Carbazole-based Schiff base: A sensitive fluorescent ‘turn-on’ chemosensor for recognition of Al(III) ions in aqueous-alcohol media

2.1 Chemicals

Ethylenediaminotetraacetic acid disodium salt dihydrate (Na₂EDTA), the solvents, the metal salts and the sodium salts of F⁻, Cl⁻, Br⁻, I⁻, CO₃^2–, HCO₃^– and HPO₄^2- ions were purchased from Merck Chemical Co (Germany). CrCl₃ was obtained from Riedel-de Häen (Germany). 2-hydroxy-1-naphthaldehyde, 9-ethyl-9H-carbazol-3-amine PbCl₂, SnCl₂·2H₂O, K₂Cr₂O₇ and tetrabutylammoniumhexafluorophosphate (TBAPF₆) were supplied by Sigma Aldrich (Germany). All chemical reagents procured from commercial suppliers were used without purification in the synthesis and measurements.

2.2 Synthesis of Schiff base (L) and structural characterization

The condensation reaction between 9-ethyl-9H-carbazol-3-amine (0.21 g, 1 mmol) and 2-hydroxy-1-naphthaldehyde (0.172 g, 1 mmol) in a 100 mL flask containing 15 mL of EtOH was maintained for 5 h at 60 °C under reflux with continuous stirring. Then, the solvent was evaporated, and the product of 1-(((9-ethyl-9H-carbazol-3-yl)imino)methyl)naphtalen-2-ol was dried in vacuum to obtain an orange solid (yield 86%). The ligand was abbreviated as L. The synthesis procedure of L was shown in Scheme 1.

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Scheme 1

Synthesis of Schiff base (Ligand = L).

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FT-IR (cm⁻¹): 3319v (O–H stretching), 3053–2962 v(aromatic C–H stretching), 2918–2865 (aliphatic C-H stretching), 1613 (C = N), 1543, 1488 v(C = C, aromatic ring), 1323 v(C-N, aromatic amine stretching), 1232 v(C-O stretching). ¹H NMR (DMSO‑d₆ δ_H ppm, 9.83 (s, –OH), 8.56 (–CH = N-), 8.56 (d, Hg), 8.26 (d, Ha), 7.90 (d, Hj), 7.79 (d, Hk, Hn), 7.76 (d, Hf)), 7.72 (d, He), 7.62 (d, Hd), 7.56 (t, Hm), 7.48 (t, Hl), 7.35 (t, Hc), 7.23 (t, Hb), 7.05 (d, Hi), 4.48 (q, –CH₂), 1.32 (t, –CH₃). ¹³C NMR (DMSO‑d₆): δ ppm, 163.23 (C7), 153.09 (C1), 150.42 (C3), 130.26 (C10), 129.56 (C9), 129.41 (C4), 122.23 (C5), 122.11 (C8), 121.55 (C2), 121.29 (C6). ¹³C NMR (150 MHz, DMSO‑d₆, δ (ppm)) δ: 159.61, 149.28, 143.01, 140.22, 140.73, 139.72, 139.72, 138.44, 137.12, 132.83, 129.78, 128.00, 125.71, 125.99, 125.21, 122.41, 122.20, 116.96, 115.55, 112.76, 110.23, 59.45, 14.20. LC-MS/MS, m/z: [M + H]⁺ for L, calc.: m/z = 364, found: m/z = 365 [M + H]⁺. LC-MS/MS, for L-Al³⁺, calc.: M = 409, found: M = 410 [M + Al^(III) + H₂O + H]⁺.

