Polarized Raman spectra measured in Gly:CA crystals of both pyramidal (Figure 4a) and plate-like shapes (Figure 4b) are like those of nominally pure α- and γ-Gly [56–58]. They consist of many lines, originated predominantly from intramolecular vibrations of different types. Analysis of the Raman and FTIR spectra makes it possible to ascertain the chemical structure and polymorphous modification of the molecular crystals studied. Tables 8 and 9 portray the position of the lines in the polarized Raman and FTIR absorption spectra in pyramidal [γ-Gly:CA (80:20)] and plate-like [α-Gly:CA (90:10)] Gly:CA crystals, correspondingly. The positions of the lines in pyramidal Gly:CA crystals coincide with the positions of most lines in both nominally pure γ-Gly, and in plate-like α-Gly:CA crystals [56–58]. The shifts in the positions of high-frequency spectral lines at high frequency in each pair of crystals (α-Gly, α-Gly:CA) and (γ-Gly, γ-Gly:CA) do not exceed several cm−1. This indicates that the types and frequencies of molecular modes active in in Raman spectra of α- and γ-Gly doped with CA are close to those in pure Gly crystals. This is in agreement with the results of XRD analysis of Gly:CA single crystals and powders, which demonstrate very small changes in the crystal structure and position of atoms caused by doping with CA. Weak lines observed in α-Gly:CA spectrum portrayed in Figure 4c by red arrows are absent in pure Gly crystals and can be attributed to CA ions [59] (see Table 9).

**Table 8.** Positions of lines (*ν*, cm−1) observed in polarized Raman and FTIR spectra of γ-Gly:CA (80:20) crystals and assignments of analogous lines in nominally pure γ-Gly from Ref. [58].

**Table 8.** *Cont.*

**Table 9.** Positions of lines (*ν*, cm−1) observed in polarized Raman and FTIR spectra of α-Gly:CA (90:10) crystals and assignments of analogous lines in nominally pure α-Gly from Ref. [58].

**Table 9.** *Cont.*

The difference in polarized Raman spectra of γ- and α-Gly is most evident in the position of strong lines in the region of low (0–300 cm−1) and high (2800–3100 cm−1) frequencies of the spectrum. This difference is clearly observed in the Raman spectra of pyramidal and plate-like Gly:CA crystals (Figure 4a,b). At high frequencies (2800–3100 cm−1) the stretching symmetric and antisymmetric vibrations of CH2 group have been observed in α-Gly:CA at 2973 cm−1 and 3007 cm−1 and in γ-Gly:CA at 2963 cm−1 and 2999 cm−1 (Figure 5b), respectively, which corresponds to literature data related to pure α- and γ-Gly [58]. At low frequencies (0–300 cm−1), positions of lines in α- and γ-Gly:CA spectra are slightly shifted in compared to pure Gly crystals [56,57] (Tables S5 and S7 in Supplementary Materials), which may be attributed to CA doping.

**Figure 4.** Polarized Raman spectra of pyramidal (**a**) and plate-like (**b**) Gly:CA crystals after subtracting the photoluminescence background. Fragment of the plate-like Gly:CA spectrum on an enlarged scale (**c**). For pyramidal crystals, the Z-axis is parallel to the polar axis (the height of the pyramid); for plate-like crystals, the Y-axis is parallel to the *C*2 axis. The red arrows in (**c**) indicate positions of lines observed in CA crystals. *λ*exc = 532 nm.

**Figure 5.** Comparison of Raman spectra of Gly:CA crystals of pyramidal (blue line) and plate-like (red line) shapes in low-energy (**a**) and high-energy (**b**) spectral regions.

As in the case of Raman spectra, a comparison of the FTIR absorption spectra of doped CA and nominally pure Gly indicates their close similarity (Tables 8 and 9). However, in the case of α-Gly:CA (90:10) plates, additional lines in the photon energy range *E*ph = 0.5–0.6 eV (4000–5000 cm−1) were observed in the FTIR absorption spectra measured in transmission geometry. Note that similar lines have been observed in the FTIR spectra of TGS crystals doped with CA [31] (Figure 6). No additional lines were observed in the reflection geometry, most likely due to their low intensity. Note that we did not measure FTIR absorption spectra of γ-Gly crystals in transmission geometry because of their pyramidal shape.

**Figure 6.** FTIR absorption spectra in spectral region *E*ph = 0.5–0.6 eV in Gly:CA (90:10) measured in this work and in previously studied TGS:CA (90:10) (see text).

So, polarized Raman and FTIR absorption spectra of Gly:CA single crystals indicates that addition of croconic acid to the aqueous solution which was used to grow Gly:CA crystals does not significantly affect the polymorphic modification of the crystals grown. It has been established that γ-Gly forms pyramidal crystals, while α-Gly forms platelike crystals. The incorporation of CA molecules in a glycine crystal does not noticeably change the spectral positions of Raman lines in the high-energy region of the spectrum (2900–3100 cm−1), but leads to modest shift of several low-energy lines, which characterizes the effect of CA doping on the lattice vibration spectrum [32].

#### *3.4. UV-Vis Absorption*

RT absorption spectra of α-Gly:CA (90:10) and γ-Gly:CA (80:20), are portrayed in Figure 7. Colorless α- and γ-Gly crystals are transparent in wide spectral range. The energy band gaps of different polymorphic modifications of Gly crystals practically coincide and are about 5.1 eV [8,11,12]. Significant optical absorption is observed only for photons with energies *E*ph > 5 eV, which manifests itself in a sharp increase of absorbance at *λ* < 240 nm. The doping of Gly with CA is accompanied by the appearance of absorption in the transparent region of pure Gly. This absorption manifests itself in the form of a structured long-wavelength tail adjacent to the edge of the fundamental absorption of a Gly crystal. The traits of the absorption are clearly observed when considering the absorption in Tauc coordinates (*αE*ph) 2, *E*ph (Figure 7c,d) (where *α* is the absorption coefficient of the crystal). The shortest wavelength part of the absorption spectrum in region *Eph* = (5.8–6.2) eV in Figure 7c,d is well approximated by the linear dependence (solid black line), which can be regarded as the Tauc plot related to direct allowed interband transitions in crystals. The Tauc plot yields an estimation of the value of the optical bandgap as ~5.3 eV, which is close to the *E*g value in pure Gly (~5.1 eV) (Figure 7c,d). Note that in Tauc coordinates, the shape of the absorption edge of undoped Gly is indeed well approximated by a straight line [8]. The presence of the additional absorption in the Gly:CA spectra (as compared with

undoped Gly) can be associated with electronic transitions involving defects in the Gly lattice induced by the introduction of CA molecules. The contribution of these transitions to the absorption spectrum can be estimated as the difference between the absorption spectrum of Gly:CA (line 1 in the insets in Figure 7e,f) and the fragment of its fundamental absorption spectrum modeled by the Tauc plot (line 2 in the insets in Figure 7e,f). The absorption spectra thus obtained are portrayed in Figure 7e,f, which includes at least two absorption bands with maxima at ~5.2 and ~3.9 eV. It should be noted that similar structure is observed in the absorption spectrum of CA solution in ethanol (olive line in Figure 7e,f) or in water [60]. In Gly:CA crystals the absorption bands can presumably be associated with the creation of excitons localized at the lattice defects, including impurity CA molecules or neighboring Gly ones.

**Figure 7.** Absorbance spectra of (**a**) α-Gly:CA (90:10), (**b**) γ-Gly:CA (80:20), (**c**) α-Gly:CA (90:10), and (**d**) γ-Gly:CA (80:20) in Tauc coordinates. (**e**,**f**) Change in the Gly crystal absorption caused by incorporating CA molecules into the Gly crystals. The olive lines represent the absorption spectrum of CA solution in ethanol [60]. The inset portrays the absorption spectra of doped and pure Gly crystals (see text).

Table 10 presents a comparison of the *Eg* values, and high and low frequency local bands in α-Gly:CA (90:10), γ-Gly:CA (80:20), and TGS:CA (80:20) [31]. It is observed that CA doping results in practically the same changes of absorption spectra in these crystals.

**Table 10.** Energy gap *Eg*, eV, photon energy *E*ph of high- and low-energy local bands and normalized amplitude of low energy band in absorption spectra of α-Gly:CA (90:10), γ-Gly:CA (80:20) and TGS:CA (80:20) [31].

Thus, doping of crystalline Gly with CA significantly affects the shape of the absorption edge of Gly, which manifests itself in smearing of the sharp absorption edge and the appearance of noticeable absorption at frequencies *hν* < *Eg*. Note that the same changes of absorption spectrum have been observed earlier in CA-doped triglycine sulfate crystals [31].

#### *3.5. Photoluminescence*

The photoluminescence (PL) of "pure" glycine crystals is very weak. As a result, in the emission spectrum of "pure" γ-Gly narrow Raman lines are clearly observed, whose intensity is comparable to that of the luminescence band (Figure 8a). Like pure glycine, solid croconic acid is also practically non-luminescent [61]. However, doping glycine crystals with croconic acid leads to a dramatic increase in the emission intensity, while the intensity of Raman scattering does not change (Figure 8b). Thus, the relatively bright luminescence of γ-Gly:CA is a distinctive characteristic of the doped crystals. A similar situation also occurs in the case of α- Gly:CA.

The RT PL spectra of α- and γ-Gly:CA crystals excited by light with *λ*exc = 325 nm are portrayed in Figure 8b. The spectra of both γ- and α-modifications consist of broad structured emission bands with their maxima at ~2.50 eV (γ-Gly:CA) and ~2.56 eV (α-Gly:CA), respectively. Comparison of the PL spectra of pure and doped Gly crystals (Figure 8) portrays that the shapes of the PL bands are practically identical, but doping Gly with croconic acid leads mainly to a dramatic increase in the PL intensity. Considering that the band gap of γ-glycine crystals is about 5 eV [8,11–13] the optical electron transitions responsible for the emission involve localized electron states whose energy levels are located within the crystal band gap.

Figure 8c,d portrays the decomposition of the emission band profile into four Gaussian components both in the case of α-Gly:CA and γ-Gly:CA. The positions of the Gaussian components and their relative intensity in the PL spectra of α-Gly:CA (90:10), γ-Gly:CA (80:20), and TGS:CA (90:10) [31] are presented in Table 11. The observed emission-band structure implies the presence of at least four different emitting centers that contribute to the emission of Gly:CA crystals. Note that the expansion of the TGS:CA (90:10) PL spectrum in terms of the Gaussian component presented in our previous paper [31] contains only three components. Here, we present the results of more accurate calculations that include four Gaussian components which provide a better fit. Both in Gly:CA crystals and in TGS:CA crystals, the positions and normalized intensities of the Gaussian components in both Gly:CA and TGS:CA crystals are close, which demonstrates that in all the crystals compared the PL originates from the same emission centres. The similar PL behavior in CAdoped Gly and TGS crystals clearly indicates that the appearance of PL is not associated with the SO4 groups, which are present in TGS but absent in Gly crystals. The PL is probably the result of the interaction of CA with glycine ions and, most likely, with glycine

zwitterions, since, as XRD phase analysis demonstrates, the structural parameters of these ions are affected by CA doping.

