Single Crystal, Vibrational Spectroscopy, Hirshfeld Surface Analysis, DFT Studies, Antioxidant Activity of a New Single Hybrid Material EDTAH₂²⁺.2ClO^4-.4H₂O

1. Introduction

Inorganic-organic hybrids materials can be applied in many branches of materials chemistry because they are simple to process and are amenable to design on the molecular scale. The main idea in development of the hybrid materials was to take advantage of the best properties of each component that forms a hybrid, trying to decrease or eliminate their drawbacks getting in an ideal way a synergic effect; that results in the development of new materials with new properties. Hybrid materials do not represent only a creative alternative to design new materials and compounds for academic research, but their improved or unusual features allow the development of innovative industrial applications. In last few decades, these materials provide a large range of enhanced and various physico-chemical properties can lead to technological innovations and making them ideal for applications in various fields, such as energy storage, electronics, optical, thermal stability, environmental remediation, and biomedical engineering [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24].

A fascinating class of materials known as supramolecular hybrid materials combines the functionality of inorganic or organic components with the principles of supramolecular chemistry [25,26,27]. The supramolecular hybrid materials are held together only by non-covalent interactions such as van der Waals forces, hydrogen bonding, short contacts and π-π stacking, enabling the development of new structures with improved properties[28,29] . The combination of supramolecular chemistry’s molecular recognition and hybrid materials’ adaptability creates new opportunities for a variety of applications, from energy storage and drug delivery to catalysis and sensing [30,31], as well as, their outstanding potential for sustainability and environmental remediation has attracted increasing interest, these materials can be modified for effective recycling and pollution removal. [32,33,34]. Furthermore, recent researches in the design of novel supramolecular systems have generated prospects for intelligent, adaptive materials in nano-technological and biomedical applications [35,36,37]. This paper aims to provide the synthesis of a new supramolecular hybrid crystal, based on Ethylenediaminetetraacetic acid (EDTA) as organic part.

EDTA is a chemical used for both industrial and medical purposes. It has worked as a powerful chelate that used in analytic chemistry and industrial processes, it is a molecule that is known as a chelating agent, used something that has a claw-like structure that is use to stick and grab other molecules while, it is used to sequester and decrease the reactivity of metal ions that may be present in a product. Moreover, doctors prescribe them to clear toxins from the body. Also, it is one of the key ingredients in cancer-fighting medicines. Moreover, this material is being investigated for use in biomedical applications, specifically in drug delivery systems, where the chelation capabilities of EDTA can improve the medicines’ controlled release. EDTA-functionalized hybrid materials are useful for environmentally friendly and sustainable applications because they have shown great promise for environmental remediation, including the removal of heavy metal ions from contaminated water. With respect to its adaptability, EDTA is a promising component for creating multifunctional supramolecular hybrid materials with a wide range of other applications [38,39,40,41,42,43,44,45,46,47,48,49,50].

The new hybrid material crystal EDTAH₂²⁺.2ClO₄^-.4H₂O, the characterization of this material was carried out exploring single crystal X-ray diffraction, infrared and UV-visible spectroscopies. Through X-ray diffraction data, the 3D-supramolecular network was discussed, the Hirshfeld surface analysis provide supplementary discussion of the short-contacts and weak interactions within the supramolecular structure. In addition, the infrared spectroscopy divulges the vibrational frequencies, in which they were all attributed to the various functional group of the material. The electronic transitions were studied using UV-visible spectroscopy. Finally, the antioxidant activity of the synthesized hybrid material was evaluated in vitro using the β-carotene bleaching assay, with BHT used as a standard reference compound. In addition, Density Functional Theory (DFT) calculations were carried out to gain insight into its electronic properties and to rationalize its antioxidant behavior.

2. Experimental

2.1. Synthesis and Crystal Growth

EDTAH₂²⁺.2ClO^4-.4H₂O synthesized by the chemical reaction of a commercially with EDTA (Aldrich, 99%) and perchloric acid (Aldrich, 65%). The acid was added to the hot aqueous solution of EDTA with the help of a dropper, in the 1:2 molar ratio. Then the reaction mixture was brought to reflux under magnetic stirring. After 6 h of refluxing, the solution was cooled to room temperature, it remained clear, without any precipitants. The solution slowly evaporated during a few days till the white and single crystals appeared, collected by filtration, then dried in air. The chemical structure of the hybrid ionic material obtained (Scheme 1) was identified by single-crystal X-ray diffraction analysis.

