Innovative sodium benzoate-modified hydroxyapatite for enhanced dye removal using a combined experimental and DFT approach

Abstract

This study presents the development of a novel adsorbent based on hydroxyapatite (CaHAp) grafted with sodium benzoate (SB) at varying proportions (5%, 10%, 15%) via a double decomposition method. Structural and chemical characterizations (XRD, FTIR, SEM, TGA/DTA) confirmed the successful integration of SB, with noticeable improvements in crystallinity (+ 10%), porosity, and thermal stability compared to pristine CaHAp. Adsorption experiments using methylene blue (MB⁺) as a model pollutant showed that the CaHAp-(SB)15 composite achieved an adsorption efficiency of 90%. The adsorption process followed Langmuir isotherm (R² > 0.96) and pseudo-second-order kinetics (R² > 0.99), indicating monolayer chemisorption as the dominant mechanism. Increasing SB content correlated with a significant rise in adsorption capacity from 5.41 to 9.42 mg·g⁻¹, demonstrating the role of –CO₂⁻ groups in enhancing electrostatic interactions with cationic MB⁺. DFT simulations supported the experimental findings, showing favorable interactions between SB and CaHAp at both molecular and atomic levels, particularly through non-covalent forces such as van der Waals and electrostatic interactions. This combined theoretical–experimental approach highlights CaHAp-(SB)15 as a promising, low-cost, and eco-friendly adsorbent for dye-contaminated wastewater, offering high efficiency, reproducibility, and environmental compatibility.

Introduction

Water pollution resulting from industrial, agricultural, and domestic activities poses a significant challenge in many countries. This pervasive issue endangers the environment, human health, and aquatic ecosystems^1,2. Among the various pollutants, dyes from industries such as textiles, photography, pharmaceuticals, cosmetics, food production, paint, paper, and pulp are particularly concerning.

Remarkably, 10% of the 749 tons of dyes used annually by the textile industry wind up in natural water systems^3,4. These dyes are often non-biodegradable or poorly biodegradable, and environmental science research has increasingly focused on developing new treatment processes to address these pollutants⁵.

The impact of dye pollution extends beyond mere discoloration of water bodies. It significantly reduces dissolved oxygen (DO), increases chemical oxygen demand (COD), and heightens turbidity, all of which degrade water quality and harm aquatic life^6–8. Methylene blue (MB), a widely used dye, exemplifies these concerns. Exposure to MB can cause severe health effects, including eye burns leading to irreversible damage in both humans and animals. Additionally, inhalation can result in respiratory complications, while ingestion may cause burning sensations, nausea, vomiting, excessive sweating, and cold sweats⁹.

Addressing these challenges requires innovative wastewater treatment processes. Current techniques include sorption and ion exchange, precipitation, and membrane technologies¹⁰. Of these, sorption processes are particularly effective and cost-efficient for removing dyes, making them a preferred method in many applications^11,12.

The selection of materials with high adsorption capacities is a critical aspect of developing effective sorption processes. Phosphocalcic hydroxyapatite (CaHAp, Ca₁₀(PO₄)₆(OH)₂) has emerged as a highly suitable material due to its biocompatibility, bioactive properties, surface functionalities, ease of processing, and low cost^13,14. Comprising 18.5% phosphorus, 39% calcium, and 3.38% hydroxide¹⁴, CaHAp constitutes approximately 50% of bone weight and is a key factor in bone hardness¹⁵. Additionally, carbonate-apatite, a variant of hydroxyapatite, forms the majority of hard tissues such as tooth enamel¹⁶, and is widely used in bone implants, prosthetic coatings, and bone regeneration devices due to its similarity to natural bone mineral¹⁷.

Over the past decade, calcium phosphate hydroxyapatite has attracted growing interest beyond its traditional biomedical applications, particularly in the field of environmental sorption technology. This interest is due to its stable hexagonal crystal structure, low water solubility, and chemically rich surface, consisting of calcium ions (Ca²⁺), phosphate groups (PO₄³⁻), and hydroxyls (OH⁻), which are capable of effectively interacting with a wide variety of pollutants in aqueous environments. These interactions are based on several complementary mechanisms, including ion exchange, surface complexation, electrostatic attraction, and co-precipitation of poorly soluble phases, which give CaHAp great versatility in sorption¹⁸.

Numerous recent studies have demonstrated the effectiveness of CaHAp and its derivatives in removing toxic heavy metals (Pb²⁺, Cd²⁺, Zn²⁺), radionuclides, hazardous anions such as fluoride, and organic pollutants, including synthetic dyes, from contaminated water^19–21. Hydroxyapatite-based materials often exhibit fast adsorption kinetics and high capacities, making them particularly attractive for large-scale wastewater treatment applications²².

The microstructural characteristics of hydroxyapatite, including its shape, crystallinity, porosity, and grain size distribution, play a vital role in determining its effectiveness as an adsorbent²³. Efforts to enhance its adsorption capacity have focused on increasing the number of active sites through surface modification. One such approach involves grafting organic molecules onto the solid’s surface, enabling it to bind a broader range of organic and inorganic pollutants^24,25.

In the case of cationic dyes such as methylene blue, the organic functionalization of CaHAp appears particularly relevant. It strengthens the specific interactions between the adsorbent and the dye through electrostatic attraction and π–π interactions when aromatic groups are introduced to the surface of the material. These mechanisms help improve the selectivity, stability of the adsorbed species, and overall efficiency of the sorption process, while being part of a sustainable approach based on the use of abundant, inexpensive, and environmentally friendly materials²⁶.

In this study, sodium benzoate (SB) was selected for grafting onto CaHAp. This modification represents a significant innovation as it not only enhances the material’s physicochemical properties but also targets the specific challenges posed by methylene blue (MB) dye. By incorporating sodium benzoate, the study introduces a material with improved adsorption performance, higher selectivity, and enhanced stability under operational conditions. This approach leverages the unique chemical structure of sodium benzoate to optimize interactions with Ca²⁺ cations and maximize adsorption efficiency. Furthermore, the process is designed to be environmentally friendly and cost-effective, addressing key concerns in practical wastewater treatment applications. By developing a CaHAp-SB composite, this study seeks to provide a material with superior adsorption performance, addressing the critical need for effective and sustainable solutions for dye removal in wastewater treatment⁴. This innovative approach highlights the potential for combining biocompatible materials with functional modifications to tackle environmental challenges more effectively.