**Figure 8.** (**a**) Emission spectrum of "pure" γ-glycine wih a small admixture of citric acid. Here, R labels the Raman lines. (**b**) Emission spectra of γ- and α-glycine crystals doped with croconic acid. (**c**) Decomposition of the α-Gly:CA emission band profile into four Gaussian components. *hν*exc = 3.8 eV. *T* = 300 K. (**d**) Decomposition of the γ-Gly:CA emission band profile into four Gaussian components. The Gaussian components in (**c**,**d**) are shown by different colors.

**Table 11.** Resonance photon energies *E*ph, eV, normalized intensity *Ai*/*A*2 obtained after decomposition of PL spectra in α-Gly:CA, γ-Gly:CA and TGS:CA in four Gauss components.

From Table 11 we observe that the normalized intensities of Gauss components may depend on crystal symmetry because bands 3 and 4 in non-centrosymmetric γ-Gly, and TGS:CA are more intensive than in centrosymmetric α-Gly:CA and band 1 has higher amplitude in α-Gly:CA.

Although the specific structure of these centers is currently not clear, it can be assumed that they include molecules of both glycine and croconic acid (glycine and CA crystals are not luminescent by themselves). The presence of an analogous emission band in the luminescence spectrum of "pure" γ-Gly (Figure 8a) with a small admixture of citric acid demonstrates that not only CA but also other organic acids can promote the formation of emitting centers (glycine lattice defects) with a similar structure of radiative transitions.

#### **4. Conclusions**

Crystals of α- and γ-glycine doped with croconic acid were grown from aqueous solutions of glycine and croconic acid. α-Gly crystals grown from a solution with a relative weight content Gly and CA of 90:10 have a plate shape. Pyramid-shaped γ-Gly crystals were obtained from a solution with a relative weight content of 98:2 and 80:20. A study of the structural and optical properties of the grown crystals has been performed. The study indicates that the crystal symmetry and morphology of CA-doped-doped glycine single crystals grown from aqueous solutions with different components weight ratios correspond well to well-known α- and γ-isomorphs of nominally pure glycine. Incorporation of CA molecules in the host glycine crystal structures does not change remarkably the parameters of the unit cells, but results in small changes of unit cell volumes, and bond angles and lengths of glycine zwitterions.

In many aspects, this situation is quite similar to that observed in CA-doped TGS single crystals [31]. CA doping both Gly and TGS crystals does not significantly change the frequencies of molecular vibrations in Raman and FTIR spectra. Also, it does not remarkably influence the magnitude of forbidden gap *E*g, caused by direct allowed electronic transitions.

The most significant changes caused by CA doping glycine (Gly:CA), as in the case TGS crystals, are associated with absorption and photoluminescence spectra. In the UV Vis region CA doping results in appearance of a strong absorption band caused by local electronic transitions near the band gap (*E*ph ≈ 5.2–5.4 eV), and a broad band of lower intensity with a maximum in the region of *E*ph ≈ 3.7–3.9 eV. Weak absorption bands were detected by FTIR in TGS:CA and Gly:CA in the region of *E*ph ≈ 0.5–0.7 eV.

Strong PL spectra in α-, γ-Gly:CA, and TGS:CA crystals demonstrate the contributions of four emission centers emitting photons of close energies (~2.2 eV, 2.57 eV, 2.86 eV, 3.18 eV) and intensity ratios. It can be assumed that the appearance of these centers is due to the incorporation of small CA ions into the pores of the host crystal structure and the formation of hydrogen bonds between the CA molecule and glycine zwitterions.

In α- and γ-Gly:CA, in contrast to TGS:CA, different phases (regions) with a crystal structure of the same symmetry but slightly different unit cell parameters arising from the inhomogeneous doping distribution of the CA molecule in the host crystal were revealed by powder X-ray diffraction methods. The presence of twins in non-centrosymmetric γ-Gly:CA was detected using single-crystal XRD. These crystal structure details do not appear in optical spectroscopy experiments due to the integral nature of these methods.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/cryst12101342/s1. Table S1: Results of refinement of the α-Gly:CA (90:10) structure (space group *P*21/*n* (14)) using single crystal XRD data. Table S2: Atomic displacement parameters for the α-Gly:CA (90:10) (Å2). Table S3: Results of refinement of the γ-Gly:CA (80:20) structure (space group *P31* (N 144)) using single crystal XRD data. Table S4: Atomic displacement parameters for the γ-Gly:CA (80:20) (Å2). Figure S1: Structures of (a) α-Gly:CA (90:10) and (b) γ-Gly:CA (80:20) with atoms represented by thermal ellipsoids by means of program ORTEP using, correspondingly, the data presented in Tables S1–S4. Figure S2: (a), (c) WHP and (b), (d), (f) SSP of α1 and α2 modifications of α-Gly. Figure S3: (a), (c) WHP and (b), (d), (f) SSP of γ1 and γ2 modifications of γ-Gly. Figure S4: LB fit results for (a) α-Gly:CA (90:10 and (b) γ-Gly:CA (80:20). Figure S5: Rietveld fit results for α-Gly:CA (90:10). Table S5: Low frequency intermolecular modes and symmetry modes in Gly:CA plate in different experimental geometries, and in nominally pure α-Gly [5]. Table S6: High frequency intramolecular modes in Gly:CA plate in different experimental geometries, and in nominally pure α-Gly [6]. Table S7: Low frequency intermolecular modes in Gly:CA pyramid in different experimental geometries, and in nominally pure γ- Gly [7]. Table S8. High frequency intramolecular modes in Gly:CA pyramid.

**Author Contributions:** Formal analysis, E.B., A.Z., A.A.L., B.K., V.D., A.S. (Alexander Smirnov), A.S. (Anatoly Starukhin), S.P., H.Z., F.L. and H.K.; Investigation, E.B., A.Z., A.A.L., V.D., A.S. (Alexander Smirnov), A.S. (Anatoly Starukhin), S.P., H.Z., F.L. and H.K.; Writing—original draft, E.B., A.Z., A.A.L., A.S. (Anatoly Starukhin), B.K., H.Z., F.L. and H.K.; Writing—review and editing, E.B., B.K. and H.K.; Funding Crystals 2022, 12, 679 23 of 25 acquisition, E.B. and H.K.; Conceptualization, E.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by RFBR (project number 21-52-53015) and NSFC (project number<PHONE_NUMBER>0).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The X-ray experiment for TGS:CA (90:10) was carried out using facilities of the X-ray diffraction Resource Centers of St. Petersburg University. The XRD powder characterizations were performed using equipment and software of the Joint Research Center "Material science and characterization in advanced technology" (Ioffe Institute, St.-Petersburg, Russia).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Study on Precipitation Processes and Phase Transformation Kinetics of Iron Phosphate Dihydrate**

**Huiqi Wang 1, Mingxia Guo 2, Yue Niu 1, Jiayu Dai 1, Qiuxiang Yin 1,3 and Ling Zhou 1,\***

**Abstract:** The process of the phase transformation from amorphous to crystalline FePO4·2H2O was studied in this research. It was found that Fe and P are predominantly present as FeHPO4 + and FeH2PO4 2+ and an induction period exists during the transition from amorphous to monoclinic form. The induction period and the time required for phase transformation were shortened with the increased temperature. Phase transformation could be kinetically described by the Johnson–Mehl– Avrami (JMA) dynamics model. The dissolution rate of amorphous FePO4·2H2O is the rate-limiting step of this process. the activation energy of phase transformation is calculated to be 9.619 kJ/mol. The results in this study provided more guidelines for the regulation of FePO4·2H2O precursors by precipitation method.

**Keywords:** precipitation process; iron phosphate dihydrate; Johnson–Mehl–Avrami (JMA) dynamics model

#### **1. Introduction**

At present, the demand for rechargeable lithium batteries for electric vehicles is growing with increased energy efficiency requirements. Creating composite electrode materials with the required properties is a promising direction in battery technology and engineering [1–4]. Researchers have proposed numerous techniques for fabricating the positive electrode LiFePO4 of reversible lithium batteries [5]. So far, the precursor of LiFePO4—FePO4 was mainly prepared by the: (1) solvent gel method [6], (2) hydrothermal method [7], (3) coprecipitation method [8], and others. Zhang et al. [9] synthesized nanosized FePO4 by a modified sol–gel method, which is convenient for controlling the carbon content and size of LiFePO4. Chen et al. [10] produced FePO4 microspheres with carbon nanotube embedded (FePO4/CNT), which enhanced their electronic conductivity by a hydrothermal process. Tong et al. [11] prepared iron phosphate with various morphologies and crystalline structures by coupling precipitation and aging. Coprecipitation is the most advantageous method for synthesizing iron phosphate owing to its simple operation, economic efficiency, and environmental friendliness among these methods [5].

FePO4·2H2O acts as a transient precursor in the coprecipitation method to obtain FePO4. Many researchers have reported the preparation of FePO4·2H2O with various structures and morphologies, which were achieved by altering the sources of Fe and P as well as reaction conditions [11,12]. For example, Guo et al. [13] found that iron phosphate precursor with different morphologies can be obtained at low temperature using Fe2(SO4)3, H3PO4 and different additives. Jiang et al. [14] synthesized irregular polygon shapes of amorphous FePO4·2H2O, and elongated sticks of crystalline FePO4·2H2O by refluxing amorphous FePO4·2H2O at 100 ◦C for 2 h using Fe(NH4)2(SO4)2·6H2O, NH4H2PO4, and

**Citation:** Wang, H.; Guo, M.; Niu, Y.; Dai, J.; Yin, Q.; Zhou, L. Study on Precipitation Processes and Phase Transformation Kinetics of Iron Phosphate Dihydrate. *Crystals* **2022**, *12*, 1369. https://doi.org/10.3390/ cryst12101369

Academic Editor: Yanfei Wang

Received: 5 September 2022 Accepted: 22 September 2022 Published: 27 September 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

H2O2 as raw materials. As the important precursor of LiFePO4 cathode material, the properties of FePO4 have to be controlled in the preparation process, such as the specific amount of iron–phosphorus ratio, high specific surface area, and tap density [15]. By understanding the mechanism and kinetics of the preparation process, the properties of FePO4·2H2O can be better controlled and the quality of FePO4 can be further improved. Many studies have demonstrated that the crystal structure of FePO4 significantly impacts the morphology of FePO4 [16–18]. However, insufficient literature exists regarding the phase transformation on this process.

