Novel Nano Zn +2 -Compound from LA Ligand as an Acid-Base Indicator: Synthesis, Characterization, pH Sensor, and Fluorescent Study

The main goal of our research is to synthesize a novel nano Zn +2 complex with the 8-(2-(dansyl chloride)azo)adenine (LA) ligand as a pH sensor because of its advantageous properties. The molecular formulas of the ligand and Zn +2 -LA complex were confirmed by studying the elemental analyses (C.H.N.), spectral methods, XRD, and thermal analyses (TGA). The molar ratio of Zn +2 -LA was studied as a ratio (1:2) (M:L). Through the survey in FT-IR and UV-Vis spectra, it was found that the complex has a tetrahedral geometry shape with a dark purple color (552 nm), obtained from a single molecule. The optimum concentration is 0.3 mM, pH = 5.5, and time = 0-3 hours, which in the studies followed Lambert-Beer’s law. Linearity ranged from 0.025 to 0.4 mM for the Zn +2 -LA complex, with a linear correlation coefficient of 0.9963. The detection limit was 0.0219 μg/mL, and the limit of quantification was 0.0663 μg/mL. LA could be used as a titration indicator of sodium hydroxide (NaOH) by oxalic acid (H 2 C 2 O 4 ) with a maximum concentration of 0.1 M of H 2 C 2 O 4 . Finally, the characteristic fluorescence peaks of the solutions were identified at 462 nm and 554 nm for the ligand LA and its complex respectively, which were dependent on pH = 5.5 and at 1 minute. The type of emission fluorescence is 𝜋∗ → 𝑛 transition.


Introduction
Acid-base indicators, also referred to as pH indicators, are weak acids or bases with a chromic halo that change their structural composition as the solution's pH changes.Such indicators should, in theory, be sharp visual shifts, selective, sensitive, stable, infrequently specific, and free from interference with the solution's constituents.Examples include color, luminescence, fluorescence, or the lowest possible concentration of turbidity.The presence of a pH indicator has no bearing on whether a chemical solution is acidic or alkaline when employed as a diluted solution [1].Numerous signs can be found in nature.A natural pH indicator built from a variety of lichens is called litmus, for instance.Flowers and vegetables include anthocyanins, which serve as pH indicators, and carotenoids [2].Several azo compounds, including methyl red, methyl orange, and congo red, exist in three tautomeric forms; each one has a unique hue and can transform from one shape to another based on the pH of the environment.As a result, they are employed as pH indicators.Methyl red turns red at pH values below 4.4, yellow at pH values above 6.2, and orange at pH values in the middle [3].Methyl orange is typically employed in the titration of acids.It is colored yellow in basic media and red in acidic media [3].Researchers in many different domains have extensively exploited this property for detecting pH levels; in analytical chemistry, it is employed to establish the end point of an acid-base titration [3] and calculate the acidity of the living medium for biological research [4].Additionally, azo compounds have been thoroughly studied in applications like inkjet printing and optical recording mediums due to their superior thermal and optical properties, oil-soluble light-fast dyes [5], organic photoconductors [6], and molecular memory storage [7].Recent studies have found a tremendous increase in interest in identifying the most extensively used azo compounds as markers due to the existence of delocalization and hues, especially yellow, red, and orange.Organic azo compounds such as p-dibenzothiophen azo-8-naphthol derivatives [8] is are colorless to orange in basic media and from orange to colorless in acidic media, and the pH range of color change was (6-10); mercurial azo compounds [9], 1,8-naphthalimide derivatives [10], azo ligands as 2-amino-6-ethoxybenzothiazole and 4-chloro-3,5-dimethyl phenol derivatives [11], 3,4-dihydroxyazobenzene (DHAB), 4'sulfo3,4dihydroxyazobenzene (SDHAB), calcon (mordant black 17, CLN), 4-(thiazolylazo)resorcinol (TAR), 4(2thiazolylazo) 5 diethyl maminophenol (TAAP) azo compounds [12], 8-[5-(2-sulfonic acid naphthyl)azo] Caffeine (SNAC) is brown in acidic medium and red in basic medium [13], [4-(5-(7-Bromo-8hydroxyquinoline)azo)sulfamethoxazole] (HAS) is orange in acidic medium and red in basic medium [14], and 2-((9H-prin-6-yl)diazenyl)-5-(dimethylamino) naphthalene-1-slfonyl chloride (DAD) is purple color in basic medium and yellow color in acidic medium [15], and for the fluorescence spectroscopic analysis [16], sensor activity of three N-phthalimide azoazomethine derivatives toward metal ions in environmental water samples [17], an azo dye (E)-1-((5-methoxythiazolo [4,5-b]pyridin-2-yl)diazenyl)naphthalen-2-ol (TPN1) based sensor in a purple color dye for selective and sensitive detection of Cu +2 , Sn +2 , and Al +3 ions [18].In our work, novel nano Zn+2-LA complexes will be synthesized and characterized by spectroscopic techniques (FT-IR, UV-Vis, and 1 H NMR), thermal analysis (TGA), elemental analysis (C.H.N), XRD, and molar conductivity, before investigation of their dyeing properties, antioxidants, and anticancer applications.

