A New Azo-Schiff Base Dual-mode Chemosensor: Colorimetric Detection of Cobalt Ions and Fluorometric Detection of Aluminum Ions in Aqueous Ethanol Solution

A new Azo-Schiff base ligand (H2L) was designed and synthesized as a cation chemosensor. The chemosensor H2L as dual chemosensor showed selective fluorescence recognition of Al3+ with a noticeable fluorescence enhancement and colorimetric detection of Co2 + in aqueous ethanol solution. The H2L exhibits a linear response toward Al3+ ions in the concentration range of 1.91 × 10–8 M to 4.8 × 10–6 M with a limit of detection of 1.91 × 10–8 M. The sensing mechanism of sensor H2L toward Al3+ was investigated by 1H NMR and IR spectroscopies. Fluorescence switch based on the control of EDTA and Al3+ proved H2L could act as a reversible chemosensor. The molecular structure of [NiL] complex has been determined by X-ray crystallography.


Introduction
In recent decades, the field of chemosensing has become an interdisciplinary field that consists of researchers from a variety of different backgrounds, including: Inorganic chemistry, analytical chemistry, synthetic organic chemistry, photochemistry, biosciences, physical and materials sciences [1]. Chemosensors have been widely applied for sensing and detection of environmentally and/or biologically important anions, cations, small neutral molecules and biomacromolecules, due to operational simplicity, high sensitivity and selectivity.
Fluorescent and colorimetric chemosensors that detect metal ions have attracted considerable attention due to their wide area of applications. There are increasing number of probes which can detect more than one metal ion. Exploration on fluorescence dual/multiple chemosensors is drawing continuous attention of the researchers for last few years [1][2][3][4]. As a dual chemosensor is able to identify two analytes, time and labor can be saved for the generation of chemosensors for each of the analytes. Aluminum is the third most abundant of all elements after oxygen and silicon and it is the most abundant metallic element in the earth's crust (8.1% by weight). Human exposure to aluminium because of widespread use of aluminum in modern society, such as food additives, production of light alloy and medicines. However, exposure to high levels of Al 3+ ions can cause serious health problems such as Parkinson's disease [5] and Alzheimer's disease [6]. Furthermore, nearly forty percent of the world's acid soils are exposed by the effects of aluminum toxicity that is the significant factor for hampering plant growth on the acid soils [7,8]. Co 2+ is an essential trace element for human body with significant impact on the processes of haematopoiesis-stimulation of erythropoietin production and haemoglobin synthesis [9]. On the other hand, higher concentrations of cobalt result in toxicological effects, such as cardiomyopathy, vasodilatation and flushing [10,11].
Therefore, detection of Al ‫+3‬ and Co 2+ is very significant to monitor the concentration level in the environment their direct impact on human health. There are several different types of chemosensors developed for Al 3+ and Co 2+ detection  but the chemosensors which can both detect Al 3+ and Co 2+ are scarcely reported. In the other hand, the Azo-Schiff base chemosensors for Al 3+ were scarcely reported [35,36]. In this work, an Azo-Schiff base-type Al 3+ and Co 2+ chemosensor (H 2 L) was prepared and then its photophysical properties were studied. The studies showed that H 2 L can be used as a turn-on fluorescence chemosensor for Al 3+ and a colorimetric chemosensor for Co 2+ in aqueous ethanol solution.

Synthesis of [NiL] Complex
To a solution

X-ray Crystallography
X-ray data for [NiL] complex were collected at room temperature with a Bruker APEX II CCD area-detector diffractometer using Mo Kα x-ray radiation (k = 0.71073 Å). Other crystallographic data are summarized in Table 1.

Synthesis
As shown in Scheme 1, The Azo-Schiff base ligand (H 2 L) can be facilely synthesized according to the literature method [37]. The IR spectrum of H 2 L shows the imine stretching vibration at 1640 cm −1 as a strong band, while there are no bands that can be attributed to the primary amine and carbonyl groups, indicating that reaction of the aldehyde with the amine has completely occurred. The O-H stretching frequency was not observed due to its involvement in hydrogen bonding (Intra-ligand hydrogen bond) with imine nitrogen atom (O-H……N). The nickel complex was synthesized by direct reaction between nickel(II) chloride hexahydrate with Azo-Schiff base ligand (Scheme 2). The stretching frequency of (C-O) bond shifted to lower frequency (14 cm −1 ) compared to free ligand. This indicates that the phenolic oxygen atom participates in coordination with nickel ions after losing its proton. The (C = N) stretching frequency in the complex shifted to lower frequency (10 cm −1 ) compared to the free ligand. This evidence also confirms the participation of imine group in coordination with nickel ions. New vibration bands of 564 and 433 cm −1 assigned to υ(M-O) and υ(M-N) respectively, have been appeared in the spectrum of [NiL].
In the 1 H NMR spectrum of H 2 L, the peak related to imine proton was observed at 8.38 ppm. The related peaks to other groups are matched with the structure of the ligand.

