A hydroxypropiophenone-based fluorescent probe for the selective determination of Al(III) ions in aqueous ethanol European Journal of Chemistry 14 (1) (2023) 99-108 European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2023 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. https://dx.doi.org/10.5155/eurjchem.14.1.99-108.2360 European Journal of Chemistry View Journal Online View Article Online A hydroxypropiophenone-based fluorescent probe for the selective determination of Al(III) ions in aqueous ethanol Chandni Singh 1, Divya Pratap Singh 2,*, Sunil Kumar Singh 1,*, Romi Dwivedi 3, Ashish Kumar Singh 1,* and Vinod Prasad Singh 3 1 Department of Chemistry, Guru Ghasidas Vishwavidyalaya, Bilaspur-495009, India 2 Department of Chemistry, Rajkiya Engineering College, Azamgarh-276201, India 3 Department of Chemistry, Institute of Science, Banaras Hindu University, Varanasi-221005, India * Corresponding author at: Department of Chemistry, Rajkiya Engineering College, Azamgarh-276201, India. e-mail: dpsingh_011@yahoo.co.in (D.P. Singh), singh.skumar@gmail.com (S.K. Singh), ashish.bhuchem@gmail.com (A.K. Singh). 10.5155/eurjchem.14.1.99-108.2360 Received: 20 November 2022 Received in revised form: 03 January 2023 Accepted: 12 January 2023 Published online: 31 March 2023 Printed: 31 March 2023 In this work, we have synthesized a novel dihydrazone-based fluorescent probe N'1,N'2- bis{1-(2-hydroxyphenyl)propylidene}oxalohydrazide (H2hpoh)for Al3+ ions by a simple condensation reaction. The prepared organic probe has been characterized by different physicochemical and spectroscopic techniques. The single-crystal structure of the receptor has also been reported. Crystal data for C20H22N4O4: monoclinic, space group P21/c (no. 14), a = 6.0747(15) Å, b = 11.621(5) Å, c = 13.453(4) Å, β = 94.61(3)°, V = 946.6(5) Å3, Z = 2, T = 293(2) K, μ(MoKα) = 0.096 mm-1, Dcalc = 1.342 g/cm3, 4046 reflections measured (6.076° ≤ 2Θ ≤ 58.05°), 2149 unique (Rint = 0.0876, Rsigma = 0.2223) which were used in all calculations. The final R1 was 0.0972 (I > 2σ(I)) and wR2 was 0.2316 (all data). The ethanolic aqueous solution of the probe shows enhanced fluorescence in the presence of Al3+ ions, whereas no appreciable change in the spectral pattern is observed in the presence of other cations, i.e., Na+, K+, Ca2+, Ba2+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cr3+, Cd2+ and Hg2+. The binding mode of the receptor with Al3+ ions was studied using various spectral titration techniques such as UV-visible, fluorescence, and 1H NMR. The receptor acts as a dibasic hexadentate ligand and interacts with two Al3+ ions with a high binding constant KB = 8.99×1010 1/M. The lowest detection limit for the Al3+ complex of H2hpoh was determined to be 7.8×10−5 M. With the help of DFT calculations, the mechanism of fluorescence enhancement has been explained. Dihydrazone UV-vis study Chemical sensor Fluorescence study Density functional theory Chelation-enhanced fluorescence Cite this: Eur. J. Chem. 2023, 14(1), 99-108 Journal website: www.eurjchem.com 1. Introduction The selective identification of chemical and ionic species that are important to the environment using molecular sensors has received great attention [1-7]. Effective chemical sensors have been designed and developed using non-covalent interac- tions such as Vander Waal’s contact, hydrogen bonding, electro- static force, metal-ligand coordination, and hydrophobic inte- ractions [8-10]. Govindaraju et al. suggested that 'Photoinduced electron transfer (PET) based sensors exhibit changes in emission intensity with little or no spectral shift, whereas internal charge transfer (ICT) sensors exhibit both intensity changes and spectral shifts' [9]. The design of receptors for the detection of aluminum ions may be significantly influenced by the use of Schiff bases in homogeneous or heterogeneous environments. Aluminum is widely used for industrial and domestic purposes. The toxicity of aluminum towards a diversity of living beings, including humans, is also well discussed in the literature from