To complement the experimental adsorption study, molecular docking was employed as a theoretical tool to gain molecular-level insight into interaction mechanisms at calcium-rich hydroxyapatite surfaces. Osteopontin, a well-known hydroxyapatite-binding protein, was selected as a representative model to probe surface interaction behavior relevant to calcium phosphate-based adsorbents. The surface modification with benzoate introduces additional aromatic and carboxylate functionalities, which can indirectly affect molecular interactions at calcium-rich sites. Accordingly, molecular docking was used to investigate interaction trends within this modified binding environment rather than to explicitly model benzoate–ligand complexes.

Experimental

Materials and methods

Materials used

Calcium nitrate tetrahydrate (Ca(NO₃)₂·4 H₂O), ammonium hydrogen phosphate ((NH₄)₂HPO₄), sodium benzoate (NaC₆H₅CO₂), and ammonium hydroxide (NH₄OH, 25%) were of analytical grade (≥ 99%, Sigma-Aldrich) and used without further purification. Distilled water was used throughout.

Synthesis of hydroxyapatite, and hydroxyapatite- sodium benzoate

Hydroxyapatite (CaHAp) was synthesized by a double decomposition method using calcium nitrate tetrahydrate and ammonium hydrogen phosphate as Ca and P precursors, while maintaining a stoichiometric Ca/P molar ratio of 1.67. The calcium solution was added dropwise to the phosphate solution under continuous stirring, and the pH was adjusted to 10 using NH₄OH (25%). The reaction mixture was then magnetically stirred at 85 °C for 90 min to promote precipitation. The resulting solid was filtered, thoroughly washed with distilled water, dried at 80 °C for 24 h, and finely ground to obtain a homogeneous powder^27,28.

Sodium benzoate–grafted hydroxyapatite (CaHAp–SB) was prepared using the same synthesis route, with sodium benzoate added to the calcium solution at 5, 10, and 15 wt% relative to calcium. The obtained samples were labeled CaHAp-(SB)5, CaHAp-(SB)10, and CaHAp-(SB)15 (Fig. 1).

Fig. 1.

Schematic representation of CaHAp-SB synthesis by the double decomposition method⁹.

Technical characteristics

FTIR measurements

FTIR was utilized to examine the development of CaHAp. The spectra were obtained using an FT/IR-4700 spectrometer (JASCO International LTD., Tokyo, Japan). Each spectrum consisted of an average of 32 scans at a resolution of 4 cm^-1, and the spectral range was 400–4000 cm^-1. Each analysis was preceded by a background check²⁸.

XRD measurements

The crystal structure and elemental composition of the composites were examined using the XRD-EDX. Using a Cu Kα radiation source at 40 kV and 30 mA, as well as a scan rate of 2°/min, the Panalytical X’Pert Pro equipment (Malvern Panalytical GmbH, Kassel, Germany) was utilized to record the XRD patterns. The same instrument’s associated detector was used to obtain the EDX spectra.

Thermogravimetric analysis

The TA Instrument TGA Q500 and Q50 are standard thermogravimetric analyzers that have a temperature range of 20 to 900 °C and a heating rate of 10 °C/min. On a DSC Q2000 V24.4 Build 116, the differential scanning calorimetry (DSC) curves were recorded. Ten milligram samples were put inside aluminum DSC pans and sealed before being added to the DSC cell. The Shimadzu GCMS-QP2010 (Shimadzu, Japan) chromatography/mass spectrometry apparatus, along with the GCMS solution 2.5 software²⁹.

Morphological studies

A Thermo ScientificTM Quattro ESEM (ThermoFisher Scientific, Paisley, United Kingdom) operating at an accelerating voltage of 15 kV was used to capture the SEM pictures. Prior to imaging, the films underwent sputter coating with gold to enhance their electron beam conductivity.

Adsorption

Understanding the interaction mechanisms between adsorbents and adsorbates requires an investigation of the surface reactivity of CaHAp–SB composites, which governs their interfacial properties. Methylene blue (MB) was selected as a model pollutant to evaluate the adsorption performance of the synthesized materials²⁹. The influence of key parameters, including solution pH, initial MB concentration, contact time, adsorbent dose, and kinetic and isotherm behavior, was examined to determine the optimal adsorption conditions³⁰.

Analytical-grade MB powder was dissolved in distilled water to prepare stock solutions with concentrations ranging from 5 to 140 mg L⁻¹. Batch adsorption experiments were performed by adding 100 mg of CaHAp–(SB)ₙ (0 ≤ n ≤ 15%) to 100 mL of MB solution, followed by magnetic stirring at 150 rpm and 25 °C for 3 h, which was sufficient to reach pseudo-equilibrium (Fig. 2).

Fig. 2.

Illustration of the Global Adsorption Process of (MB⁺) onto (CaHAp-SB).

MB concentrations were monitored by UV–Vis spectroscopy, and the adsorption capacity of the composites was calculated using Eqs. (1) and (3)⁹:

1

2

Where C₀ and Ce represents the initial concentration and the equilibrium concentration, respectively of MB in mg/L. Q_e is the equilibrium adsorption capacity in ppm, m is the mass of the absorbent (mg), and V represents the volume of the solution in liters (L).

Isotherm of the adsorption process

In the investigation of the adsorption process, the Langmuir and Freundlich isotherm models were employed to characterize the adsorption behavior of MB on CaHAp. The Langmuir isotherm model is delineated by Eqs. (3) and (4)⁹:

3

Where, q_max represents the maximum single-layer adsorption capacity of the adsorbent in (mg.g^-1), and K_L is the Langmuir constant.in (L.mg^-1).

The Langmuir isotherm model enables the prediction of whether adsorption will be favorable or unfavorable by employing the dimensionless constant separation factor as presented in Eq. (4)⁹:

4

If the R_L value surpasses one, the adsorption is deemed unfavorable; otherwise, it is regarded as favorable or linear if it falls between one and one.

