DFT-Experimental Study of Glycine-Influenced Strontium Formate Dihydrate for Optical and Antimicrobial Applications

1. Introduction

Strontium formate dihydrate (SFD), with the chemical formula [Sr(HCOO)₂·2H₂O], is an intriguing crystalline material that crystallizes in the orthorhombic crystal system and belongs to the space group P2₁2₁2₁, containing four formula units per unit cell. The crystal and molecular structure of (SFD) was initially determined by Caligne [1], who reported the lattice parameters as a = 7.332 Å, b = 12.040 Å, and c = 7.144 Å. Subsequent studies by Greena et al. [2,3,4,5] have examined the influence of transition-metal doping-specifically with Cu, Ni, Zn and Mg - on the structural, optical and biocidal properties of (SFD). More recently, Muthupoongodi et al. [6,7] demonstrated that glycine doping significantly enhances the functional properties of (SFD), making it a promising material for photonics and electronic device applications. Furthermore, their thermal studies revealed that pure (SFD) crystals exhibit high thermal stability up to approximately 72 °C and contain less than two molecules of water of crystallization, with an estimated value of 1.66 molecules.

The molecular conformation and frontier molecular orbital (FMO) characteristics are crucial for understanding the functional properties and potential applications of molecular systems; however, obtaining such information through experimental techniques is often challenging. These data can instead be reliably derived using density functional theory (DFT), which explains the electronic structure of atoms, molecules, and materials based on their three-dimensional electron density distribution. DFT is widely regarded as a powerful computational tool for predicting vibrational spectra, molecular geometries, and electronic properties with high accuracy [8].

In recent years, there has been increasing interest in exploring organic and semi-organic materials for nonlinear optical (NLO) applications, primarily due to their large second-order electric susceptibilities, which are directly associated with the first-order hyperpolarizability. Among these, amino acids have attracted considerable attention owing to their crucial role in biochemical processes, particularly in influencing the structural and functional properties of biomolecules such as metaloproteins. Research on amino acid-doped semi-organic crystals has expanded significantly due to their therapeutic importance in applications such as radiopharmaceuticals, chemotherapy, and the treatment of metal toxicity [9,10,11,12,13]. The donor atoms present in amino acids, primarily oxygen (O) and nitrogen (N), from their chelating groups, exert opposite electronic effects [14], which make them highly relevant in biochemistry and pharmacology due to their potential antimicrobial, antineoplastic, and anticancer activities [15,16,17,18,38,39,40].

Although several studies have investigated transition-metal and amino acid-doped strontium formate dihydrate crystals, systematic studies correlating glycine incorporation with the electronic structure, vibrational behavior and antimicrobial activity of the crystal remain limited. In particular, the influence of glycine incorporation on charge distribution, frontier molecular orbitals, nonlinear optical response and biological activity has not been comprehensively explored using combined experimental and DFT approaches. Glycine was selected as a dopant due to its simple molecular structure, biocompatibility, zwitterionic nature and the presence of amino and carboxyl functional groups capable of interacting with the host crystal lattice through hydrogen bonding and electrostatic interactions.

Greena et al. [3] demonstrated the antimicrobial properties of metal-doped (SFD) crystals using Mg, Cu, Ni and Zn ions. However, the antimicrobial potential of amino acid-doped (SFD) crystals has not yet been explored and investigated. Since and then, we have synthesized glycine-doped (SFD) single crystals and aimed to explore their antimicrobial efficacy against selected pathogens. Interestingly, we examined the influence of glycine concentration as well as the incorporation on the molecular geometry, frontier molecular orbitals, electronic properties and antimicrobial properties of strontium formate dihydrate crystals using combined spectroscopic and theoretical investigations.

2. Experimental Section

2.1. Methodology

All the model systems were fully optimized using Becks three parameterized Lee, Yang and Parr nonlocal correlation functional (B3LYP) level of theory and 6-311G** quality basis set, as they are available in Gaussian-09 package [19,20,21].