Structure of [NiL]
Unfortunately, we were not able to obtain the suitable single crystals for the related Al 3+ complex. Instead, we obtained suitable crystals for Ni 2+ ions. A representation of molecular structure of The [NiL] complex as determined by x-ray diffraction is shown in Fig. 1. Selected angles and bond length are listed in Table 2. The nickel atom is surrounded by two nitrogen atoms, a phenolic oxygen atom and a sulfur atom from the methyl dithiocarboxylate residue. The delocalization of the p-electron density through the six member rings was observed by analysis of the bond length within the metallocyclic part of the complex. A high delocalization of the p-electron density in the cyclopentene fragment were also observed. the mean value for the S1-C15 bond length is 1.693(6) Å and which is considerably lower than what is usually observed for a single S-C sp3 bond, 1.808(10) Å, and similar to the 1.712(17) Å attributed to the S-C sp2 bond in thiophene [33]. Furthermore, in the cyclopentene fragment, the bond length observed between the C10-C14 carbon atoms has a mean value 1.421 (7) Å, that is similar to the mean value for the C sp2 -C sp2 bond in conjugated systems [38]. Finally, the similarity of the N1-C7 and N2-C10 bond length values is indicative that this fragment coordinates in a Schiff base mode (1.276(7) and 1.309 (7), respectively). The complex is tetracoordinated and in a square planar geometry. The degree of the distortion will be quantified by measuring the dihedral angle between planes formed by the N1/Ni/O and N2/Ni/S atoms of the two semi-coordination spheres shows the degree of the distortion that for The complex presents is 0.00°. The plane containing S1, N1, N2, O1 has r.m.s. values of 0.002 Å and the sum of the angles subtended by the donor atoms at Ni(II) for [NiL] is 360°. The nickel atom for [NiL] is close to co-planarity, being only 0.001 Å out of the plane containing S1, N1, N2, O1.

Spectral Properties of Fluorescent H 2 L
It is known that Schiff base ligands are good compounds for fluorescence studies and used to develop the field of chemosensing. The ligand of H 2 L (10 μM) exhibits a fluorescence emission peak at 419 nm when excited at 252 nm.  Fig. 2.
As shown in Fig. 2, the fluorescence emission intensity of H 2 L is only increased in the presence of Al 3+ ions. the fluorescence emission is completely quenched in the presence of other tested metal ions (K + , Mg 2+ , Sn 2+ , Hg 2+ , Cr 3+ , Fe 2+ , Fe 3+ , Mn 2+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , Cd 2+ and Pb 2+ ions. The highest FEF (fluorescence enhancement Factor) for H 2 L was observed in the presence of Al 3+ ions (Fig. 3). The FEF was calculated by using FEF = (Ix-I H2L )/I H2L that I H2L and Ix are the fluorescence intensities of H 2 L before and after addition of metal ions at 419 nm, respectively. The FEF values of H 2 L related to different metal ions seen in Fig. 3.
To investigate practical applicability of H 2 L as a selective fluorescence chemosensor for Al 3+ ions, the competition experiments were also performed in the presence of other metal cations (Fig. 4). The results show that the fluorescence emission of [AlL] + (complex of H 2 L with Al 3+ ) do not significantly change in the presence of K + , Mg 2+ , Al 3+ , Sn 2+ , Hg 2+ , Cr 3+ , Fe 2+ , Mn 2+ , Co 2+ , Ni 2+ , Zn 2+ , Cd 2+ and Pb 2+ + with a concentration up to 100 mM. In the presence of Cu 2+ and Fe 3+ ions had a disturbance under their much excess.
The fluorescence titration of receptor H 2 L with Al 3+ was performed by mixing a constant concentration of H 2 L with various amounts of Al 3+ ions. Figure 5 shows changes of the fluorescence intensity of H 2 L after the addition of various concentrations of Al 3+ ions. The fluorescence emission intensity at 419 nm enhanced gradually along with the increase of concentration of Al 3+ . It reaches to maximum value when the concentration of Al 3+ achieved to 1 equiv, indicating the 1:1 binding of H 2 L to Al 3+ ‫‬ (Fig. 5).  The binding stoichiometry of H 2 L and Al 3+ was studied by the job plot method. The molar ratio was selected by [Al 3+ ]/([ Al 3+ ] + [H 2 L]) and measured from 0 to 0.9. The entire concentrations were kept at 10 µM. The maximum value was observed when the molar ratio was 0.5 (Fig. 6). This means that there is a 1:1 molar ratio for H 2 L-Al 3+ complex in the binding stoichiometry. The fluorescence quantum yields (fc) for the ligand and H 2 L-Al 3+ complex were measured using the ratio method. The values of fc were 0.03013 and 0.14428 for H 2 L and Al-H 2 L complex, respectively.
On the basis of analytical and spectroscopic studies, a sensing mechanism of receptor H 2 L to Al 3+ ion is proposed. The both imine and hydroxyl groups of the receptor H 2 L are involved in the complexation with Al 3+ ions. Therefore, the receptor H 2 L acts as a fluorescence "off -on" sensor with respect to Al 3+ . The weak fluorescence of the receptor H 2 L may be largely due to the photo-induced electron transfer (PET) process from the imine nitrogen or hydroxyl oxygen to diphenyldiazene derivative. Moreover, the receptor H 2 L was freely movable along the C-C covalent linkage between diphenyldiazene derivative moiety and the imine bond. Since, the absence of structural rigidity in the receptor molecule because of the intramolecular rotation, therein the charge transfer-excited singlet state is abruptly deactivated. However, the strong fluorescence exhibited upon the complexation of receptor H 2 L with Al 3+ (1:1) due to the chelation-enhanced fluorescence (CHEF) effect, which resulted in the arrest of both PET process as well as C-C rotation that further increases the rigidity of the molecular assembly.