The Freundlich isotherm model, delineated by Eqs. (5) and (7), symbolizes the heterogeneous surface energy of a non-ideal adsorption process⁹:

5

6

where K_F stands for the relative adsorption capacity and 1/n for the adsorption intensity. Adsorption is not favorable if 1/n is less than 0.1; it is beneficial if 1/n is between 0.1 and 0.5.

Kinetics of the adsorption process

The rates of MB adsorption on the surfaces of CaHAp were investigated using the pseudo first-order and second-order kinetic models, which are detailed below. To calculate the linearized versions of the rate equations, Eq. 7 through 12 were utilized⁹:

7

8

9

10

11

where q_e represents the equilibrium adsorption capacity (mg g^-1) and Q_t stands for the temperature-dependent adsorption capacity. The rate constant for the pseudo-first-order reaction (in minutes) is denoted as K₁, while the rate constant for the pseudo-second-order reaction (in grams per milligram per minute) is represented by K₂. The boundary layer thickness can be ascertained using Z (in milligrams per gram), where Kid stands for the diffusion rate constant expressed in milligrams per gram per minute to the power of 1/2.

The rate of film diffusion is indicated as K_fd (in minutes) in Eq. (12), and the fraction of equilibrium attainment is represented by F (where F = q_t/q_e).

Equation (14) were utilized to determine the entropy (S°), enthalpy (H°), and Gibbs free energy (G°)⁹:

12

13

14

where T stands for the temperature of the solution measured in Kelvin (K), R denotes the ideal gas constant in joules per mole per Kelvin (J mol K^-1), Cads represents the amount of MB adsorbed at equilibrium measured in milligrams per liter (mg.L^-1), and Ce represents the equilibrium concentration also measured in milligrams per liter (mg L^-1)⁹.

Density functional calculations

We used the density functional theory at the restricted orbitals B3LYB level and 6-311G (d, p) electronic basis set³¹. Van-der-Waals interaction were introduced through the Grimme’s D3 dispersion corrections³¹. GeomeTRIC algorithm was applied for rapid geometry optimizing³² as it is implemented in GPU-oriented TeraChem software. the solvent effects were introduced in the frame of the COSMO solvent model³¹ implemented in TeraChem. The dielectric constant of the solvent (water) was ε = 78.4. Conceptual DFT provide useful equations and electronic structure principle to highlight the local and global reactivities of the chemical systems through well-known reactivity descriptors chemical potential (µ), electronegativity (χ), hardness (η) and softness (σ). For the mathematically defining of these descriptors, in CDFT, the following relations are used³¹.

15

16

17

In the given relations, E and N stand for total electronic energy and total number of the electrons of the studied chemical system, respectively.

Chemical hardness is one of the widely used parameters in chemical reactivity analysis of atomic and molecular systems. To compute the chemical hardness based on ground state ionization energy (I) and electron affinity (A) of atoms, ions and molecules, the following equation is used³¹.

18

For the approximately prediction of ionization energy and electron affinity of molecules, we considered the Koopmans Theorem (KT)³² giving the following relations based on frontier orbital energies.

19

20

Here, E_HOMO and E_LUMO are the energies of HOMO and LUMO orbital, respectively.

Theoretical calculations

Density functional theory (DFT) is a crucial theoretical method for studying the reactivity of organic molecules and understanding the adsorption mechanism of these molecules on the hydroxyapatite surface³³. In this study, we have used this method to examine and investigate the global and local reactivity of sodium benzoate (SB) molecules. HOMO and LUMO energies, energy gap (ΔEgap), percentage of electrons transferred (ΔNmax), and dipole moment (µ) are among the important theoretical parameters that were calculated using Eqs. (1, 2)³⁴. DFT was also used to find the lowest unoccupied molecular orbitals (LUMO) (Eq. 20) and most occupied molecular orbitals (HOMO) (Eq. 19) of these organic molecules. The Digital Double Plus polarization (DNP) basis set, specified in Material Studio 6.0 software, was used in this work with the GGA/PBE function and the applicable Dmol3 module. The electrophilic and nucleophilic sites in the structure of this adsorbate (SB) were determined by calculating Fukui indices (Fukui (+) and Fukui (-)) using Hirshfeld population analysis and Eqs. (3 and 4)^1,31. In addition, the reduced density gradient (RDG) and non-covalent interaction (NCI) isosurfaces of this compound were examined using visualization tools such as VMD and Gnuplot. In addition, the DFT method was used to optimize the structures of sodium benzoate molecules to better understand and study intra- and intermolecular interactions. To investigate the adsorption of SB onto the hydroxyapatite (CaHAp) surface, we first optimized the hydroxyapatite geometry along the (111) direction using CASTEP calculations. Then, we positioned the SB molecules on the hydroxyapatite surface using a “fine” quality adjustment to obtain the most stable adsorption geometry³³.

Ligand preparation

The Molecular Operating Environment (MOE) software was utilized to evaluate the hydroxyapatite ligand. Initially, the 3D structure of the ligand was prepared in an SDF (structure-data file) format and then chosen for molecular docking investigations. This compound screening process employed two distinct approaches: (a) pharmacophore-based screening and (b) molecular docking. In the pharmacophore-based virtual screening, the MOE Compute option was employed to identify specific pharmacophoric features within the active site. These features included anionic and cationic atoms, H-bond donors and acceptors, aromatic centers, Pi-ring centers, and hydrophobic centroids³⁴.

After selecting these ligand features, the software was used to filter compound positions based on their compatibility with the selected features within the active site. Furthermore, energy minimization was performed on the ligand studied in MOE before the docking process. The resulting output file, which contained the chosen compound, was then used for further molecular docking analyses³⁵.

Molecular docking

In a molecular docking approach, the ligands that passed pharmacophore-based screening were subjected to docking simulations with the three-dimensional structure of the target protein. The investigation centered on identifying the ligand with the most favorable biological activity, utilizing the protein structure obtained from the Research Collaboratory for Structural Bioinformatics (RCSB), specifically the Crystal structure of anti-osteopontin antibody 23C3 in complex with its epitope peptide with 2.80 Angstrom resolution (PDB ID: 3CXD)³⁶, as depicted in Fig. 3. This protein structure was prepared for docking within the Molecular Operating Environment (MOE).

Fig. 3.