Materials
All chemicals (ZnCl 2 , NaNO 2 , HCl, Adenine, Dansylchoride, and Ethanol) were obtained from Sigma Aldrich and used without further purification.

Chemistry 2.2.1. Synthesis of [8-(2-(dansyl chloride)azo)adenine] (LA) ligand
In a round-bottomed flask, adenine (1 mmol) and sodium nitrite solution (15 mL, 10%) at 0-5 °C were stirred for 30 minutes to afford the diazonium salt of adenine.This salt was then slowly added with continuous stirring to the cooled basic solution of adansyl chloride, which was used as a coupling reagent.The solid crude material was then filtered, washed with ethanol and water several times, and dried to give the desired ligand [19].

Synthesis of Zn +2 -LA complex
A solution of the ligand (0.002 mol) in a minimal amount of ethanol was slowly added to a stirred ZnCl 2 solution (100 mL, 0.001 mol/L).The reaction mixture was then stirred until a deep violet-colored precipitate was formed.The solid crude material was then filtered, washed several times with ethanol and water, and dried in air to give the Zn +2 -LA complex (Scheme 1) [14].

Statistical analysis
The data were presented as averages, and the standard deviation (SD) was gathered from studies done in triplicate.Data were evaluated using the "Origin 5.0" ANOVA to analyze differences, and significance was shown at p<0.005 [15].

Results and discussion
Dansyl chloride and adenine were used to synthesize a novel azo ligand (LA) over a reaction (diazo-coupling).This ligand was reacted with ZnCl 2 to form a Zn complex.The results taken from elemental analysis and magnetic susceptibility for the LA and Zn ⁺ 2 -LA complex satisfying approval with calculated values are shown in Table 1.The proposed structural formula for the ligand (LA) and the Zn ⁺ 2 -LA complex was confirmed by the microelemental analysis (C.H.N), magnetic moment, thermal gravimetric analysis (TGA), and molar conductivity measurements; however, the Zn ⁺ 2 -LA complex has an ionic character with a (1:2) electrolyte.Also, the synthesized compounds become stale with time (Figure 1).Unaffected by the atmosphere and moisture, as well as being dissolved in ethanol, methanol, DMSO, and DMF.

FT-IR spectroscopy
The identification vibration bands of the free ligand (LA) and Zn ⁺ 2 -LA complex were compared.The FT-IR spectrum of the ligand (LA) was compared to the Zn ⁺ 2 -LA complex spectrum in order to confirm the cheating mode with the LA to the Zn⁺ 2 in the complex.The FT-IR spectrum of the ligand (LA) showed two absorption bands at 1598 and 1620 cm -1 belonging to the C=N group at the pyrimidine ring (Figure 1).This band was then shifted to lower frequencies (1614 cm -1 ) in the spectrum of Zn⁺ 2 -LA complex, with sharpness due to the chelating of the zinc with nitrogen atoms (Figure 2) [20].The most characteristic band in the LA spectrum is for the N=N group, which appeared at 1436 and 1402 cm -1 , and with chelating to Zn ⁺ 2 , these bands changed to 1442 and 1409 cm -1 [21].The other absorption bands, such as N-H and SO 2 Cl, were not unaltered in shape and position, which is a good indication of their nonparticipation in the chelation with Zn ⁺ 2 .The FT-IR spectrum of Zn ⁺ 2 -LA complex revealed new bands at 305 and 305 cm 1 attributed to the M-N (Zn ⁺ 2 with an azo group) and M-N (Zn ⁺ 2 with a pyrimidine ring), respectively [22,23].From these results, we can conclude that the ligand LA behaves neutrally as N,N-bidentate.All the details of the FT-IR spectral data for the ligand complex are shown in Table 2.