time to time [11-14]. Aluminum in its ionic form (Al3+) can react with biological species by altering or suppressing their function, leading to harmful effects. The Al3+ion is neurotoxic and causes neurofibrillary, enzymatic, neurological disorders such as Parkinson’s disease, Alzheimer’s disease, and neurotransmitter changes in the central nervous system [15-17]. Schiff bases create an environment similar to the one present in biological systems, usually by making coordination through oxygen and nitrogen atoms [16-18]. Several significant properties of carbonic acid hydrazides, along with their appli- cations in medicine and analytical chemistry, have led to increased interest in their complexation characteristics with transition metal ions [19,20]. Schiff bases offer several attract- tive structural features, such as the degree of rigidity, a conjuga- ted π-system, and an NH unit that readily participates in hydrogen bonding and maybe a protonation-deprotonation site [21,22]. 2-Hydroxy hydrazone ligands are of interest mainly due to the existence of OH···N or O···HN type hydrogen bonds and tautomerism between the forms of phenol-imine and keto- amine[18,19].It is well recognized that the formation of metal complexes plays an important role to improve the biological activity of free hydrazones [23]. Various Al3+ion sensors based on o-hydroxyphenyl hydrazone have already been reported by our group, as well as other scientists around the world [16-19,24-38]. The selected receptor N'1, N'2-bis{1-(2-hydroxyphenyl) propylidene}oxalo ABSTRACT RESEARCH ARTICLE KEYWORDS https://dx.doi.org/10.5155/eurjchem.14.1.99-108.2360 https://www.eurjchem.com/ https://dx.doi.org/10.5155/eurjchem.14.1.99-108.2360 mailto:dpsingh_011@yahoo.co.in mailto:singh.skumar@gmail.com mailto:ashish.bhuchem@gmail.com http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.14.1.99-108.2360&domain=pdf&date_stamp=2023-03-31 100 Singh et al. / European Journal of Chemistry 14 (1) (2023) 99-108 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.99-108.2360 H2N H N O O N H NH2 Ethanol Reflux, 4 hrs HO N N H O O H N N OH OH O + Scheme 1. Synthesis of receptor. N H N O N H O N HO OH N N OH N OH N HO OH Scheme 2. Tautomerization in H2hpoh. hydrazide (H2hpoh) for the present study can easily be synthesized by a simple and one-step condensation reaction (Scheme 1). In the present work, the fluorometric property of a dihydrazone-based Schiff base, derived from the condensation of oxalic acid dihydrazide with 2-hydroxypropiophenone, is reported for the selective detection of Al3+ ions in aqueous ethanol. Synthetic ease, low cost, and low detection limit may establish H2hpohas a promising molecular sensor for the in vitro or in vivo detection of Al3+ ions. 2. Experimental 2.1. Materials and methods Analytical grade chemicals were obtained from commercial sources. Chloride salts of all metals, 2-hydroxypropiophenone, oxalic acid dihydrazide, and solvents were purchased from Merck Chemicals, India, or SD Fine Chemicals Limited and used as such. Ethanol is distilled in the presence of limestone and is used after thoroughly checking its purity using UV-vis and fluorescence spectral techniques [39]. 2.2. Synthesis of H2hpoh To prepare H2hpoh, a 30 mL aqueous solution of oxalic acid dihydrazide (5 mmol, 0.59 g) were mixed with 20 mL of ethanolic solution of 2-hydroxypropiophenone (10 mmol, 1.50 mL) in a 1:2 molar ratio in a round bottom flask (Scheme 1). The reaction mixture was refluxed for 4 h and a cream-colored crystalline product was obtained upon cooling of the above solution at room temperature. The product was filtered using a Buchner funnel and purified by washing several times with water followed by ethanol to remove the unreacted compo- nents. The pure compound was dried in a desiccator over anhydrous calcium chloride at room temperature. N'1, N'2-bis((E)-1-(2-hydroxyphenyl)propylidene)oxalo- hydrazide (H2hpoh): Color: Cream. Yield: 75%. M.p.: > 300 °C. FT-IR (KBr, ν, cm-1): ν(O-H),3359 (br); ν(N-H), 3285 (br); ν(C=O), 1658; ν(C=N), 1568; ν(C-OH), 1338; ν(N-N), 967. 