The 3D Crystal Structure of Osteopontin (PDB ID: 3CXD).

To anticipate the residues constituting the active site within the binding pocket, several preparatory steps were undertaken. This included 3D protonation, energy minimization of the protein, and site finder analysis. The docking grid was centered on the predicted binding pocket with Cartesian coordinates X = − 8.15, Y = 15.57, and Z = − 28.16.

The structural preparation involved tasks such as the addition of missing hydrogen atoms, rectification of bond order assignments, adjustment of charge states and orientations of various chemical groups, and the execution of restrained minimizations that allowed for the free optimization of hydrogen atom positions³⁷.

Point of zero charge (PZC) determination

The point of zero charge (PZC) of CaHAp and CaHAp-(SB) samples was determined using the pH drift method. A series of 0.01 M NaCl solutions (50 mL) were prepared and their initial pH values were adjusted in the range of 2–12 using 0.1 M HCl or 0.1 M NaOH. A known mass of 50 mg of adsorbent was added to each solution, and the suspensions were agitated for 24 h at room temperature. After equilibration, the final pH values were measured. The PZC was defined as the point at which the curve of ΔpH (pH_final – pH_initial) versus pH_initial crossed the x-axis (ΔpH = 0).

Results and discussion

X-ray diffraction

According to the analysis of these diffractograms (Fig. 4), the hydroxyapatite structure is achieved for all materials synthesized in the absence of secondary phase. Moreover, the hydroxyapatite crystalline phase is unaffected by SB alteration of the CaHAp surface. The hexagonal system of the P63/m space group is present in all peaks. The lattice parameters acquired for all materials created using the Match program are set out in Table 1.

Fig. 4.

X-ray diffraction patterns of CaHAp-(SB) product.

Table 1.

Crystalline parameters and crystallinity of hydroxyapatites before and after grafting with SB.

Samples	a (Å)	c (Å)	Crystallinity (%)
CaHAp	9.4202	6.9157	54.66
CaHAp-(SB)5	9.5075	6.9187	60.14
CaHAp-(SB)10	9.4202	6.9157	57.42
CaHAp-(SB)15	9.4655	6.9068	58.92

These results demonstrate the impact of sodium benzoate on the lattice parameters (a) and (c) of the hydroxyapatite structure. In addition, increasing the level of this graft leads to a more marked increase in the constant “a”. This demonstrates the integration of the (NaC₆H₅CO₂) ion into the apatite structure²⁷.

However, all the lines are visible, and their intensity increases with the grafting rate. The increase in crystallinity (Table 1) and crystallite size may explain this phenomenon²⁸. Consequently, a privileged interaction of the (CO₂⁻) groups of SB with the Ca²⁺ ions of CaHAp³⁸ could form a nucleus required to trigger crystallization of the materials, depending on the grafting rate, which we will later confirm by scanning electron microscope (SEM).

FT-IR spectra

From Fig. 5, we can distinguish the primary bands that indicate the phosphate and hydroxyl groups of the apatitic structure. These results were obtained for all CaHAp and CaHAp-SB samples³⁹.

Fig. 5.

FTIR spectra of SB and CaHAp-(SB) with different SB contents.

The band located at 3400 cm^− 1 is attributed to water molecules, while the other vibrational bands in the range 1361 and 1423 cm^− 1 correspond to residual carbonate groups that could result from the dissolution of surrounding CO₂ at the time of aqueous precipitation. Their relative intensities decrease with increasing SB^40,41.

The carboxylic ion bands associated with CaHAp-SB are located at 1411 and 1554 cm^− 1, and their relative intensities increase progressively as the proportion of benzoate in the apatite phase increases.

Beyond the vibrational characteristics, the bands between 1400 and 1600 cm⁻¹ highlight the interaction between sodium benzoate and hydroxyapatite, via the symmetric and asymmetric vibrations of the –COO⁻ groups. These bands indicate strong interactions between carboxylate groups and surface calcium ions (Ca²⁺), mainly electrostatic and coordination interactions^42,43. Slight shifts and variations in intensity suggest differences in surface coverage and interaction strength, while hydrogen bonds with hydroxyl or phosphate groups may stabilize SB on CaHAp⁴⁴. The absence of significant changes in the characteristic bands of apatite confirms that these interactions are superficial, with no alteration of the crystal lattice.

The introduction of benzoate improves the resolution of the band’s characteristic of the PO₄ and OH phosphate groups present in synthetic apatite’s. The band attributed to the hydroxyl vibration appears at 3573 cm^− 1. The bands corresponding to the PO4³⁻ ion, located between 1100 and 900 cm^− 1, with particular bands at 1087, 1023 and 961 cm^− 1 and also at 873 cm^− 1 for HPO₄²⁻, relate to vibrations of the P-O bond, with these bands becoming increasingly easy to observe^45,46. This evolution is due to the increase in crystallinity.

Thermal analysis

The thermogravimetric (TGA) and derivative thermogravimetric (DTG) curves of pristine CaHAp and sodium benzoate grafted samples (CaHAp–(SB)5, CaHAp–(SB)10, and CaHAp–(SB)15), presented in Fig. 6a and b, reveal two main stages of mass loss.

Fig. 6.

a TGA curves for CaHAp grafted, and b DTA curves for CaHAp grafted.

The first stage, observed between 25 and 200 °C, corresponds to the removal of physically adsorbed and weakly bound water. As shown in Fig. 6a, all samples display comparable weight-loss profiles in this region, indicating that sodium benzoate grafting does not significantly affect the initial dehydration behavior of hydroxyapatite. The absence of distinct DTG peaks in this range in Fig. 6b further confirms that this process is dominated by water desorption.

The second stage, extending from 200 to 800 °C, is associated with the decomposition of organic species grafted onto the hydroxyapatite surface, progressive dehydroxylation of the apatite lattice, and structural rearrangements. In this temperature range, the effect of sodium benzoate content becomes evident. As illustrated in Fig. 6a, the CaHAp–(SB)15 sample exhibits the lowest total mass loss and the highest residual mass, demonstrating superior thermal stability compared with pristine CaHAp and samples grafted with lower SB contents. This enhanced stability is also reflected in Fig. 6b, where CaHAp–(SB)15 shows reduced DTG peak intensity, indicating slower decomposition kinetics.