This paper studied the precipitating process of FePO4·2H2O using ferrous sulfate heptahydrate, phosphoric acid, and hydrogen peroxide as reactants, and the morphology and phase transformation kinetics of FePO4·2H2O from amorphous to monoclinic in acidic solution (pH < 1) were investigated. The changes in the morphology, structure, and composition of the precipitate during the phase transformation were also tracked. These studies provide ideas and a basis for further improvement in the properties of iron phosphate as a potential electrode material precursor.

#### **2. Materials and Methods**

*2.1. Chemicals*

All the reagents used in experiments containing ferric sulfate septihydrate (FeSO4·7H2O), hydrogen peroxide (H2O2), monoclinic iron phosphate dihydrate (FePO4·2H2O), and phosphoric acid (H3PO4) were of analytical reagent grade. Deionized water was used throughout the process. All these chemicals were purchased from Kaimart Tianjin Chemical Technology Co., Ltd (Tianjin, China). and were used directly without further purification. Detailed information could be seen in Table 1.

#### *2.2. Experimental Procedure*

Ferrous sulfate septihydrate and phosphoric acid at a molar ratio of 1:1.05 were dissolved with deionized water in a 500 mL jacketed glass reactor coupled with a thermostatic bath. Excess phosphorus was used to make sure the iron precipitated completely during the reaction. The mixture was then stirred by a stirrer module until ferrous sulfate septihydrate dissolved completely. The peristaltic pump added a certain amount of hydrogen peroxide, the mole of which was round 0.6 time the ferrous, to make Fe2+ iron oxidized completely. When the solution temperature was raised to 60 ◦C the precipitation was generated from the solution. The solution temperature was kept at 90 ◦C for 8 h and a phase transformation occurred during this process. The precipitation was filtered out at the end of the experiment. The experimental setup is shown in Figure 1 and detailed information of the solution can be seen in Table 2.

**Table 2.** Compositions of the solution.

**Figure 1.** Experimental apparatus utilized in the experiments.

#### *2.3. Preparation of Standard Solution for RAMAN Analysis*

The difference of the two polymorphs in solution can be shown by Raman spectra through the characteristic peaks. Constructing a standard line using Raman spectra of the prepared polymorphic mixtures to quantitatively analyze changes of monoclinic FePO4·2H2O during the phase transformation process: the standard solution was a mixture of amorphous and monoclinic FePO4·2H2O at a predetermined concentration of H2SO4. The mole fraction of monoclinic FePO4·2H2O ranged from 25% to 75%. The temperature of solution was raised to 90 ◦C. Each measurement was carried out in three times. There was no polymorphic transformation that occurred for 1–3 min during detection, which can be indicated by Raman spectra. The function that connected the corresponding peak intensity to the mass fraction of monoclinic FePO4·2H2O could then be determined.

#### *2.4. Characterization*

#### 2.4.1. Powder X-ray Diffraction (PXRD) Analysis

The samples collected during the process were analyzed by X-ray diffraction (Rigaku D/MAX-2500) with Cu Kα radiation (λ = 1.5405 Å) by Ni filter. The scanning angle ranged from 2◦ to 40◦ at a rate of 8◦/min. The characterization was carried out at a voltage of 45 kV and a current of 40 mA. The melting properties of FePO4·2H2O are measured by thermal gravimetric analyzer (TG, MettlerToledo, Zurich, Switzerland). The sample (5−10 mg) was heated at a rate of 10 K/min from 303.15 K to 1073.15 K under a nitrogen atmosphere.

#### 2.4.2. Morphology Characterization

The morphologies of products were observed by scanning electron microscopy (SEM; JSM-7401F, 3 kV) after being sputter-coated with Au/Pd and transmission electron microscopy (TEM; JEM-2010, 120 kV) using an accelerating voltage of 100 kV.

#### 2.4.3. Spectra Characterization

The changes of functional groups in precipitation during the reaction were detected by FTIR (Bio-rad FTS 6000), which scanned with wavenumber from 400 to 4000 cm−1; Raman analysis (RFS 100/S) used a 1064 nm Nd-YAG laser and the scanning step was 2 cm−1 with a 50 kHz scanning frequency, utilizing fiber-coupled probe optic technology for in situ monitoring. This system was equipped with a probe head for direct insertion or non-contact sampling.

#### **3. Results and Discussion**

#### *3.1. The Involved Precipitation Reactions Analysis*

The complexation of Fe3+ with phosphate is complicated in solution [19,20]. In this work, complexation ions such as Fe(PO4)2 3−, Fe(OH)PO4 −, FeH3(PO4)3 3−, Fe(PO4)2 3−, Fe2PO4 3+, etc., were not taken into account owing to small equilibrium constants (<10−8). The possible reactions in solution and corresponding equilibrium constants are listed in Table 3 [21–23]. On the basis of mass conservation and reactions in Table 3, the mass balance of Fe and P can be expressed, as given in formulas (1) and (2). The total concentration of Fe and P was measured by redox titration of potassium dichromate and the gravimetric method. According to pH and the equilibrium constants of possible reactions, the corresponding concentration of complexation ions can be represented by free Fe3+ ion and H3PO4 molecules.

$$\begin{aligned} \text{[Fe]}\_{\text{T}} &= \text{[Fe}^{3+}\text{]} + \text{[FeHPO}\_4^{+}\text{]} + \text{[FeH}\_2\text{PO}\_4^{2+}\text{]} + \text{[FeH}\_2(\text{PO}\_4)^{2-}\text{]} + \text{[FeH}\_4(\text{PO}\_4)^{2+}\text{]} \\ &+ \text{[FeH}\_8(\text{PO}\_4)\_4^{-}\text{]} + 2 \times \text{[Fe}\_2\text{HPO}\_4^{4+}\text{]} + 2 \times \text{[Fe}\_2\text{H}\_3(\text{PO}\_4)\_2^{3+}\text{]} + 3 \times \text{[Fe}\_3\text{H}\_6(\text{PO}\_4)\_4^{3+}\text{]} \end{aligned} \tag{1}$$

$$\begin{aligned} \text{[P]}\_{\text{T}} &= \text{[H}\_{3}\text{PO}\_{4}\text{]} + \text{[H}\_{2}\text{PO}\_{4}\text{]} + \text{[HPO}\_{4}\text{2}^{2-}\text{]} + \text{[PO}\_{4}\text{3}^{3-}\text{]} + \text{[FeHPO}\_{4}\text{4}^{+}] + \text{[FeH}\_{2}\text{PO}\_{4}\text{2}^{2+}\text{]} \\ &+ 2 \times \text{[FeH}\_{2}\text{(PO}\_{4}\text{)}\_{2}\text{2}^{-}\text{]} \text{]} + 2 \times \text{[FeH}\_{4}\text{(PO}\_{4}\text{)}^{2+}\text{]} + 4 \times \text{[FeH}\_{8}\text{(PO}\_{4}\text{)}\_{4}^{-}\text{]} + \text{[Fe}\_{2}\text{H}(\text{PO}\_{4})^{4+}\text{]} \text{]} \\ &+ 2 \times \text{[Fe}\_{2}\text{H}\_{3}\text{(PO}\_{4}\text{)}\_{2}^{3+}\text{]} + 4 \times \text{[Fe}\_{3}\text{H}\_{6}\text{(PO}\_{4}\text{)}\_{4}^{3+}\text{]} \end{aligned}$$

where [Fe]T and [P]T is the total concentration of Fe and P in solution; [*x*] is the concentration of *x*; *x* is the species of complexation.

**Table 3.** Possible complexation reactions for Fe3+ and phosphate.

The unit of equilibrium constants K is (mol·L−1)ˆΔν, Δν is sum of stoichiometric numbers in reaction.

And the concentration of free Fe3+ ion and H3PO4 molecules, under pH ranging from 0.2 to 1.3, can be calculated by the total concentration and mass balance of Fe and P. The equations for the concentration of Fe3+ and H3PO4 were presented in Formulas (3) and (4). In addition, the concentration of various complexation species with phosphate groups under different pH environments is listed in Tables S1–S4 (Supporting Information). The distribution of the various complexation species is presented in Figure 2.

$$\mathbf{x} + (A+B)\mathbf{x}\mathbf{y} + (\mathbb{C}+D)\mathbf{x}\mathbf{y}^2 + E\mathbf{x}\mathbf{y}^4 + 2F\mathbf{x}^2\mathbf{y} + 2G\mathbf{x}^2\mathbf{y}^2 + 3H\mathbf{x}^3\mathbf{y}^4 = 0.293 \tag{3}$$

$$\begin{array}{c}(1+a+b)y + (A+B)xy + 2(C+D)xy^2 + 4Exy^4 + Fx^2y + 2Gx^2y^2\\ +4Hx^3y^4 = 0.2978\end{array} \tag{4}$$

where *x* is the concentration of Fe3+ and *y* is the concentration of H3PO4; *a*, *b*, *A*, *B*, *C*, *D*, *E*, *F*, *G*, *H* are the constants in different pH environment based on the equilibrium constants of possible reactions respectively.

It can be found that Fe and P are predominantly presented as FeHPO4 + and FeH2PO4 2+ in the pH range. The pH of the mixture at room temperature in this work is around 0.85 and the proportion of main complexation ions are calculated to be 53.82% FeHPO4 +, and 9.79% FeH2PO4 2+. Therefore, the main precipitation reactions, even during the process of raising the temperature, can be written as the following reactions in Table 4 (the pH decreased with the increase of temperature).

**Figure 2.** Distribution of the various complexation species under different pH environments: (**a**) shows distribution of H+, H2PO4 −, HPO4 2−, PO4 3− and Fe3+; (**b**) shows distribution of the rest of the ions.

**Table 4.** Possible reactions in solution with an initial pH value of 0.85.

#### *3.2. Insight into the Evolution of the Precipitate*

#### 3.2.1. The Change of Structure

The crystal structure and phase purity of the samples were analyzed by PXRD. The powder X-ray diffraction pattern was shown in Figure 3. There were not any intensive diffraction peaks detected before 235 min, indicating that the precipitation initially produced by the reaction was amorphous. After 235 min, new characteristic diffraction peaks appeared (17.18◦, 18.88◦, and 19.94◦, respectively), and the intensity of these peaks increased with the evolution of time. The results demonstrated that there was a phase transition during the reaction and the crystallinity gradually increased over time. Since the PXRD pattern of samples after 235 min is consistent with standard data of JCPDS file No. 15-0390, it is found that the transformed product possessed monoclinic structure and a space group of P21/n [15,24–26]. The crystal structure of monoclinic FePO4·2H2O was shown in Figure 4.

**Figure 3.** The Powder X-ray diffraction pattern of samples taken at 0 min (**a**), 180 min (**b**), 205 min (**c**), 225 min (**d**); 235 min (**e**), 245 min (**f**); 255 min (**h**), 450 min (**g**), respectively.