Limit of Detection and Response Time
The limit of detection of H 2 L was calculated using the fluorescence titrations. limit of detection was obtained based on the equation of 3σ/s that s is the slope of the calibration curve and σ is the standard deviation of the blank  The response time of the chemosensor toward Al 3+ was studied by the kinetics of fluorescence enhancement at 419 nm upon analyzing different concentrations of Al 3+ (Fig. 8). The response time of H2L toward Al 3+ is concentration-dependent, as the time required to reach equilibrium increases with the increase of Al 3+ concentrations. However, in all cases, the stable reading could be obtained in less than 20 s. Therefore, this chemosensor could be used for real-time tracking of Al 3+ . As seen in Fig. 8 once a plateau is reached, the fluorescence intensity at 419 nm stays almost unchanged the rest of the time, indicating that the chemosensor is photostable under irradiation with visible light.

Binding Constants Measurements
The Benesi-Hildebrand plot was used to calculate the association constant (K a ) for H 2 L with Al 3+ at room temperature. The fluorescence titration spectra of H 2 L (10 µM) were performed in the presence of the various concentrations of Al 3+ (0-1.2 µM). The emission intensity of fluorescence was gradually increased when various concentrations of Al 3+ ions were increased (Fig. 9). As seen in Fig. 9, at concentrations of more than 10 µM of Al 3+ , the fluorescence emission reached to the constant value.
I and I 0 represent the fluorescence intensity of H 2 L in the presence and absence of Al 3+ ions, respectively, I max is the fluorescence emission of H 2 L in the presence of the excess amount of Al 3+ .
A linear relationship was observed when emission intensity [1/(I − I 0 )] was drawn versus a function of 1/[ Al 3+ ] (R 2 = 0.99). The resulting Benesi-Hildebrand plot, shown in Fig. 8, had a K a of 1.76 × 10 6 M −1 . The K a obtained illustrates a strong binding ability of Al 3+ with the H 2 L.

The Chemical Reversibility Behavior
The chemical reversibility behavior of the binding of H 2 L and Al 3+ was studied. It could be expected that the addition of EDTA will liberate Al 3+ from the [AlL] + complex, releasing H 2 L, because of the high stability of the EDTA-Al 3+ complex. Therefore, excess of EDTA was added to the [AlL] + complex in buffered solution, which shows a remarkable decrease of fluorescence signal at 418 nm, whereas readdition of excess Al 3+ could recover the fluorescence signal (Fig. 10). These finding show that binding of Al 3+ with H 2 L is chemically reversible, which can be used for the dynamic monitoring of Al 3+ in various samples.  The reported LOD values for Al 3+ in some of literatures and in the present work have been listed and compared (Table 3). As seen in Fig. 3, H 2 L has suitable situation among the other chemosensors in terms of low LOD value [12-21, 35, 36].

Infrared Studies
The IR spectrum of H 2 L shows a band at 1640 cm −1 related to stretching vibration of imine group (Fig. 11). As seen in Fig. 10, the imine band of H 2 L has been shifted to lower frequency 1637 cm −1 in the aluminum complex. This shift can be associated to the coordination of imine nitrogen atom to the metal atom. The coordinate covalent is formed between nitrogen and aluminum by the donation of electrons from C = N bonding orbital to the aluminum atom.

NMR Studies
The 1 H NMR spectra of H 2 L were recorded in the absence and presence of and Al 3+ ions ( Fig. 12) As shown in Fig. 11, upon the addition of Al 3+ , the resonance signals corresponding to imine protons shift downfield from 8.41 to 8.98 ppm. When Al 3+ was added, the signals corresponding to hydroxyl protons (-OH) and secondary amine (NH) were disappeared duo to deprotonation of the hydroxyl groups and secondary  amine to strongly coordinate to Al 3+ . This result indicates that a more efficient coordination of Al 3+ ion occurs with the imine nitrogen (C = N), the nitrogen (-NH) of secondary amine and hydroxyl groups. When Al 3+ ion is coordinated to H 2 L, the charge transfer was arrested due to the strong complexation of Al 3+ with H 2 L.

Molar Conductivity Measurements
The molar conductivities (Ʌ m ) of the AL(III) complex in DMF solution at 25 C were found to be in the range 94-105 Ω −1 cm 2 mol −1 . The Ʌ m value is in the range reported for 1:1 electrolytes. The [H 2 L] is coordinated to the Al 3+ ions as a doubly negatively charged anions. Therefore, it seems two phenolic OH have been deprotonated and bonded to the aluminums ions.

Colorimetric Sensing for Co 2+
The colorimetric sensing ability of H 2 L was investigated in EtOH/H 2 O (9:1, v/v) upon the addition of a variety of metal ions such as K + , Mg 2+ , Al 3+ , Sn 2+ , Hg 2+ , Cr 3+ , Fe 2+ , Fe 3+ , Mn 2+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , Cd 2+ and Pb 2+ . The changes in the UV-Vis spectra of H 2 L in the presence of the various metal cations is shown in Fig. 13. The prominent band appearing at around 350 nm is because of electron migration along the entire conjugate system of the ligand. The entire conjugate system is consist of both azo group and the aryl rings. For azomethine or azo dyes that a hydroxyl group is in opposition to the N-N or C-N bond on the aromatic ring the intramolecular CT band is generally broadness [39]. The band observed at around 470 nm can be assigned to an n-π* electronic transition of azo-aromatic chromophore [39,40]. When Co 2+ was added, the major absorption peak at 350 was red shifted to 390 nm and a new peak at 530 nm was generated in the visible region (Fig. 14). However, no such change for H 2 L in the presence of other test metal cations was observed (Fig. 13). Consistent with the spectral changes, the color of H 2 L solution changed from intense yellow to pale yellow on addition of Co 2+. The noteworthy visible color change indicated that the compound H 2 L could serve as a potential candidate of a "naked-eye" chemosensor for Co 2+ .
Application H 2 L can be used as a fluorescence probe for measuring of Al 3+ concentration in drinking water. As seen in Table 4, the results were agreeing and satisfactory with AAS (atomic absorption spectroscopic) method. The relative errors were less than 6.6%.

Logic Gate Operation
H 2 L molecule can be perform as a molecular logic gate switch based on fluorescence emission as output and  metal ions as input. The inputs were selected as Al 3+ ions (input1) and Cu 2+ ions (input 2) and the fluorescence emission intensity at 419 nm was selected as the output. When H 2 L was exposed to Al 3+ (input 1), The fluorescence emission intensity was increased at 419 nm. The fluorescence emission intensity was completely quenched, upon addition of Cu 2+ (input 2, in the presence of Al 3+ . The fluorescence band of H 2 L was also disappeared when titration was carried out with Cu 2+ alone. As discussed above, the fluorescence emission intensity is high enough (output 1 = 1) only under the addition of Al 3+ (input 1 = 1 and input 2 = 0). According to the truth table of Fig. 15, the fluorescence responses of H 2 L satisfy the INHIBIT type logic gate function at the molecular level with the two chemical inputs Al 3+ and Cu 2+ .

Conclusion
In conclusion, a new fluorescent turn-on chemosensor was added into the library of Al(III) selective chemosensors. This Azo-Schiff base receptor detects Al(III) in the aqueous ethanolic medium by giving significant fluorescent enhancement at 419 nm due to the photo-induced electron transfer (PET) and the C = N isomerization mechanism. The association constant of the [H 2 L-Al 3+ ] complex is 1.76 × 10 6 M −1 . The lower detection limit of Al 3+ is 1.91 × 10 -8 M and indicate on suitable situation of H 2 L among the other chemosensors in terms of low LOD value. The chemosensor also displayed high colorimetric sensitivity for Co 2+ ions. Molecular logic gates INHIBIT is proposed using Al 3+ and Cu 2+ as inputs.