The improved thermal resistance of CaHAp–(SB)15 can be attributed to strong electrostatic and coordination interactions between carboxylate groups of sodium benzoate and Ca²⁺ ions in the hydroxyapatite structure. These interactions restrict molecular mobility, reduce water release, and delay the onset of thermal degradation. Consequently, 15 wt% sodium benzoate grafting represents the optimal proportion for enhancing thermal stability, with the material remaining structurally stable up to approximately 700–800 °C.

Overall, the TGA/DTG results presented in Fig. 6 clearly demonstrate that sodium benzoate grafting improves the thermal stability of hydroxyapatite in a composition-dependent manner, with CaHAp–(SB)15 showing the most pronounced stabilization effect.

SEM analysis

The sizes and morphologies of CaHAp and CaHAp-(SB) particles were examined using a scanning electron microscope (SEM) (Fig. 7). According to the micrographs, the morphology and shape of CaHAp (picture a) differ from the others due to the presence of organic materials in all three formulations.

Fig. 7.

Scanning electron microscopy images of the CaHAp grafted (a CaHAp, b CaHAp-(SB)5, c CaHAp-(SB)10, d CaHAp-(SB)15).

However, there was a minor shift in morphology in the later. Furthermore, particle homogeneity and porosity increase proportionally with grafting rate, producing a microporous surface, particularly in picture d, and explaining apatite’s better physicochemical attributes.

Theoretical calculations

Reactivity and non-covalent interaction of sodium benzoate

Figure 8 this can promote adsorption of the molecule onto the CaHAp surface, forming adsorptive bonds with hydroxyapatite atoms.

Fig. 8.

a optimized geometry, b highest occupied molecular orbitals (HOMO), c lowest unoccupied molecular orbital (LUMO), d ESP isosurface distributions, e non-covalent interactions (NCI), and f Reduced Density Gradient (RDG) of the sodium benzoate (SB) computed by CASTEEP calculations in material studio 6.0.

The SB molecule has positive (electrophilic) sites colored blue and negative (nucleophilic) sites colored red, as shown in Fig. 8d⁴⁷. These sites are listed in Table 2. From this table, Electrophilic atoms in the SB molecule, such as C(2), C(5), and Na(9), have higher Fukui (+) indices. The molecule also includes nucleophilic atoms such as C(3), O(8) and O(10). The results of non-covalent interactions obtained using the two theoretical approaches, NCI and RDG, are also shown in Fig. 8e, f. We found that the SB molecule exhibited intramolecular interactions such as van der Waals bonds and steric influence in the aromatic ring of both isosurfaces⁴⁸.

Table 2.

Calculated Hirshfeld charges of compounds SB computed by DFT/GGA calculations in material studio 6.0 software.

SB
S. No.	Atom	Fukui (+)	Fukui (-)
1	C	0.056	0.057
2	C	0.12	0.108
3	C	0.056	0.058
4	C	0.071	0.072
5	C	0.079	0.076
6	C	0.072	0.072
7	C	0.102	0.102
8	O	0.116	0.125
9	Na	0.04	0.038
10	O	0.056	0.064

These results, which derive from the reactivity of this molecule and the types of interaction, show that the atoms of the SB molecule can easily connect to those of the hydroxyapatite molecule (adsorbent).

The HOMO–LUMO distributions shown in Fig. 8b, c provide insight into the feasibility of charge transfer during adsorption. The HOMO is mainly localized over the carboxylate and aromatic regions, indicating electron-donating capability, whereas the LUMO is distributed over electron-deficient regions, favoring electron acceptance. The relatively moderate HOMO–LUMO energy gap suggests that electron redistribution between sodium benzoate–functionalized CaHAp surfaces and methylene blue molecules can occur with a low energetic barrier. This facilitates interfacial charge transfer and strengthens donor–acceptor interactions, which contributes to the rapid adsorption kinetics observed experimentally. It should be emphasized that this charge-transfer process is related to adsorption-driven surface interactions rather than photocatalytic degradation, as no photo-induced mechanisms are involved.

Interactions between sodium benzoate and hydroxyapatite

The application of computational chemistry methods (DFTB, CASTEP, molecular dynamics (MD)) to the realization and study of the adsorption of organic molecules on the surface of hydroxyapatite has aroused the interest of researchers in the field of adsorption⁴⁹.

The adsorption of sodium benzoate (SB) on the hydroxyapatite surface was studied in the present work using CASTEP calculations. The adsorption of the SB molecule on the hydroxyapatite surface is shown in Fig. 8.

These data allowed us to validate previous conclusions on the local and global reactivity of the SB molecule. The fact that the carbon and oxygen atoms of the SB molecule make bonds with the hydroxyapatite atoms in Fig. 10 indicates that hydroxyapatite is a suitable adsorbent material (Fig. 9).

Fig. 10.

The 2D intermolecular interactions of the best result between the hydroxyapatite complex and the osteopontin protein.

Fig. 9.

Interactions between sodium benzoate (SB), and hydroxyapatite (CaHAp) computed by CASTEEP calculations in material studio 6.0 software.

Molecular Docking studies

The molecular docking approach is a fundamental technique for simulating atomic-level interactions between a small molecule and a protein. It enables a comprehensive characterization of small molecule behavior as it engages with the binding sites of target proteins, concurrently shedding light on the fundamental biochemical processes that underlie these critical interactions³².

In the context of bone development, hydroxyapatite assumes a central role in bone composition, strength, and functionality. It contributes to bone hardness and mechanical resilience, acts as a reservoir for essential minerals, participates in the regulation of bone metabolism, and provides a substrate for bone cell adhesion, thus serving as a pivotal constituent of bone tissue. More specifically, osteopontin is a protein that facilitates the adhesion of bone cells to hydroxyapatite. It plays a crucial role in the regulation of bone mineralization and holds potential for investigation in the realm of bone regeneration. Within the scope of our research, we have conducted molecular docking studies to assess the interaction between hydroxyapatite and the osteopontin protein.