2.2. Growth of Pure and Glycine-Doped Strontium Formate Dihydrate Crystals

Analytical reagent (AR) grade strontium carbonate (SrCO₃), formic acid (HCOOH) and glycine (NH₂CH₂COOH) with purity greater than 99% were used without further purification. Pure strontium formate dihydrate crystals were synthesized by slowly adding stoichiometric amounts of strontium carbonate to aqueous formic acid under continuous stirring at room temperature according to the reaction:

SrCO₃ + HCOOH → Sr(HCOO)₂ + CO₂ + H₂O

The obtained solution was filtered using Whatman filter paper to remove insoluble impurities and allowed to evaporate slowly at room temperature (30 ± 2 °C). For glycine-doped crystals, glycine was added to the supersaturated solution at concentrations of 0.005 M, 0.05 M and 0.1 M under constant stirring for 3 h to ensure homogeneous mixing. The prepared solutions were filtered and kept undisturbed for slow evaporation. Transparent crystals suitable for characterization were harvested after approximately 10 – 15 days.

2.3. Evaluation of Antimicrobial Activities

The antibacterial and antifungal activities of the synthesized pure and glycine-doped (SFD) single crystals were evaluated using the disc diffusion method. The biological screening was performed by dissolving the test crystals in dimethylformamide (DMF), which served as the solvent for preparing the required concentrations. Fresh microbial suspensions were prepared from 24-hour cultures of bacteria and fungi using sterile peptone water and adjusted to a turbidity equivalent to 0.5 McFarland standard to ensure uniform inoculum density.

Muller-Hinton agar plates (90 mm Petri dishes) were inoculated evenly using the prepared microbial suspensions. Sterile paper discs (6 mm in diameter) were impregnated with 10 μL of the respective test solutions and carefully placed in a circular arrangement on each inoculated plate. Discs loaded with DMF alone were used as negative controls to ensure solvent compatibility. Control experiments confirmed that the concentration of DMF used in the assay did not exhibit any inhibitory effect on microbial growth, validating its suitability for the antimicrobial and antifungal activity tests.

Antimicrobial experiments were performed in triplicate, and the obtained inhibition zone values are presented as mean ± standard deviation. Statistical significance between pure and doped samples was evaluated using one-way ANOVA with p < 0.05 considered statistically significant.

3. Results and Discussion

3.1. Optimized Molecular Structure

The structural optimizations of the (SFD) model systems were carried out using the Gaussian 09 package to determine the lowest-energy conformations. To ensure that the obtained geometries correspond to true minima on the potential energy surface, vibrational frequency analyses were performed. The fully optimized molecular structure of (SFD), along with the corresponding atom numbering scheme, is depicted in Figure 1.

The calculated bond lengths and bond angles are summarized in Table 1, and the obtained structural parameters show good agreement with the previously reported literature values [1].

The optimized molecular geometry also corresponds well with the X-ray crystallographic data of (SFD). In the optimized structure, the Sr atom is coordinated by two water molecules and two formate groups, with all four Sr–O bonds exhibiting nearly identical lengths in the DFT analysis (~2.45 Å). However, in the reported crystal structure [1], the two water molecules appear to interact more weakly with the Sr center compared to the formate groups. This indicates the presence of slight structural defects associated with the water molecules in the crystalline phase. Furthermore, the analysis suggests that the actual crystal contains fewer than two fully coordinated water molecules, which is consistent with the earlier findings reported in the literature [6].