2.2. Single Crystal X-Ray Diffraction (SXRD)

The single-crystal X-ray diffraction (SXRD) was performed at 150 K, using Bruker D8 Venture photon diffractometer, equipped with a Mo(Kα) anticathode and a graphite filter monochromator giving λ= 0.71073Å. The refinement was performed by the full-matrix least square method using SHELXL-2017 (Sheldrick, 2017) programs [51].

All observed reflections and data reduction were used for the unit cell refinement, by the mean of the CrysAlisRed program [52]. At the end, the structures were plotted by using Mercury software [53]. The X-ray crystallographic data for the structure reported in this paper have been deposited at “Cambridge Crystallographic Data Centre” (CCDC) with the reference number 2412884.

2.3. Hirschfield Surface Analysis

The 3-Dimensional Hirshfeld surfaces are mapped for the affined molecule with dnorm, shape-index, curvedness and 2-Dimensional fingerprint plots were generated using CrystalExplorer-17.5 [54].

2.4. UV-Vis. and FT-IR Spectroscopies

The UV-Visible spectra were performed in two different solvents: distilled water and methanol in the range 200-800 nm at room temperature, using JASCO V680 Spectrophotometer. The FT-IR spectrum was recorded in the range 4000-600 cm^-1 by SHIMADZU IRSpirit equipped with the diffuse reflectance attachment (Miracle Attenuated Total Reflectance Attachment) at a 4 cm^-1 resolution in the wave number’s region of 4000-400 cm-1

2.5. Antioxidant Activity Test in Vitro

2.5.1. β-Carotene Bleaching

The β-carotene bleaching assay is a widely used method in food chemistry for estimating the antioxidant activity of compound [55]. This assay relies on the discoloration of the yellowish β-carotene solution, which occurs due to the disruption of its π-conjugation system. This disruption results from the addition of lipid or lipid peroxyl radicals to the C=C double bonds of β-carotene, leading to its degradation. In this method [56], a β-carotene/linoleic acid emulsion in water serves as the reaction medium, where peroxyl radicals are generated. The reduction in β-carotene concentration is monitored in both a control sample and a sample containing the tested antioxidant. The antioxidant activity of the examined substance is then determined by spectrophotometric measurement at 490 nm, following a slightly modified version of a previously established protocol.

2.5.2. Antioxidant Activity Test in Vitro

The stock solution of the β-carotene/linoleic acid emulsion was prepared by dissolving 0.5 mg of β-carotene in 1 ml of chloroform. Next, 25 μl of linoleic acid and 200 mg of Tween 40 were added to the mixture. The chloroform was evaporated at 40 °C using a rotary evaporator, and 100 ml of oxygen-saturated distilled water was added to complete the emulsion preparation. Afterward, 350 μl of test material (2 mg/ml) dissolved in DMSO, along with the BHT standard (soluble in methanol), were mixed with 2.5 ml of the emulsion. A negative control was prepared using only the emulsion (containing methanol, H₂O, and DMSO) without test samples, while the blank contained only the test samples without the emulsion. The emulsion system was incubated in the dark at room temperature, and absorbance was measured at 490 nm at specific time intervals (t = 0, 1, 2, 4, 6, 24 and 48 hours). The β-carotene bleaching rate, indicating the antioxidant activity (AA), was calculated using the equation:

$$\mathrm{A}\mathrm{A}\mathrm{\%}={\displaystyle \frac{Ae}{Ac}}\times 100$$

Where: Ae is the absorbance of β-carotene in the presence of the test samples, and Ac is the absorbance of the control (BHT).

2.6. Quantum Calculations Details

The full geometry optimization were carried out using the Gaussian 9 program, based on density functional theory (DFT) [57], using Beck’s three parameter hybrid exchange functional [58], with Lee-Yang-Parr correlation functional (B3LYP) and 6-31G (d, p) basis set [59,60].

3. Results and Discussion

3.1. XRD Study

The asymmetric unit of the synthetized hybrid material crystal is shown in the Figure 2(a), it is formed by one water molecule, one perchloric acid anion and one half EDTA cation. In addition, the related detailed crystal data and data collection parameters are collected in the Table 1. Furthermore, any abnormal measurement of bond length or angle was noted on the studied crystal, all are in general order [61,62,63,64,65].