1H NMR (300 MHz, DMSO-d6, δ, ppm): 13.019 (s, 2H, Enolic-OH), 11.990 (s, 2H, Ar-OH), 9.992 (s, 2H, NH), 7.681-6.842 (8H, Ar- H), 4.511 (s, 4H, CH2), 1.140 (s, 6H, CH3). 13C NMR (75 MHz, DMSO-d6, δ, ppm): 164.80 (C=O), 159.69 (C-OH), 158.66 (C=N), 158.35 (C=N), 132.30-118.12 (Aromatic carbons), 20.11 (CH2), 11.79 (CH3). MS (ESI, m/z (%)): 383.1, (calc: 382.41). 2.3. Instrumentation The 1H and 13C NMR spectra were recorded on a Bruker Avance IIIHD FT-NMR spectrophotometer (300 MHz). A JEOL AL-300 FT-NMR (300 MHz) multinuclear spectrometer is used for NMR titration, for which tetramethylsilane (TMS) is used as an internal standard. The PerkinElmer FT-IR spectrophoto- meter is utilized to record the infrared spectrum in the spectral range of 400-4000 cm-1 using the KBr Pallet method. A Shimadzu Pharmaspec UV-1700 spectrophotometer is used to record the electronic absorption spectra in a water-ethanol mixture (1:4). Fluorescence spectra were recorded on a Horiba Jobin-Yvon Fluorolog3 spectrofluorometer (Model FL3-11). The Oxford Diffraction Gemini Diffractometer equipped with a graphite monochromated MoKα (λ = 0.71073 Å) radiation source is utilized for the collection of single-crystal X-ray diffraction data of H2hpoh at 293(2) K. The structure was solved by the direct method (SHELXL-97) and full-matrix least- squares on F2 using anisotropic displacement parameters for all non-hydrogen atoms is applied for refinement of all data. All hydrogen atoms were included in the refinement at a geometrically ideal position and refined with a riding model [40,41]. The structures of H2hpoh were generated using Mercury and ORTEP-3 software packages [42,43]. 3. Results and discussion As is well known about the existence of amide/hydrazide in keto-amine and enol-imine tautomeric structures, the prepared probe may exist in the following tautomeric structures (Scheme 2). From the chemical structure, it is clear that the enol-imine form is highly conjugated compared to the keto-amine form. Hence, in the keto-amine form, various rotational states are possible for non-radiative decay during electronic transitions, whereas such states are not possible in the enol-imine form. The solution of H2hpoh is weakly fluorescent, suggesting a dynamic tautomerism with a major contribution of keto-amine form. The change in fluorescence properties upon reaction with metals may be due to the interaction of metal ions with H2hpoh in the particular tautomeric form which is discussed later. In the FT-IR spectrum of the H2hpoh receptor, the charac- teristic stretching vibrations of OH and NH, C=O, C=N, and C-O appear at 3359, 3285,1658, 1568 and 1338 cm-1, respectively [25-28]. Characteristic signals of Ar-OH and NH protons in the 1H NMR of H2hpoh appear at δ 11.990 and 9.992 ppm, respectively. The additional signal appears at δ 13.019 ppm for the enolic OH proton due to the keto-enol tautomeric resonance. Whereas, aromatic protons resonate between δ 7.681-6.842 ppm and the aliphatic CH2 and CH3 protons of propiophenone resonates at δ 4.511 and 1.140 ppm, respectively. The peaks appear with shoulder not multiplets in the aromatic and aliphatic region due to dynamic keto-enol tautomeric resonance. Singh et al. / European Journal of Chemistry 14 (1) (2023) 99-108 101 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.99-108.2360 Table 1. Crystal data and structure refinement for H2hpoh. Empirical formula C20H22N4O4 Formula weight (g/mol) 382.41 Temperature (K) 293(2) Crystal system Monoclinic Space group P21/c a, (Å) 6.0747(15) b, (Å) 11.621(5) c, (Å) 13.453(4) α (°) 90 β (°) 94.61(3) γ (°) 90 Volume (Å3) 946.6(5) Z 2 ρcalc(g/cm3) 1.342 μ (mm-1) 0.096 F(000) 404.0 Crystal size (mm3) 0.24 × 0.22 × 0.19 Radiation MoKα (λ = 0.71073) 2Θ range for data collection (°) 6.076 to 58.05 Index ranges -8 ≤ h ≤ 5, -15 ≤ k ≤ 15, -16 ≤ l ≤ 12 Reflections collected 4046 Independent reflections 2149 [Rint = 0.0876, Rsigma = 0.2223] Data/restraints/parameters 2149/0/131 Goodness-of-fit on F2 0.997 Final R indexes [I≥2σ (I)] R1 = 0.0972, wR2 = 0.1488 Final R indexes [all data] R1 = 0.2993, wR2 = 0.2316 Largest diff. peak/hole (e.