2.3 Stock solution preparation and instrumentation

1.0 × 10^-4 mol L^-1 solution of L (0.0364 g, 1.0 mol) was prepared in EtOH. 5.0 × 10^-3 mol L^-1 solutions of K⁺, Ag⁺, Ba²⁺, Mn²⁺, Mg²⁺, Sn²⁺, Hg²⁺, Ca²⁺, Co²⁺, Zn²⁺, Cu²⁺, Ni²⁺, Pb²⁺, Al³⁺, Fe³⁺, Cr³⁺ and Cr⁶⁺ were prepared by solubilizing the metal salts and anions in deionized water. The anions, F⁻, Cl⁻, Br⁻, I⁻, CO₃^2–, HCO₃^– and HPO₄^2- (1 mM) solutions were also prepared in deionized water. The UV–vis and fluorescence selectivity experiments were performed by taking 1.5 mL of L (1.0 × 10–4 M) and 1.5 mL of different metal ions and anions (5.0 × 10^-3 M) in a quartz cell to maintain the solvent ratio EtOH:H₂O (1:1, v/v).

Characterization studies consisted of the Perkin Elmer Frontier FT-IR Fourier Transform Infrared spectroscopy method using Attenuated Total Reflection accessory for functional groups analysis between 4000 cm⁻¹ and 450 cm⁻¹, ¹H-600 MHz and ¹³C NMR-150 MHz spectroscopy with Agilent technologies (DMSO‑d₆: solvent and TMS: internal standard), UV–Vis spectroscopy with Analytikjena Specord 210 Plus spectrophotometer, PL emission spectra with Shimadzu R5301 PC spectrofluorophotometer equipped with a Xenon lamp as the excitation source and CV measurements with CHI 660C Electrochemical Analyzer (USA). Optical band gap (E_g) was obtained using the formula of 1242/λ_onset (eV) (Colladet et al., 2004). Mass spectrometry data were measured using Finnigan LCQ ion-trap mass spectrometer (Shimadzu LC-MS/MS-8040, Japan). Ligand and ions were combined in a mixture of EtOH: H₂O (1:1, v/v).

2.4 Fluorescence spectroscopic study

Photoluminescence (PL) emission spectrum of the ligand (L) in EtOH was monitored by spectrofluorophotometer. Upon addition of K⁺, Ag⁺, Ba²⁺, Mn²⁺, Mg²⁺, Sn²⁺, Hg²⁺, Ca²⁺, Co²⁺, Zn²⁺, Cu²⁺, Ni²⁺, Pb²⁺, Al³⁺, Fe³⁺, Cr³⁺, Cr⁶⁺, F⁻, Cl⁻, Br⁻, I⁻, CO₃^2–, HCO₃^– and HPO₄^2-, the fluorescence spectral behavior was monitored to study influences of the metal ion and anion. A series of Al³⁺ solution (in the range of 0.25 mM-0.195 μM) was prepared on account of observing the concentration efficacy of Al³⁺ ions on the PL property of L.

Fluorescence PL quantum yield ${(QY}_s)$ is defined as the ratio of the number of photons emitted to the number of photons absorbed through fluorescence of the fluorophore can be determined using the comparative method according to equation (1) (Lakowicz, 1999):

where A, F and n denote absorption at the maximum excitation wavelength, integrated area underneath the fluorescence emission spectrum and refractive index of the solvent, respectively. Excitation and emission slit width values for both the sample and the standard were adjusted as 5 nm. The fluorescence quantum yield of standard fluorophore was measured using fluorescein in 0.1 M of aqueous NaOH ( ${\mathit{QY}}_f$  = 0.97).

3.1 Structural characterization

Solubility test for the ligand synthesized was carried out at room conditions (L/solvent, 1 mg 1 mL⁻¹). L was soluble in EtOH, DMSO, ethyl acetate, acetonitrile, and acetone, whereas partly soluble in MeOH. In addition, L was insoluble in toluene and hexane as apolar solvent.

FT-IR and spectroscopy verified the synthesized Schiff base’s characteristic substituents. The signal at 3319 cm⁻¹ was associated with O-H stretching mode (Fig. S1). The aromatic C-H and the aliphatic C-H stretching vibrations were monitored in the region of 3053–2962 cm⁻¹ and 2918–2865 cm⁻¹, respectively. In the IR spectrum of L, absence of the peak related to the amine group (–NH₂) and the peak for the aldehyde carbonyl group (-C = O) indicated the formation of a Schiff base compound, with concomitant characteristic stretching vibration pronounced at 1613 cm⁻¹ for the imine (–HC = N-) group. The peaks observed at 1543 cm⁻¹ and 1488 cm⁻¹ were attributed to the vibration bands of C = C bonds in the benzene ring. The C-N and C-O stretching modes were noticeable at 1323 cm⁻¹ and 1232 cm⁻¹, respectively. The sharp peaks between 807 cm⁻¹ and 719 cm⁻¹ could be related to the C = C and C-H bending modes.