The Table 2 reports some characteristic bond lengths, angle and torsion measurements of the title material. Another common point that, the protonated EDTA molecules in the crystal possess a cis-conformation (Figure 1 (a)), maintained by two intramolecular hydrogen bonds of the type O-H∙∙∙O, also it is centroid and characterized by a punctual symmetry group C2, noting that all carboxylic acid groups have a similar steric environment of slightly deformed sp2-hybridization (Table 1).

In addition, the synthetized ionic material crystalizes in monoclinic system with P2/n space group, with the unit cell filling (Z=2), that having the parameters a=5.5183(2), b=13.9178(5), c=14.3894(5)Å and β=99.458(2)°. In addition, the unit cell is characterized by identity symmetry, an inversion center at the origin (0, 0, 0), 2-fold rotation axis along b-direction at (0, y, ¼) with a screw component [0, ½, 0], and a glide plane perpendicular to b-direction with glide component [0, 0, ½].

Furthermore, the junction between the different components is ensured by weak interactions, forming consequently a hybrid type-I material. These weak interactions are mainly hydrogen bonds type, and counted to be twelve inter and intra-hydrogen bonds for one formal unit cell, and all are of the types O-H∙∙∙O and N-H∙∙∙O, this richness of hydrogen bonds can find interest in supramolecular chemistry field[18,19,20].

Table 3. Hydrogen bond length and angles obtained from SXRD considering 3Å as maximum donor acceptor distance.

D-H∙∙∙A	D-H (Å)	H∙∙∙A (Å)	D-A (Å)	D-H∙∙∙A (°)
O2-H∙∙∙O10	0.840	2.820	2.983	92.91
N5-H∙∙∙O13	0.864	2.171	2.815	131.14
O2-H∙∙∙OW1	0.840	1.724	2.553	168.88
OW1-H∙∙∙O3	0.843	2.010	2.829	163.83
O10-H∙∙∙OW2	0.854	2.251	2.617	135.22
O14-H∙∙∙OW1	0.845	2.015	2.858	174.92
O12-H∙∙∙OW2	0.851	2.175	2.668	116.69
O11-H∙∙∙OW2	0.854	2.251	2.919	135.22

Table 3. Hydrogen bond length and angles obtained from SXRD considering 3Å as maximum donor acceptor distance.

D-H∙∙∙A	D-H (Å)	H∙∙∙A (Å)	D-A (Å)	D-H∙∙∙A (°)
O2-H∙∙∙O10	0.840	2.820	2.983	92.91
N5-H∙∙∙O13	0.864	2.171	2.815	131.14
O2-H∙∙∙OW1	0.840	1.724	2.553	168.88
OW1-H∙∙∙O3	0.843	2.010	2.829	163.83
O10-H∙∙∙OW2	0.854	2.251	2.617	135.22
O14-H∙∙∙OW1	0.845	2.015	2.858	174.92
O12-H∙∙∙OW2	0.851	2.175	2.668	116.69
O11-H∙∙∙OW2	0.854	2.251	2.919	135.22

Moreover, the 3D-network of title material is constituted by an alternation of organic and inorganic supramolecular chains can be seen in the direction (Oc), Alternated supramolecular chains of protonated EDTA and water molecules disposed along the (Oc) direction (Figure 2 (a)), two water molecules join two protonated EDTA molecules via hydrogen bonds of the type O-H∙∙∙O, in which, each oxygen atom of the two water molecules acts as donor and acceptor. Along the same direction, a supramolecular inorganic chain constituted by an alternation of one perchlorate and one water molecules, interconnected by O-H∙∙∙O hydrogen bonds type. In addition, the organic and the inorganic chains are joined by hydrogen bonds of the type N-H∙∙∙O, while the organic chains are related between them by hydrogen bonds of the type O-H∙∙∙O (Figure 2 (a)). Along the perpendicular direction (Oa), an almost parallel supramolecular layers form the 3D network, that can be seen as a superposition of unit cell, which each unit cell contains one layer part.