3.2. Frontier Molecular Orbitals (HOMO - LUMO)

The frontier molecular orbitals (HOMO and LUMO) play a crucial role in understanding the chemical reactivity and stability of a molecule. The energy of the (HOMO) is directly associated with the ionization potential, while the (LUMO) energy corresponds to the electron affinity [22,23]. The energy difference between (HOMO) and (LUMO) is widely used as an indicator of kinetic stability and chemical reactivity [24,25]. A larger (HOMO – LUMO) energy gap generally signifies higher kinetic stability and lower chemical reactivity, as it becomes energetically unfavorable to transfer electrons from the low-lying (HOMO) to the high-lying (LUMO) [23]. Variations in the (HOMO – LUMO) gap caused by interaction with noble metals, non-metallic species, semiconductor materials, or other molecular systems can lead to significant changes in charge transfer characteristics, peak intensities, and spectral positions. For (SFD), the calculated (HOMO – LUMO) energy separation is 7.111 eV, and the graphical representation of its frontier molecular orbitals is provided in Figure 2.

3.3. Molecular Electrostatic Potential

The molecular electrostatic potential (MEP) is a powerful tool for visualizing and understanding the distribution of electronic charge on the molecular surface, thereby providing insights into the relative polarity of different regions (positive and negative) [26,27]. It is particularly useful for predicting reactive sites, understanding molecular interactions with the surrounding environment, and studying biological recognition processes. Mapping the electrostatic potential onto an iso-electron density surface offers a clear visual representation of the polarity distribution [28]. On the (MEP) surface, different colors indicate varying electrostatic potential values: red corresponds to regions of highest negative potential, blue represents areas of highest positive potential, and green indicates regions of near-zero potential.

The distinction between nucleophilic and electrophilic regions directly influences the molecule’s ability to donate or accept electrons [29]. The total electron density surface mapped with the electrostatic potential surface (EPS) for the (SFD) crystal is illustrated in Figure 3(a). Regions of negative potential, highlighted in red and yellow, correspond to electron-rich areas that attract protons, whereas positive potential regions, shown in blue, represent electron-deficient zones where protons experience repulsion due to low electron density and incomplete shielding of nuclear charges.

The electrostatic potential contour map, representing both positive and negative potentials, is shown in Figure 3(c). Dense regions indicate slightly electron-rich zones, while sparsely colored areas represent regions of nearly zero potential. As depicted in Figure 3(a), the area around the oxygen atoms in the formate group (HCOO^-) exhibits the most negative potential (red) with a minimum value of −0.102 a.u. In contrast, the strontium (Sr) atom shows the highest positive potential (blue) with a maximum value of +0.102 a.u.; Notably, the intense positive region is primarily localized on the strontium center and hydrogen atoms, which is attributed to their bonding with highly electronegative oxygen atoms.

3.4. Mulliken Atomic Charges

The atomic charges play a significant role in determining several molecular properties, including dipole moment and polarizability. The Mulliken population analysis [30] was used to calculate the atomic charges of (SFD), and the results are summarized in Table 2(a). The calculated values reveal that hydrogen atoms 12H, 13H, 14H, and 15H carry comparatively higher positive charges than hydrogen atoms 10H and 11H. This difference arises due to the influence of nearby electronegative oxygen atoms. In contrast, hydrogen atoms 10H and 11H are bonded to carbon atoms 2C and 3C, which are themselves connected to two highly electronegative oxygen atoms each. As a result, the positive charge on these two hydrogen atoms is relatively reduced, although they still retain a slight positive nature.

Similarly, the carbon atoms 2C and 3C exhibit lower positive charges due to their direct bonding with the electronegative oxygen atoms 4O, 5O, 6O, and 7O. Consequently, the hydrogen atoms 10H and 11H, which are attached to these carbon centers, possess lower atomic charges (0.199 and 0.199) compared to other hydrogen atoms in the molecule. Furthermore, all oxygen atoms in (SFD) are found to carry negative charges, confirming their role as electron-donor sites. The relatively large calculated dipole moment of (SFD) (2.058 Debye) suggests the presence of strong intermolecular attractions, primarily governed by dipole – dipole interactions and van der Waals forces within the crystal lattice. The detailed crystallographic parameters obtained from this analysis are presented in Table 2(b).