¹H NMR spectra of the Schiff base (L) and the coordinated L with Al³⁺ are displayed in Fig. S2. The peaks at 9.83 ppm and 8.56 ppm were attributed to –OH and –CH = N- protons of L, respectively. The aromatic protons of L were observed between 8.56 ppm and 7.05 ppm. Owing to the positive mesomeric effect of –OH group, the ortho positioned Hi came out up field at 7.05 ppm as doublet and the meta positioned Hj moved downfield to 8.07 ppm as doublet (Fig. S2a). The signals of the protons in the aliphatic region were observed at 4.48 ppm (quartet) and 1.32 ppm (triplet) for –CH₂ and –CH₃, respectively. Slight changes in the δ values upon coordination with Al³⁺ ions were depicted in Fig.S2b. Since the coordination took place from both –OH and –CH = N- sides, the regarding protons of –OH and –CH = N- moved up field and recorded shift of Δδ 0.09 ppm and Δδ 0.14 ppm, respectively. A lowered intensity for the –OH peak was observed. Aromatic protons showed slightly up field shift upon chelation. It could be inferred that the binding of L to Al³⁺ resulted in a formation of a rigid system along with imine-nitrogen and hydroxyl group of L as binding site.

¹³C NMR spectrum of the synthesized Schiff base (L) is displayed in Fig.S3. The highest chemical shift value of azomethine carbon (–CH = N-) appeared at 169.83 ppm. Since –OH group of naphthyl moiety pulled electrons away from the ring inductively, the peak of C14 was deshielded to 154.28 ppm. Due to the electronegative element of nitrogen, C1 of carbazole moiety also appeared downfield at 140.69 ppm. The aromatic carbons were observed in the range of 138.84–109.06 ppm. Alkyl –CH₂ and –CH₃ asserted themselves at 37.66 ppm and 14.13 ppm, respectively. In consideration of (+) mesomeric effect of OH group towards the ring inducing a negative charge formation at the ortho and para corners of the naphthyl moiety, the peaks at 129.1 ppm, 120.75 ppm and 109.92 ppm were attributed to C17, C15 and C13, respectively. The peak of the meta positioned C16 was observed at 138.85 ppm. C3 and C7, which were the ortho and para positioned carbons appeared at 126.78 and 115.19 ppm, respectively. ¹H NMR was integrated with ¹³C NMR spectral measurement for the structural confirmation of L.

