Schiff Base Ligand 3-(-(2-Hydroxyphenylimino) Methyl)-4H-Chromen-4-One as Colorimetric Sensor for Detection of Cu2+, Fe3+, and V5+ in Aqueous Solutions

The ligand 3-(-(2-hydroxyphenylimino) methyl)-4H-chromen-4-one (SL) has been synthesized and examined as a chemosensor for some metal ions in aqueous solutions based on colorimetric analysis. Color changes were monitored using UV–visible spectroscopy. Binding stoichiometry and limit of detection (LOD) were estimated using titration experimentation based on UV–visible absorbance and Job's plot. The synthesized ligand was tested for selectivity in the presence of several cations and was examined for possible utility as a chemosensor in real water samples. The results indicated sensing ability and selectivity for Cu2+, Fe3+, and V5+. Stable complexes were formed between SL and Cu2+, Fe3+, and V5+, and the ligand-to-metal binding stoichiometry was found 2 : 1 in the SL-Cu2+ and SL-Fe3+ complexes, and 1 : 1 in the SL-V5+ complex. The results of LOD and bending constant were (7.03 μM, 1.37 × 104 M−1), (5.16 μM, 2.01 × 104 M−1), and (5.94 μM, 1.82 × 104 M−1) for Cu2+, Fe3+, and V5+, respectively.


Introduction
It is well established that some metal ions have important benefcial roles and functions in biological and chemical processes, and on the other hand, overexposure to some metal ions causes toxicity. Transition metals are abundant in nature, particularly in soils and aquatic systems and releases of these elements, to the environment are associated with industrial processes. However, their extreme presence in water resources causes serious contamination and can be considered hazardous pollutants at some levels, and creates a threat to human beings and other biota because of their bioaccumulation in food chains and toxicity efects [1,2]. Terefore, it is very necessary to determine and detect their presence in a water medium to ensure water quality and avoid their adverse efects.
Among the most commonly occurring transition metal ions that may be present in water systems and could potentially cause serious health problems are Cu 2+ , Fe 3+ , and V 5+ . Information of the high levels of exposure to copper from diet and drinking water results in gastrointestinal and hepatic systems illness as it is absorbed rapidly by the stomach and intestine [3]. Iron toxicity is hazardous to children, and subchronic and chronic exposure to iron results in excessive iron absorption and causes hemochromatosis, and studies strongly suggest iron is noxious to tissues [4]. Vanadium is also a toxic metal and a potent environmental pollutant. Te primary targets of exposure to vanadium are the gastrointestinal and hematological systems [5].
Te detection and recognition of heavy metal ions in diferent sources is an active feld of research and has been a focus of researchers in recent years. Enormous attempts have been devoted for obtaining and developing practical, easy, selective, sensitive, and rapid sensors for the detection of metal ions [1,2].
Colorimetric sensors that rely on color changes on bounding with the analytes are believed to be one of the most efcient and suitable techniques for detecting inorganic ions and other chemical species [6,7]. Colorimetric chemosensors-based techniques are one of the interesting tools that can be used for this purpose because of their advantages, such as their ability to produce a desirable onsite"naked-eye" response, a reasonable cost, simplicity, selectivity, a low detection limit, immediate response, and high sensitivity [6][7][8]. Chemosensor molecules that can be used for sensing the presence of metal ions must have the ability to bind to metal ions and produce detectable physicochemical changes easily and promptly. Recently, there has been a large body of research articles and reviews published reporting the use and development of many molecules that can be used as colorimetric chemosensors for detecting metal ions in diferent sources [8][9][10]. One of these interesting compounds is Schif base ligands, which can coordinate easily with metal ions, forming specifc color changes that indicate the presence of specifc metal ions [8,11]. In general, the Schif base ligands on binding with metal ions produce colored complex compounds that can be recognized easily by the naked eye. Based on this character, these coordination entities can be developed to be used as colorimetric sensors for detecting the presence of metal ions in diferent sources [1]. Te Schif base ligand 1-(2-thiophenylimino)-4-(N-dimethyl) benzene was investigated by our research group for the detection of multiple metal ions in solutions and showed remarkable selectivity and sensitivity response toward four metal cations: Fe 2+ , Fe 3+ , Cr 3+ , and Hg 2+ [1]. Te Schif base 2-(3-(2-hydroxyphenylamino)-1,3-diphenylallylidene) amino) phenol was reported by Mergu and Gupta as a highly selective and sensitive chemosensor towards Cu 2+ ions over other metal ions in a wide pH range [12]. A simple and reversible pyrrole-based Schif base, (E)-N′-((1H-pyrrol-2-yl) methylene), isonicotinohydrazide chemosensor for the detection of Cu +2 ions was reported by Sidana et al., which showed high selectivity and sensitivity by the colorimetric and spectroscopic method in an aqueous organic solvent over other possible interfering ions in a wide pH range. Moreover, the Cu +2 detection limit was lower than the WHOrecommended guidelines for drinking water, and practical application for real sample analysis was observed via naked eye color change [13].
Two colorimetric pyrrole-based Schif base chemosensors were investigated in an aqueous analyte solution by Udhayakumari and Velmathi, which showed highly sensitive probes towards multimetal ions (Fe +3 , Cu +2 , Hg +2 , and Cr +3 ) in water solutions by a distinct color change and exhibited a low detection limit of micromolar levels [14].
Schif bases derived from 3-formyl chromone have been studied signifcantly and have attracted the attention of chemists and researchers as they can easily bind with metal ions, forming metal complexes that have many applications [15,16]. Chromone-based azomethine colorimetric chemosensors were reported by Rezaeian et al. and successfully used to detect and recognize Cu 2+ , Zn 2+ , and CN − ions [17].
In continuation to our research work, herein this investigation we present the design and preparation of the multidentate Schif base ligand named 3-(-(2-hydroxyphenylimino) methyl)-4H-chromen-4-one by condensing 3-formyl chromone with 2-aminophenol that have the ability to bind to metal ions forming colored complexes and testing it as a colorimetric chemosensor for some metal ions in aqueous solutions.