3.2. Hirshfeld Surface Analysis (HS)

An important technique for gaining more definition in the intermolecular interaction of molecular crystals is the study of the Hirshfeld surface (HS). The size and shape of the HS allow the qualitative and quantitative investigations and visualization of near intermolecular contacts in molecular crystals [66]. Furthermore, a collection of 3D points in which the target molecule’s electron density contribution equals that of every other molecule determines the HS. The distance between any HS point and the inner (di) or externally surface (de) of the nearest nucleus is known as the conventional contact distance mapping (d_norm), and it is used to categorize molecular interactions (Formula 1) [67].

$${{\displaystyle d}}_{norm}={\displaystyle \frac{{{\displaystyle d}}_i{{\displaystyle -r}}_i^{\upsilon dW}}{{{\displaystyle r}}_i^{\upsilon dW}}}+{\displaystyle \frac{{{\displaystyle d}}_e{{\displaystyle -r}}_e^{\upsilon dW}}{{{\displaystyle r}}_e^{\upsilon dW}}}$$

(1)

Where r_e^vdW and r_i^vdW are the van der Waals radius of the appropriate atom internal and external respectively. Using a red-blue-white color scheme, the d_norm rates were mapped onto the HS as follows: the closer distance and negative d_norm values are represented by the red regions; the longer contacts and positive d_norm values are represented by the blue domains; and the contact lengths equal to the VdW separation and zero d_norm values are represented by the white regions. However, the two-dimensional fingerprints show the distance between molecules in the crystal, which improves the HS analysis [68]. When, the shape index (S), which is a dimensionless measurement of the local form of the surface that changes from red to blue throughout the interval [−0.99, + 0.99], can be written as follows using formula (2):

$$S={\displaystyle \frac{2}{\pi }}\arctan \left({\displaystyle \frac{{{\displaystyle k}}_1+{{\displaystyle k}}_2}{{{\displaystyle k}}_1-{{\displaystyle k}}_2}}\right)$$

(2)

Where, the primary curvatures of the Hirshfeld surface at the site of study, denoted by k1 and k2. On the other hand, curvedness © expresses le the root of the average square of the surface curvature, it is defined by the formula (3):

$$C={\displaystyle \frac{2}{\pi }}\ln \sqrt{({{\displaystyle k}}_1^2+{{\displaystyle k}}_2^2)/2}$$

(3)

The values of © are contain in the domain ([-4.0, +0.4]), which the domain’s extremities are commonly indicated by red and blue colors [69].

The molecular HS mapped with d_norm, shape index, and curvedness is shown in Figure 3. The form index is between -0.99 and 0.99Å in color, the curvedness is between -3.6 and +0.48Å, and the d_norm surfaces are mapped between -0.74 and 1.34Å. Furthermore, the d_norm surface exhibits extremely intimate intermolecular interactions, and the information from the shape index is compatible with 2D fingerprint plots and is especially sensitive to even little variations in the surface shape. The degree of curvature of the crystal depends on the quantity of its forms. Curved surface areas are associated with low curvedness values. Conversely, the sharp curvature zones tend to link with significant curvedness and divide the surface into patches, indicating interactions. The nearest neighbor coordination environment of a molecule is identified from the color patches on the Hirshfeld surface depending on their closeness to adjacent molecules (Figure 3 (d)).

Table 4. lists the results of the HS analysis, which also determines several quantitative measurements including asphericity, globularity, SH area, and SH-volume. The globularity, which is less than one [70], indicates that the HS is far from spherical, and the asphericity [71,72], indicates structural anisotropy.

Table 4. Quantitative measures of Hirshfeld surfaces.

Molecular volume VH (Å3)	Surface area SH (Å2)	Globularity (G)	Asphericity (Ω)
418.77	396.33	0.683	0.198

Table 4. Quantitative measures of Hirshfeld surfaces.

Molecular volume VH (Å3)	Surface area SH (Å2)	Globularity (G)	Asphericity (Ω)
418.77	396.33	0.683	0.198

The 2D fingerprints allow for the discussion of weak contacts and the differentiation of contributions from different types of interactions that overlap throughout the fingerprint. O···H connections constitute the primary intermolecular contact within the investigated crystal. The primary contributions were determined to be H···O, H···H, O···O, H···C and O···C contacts of 74.4, 14.2, 9.7, 1.2, and 0.5% respectively. The azote atom exhibits no contact in the crystal packing, as is typical in pentahedral coordination. The H∙∙∙O and O∙∙∙H contacts predominate, followed by the H∙∙∙H and O∙∙∙O contacts.