Fig. 1 shows the optical behavior of L recorded by UV–vis spectroscopy. The optical measurements were implemented at room temperature in the range of 260–800 nm, versus a blank solution in EtOH:H₂O (1:1). The normalized absorption spectrum of the free sensor L displayed three absorption bands centered at 297 nm, 318 nm and 330 nm, corresponding to the intra-ligand aromatic π → π* electron transitions. The prominent band of L displayed at 462 nm could be ascribed to the electronic transitions from nonbonding orbitals on the heteroatoms to ligand π* orbitals, namely, n → π* electronic transition. Upon addition of Al³⁺ ions, the absorption bands of aromatic π → π* electron transitions were observed at 298 nm, 318 nm and 330 nm. The absorption band at a maximum wavelength of 462 nm indicated a bathochromic shift to 479 nm. A new band at 531 nm arose upon complexation with Al³⁺ in EtOH:H₂O (1:1, v/v) via the donation of lone pairs of electrons on nitrogen/oxygen of the ligand to metal ion charge transfer. λ_onset is the onset wavelength which can be identified by intersection of two tangents on the absorption edges, indicating the starting wavelength for the electronic transition (Colladet et al., 2004). λ_onset values obtained from the UV–Vis spectra were obtained as 519 nm and 580 nm, respectively. The calculated optical band gap (E_g) value of L was 2.39 eV in EtOH:H₂O. Having coordinated to Al³⁺ ions, this value lowered to 2.14 eV as a result of a chelation between L and Al³⁺. Bathochromic shift observed in UV–vis spectra was ensued from the binding of Al³⁺ ions to -C = N- bridge and –OH of 2-hydroxynaphthalene unit (Das and Goswami, 2017; Yoon et al., 2007). On account of charge transfer from L towards Al³⁺, the yellow solution of L turned into white upon addition of Al³⁺ ions under 366 nm of UV light, as shown in Fig. 2. The addition of other metal ions and anions (K⁺, Ag⁺, Ba²⁺, Mn²⁺, Mg²⁺, Sn²⁺, Hg²⁺, Ca²⁺, Co²⁺, Zn²⁺, Cu²⁺, Ni²⁺, Pb²⁺, Al³⁺, Fe³⁺, Cr³⁺, Cr⁶⁺, F⁻, Cl⁻, Br⁻, I⁻, CO₃^2–, HCO₃^– and HPO₄^2-) did cause no alterations on metal-complexation, which was also confirmed by detecting no color change under 366 nm light, as seen in Fig. 2A and Fig. 2B.

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Fig. 1

UV–vis absorption spectra of L (0.1 mM) and L + Al³⁺ (0.5 mM) in EtOH.

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Fig. 2

Digital camera photograph of L in the presence of metal ions (A) and anions (B) under 366 nm UV light.

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3.2 pH effect

To determine useful pH range where the ligand can perform without being affected by hydrogen ions, which cause interference in the performance of the ligand is necessary. Thus, pH dependence of the ligand was investigated and the results obtained are given in Fig. 3. The emission intensity of L (0.1 mM) was stable in the pH range from 2.0 to 12.0, concluding with a pH-independent ligand. Being present of Al³⁺ (20 eqv.) enhanced the PL emission intensity, which reached to maximum value at midpoint, therefore, it was better to make all measurements at pH 7.5 and 8.0 (Fig. 3). The change in potential above pH 8.0 resulted in the formation of Al(OH)₃ concomitant with the breakdown of L-Al³⁺ complex, hence fluorescence was quenched. Consequently, L could be used for sensing Al³⁺ ions in aqueous medium under neutral conditions.

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Fig 3

pH dependence of L and L + Al³⁺.

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3.3 Selectivity of L to Al³⁺ and competitive study

The selectivity of L (0.1 mM) in EtOH was investigated towards metal ions (K⁺, Ag⁺, Ba²⁺, Mn²⁺, Mg²⁺, Sn²⁺, Hg²⁺, Ca²⁺, Co²⁺, Zn²⁺, Cu²⁺, Ni²⁺, Pb²⁺, Al³⁺, Fe³⁺, Cr³⁺ and Cr⁶⁺, 1.0 eqv. in H₂O) and anions (F⁻, Cl⁻, Br⁻, I⁻, CO₃^2–, HCO₃– and HPO₄^2-, 1.0 eqv. in H₂O). The sensor L possessed a weak fluorescence emission at 533 nm with 73 a.u. intensity under excitation at 320 nm. The excited-state intramolecular proton transfer (ESIPT) eventually the phenolic proton and C = N isomerization, resulting in non-fluorescent ligand (Tang et al., 2011; Chen et al., 2021). It was noted that only Al³⁺ ion exhibited a drastic enhancement in the PL emission spectra. The excitation at 320 nm gave results for the emission intensities of L-metal ion and L-anion solutions in the presence of K⁺, Ag⁺, Ba²⁺, Mn²⁺, Mg²⁺, Sn²⁺, Hg²⁺, Ca²⁺, Co²⁺, Zn²⁺, Cu²⁺, Ni²⁺, Pb²⁺, Al³⁺, Fe³⁺, Cr³⁺, Cr⁶⁺, F⁻, Cl⁻, Br⁻, I⁻, CO₃^2–, HCO₃^– and HPO₄^2-, which were found to be 74, 98, 78, 73, 101, 102, 79, 102, 101, 96, 103, 96, 95, 789, 39, 104, 74, 82, 78, 77, 91, 91, 92 and 91 a.u, respectively, at 533 nm of maximum emission wavelength of L as displayed in Fig. 4. All of the emission bands exhibit the same spectral envelope. As demonstrated in Fig. 4, the fluorescence enhancement performance in the presence of Al³⁺ observed at 533 nm was 11-fold greater than that of the control by emitting white color, indicating that L came up with a high fluorescent selectivity towards Al³⁺, while no fluorescence was monitored in the presence of other metal ions and anions.