**Figure 4.** Crystal structure of monoclinic FePO4·2H2O.

#### 3.2.2. Change of Morphology

The morphology during the reaction was studied by SEM and TEM as shown in Figures 5 and 6. At the initial stage of the reaction, the sample mainly exists in the form of amorphous microsheet agglomeration and the average size is about 2 μm to 4 μm, as shown in Figures 5a and 6a. The size of microsheets gradually decreased and the extent of agglomeration increased slightly. They grew and agglomerated together by continuous dissolution–recrystallization. Figure 5c shows that plentiful nanoparticles formed on the surface of agglomerates at 150 min, which were found to be a mixture of mostly amorphous and little crystalline FePO4·2H2O by selected area electron diffraction (SAED)—which shows an inconspicuous diffraction pattern, as shown in Figure 6b. But there is still no obvious diffraction peak of powder X-ray diffraction pattern at this time, at which the content of crystalline FePO4·2H2O is lower than the minimum content standard of powder X-ray diffraction analysis. The monoclinic FePO4·2H2O nuclei on the active surface of amorphous agglomerates gradually grew and agglomerated accompanying the dissolution of amorphous FePO4·2H2O into ions. Ultimately, dense spheroid-like monoclinic FePO4·2H2O particles were produced continuously by dissolution–recrystallization of agglomerates. It was proven that the spheroid-like particles are comprised of long flakes of monoclinic FePO4·2H2O as illustrated in Figure 6c.

**Figure 5.** SEM images of morphology evolvement of FePO4·2H2O during the experiment: (**a**) 0 min; (**b**) 30 min; (**c**) 150 min; (**d**) 450 min; (**a'**–**d'**) are images with the higher magnification at the corresponding times (0 min, 30 min, 150 min, 450 min).

**Figure 6.** TEM images of FePO4·2H2O at (**a**) initial, (**b**) 150 min and (**c**) end of reaction, respectively; (**d**) typical EDS spectrum of initially produced precipitate.

#### 3.2.3. Changes in the Process of Reaction

The samples in the different reaction stages were also analyzed by TG, as shown in Figure 7. The heating process was conducted in an N2 environment and the weight loss was on account of the dehydration [27,28], which was around 19.30%. With the increase of crystallinity, the endothermic peak for dehydration is shifting in the higher temperature direction gradually [29] (115.417 ◦C→135.063 ◦C→161.769 ◦C→176.833 ◦C), which proved that water molecules gradually enter into the lattice monoclinic FePO4·2H2O as the phase transformation progresses.

**Figure 7.** Heat-flow and mass change of precipitation at different reaction time during heating.

The reaction process was characterized by off-line FTIR, as shown in Figure 8. The characteristic peaks of the O-H stretching and bending vibrational mode are at 3500–3000 cm−1 and about 1600 cm−1 respectively [30,31]. The peak band at 513 cm−1 is attributed to the bending vibration mode and stretching band of O-P-O bonds. The P-O-P bonds are also reflected at 980 cm−1 to1100 cm−1, which corresponds to the tetrahedral PO4 3− anions.

**Figure 8.** FTIR spectra of the precipitates at different reaction times.

After 180 min, a new peak at 750 cm−1 appeared, corresponding to the P-O vibration caused by the coupling effect between PO4 3− polyanion and Fe-O within the structure [32–35]. The symmetrical vibration characteristic peaks of PO4 3− anions at 997 cm−1 disappeared, and asymmetrical vibration peak at 1165 cm−1 appeared over time, as shown in Figure 8. These changes indicate that the FePO4·2H2O orderly rearranged and monoclinic FePO4·2H2O formed.

The result shown in Figure 9 demonstrated that the proportion of Fe in the precipitate increased at the early stage of phase transformation and then decreased with the increase of crystallization, which can also be proven by the energy dispersive spectrometer (EDS) information in Table 5. At the highest proportion of iron, the corresponding precipitation is amorphous, and the characteristic vibration peaks of iron binding with other functional groups are not found in the FTIR spectrum, indicating that there is not any intermediate during transformation.

**Figure 9.** The mass fraction trend of Fe in precipitation at different reaction time.

**Table 5.** Composition of produced precipitate under different time.

The solubility of the amorphous form of FePO4·2H2O is higher than that of the monoclinic form [36]. During the rearrangement of iron phosphate dihydrate, the solution became supersaturated again with the dissolution of amorphous iron phosphate. The iron ions in solution adsorbed on the surface of the agglomerate (were partially neutralized by sulfate ions) or encased in the interior of the agglomerate, leading to a rise in the iron mass fraction (Supporting Information, Table S1). Then iron mass fraction decreased with the nucleation and growth of monoclinic FePO4·2H2O. The rise in mass fraction following transformation could be attributable to the impurity removal due to the decreased content of the S in precipitation.

#### *3.3. Kinetic of Phase Transformation*

Raman was used to monitor the transformation of amorphous FePO4·2H2O, as shown in Figure 10. Transformation of amorphous FePO4·2H2O was followed by the appearance of monoclinic FePO4·2H2O characteristic peaks and the changes in height of the characteristic peaks: a new peak at 303 cm−1 appeared in the Raman spectra and its intensity increased gradually as the monoclinic FePO4·2H2O content increased during transformation.

**Figure 10.** Raman spectra of the precipitates at different reaction times.

The corresponding characteristic peak intensity of the standard solution was measured by Raman spectroscopy (shown in Figure 11) and the standard curve could be obtained according to the relationship between intensity of characteristic peak and the mass fraction of monoclinic FePO4·2H2O, as shown in Figure 12. The transformation process was quantitatively evaluated by the relationship, which can be seen in Figure 13. It shows the changes of polymorphic composition during transformation. The mass fraction of the amorphous FePO4·2H2O decreased gradually after the induction time of 101 min, which could be attributed to the nucleation of monoclinic FePO4·2H2O and the transformation of the amorphous FePO4·2H2O. The curves are stable in zone 3 for the completion of transformation.

The Johnson–Mehl–Avrami (JMA) dynamics model was used for describing the phase transformation, as shown in equation 4. It was considered that the solution is well-mixed and the growth rate of crystals is independent of time. It assumed that the nucleation sites were located in the well-mixed reactant bases on solid-state reactions [37,38]. The calculated parameters were shown in the Table 6 and the fitting relevance value of R2 was over 0.97 indicating that the fitting result was great consistent with experimental results.

$$\ln x(t) = 1 - \exp\left\{-\mathbf{K} \times (t - t\_{\rm ind})^{\mathbf{n}}\right\} \tag{5}$$

where *x* is the percentage content of monoclinic FePO4·2H2O at reaction time *t*; *t*ind is the induction period of FePO4·2H2O transformation; K is the rate constant of transformation; n (called as Avrami exponent) is a constant related to the behaviors of nucleation and growth of FePO4·2H2O; if the value of n is over 1, the nucleation is the key factor for phase transformation or the migration of the chemicals to the nucleation point is dominant.

**Figure 11.** Raman spectra of slurries of monoclinic and amorphous FePO4·2H2O during the transformation.

**Figure 12.** The standard curve of monoclinic FePO4·2H2O.

**Figure 13.** In situ polymorphic mass fraction profiles of monoclinic and amorphous FePO4·2H2O through the solvent-mediated transformation process.

**Table 6.** Fitting parameters of JMA model at 90 ◦C.

*T* is the time required for transformation.

According to the spectra presented in Figure 13 and the Equation (5), the relationship of time and crystallinity and the parameters of JMA dynamics model were obtained, which were shown in Figure 14 and Table 6. The time required for transformation was 56.5 min and the induction period of this process was 101 min. Moreover, the concentration of Fe in the solution and the intensity of the corresponding characteristic peak at 303 cm−1 changed almost simultaneously: the concentration of Fe in solution decreased accompanying by the increase of the corresponding peak intensity with the time evolution (Supporting Information, Figure S2). It was indicated that the dissolution rate of amorphous FePO4·2H2O was slowest during phase transformation. Therefore, the dissolution rate of amorphous FePO4·2H2O is considered to be the control step of the transformation rate.

**Figure 14.** The percentage content of monoclinic FePO4·2H2O vs. time at different temperatures.

The experiments at different temperatures were carried out in order to evaluate the kinetics of the transformation process. The fitting results at different temperatures evaluated by the JMA model between temperature and the rate constant of transformation are shown in Table 7, and the changes trends of characteristic peak under different temperatures were presented in Figure S2 in Supporting Information. The activation energy of the transformation process was obtained by the Arrhenius equation, which was shown as Equation (6):

$$
\ln(\text{K}) = -\text{E}\_{\text{a}} \times \text{(R} \times T)^{-1} + \ln(\text{A})\tag{6}
$$

where *K* is the rate constant of transformation; R is the molar gas constant; *T* is the thermodynamic temperature; Ea is the apparent activation energy and A is the pre-exponential factor which is also called the frequency factor.

**Table 7.** Fitting parameters of JMA model at different temperature.

*T* is the time required for transformation.

The parameters of reaction calculated by the JMA model are listed in Table 7. With the increase of reaction temperature, the induction period and the time required for transformation decreased, and rate constant of transformation increased. Increasing temperature can enhance molecular movement and reduce the interfacial energy of solid–liquid interface. Therefore, the nucleation rate of monoclinic FePO4·2H2O was accelerated and correspondingly the induction period and the time required for transformation was shortened. The value of the apparent activation energy Ea in the transformation process can be derived by plotting the logarithm of *K* against 1/*T* and analyzing the slope and intercept (Figure S3 in Supporting Information). The value of the activation energy was calculated to be 9.619 kJ/mol.

#### **4. Conclusions**

The phase transformation from amorphous to monoclinic FePO4·2H2O was investigated. It was found that Fe and P are predominantly present as FeHPO4 + and FeH2PO4 2+ and the transformation process of FePO4·2H2O from amorphous form to monoclinic form was determined by the nucleation rate of the monoclinic form. The corresponding Raman spectroscopy results indicated that the induction period and the time required for transformation reduced as the reaction temperature rose. The kinetic parameters of the FePO4·2H2O transformation were calculated by the JMA model and the findings demonstrated that the transformation reaction constant increased as the reaction temperature increased. The activation energy of the transformation was 9.619 kJ/mol. These studies were beneficial to controlling and improving properties of FePO4 for meeting the requirements as potential electrode material precursors.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cryst12101369/s1, Table S1: The value of different parameter at pH range in equations; Table S2: The concentration of different ions at pH range; Table S3: The concentration of different ions at pH range; Table S4: The concentration of different ions at pH range; Table S5: The content of S and O in precipitation at different time; Figure S1: The mass fraction trend of Fe in solution at different time; Figure S2: Raman spectra of the precipitates at 70 °C (a), 75 ◦C (b), 80 ◦C (c), 85 ◦C (d), 95 ◦C (e); (f) the change of characteristic peak at 303 cm−1 over time under different temperature; Figure S3" The linear relationship between ln K and (1/T).