Å-3) 0.23/-0.23 Figure 1. ORTEP diagram of H2hpoh with atom labeling. The dotted bonds are intramolecular H-bonding. Figure 2. Intermolecular C-H∙∙∙O interaction between H2hpoh molecules. Similarly, characteristic peaks of >C=O, C–OH, >C=N–, >CH2 and –CH3 in the 13C NMR spectrum of H2hpoh appear at δ 164.80, 159.69, 158.66 & 158.35, 20.11 and 11.79 ppm, respect- tively, and aromatic carbons resonate between δ 132.30-118.12 ppm. In the electrospray ionization mass spectra (ESI-MS) of H2hpoh, the peak of the molecular ion observed at m/z = 383.1 corresponds to [M+H]+. Finally, the structural characterization of H2hpoh was performed using a single crystal XRD study and the ORTEP diagram with atom labeling scheme is presented in Figure 1. This probe crystallizes in the monoclinic P21/c space group (Table 1). The bond distance O(1)-C(1) (1.208 Å) and N(2)-C(4) (1.297 Å) corresponds to the double bond character of the >C=O and >C=N– bond (Table 2). It suggests that H2hpoh exists as a keto-amine form in the solid state. The presence of an intramolecular hydrogen bond O(2)- H(2)∙∙∙N(2) between O(2)-H(2) of the phenyl ring and the imine-N atom in the crystal structure of H2hpoh indicates the stabilization of the keto-amine form (Table 3) [25,28]. Along with intramolecular hydrogen bonds, intermolecular C-H∙∙∙O led to a one-dimensional chain (Figure 2) and the inter- molecular C∙∙∙C interaction between phenolic and carboxylic carbons of adjacent molecules led to a stacked structure (Figure 3). 102 Singh et al. / European Journal of Chemistry 14 (1) (2023) 99-108 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.99-108.2360 Table 2. Bond length, bond angle, and torsion angle for H2hpoh. Atom Atom Length (Å) Atom Atom Length (Å) O1 C1 1.208(6) C6 C7 1.367(6) O2 C10 1.346(6) C7 C8 1.362(7) N2 N1 1.370(6) C3 C4 1.527(7) N2 C4 1.297(6) C3 C2 1.487(7) N1 C1 1.343(6) C10 C9 1.391(6) C5 C6 1.410(6) C9 C8 1.381(7) C5 C10 1.392(7) C1 C11 1.528(10) C5 C4 1.463(6) Atom Atom Atom Angle (°) Atom Atom Atom Angle (°) C4 N2 N1 119.9(5) C9 C10 C5 119.4(5) C1 N1 N2 117.0(5) N2 C4 C5 114.6(5) C6 C5 C4 121.2(5) N2 C4 C3 124.7(5) C10 C5 C6 117.4(5) C5 C4 C3 120.7(5) C10 C5 C4 121.4(5) C8 C9 C10 121.7(6) C7 C6 C5 122.0(5) O1 C1 N1 126.3(5) C8 C7 C6 120.2(6) O1 C1 C11 122.5(6) C2 C3 C4 110.1(5) N1 C1 C11 111.2(6) O2 C10 C5 124.4(5) C7 C8 C9 119.2(6) O2 C10 C9 116.2(5) A B C D Angle (°) A B C D Angle (°) O2 C10 C9 C8 -179.9(5) C6 C7 C8 C9 -0.6(9) N2 N1 C1 O1 -3.1(9) C10 C5 C6 C7 -0.9(8) N2 N1 C1 C11 178.8(5) C10 C5 C4 N2 -1.7(7) N1 N2 C4 C5 -179.9(4) C10 C5 C4 C3 175.1(5) N1 N2 C4 C3 3.3(8) C10 C9 C8 C7 1.0(10) C5 C6 C7 C8 0.6(8) C4 N2 N1 C1 -173.8(5) C5 C10 C9 C8 -1.4(9) C4 C5 C6 C7 -180.0(5) C6 C5 C10 O2 179.7(5) C4 C5 C10 O2 -1.3(8) C6 C5 C10 C9 1.3(8) C4 C5 C10 C9 -179.6(5) C6 C5 C4 N2 177.3(5) C2 C3 C4 N2 -98.5(6) C6 C5 C4 C3 -5.8(8) C2 C3 C4 C5 85.0(6) 1 Symmetry code: 2-x, -y, -z. Table 3. Hydrogen bond parameters (Å and °) in the H2hpoh. D−H∙∙∙A D−H H∙∙∙A D∙∙∙A < (DHA) O(2)−H(2)∙∙∙N(2) 0.82 1.79 2.503(6) 144 N(1)−H(1)∙∙∙O(1) 0.80(5) 2.27(6) 2.673(6) 111(5) C(3)−H(3B)∙∙∙N(1) 0.97 2.51 2.849(8) 100 C(6)−H(6)∙∙∙O(2) 0.93 2.43 3.325(7) 161 C(7)−H(7)∙∙∙O(1) 0.93 2.59 3.493(7) 164 Figure 3. Intermolecular C∙∙∙C interaction between phenolic and carboxylic carbons. The sensing properties of H2hpoh have been investigated by means of UV-visible/fluorescence aqueous ethanol titration (water:ethanol mixture, 1:4) and 1H NMR titration in DMSO-d6 at room temperature. The absorption spectral band that appears at 335 nm corresponds to a yellow-green solution. A solution of H2hpoh (50 µM) in aqueous ethanol was used for UV- visible titration experiments and triple distilled water is used to prepare solutions of metal chloride salts. The addition of an aqueous solution of Al3+ ions (100 µM) to aqueous ethanolic solutions, each of H2hpoh (50 µM) led to a shift in the absorption band from 335 to 382 nm (bathochromic shift of ~47 nm) with a decrease in the intensity of absorption. As a result, the