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Fig. 4

Fluorescence emission spectra of L (0.1 mM) treatment with various metal ions and anions such as Ag⁺, K⁺, Mg²⁺, Ba²⁺, Mn²⁺, Hg²⁺, Sn²⁺, Co²⁺, Ca²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Pb²⁺, Al³⁺, Fe³⁺, Cr³⁺, Cr⁶⁺, F⁻, Cl⁻, Br⁻, I⁻, CO₃^2–, HCO₃– and HPO₄⁻ (0.5 mM) at room temperature in EtOH: H₂O (1:1, v:v, λ_ex = 320 nm, excitation and emission slit widths = 5 nm).

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Due to the isomerization of the C = N bond in the excited state via the excited state intra molecular proton transfer (ESIPT) (Manna et al., 2020), the ligand showed weak fluorescence. Specific metal ion binding through O donor site and the C = N moiety of L restricted C = N isomerization and thus, emission intensity could be enhanced due to the chelation enhancement fluorescence (CHEF) effect (Gupta and Kumar, 2016). The participation of Al³⁺ in the ethanol solution of L eventuated with an emission enhancement at 533 nm due to chelation-enhanced fluorescence (CHEF) (Kim et al., 2003) on account of binding with Al³⁺, which could be related to the formation of a rigid system by the coordination of Al³⁺, leading to the disruption of the ESIPT feature (Salarvand et al., 2019; Li et al., 2013). The fluorescence enhancement happens due to the non-bonded electrons of N atom from C = N took part in coordination with Al³⁺ ion to inhibit the isomerization process. The coordination of L with Al³⁺ ion hindered the rotation around the C = N bond and prevented the C = N isomerization. The result was the inhibition of ESIPT in the ligand, resulting in a fluorescence enhancement and inducing chelation-enhanced fluorescence, thereby generating white fluorescence in the ligand–metal product. The selectivity and sensitivity took part in fluorescence enhancement by Al³⁺ ion capturing and the PL emission change through CHEF effect (Sahana et al., 2013; Shyamal et al., 2016; Manna et al., 2020; Shyamal et al., 2016) due to retardation of C = N isomerization. The presence of C = N and O sites of L, which showed strong affinity towards a hard metal like Al³⁺, would form the chelation with Al³⁺ and, thus, would be the essence of a promising turn-on fluorescent sensor for Al³⁺ ion, as seen in Fig. 5.

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Fig. 5

Suggested mechanism of binding of L with Al³⁺.

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The fluorescence quantum yield (QY) of L-Al³⁺system was calculated as 21.3%. Time-dependent (0–3600 sec) fluorescence measurement pointed out that irradiation at 320 nm within 3600 sec eventuated with no considerable changes in fluorescence emission intensity as displayed in Fig.S4, showing that the L-Al³⁺ system possessed a remarkable photostability in EtOH:H₂O (1:1, v/v) media.