Figure 4. 2D-fingerprint plots of EDTA²⁺·2ClO₄⁻·4H₂O: showing the percentages of contacts contributing to the total Hirshfeld surface area of the molecule.

Figure 4. 2D-fingerprint plots of EDTA²⁺·2ClO₄⁻·4H₂O: showing the percentages of contacts contributing to the total Hirshfeld surface area of the molecule.

3.3. FT-IR Analysis

The experimental and DFT-calculated FT-IR spectra of the hybrid crystal EDTAH₂²⁺·2ClO₄⁻·4H₂O are presented in Figure 5, while the corresponding vibrational assignments are summarized in Table 5. Overall, an excellent agreement is observed between the experimental spectrum and the scaled theoretical frequencies, confirming the reliability of the optimized molecular structure and the DFT model. The small deviations between calculated and experimental band positions originate from the harmonic approximation adopted in the calculations, together with the neglect of crystal packing effects and anharmonic vibrational contributions, which commonly affect condensed-phase spectra.

The high-wavenumber region is dominated by vibrations associated with the extensive hydrogen-bond network. The broad experimental band at 3524 cm⁻¹, reproduced theoretically at 3432 cm⁻¹, is assigned to the asymmetric O–H stretching vibration of lattice water molecules. The band at 2750 cm⁻¹ (calculated 2790 cm⁻¹) is attributed to N⁺–H stretching of the protonated amine groups overlapping with asymmetric C–H stretching, further confirming the protonation state of the EDTA molecule within the crystal lattice. The band observed at 3010 cm⁻¹ (calculated 2865 cm⁻¹) is assigned to the O–H stretching of the carboxylic group interacting with water oxygen atoms, reflecting the strong involvement of the carboxylic functions in the hydrogen-bonding network.

The intense absorption at 1733 cm⁻¹, well reproduced by the DFT calculation (1718 cm⁻¹), originates from the C=O stretching vibration of the carboxylic group. The band at 1629 cm⁻¹ (calculated 1591 cm⁻¹) is assigned to the bending vibration of water O–H together with the H₂O in-plane bending mode, in line with the important role played by lattice water in the hydrogen-bond network. Intermediate-frequency bands located at 1457 and 1412 cm⁻¹ arise from coupled C–H, C–N, and C–C stretching modes together with O–H bending of the carboxylic group, and from C–N stretching coupled with N–H bending, respectively. A closely related band at 1452 cm⁻¹ is attributed to C–O and C–C stretching of the carboxylic group together with C–N and C–C bending, illustrating the strong vibrational coupling within this spectral region. The bands at 1339 and 1258 cm⁻¹ are assigned to C–N and C–H bending combined with O–C–O bending of the carboxylic group, and to the C–C asymmetric stretching vibration of the carboxylic acid function, respectively, while the band at 1195 cm⁻¹ corresponds to a strong O–C–O bending vibration of the carboxylic group [73,74]. The calculated spectrum reproduces these bands with very good accuracy, demonstrating that the optimized geometry correctly describes the local environment of the EDTA framework.

In the fingerprint region, the band at 1044 cm⁻¹ is assigned to the O–H vibration of water interacting with the O–H of the carboxylic group, coupled with the C–O symmetric stretching vibration, while the band at 949 cm⁻¹ corresponds to the asymmetric stretching vibration of the perchlorate anion. The absorptions at 888, 797, 679, and 544 cm⁻¹ are attributed, respectively, to vibrations of the hydrogen-bond network involving ClO₄⁻ and O–H groups together with the ClO₄⁻ symmetric stretching mode, to the ClO₄⁻ bending vibration, to the O–H vibration of water interacting with water O–H combined with an asymmetric bending-type vibration of ClO₄⁻, and to a C–N bending vibration. Most of these low-frequency vibrations are strongly coupled with hydrogen-bond network interactions, indicating that the lattice water molecules and perchlorate ions actively participate in the vibrational dynamics of the crystal, in particular through the characteristic ClO₄⁻ stretching and bending modes, which confirm the structural integrity of the perchlorate tetrahedra.