Materials and Characterization.
All chemicals used in synthesis and experimentation were of analytical grade, purchased from Sigma-Aldrich and BDH chemical companies, and were used without purifcation. Te absolute ethanol solvent used in experiments was of HPLC/spectrophotometric grade (97.8%). Te precursor compound, 3formyl chromone, and the Schif base ligand were synthesized in our laboratory. Te synthesized Schif base ligand was characterized using mass spectroscopy, FT-IR, and 1 H NMR. Te mass spectrum was recorded on a TermoFisher Scientifc-LCQ feet ion trap mass spectrometer with high resolution using the electrospray ionization (ESI) method. Infrared measurements were made in the 4000-400 cm −1 region on a TermoFisher Scientifc Nicolet iS50 FT-IR spectrophotometer using the attenuated total refection (ATR) method for solid powder. Te 1 H NMR spectrum was recorded on a Bruker-400 MHz and TMS as an internal standard working in DMSO d 6 , respectively. UV-vis absorption spectra were recorded with a TermoFisher Scientifc Evolution 300 UV-visible double beam spectrophotometer in the range of 200-800 nm.

Preparation of the Precursor Compound 3-Formyl
Chromone. 3-formyl chromone was prepared by formylation of 2-hydroxyacetophenone using the Vilsmeier-Haack reaction as reported with a slight modifcation (scheme 1) [18]. 2-hydroxyacetophenone (8.7 g, 0.05 mole) was added into a 500 mL clean and dry round-bottom fask, and then added 150 mL of dimethylformamide (DMF) was added. Te reaction mixture was cooled to 0-5°C and phosphorus oxychloride (7.0 mL, 0.075 mole) was added dropwise at 0-10°C with continuous stirring of the reaction mixture for 30 minutes under cooling conditions. Te reaction mixture was further stirred at 20-30°C for 10 hours. Finally, the thick reaction mixture was poured onto the 300gram crushed ice and stirred for 2 hours. Te light-yellow solid that precipitated out was fltered, washed with water, which was then dried to get 3-formyl chromone, and recrystallized using acetone. Te yield was 82.3% and the melting point was 151°C (standard 151-153°C).