**Author Contributions:** Writing—original draft preparation, H.W.; writing—review and editing, H.W., M.G., Y.N., J.D. and L.Z.; visualization, H.W. and L.Z.; supervision, Q.Y. and L.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Natural Science Foundation of Tianjin, grant number 21JCY-BJC00600.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors are grateful for the financial support of the Special project for the transformation of major scientific and technological achievements of Guizhou Province.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Electroextraction of Ytterbium on the Liquid Lead Cathode in LiCl-KCl Eutectic**

**Zhuyao Li 1,2, Liandi Zhu 2, Dandan Tang 1,\*, Ying Dai 2, Feiqiang He 2, Zhi Gao 2, Cheng Liu 2, Hui Liu 2, Limin Zhou 2, Zhirong Liu 1,2 and Jinbo Ouyang 2,\***

**Abstract:** The reduction mechanisms of Yb(III) on W electrodes in molten LiCl-KCl-YbCl3 were explored at 773 K, and the diffusion coefficient of Yb(III) was determined. Then, various electrochemical techniques were employed to investigate the electroreduction of Yb(III) in molten LiCl-KCl on a liquid Pb film and Pb electrode. Electrochemical signals were associated with forming Pb3Yb, PbYb, Pb3Yb5, and PbYb2. The deposition potentials and equilibrium potentials of four Pb-Yb intermetallics were obtained through open-circuit chronopotentiometry. Metallic Yb was extracted by potentiostatic electrolysis (PE) on a liquid Pb electrode, and XRD analyzed the Pb-Yb alloy obtained at different extraction times. The recovered Yb was found in the form of Pb3Yb and PbYb intermetallics. The extraction efficiency of Yb was calculated according to ICP analysis results, and extraction effectivity could attain 94.5% via PE at −1.86 V for 14 h.

**Keywords:** electrochemical extraction; ytterbium; liquid Pb electrode; Pb-Yb alloy; electrochemical behavior

#### **1. Introduction**

The increasing demand of human beings for energy and the promotion of global low-carbon energy has increased the number of nuclear power stations in the world [1,2]. However, the management, storage, and disposal of used nuclear fuel (UNF) discharged from nuclear power plants has become a worldwide issue [3,4]. A 1000 MWe nuclear reactor generates about 25–30 tons of UNF annually [5,6]. Based on the International Atomic Energy Agency (IAEA) estimate, around 450 operating nuclear power plants have generated a global inventory of some 270,000 metric tons of UNF [7,8]. Pyroprocessing is one of the most promising technologies for treating UNF, and electrorefining is an essential unit operation in the pyroprocessing technique [9–12].

In electrorefining of UNF in molten LiCl-KCl, the standard potentials of lanthanide elements (Lns) are more negative than those of actinide elements (Ans) [13–15]. U and transuranic elements (TRUs) are deposited onto the cathode, and various lanthanide fission products contained in UNF dissolve in the molten salt. During the electrolysis process, Lns accumulate, which makes difficult the separation of Ans and Lns [16–23]. Therefore, proper electrode materials have a good effect on the separation process of Ans and Lns. Compared with the solid cathode, the liquid metal cathode has been widely studied because of its constant surface area and easy diffusion of deposits to liquid metal. Jiao et al. [24] summarized liquid metals from the aspects of properties, depolarization effects, alloy preparation, etc., such as liquid Bi [25–28], Pb [29–32], Sn [33–36], and Ga [37–40], which provided a reference for molten salt electrorefining.

In addition, lead has a low melting point (327 ◦C), and through evaluating the vapor pressures of various liquid metals, we discovered that the order of separating Ans and Lns from the liquid metal cathode via distillation is: Cd > Zn > Pb > Bi > Al > Ga > Sn. Liquid

**Citation:** Li, Z.; Zhu, L.; Tang, D.; Dai, Y.; He, F.; Gao, Z.; Liu, C.; Liu, H.; Zhou, L.; Liu, Z.; et al. Electroextraction of Ytterbium on the Liquid Lead Cathode in LiCl-KCl Eutectic. *Crystals* **2022**, *12*, 1453. https://doi.org/10.3390/ cryst12101453

Academic Editor: Stefano Carli

Received: 15 September 2022 Accepted: 11 October 2022 Published: 14 October 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Pb appears to be a proper candidate for electrode material in separating and extracting Ans and Lns from molten salt by electrowinning. After electrowinning, a Pb-Ans/Lns alloy can be formed on the liquid Pb electrode. Finally, the cathode deposits can be separated from the liquid Pb cathode by Pb distillation. Therefore, lead was employed as the cathode for ytterbium extraction in this work. In our previous work, liquid lead was used as a cathode due to the underpotential deposition of Dy(III) on the liquid Pb electrode. The reduction potential of Dy on the liquid Pb electrode is −1.35 V (vs. Ag/AgCl), which is more positive than that on the W electrode. Dysprosium was extracted from molten LiCl-KCl-DyCl3, and the extraction efficiency of dysprosium reached 97.2% [41,42]. Ytterbium is a variable valence element, one of the typical rare earth fission elements. Ji et al. [43] studied the electrochemical properties of ytterbium on liquid Zn electrodes in LiCl-KCl melts. Li et al. [44] studied the electrochemical behavior of Yb(III) on Cu electrodes in LiCl-KCl melts. Yan et al. [45] researched ytterbium and zinc co-deposition in LiCl-KCl-ZnCl2-YbCl3 melts on a W electrode. However, there is little information about the extraction of ytterbium from molten chlorides on liquid lead cathodes.

In the present study, different electrochemical techniques were used to explore the reduction mechanisms of Yb(III) on W, liquid Pb film, and liquid Pb electrodes. Moreover, the recovery of Yb from molten salt was executed by PE, and the products were characterized by SEM-EDS and XRD.

#### **2. Experimental**

All electrochemical measurements were carried out in an alumina crucible with LiCl-KCl (55.8 wt%) melts in an electric furnace. Yb was introduced into the LiCl-KCl melts as dehydrated YbCl3 powder (analytical grade). The reference electrode consisted of a silver wire (1 mm in diameter) immersed in a 1.5 wt% AgCl solution. A spectral pure graphite rod (6 mm in diameter) was used as a counter electrode. A tungsten wire (1 mm in diameter, 99.95%), a Pb film electrode (Pb coated on a W wire), and liquid lead (99%) were used for the working electrodes. PE and electrochemical measurements were performed (Metrohm, Ltd., Autolab PGSTAT 302N with Nova 1.10 software). The resistance between the reference electrode and the working electrode in molten salt was calculated by electrochemical impedance spectroscopy(EIS). The EIS was performed under the open circuit potential of the LiCl-KCl molten salt system under the condition of 10 mV amplitude over a 0.01-10000 Hz frequency range, as shown in Figure 1. According to Figure 1, the solution resistance(Rs) of the molten LiCl-KCl was 0.62 Ω de-termined at 773 K. After the ohmic potential(IR) compensation, cyclic voltammograms (CVs) were obtained. The phase composition of extraction products was characterized using XRD (Philips, Amsterdam, The Netherlands). The metal ion concentration in the melts was analyzed by ICP-AES (USA Thermo Elemental, IRIS Intrepid II XSP). Figure 2 shows the schematic setup of three-electrode electrochemical cells inside the furnace.

**Figure 1.** EIS data on the W electrode in molten LiCl-KCl at temperatures of 773 K.

**Figure 2.** The schematic setup of three-electrode electrochemical cells inside the furnace.

#### **3. Results and Discussion**

#### *3.1. Electrode Reaction of Yb(III) on the W Electrode*

Figure 3 shows the CVs attained in LiCl-KCl and LiCl-KCl-YbCl3 melts on the W electrode. The redox couples RA/OA (−2.39/−2.28 V) are considered as the redox of Li(I)/Li(0) on the W electrode. When YbCl3 is added, the newly added electrochemical signal RB/OB (−0.47/−0.32 V) corresponds to Yb(III)/Yb(II). No electrochemical signal of Yb(II) reduction to Yb was found, which indicates that the reduction potential of Yb(II) is more negative than that of Li (I) [43,44]. In this system, Yb(III) first becomes a low valence ion and is then oxidized to a high valence state. No metal Yb was obtained on the W electrode. They are thought to correspond to the following reaction.

$$\text{Yb}(\text{III}) + \text{e}^- \rightarrow \text{Yb}(\text{II}) \tag{1}$$

**Figure 3.** CVs obtained on the W electrode (*S* = 0.314 cm2) in the absence and presence of YbCl3 (1.55 × 10−4 mol cm−3) in eutectic LiCl-KCl.

Typical CVs of molten LiCl-KCl-YbCl3 recorded on the W electrodes at various scan rates are shown in Figure 4a. What can be observed is that with an increase in scan rate, the peak potential for RB was constant (Figure 4b). Therefore, it can be decided that the electrode reduction reaction RB is a reversible process on scan rate in a range of 0.08–0.28 V s−1. Figure 4b illustrates the linear relationship of *I*p and *v*−1/2 of Yb(III) in molten LiCl-KCl-YbCl3, indicating that the redox reaction is controlled by diffusion. In a soluble/soluble system, for the chemical reaction controlled by the mass transfer rate step, there is a relationship between the cathode peak current and the square root of the sweep rate, which can be described by Randles–Sevcik equation as follows [43]:

$$I\_{\rm P} = 0.4463(nF)^{3/2}(RT)^{-1/2}\text{SCD}^{1/2}v^{1/2} \tag{2}$$

where *S* represents the surface area of the working electrode, *C*0 represents the bulk concentration of the Yb(III) ion, *D* corresponds to the diffusion coefficient, *I* corresponds to the applied current, *n* corresponds to the number of exchanged electrons, *F* corresponds to the Faraday constant, and *v* represents the potential scan rate.

**Figure 4.** (**a**) CVs obtained on the W electrode at different scan rates in molten LiCl-KCl-YbCl3; (**b**) peak potential of the cathodic RB vs. log*v* derived from (**a**); peak current of the cathodic RB vs. the square root of scan rate derived from (**a**).

The calculated diffusion coefficient of Yb(III) at 773 K was 1.86 × 10−5 cm2 s−1. The diffusion coefficient of the Yb(III) ion in LiCl KCl molten salt calculated by Smolenski et al. at 848 K is 2.7 × 10−5 cm2 s−1, which is consistent with our research results [46].

Square wave voltammetry (SWV) was carried out to measure the number of electrons transferred during the reduction process of Yb(III) on the W electrode.