3.5. Vibrational Spectra

The DFT-simulated FT-infrared (FT-IR) and (FT-Raman) spectra of (SFD) obtained in the present study were compared with the experimental spectra reported in the literature [6], as shown in Figure 4(a) & (b). The assignment of vibrational bands in the solid-state (FT-IR) and (FT-Raman) spectra was carried out using theoretically predicted frequencies and further validated by comparison with previously reported experimental data [6]. Overall, the calculated vibrational frequencies show excellent agreement with the experimental observations Figure 4(a).

The water molecule in the (SFD) crystal exhibits C₂V symmetry, with the normal modes 2a₁ and b₂ being active in both the IR and Raman spectra. The stretching vibrations associated with free H₂O molecules generally appear in the region 3000 - 3800 cm⁻¹ [30]. In the experimental FT-IR spectrum, a broad absorption band is observed in the range 3000 - 3500 cm⁻¹, corresponding to the presence of water molecules within the (SFD) crystal [6]. In contrast, the theoretical spectrum predicts the H₂O stretching modes in the higher wavenumber range of 3500 - 3800 cm⁻¹. The upward shift of these sharp and intense bands in the simulated spectrum can be attributed to strong intermolecular hydrogen bonding between the water molecules and the formate anion. Furthermore, the intensity of these peaks in the theoretical spectrum suggests the presence of two water molecules within the optimized structure.

The Raman spectrum calculated using DFT methods is presented in Figure 4(b). However, certain vibrational bands could not be directly correlated between the experimental and theoretical spectra. This discrepancy arises from the fact that the DFT calculations were performed in the gas phase, which does not fully account for the intermolecular interactions present in the real crystal. Consequently, some vibrational modes associated with hydrogen bonding between water molecules in the crystal, particularly those expected in the 2000 - 2700 cm⁻¹ region, are absent in the theoretical spectrum but appear in the experimental results. On the other hand, water-related vibrational modes are still observed in the theoretical spectrum within the 2700 - 3200 cm⁻¹ range.

Additionally, the theoretical analysis reveals a few stretching vibrations in the 1000 - 500 cm⁻¹ region that are not detected experimentally [6], while previous reports indicate that water molecule vibrations are expected around 500 - 400 cm⁻¹ [31]. The low-frequency region below 200 cm⁻¹ in the Raman spectrum corresponds to lattice vibrations of the crystal.

The correlation between theoretical and experimental vibrational spectra demonstrates that the optimized molecular structure obtained from DFT calculations adequately reproduces the principal vibrational features of the crystal system. Minor deviations in peak positions arise primarily from the fact that theoretical calculations were performed in the gas phase, whereas the experimental spectra correspond to the solid crystalline state where intermolecular hydrogen bonding and lattice interactions significantly influence vibrational behavior. In particular, the broadening and shifting of O-H stretching bands in the experimental spectra indicate strong hydrogen-bond interactions involving water molecules and glycine functional groups within the crystal environment.

Overall, the comparative analysis of experimental and simulated FT-IR and FT-Raman spectra suggests a slight reduction in the number of water molecules within the crystal lattice. The findings from this study indicate that the hydrous strontium formate crystal likely contains fewer than two fully coordinated water molecules.

3.6. Nonlinear Optical Properties

The theoretical evaluation of molecular polarizability and hyperpolarizability, along with their interpretation in terms of structure–property relationships, has become an area of significant research interest due to its crucial role in the design of novel materials for nonlinear optical (NLO) applications and in understanding the interaction between electromagnetic fields and matter [32].

In the present study, the first-order hyperpolarizability (β) and total dipole moment (μ) of (SFD) were computed using the B3LYP functional combined with the 6-311G** basis set, a widely adopted approach for predicting NLO properties. The calculated static dipole moment (μ) and mean first-order hyperpolarizability (β₀) were obtained from the x-, y-, and z- components of the corresponding tensors, as defined by the following relations:

µ_x = 0.0006, µ_y = 0.0017 and µ_z = 2.0578

$${\hat{\mathrm{I}}}_{\mathit{total}}^2={({\mu }_x^2+{\mu }_y^2+{\mu }_z^2)}^{1/2}$$

