there 10.0 μL solutions from the stock solution was diluted with different pH solutions (2.0 mL) to make the final concentration 50 × 10–6 M. The absorption spectra were recorded at rt without giving any incubation time. For the pH study, 2.0 μL solutions were diluted with various solvents (2.0 mL) to prepare the final concentration 10 × 10–6 M. The absorption spectroscopic data were recorded at rt after incubating for 1 h.

Solution Preparation for Absorbance and Emission Study

It is to be mentioned that, in this work, the absorbance and fluorescence spectra were analysed at room temperature without giving any incubation time, unless otherwise indicated.

From the prepared stock solution of FPA, 250.0 μL solutions were taken out and diluted with 50.0 mL of water to achieve a final concentration of 50.0 μM for absorption spectroscopy. Similarly, for fluorescence study, 50.0 μL was taken out from a stock solution of FPA and diluted with 50.0 mL of water to make the final concentration 10.0 μM. The aqueous solutions of anions and cations with a concentration of 1 × 10–2 M were prepared from their respective perchlorate (alkali alkaline earth and transition metals) or nitrate salts (lanthanide metals).

For ion selectivity study, the stock solution of cations was prepared in 2.5 × 10–4 M concentration. The salt solutions (1.5 mL) were added to the FPA solution (1.5 mL) to adjust the 5:1 (ion/receptor) ratio for scanning of various ions. For emission study, the salt solutions (1.5 mL) were added to the receptor FPA solution (1.5 mL) to prepare the 10:1 (ion/receptor) ratio for scanning of various ions.

LOD Determination

To determine the LOD for strongly interacting cations (Hg2+ ions), the absorbance titration was performed by adding an incremental amount of mercury ions of known concentrations from 0.1 to 5.0 equiv into 25.0 μM aqueous FPA solution. To determine LOD, the fluorescence titration was performed by adding an incremental amount of mercury ions of known concentrations from 0.01 to 15.0 equiv into the 5.0 μM aqueous FPA solution.

Job Plot Measurements

The same concentration of the receptor FPA and Hg2+ ions (50 × 10–6 M) was prepared in water. The different volumes (3000–300) × 10–6 L of FPA were transferred into vials and made up with the equal concentration of Hg2+ ion solution to prepare a total volume of 3.0 mL and a mole fraction ratio of Hg2+ ions from 0 to 0.90.

Mass Spectroscopy

HRMS (ESI-TOF)] m/z: [FPA + Hg2+ + H2O + H+], 714.1416; calcd, 714.3174; C30H23ClHgNO6, 730.0927, calcd, 730.0920 (Figure S16a).

In LCMS (m/z): [FPA-benzyl group + Hg2+ + 2H2O], 640.0517; calcd, 640.0884; [FPA + Hg2+ + 2OH– – H+], 728.1855, calcd, 728.1197 (Figure S16b).

1H NMR Titration of FPA with Hg2+ Ion

FPA (4) (5 × 10–3 M) was dissolved in DMSO-d6 (0.50 mL). The 2.5 μL of 0.50 M Hg(ClO4)2 solution in DMSO-d6 was added incrementally into 5 × 10–3 M solution of FPA to prepare the concentration of Hg2+ ions in an NMR tube ranging from 0.5 to 3.0 equiv with respect to FPA. After shaking them for a minute, their 1H NMR spectra were recorded at room temperature.

Hg2+ Ion Detection at Different pH values

From the stock solution of FPA (1 × 10–2 M), 50.0 μL was taken out and diluted with 10.0 mL of solution of respective pH to prepare final concentration 50.0 μM. The FPA (1.5 mL) solution was diluted with the same pH solution and incubated for 1.0 h, and the absorption spectra were measured. From the 1 × 10–2 M Hg2+ ion stock solution, 250.0 μL of solutions were taken out and diluted with 10.0 mL of solution of respective pH to prepare final concentration 250.0 μM. The 1.5 mL of the prepared Hg2+ solution was added to the 1.5 mL FPA (50.0 μL) solution and absorption spectra were recorded.

Detection of Hg2+ in IEs

Solution Preparation of IE

IE was collected from the Vapi industrial effluent plant, Gujarat, India. The collected sample was filtered, and the pH of the effluent was measured and found to be basic (pH 8). The collected IE was acidified with HCl to maintain the acidic pH 5 to measure the mercury ion concentration. The IE sample was diluted 10 times for further use at pH 5.0.

Solution Preparation of FPA

FPA (4) stock solutions (50.0 μM) were prepared at pH 5 in Millipore water.

Absorbance Study of IE

The FPA solution (50.0 μM, 1.0 mL) was taken into a vial and a blank solution of pH 5 (1.0 mL) was added to make the final concentration 25.0 μM. The absorbance spectra of the receptor solution were measured. To measure absorbance response of receptor upon addition of IE, the same FPA solution (50.0 μM, 1.0 mL) was taken into the vial and IE (1.0 mL) was added to it, and absorbance spectra were recorded. To study the linearity and LOD of Hg2+ ion detection, the spiking IE was prepared with different concentrations of Hg2+, and absorbance spectra of these solutions were measured by adding IE (1.0 mL) to FPA solution (1.0 mL) at pH 5.