To further corroborate the high selectivity of L sensor towards Al³⁺, competitive tests were conducted in the presence of selected metal ions and anions. The selectivity of L towards Al³⁺ was examined in a ternary mixture containing L (0.1 mM), Al³⁺ (0.5 mM) and other interfering metal ions (K⁺, Ag⁺, Ba²⁺, Mn²⁺, Mg²⁺, Sn²⁺, Hg²⁺, Ca²⁺, Co²⁺, Zn²⁺, Cu²⁺, Ni²⁺, Pb²⁺, Al³⁺, Fe³⁺, Cr³⁺ and Cr⁶⁺, 0.5 mM) and anions (F⁻, Cl⁻, Br⁻, I⁻, CO₃^2–, HCO₃– and HPO₄^2-, 0.5 mM), followed by excitation at 320 nm. Fig. 6 and Fig. 7 revealed that the fluorescence intensity of L-Al³⁺ at 533 nm had no obvious fluorescent alterations in the presence of the other metal ions and anionic species, concluding with the fact that the tested ions had no prominent impact on fluorescence intensity of the L-Al³⁺ system.

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Fig. 6

(blue bar): metal ion selectivity profile of the sensor L (0.1 mM); (orange bar): change in emission of L + Al³⁺ (0.5 mM); (purple bar): change in emission of L + Al³⁺ + Mⁿ⁺ (0.5 mM) at 533 nm.

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Fig. 7

(blue bar): anion selectivity profile of the sensor L (0.1 mM); (orange bar): change in emission of L + Anion^n- (0.5 mM); (pruple bar): change in emission of L + Al³⁺ + Anion^n- at 533 nm.

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3.4 Fluorescence titration study and limit of detection (LOD) of aluminum ions

Non-fluorescence property of the ligand (L) was altered by the addition of Al³⁺ to the yellow colored L solution resulting in a bright white fluorescence under excitation at 320 nm. The fluorescence titration of L upon the addition of different concentrations of Al³⁺ from 0.25 mM to 0.195 μM at 533 nm of an emission wavelength revealed an increase in the fluorescence emission intensity, as seen in Fig. 8.

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Fig. 8

Fluorescence emission spectra recorded for L (0.1 mM) upon increasing concentration of Al³⁺ ion (2.50 × 10^-4, 2.25 × 10^-4, 1.75 × 10^-4, 1.50 × 10^-4, 1.25 × 10^-4, 1.0 × 10^-4, 5.0 × 10^-5 1.25 × 10^-5, 6.125 × 10^-6, 3,125 × 10^-6, 1,56 × 10^-6, 7.8 × 10^-7, 3.9 × 10^-7, 1.95 × 10^-7 M).

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To make a detailed investigation about the binding mode, limit of detection (LOD) of L for Al³⁺ was determined using fluorescence titration results. Fig. 9 represents the fluorescence emission intensity of L (I) in the presence of Al³⁺ at 533 nm versus the concentration of Al³⁺ ions ranged from 0.25 mM to 0.195 μM. From the changes in Al³⁺ dependent fluorescence intensity (I_{533 nm}), the LOD in EtOH:H₂O (1:1, v/v) was estimated to be 2.59 × 10^-7 M, using the formula of LOD = 3σ/k formula (Analytical Methods Committee, 1987), where σ was standard deviation of ten control measurements of L, and k was the slope of the line created by plotting the fluorescence intensities versus [Al³⁺] values. The calculated LOD value was below the tolerable concentration of Al³⁺ in drinking water (2.41 μM) as defined by WHO, and Al³⁺ levels higher than 2.4 μM may cause encephalopathy as reported in the literature (Kaur, and Kaur, 2017), indicating that L was highly sensitive to the recognition of Al³⁺ ions at the sub-micromolar concentration.

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Fig. 9

Plot of the fluorescence intensity at the PL emission peak of L at 533 nm versus the concentration of Al³⁺ (0.25 mM – 0.195 μM).