Figure 5a depicts the SWV curves gained in LiCl-KCl-YbCl3 molten salt at different frequencies. One reduction signal, RB (−0.47 V), correlated with the reduction process of Yb(III) to Yb(II). Furthermore, the shape of RB was flat and symmetrical, which indicates that the electrode reactions were reversible in a soluble/soluble system. The linear relationship between *I*pc and *f* 1/2 suggests that the reduction of Yb(III) on the W electrode was controlled by diffusion. Therefore, the number of electrons transferred was calculated from *W*1/2 using Equation (3) [42]:

$$W\_{1/2} = 3.52 \frac{RT}{nF} \tag{3}$$

where *W*1/2 is the half-peak width of the SWV curve, and the calculated number of electrons transferred from the reduction of Yb(III) to Yb(II) is close to 1 (Table 1).

**Figure 5.** (**a**) SWV curves for the reduction of Yb(III) on the W electrode at 773 K; pulse height: 100 mV; step potential: 5 mV; frequency: 5–40 Hz. (**b**) The linear relationship of *I*p versus *f* 1/2.

**Table 1.** Half-peak width and transfer electron number of Yb(III) calculated on a liquid Pb electrode in molten LiCl-KCl-YbCl3 under different frequencies.

#### *3.2. Electrochemical Properties of YbCl3 on a Pb Film Electrode*

The Pb film was prepared on the surface of the W electrode through PE at −1.0 V for 1 s. Figure 6 shows the CVs on the W electrode before and after adding YbCl3 and PbCl2 to LiCl KCl molten salt. The peaks RB and RC in the inset of Figure 6 are related to the reduction of Pb(II) to Pb and Yb(III) to Yb(II), respectively. Based on the binary phase diagram of the Pb-Yb system [47] shown in Figure 7, there are four intermetallics (Pb3Yb, PbYb, Pb3Yb5, and PbYb2) in the study temperature range. Therefore, four reduction peaks (RI, RII, RIII, and RIV) in the inset of Figure 6 correspond to four different Pb-Yb intermetallics. Pb(II) is first deposited on the W electrode to form a Pb film electrode. Then, Yb(II) is reduced at the corrected potential on the Pb film electrode due to depolarization to form Pb-Yb intermetallics. The reaction is as follows:

$$\text{Yb}(\text{II}) + 2\text{e}^- + \text{xPb} = \text{Pb}\_{\text{x}}\text{Yb} \tag{4}$$

**Figure 6.** CVs of blank eutectic LiCl-KCl (black line) and molten LiCl-KCl-YbCl3-PbCl2 salts (red line) on the W electrode at 773 K.

**Figure 7.** The binary phase diagram of the Pb-Yb system.

To confirm the attribution of the peaks, Figure 8 shows the SWV curves at different frequencies on the W electrode in molten LiCl-KCl-YbCl3-PbCl2. Six reductive peaks were observed clearly in Figure 8. Two reduction signals RB and RC pertain to the reduction of Yb(III) to Yb(II) and Pb(II) to Pb, respectively. Four reduction peaks RI, RII, RIII, and RIV are associated with four Pb-Yb intermetallics. The reduction peak potentials are listed in Table 2.

**Figure 8.** SWV curves of molten LiCl-KCl-YbCl3-PbCl2 on the W electrode at 773 K under different frequencies; potential step: 5 mV; frequency: 20-30 Hz.

**Table 2.** Summary of the results obtained on a Pb film electrode through CV, SWV, and OCP in molten LiCl-KCl-YbCl3.

To explore the electroextraction of Yb, open circuit chronopotentiometry (OCP) was used in LiCl-KCl-YbCl3-PbCl2 melts at various temperatures. Figure 9 displays the OCP curves measured on the Pb film electrode after deposition at −2.5 V for 100 s in molten LiCl-KCl-YbCl3-PbCl2 at 773–833 K.

**Figure 9.** OCP curves recorded on the W electrode in molten LiCl-KCl-YbCl3-PbCl2; deposition potential: −2.5 V; time: 100 s.

Plateau RB and RC were observed at −0.43 V and −0.33 V and were assigned to Yb(III)/Yb(II) and Pb(II)/Pb(0) redox couples, respectively. The four plateaus RI, RII, RIII, and RIV were correlated to the equilibrium of Pb-Yb intermetallics. We speculated that the potential plateaus should be related to the following equilibriums:

$$\text{Plateau } \mathrm{R}\_{\mathrm{A}} \text{ : Li}(\mathrm{I}) + \mathrm{e}^- \rightleftharpoons \mathrm{Li} \tag{5}$$

$$\text{Plateau R}\_{\text{I}} \colon \text{ 3Pb} + 2\text{e}^- + \text{Yb}(\text{II}) \rightleftharpoons \text{Pb}\_3\text{Yb} \tag{6}$$

$$\text{Plateau } \mathrm{R\_{II}} \colon \mathrm{Pb\_3Yb} + 4\mathrm{e}^- + 2\mathrm{Yb}(\mathrm{II}) \rightleftharpoons 3\mathrm{PbYb} \tag{7}$$

$$\text{Plateau } \mathsf{R}\_{\text{III}} \text{ : } \text{ 3PbYb} + 4\text{e}^- + \text{2Yb}(\mathsf{II}) \rightleftharpoons \mathsf{Pb}\_3\mathsf{Yb}\_5 \tag{8}$$

$$\text{Plateau } \mathsf{R}\_{\text{IV}}: \text{ Pb}\_3\mathsf{Yb}\_5 + 2\mathsf{e}^- + \mathsf{Yb}(\Pi) \rightleftharpoons \text{3PbYb}\_2 \tag{9}$$

$$\text{Plateau } \mathsf{R\_B} \text{ : } \mathsf{Yb(III)} + \mathsf{e}^- \rightleftharpoons \mathsf{Yb(II)} \tag{10}$$

$$\text{Plateau} \ R\_{\text{C}} : \text{Pb(III)} + 2\text{e}^- \rightleftharpoons \text{Pb Pb(II)} + 2\text{e}^- \rightleftharpoons \text{Pb} \tag{11}$$

#### *3.3. Electrochemical Property of Yb(III) Ions on the Liquid Pb Electrode*

Figure 10 is the cyclic voltammogram of LiCl-KCl-YbCl3 molten salt on a liquid Pb electrode. Signal C relates to the reduction of Pb(II) to Pb, and signal RB relates to the reduction of Yb(III) to Yb(II). The conclusion is consistent with that discussed above. This indicates that Yb(III) is first reduced to Yb(II) on the liquid Pb electrode, and then Yb(II) is reduced to Yb. One signal that appears at −1.71 V corresponds to the formation of a solid Yb(Pb) solution.

**Figure 10.** CVs obtained in LiCl-KCl-YbCl3 (1.55 × 10−4 mol cm−3) melt on liquid Pb cathodes (*S* = 0.949 cm2) at 773 K.

According to the CV results above, Yb was extracted by PE at −1.86 V on a liquid Pb electrode. Figure 11 shows the alloy's XRD pattern obtained by electrolysis at constant potential −1.86 V for 6, 10, and 14 h, respectively, on the liquid Pb electrode in molten LiCl-KCl-YbCl3 at 773 K. The alloy electrolyzed for 6 h was the Pb3Yb phase, the alloy electrolyzed for 10 h was the Pb3Yb phase, and the alloys electrolyzed for 14 h were Pb3Yb and PbYb phases. This shows that with the increase of electrolysis time, the content of Yb in liquid lead gradually increased, and part of Pb3Yb began to transform into the PbYb phase.

**Figure 11.** XRD patterns of the cathode products on a liquid Pb electrode in molten LiCl-KCl-YbCl3 salts through PE at −1.86 V for 14 h at 773 K.

After electrolysis for 14 h, the alloy was analyzed by SEM-EDS, and the SEM photograph and element distribution surface scan of the alloy were obtained, as shown in Figure 12. Two zones, dark and bright grey, were on the deposit's surface. From the results of the EDS mapping of the sample (Figure 12b), the element Yb mainly distributed in the dark grey zone. The upper salt was detected by inductively coupled plasma atomic emission spectrometry (ICP-AES). It was calculated that after electrolytic extraction for 14 h, the extraction rate of Yb was 94.5%.

**Figure 12.** SEM image (**a**) and EDS mapping (**b**) analysis of the deposit obtained by PE at −1.86 V on a liquid Pb electrode in molten LiCl-KCl-YbCl3 salts for 14 h at 773 K.

#### **4. Conclusions**

In this work, the electroreduction of Yb(III) ions on the W cathode was explored through CV and SWV. The electroreduction of Yb(III) to Yb(II) was found to be a diffusioncontrolled process with one electron exchanged, and the reduction of Yb(III)/Yb(II) was a reversible process. The diffusion coefficient of Yb(III) was 1.86 × 10−5 cm2 s−1. The electrochemical behavior of Yb(III) on a liquid Pb film cathode was explored by CV, SWV, and OCP. Then, equilibrium potentials and the deposition potentials of four Pb-Yb intermetallics (Pb3Yb, PbYb, Pb3Yb5, and PbYb2) were determined. The reduction of Yb(III) on liquid Pb and liquid Pb film electrodes was proven to be a two-step mechanism.

At last, the electrochemical extraction of Yb(III) on a liquid Pb electrode was studied by CV. Meanwhile, ytterbium was extracted on a liquid Pb electrode from a LiCl-KCl melt by PE at −1.86 V. The extraction product was detected by SEM-EDS and XRD. XRD analysis of the extracted products obtained at different electrolysis times shows that the extracted products gradually changed from a Pb-rich alloy (Pb3Yb) to a Yb-rich alloy (Pb3Yb and PbYb) with the increase in electrolysis extraction time. The extraction efficiency of Yb reached up to 94.5% by PE at −1.86 V for 14 h, which indicates that it is feasible to electrolytically extract Yb from LiCl-KCl melts on a liquid Pb electrode.

**Author Contributions:** Conceptualization, Investigation, Writing—original draft, Z.L. (Zhuyao Li); Investigation, Software, L.Z. (Liandi Zhu); Methodology, Resources, D.T. Supervision, Y.D.; Formal analysis, Investigation, F.H. and Z.G.; Data curation, C.L. and H.L.; Supervision, L.Z. (Limin Zhou) and Z.L. (Zhirong Liu); Project administration, J.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** The paper was financially co-supported by the National Natural Science Foundation of China (11905029, 22176032, and 11875105) and the Independent fund of State Key Laboratory of Nuclear Resources and Environment of East China University of Technology (2020Z19). Academic and technical leader training program for major disciplines in Jiangxi Province (20212BCJ23001).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

**Yuxiao Zhang, Xuntao Jiang, Xia Wu, Xiaoqiang Wang, Fang Huang, Kefei Li, Gaoyang Zheng, Shengzhou Lu, Yanxu Ma, Yuyu Zhou and Xiaoxi Yu \***

> State Key Laboratory of Heavy Oil Processing, College of Chemistry and Chemical Engineering, China University of Petroleum (East China), Qingdao 266580, China

**\*** Correspondence<EMAIL_ADDRESS>Tel.: +86-17606393651

**Abstract:** This study aimed to explore the effects of nucleate agent sizes on lysozyme crystallization. Silica nanoparticles (SNP) with four different particle sizes of 5 nm, 15 nm, 50 nm, and 100 nm were chosen for investigation. Studies were carried out both microscopically and macroscopically. After adding SNP, the morphological defects of lysozyme crystals decreased, and the number of crystals increases with the size of the SNP. The interaction between SNP and lysozyme was further explored using UV spectroscopy, fluorescence spectroscopy, and Zeta potential. It was found that the interaction between SNP and lysozyme was mainly electrostatic interaction, which increased with the size of SNP. As a result, lysozyme could be attracted to the surface of SNP and aggregated to form the nucleus. Finally, the activity test and circular dichroism showed that SNP had little effect on protein secondary structure.

