RIF-Mnt Model

The structural model of the RIF-Mnt complex was proposed to involve the possible species present in the interlayer space, particularly the RIF moiety and residual water, which is almost always present in Mnt. The computational cell of the Mnt structural model was taken from previous work (Scholtzová et al., Reference Scholtzová, Madejová and Tunega2014). This cell has a lateral size of 4a2b of the elementary cell a and b vectors. In the model, two Mg^II/Al^III substitutions are in the octahedral sheet resulting in the Mnt formula of Na_0.25(Si₈)(Al_3.75Mg_0.25)O₂₀(OH)₄. This composition produces a smaller excess layer charge than the montmorillonite probe used in the experiment with a composition of Na_0.64Ca_0.03K_0.02(Si_8.00)(Al_3.13Fe_0.09Mg_0.78)O₂₀(OH)₄. It was necessary to use the composition of the Mnt model with the smaller concentration of Mg^II/Al^III substitutions because it allowed the size of the computational cell (451 atoms) to be kept reasonable and also allowed us to perform computationally very demanding AIMD simulations. The Mnt model used is representative enough to describe all important interactions of RIF in the interlayer space. The negative layer excess charge (–2) was compensated by one protonated rifampicin structure (RIF⁺) and one hydrated Na⁺ cation coordinated by four H₂O molecules, both localized in the interlayer space of Mnt. The RIF structural data were taken from the experimental work of Ibiapino et al. (Reference Ibiapino, Seiceira, Pitaluga, Trindade and Ferreira2014). The RIF molecule was protonated on the N50 atom (Fig. 1) as the protonated form of RIF was used in the experimental preparation of the RIF-Mnt intercalate. The hydrated Na⁺ cation was used successfully in previous work, where water molecules represented a residual water content in the Mnt samples (Moreno-Rodríguez et al., Reference Moreno-Rodríguez, Jankovič, Scholtzová and Tunega2021).

Fig. 1

The structure of RIF. Its heterocyclic structure containing a naphthoquinone core is spanned by an aliphatic ansa chain (upper part of the RIF structure); the red arrow highlights the protonated site (N50)

The summary formula of the final structural model of the RIF-Mnt intercalate was then Na(H₂O)₄(Si₆₄)(Al₃₀Mg₂)O₁₆₀(OH)₃₂)(C₄₃N₄O₁₂H₅₉) with the lattice vectors 4a = 20.966 Å, 2b = 18.176 Å, and c = 30.00 Å at the starting point of calculations (Fig. 2).

Fig. 2

Proposed model of the RIF-Mnt structure; the red arrow highlights the protonated site (N50)

Structural Optimization

Structural relaxation of the model revealed the influence of the environment (montmorillonite layers) on the deformation of the RIF structure after intercalation into the interlayer space of Mnt. The optimized interlayer distance, d ₀₀₁, achieved a value of 16.4 Å, in good agreement with an experimental value of 15.6 Å. The RIF molecule was flattened in the confined space between montmorillonite layers. The extent to which the flexible ansa chain of the RIF structure was deformed in comparison with the original structure of RIF taken from the molecular crystal is shown in Fig. 3.

Fig. 3

Comparison of the RIF structure in the molecular crystal (black) with the optimized RIF structure in the interlayer space of montmorillonite (orange); the red arrow highlights the protonated site (N50)

Hydrogen Bonds

The RIF molecule was anchored on the montmorillonite surface through many hydrogen bonds (Fig. 4). Besides the stabilizing intra-hydrogen bonds of strong to moderate strength in the RIF molecule, further cross-linking hydrogen bonds of moderate to weak strength (Desiraju & Steiner, Reference Desiraju and Steiner2006) were determined (Table 1). These hydrogen bonds formed between the RIF molecule, water molecules (hydrated Na⁺), and basal oxygen atoms of the montmorillonite surface. The strongest intramolecular hydrogen bonds were of the O_RIF-H···O_RIF type, while the weakest were of the C–H···O_RIF type. The RIF was anchored into the Mnt surface mainly through multiple hydrogen bond types such as C–H···O_b (b- basal) and O_RIF–H···O_b (moderate to weak), and strong N–H···O1_b and very weak N–H···O2_b of the protonated piperazine ring and two different basal oxygen atoms (O1 and O2) of the Mnt surface. Similar hydrogen bonds were also identified in a study of interactions of palygorskite with RIF (Damasceno et al., Reference Damasceno, de Almeida, Silva, de Assis, dos Santos, Dias and da Silva2020).