color of the solution turned from yellow-green to intense green, which is visible with the naked eye. Not only have, we performed UV-visible titration of H2hpoh with direct addition of two equivalents of aqueous Al3+ ions (Figure 4a), but we also performed UV-visible titration of H2hpoh by gradual addition of aqueous Al3+ in 0-5 equivalents (Figure 4b). Furthermore, to check the interaction of other metals with H2hpoh, UV-vis spectra of H2hpoh with the addition of other metal ions viz. Na+, K+, Ca2+, Ba2+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cr3+, Cd2+ and Hg2+ (as chloride) are also performed. Among all metal ions, alkali/alkaline earth metal ions and Mn2+ do not produce a significant change in the UV-vis spectra of H2hpoh, suggesting that only electrostatic interactions are present between metal ions and H2hpoh. On the other hand, spectral changes are observed in the case of different transition metal ions, which is an indication of metal ions-H2hpoh binding. Singh et al. / European Journal of Chemistry 14 (1) (2023) 99-108 103 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.99-108.2360 (a) (b) Figure 4. UV-visible spectra of the H2hpoh (50 µM) water-ethanol mixture (1:4) without and with the addition of Al3+ ions (a) direct addition of 2 equivalents of aqueous Al3+ ions, and (b) gradual addition of aqueous Al3+ ions (0-5 equivalents). Figure 5. Fluorescence spectra of H2hpoh (5 µM) + metal ions (100 µM) at λem: 478 nm (λex: 382 nm) in the water-ethanol mixture (1:4), where L = H2hpoh, Mn+ = Na+, K+, Ca2+, Ba2+, Al3+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cr3+, Cd2+ and Hg2+. Inset (Solution of H2hpoh in the presence of Na+, Fe3+, Mg2+, Al3+, and Cu2+ in visible and UV light). To determine the binding stoichiometry of H2hpoh and Al3+ ions, electronic absorption spectra are recorded for solutions of H2hpoh and Al3+ ions mixed in varying mole fractions. Finally, from Job’s plot, it is clear that the complex formation of Al3+ with receptor H2hpoh is 2:1 stoichiometry, respectively [44]. From the UV-vis study, it is clear that the selectivity for sensing to any metal ion under investigation is not possible employing electronic absorption spectroscopy. Hence, to obtain the selective response of receptor H2hpoh among metal ions, i.e., Al3+, Na+, K+, Ca2+, Ba2+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cr3+, Cd2+ and Hg2+, electronic emission studies were also performed (Figure 5). The solution of the pure receptor H2hpoh shows weak fluorescence at λem = 478 nm when excited at λex = 382 nm, no significant change in the fluorescence intensity is observed upon the addition of other metals. However, the solution of H2hpoh with two equivalent aqueous solutions of Al3+ ions exhibit very high intensity fluorescence spectra (Figure 6). This distinct change in color from light yellow-green to intense green is visible under UV light, suggesting the formation of a strong fluorescent complex of H2hpoh with Al3+.The gradual addition of Al3+ to H2hpoh in fluorescence titration also supports the fluorescence turn-on of H2hpoh by Al3+ (Figure 6b). Among the various possibilities for quenching fluorescence in pure H2hpoh, cis-trans isomerization across the >C=N- bond and intramolecular charge transfer (ICT) in hydrazone-based receptors are the most probable [45]. Fluorescence enhance- ment upon addition of Al3+ ions to H2hpoh may be due to inhibition of cis-trans isomerization [46] and/or reduction in the ICT effect due to the chelation of H2hpoh with Al3+. 