**Keywords:** nanoparticle size; lysozyme crystallization; electrostatic interaction

#### **1. Introduction**

Single crystal X-ray diffraction technology [1] is a primary technical means to analyze the atomic-level structure of biological macromolecules. The successful application of Xray [2] crystallography in determining the 3-D structure of proteins relies on the cultivation of high-quality protein crystals [3–5]. However, protein crystallization is more complicated than small molecules due to the complexity and instability of protein molecules. Therefore, conducting in-depth research on the crystallization process of proteins is necessary.

Similar to small molecules, the nucleation process of protein crystallization [6,7] goes in two ways: homogeneous nucleation and heterogeneous nucleation [8]. Kordonskaya et al. [9–11] studied the growth of tetragonal crystals under homogeneous nucleation. It was found that lysozyme forms oligomers prior to crystal formation, which may be intermediates and can serve as growth units in crystal growth. Due to a high potential barrier of oligomer formation, homogeneous nucleation can only occur when the supersaturation in protein solution is high enough, which easily causes precipitates and is limited in low protein concentration systems. In contrast, when foreign substances exist as nucleating agents in the crystallization solution, the nucleation potential barrier will be significantly reduced due to protein adherence to the nucleating agent. As a result, heterogeneous nucleation could occur at relatively low protein concentrations. Therefore, it is beneficial for improving the quality of protein crystals and obtaining better diffraction data.

Nanomaterials refer to materials that have at least one dimension of nanometer size (1–100 nm) in three-dimensional space. Due to their excellent biocompatibility, nanomaterials have attracted much attention in studying the heterogeneous nucleation of proteins. Gold nanoparticles [12], platinum nanoparticles [13] and functionalized carbon nanoparticles [14] were proven to be effective in promoting protein crystallization and increasing crystal quality. Another commonly studied nanomaterial was silica nanoparticles (SNP),

**Citation:** Zhang, Y.; Jiang, X.; Wu, X.; Wang, X.; Huang, F.; Li, K.; Zheng, G.; Lu, S.; Ma, Y.; Zhou, Y.; et al. Silica Nanoparticles-Induced Lysozyme Crystallization: Effects of Particle Sizes. *Crystals* **2022**, *12*, 1623. https://doi.org/10.3390/ cryst12111623

Academic Editors: Jingxiang Yang and Xin Huang

Received: 31 October 2022 Accepted: 9 November 2022 Published: 12 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

which were also confirmed to assist protein crystallization. Compared with other nanomaterials, SNP is cheaper and easier to synthesize. Therefore, SNP has better application potential for the research and industrialization of protein crystallization.

The size of nanoparticles is an important factor affecting its properties. Peukert et al. carried out lysozyme crystallization using silica nanoparticles (SNP) ranging from 10 to 200 nm. The addition of SNP brought about a clear extension in the crystallization window, especially when larger seed particles were used [12]. Interestingly, the work of Delmas investigated the effect of silica particles with sizes from 230 nm to 698 nm and found that the optimal nucleation occurred when particles sizes were 432 nm [15]. Up to now, the mechanism of SNP size on inducing an effect on protein crystallization is still unclear. In addition, the different preparation methods of SNP [16,17] will also lead to differences in their surface properties, affecting the interaction between SNP and proteins. Therefore, it is necessary to deeply explore the effect of silica with different particle sizes on protein crystallization.

In this work, SNP with sizes of 5 nm, 15 nm, 50 nm, and 100 nm were utilized as the nucleating agent, and lysozyme was chosen as the model protein. The influences of SNP sizes on lysozyme crystallization were studied using crystal morphology in the first place. To further reveal the intermolecular interactions between SNP and lysozyme, UV spectroscopy, fluorescence spectroscopy, and Zeta potential were applied. Finally, activity tests and circular dichroism were carried out to determine the effect of SNP on the secondary structure and function of proteins.

#### **2. Materials and Methods**

#### *2.1. Materials*

Hen egg white lysozyme (HEWL) with a purity of 99% was purchased from Beijing Solarbio Technology of China (Beijing, China) and used directly without further purification. *Micrococcus lysodeikticus* (ATCC No. 4698, Manassas, VA, USA), used for the lysozyme activity assay, was purchased from Sigma-Aldrich. Analytical grade reagents, such as acetic acid, sodium hydrate, sodium chloride, and anhydrous sodium acetate, were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). SNP of different sizes were purchased from Shanghai Jiute Nano Material Technology Co. Ltd. (Shanghai, China). The sizes of SNP were evaluated using transmission electron microscopy (See Supplementary Materials Figure S1). The information of SNP is shown in Table 1. Ultrapure water was used in all experiments.

**Table 1.** Properties of the SNP used in this investigation.

#### *2.2. Lysozyme Crystallization*

Lysozyme crystallization experiments were carried out in 96-well plates. The concentration of each crystallization solution contained: 15 or 20 mg/mL lysozyme and 3 wt.% sodium chloride. SNP with four different sizes were used as the nucleating agent, respectively, and the concentration was kept at 1 mg/mL. All materials were dissolved in a pH = 4.5, 0.05 M sodium acetate buffer. After gentle mixing, the 96-well plates were sealed with parafilm to prevent evaporation and placed in a refrigerator atT=4 ◦C. After every 24 h, the plates were taken out and analyzed using a microscope.

#### *2.3. UV Spectroscopy Experiment*

UV spectroscopy was carried out using a lysozyme and SNP solution mixture. The UV spectrophotometer was preheated for 30 min before measuring. At ambient temperature, a UV cell with a 1 cm light path was selected to measure the UV absorption spectrum of the prepared solution in the 200–800 nm band. Sodium acetate buffer was used for calibration before measuring.

#### *2.4. Fluorescence Spectroscopy Experiment*

The samples containing 10 mg/mL lysozyme and SNP with different sizes were incubated for 24 h. Afterward, the solution was added to a 1 cm light path cuvette. The scan mode was set to Emission, and the data mode was selected as Fluorescence [18]. The fluorescence emission spectrum of lysozyme was carried out with an excitation wavelength of 280 nm and a slit of 3 nm.

#### *2.5. Zeta Potentials of Lysozyme and SNP*

Zeta potential was measured for both lysozyme and SNP solutions. The concentrations were all 5 mg/mL. A DTS1060 sample cell was used for the measurement. The measurement parameters were the Zeta interface and manual measurement, respectively. The measurements were repeated several times until the data became stable.

#### *2.6. Enzyme Activity Experiment*

Lysozyme activity was measured using *Micrococcus lysodeikticus* as the substrate. A cuvette with an optical path length of 1 cm and a volume of 4 mL was used. A 2.5 mL bacterial suspension with an absorbance value (at 450 nm) of about 1.3 was added to the cuvette. Then, 200 μL of lysozyme solution (0.1 mg/mL) was added to the cuvette and mixed immediately. The absorbance values A1 and A2 at 450 nm at 1 min and 2 min were recorded at 25 ◦C. The activity of lysozyme (0.1 mg/mL) and SNP solutions with different particle sizes was also evaluated.

The enzymatic activity *EA* was calculated by the following formula [19]:

$$E\_A = \frac{\Delta E\_{450\text{nm}}}{0.001 \times E\_W} \tag{1}$$

where Δ*E*450nm is the change of absorbance per minute at 450 nm, namely |A1 − A2|; *EW* is the mass of the original enzyme contained in the 0.5 mL detection enzyme solution, mg; 0.001 is a unit in which the absorbance drops; and *EA* is the specific activity of the enzyme, with a unit of U/mg.

#### **3. Results and Discussion**

#### *3.1. Effect of SNP Sizes on Lysozyme Crystal Morphology*

The microscopy images of the lysozyme crystals obtained are shown in Figure 1. Lysozyme crystals were analyzed at different times. Within 24 h, the solution without SNP did not form crystals (Figure 1a). While in crystallization solutions with SNP, tiny crystals formed (Figure 1b–e). When the crystallization time was extended to 48 h and 72 h, the crystals in solutions containing SNP continued to grow (Figure 1g–j,l–o). No crystals could be observed under homogeneous nucleation conditions until 72 h (Figure 1k).

It can be further seen from Figure 1 that the number of crystals changed regularly with the SNP sizes. The numbers of crystals were estimated by image processing and are displayed in Figure 2. The number of crystals increased rapidly within 48 h. The addition of SNP could promote lysozyme nucleation, which agrees with the work of Yamazaki et al. [20]. After 48 h, the increase in the number of crystals slowed down due to the decrease in supersaturation. During the crystallization time investigated, the number of crystals increased with the size of SNP. The size distribution of lysozyme crystals after 48 h and 72 h was estimated and is shown in Figures S2 and S3. It can be found that the particle size of SNP showed no significant effect on the size of lysozyme crystals within 48 h. After 72 h, the crystal size of lysozyme decreased with the increase in SNP size, due to the larger supersaturation consumed. Lysozyme crystallization was further carried out

at protein concentrations of 20 mg/mL with SNP of different sizes. A similar phenomenon was observed, as shown in Figure 3.

**Figure 1.** Microscopy photos of crystals obtained with SNP of different sizes. Conditions: 0.05 M, pH 4.5 sodium acetate buffer solution, 3 wt.% sodium chloride, 15 mg/mL lysozyme at 4 ◦C. Scale bar: 200 μm. (**a**): Crystal micrograph obtained under 24 h without SiO2; (**b**): Crystal micrograph obtained at 24 h with 1 mg/mL 5 nm SiO2; (**c**): Crystal micrograph obtained at 24 h with 1 mg/mL 15 nm SiO2; (**d**): Crystal micrograph obtained at 24 h with 1 mg/mL 50 nm SiO2; (**e**): Crystal micrograph obtained at 24 h with 1 mg/mL 100 nm SiO2; (**f**): Crystal micrograph obtained under 48 h without SiO2; (**g**): Crystal micrograph obtained at 48 h with 1 mg/mL 5 nm SiO2; (**h**): Crystal micrograph obtained at 48 h with 1 mg/mL 15 nm SiO2; (**i**): Crystal micrograph obtained at 48 h with 1 mg/mL 50 nm SiO2; (**j**): Crystal micrograph obtained at 48 h with 1 mg/mL 100 nm SiO2; (**k**): Crystal micrograph obtained under 72 h without SiO2; (**l**): Crystal micrograph obtained at 72 h with 1 mg/mL 5 nm SiO2; (**m**): Crystal micrograph obtained at 72 h with 1 mg/mL 15 nm SiO2; (**n**): Crystal micrograph obtained at 72 h with 1 mg/mL 50 nm SiO2; (**o**): Crystal micrograph obtained at 72 h with 1 mg/mL 100 nm SiO2.