Fig. 4

Network of hydrogen bonds in the RIF-Mnt structure. Intra hydrogen bonds: O_RIF–H···O_RIF (lime), N–H···N (light blue), C–H···N (olive) and C–H···O_RIF (orange); O_w–H···O_w (magenta), and inter hydrogen bonds O_w–H···O_b (blue), O_RIF–H···O_b (cyan), C–H···O_b (black), N–H···O_b (petrol) and O_w–H···O_RIF (mint). The red arrow highlights the protonated site (N50)

Table 1

The D–H···A hydrogen bond lengths (min, median, max) [Å] present in the RIF-Mnt structure (RIF – rifampicin, w – water, b – basal, D – donor, A – acceptor)

The underlined numbers are medians

The N–H···O_b hydrogen bond had an essential importance in the anchoring of RIF onto the clay mineral, as the unprotonated form of rifampicin is difficult, even impossible, to intercalate into the interlayer space of montmorillonite.

The hydrated sodium cation supported anchoring of the RIF molecule into the Mnt siloxane surface through the net of hydrogen bonds formed by the coordinated water molecules. One water molecule was moving away from the coordination sphere of the Na cation, interacting with oxygen from the carbonyl C = O group of RIF (Fig. 4). Outgoing water (w) interacted through moderate hydrogen bonds with the Mnt surface (O_w–H···O_b) and through strong hydrogen bonds with other water molecules (O_w–H···O_w). These H₂O molecules interacted further with the rifampicin molecule (O_w–H···O_RIF), which then interacted with the opposite siloxane surface (O_RIF–H···O_b) of Mnt. These interactions contributed to the stability of the RIF–Mnt system (Table 1, Fig. 4). The strength of hydrogen bonds was in full accordance with the hydrophobicity of the siloxane surface of montmorillonite. The siloxane surface of montmorillonite is hydrophobic due to the character of the substitutions in the octahedral sheet and low humidity (Szczerba et al., Reference Szczerba, Derkowski, Kalinichev and Środoń2015, Reference Szczerba, Kalinichev and Kowalik2020); therefore, O_w–H···O_w hydrogen bonds were stronger than O_w–H···O_b.

The stability of the RIF-Mnt structure was expressed by the calculated intercalation energy as a difference between the sum of total energies of products and the sum of total energies of reactants (ΔE _int = ƩE _products – -ƩE _reactants) for the reaction:

(1) $[\mathrm{Na}{{{\left(H_2,O\right)}_4]}^{+}}_2-\mathrm{Mnt}+{\mathrm{RIF}}^{+}\to {\mathrm{RIF}}^{+}{\left(\mathrm{Na},{\left(H_2,O\right)}_4\right)}^{+}-\mathrm{Mnt}+{\left[\mathrm{Na}{\left(H_{2}O\right)}_4\right]}^{+}$

where [Na(H₂O)₄]⁺₂–Mnt is pure model montmorillonite, RIF⁺ is a protonated rifampicin molecule, and RIF⁺[Na(H₂O)₄]⁺–Mnt is the complex. The hydrated [Na(H₂O)₄]⁺ cation and RIF⁺ were calculated as isolated species in the computational cell of the RIF-Mnt complex. The calculated intercalation energy accounted for –4300 kJ mol^–1, indicating that the synthesized RIF-Mnt complex is stable; thus, montmorillonite could be a suitable carrier for RIF for postponed stimuli-induced release of this drug.

FTIR and Calculated Spectra

The total vibrational density of states (VDOS) of the RIF-Mnt model calculated from AIMD showed similar features to the measured FTIR spectrum of the synthesized RIF-Mnt intercalate (Fig. 5). The contributions of the individual functional groups were identified by the calculated projected spectra (PVDOS). The vibrational spectrum of the RIF-Mnt structure can be divided into two main frequency regions.