(1)

The dipole moment (μ) was calculated using Gaussian 09 software and it is found to be

µ = 2.0578 Debye. The first order hyperpolarizability (β) was also calculated were determined using the finite field approach theory. The components of first order hyperpolarizability can be obtained using the following equation (Equ. 2):

β_i = β_ijk + ⅓ ∑(β_ijj + β_jij + β_jji), (I ≠j)

(2)

Using the x, y and z components, and the magnitude of the first hyperpolarizibility tensor can be calculated. The output from Gaussian 09 provides 10 components of this matrix as β_xxx, β_yxx, β_xyy, β_yyy, β_zxx, β_xyz, β_zyy, β_xzz, β_yzz, β_zzz, respectively. The components of the first hyperpolarizability can be calculated using the following equations (Equ. 3):

$${\beta }_{\mathit{total}}={({\beta }_x^2+{\beta }_y^2+{\beta }_z^2)}^{1/2}\dots$$

(3)

where, ${\beta }_x$ = ${\beta }_{xxx}$ + ${{\hat{\mathrm{I}}}^2}_{xyy}$ + ${\beta }_{xzz}$

${\hat{\mathrm{I}}}_y^2$ = ${\beta }_{yyy}$ + ${\beta }_{yzz}$ + ${\beta }_{yxx}$

${\beta }_z$ = ${\beta }_{zzz}$ + ${\beta }_{zxx}$ + ${\beta }_{zyy}$,

The above tensor values are presented in Table 3.

The first order hyperpolarizability of the crystal is calculated and it is found to be 6973.787 a.u.; The calculated hyperpolarizability of the crystal is 6973.7 a.u. times that of the standard (NLO) material urea (0.13 × 10^-30 e.s.u.) [32]. We concluded that the title crystal and its derivatives are potential molecules for future studies of nonlinear optical applications.

3.7. Antimicrobial Activity

3.7.1. Determination of (MIC)

The in vitro antimicrobial activity of the as-grown pure and glycine-doped (SFD) single crystals was evaluated against four bacterial strains: Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Both standard reference drugs and the test samples were dissolved in dimethylformamide (DMF) to obtain a final concentration of 100 μg/mL. The minimum inhibitory concentration (MIC) was determined using the broth microdilution method [33]. Serial dilutions of the test samples and standard compounds were prepared using nutrient broth for bacterial strains and Sabouraud dextrose broth for fungal strains [34]. The inoculated samples were incubated under controlled conditions at 37 °C for 24 h for bacterial cultures and at 25 °C for 48 h for fungal cultures. The (MIC) values were recorded as the lowest concentration of the test compound capable of completely inhibiting microbial growth.

3.7.2. Antibacterial Activity

The glycine-doped (SFD) single crystals synthesized with varying concentrations of glycine (0.005 M, 0.05 M, and 0.1 M) exhibited significant antimicrobial activity against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The minimum inhibitory concentration (MIC) values obtained for the tested crystals are presented in Table 4.

Among the samples, the crystals doped with a higher concentration of glycine demonstrated enhanced antibacterial efficiency. This improvement can be attributed to the enrichment of heteroatoms such as nitrogen (N) and oxygen (O) within the crystal lattice, which facilitate strong chelating interactions with microbial components. Although all doped crystals exhibited moderate antimicrobial activity, the (SFD) crystals with a higher glycine content showed comparatively better inhibition than undoped (SFD). The significant enhancement in antimicrobial performance is likely due to the presence of amino (-NH₂) and carboxylic (-COOH) functional groups in glycine, which may induce specific biochemical transformation mechanisms within microbial systems.

These functional groups can improve cell permeability by interacting with the lipid membrane, thereby facilitating the transport of bioactive components. Since the lipid membrane preferentially allows the passage of lipid-soluble compounds, lipophilicity plays a critical role in controlling antimicrobial efficiency. However, despite the improved activity of glycine-doped (SFD) crystals, their antimicrobial efficacy remained lower than that of standard drugs, as illustrated in Figure 5.