104 Singh et al. / European Journal of Chemistry 14 (1) (2023) 99-108 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.99-108.2360 (a) (b) Figure 6. Fluorescence of H2hpoh (50 µM) in a water-ethanol mixture (1:4) without and with the addition of Al3+ ions (a) direct addition of two equivalents of aqueous Al3+ ions, and (b) gradual addition of aqueous Al3+ ions (0-2 equivalents) (λem: 478 nm, λex: 382 nm). Figure 7. Interference study in a binary solution of H2hpoh (5 µM) + Al3+ (100 µM) with other metal ions Mn+ (100 µM), (λem: 478 nm, λex: 382 nm) (where Mn+ = Al3+, Na+, K+, Ca2+, Ba2+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cr3+, Cd2+ and Hg2+). Chelation of Al3+ ions with H2hpoh not only reduces the ICT effect, but also restricts the free rotation around the carbon of azomethine attached to the aromatic ring. As a result of rotation restriction, nonradiative decay processes are hindered, resul- ting in a significant enhancement of the fluorescence intensity. Such enhancement in fluorescence intensity upon chelation of metal ions with the receptor is known as chelation enhanced fluorescence (CHEF) [47-49]. Most of the metal ions do not show interference in the detection of Al3+ by H2hpoh except Fe3+ and Cu2+ in aqueous ethanol. Although the direct addition of Fe3+ and Cu2+ to the solution of H2hpoh does not cause a significant change in the fluorescence spectra, the addition of these ions to the binary solution of H2hpoh (5 µM) + Al3+ (100 µM) results inhibition of the fluorescence intensity (Figure 7). The smaller ionic radii (0.5 Å) of the Al3+ ion may be responsible for the selectivity of H2hpoh towards Al3+ by along with appropriate coordination geometry for the chelating receptor H2hpoh and the higher charge density (R = 4.81) of the Al3+ ion may be responsible for the strong coordination ability of Al3+ with H2hpoh [49]. Fluorescence responses are very fast after the addition of Al3+ to H2hpoh and the maximum fluorescence has been observed after the addition of two equivalents of aqueous Al3+in a 5 µM aqueous ethanolic solution of H2hpoh. Consequently, the binding constant (KB) is obtained from the linear fitting of the fluorescence titration intensities at various concentrations of Al3+ ions in the modified Benesi-Hildebrand equations for the stoichiometry of the complex 2:1 (M: L) (Equation (1)) [50]. Io I−Io = � a b−a � 2 ( 1 𝐾𝐾𝐵𝐵[substrate]2 + 1) (1) where Io and I are the fluorescence intensity of H2hpoh at 478 nm in the absence and in the presence of various concentrations of Al3+; a, b are constants; [substrate] is the concentration of Al3+. Singh et al. / European Journal of Chemistry 14 (1) (2023) 99-108 105 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.99-108.2360 Table 4. Comparison of binding constant and detection limit. Compound M:L Binding constant Kb (M-1) Detection limit (M) References N,N’-bis((2-hydroxynaphthalen-1-yl)methylene)oxa-lohydrazide(H2ohn) with Al3+ 2:1 2.62×1011 8.56×10-10 [25] o-Hydroxypropiophenone-2-thiophenoyl hydrazone,tcph with Al3+ 1:1 5.00×106 1.35×10−9 [27] N,N’-bis((2-Hydroxynaphthalen-1-yl)methylene)malo-nohydrazide (H2nmh) with Al3+ 2:1 5.74 × 109 5.78×10−8 [28] (E)-2-(2-Aminothiazol-5-yl)-N'-((2-hydroxynaphthalen-1-yl)methylene)acetohydrazide (NTH) with Al3+ 1:1 3.65×109 1.09×10−9 [36] β-Pinene-based fluorescent probe (6,6-dimethyl-3-(pyridin-2-yl)-4,5,6,7-tetrahydro-2H-5,7- methanoindazole) 1:1 1.89×103 8.10×10−8 [37] 2-((E)-1-(2-(Dimethylamino) ethylimino)ethyl)phenol (HL) with Al3+ 2:1 2.10×104  4.32×10−6 [38] N'1,N'2-bis{1-(2-Hydroxyphenyl) propylidene}oxalohydrazide(H2hpoh) with Al3+ 2:1 8.99×1010 7.80×10−5 This work Figure 8. Benesi-Hildebrand plot for H2hpoh with Al3+, considering the 2:1 complexation. The goodness of fit is shown by the R2 value. O O N N N N O AlO Al Cl OH Cl Cl HO Cl O O H N N H N N HO OH Al3+ Scheme 3. Proposed reaction between theH2hpoh and Al3+ ions. Observation of the linear fit in Io/I-Io vs 1/[Al3+]2 graph and the close agreement of the experimental value of KB = 8.99×1010 M−1 to the theoretical fit, support 2:1 (M:L) complex stoichio- metry (Figure 8). The high binding constant is consistent with the highly selective sensing of Al3+ with H2hpoh without the interference of most metal ions. The detection limit (LOD) of H2hpoh can be calculated based on Equation (2) [51]. Detection limit = 3σ slope (2) where standard deviation (σ is calculated by recording the fluorescence intensity of H2hpoh without Al3+ by 10 times. A plot of fluorescence intensity as a function of the concentration of the added metal ion provides the value of slope. The detection limit was found to be 7.8×10−5 M in the linearity range of 4.99×10-7-7.99×10-6 M (R2 = 0.99824). A comparative table of detection limit and binding constants for some receptors of Al3+ with different binding ratios is provided in Table 4. From Table 4, it is clear that although the binding constant of H2hpoh with Al3+ is very high, the detection limit is in order of µM only. The interaction of the H2hpoh receptor with Al3+ is also supported by the NMR study (Figure 9). 