The FT-IR results are fully consistent with the single-crystal X-ray diffraction analysis, which revealed a three-dimensional supramolecular architecture stabilized by numerous O–H···O and N–H···O hydrogen bonds, as well as with the Hirshfeld surface analysis showing that H···O/O···H contacts account for 74.4% of the total intermolecular interactions. Such a dominant contribution explains both the broad O–H stretching region and the extensive vibrational coupling observed throughout the spectrum, particularly the strong involvement of the carboxylic and water O–H groups highlighted by the revised assignments.

From a functional viewpoint, the coexistence of protonated amine groups, carboxylic functionalities, lattice water molecules, and a dense hydrogen-bonding network provides numerous interaction sites capable of participating in proton transfer, molecular recognition, and hydrogen-bond-mediated stabilization. These structural characteristics are particularly relevant for biological systems, where hydrogen bonding governs biomolecular recognition and host-guest interactions. Furthermore, the presence of multiple oxygen- and nitrogen-containing functional groups may facilitate interactions with reactive oxygen species and biomolecules, which is consistent with the significant antioxidant activity observed for the material (82.6 ± 1.9% inhibition after 24 h). Although FT-IR spectroscopy alone cannot establish the antioxidant mechanism, the vibrational analysis confirms the preservation of the functional groups responsible for intermolecular interactions, supporting the potential of this supramolecular hybrid material for future biomedical and bioactive applications.

3.4. UV-Visible Spectra

The UV–Vis spectra of EDTAH₂²⁺.2ClO₄⁻.4H₂O (Figure 6) exhibit a pronounced solvent dependent behavior. In methanol, well resolved absorption bands at 207.51 and 225.33 nm are assigned to π→π* transitions, while those at 309.13 and 351.87 nm correspond to n→π* transitions, in agreement with previously reported data for polyaminocarboxylic systems [75]. In aqueous solution, the absorption bands appear at 199.93, 238.20, 301.75, and 343.07 nm, accompanied by significant band broadening. The overall spectral changes indicate a clear solvatochromic effect arising from differential stabilization of the ground and excited states by the solvent. The observed hypsochromic shifts are consistent with stronger hydrogen bonding interactions in water, leading to preferential stabilization of the ground state [76,77]. The pronounced band broadening suggests the presence of multiple solvation environments, as well as possible ion pairing with ClO₄⁻, contributing to spectral inhomogeneity and intermolecular interactions in solution [78].

3.5. Antioxidant Activity

To evaluate the antioxidant activity of the synthesized material, the β-carotene bleaching assay was performed in vitro. The assessment of its antioxidant capacity, based on absorbance measurements recorded over a 48-hour period, revealed a significant activity compared to the reference control. The experiment was conducted in triplicate to ensure the reliability and reproducibility of the results. This activity was reflected in better preservation of the β-carotene color, indicating effective protection against oxidation.

3.5.1. β-Carotene Bleaching Test

The inhibitory power of lipid peroxidation by the hybrid material was evaluated using the β-carotene/linoleic acid bleaching assay. During incubation, the β-carotene/linoleic acid emulsion generates hydroperoxide free radicals [79], which oxidize β-carotene, leading to its discoloration. Negative controls, such as DMSO, MeOH, and H₂O (without samples), unambiguously promote β-carotene oxidation. Among them, water exhibited the weakest inhibitory effect compared to the organic solvents MeOH and DMSO, which were used to dissolve the tested compound. In general, these solvents showed lower inhibition levels than BHT and the tested material. After 24 hours of storage in the dark, the inhibition results, represented in graphical curves and histograms (Figure 7), indicate that the antioxidant efficiency of the hybrid material is lower than that of BHT. However, the inhibition remains significant, suggesting that the hybrid material could be considered for applications requiring moderate to high antioxidant activity. The results, presented in Table 6, allow the antioxidant activity percentages to be ranked in ascending order as follows: H₂O < MeOH < DMSO < Hybrid material < BHT.

3.6. Frontier Molecular Orbital (FMO) Analysis and Global Reactivity Descriptors

To obtain further insight into the electronic structure and global reactivity of the synthesized hybrid compound, the frontier molecular orbitals were analyzed using the optimized geometry. The HOMO-2, HOMO-1, HOMO, LUMO, LUMO+1, and LUMO+2 orbitals are presented in Figure 8, while the corresponding frontier-orbital energies, conceptual-DFT descriptors, and thermodynamic parameters are summarized in Table 7. The FMO analysis identifies the spatial regions involved in electron donation and acceptance, whereas the global descriptors provide complementary quantitative information on electronic stability, polarizability, and the propensity for charge redistribution. The thermodynamic functions, in turn, describe the energetic and thermal characteristics of the optimized molecular model at 298 K. These three levels of analysis should therefore be considered complementary rather than interpreted independently.

The spatial distribution of the frontier orbitals highlights the electron-donor regions associated with the occupied orbitals and the electron-acceptor regions associated with the unoccupied orbitals, providing a qualitative representation of the sites potentially involved in intramolecular or intermolecular charge redistribution.

Figure 9. Temperature dependence of the calculated thermal enthalpy, entropy, and constant-volume heat capacity of the optimized EDTAH₂²⁺·2ClO₄⁻·4H₂O molecular mode.

Figure 9. Temperature dependence of the calculated thermal enthalpy, entropy, and constant-volume heat capacity of the optimized EDTAH₂²⁺·2ClO₄⁻·4H₂O molecular mode.

The calculated HOMO and LUMO energies are -6.339 and -5.445 eV, respectively, giving a HOMO-LUMO separation of 0.894 eV. Within the adopted molecular DFT model, this relatively small orbital-energy separation indicates that electronic redistribution between the occupied and unoccupied frontier levels may occur with a comparatively low excitation-energy requirement. It is therefore consistent with an electronically polarizable and potentially reactive system. However, the HOMO-LUMO gap should not be interpreted alone as direct proof of antioxidant activity, because radical scavenging also depends on the reaction mechanism, solvent environment, proton-transfer ability, bond-dissociation energies, and the stability of the generated radical species [80,81,82]. According to Koopmans-type approximations, the vertical ionization potential and electron affinity were estimated as IP = 6.339 eV and EA = 5.445 eV, respectively. The ionization potential reflects the energy required to remove an electron, whereas the relatively high electron affinity indicates a marked ability of the system to accept electron density. Taken together, these values suggest an amphielectronic response in which the hybrid molecular unit can participate in charge-transfer interactions, although the balance between electron donation and acceptance must be interpreted together with the global reactivity descriptors.

The calculated chemical hardness (η = 0.447 eV) and global softness (S = 1.119 eV⁻¹) further support the relatively reactive nature of the system. In general, soft molecular systems exhibit higher electronic flexibility, facilitating charge redistribution during interactions with free radicals or biological receptors [83,84,85,86,87]. This behavior is fully consistent with the experimentally observed antioxidant activity. The negative chemical potential (μ=−5.892 eV) indicates that electron loss from the optimized neutral system is energetically unfavorable within the adopted approximation; however, this quantity should not be interpreted by itself as a direct measure of thermodynamic stability. In parallel, the relatively high electrophilicity index (ω=38.85 eV) reflects a strong tendency to accept and stabilize additional electron density following charge transfer [88,89]. Such an electronic profile is often associated with molecular systems capable of interacting with biological macromolecules through charge-transfer processes [90,91,92,93,94,95,96]. Overall, the calculated descriptors consistently characterize the molecular model as electronically soft, strongly electrophilic, and capable of substantial charge redistribution. Inspection of the frontier molecular orbitals shows that the occupied orbitals are predominantly distributed over oxygen- and nitrogen-containing regions of the protonated EDTA unit, with contributions from the carboxylic groups, amine functions, perchlorate ions, and lattice-water environment depending on the orbital considered. These electron-rich regions represent the most plausible sites for electron donation and for the establishment of hydrogen-bonding interactions. The unoccupied orbitals exhibit a different and, for some levels, more extended distribution over the ionic supramolecular unit, identifying the regions that can accommodate additional electron density. The spatial complementarity between the occupied and unoccupied orbitals is therefore consistent with the low calculated hardness and high softness, since both indicate that the electron density can be redistributed across several chemically distinct sites [97]. The simultaneous presence of multiple hydrogen-bond donor and acceptor sites revealed by the crystal structure, Hirshfeld surface analysis and frontier orbital distribution demonstrates that the biological response of the material is not governed by a single functional group but rather by the entire supramolecular hydrogen-bond network. Therefore, the small HOMO–LUMO separation, low hardness, high softness, and spatial distribution of the frontier orbitals provide a coherent description of an electronically responsive molecular system. These properties may facilitate charge redistribution during interactions with radical species and are qualitatively compatible with the experimentally observed antioxidant response. Nevertheless, the present descriptors do not establish a unique radical-scavenging mechanism. Additional calculations, such as O–H or N–H bond-dissociation enthalpies, ionization energies in solution, proton affinities, and the stability of the corresponding radical species, would be required to discriminate among hydrogen-atom transfer, single-electron transfer, and sequential proton-loss electron-transfer pathways [98,99,100].

The electronic-structure results are consistent with the experimental structural characterization. Single-crystal X-ray diffraction revealed a three-dimensional network involving protonated amine groups, carboxylic functions, perchlorate ions, and lattice-water molecules, while the Hirshfeld-surface analysis showed that H···O/O···H contacts dominate the intermolecular-contact distribution. FT-IR spectroscopy independently confirmed the participation of O–H, N–H, C=O, and perchlorate groups in the hydrogen-bonded environment. The FMO maps indicate that these same oxygen- and nitrogen-containing regions contribute significantly to the frontier electronic density. This correspondence links the experimentally observed interaction sites to the calculated donor and acceptor regions. Furthermore, the delocalization of the frontier orbitals over several functional groups is consistent with the small energy gap, low hardness, and high softness, all of which indicate that electronic perturbation is not restricted to a single localized site but can be distributed over the ionic molecular assembly. The thermodynamic quantities calculated from the vibrational analysis provide a complementary description of the optimized molecular model. At 298 K, the total electronic energy is -39.865 keV, whereas the reported thermal energy, heat capacity, and entropy are 148.153 kcal mol⁻¹, 76.014 cal mol⁻¹. K⁻¹, and 170.949 cal mol⁻¹. K⁻¹, respectively. The total electronic energy defines the energetic reference of the optimized structure at the selected DFT level, while the thermal energy includes translational, rotational, and vibrational contributions at the specified temperature. The heat capacity reflects the amount of thermal energy required to increase the temperature of the molecular system, and the entropy describes the number of thermally accessible molecular states. These quantities should not be directly equated with the conceptual-DFT descriptors: the latter are derived from frontier-orbital energies and describe electronic response, whereas the thermodynamic functions are obtained from the molecular partition function and characterize thermal population and molecular motion [101,102,103].

Nevertheless, the two analyses provide complementary information. The low hardness and high softness indicate that the electron density can respond readily to an electronic perturbation, whereas the finite heat capacity and relatively high entropy reflect the contribution of numerous vibrational and conformational degrees of freedom associated with the EDTA framework, lattice-water molecules, and hydrogen-bonded ionic environment. As temperature increases, the calculated entropy, heat capacity, and thermal enthalpy are expected to increase because a larger number of translational, rotational, and vibrational states become populated. This thermal activation may enhance structural fluctuations and intermolecular reorganization, but it does not directly modify the zero-temperature HOMO–LUMO descriptors reported in Table 7. The FMO, global-reactivity, and thermodynamic results therefore describe distinct but complementary aspects of the material: spatial electron donation and acceptance, susceptibility to electronic redistribution, and thermal accessibility of molecular degrees of freedom, respectively.

4. Conclusions

In this work, a new single-crystal hybrid ionic material, EDTAH₂²⁺·2ClO₄⁻4H₂O, was successfully synthesized and fully characterized. Its molecular and crystal structures were determined by single-crystal X-ray diffraction, while Hirshfeld surface analysis revealed that the crystal packing is mainly governed by O···H/H···O interactions, highlighting the key role of hydrogen bonding in stabilizing the supramolecular architecture. The UV–visible spectrum indicates good transparency in the visible region, and the FTIR results are consistent with the crystallographic data, confirming the formation of the hybrid structure. The β-carotene bleaching assay revealed that the material exhibits notable antioxidant activity, demonstrating its ability to inhibit lipid peroxidation in comparison with BHT as a reference standard. DFT calculations not only showed good agreement with the experimental geometry but also provided insights into the electronic properties of the material, supporting its potential reactivity and contribution to the observed antioxidant behavior. Overall, these findings suggest that the synthesized hybrid material may be a promising candidate for antioxidant applications.