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Some reported similar studies about Schiff base sensors for the recognition of Al³⁺ and the calculated LOD, K_a values are arranged in Table 1. Based on the aforementioned findings, low LOD value of L for Al³⁺ specified that the Schiff base synthesized in this study demonstrated a distinctive selectivity compared to other Schiff base ligands sensing aluminum in the literature (Roy, 2021; Salarvand et al., 2019; Kaur, and Kaur, 2017; Zhu et al., 2016; Singh et al., 2013; Kumar et al., 2015; Huang et al., 2017; Wang et al., 2015, Manna et al., 2020; Ghorai et al., 2016; Chandra et al. 2020). Although Schiff base sensor with 2-hydroxy-1-benzaldehyde and anthracene moieties possessed a fluorescence response towards both Hg (II) and Al (III) in the presence of interfering metal ions (Kaur, and Kaur, 2017), the ligand synthesized in this study formed a chelate between only Al (III) in the presence of competing metal ions and anions, indicating a specific selectivity of L. The lowest LOD value for the Schiff base chemosensor synthesized by Ghorai et al. had a fluorescence response towards both Hg (II) and Al (III) (Ghorai et al., 2016). Another Schiff base containing pyrene and o-vanillin units synthesized by Shyamal et al. had 8.64 nM of LOD for Al³⁺ in CH₃CN: H₂O (95:5, v:v) (Shyamal et al., 2016a).

Table 1 Comparison of same Schiff base ligands for the selectivity of Al³⁺.

Structure of chemosensor	Media	LOD (mol L-1)	Ka (M−1)	Reference
	EtOH/HEPES buffer (95:5, v/v)	1.08 x10-7	6.53 x103	(Zhu et al., 2016)
	MeOH-H2O (9:1 v/v)	6.4 × 10-7	1.546 × 105	(Salarvand et al., 2019)
	CH3CN	3.2 × 10-7	3.148 × 104	(Singh et al., 2013)
	MeOH-H2O (1:1 v/v)	5.0 × 10-5	4.0 × 106	(Kumar et al., 2015)
	CH3CN: H2O (1:1, v:v)	1.98 × 10-6	4.35 × 104	(Huang et al., 2017)
	EtOH/ H2O (1:1, v:v)	2.2 × 10-7	1.89 × 104	(Wang et al., 2015)
	H2O:DMSO (1:9, v/v)	1.27 × 10-6	6.4 × 104	(Roy et al, 2021)
	MeOH-H2O	5.2 × 10-6	1.05 × 104	(Kaur and Kaur, 2017)
	MeOH-Tris-HCl buffer (1:1, v/v)	1.15 × 10-7	1.76 × 102 M-1/2	(Manna et al., 2020)
	MeOH-water (2:1, v/v)	1.26 × 10-8	1.23 × 105	(Ghorai et al., 2016)
	CH3CN: H2O (1:1, v:v)	1.17 × 10-7	2.35 × 105	(Chandra et al., 2020)
	EtOH/deionized H2O (1:1, v/v)	2.59 × 10-7	5 × 104	This study

Since the analysis of an analyte in a high water content solvent was more suitable for the analysis of real samples, the water content was 50% for the complete dissolution of the synthesized Schiff base in this study.

3.5 Binding affinity of L towards Al³⁺

A meticulous study was carried out to comprehend the binding behavior of L towards Al³⁺ ions using Job’s plot analysis based on fluorescence measurement (Likussar and Boltz, 1971; Job, 1984). In this method, standard nine mixtures were prepared by adding aliquots of Al³⁺ equivalent to 9–1 µmol into a series of 10 mL volumetric flasks containing aliquots of L equivalent to 1–9 µmol so that each flask contained a total number of 10 µmol. Job’s plot was acquired by plotting PL emission values at 533 nm versus the mole fraction of ligand (L) where the transition point for fluorescence intensity was observed at the molar fraction of 0.5, specifying that 1:1 stoichiometric complexation of L with Al³⁺ (Fig. S5). To support 1:1 binding mode, LC-MS/MS of L and L + Al³⁺ were presented. The LC-MS/MS spectrum of L revealed an intense peak at m/z 365 corresponding to [L + H]⁺ (Fig S6). Upon addition of 20 eqv. of Al^3+, a main peak at m/z 410 appeared and could be assignable to the 1:1 formation of [L + Al (III) + H₂O + H]⁺ (Fig. S7). Al³⁺ used two 3-level orbitals to make chelation with L and one 3-level orbital to accept lone pair from one water molecule. The result was to obtain an isotopic peak at m/z 410 of [L + Al (III) + H₂O + H]⁺.