Synthesis of 3-(-(2-hydroxyphenylimino) Methyl)-4H-
Chromen-4-One. Te Schif base ligand (SL) named 3-(-(2hydroxyphenylimino) methyl)-4H-chromen-4-one was synthesized following the reported procedure by condensation reaction between 3-formyl chromone and 2aminophenol in 1 : 1 stoichiometric ratio using absolute ethanol as solvent and refuxing for nearly six hours with continuous stirring at 50-60°C using a hot stage magnetic stirrer (Scheme 1) [16,19,20]. Te progress of the reaction was monitored by TLC. Finally, the reaction mixture was concentrated to nearly 20 mL and then poured into crushed ice and stirred to get a light-yellow solid mass, which was fltered and washed with water. Te obtained material was purifed by recrystallization using absolute ethanol and dried in a desiccator over anhydrous calcium chloride. Te yield obtained was 68.2%, and the melting point was 202°C (reported 198-200°C) [21].

Preparation of Stock Solutions of Metal Ions and the
Ligand Chemosensor. Te solutions of metal ions were prepared by dissolving metal chloride salts of the following ions: Cu 2+ , Cr 3+ , Fe 2+ Ni 2+ , Ca 2+ , Co 2+ , Mg 2+ , Zn 2+ , Fe 3+ , NH 4 VO 3 (V 5+ ), Mn 2+ , Hg 2+ , Pb 2+ , Ba, and Al 3+ with 1 × 10 −2 M concentration in double-distilled water, and the pH was between 6.20-6.50. Te stock solution of the targeted ligand chemosensor was prepared with a concentration of 1.0 × 10 −2 M by dissolving in absolute ethanol, and the pH was 7.84. Te UV-vis spectra measurements were performed using a solution of 1 × 10 −3 M concentration for the ligand compound in absolute ethanol in the range of 200-800 nm.

Cationic Selectivity Recognition.
In this experiment, 1 mL of solution with a concentration of 1 × 10 −2 M of each metal ion was mixed with 2 mL of the examined Schif base ligand (SL) with a concentration of 1 × 10 −2 M in a test tube, and the fnal volume of the mixture was completed to 10 mL by adding absolute ethanol. Te fnal pH was measured at 7.37, and the room temperature was 26°C. Te mixtures were left for 20 minutes at room temperature to allow complete complexation. Color changes were monitored using UV-visible spectroscopy.
2.6. Limit of Detection. Te limit of detection (LOD) was estimated based on a UV-visible titration experiment. For this purpose, a plot of the measured absorbance intensity at a wavelength of 470 nm (in the case of Cu 2+ and Fe 3+ ) and 466 nm (in the case of V 5+ ) versus the concentration of each metal ions (Cu 2+ , Fe 3+ , and V 5+ ) was constructed. To perform the titration experiment for each of Cu 2+ , Fe 3+ , and V 5+ , a solution (fxed volume � 1 mL) of the ligand SL with a concentration of 0.001 M in ethanol was taken into a test tube, and to it, a series of 10 solutions (ranging from 0.01 to 1.0 mL of 0.001 M) of each metal ion in deionized water was added. After well mixing, the UV-visible spectra were scanned at room temperature in the range of 200-800 nm for each sample. Te limit of detection was estimated based on the literature reported procedure [22,23].

Binding Constant.
Te binding constant for the SL-M n+ complex was determined by colorimetric titration of metal with diferent concentrations of LS. Te binding has also been verifed using the aid of the Benesi-Hildebrand equation [24].