These investigations aim to enhance our comprehension of the molecular mechanisms governing bone resorption and contribute to the development of more effective biomedical materials for bone regeneration. The strength of the ligand-receptor interaction was evaluated utilizing docking scores, where a more negative score signifies a higher binding affinity between the ligands and targets. The results indicated that the complex exhibited a strong affinity, characterized by a highly favorable binding energy estimated at -6.768 kcal.mol^− 1.

Consequently, the complex exhibits a notably low binding energy and is considered stable, engaging with surrounding residues through diverse interactions, as depicted in Fig. 10. Our research serves as a bridge between molecular interactions and biomedical applications, thus advancing materials with the potential to revolutionize bone regeneration therapies. Further comprehensive research and experimental validations will be imperative to corroborate and harness these promising outcomes for practical clinical applications⁵⁰.

Adsorption studies of MB on non-functionalized and functionalized hydroxyapatite

Point of zero charge (PZC) analysis

The PZC values of CaHAp and CaHAp-(SB) composites are presented in Fig. 11. The curves in Fig. 11 illustrate the variation of ΔpH (pH_final – pH_initial) as a function of the initial pH for CaHAp and CaHAp-(SB) composites. The point of zero charge (PZC) corresponds to the pH value at which ΔpH = 0, i.e., where the surface of the adsorbent carries no net charge.

Fig. 11.

Point charge zero (PZC) of CaHAp and CaHAp-(SB) composites.

For pure CaHAp, the PZC is located around 7.8, while progressive incorporation of sodium benzoate (SB) shifts the PZC toward higher values: approximately 8.2 for CaHAp-SB5, 8.5 for CaHAp-SB10, and 8.9 for CaHAp-SB15.

This trend demonstrates that SB functionalization increases the basicity of the surface and enriches it with negatively charged functional groups (–CO₂⁻). From an adsorption perspective, this shift has a direct impact on the interaction with methylene blue (MB), a cationic dye. When the solution pH is below the PZC, the surface is positively charged, leading to electrostatic repulsion with MB molecules and thus lower adsorption. Conversely, when the solution pH is higher than the PZC, the surface acquires a negative charge, which favors strong electrostatic attraction with MB.

The fact that CaHAp-SB15 exhibits the highest PZC indicates that, under typical adsorption conditions (pH ≈ 9), its surface is more negatively charged than that of pure CaHAp. This explains the superior adsorption capacity of SB-modified hydroxyapatite compared to unmodified CaHAp.

Adsorbent mass effect

The effect of adsorbent weight was studied between 50 and 200 mg (Fig. 12). With a mass of 100 mg of material, a maximum of 90% MB can be set, as shown by the curves in the figure. To ensure an equal number of adsorption sites, the quantities of dye fixed must be consistent with the doses of adsorbent in solution.

Fig. 12.

Mass effect of hydroxyapatites on MB adsorption in aqueous solution. Volume of solution = 100 mL; Concentration = 20 mg L^− 1; Time = 3 h; pH = 9; Stirring speed = 150 rpm; Temperature = 25 °C.)

The surface of our material has an increasing number of adsorption sites as the dose of adsorbent increases. When the active sites on the surface reach a certain degree of saturation due to particle aggregation, the adsorption capacity is limited. To avoid ineffective overdosing, it is useful to use adsorbent doses of less than 100 mg. To this end, 100 mg of adsorbent mass in 100 ml of MB solution was applied, resulting in an optimum adsorbent dose of 1 g.L^− 1.

Effect of initial dye concentration

Figure 13 illustrates the effect of the initial dye concentration on the adsorption capacity of the apatite matrix we prepared, varying the initial methylene blue concentration from 0 to 140 mg L^− 1.

Fig. 13.

Effect of initial dye concentration on adsorption efficiency (Mass of adsorbent = 0.1 g; volume of solution = 100 mL; Time = 3 h; Stirring speed = 150 rpm; pH = 9; Temperature = 25 °C).

We report that the adsorption capacity increases progressively with increasing initial methylene blue concentration, in view of the greater accessibility of the MB next to the adsorbent as well as the strong mass transfer prior to adsorption-desorption, In order to reach a maximum adsorbed amount of 34 mg g⁻¹ that could be attributed to the CaHAp-(SB)15 composite for an initial concentration of 100 mg L⁻¹, even if we increase the adsorbate concentration, we will still obtain the same value.

Overall, the results suggest that the initial MB concentration exerts a significant impact on the adsorption process and should be carefully considered in the development of CaHAp-based adsorbents to treat pollutants present in wastewater.

Effect of contact time

The adsorption of MB on CaHAp-SB was examined with different grafting rates (0% − 15%) at different contact times, as shown in the Fig. 14. The quantities of MB adsorbed on CaHAp-SB composites develop rapidly with increasing time, then slow down until equilibrium is reached.

The results obtained for all compounds indicate that equilibrium is reached most precisely after 20 min. Furthermore, the adsorption capacity of MB increases as the rate of benzoate grafting into the apatite phase rises from 0% to 15%. This evolution proves that the presence of benzoate in the apatite phase led to an improvement in MB retention capacity. Subsequently, the CaHAp-(SB)15 compound was qualified for further adsorption experiments²⁷.

Fig. 14.

Effect of contact time on methylene blue uptake by different CaHAp (Mass of adsorbent = 0.1 g; volume of solution = 100 mL; Concentration = 20 mg.L^− 1; Stirring speed = 150 rpm; pH = 9; Temperature = 25 °C).

Kinetic studies

Figure 15a shows linear plots of log (q_e-q_t) versus time for MB adsorption on the grafted apatites concerned. The rate constants K₁ were deduced from the slopes of these lines.

Fig. 15.

Linear representations of a pseudo first-order and b pseudo second-order kinetic models for MB adsorption by CaHAp-(SB) hybrid apatite’s.

In Fig. 15b, the constants K₂ and q_e,2 is determined from the y-intercept and slopes of these lines respectively.

The results presented in the table show that the pseudo-second-order model (k₂ and q_e,2) is perfectly suited to ensure the reproduction of kinetic data for MB adsorption on CaHAp-(SB). Similarly, the theoretical values q_{e, th} produced by the second-order equation are very close to q_{e, exp}, although the pseudo-second-order model is able to correctly describe MB adsorption on grafted materials.