Thermogravimetric analysis (TGA)
The thermal decomposition behavior of the ligand (LA) and Zn ⁺ 2 -LA complex was discussed by the thermogravimetric technique.The TGA thermograms are shown in Figures 3  and 4, while the outcomes are listed in Table 3.Generally, the ligand and its complex display for steps in the range 25-800 °C with argon gas.For the Zn ⁺ 2 -LA complex, the first step of decomposition started at 25 °C and ended at 50 °C with a corresponding weight loss of 2.90%, which is in indication of the presence of lattice water.The second stage of decomposition was noticed at temperatures ranging from 50 to 285 °C (25.96% weight loss).The third and fourth stages of decomposition lost weights of 25.96 (285-590 °C) and 9.12% (590-800 °C), respectively.The Zn ⁺ 2 -LA complex is more thermally stable than the ligand (LA) due to the complex residue (35.41%), while the residue of the LA ligand is 5.29% [15,24].

Scanning electron microscopy analysis (SEM)
The forms of the surface for the Zn ⁺ 2 -LA complex were confirmed by the studying SEM technique.Figure 5 displays the SEM image for the complex, which is non-uniform with a smooth surface and coral shape (D = 53.83nm).The image tends to be white, which indicates the large molecule [25].

X-Ray diffraction (XRD) analysis
The X-ray diffraction patterns in the range 5 ≤ 2θ ≤ 65 (for the LA ligand and Zn ⁺ 2 -LA complex with an X-ray source crystal (CuKu), which was examined, indicated the degree of crystallinity of these compounds.Figures 6 and 7 showed the three strongest reflection peaks for the LA ligand and Zn ⁺ 2 -LA complex in the range of 2θ (11.3495-13.971)and (11.3491-16.5335),respectively.The main characteristic reflection peak for the LA ligand occurs at 11.3491, and the band at 16.5335 is for Zn ⁺ 2 -LA complex.According to this information, the semi-crystalline nature and particle size of the LA ligand and Zn ⁺ 2 -LA complex were estimated from XRD patterns based on the highest intensity value compared with the other bands employing the renewed Debye-Scherrer equation (D = (k λ/β cos θ) [26], where D is the mean size of crystalline domains, which may be equal to or smaller than the crystal grain size (the apparent particle size of the grains) and was represented as volume function by A or nm unit.K is a constant that represents a factor without unit and typical value at a bout (K = 0.94), λ is the X-ray wavelength (0.15406 nm), β is the full width at half maximum (FWHM) of the X-ray diffraction peak, whereas θ is the Bragg angle.The particle sizes of the LA ligand and Zn ⁺ 2 -LA complex are 0.1679 and 0.3480 nm, respectively.These values emphasize that the particle size is located within the nanoscale range [27].The interplanar spacing (d) was calculated from the status of the intense peak according to Brag's equation (nλ = 2d sin θ).Where λ = 1.5406A 0 ; n = an integer number].The d dot was calculated and observed in the LA ligand at 7.781; 7.790 Å, and in the Zn ⁺ 2 -LA complex at 5.355; 5.357 Å.

. Electronic absorption spectra and magnetic moment measurements
The azo ligand (LA) and Zn ⁺ 2 -LA complex were examined in the UV-Vis range.The free ligand appeared at  →  * transition due to intramolecular charge transfer of heterocyclic and the aromatic moieties were assigned at 462 nm (21645 cm -1 ) [28].The electronic spectrum of Zn ⁺ 2 -LA complex showed a shift towards a high wave length of the absorption band at 554 nm (18050 cm -1 ) and conformed the chelating of the ligand (LA) to the Zn ⁺ 2 (Figure 8) [29].

Figure 8 :
The UV-Vis spectrum of the LA ligand and Zn +2 -LA complex

Optimization of experimental conditions for the Zn⁺ 2 -LA complex
The complex's optimum performance was attained using a variety of experimental settings, including pH reaction, concentration of the Zn ⁺ 2 -LA complex, and time.The studies' pH was controlled using the Briton-Robinson buffer (BR) during this process.The results of interference investigations showed that when there is a Zn ⁺ 2 -LA and BR buffer solution, less interference is present.But in the absence of Zn +2 , the LA pH values had no impact on the hue's transition to yellow [15].The Zn +2 -LA solutions displayed a deep purple color, and the peaks in the absorption spectra were visible at 554 nm using the BR buffer solution with a pH range of 2-5.5 and for the LA ligand at 464 nm.The hue of the solutions was unaffected by changing the BR buffer solution, and thus for further studies.The BR buffer solution specified a concentration of 40 mM.The Zn +2 salt is mixed immediately with the LA ligand at 25 °C to synthesize Zn ⁺ 2 -LA complex, and the maximal absorbance of the substance is 554 nm, as shown in Figure 9.The dark purple color gets more intense at pH 5.5.

Figure 9 :
The effects of pH buffer solutions on the color development of Zn +2 -LA complex 0.3 mM, BR buffer solution 40 mM, and 25 °C at 0 minutes As shown in Figure 10, estimating the optimal concentration for the Zn⁺ 2 -LA complex is necessary.As anticipated, the reaction was failed when the Zn +2 -LA combination present at a lower concentration (less than 0.02 mM), which reduced the sensitivity of the Zn⁺ 2 -LA complex.When the concentration of the Zn +2 -LA complex is greater than 0.025 mM, as procedure becomes increasingly sensitive, and the developed solution's color deepens.Therefore, the optimum Zn +2 -LA complex concentration was determined to be 0.3 mM.In the presence of various concentrations of Zn⁺ 2 -LA complex, under all ideal experimental settings, Abs.pH systems for identifying colors in UV-Vis absorption spectra were used, and the solution's absorbance at 554 nm significantly increased with the concentration of Zn⁺ 2 -LA complex.

Figure 10 :
The effects of the concentrations on color development of the Zn +2 -LA complex, 0.025-0.35mM, pH = 5.5, 40 mM BR buffer solution and 25 °C at 0 minutes The solution's color gradually changed from yellow to deep purple.With the bare eye, it is easy to see how the solution's color changes (i.e. the experiment's visual limit) when the concentration of Zn +2 -LA complex is higher than 0.025 mM.For speedy and portable colorimetric detection, it was also necessary to take the Zn +2 -LA complex reaction time into account.The proposed methodology's accuracy and repeatability are impacted by the response temperature's tendency to degrade, which was chosen at 25 °C.Figure 11 showed that the Zn +2 -LA complex reaction timings were completed more quickly than it would have taken for the reaction to finish.

The analytical performance for the Zn +2 -LA complex
The Zn +2 -LA complex solution's absorbance at 554 nm increased noticeably as the Zn +2 -LA complex concentration increased from 0.025-0.35mM.The outcome was that the color of the solution progressively shifted from yellow to deep purple.With the bare eye, it is easy to see how the solution's color changes (i.e., the experiment's visual limit) when the Zn +2 -LA complex concentration is higher than 0.025 mM.Absorption of the Zn +2 -LA complex with excellent linearity with LA concentrations was seen at 554 nm in the range of 0.025-0.35mM, as shown in Figure 12.Abs.

Time (min.)
Figure 12 : The colorimetric methods successfully used all of the ideal conditions to achieve the linear calibration curves for Zn +2 -LA complex determination, 0.025-0.35mM for the Zn +2 -LA complex at 25°C, 0 minutes, pH 5.5, and 40 mM BR buffer solution.
The resulting regression equation was: A 554 = with a correlation coefficient (R 2 ) of 0.9963, where x is the concentration expressed in mM units.The limits of detection (LOD) were determined using the formulas (3.3σ/S) in mM, and quantification (LOQ) was determined using the formulas (10σ/S) in M, where S is the regression equation's slope and σ is the intercept's standard error.By measuring the relative standard deviation (RSD), the reproducibility of the Zn +2 -LA complex was confirmed, and it was determined to be less than 3% at n = 5, demonstrating the method's excellent parameter.The concentration of the Zn +2 -LA complex is 0.3 mM, which shows very good repeatability and reproducibility, as shown in Figure 10.For the suggested methods, all regression equation statistics are given in Table 4.

Proposition the mole ratio
Through the spectroscopic study of the solutions of mixing Zn +2 with LA ligand, the molar ratio method was applied by utilizing the optimal concentration (0.3 mM). Figure 13 shows the relationship that was obtained.The mole ratio of LA ligand and Zn +2 -LA complex while the data are listed in Table 5.The result indicates the formation of a M:L (1:2) ratio.

Stability constant and Gibbs free energy (ΔG)
To find the stability constant (K) for a Zn +2 -LA complex with a molar ratio of 1:2 of M:L, we follow the following equations [28].
; Where Degree of dissociation C = Molar concentration for the complexes in molar (c = 0.3 mM) Absorbance of a solution containing an excess of ligand Absorbance of a solution containing a stoichiometric amount of ligand and metal ions.It was found that it possesses a high stability constant (K) (26.04 × 10 6 Mol -2 .L 2 ).The high value of (K) gave good information about the stability of the complex.

Mole ratio
Based on the sharp color change from yellow to bright purple with indicator LA ligand examination, LA ligand will be further examined for acid-base titration indicator compounds.LA ligand will be tested for equivalent points in acid-base at pH 3-11.

Color stability test
Color stability was determined by measuring the discoloration time at an equivalent point in the titration.Since the pH range that causes a color shift for LA ligand titration is between 3 and 11, for the purpose of examining color stability at an equivalent point, we shall select the NaOH solution with H 2 C 2 O 4 solution at different concentrations.Table 5

Applications of LA ligand as a titration indicator for acid-base
We shall contrast the use of the LA ligand as a titration indication with that of the reference indicator phenolphthalein during the titration of the NaOH solution with the H 2 C 2 O 4 solution (0.1 M). Figure 14 depicts the color change during titration, and Table 6 contains the results of the titration.According to Table 7, the target compound LA ligand can be used to titrate tacid-base with the same accuracy as phenolphthalein.

Fluorescent spectra of compounds
The emission spectra of the LA ligand and the Zn +2 -LA complex were recorded in EtOH at room temperature, as seen in Figures 15 and 16, respectively.The ligand exhibits an excitation maximum wavelength at 462 nm and is characterized by an emission maximum (λ em , max) at 700.723 nm and a low intensity (72.346 nm), which is shifted upon binding to metal ions.While the Zn +2 -LA complex has maximum fluorescence emission wavelengths (λ em ,max) at 700.372 nm, excitation wavelengths at 552 nm show a strong and sharp intensity of 372.346, which is slightly red shifted when compared to the ligand.It also exhibited a broad peak at 536 nm with an intensity of 21.5, while the Zn +2 -LA complex exhibits long wavelengths at 667 nm with an intensity of 21.57, indicating that the ligand successfully chelated metal ions.The type of emission fluorescence is the  * →  transition.

Dyeing properties
Azo dyes are highly colored and have been utilized as dyes and pigments for a long time [15].The LA ligand and Zn +2 -LA complex on a wool fabric were applied.These dyes provided colors from orange to pomegranate (Figure 17), with good brightness and depth on fabric (Table 8).As well as standing, the color fastness of the textiles was examined for washing with soap powder at a concentration of 2%.So provided excellent fastness to both wet and dry robbing and excellent light fastness for defiled and color shift, all of this is subject to Iraqi standards for woolen textile No. 3616.Grinding: 1-2 (not good), 3 (moderate), 5-4 (good) according to Iraqi specifications No. 3616 for woolen fabric.

Figure 17 :
The fiber textile dyeing of LA ligand and Zn +2 -LA complex

Antioxidant Activity
Figures 18 and 19 depict the scavenging effect of LA ligand and the Zn +2 -LA complex at various doses.As they high inhibition power compared to ascorbic acid, which was used as a control.Five concentrations (12.55, 25, 50, 100, and 200 µg/mL) of the LA ligand and Zn +2 -LA complex were more effective in DPPH radical scavenging activity than ascorbic acid.Table 9 shows the scavenging activity that increased dramatically with increasing concentration.

Study of the cytotoxic effects
The cytotoxic effectiveness and mechanism of behavior of the LA ligand and Zn +2 -LA complex were investigated against liver carcinoma cell lines (HepG-2) and normal cell lines (HdFn) by MTT assay after incubation for 24 hours at 37 °C with concentrations of 6.25-400 µg/mL.It was found that these compounds had different growth inhibitory effects on HepG-2 and HdFn cell lines.The data from Tables 10 and 11 and Figures 20 and 21 showed that the cell viability of the HepG-2 cell line was reduced by increasing the concentration of these compounds, but we notice the opposite in the case of the normal cell line (HdFn).It can be concluded that the greatest cytotoxic effect occurs at a concentration of 400 µg/mL.The half maximal inhibitory concentration (IC 50 ), which was utilized to measure drug efficacy, and also the p-value are used in hypothesis testing.The IC 50 is lower; therefore, a lower concentration of drugs is required to stimulate carcinoma cell death and thus can be tolerated.In this study, we demonstrate that a straightforward, economical, and precise novel nanoazo complex made up of Zn +2 ions with LA ligand works with a pH of 4. The LA might be utilized as a titration indicator instead of the phenolphthalein indicator for titration of NaOH with H 2 C 2 O 4 with the same result.Finally, we see a shift to a longer wavelength in complexes when compared with ligands, which indicates that the ligand successfully chelated metal ions.

Figure 5 :
Figure 5 : SEM image for the Zn ⁺ 2 -LA complex

Figure 6 :
Figure 6 : X-Ray diffraction of the LA ligand Figure 7 : X-Ray diffraction of the Zn ⁺ 2 -LA complex 3.5.Electronic absorption spectra and magnetic moment measurementsThe azo ligand (LA) and Zn ⁺ 2 -LA complex were examined in the UV-Vis range.The free ligand appeared at  →  * transition due to intramolecular charge transfer of heterocyclic and the aromatic moieties were assigned at 462 nm (21645 cm -1 )[28].The electronic spectrum of

Figure 11 :
Figure 11 : The effects of the reaction time of the Zn +2 -LA complex, 0.3 mM, pH = 5.5, 40 mM BR buffer solution, and 25 °C at 0-250 minutes

Figure 13 :
Figure 13 :The mole ratio of LA ligand and Zn +2 -LA complex while the data are listed in Table5.The result indicates the formation of a M:L (1:2) ratio.

Figure 14 :
Figure 14: Color change with indicator compound LA

Figure 18 :
Figure 18 : Scavenging activity of the LA ligand Figure 19 -Scavenging activity of the Zn +2 -LA complex

Figure 20 : 21 :
Figure 20 : Cytotoxic effect of the LA ligand Figure 21 : Cytotoxic effect of the Zn +2 -LA complex 4. ConclusionIn this study, we demonstrate that a straightforward, economical, and precise novel nanoazo complex made up of Zn +2 ions with LA ligand works with a pH of 4. The LA might be utilized as a titration indicator instead of the phenolphthalein indicator for titration of NaOH with H 2 C 2 O 4 with the same result.Finally, we see a shift to a longer wavelength in complexes when compared with ligands, which indicates that the ligand successfully chelated metal ions.

Table 1 :
Some physicochemical properties and elemental analysis of the ligand and Zn ⁺ 2 -LA complex

Table 3 :
TGA data for the ligand (LA) and Zn ⁺ 2 -LA complex

Table 4 :
The linear regression lines' summary statistic for determining the Zn +2 -LA complex

Table 5 :
Absorbance of a LA-metal ion solution versus mole ratio

Table 6 :
contains the results of the color stability of the equivalent point for the titration of NaOH solution with H 2 C 2 O 4 solution.According to Table 6, the range of H 2 C 2 O 4 concentrations is from 0.025 to 0.5 M. The analogous point for NaOH solution titrated by H 2 C 2 O 4 solution

Table 7 :
The results of H 2 C 2 O 4 solution (0.05 M) titration with NaOH using phenolphthalein and LA ligand as titration indicators

Table 8 :
Dying properties of LA ligand and Zn +2 -LA complex

Table 9 -
Radical scavenging activity of the LA ligand, the Zn +2 -LA complex, and ascorbic acid.

Table 10 :
Inhibition effect of the LA ligand on HepG-2 and HdFn cell lines after incubation for 24 hours at 37 °C

Table 11 :
Inhibition effect of the Zn +2 -LA complex on HepG-2 and HdFn cell lines after incubation for 24 hours at 37 °C