Estimation of Coordination Stoichiometry.
In order to estimate the binding stoichiometry between the ligand chemosensor and the metal ions, we used Job's plot method [1]. For this purpose, 0.001 mmol (0.0265 mg) of SL ligand was dissolved in 100 mL of absolute ethanol. A series of ligand solutions ranging from 0.1 mL to 0.9 mL were transferred to diferent fasks. Ten, 0.001 mmol of each metal ion of Cu 2+ , Fe 3+ , and V 5+ was dissolved in 10 mL of distilled water, and a series of each metal solution ranging from 0.1 mL to 0.9 mL was added to each fask of the SL ligand solution, respectively. Each mixture was completed to a volume of 10 mL by adding absolute ethanol solvent, shaken properly, and UV-visible spectra were recorded. Job's plot was then constructed by plotting the molar fraction of the chemosensor (SL) against absorbance intensity at the strongest wavelength for each metal ion, which was 456, 461, and 465 nm for Cu 2+ , Fe 3+ , and V 5+ , respectively.

Application of SL Chemosensor in Real Water Samples.
To examine the possible utility of the tested SL chemosensor in real samples, three water samples, including distilled water, tap water from domestic water supplies, and a water sample from the Al-Aqiqwater-reservoir dam located at Al-Baha region, KSA, were collected, and each sample was spiked with a known concentration (70 µM) of each Cu 2+ , Fe 3+ , and V 5+ solution and analyzed with the SL chemosensor (140 µM). All the real water samples were fltered to remove solid impurities using Whatman medium fow flter paper (Grade 1: 11 µm) and centrifuged using a benchtop centrifuge at 16,000 rpm (the pH was in the range of 6.4-7.8).
UV-visible absorption spectra were recorded at 470 nm in the case of Cu 2+ and Fe 3+ and at 466 nm in the case of V 5+ for each sample, and the amount of Cu 2+ , Fe 3+ , and V 5+ in each sample was calculated using the calibration curve. Each experiment was repeated three times, and the mean values were recorded.

Molecule Design and Preparation of the Schif Base Ligand
(SL) Chemosensor. Te selected Schif base ligand 3-(-(2hydroxyphenylimino) methyl)-4H-chromen-4-one was successfully prepared by a one-step condensing reaction between 3-formyl chromone and 2-aminophenol, and its structure was characterized and confrmed using mass spectroscopy, IR, and 1 H NMR spectroscopy and was similar to those reported in the literature [20,25]. Tis compound is characterized by the presence of three adjacent coordination sites (the N atom of the azomethine group, the O atom of the phenolic group, and the O atom of the ketonic group), which can bind to metal ions in 1 : 1 or 1 : 2 (M: L) stoichiometric ratio easily and form diferent colored metal complexes with diferent metal ions that can be recognized easily with the naked eye [15,25]. Moreover, this compound is soluble in ethanol, DMF, and DMSO, which are miscible with water. For this reason, we have selected this ligand (SL) to be investigated as a sensor for the presence of metal ions in water solutions.

Colorimetric Sensing Properties of (SL) Compound and
Selectivity. Te UV-visible maximum absorption spectra (λ max ) for the compound (SL) were recorded in absolute ethanol at room temperature with a 1 × 10 −3 M concentration over the wavelength range of 200-800 nm. Te strongest absorption band (Figure 1(a) inset) appeared in the visible wavelength period at 400 nm attributed to n ⟶ π * allowed transitions of the azomethine (-CH�N-) group, which is considered λ max and used for further investigation. Another weak band at lower energy appeared at 311 nm and can be assigned to π ⟶ π * transitions due to π-orbital localization on the aromatic ring [17,26].
To investigate the recognition ability and examine the selectivity of the prepared SL compound, several cationic metal ions (Cu 2+ , Cr 3+ , Fe 2+ , Ni 2+ , Ca 2+ , Co 2+ , Mg 2+ , Zn 2+ , Fe 3+ , NH 4 VO 3 (V 5+ ), Mn 2+ , Hg 2+ , Pb 2+ , Ba 2+ , and Al 3+ ) were used. In this experiment, 1 equiv. of each metal ion was used to prepare the solutions in doubledistilled water and was added to the ethanolic solution of the SL compound, and color change was monitored by the naked eye and through the recording of UV-visible absorption spectra in the range of 200-800 nm. No signifcant color change was noticed on the addition of the screened metal ion solutions to the tested SL chemosensor solution except for Cu 2+ , Fe 3+ , and V 5+ , which showed a signifcant color change from yellow to brown, red, and orange, respectively (Figure 1(b)). Moreover, it was noticed that with the addition of Cu 2+ , Fe 3+ , and V 5+ ion solutions, there was a decrease in absorbance intensity (hypochromic efect) and an emergence of a new band in the range of 450-700 nm with the absorption maxima at 472 nm (Figure 1(a)). While on the addition of other metal ion solutions, we did not observe any efect on the absorption intensity at 472 nm (Figure 1(a)).
Te obtained color change, hypochromic shift, and bathochromic shift (red-shift of about 72 nm) of the UV-visible band of the free ligand may be due to deprotonation of the phenolic group in the chemosensor SL ligand and complexation of the ligand with Cu 2+ , Fe 3+ , and V 5+ metal ions, forming colored metal complexes. Additionally, the ligand-to-metal charge transfer (LMCT) due to complexation is another factor that afects the color change [27]. Te selectivity of the examined SL chemosensor towards various tested common metal ions based on absorbance intensity was plotted as a bar graph in Figure 2. .