This suggests that the kinetics of MB adsorption on CaHAp-(SB) is based on the availability of active sites, and that chemical interactions between MB and CaHAp-(SB) are dominant⁵¹. Unlike electrostatic interactions, which may be involved in the adsorption mechanism.

To gain deeper insight into the adsorption mechanism, particularly the diffusion steps, the intraparticle diffusion (IPD) model was applied. The analysis revealed a two-step process for both the raw CaHAp and the CaHAp modified with sodium benzoate. In the first phase, high-rate constants and strong correlation coefficients were observed (Table 3), indicating that this step is mainly governed by external mass transfer toward the surface of the adsorbents. The second phase, characterized by lower rate constants and reduced correlation values, is attributed to the gradual diffusion of MB molecules into the internal pores of the adsorbent particles (Fig. 16; Table 4).

Table 3.

Kinetic parameters for MB dye adsorption on CaHAp and CaHAp-(SB).

	CaHAp	CaHAp-(SB)5	CaHAp-(SB)10	CaHAp-(SB)15
qe, exp (mg g−1)	5.41	6.60	8.0	9.42
Pseudo first order
R2	0.9528	0.9966	0.9446	0.9691
qe,1 (mg g−1)	5.64	6.04	4.79	5.99
k1 (min−1)	0.090	0.127	0.090	0.112
Pseudo seconde order
R2	0.9966	0.9966	0.9993	0.9994
qe,2 (mg g−1)	5.65	6.74	8.14	9.57
K2 (mg min−1)	0.091	0.204	0.140	0.120

Fig. 16.

Intraparticle diffusion modeling of MB adsorption onto CaHAp and CaHAp-(SB): two-stage analysis using the Weber–Morris model.

These results suggest that a biphasic diffusion mechanism governs the retention of MB. The first phase is dominated by diffusion through the external boundary layer (film diffusion), while the second phase involves slower intraparticle diffusion, gradually leading to equilibrium (Table 4)⁵².

Table 4.

Intraparticle diffusion model (Weber–Morris) kinetic parameters for MB adsorption onto CaHAp and CaHAp-(SB).

Adsorbents	Phase I			Phase II
	K1 (mg g −1min− 1/2)	C (mg g− 1)	R 2	K2 (mg g −1min− 1/2)	C (mg g− 1)	R 2
CaHAp	1.051	0.966	0.986	0.002	5.372	0.001
CaHAp-(SB)5	0.710	3.713	0.988	0.032	6.407	0.589
CaHAp-(SB)10	0.758	4.587	0.982	0.005	7.927	0.021
CaHAp-(SB)15	1.056	4.966	0.993	0.026	9.727	0.727

Acid-base properties of solutions during MB adsorption by hybrid apatite’s

These acid-base tests aim to determine the pH fluctuation that occurs during MB adsorption on the apatite matrices used. This enables us to propose a binding mechanism between adsorbent and adsorbate. In the absence of dye, the pH of the solution increases as a function of time until it reaches an equilibrium pH of between 8.08 and 9.10, in relation to the rate of SB grafting, and then stabilizes (Table 5).

Table 5.

Equilibrium pH in aqueous medium with and without MB taking into account different SB grafting rates.

Grafting rate (%)	0	5	10	15
Without MB	8.08	8.27	8.60	9.10
With MB	7.10	7.60	7.90	8.14

The release of basic species into the solution from hydroxyapatite, particularly after grafting with sodium benzoate, is responsible for the increase in pH (Fig. 17).

Fig. 17.

pH evolution of CaHAp-(SB) hybrid apatite’s a in aqueous medium, b during MB adsorption process.

A pH increase can also be observed in proportion to the rate of grafting in the presence of MB, with the pH stabilizing after 20 min.

Adsorption isotherms

Figure 18 shows the isotherm for the adsorption of MB in aqueous solution by CaHAp. The models most frequently used by researchers to express isotherms of dyes on biomass^53,54. They are given below in Eqs. (9) and (10). After analyzing the supernatants and determining the residual concentrations, we studied the evolution of C_e/q_e as a function of C_e according to the Langmuir model, as well as the evolution of log q_e as a function of log Ce according to the Freundlich model⁵⁵.

Fig. 18.

Linear representations of a the Langmuir model, b the Freundlich model for MB adsorption on CaHAp-(SB) hybrid apatites.

Linear representations of the experimental values have enabled us to determine the equilibrium parameters and Langmuir and Freundlich constant values by linear regression, with satisfactory statistical quality, as shown in Table 6.

Table 6.

Langmuir and Freundlich model constants for MB adsorption on CaHAp-(SB) hybrid apatite’s.

	CaHAp	CaHAp-(SB)5	CaHAp-(SB)10	CaHAp-(SB)15
qe,exp (mg g− 1)	24.10	27.71	31.50	34.19
Langmuir
qm (mg g− 1)	28.44	31.75	35.50	37.91
KL	0.062	0.083	0.102	0.122
R2	0.8783	0.9227	0.9391	0.9605
Freundlich
1/n	0.9539	0.7997	0.7278	0.5471
log KF	-0.303	0.082	0.299	0.659
R2	0.9362	0.9106	0.8996	0.8801

Based on the correlation coefficient (R²) of the adsorption isotherm lines of the two models, we can conclude that the Langmuir model describes the experimental data well and is the most suitable for characterizing MB dye adsorption by CaHAp-(SB) composites (Fig. 19).

Fig. 19.

Proposed mechanism of the interaction between SB and the CaHAp surface.

Hydroxyapatite (CaHAp) is a porous mineral material composed of calcium, phosphorus, and oxygen, with a surface exposing numerous calcium ions (Ca²⁺) that can interact with other molecules.

When sodium benzoate is added to a solution, it dissolves into sodium ions (Na⁺) and benzoate ions (C₆H₅COO⁻). These benzoate ions, carrying a negative charge, are naturally attracted to the positively charged calcium ions on the CaHAp surface. They bind firmly to this surface through electrostatic interactions and sometimes coordination bonds, forming a thin organic layer around the hydroxyapatite particles. This layer significantly modifies the surface properties: it introduces hydrophobic aromatic (benzene) groups and negative charges, making the material much more reactive toward certain dye molecules. When this material is placed in a solution containing dyes, the dye molecules are strongly attracted to the surface through several types of interactions: electrostatic forces (between opposite charges), hydrogen bonds (between the functional groups of the dyes and the surface groups), and π–π interactions (between the aromatic rings of the benzoate and those of the dyes).