The calculation of the binding constant (K_a) for the L + Al³⁺ chelation was obtained according to Benesi-Hildebrand expression (Benesi and Hildebrand, 1949). Fluorescence titration study provided a plot for the calculated values of [1/(I-I₀)] at 533 nm depending on 1/[Al³⁺], as seen in Fig. 10. The slope of the line drawn by using Benesi–Hildebrand equation led us to calculate the association constant (K_a) of the L + Al³⁺ complex, which was found to be 5 × 10⁴ M⁻¹. I and I₀ were the fluorescence emission intensities of L in the presence and in the absence of Al³⁺ ion, respectively.

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Fig. 10

Benesi-Hildebrand plot of 1/I-I₀ versus 1/[ Al³⁺].

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3.6 Reversibility process using electrochemical techniques

The reversibility of the chemosensor to target ion binding is a very important aspect in practical application. Accordingly, the reversibility of L + Al³⁺ chelation was studied using electrochemical techniques (Patil et al., 2019). To estimate oxidation–reduction behavior of L, redox behavior was investigated by the cyclic voltammetry (CV) technique with a predictable three-electrode cell assembly with Pt wire as counter-electrode, glassy carbon as a working electrode and Ag/AgCl as the reference electrode. The supporting electrolyte was 0.1 M TBAPF₆ in acetonitrile solution. The CVs were monitored at a scan rate of 100 mV s⁻¹ in the potential range from + 2.0 V to -2.0 V to detect peaks in both forward and reverse scans at room temperature and under Argon atmosphere. Two quasi-reversible oxidation CV waves representing the oxidation–reduction process were apparent in Fig. S8. The HOMO-LUMO energy levels and the calculated band gap values are tabulated in Table 2. Initial oxidation corresponded to carbazole oxidation at a potential of 1.15 V, forming the cation radical (polaron) in one-electron process (Karon and Lapkowski, 2015). The first oxidation peak was reversible. Since each discrete oxidation process was also assigned to a unique one-electron transfer, the other oxidation then formed at around 1.54 V associated with hydroxyl group, forming polaron structure when scanning to higher anodic potentials (E_pa) (Seo et al., 1966). The cathodic reduction peak (E_pc) of L, seen at −1.55 V, was due to the reduction process observed via protonation of azomethine nitrogen of C = N linkage (Kolcu and Kaya, 2016). The HOMO and LUMO energies designating the ability to donate an electron and the capability to accept the electron were calculated using the formulas of E_HOMO= -(4.39 + E_pa) eV and E_LUMO= -(4.39 + E_pc), respectively. To gain knowledge about the electron transition to take place in the ligand and in the ligand-Al³⁺ complex, the calculation of band gap (E'_g) energy between HOMO and LUMO was fulfilled. The energy gap had control over the electron transition to occur in the ligand and in the ligand bound to metal ion. E'_g was found to be 3.10 eV and decreased to 2.52 eV after addition of Al³⁺ ion, concluding with the fact that the formation of L + Al³⁺ system lowered the energy required for the electronic transition. These results were also in conformity with the result obtained from UV–vis spectral studies, in which a decrease in the calculated optical band gap (E_g) value of L after coordination to Al³⁺ ions, as a result of a chelation between L and Al³⁺. The disappearance of the reduction peak of L at −1.55 V, as seen in Fig. 16, in presence of Al³⁺ confirmed that chelation took place between Al³⁺ ions and L. The reversibility feature, which was essential in developing a sensor (Rana et al., 2017), could be tested by titration of L-Al³⁺ complex with 1.0 equivalent of Na₂EDTA, which resulted in the recovery of the original C = N reduction peak of L in the voltammogram, as displayed in Fig. 16. The reappearance of the original -C = N- reduction peak of L revealed that EDTA displaced the metal ion in L-Al³⁺ system, confirming decomplexation of the L-Al³⁺ adduct; in other words, L could be successfully recovered.