Estimation of Coordination Stoichiometry and
From Job's plot (Figures 3(a)-3(c), 4(a), 4(b)-6(a), 6(b)), it is noticed that the maximum absorbance value obtained when the molar ratio was 0.66, 0.66, and 0.5 for Cu 2+ , Fe 3+ , and V 5+ , respectively, which suggests that the binding stoichiometry between the SL chemosensor and the metal ions Cu 2+ , Fe 3+ , and V 5+ is 1 : 2 (M: L) in case of SL-Cu 2+ and SL-Fe 3+ complexes, while the molar ratio in the SL-V +5 complex was 1 : 1 in the binding stoichiometry. Accordingly, we proposed the possible complexation structure shown in Figure 7 between the SL ligand and the targeted metal ions.

UV-Visible Titration Studies and Limit of Detection (LOD)
. UV-visible titration experiments were performed in order to determine the limit of detection and further investigate the chemosensing properties of SL towards the targeted Cu 2+ , Fe 3+ , or V 5+ metal ions. As shown in Figures 4(a)-6(a), the absorption band increased gradually for Cu 2+ , Fe 3+ , or V 5+ ions. A calibration curve was made at 470 nm in the case of Cu 2+ , Fe 3+ , and 466 nm for V 5+ ions.

Elucidation of SL-Cu 2+ , SL-Fe 3+ , and SL-V 5+ Complexes
Structure. In order to confrm the formation of metal complexes and the observed stoichiometric ratio for the formation of metal complexes, as well as the possible binding modes of the synthesized SL ligand with the metal ions, the metal complexes were prepared and characterized using FTIR, mass spectroscopy, UV-visible, and 1 H NMR spectroscopy analysis.    a sign of the involvement of the nitrogen atom of the azomethine group in coordination with the metal ion. Also, the band observed at 1693 cm −1 due to the ketonic ](C�O) group of the chromone moiety in the free ligand (SL) suffered a blue shift on complex formation with metal ions and appeared in the range of 1635-1645 cm −1 , which indicates involvement of the oxygen atom of this group in binding to metal ions [15,25,28]. Te broadband due to the phenolic group (-OH) appeared at 3197 cm −1 in the spectrum of the free organic molecule (SL) and disappeared in the spectra of the metal complexes, indicating bonding of the oxygen atom after deprotonation of the phenolic group with metal ions [29]. Te binding of oxygen atoms and nitrogen atoms of the ligand SL with metal ions was supported by the appearance of new bands in the range 487-489 cm −1 and 549-585 cm −1 may be due to ](M-N) and ](M-O), respectively, in the IR spectrum of the metal complexes [1]. Terefore, the synthesized SL ligand is considered a tridentate ligand and has the ability to bind through ONO atoms with metal ions. In the IR spectrum of vanadium complexes, a sharp band was observed in the range 911-960 cm −1 and another broadband in the region 845-830 cm −1 can be assigned to ] asym and ] sym vibrations of cis-VO 2 groups [30]. Moreover, a broadband was observed in the range 3258-3400 cm −1 in the IR spectra of metal complexes that can be attributed to the presence of water molecules [31].

1 H-NMR Spectra.
In order to confrm the complexation of the SL ligand with Fe 3+ and V 5+ , we have recorded the 1 H-NMR spectra of the free ligand and its metal complexes. Te observations showed a downfeld proton appeared at 12.04 ppm and may be assigned to the phenolic -OH group, and an azomethine (HC�N) proton appeared at 8.15 ppm [32]. Te peak due to the phenolic group disappeared in the spectra of Fe 3+ and V 5+ complexes, indicating deprotonation of the OH group and bonding to the metal ions [29]. Moreover, the peak due to azomethine proton sufered a chemical shift and appeared at 8.945, 8.68,   Te diference between the known concentration spiked into the sample and the metal ion detected using the examined sensor may be due to the formation of stable metal complexes with Cu 2+ , Fe 3+ , or V 5+ metal ions.
Te real water samples analysis data ( Table 2). Te calculated recoveries for known amounts of Cu 2+ , Fe 3+ , or V 5+ added ranged from 400.1-657.0%, 42.85-100%, and 57.14-97.14%, respectively. Tese results indicated that SL could be suitable and useful for the sensitive detection of Cu 2+ , Fe 3+ , or V 5+ in real water samples with good precision and accuracy in the case of Cu 2+ and V 5+ and moderate precision for Fe 3+ ions.

Comparison with Other Studies.
Tere are many reported studies that have identifed chromone-based Schif base molecules that are used as colorimetric probes towards some metal ions. Fan et al. reported the use of a novel and simple Schif base receptor based on a chromone derivative called 7-methoxychromone-3-carbaldehyde-(indole-3-formyl) hydrazone as a selective and sensitive probe for Al 3+ with colorimetric and fuorescent responses [33]. Te reported molecule had a sensing limit for recognizing aluminum ions as low as 1.0 × 10 −7 M, which is sufcient for sensing Al 3+ that may be available in environmental and biological systems.
Another chromone-based colorimetric sensor was developed and synthesized by the condensation reaction between 3-formyl-6-methylchromone and 2-aminobenzamide for highly selective detection of Cu 2+ ions in semiaqueous media, which showed sensing ability for copper ions with    diferent concentrations ranging from 10 −3 to 10 −7 in aqueous solutions and showed excellent applicability in real water samples [34]. Colorimetric chemosensors based on the Schif base 2-hydroxy-5-[(2-hydroxy-1-naphthyl) methylideneamino]benzoic acid have been reported to exhibit high selectivity and sensitivity for detecting Cr 3+ , Cu 2+ , Fe 3+ , and Al 3+ ions simultaneously in DMF/H 2 O (v/v � 1/1) solution with LOD of 3.37 × 10 −7 M, 4.65 × 10 −7 M, 3.58 × 10 −7 M, and 4.89 × 10 −7 M, respectively [32]. A chromone-based azomethine chemosensor synthesized by the condensation reaction of 3-formylchromone with 2,6pyridinedicarbohydrazide was successfully used as an immediate and naked-eye sensor for Cu 2+ , Zn 2+ , and CN − ions in aqueous media with excellent accuracy and precision with detection limits of 5.50 × 10 −7 , 8.70 × 10 −7 and 1.56 × 10 −6 M, respectively, which are lower than the permissible levels recommended by WHO for safe drinking water [24]. A chromone-based Schif base molecule synthesized by the condensation reaction between 3-formyl chromone and octopamine was reported by Lee and Kim as sensor probe for Cu 2+ ions only by fuorescence quenching with a LOD of 3.95 × 10 −6 M [35]. Compared with the abovereported chromone-based Schif base colorimetric sensors, our synthesized chromone-based Schif base sensor can be considered selective for detecting three metal ions, namely Cu 2+ , Fe 3+ , and V 5+ ions, in aqueous media with excellent accuracy and precision with detection limits of 3.322 × 10 −5 M, 2.065 × 10 −5 M, and 1.782 × 10 −5 M, respectively, which are lower than the permissible level recommended by WHO for safe drinking water.