Through this mechanism, sodium benzoate-modified hydroxyapatite can efficiently capture and retain dyes, making it a highly promising material for the treatment and purification of colored wastewater.

Dissociation of sodium benzoate

In aqueous solution, sodium benzoate dissociates as follows:

• The benzoate ion (C₆H₅COO⁻) is the active species that will interact with the hydroxyapatite surface.

• The sodium ion (Na⁺) remains solvated and does not play a major role.

Hydroxyapatite (CaHAp) surface structure

The chemical formula of hydroxyapatite is Ca₁₀(PO₄)₆(OH)₂. Its surface exposes numerous calcium ions (Ca²⁺), which are available for interactions with other molecules. In addition, the surface contains hydroxyl groups (–OH) and phosphate groups (PO₄²⁻), which contribute to its chemical reactivity and ability to form hydrogen bonds or coordinate with anionic species. This combination of exposed ions and functional groups makes CaHAp a versatile material for adsorption, catalysis, and surface modification.

The variation in pH observed before and after the adsorption of methylene blue (MB⁺) highlights the influence of grafting hydroxyapatite (CaHAp) with sodium benzoate on the adsorption mechanism (Fig. 20)⁵⁶.

Fig. 20.

Schematic representation of the interaction mechanism between methylene blue (MB) and a CaHAp or b CaHAp-(SB) during dye adsorption from wastewater.

In the absence of grafting, MB⁺ adsorption mainly relies on hydrogen bonding interactions between the P–OH groups of CaHAp and the nitrogen atoms of MB molecules. Additionally, Lewis acid–base interactions between the nitrogen atoms of MB and Ca²⁺ ions on the surface of hydroxyapatite induce proton (H⁺) exchange, contributing to a significant decrease in the solution pH⁵⁷. This mechanism represents one of the dominant interaction pathways when grafting is not applied.

Conversely, the introduction of carboxylate groups (CO₂-) through grafting imparts a negative surface charge to CaHAp-SB, promoting MB⁺ adsorption via electrostatic interactions. This pathway does not involve substantial proton release into the medium, explaining the smaller pH drop after adsorption⁵⁸. Therefore, the CO₂⁻groups act as electrostatic active sites capable of effectively capturing organic cations such as MB⁺, while maintaining relative pH stability (Fig. 15).

When a dye, such as a cationic dye (D⁺), is present in solution, it interacts with the sodium benzoate-modified hydroxyapatite (CaHAp) surface. The negatively charged benzoate groups anchored on surface Ca²⁺ ions attract the positively charged dye molecules, forming stable complexes. In addition to electrostatic interactions, hydrogen bonds can form between functional groups on the dye (–OH, –NH) and the hydroxyl or phosphate groups on the CaHAp surface. Aromatic rings of the benzoate molecules also engage in π–π stacking interactions with the aromatic structures of the dye molecules. These combined interactions electrostatic, hydrogen bonding, and π–π stacking enable efficient adsorption and retention of dye molecules, making sodium benzoate-modified hydroxyapatite a highly effective material for removing dyes from aqueous solutions.

Conclusion

This study has successfully developed and characterized an innovative adsorbent material, sodium benzoate-modified hydroxyapatite (CaHAp-SB), for the effective removal of methylene blue (MB) dye from aqueous solutions. By employing an in-situ grafting approach, sodium benzoate was incorporated into the hydroxyapatite structure in varying proportions (5%, 10%, and 15%), resulting in nanoscale materials with significantly enhanced porosity, crystallinity, and adsorption capacity. The CaHAp-(SB)15 composite, in particular, demonstrated an impressive dye removal efficiency of up to 90%, positioning it as a promising candidate for wastewater treatment applications.

Comprehensive material characterization through XRD, FTIR, SEM, TGA, and DTA confirmed the structural and chemical integration of sodium benzoate, highlighting the material’s stability and superior physicochemical properties. Adsorption studies revealed a strong alignment with Langmuir’s isothermal model, indicating a monolayer adsorption mechanism, while pseudo-second-order kinetics confirmed that chemisorption governs the process. Theoretical simulations using density functional theory (DFT) provided valuable insights into the molecular interactions between MB and the CaHAp-SB composites, emphasizing the role of sodium benzoate in enhancing the affinity and efficiency of the adsorption process.

The findings of this research are highly significant for addressing the pressing environmental challenge of dye pollution in industrial wastewater. The innovative integration of sodium benzoate not only enhances the adsorption capacity of hydroxyapatite but also offers a cost-effective, scalable, and environmentally friendly solution. The use of CaHAp-SB composites can be particularly impactful in industries where large-scale dye pollution is a critical concern, such as textiles, pharmaceuticals, and cosmetics.

Looking forward, future research should focus on several key areas to further exploit the potential of CaHAp-SB composites. These include optimizing the synthesis process to improve scalability, exploring the material’s performance with other organic and inorganic pollutants, and assessing its reusability and long-term stability under industrial conditions. Additionally, the integration of CaHAp-SB composites into advanced wastewater treatment technologies, such as hybrid adsorption-filtration systems, could significantly enhance treatment efficiency and operational sustainability.

This study underscores the transformative potential of modified hydroxyapatite materials in environmental remediation, bridging the gap between innovative material science and practical, large-scale applications. By advancing sustainable and cost-effective solutions, CaHAp-SB composites represent a significant step forward in addressing global water pollution challenges.

Author contributions

Conceptualization, A.B.; methodology, K.A., and O.B; software, S.L; validation, S.O, and K.A.; formal analysis, M.M.; investigation, M.EK.; resources, B.H .; data curation, S.J. and S.S; writing—original draft preparation, S.F.A, and A.B.; writing—review and editing, L.R, and, H.A.A; visualization, S.S, and B.H, All authors have read and agreed to the published version of the manuscript.

Funding

Not applicable.

Data availability

The data presented in this study are available upon request from the corresponding author.

Declarations

Competing interests

The authors declare no competing interests.