1H NMR titrations have been performed by concomitant addition of Al3+ (1×10–1 M solution in D2O) to a 1×10–3 M solution of receptors in DMSO-d6. Upon addition of Al3+, the peak corresponding to the amine proton disappears and the phenolic OH proton shifts slightly downfield with the reduction in peak intensity, suggesting the enolization of the >C=O group and the interaction of Al3+ with phenolic OH. The general changes in 1H NMR spectra indicate the complexation between Al3+ and H2hpoh in the deprotonated enol-imine form through carbonylate-O, azomethine-N, and phenolic-OH. On the basis of spectroscopic characterization, the proposed structure of H2hpoh-Al3+ complex might be according to Scheme 3. The proposed structure is supported further by theoretical studies. Density functional theoretical (DFT) calculations have been employed for structural optimization and TD-DFT calculations of H2hpoh and its Al(III) complex using the C1 point group. For both H2hpoh and its Al(III) complex, Becke’s three-parame- terized Lee-Yang-Parr (B3LYP) exchange functional with basis sets 6-31G* for all C, H, N, O, and Al atoms has been used in the Gaussian-03 program [52].The geometry around each Al3+ is considered a distorted octahedral in which three coordination sites are occupied by one carbonylate-O, one azomethine-N, and one phenolic OH of H2hpoh,and the other three coordination sites are occupied by two chloride ions and one water molecule. The energy (RB+HF-LYP) was found to be -1295.6107 a.u. and -3773.4669 a.u., respectively, for H2hpoh and its Al3+ complex. The energy level of HOMO and LUMO as well as the bandgap of H2hpoh is much higher than its Al3+ complex; therefore, the thermodynamically favorable conversion of H2hpoh to H2hpoh- Al3+ (Figure 10) with a bathochromic shift in the UV-visible spectra of the H2hpoh-Al3+ complex. Based on Job’s plot, NMR titrations, and DFT calculation, it can be predicted that each molecule H2hpoh interacts with two Al3+ ions in a dibasic hexadentate mode to form the [Al2Cl4(hpoh)(H2O)2] complex. 106 Singh et al. / European Journal of Chemistry 14 (1) (2023) 99-108 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.99-108.2360 Figure 9. 1H NMR titration of H2hpoh upon adding 0-2 equivalents of Al3+ in DMSO-d6. Figure 10. Diagram of the energy level for the frontier π molecular orbitals of H2hpoh (left) and its Al3+ complex (right). 4. Conclusions In this study, we are exploring the possibility of the preparation of a highly efficient fluorescent probe H2hpoh for selective detection of the Al3+ ion. The synthetic ease, low-cost, and high efficiency of dihydrazone-based fluorescent probes may be a milestone towards the development of a fluorescence- based chemical sensor for metal ions in biological systems. Fluorescence turn-on is due to the reduction in the ICT and the restricted rotation of the >C=N- bond in H2hpoh or chelation- enhanced fluorescence after the binding of Al3+ ions. From UV- vis, fluorescence and 1H NMR titration experiments, the stoichiometry of the H2hpoh-Al3+ complex was established to be 2:1 (M:L). The binding of hexadentate H2hpoh with each Al3+is carried out through one carbonylate-O, one azomethine-N, and phenolic OH and the remaining coordination site of Al3+is completed by chloride and water ligands. From the fluorescence study, the binding constant (KB) and detection limit for Al3+ were also determined. The structure of H2hpoh is established by a single crystal X-ray diffraction study, while the structure of the H2hpoh-Al3+ complex is established by DFT calculations. Acknowledgments The authors thank the