The observed antimicrobial behavior can be correlated with two key factors: (i) The incorporation of heteroatoms such as O and N enhances the solubility of the crystals (both hydrophilic and hydrophobic) and promotes interactions with trace elements present in microbial cells, thereby inhibiting their growth; (ii) The mode of action involves the ability of the carboxylic acid and amino groups within the doped crystals to form hydrogen bonds with counter-ions, solvent molecules, and active centers of cellular constituents. This interaction disrupts essential cellular metabolism, ultimately leading to microbial growth inhibition [35].

3.7.3. Antifungal Activity

To explore the potential biological applications of glycine-doped (SFD) crystals in the field of bioinorganic chemistry, their antifungal activity was evaluated using the disk diffusion method. Pure (SFD) and glycine-doped strontium formate dihydrate crystals with different glycine concentrations were tested in vitro against five pathogenic fungal strains: Aspergillus niger, Fusarium solani, Curvularia lunata, Rhizoctonia bataticola, and Candida albicans. The results of the antifungal studies are summarized in Table 5, which clearly indicate that the glycine-doped (SFD) crystals exhibit greater antifungal efficacy compared to pure (SFD) crystals against all tested fungal species.

The enhancement in antifungal performance can be attributed to the ability of glycine to interfere with the replication process of fungal cells by blocking metabolically active sites, thereby suppressing their growth. This improved activity may also be explained by an increase in the lipophilic nature of the doped crystals, which facilitates stronger interactions between the glycine molecules and the lipid components of the fungal cell membrane. Such interactions are likely to disrupt membrane permeability and impair cellular respiration, ultimately leading to significant disturbances in normal cellular processes and effective inhibition of fungal proliferation.

The enhanced antimicrobial activity observed for glycine-doped strontium formate dihydrate crystals may be attributed to several synergistic factors. The incorporation of glycine introduces additional amino (-NH₂) and carboxyl (-COOH) functional groups capable of interacting with microbial cell membranes through hydrogen bonding and electrostatic interactions. These interactions can alter membrane permeability, resulting in leakage of intracellular components and disruption of essential metabolic pathways. Furthermore, the presence of oxygen-rich formate groups and polar glycine moieties may facilitate stronger interactions with negatively charged bacterial cell surfaces, particularly in Gram-negative bacteria. The increased polarity and surface activity of the doped crystals may also enhance reactive oxygen species generation and interfere with enzymatic processes required for microbial survival.

The improved antifungal activity may similarly arise from disruption of membrane integrity and inhibition of fungal respiration pathways. However, detailed mechanistic investigations involving membrane permeability assays, ROS measurements and electron microscopy are required to fully establish the antimicrobial mechanism of the glycine-doped crystals.

4. Conclusions

DFT calculations (B3LYP/6-311G**) were used to study the structural and electronic properties of strontium formate dihydrate. HOMO–LUMO analysis indicates a stable electronic structure with moderate charge transfer characteristics. The experimental FT-IR and FT-Raman spectra were well supported by theoretical vibrational calculations. Minor deviations are attributed to intermolecular hydrogen bonding effects in the solid state, which are not fully represented in gas-phase calculations. The calculated first-order hyperpolarizability (β = 6973.787 a.u.) suggests a strong nonlinear optical response compared to standard materials such as urea, indicating potential for (NLO) applications. Antimicrobial studies show that glycine-doped crystals exhibit improved antibacterial and antifungal activity compared to the pure sample. This enhancement is likely due to the presence of amino and carboxyl groups that promote interactions with microbial cell membranes. However, the mechanism of glycine incorporation and its structural influence cannot be conclusively confirmed in the absence of advanced crystallographic and surface analyses such as XRD refinement and XPS. Overall, the results indicate that glycine modification influences both electronic and biological properties of strontium formate dihydrate, making it a promising candidate for further investigation in optical and bioactive material applications.