Fig. 5

Calculated (red) and experimental (black) FTIR spectra of the RIF-Mnt structure and FTIR spectrum of the RIF molecular crystal (blue)

For the first high-frequency region (4000–2500 cm^–1), the stretching modes of the OH, NH, and CH groups are typical. The OH-stretching vibrations were recognized as the highest-energy band having a maximum at 3773 cm^–1 (calculated VDOS). This band consists of overlapped stretching vibrations of the OH groups of the Mnt layer, OH groups of RIF, and H₂O molecules from the coordination shell of sodium cation. H₂O modes were detected as small shoulders at the high-frequency edge of the main peak at ~3860 and ~3800 cm^–1, respectively. In the experimental FTIR spectrum, the corresponding broad band was observed at a lower-frequency range (maximum at 3634 cm^–1) compared to the band from VDOS (Fig. 5). Generally, there is an evident high-frequency shift of the calculated stretching modes compared to experimental bands. This shift is obvious when DFT-calculated and experimental values are compared. Usually, a scaling factor is recommended (Scott & Radom, Reference Scott and Radom1996) for a better match between calculated and experimental frequencies. For example, if the calculated stretching vibrations of OH groups of montmorillonite (3773 cm^–1) were to match the experimental value of 3634 cm^–1, a scaling factor of 0.9632 should be applied. However, the scaling factor could shift some bands (especially deformation and low-frequency bands) in the wrong direction. Therefore, scaling was not applied further.

The well-recognized calculated band at ~3500 cm^–1 representing the NH stretching vibration in the VDOS spectrum helped to detect the position of the N–H stretching vibrations in the experimental FTIR spectrum of the RIF-Mnt, where just a broad band of low intensity spanned 3550–3150 cm^–1. Obviously in this broad experimental band, the overlapping bands of the NH and OH stretching vibrations of water are present. The observed FTIR spectrum of pure RIF (measured as solid molecular crystal in the KBr pellet) showed the broad band spanned the region of ~3750–3250 with a maximum at ~3440 cm^–1 (Fig. 5). This band consisted of the overlapped OH- and NH-stretching modes of the RIF molecule, in which OH modes were more populated in the higher-frequency part of the band range. The NH modes were in the lower-frequency part. Comparing calculated and FTIR spectra of the RIF-Mnt and pure RIF, it was estimated that there was a red-shift of ~50–70 cm^–1 of the NH-stretching modes of RIF in the RIF-Mnt intercalate due to hydrogen bonds formed with the Mnt layer (see previous section). This assumption is in accord with a similar red shift observed in the study of RIF-nanohydroxylapatite (3425 vs. 3350 cm^–1) (Qayoom et al., Reference Qayoom, Verma, Murugan, Raina, Teotia, Matheshwaran and Kumar2020).

The CH-stretching vibrations were well recognized in both calculated and experimental spectra of the RIF-Mnt structure (Fig. 5). Calculated bands at 3096 and 3009 cm^–1 were assigned to asymmetric and calculated bands at 2941 and 2877 cm^–1 to symmetric stretching CH vibrations. These calculated bands correspond to the multiple experimental bands at 2977 and 2929 cm^–1 (asymmetric stretching CH modes) and 2889 and 2865 cm^–1 (symmetric stretching CH modes), respectively. CH-stretching vibrations were also well resolved in the FTIR spectrum of the RIF molecular crystal in the spectral region from 3100 to 2700 cm^–1 (Fig. 5).

The second low-frequency region (below 1800 cm^–1) of the calculated spectrum represents a range of the complex spectrum with overlapped bands by different functional groups and types of vibrations (bending, deformation, skeletal). Specifically, performing an unambiguous and complete assignment is extremely difficult in the experimental spectrum (mainly below ~1200 cm^–1). Fortunately, identifying individual bands was possible in the calculated VDOS spectrum by using calculated projected VDOS (PVDOS) spectra of the respective groups of atoms. Typical bending vibrations were identified for water (1630 cm^–1) together with the overlapped bands of vibrations of N19–H86 (Fig. 1) amide group (1614 cm^–1), and CH groups (1444 and 1353 cm^–1) in the calculated spectrum. Further, calculated Si–O stretching modes were assigned to bands at 1124 and 1015 cm^–1. Vibrations of the C7 = O59 unit of the acetyl and furanone groups (Fig. 1) detected at 1710 cm^–1 in the calculated spectrum could correspond to the broadened band at 1725 cm^–1 in the FTIR spectrum of the RIF-Mnt structure. The overlapped bands of stretching vibrations at 1614 cm^–1 in the calculated PVDOS was assigned to the amidic C20 = O21 group (Fig. 2). The value of 1670 cm^–1 was examined for this functional group in the RIF-palygorskite system (Damasceno et al., Reference Damasceno, de Almeida, Silva, de Assis, dos Santos, Dias and da Silva2020). The lower value in the current findings could result from an interaction of this amidic C20 = O21 group with the sodium cation, as discussed in the previous paragraph. The experimental C–O–C bending vibrations of RIF (1310 cm^–1 and range of 1166–1000 cm^–1) overlapped with Si–O–Al stretching vibrations agreed with the assignment of similar bands at 1300 and 1100–900 cm^–1 of the RIF-palygorskite complex (Damasceno et al., Reference Damasceno, de Almeida, Silva, de Assis, dos Santos, Dias and da Silva2020). Furthermore, the N50–H bending modes of the protonated nitrogen atom at 922 and 549 cm^–1, the Si–O–Al bending and bridging modes at 925, 599, and 453 cm^–1, and the Si–O–Si bending and bridging modes at 383 and 187 cm^–1 were distinguished clearly in the calculated PVDOS spectrum. The CH rocking vibrations contribute to the band at 187 cm^–1. The experimental spectrum in the region below ~1100 cm^–1 was typical of the very complex shape with distinctive bands at 1048, 975, 518, and 464 cm^–1, attributed to the naphthohydroquinone chromophore unit in the spectrum of pure RIF.

Analysis of the measured FTIR spectrum of pure RIF was focused on the low region and showed characteristic absorption bands at 1733 cm^–1 (acetyl –C = O), 1652 cm^–1 (furanone C = O), 1567 cm^–1 (–CONH₂), 1452 cm⁻¹ (–C = C–stretching), 1376 cm⁻¹ (–CH₂, –C = C), and 1253 cm^–1 (–C–O–C–). Owing to the angular deformation of methylene units, the 947 cm⁻¹ peak could be assigned to the bending vibrations of the CH and CH₂ groups (Fig. 5). The IR bands of the pure RIF at 1726, 1643, and 1566 cm⁻¹ were attributed to hydroxyl N-methyl acetyl-carbonyl, furanone-carbonyl, and amide-carbonyl groups, respectively (Agrawal et al., Reference Agrawal, Ashokraj, Bharatam, Pillai and Panchagnula2004). Additionally, the skeletal deformation modes and the ring stretching modes of the naphthohydroquinone chromophore can be observed in the 400–1300 cm⁻¹ and 1300 − 1600 cm⁻¹ regions, respectively (Favila et al., Reference Favila, Gallo and Glossman-Mitnik2007).

Adsorption Study

Adsorption isotherm data of RIF on the montmorillonite and their fit to Langmuir and Freundlich adsorption isotherm models are shown in Fig. 6a,b. The adsorption isotherm for Na-Mnt displays a progressive evolution with a rapid increase in the amount adsorbed as the equilibrium concentration increased; a steady state or plateau was observed indicating saturation. Na-Mnt is a very efficient material with the largest amounts adsorbed of 1.13 mmol g⁻¹. Surprisingly, even at high RIF concentrations, the amount adsorbed did not exceed the CEC (1.21 mmol g⁻¹) of the montmorillonite used, probably due to the lack of the intramolecular affinity or bulkier size of the molecule adsorbed. Of the two standard, non-linear isotherm models, the Langmuir model (R = 0.998) fits the measured data much better than did the Freundlich model (R = 0.901), indicating two main adsorption regimes: (1) a gradual increase in the amount adsorbed at low concentrations (< 1 mmol L^–1), and (2) a steady state at higher concentrations.

Fig. 6

Adsorption isotherm data of rifampicin on Na-montmorillonite and their fit to: (a) Langmuir (c _ads = Γ_max.K_L. c _is/(1 + K K_L.c _is)) and (b) Freundlich (c _ads = K_F. c _is ^α) isotherm models. c _ads is the adsorbed concentration; c _is is initial concentration of solutions; K_L and K_F are adsorption constants; Γ_max is the maximal saturation of RIF; and α is the exponent

The mechanism of adsorption of antibiotics on clay-based materials depends on the type of antibiotic molecule, the clay mineral used, any pre-treatments or surface modifications applied to the adsorbent material, and the adsorption conditions (mainly pH, co-existing ions, salinity, etc.). Possible driving forces in the adsorption mechanism include electrostatic attractions, which can be subdivided into coulombic attraction, and dipole interactions (e.g. hydrogen bonding, π-interactions, London forces), as well as non-specific interactions such as hydrophobic interactions. In the current study, the mechanism of adsorption of rimfapicin antibiotic onto montmorillonite was driven mainly by electrostatic interactions of the positively charged RIF and negatively charged montmorillonite layers enhanced by hydrogen bonding. Note that the theoretical model of the RIF-Mnt structure could correspond to the first RIF molecules intercalated in the structure.