Head of the Sophisticated Analytical Instrument Facility for NMR, Single-crystal XRD Institute of Science, Banaras Hindu University; Central Drug Research Institute, Lucknow, India for extending the ESI-MS facility. Ashish Kumar Singh acknowledges the Department of Science and Technology for the INSPIRE Faculty Fellowship (Award Number: DST/INSPIRE/04/2015/002001). Supporting information CCDC-985204 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/ data_request/cif, or by e-mailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44(0)1223-336033. http://www.ccdc.cam.ac.uk/%20data_request/cif http://www.ccdc.cam.ac.uk/%20data_request/cif mailto:data_request@ccdc.cam.ac.uk Singh et al. / European Journal of Chemistry 14 (1) (2023) 99-108 107 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.99-108.2360 Disclosure statement Conflict of interest: There are no conflicts to declare. Ethical approval: All ethical guidelines have been adhered. Sample availability: Samples of the compound are available from the author. CRediT authorship contribution statement Conceptualization: Chandani Singh, Romi Dwivedi; Methodology: Chandani Singh, Divya Pratap Singh, Ashish Kumar Singh; Software: Divya Pratap Singh, Ashish Kumar Singh; Validation: Divya Pratap Singh, Ashish Kumar Singh; Formal Analysis: Chandani Singh, Romi Dwivedi; Investigation: Chandani Singh, Divya Pratap Singh, Ashish Kumar Singh; Resources: Sunil Kumar Singh, Vinod Prasad Singh; Data Curation: Sunil Kumar Singh, Vinod Prasad Singh; Writing - Original Draft: Divya Pratap Singh, Sunil Kumar Singh, Ashish Kumar Singh; Writing - Review and Editing: Divya Pratap Singh, Sunil Kumar Singh, Ashish Kumar Singh, Vinod Prasad Singh; Visualization: Sunil Kumar Singh, Vinod Prasad Singh; Funding acquisition: Ashish Kumar Singh, Vinod Prasad Singh; Supervision: Sunil Kumar Singh, Divya Pratap Singh, Ashish Kumar Singh; Project Administration: Divya Pratap Singh, Ashish Kumar Singh. Funding Department of Science and Technology for INSPIRE Faculty Fellowship (Award Number: DST/INSPIRE/04/2015/002001). ORCID and Email Chandni Singh chandani0905@gmail.com https://orcid.org/0000-0002-9052-403X Divya Pratap Singh dpsingh_011@yahoo.co.in dpsbhu11@gmail.com dpsingh@gecazamgarh.ac.in https://orcid.org/0000-0001-8760-4031 Sunil Kumar Singh singh.skumar@gmail.com https://orcid.org/0000-0003-2095-6723 Romi Dwivedi dwivediromi26@gmail.com https://orcid.org/0000-0002-0485-4156 Ashish Kumar Singh ashish.bhuchem@gmail.com https://orcid.org/0000-0001-9499-5843 Vinod Prasad Singh singvp@yahoo.co.in https://orcid.org/0000-0002-2997-5333 References [1]. Roy, P. Recent advances in the development of fluorescent chemosensors for Al3. Dalton Trans. 2021, 50, 7156–7165. [2]. Gupta, A.; Kumar, N. A review of mechanisms for fluorescent ‘“turn- on”’ probes to detect Al3+ ions. RSC Adv. 2016, 6, 106413–106434. [3]. Li, B.; He, T.; Fan, Y.; Yuan, X.; Qiu, H.; Yin, S. 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W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian, Inc. , Wallingford CT, 2009. Copyright © 2023 by Authors. This work is published and licensed by Atlanta Publishing House LLC, Atlanta, GA, USA. The full terms of this license are available at http://www.eurjchem.com/index.php/eurjchem/pages/view/terms and incorporate the Creative Commons Attribution-Non Commercial (CC BY NC) (International, v4.0) License (http://creativecommons.org/licenses/by-nc/4.0). By accessing the work, you hereby accept the Terms. This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution, or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 2.1. Materials and methods 2.2. Synthesis of H2hpoh 2.3. Instrumentation 3. Results and discussion 4. Conclusions Acknowledgments Supporting information Disclosure statement CRediT authorship contribution statement Funding ORCID and Email References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField16: PrintField17: PrintField18: PrintField19: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: PrintField26: PrintField27: PrintField28: PrintField29: