Introduction

Mercury is widespread in the environment as a result of disposal of industrial and agricultural waste. Its concentration is continuously increasing in the environment due to its extensive industrial applications [1, 2]. Mercury is one of the most hazardous metal ions which can cause substantial damage to human beings even at low concentrations due to its long residence, bioaccumulation and permanent deterioration in the endocrine and central nervous systems, prion and alzheimer’s diseases vasodilatation, irritability, paralysis, blindness, result in hearing loss, mental deterioration, speech difficulty, impaired vision, vestibular dysfunction and autism, affect liver and bones upon accumulation in the body, affect stomach, and genes, can causes chromosome breakage, and birth defects, even death [3,4,5]. It can exist in ionic (Hg2+), elemental (Hg0), and organic (CH3Hg+) forms. Mercuric ion (Hg2+), more common than mercurous ion (Hg+), is a caustic and carcinogenic material with high cellular toxicity. Especially inorganic Hg2+ is a serious pollutant; thus, it must be removed properly from natural water and wastewater effluents.

The determination of trace amounts of heavy metal ions (such as Hg2+) is of interest in many fields including environmental analysis, process control, biology, and medicine. The growing awareness of environmental mercury pollution and toxicity necessitates its determination, even at very low concentrations in water. For drinking water, the allowed mercury level is 1 µgL− 1, set by World Health Organization (WHO) [6, 7].

Transition metal complexes containing Schiff base ligands have been a subject of specific interest in synthetic chemistry [8]. Compounds containing an azomethine group (–CH = N–) are known as Schiff bases. Schiff bases have different applications in various fields such as electrochemistry, bioinorganic, catalysis, metallic deactivator, separation processes, organic syntheses and environmental chemistry. For example, Schiff bases with N2O2 donor were synthesized by the reaction of different 3,5-dihalosalicylaldehyde (halo atoms equal to Cl, Br and I) with polymethylenediamines of varying chain length for antibacterial activities [9, 10]. A Schiff base bis(thiophen-2-yl-methylene)benzene-1, 4-diamine (L) was synthesized and used for selective and sensitive detection of Fe3+ [11]. Schiff bases like (E)-2-(((5-bromopyridin-2-yl)imino)methyl)-6-ethoxyphenol and (Z)-2-ethoxy-6-(((3-hydroxypyridin-2- yl)amino)methylene)cyclohexa-2,4-dien-1-one have been efficiently prepared by reaction of 2-aminopyridine derivatives with 3-ethoxysalicylaldehyde under ultrasonic irradiation [12]. Similarly, to study the influence of substitution on the DNA binding five Schiff bases were synthesized [13]. They were prepared by condensation of 2-hydroxy-5-bromobenzaldehyde with 4-aminobenzoic acid, 3-aminobenzoic acid and 3-amino-4-methylbenzoic acid with 1:1 M ratio, respectively for the first three Schiff bases and the other two Schiff bases were prepared from condensation of 4-aminobenzoate with 3-ethoxy-2-hydroxybenzaldehyde and 3-methoxy-2-hydroxybenzaldehyd with 1:1 M ratio, respectively [13]. The Schiff base N-(3-methoxy-hydroxybenzylidene) isonicotinohydrazide were synthesized from isonicotinohydrazide and 3-methoxysalicylaldehyde to a study antitubercular and antimicrobial activity [14].

New hetero-binuclear Hg-Cu Schiff base complexes were prepared by reaction of 2,2’-[1,1’-(2,2-dimethylpropane-1,3-diyldinitrilo)-diethyldyne]diphenolato copper(II) and6,6’-ethoxy-2,2’-[(2,2-dimethylpropane-1,3-diyl)bis(nitrilomethanylylidene)]diphenolatocopper(II) reacted with HgCl2 to give the corresponding binuclear complexes [15]. In other works, a new carbon paste electrode modified with N,N′-bis (5-bromo-2-hydroxy benzyli dene)-2,2-dimethylpropane-1,3-diamine Schiff base ligand was synthesised for selective and effective determination of Hg2+ ions in aqueous environmental samples using cyclic and square wave anodic stripping voltammetric methods [16].

Even though many methods have already been developed to detect Hg2+, there is still a need to develop more sensitive, selective, and simple way to detect the mercury ion existing in environmental samples. Fluorescence detection is more advantageous than other techniques due to its high sensitivity, applicability to field investigation, and low cost [17,18,19]. In this study, a Schiff base containing nitrogen and sulphur was developed as a sensor having a low detection limit for Hg2+ in aqueous samples.

To the best of the authors’ knowledge, no prior studies have estimated theoretical and experimental values by using (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl-methylene) benzenamine for fluorescence detection of Hg2+ in different water samples. In this regard, a detailed theoretical and experimental investigations into the vibrational spectra and thermodynamic data of the molecule was conducted.

Materials and methods

Synthesis of the Schiff base, (E)-2-(2-aminophenylthio)-N-(Thiophen-2-yl-methylene) Benzenamine

Reagents and chemicals were obtained from Avra Synthesis (Hyderabad India) and TCI Chemicals (Chennai India). They were used without further purification. A 20 mL ethanol solution of Bis(2-aminophenyl) Sulfide (5 mM, 1.082 g) was mixed on stirring with a 20 mL ethanolic solution of thiophene-2-carbaldehyde (5 mM, 0.468 g), followed by being refluxed at 60 °C for 8 h. After cooling in the deep freezing for 2 days the yellow product was collected washed with ethanol and dried in desiccator over CaCl2. Scheme 1 describes the procedure of synthesizing the Schiff base.

Scheme 1
scheme 1

Synthesis of the Schiff base (E)-2-(2-aminophenylythio)-N-(thiophen-2-yl-methylene) benzenamine

Full size image

Characterization and Theoretical Study of the Schiff Base

Completion of the reaction was checked by using thin layer chromatography (TLC). Proton nuclear magnetic resonance (1HNMR) and carbon-13 nuclear magnetic resonance (13 C {1 H} NMR) spectra were obtained by using a BRUKER AVACE II 400 MHz NMR spectrometer. For the Fourier transform infrared (FTIR) analysis, a Perkin Elmer 100 FTIR spectrometer was used. A Shimadzu UV-1800 spectrophotometer was employed to obtain UV-Vis spectra. Fluorescence of the sample was recorded using a Shimadzu RF 5301PC Spectrofluorophotometer. The theoretical studies were carried out using Gaussian 09 software.

Preparation of Real Water Samples

Three water samples were collected from different areas: tube well water from Mehmoodpur Araian, drinking water and tap water from Chemistry Department Punjabi University, Patiala (India). Prior to any experiment, the tube well water was centrifuged to remove the impurities such as sands. Before doing any further sample pre-treatment, all the collected ground water samples were investigated by the synthesised probe for Hg2+presence, so as to show the applicability of the reported probe under real world conditions. However, Hg2+ was not detected or was below the method detection limit in the ground water samples. The water samples were spiked with different concentrations of Hg2+. From each 10.0 mL of water sample were taken and diluted with buffer of pH = 8.0 solution in a 25 mL volumetric flask. Different amount of (0, 5, 10, 15, 20, 30, 40, 50 mL) 10 µM Hg2+ standard solution was added and filled to the mark. From the calibration curve of spike vs. volume of standard added concentration of Hg2+ ions in water samples were calculated.

Results and Discussion

Experimental Study

1HNMR and 13 C{1 H} NMR Analysis

1HNMR of the (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl-methylene) benzenamine taken at the conditions (400 MHz, dimethyl sulfoxide (DMSO-d6) solvent, δ in ppm) showed a chemical shift at 5.39 (s, 2 H of NH2), 6.5814–7.0798 (m, 8 H, of Aromatic), 7.3202–7.8765 (m, 3 H, of thiophene), and 8.77 (s, 1 H of CH = N). The presence of 14 protons at different chemical shifts indicated the formation of the Schiff base as expected. The absence of picks at around 9.0–10.0 ppm indicated that aldehyde hydrogen is replaced, instead of azomethine hydrogen appeared at 8.77 ppm. FiguresS1 and S2 shows 1HNMR and 13 C{1 H} NMR spectrum of the Schiff base, respectively.

13 C{1 H} NMR analysis also confirmed the formation of the ligand, indicating the number of carbon atoms present at correspond chemical shifts. There were 17 carbons in the ligands. Different carbons appeared at different chemical shifts. Each carbon was indicated as follows:(C-8, 111 δ), (C-12,114 δ), (C-1,116 δ), (C-10, 118 δ), (C-17, 125.25 δ), (C-3,125.79 δ), (C-16, 126 δ), (C-4, 128 δ), (C-18,131.07 δ), (C-11, 131.75 δ), (C-5, 132 δ), (C-9, 133 δ), (C-2, 137 δ), (C-19,142 δ), (C-13, 147 δ), (C-21, 150 δ), and (C-6, 153 δ).

FTIR Analysis of The Ligands

From the FTIR spectrum of the ligand (FigureS3) at 3441 and 3326 cm− 1 for N-H stretching, 3055 cm− 1 for stretching of sp2 hybrid C-H,1607 cm− 1 for N = C stretching, 1462 cm− 1 for aromatic C-C stretching, and 713 cm− 1for C-S-C stretching. The results indicated the formation of azomethine bond.

Electronic Spectroscopy Analysis Results

The UV–Vis spectra of the Schiff base were examined in the range of 200–800 nm in six solvent systems such as ethanol, methanol, DMSO, N,N-dimethylformamide (DMF), DMF/H2O (7/3; v/v), and acetonitrile/H2O (7/3; v/v). Among these solvent systems, DMF/H2O gave the best results, thereby being chosen for further spectroscopic studies. The UV–Vis spectra are shown in Fig. 1. The maximum absorptions observed in the range from 320 to 250 nm were attributed to π→π* transitions of π electrons within the structure. Absorption intensity of π→π* transitions decreased in an order: DMF/H2O > DMSO > CH3CN > ethanol > methanol > acetonitrile/H2O (hyper chromic effect). The absorption intensity at 220 nm belonged to n→π* transitions was observed in the DMF/H2O system. Absorption spectra for the complexes were also recorded in the DMF/H2O solution. In the spectra of the complexes, π→π* and n→π* transitions observed in the ligand was not affected by any kind of metal ions. The absorption at 280 nm shifted to 320 nm by complexing with Hg2+ (bathochromic shift), indicating chelation of the Schiff base with Hg2+ ions. To further confirm sensitivity of the Schiff base to Hg2+, fluorescence studies were conducted.

Fig. 1
figure 1

UV spectra of the (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl-methylene) benzenamine and its metal complexes (1 × 10− 5 M pH = 80)

Full size image

Quantum Yield

The fluorescence quantum yield (Φ) of Schiff base was noted as 0.21 in ethanol by taking anthracene as standard. The fluorescence excitation (310–365 nm) and fluorescence emission wavelength (370–450 nm) of anthracene corroborated with the excitation (310 nm) and emission wavelength (440 nm) of Schiff base. Anthracene displayed quantum yield of 0.27 in the ethanol.

Fluorescence Spectral Studies of the Schiff Base and its Metal Complexes

The fluorescence spectra of the (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl-methylene) benzenamine (1 × 10− 5 M) obtained in the DMF /H2O (7:3 v/v; pH = 8.0) system with metal ions such as Ag+, Mn2+, Fe3+, Al3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Fe2+, and Cr3+ (1.0 equiv.) are shown in Fig. 2. In free state, it was displayed a weak fluorescence emission band at 440 nm up on excitation at 310 nm. The emission intensity at 440 nm was increased with an addition of Hg2+, attributed to strong interactions between the Schiff base and Hg2+. However, no significant variation in the emission intensity was observed with additions of other metal cations. The high selectivity toward Hg2+ was likely due to a compatible ion size and a high binding affinity between the metal ions and the Schiff base. Therefore, the (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl-methylene)benzenamine could serve as a highly selective “turn-on” fluorescent chemosensor for Hg2+.

Fig. 2
figure 2

Fluorescence spectra of the (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl-methylene) benzenamine (1 × 10− 5 M) with Hg2+ and other metal ions

Full size image

Fluorescence Titrations of the Schiff Base with Hg2+

As shown in Fig. 3, an increase in fluorescence intensity was observed in the DMF/H2O (7:3 v/v) solution with increasing Hg2+concentration from 0.1 to 1.0 equi. The maximum concentration was 1.0 equi. of 1 × 10− 5 M Hg2+.

Fig. 3
figure 3

Fluorescence spectra of (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl-methylene) benzenamine (1 × 10− 5 M) with addition of increasing amount of Hg2+

Full size image

Determination of Limit of Detection and Comparison with Literature Values

Limit of detection (LOD) of a fluorescence sensor was determined as shown in Fig. 4 which illustrates a plot of emission intensity vs. concentration of Hg2+ (LOD = 3σ/k, where δ standard deviation of the blank, k is slope and found to be 3.8 × 10− 8 M). The detection limit of the sensor developed in this study was compared to previously reported values (Table 1). According to the comparison (Table 1), the (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl-methylene) benzenamine was effective to detect Hg2+ with a lower detection limit.

Fig. 4
figure 4

Linear plot of fluorescence emission intensity versus concentration of Hg2+

Full size image
Table 1 The comparison LOD of (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl-methylene) benzenamine with other Schiff bases reported in previous literature
Full size table

Determination of Binding Constant

Binding constant was calculated based on the fluorescence intensity data using the Benesi˗Hildebrand equation [20]. Figure 5 shows linear relationship between (Fmax- Fmin)/(F-Fmin) and 1/[Hg2+]. The binding constant of the complex of the (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl-methylene) benzenamine and Hg2+ was calculated as 1.5 × 104 M− 1.

Fig. 5
figure 5

Benesi-Hildebrand plot of (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl-methylene) benzenamine (1 × 10− 5 M) at different Hg2+ concentration

Full size image

Determination of Stoichiometry of The Complex

To identify stoichiometry between the (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl-methylene) benzenamine and Hg2+, the fluorescence behavior was studied by using the Job’s method (Fig. 6). Their total concentrations were kept constant, and mole fraction of Hg2+ was varied from 0 to 1.0. The maximum intensity was achieved when the molar fraction of Hg2+ reached 0.5, indicating the stoichiometry of the (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl-methylene) benzenamine to Hg2+ be 1:1.

Fig. 6
figure 6

Job’s plot of (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl-methylene) benzeneamine with Hg2+

Full size image

Selectivity Study

Selectivity was assessed through competitive experiments. The changes in fluorescence of the Schiff base in the DMF/H2O (7:3 v/v) solution were measured by the treatment of Hg2+ (1.0 equiv.) in the presence of other interfering metal cations such as Ag+, Mn2+, Fe3+, Al3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Fe2+, and Cr3+ (1.0 equiv.) (Fig. 7). No obvious change in fluorescence was observed regardless of existence of other cations. Relative error (%) was calculated using the relation: Relative error % = (ΔF/F0 × 100%) [21], where ΔF is the difference of fluorescence intensities before and after exposure to interferent cations. Note that the relative error less than ± 5% can be acceptable. The value of the relative error listed in Table 2, showing that the Schiff base had a high selectivity to Hg2+ despite the existence of the interferent ions.

Fig. 7
figure 7

Competition experiment of (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl-methylene) benzenamine selectivity toward Hg2+ in the presence of metal other ions

Full size image
Table 2 Effect of interferent cations on the fluorescence signal of the optical sensor
Full size table

Effect of pH and Solvent

Without Hg2+, the weak fluorescence intensity of the Schiff base solution could be observed from pH 1.0 to 12. Upon the addition of Hg2+ (1.0 equiv.), the fluorescence intensity was increased from 100 to 900 a.u. with an increase in pH from 6 to 8. It was recognized that at low pH values, the Schiff base binding to Hg2+ was prevented. A further increase in pH from 8 to 12 decreased the fluorescence intensity to 100 a.u (Figure S4). At high pH values, weak fluorescence was presented. In addition, as shown in FigureS5, the optimum fluorescence enhancement occurred in the presence of the DMF/H2O solvent. Therefore, pH = 8.0 and the DMF/H2O solvent were selected for further studies.

Theoretical Study

In this section, computational data of the (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl- methylene) benzenamine were compared with the experimental results discussed in Sect. 3.1. Energy of the lowest unoccupied molecular orbitals (ELUMO) indicates the tendency of a molecule to accept electrons from donor molecules. The lower this energy, the better the chance to accept electrons. A larger difference in energy between the highest occupied molecular orbitals (HOMO) and lowest unoccupied molecular orbital (LUMO) means a higher stability of molecules and complexes. Hardness (ƞ) is the measure of resistance to charge transfer. This property can be calculated from half of the difference between EHOMO and ELUMO. Chemical softness (S) is the capacity of an atom or group of atoms to receive electrons, which is half of the hardness (ƞ) [22, 23]. The binding energy between the metal cation and ligands can be calculated using the following relation [24]:

$$\triangle{E}={\text{E}}_{\text{Complex}}-({\text{E}}_{\text{cation}}+{\text{E}}_{\text{ligand}})$$

where ΔE is the change in energy; Ecomplex is the total energy of the complex; Ecation is energy of cation; and Eligand is energy of the ligand. All the investigated parameters of theoretical studies including vibrational analysis, NMR spectra, thermodynamic parameters, mulliken charge distribution, natural bond orbital analysis, natural electron configuration are presented in the Section S1 of supplementary information.

Geometrical Optimization and Frontier Molecular Orbitals (FMO)

The optimized geometries of Schiff base and Schiff base complex with Hg was obtained by using a basis set of LANL2DZ/B3LYP are shown in Fig. 8. Frontier molecular orbitals (FMO), HOMO, and LUMO play an important role in quantum chemistry. The FMO theory is useful to predict relative reactivity based on properties of the reactants [25]. The difference in energy between FMO of the (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl-methylene) benzenamine was calculated, and the results are shown in Fig. 9a (EHOMO = − 0.198 a.u. and ELUMO = − 0.067 a.u.; the change calculated as: ΔE = HOMO-LUMO, − 0.198− (− 0.0671) a.u. = −0.130 a.u.; Ƞ=1/2(HOMO-LUMO) = ½(0.130) = 0.065 a.u., S= ½(ƞ) = ½(0.065) = 0.0325 a.u.). When the Schiff base was complexed with Hg2+, the energy of the complex became lower (Fig. 9b). The energy for free (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl-methylene) benzeneamine was found to be − 1561 hartree while that of the complex with Hg2+ was − 1615 hartree. The calculated change in energy of the Hg2+ complex was–12.58 hartree using following equation:

Fig. 8
figure 8

Optimized structure of the Schiff base and Schiff base-Hg complex

Full size image
Fig. 9
figure 9

HOMO and LUMO energy diagram of the Schiff base and Schiff base-Hg complex

Full size image
$$\triangle{E}={\text{E}}_{\text{Complex}}-({\text{E}}_{\text{ocation}}+{\text{E}}_{\text{ligand}})$$
$$\triangle{E}=\text{-1615}-(\text{-41.42}+\text{(-1561))=-12.58 hartree}$$

By using the same basis set energy of different metal complexes of (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl-methylene) benzenamine were studied and compared for the relative stability. The smallest energy was obtained for the complex between the Schiff base and Hg2+. This result indicated that the Schiff base forms more stable complexes with Hg2+ than the other metal ions tested in this study. This agreed with the fluorescence study for the selectivity of the Schiff base.

Application to Real Water Samples

To verify practical application of the Schiff base, standard solution of Hg2+ was spiked to real water samples (e.g., tap water, drinking water, and tub well water) and fluorescence was monitored in situ to quantitatively detect Hg2+ ions in the samples. Table 3 shows the experimental results obtained using the real water samples. The recoveries of the real water samples were in the range from 98 to 104% with relative standard deviations (%RSDs) varied from 1.3 to 2.8%. The results showed that the developed optical sensor (i.e., the Schiff base) for Hg2+ is effective to detect mercury ion in real water, and the proposed mercury detection method may have great potential for applications to monitoring of mercury contained in water in nature.

Table 3 Determination of Hg2+ in different water samples from different sources by a standard addition method
Full size table

Conclusions

Herein, a fluorescence probe of Schiff base,(E)-2-(2-aminophenylythio)-N-(thiophen-2-yl-methylene)benzenamine was developed. The Schiff base effectively detected Hg2+ in different water samples such as tap water, drinking water, and tube well water. The method using the Schiff base was validated by recovery study, interference study, and relative standard deviation study. The Schiff base was also effective to detect Hg2+ in the presence of competing cations (e.g., Ag+, Mn2+, Fe3+, Al3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Fe2+ and Cr3+). The Schiff base, (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl-methylene) benzenamine, exhibited a detection limit of 3.8 × 10− 8 M with a binding constant of 1.5 × 104 M-1, which is lower than the detection limit of typical Hg2+ detection methods. The experimental findings were supported by theoretical studies. In the theoretical part, different metals were simulated to investigate the metal complexes based on their stability. To identify what kind of complexes of the Schiff base is stable, the structure of different metals were optimized. Based on the theoretical study results, it could be concluded that the Schiff base, (E)-2-(2-aminophenylthio)-N-(thiophen-2-yl-methylene) benzenamine, selectively forms a stable complex with Hg2+ than the other metal ions tested in this study. The results of recovery study indicated that the Schiff base is effective for the detection of Hg2+. The proposed detection method may have great potential for the application to monitoring mercury ion in real environmental samples.