**Figure 2.** Histogram of the number of crystals with SNP of different sizes as a function of time. Conditions: 0.05 M, pH 4.5 sodium acetate buffer solution, 3 wt.% sodium chloride, 15 mg/mL lysozyme at 4 ◦C.

In this work, lysozyme crystallization was carried out at 4 ◦C, in sodium acetate buffer solution near pH = 4.5 with 3 wt.% NaCl. The crystal formed at such conditions is considered to exhibit a tetragonal morphology [21–24]. As stated in other works, no significant effect on the structure of lysozyme crystals was discovered when nanoparticles such as gold particles or carbon quantum dots (GQDs) were used as nucleating agents [12,25]. Therefore, it is speculated here that the addition of nanoparticles may not alter the lysozyme crystal assembly process and that the lysozyme crystals obtained belong to a tetragonal morphology.

As shown in Figures 1 and 3, the crystals obtained with SNP are longer in the 110 face. Forsythe et al. [26] have experimentally demonstrated that the growth of different faces in lysozyme crystals is strongly dependent on the supersaturation of the protein, resulting in changes in the shape of the crystals. The 110-face of the lysozyme crystal grows faster with the increase in supersaturation. As further discovered in Figure S4, some agglomerates could be observed in solutions containing SNP of larger particle sizes and lysozyme. Sun et al. [27] found out that lysozyme could adsorb on SNP and the adsorption was related to the surface curvature of SNP. The adsorption experiments of lysozyme on SNP with different particle sizes proved that the saturated adsorption capacity of lysozyme increased with SNP sizes [28]. Therefore, it can be assumed that the addition of SNP plays a key role in protein aggregation, which improves the local concentration of lysozyme, and is beneficial to the growth of the 110-face.

In addition, as reported in previous work [28], lysozyme crystals could only be observed when the size of SNP reached 100 nm at a supersaturation of 4. Here in this work, lysozyme crystals were obtained with all sizes of SNP used at the same supersaturation, proving that the different properties of SNP could cause the difference in protein crystallization. Therefore, the mechanism of SNP inducing lysozyme crystallization needs to be further explored.

**Figure 3.** Microscopy photos of crystals obtained with SNP of different sizes. Conditions: 0.05 M, pH 4.5 sodium acetate buffer solution, 3 wt.% sodium chloride, 20 mg/mL lysozyme at 4 ◦C. Scale bar: 200 μm. (**a**): Crystal micrograph obtained under 24 h without SiO2; (**b**): Crystal micrograph obtained at 24 h with 1 mg/mL 5 nm SiO2; (**c**): Crystal micrograph obtained at 24 h with 1 mg/mL 15 nm SiO2; (**d**): Crystal micrograph obtained at 24 h with 1 mg/mL 50 nm SiO2; (**e**): Crystal micrograph obtained at 24 h with 1 mg/mL 100 nm SiO2; (**f**): Crystal micrograph obtained under 48 h without SiO2; (**g**): Crystal micrograph obtained at 48 h with 1 mg/mL 5 nm SiO2; (**h**): Crystal micrograph obtained at 48 h with 1 mg/mL 15 nm SiO2; (**i**): Crystal micrograph obtained at 48 h with 1 mg/mL 50 nm SiO2; (**j**): Crystal micrograph obtained at 48 h with 1 mg/mL 100 nm SiO2; (**k**): Crystal micrograph obtained under 72 h without SiO2; (**l**): Crystal micrograph obtained at 72 h with 1 mg/mL 5 nm SiO2; (**m**): Crystal micrograph obtained at 72 h with 1 mg/mL 15 nm SiO2; (**n**): Crystal micrograph obtained at 72 h with 1 mg/mL 50 nm SiO2; (**o**): Crystal micrograph obtained at 72 h with 1 mg/mL 100 nm SiO2.

#### *3.2. Zeta Potential of Lysozyme and SNP*

Zeta potential measurements of SNP with different particle sizes and lysozyme were further performed. Figure 4a shows that SNP with different particle sizes was negatively charged. With the increase in the SNP size, the absolute value of the potential increased. Figure 4b shows that lysozyme was positively charged. Therefore, there should be an electrostatic interaction between SNP and lysozyme, increasing with the size of the SNP.

**Figure 4.** (**a**) Zeta potential of SNP with different particle sizes. (**b**) Zeta potential of lysozyme.

#### *3.3. Effect of SNP Sizes on the UV Spectroscopy of Lysozyme*

The UV spectra of lysozyme and SNP with different particle sizes were measured. As shown in Figure 5A, the spectrum exhibited absorbance peaks at 220 nm and 280 nm, respectively, which were caused by the presence of lysozyme. The UV spectrum of SNP was measured and showed no absorbance in the measured wavelength range (Figure S5). For the convenience of observation and discussion, the spectrum from 200 to 350 nm was intercepted (Figure 5B). An apparent increase in absorbance occurred with the addition of SNP at the same lysozyme concentration. According to the Lambert–Beer law, the absorbance value protein is in positive correlation with its concentration. Hence, it is assumed here that the increase in absorbance was mainly caused by an increase in local concentration of lysozyme. Due to the electrostatic interaction between lysozyme and SNP, lysozyme could be attracted and gathered at the surface of SNP, leading to an increase in local lysozyme concentration in solution. With the increase in SNP sizes, the interaction between lysozyme and SNP was enhanced, and the amount of lysozyme absorbed was further increased, resulting in a higher UV intensity.

**Figure 5.** (**A**) UV spectrum from 200 nm to 800 nm. (**B**) UV spectrum from 200 nm to 350 nm. UV spectra of lysozyme solutions with and without SNP. From bottom to top: a, without SNP; b, 0.025 mg/mL 5 nm SNP; c, 0.025 mg/mL 15 nm SNP; d, 0.025 mg/mL 50 nm SNP; e, 0.025 mg/mL 100 nm SNP.

To verify the assumption above, the UV spectrum of pure lysozyme with different concentrations was measured (Figure S6). As shown in Figure S6, the UV absorbance increased with lysozyme concentration. The increasing trend and shape of the spectrum were consistent with Figure 5B. Hence, the presence of SNP can induce lysozyme aggregation and increase the local lysozyme concentration, which is more conducive to lysozyme crystallization.

#### *3.4. Effect of SNP Sizes on the Fluorescence Spectroscopy Experiment of Lysozyme*

Fluorescence experiments were carried out to further reveal the interaction between SNP and lysozyme. As shown in Figure 6, the fluorescence intensity of lysozyme increased after adding SNP of different sizes. For the same SNP size, the fluorescence intensity of lysozyme increased with the increase in SNP concentration. When the concentration of SNP was the same, the fluorescence intensity of lysozyme was higher after adding larger SNP.

**Figure 6.** Fluorescence spectra of lysozyme solutions with and without SNP. (**A**) 5 nm SiO2; (**B**) 15 nm SiO2; (**C**) 50 nm SiO2; (**D**) 100 nm SiO2. From bottom to top: a, no SNP; b, 1 mg/mL; c, 2 mg/mL; d, 3 mg/mL.

There may be two possible reasons for the enhancement in fluorescence intensity [29,30]. Due to the aggregation of lysozyme caused by SNP, the probability of non-radiative relaxation of the excited state was reduced. Meanwhile, the contact probability between oxygen molecules with a quenching effect in water and chromogenic groups was reduced. As a result, the fluorescence intensity was enhanced.

#### *3.5. Effect of SNP on Lysozyme Activity*

In order to clarify the influence of the SNP–lysozyme interaction on lysozyme structure and function, the lysozyme activity and secondary structure were further determined. The activity test results are shown in Figure 7, and the results from circular dichroism are displayed in Figure S7. It can be found that the addition of SNP had almost no effect on the secondary structure and function of lysozyme.

#### *3.6. Mechanism of SNP-Induced Lysozyme Crystallization*

Based on the results shown above, a mechanism of SNP-induced lysozyme crystallization is proposed and shown in Figure 8.

**Figure 8.** Mechanism of interaction between SNP with different particle sizes and lysozyme. (Note: the figure only shows the mechanism of a single SNP and lysozyme. There are multiple SNP and lysozymes in the specific crystallization process.).

Driven by electrostatic attraction, lysozyme adsorbs and aggregates on the surface of SNP, leading to an increase in lysozyme local concentration. As a result, the nucleation of lysozyme is easier when SNP is present. Moreover, the space occupied by each lysozyme molecule (asymmetric unit) in the crystal is a rectangular block with a size of 28.0 Å × 28.0 Å× 37 Å [31], which is smaller than the SNP sizes used here. It can be assumed that SNP only play the role of gathering lysozyme and do not participate in protein assembly. The initial nucleation conditions of lysozyme will not change by the addition of

SNP. With the increase in SNP size, the adsorption capacity of lysozyme increases, leading to a rise in the number of crystals.

#### **4. Conclusions**

The effects of SNP size on lysozyme crystallization were investigated. It was found that the morphological defects of lysozyme crystals were reduced after SNP addition. Further investigations showed that SNP can induce lysozyme aggregation and increase local supersaturation by electrostatic interaction, which increased with SNP size. As a result, lysozyme crystallizes easier with the addition of SNP, especially with SNP of larger size. Furthermore, the addition of SNP does not affect the secondary structure and function of lysozyme.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cryst12111623/s1, Figure S1. Transmission electron microscopy images of SNP with different particle sizes. (a): 5 nm SiO2, (b): 15 nm SiO2, (c): 50 nm SiO2, (d): 100 nm SiO2; Figure S2. Crystal size distribution of 0.05 M, pH 4.5 sodium acetate buffer solution, 3 wt. % sodium chloride solution, 15 mg/mL lysozyme at 4 ◦C for 48 h. (a) 5 nm SiO2, (b) 15 nm SiO2, (c) 50 nm SiO2, (d) 100 nm SiO2; Figure S3. Crystal size distribution of 0.05 M, pH 4.5 sodium acetate buffer solution, 3 wt. % sodium chloride solution, 15 mg/mL lysozyme at 4 ◦C for 72 h. (a) 5 nm SiO2, (b) 15 nm SiO2, (c) 50 nm SiO2, (d) 100 nm SiO2; Figure S4. (a): Corresponding microscope image after mixing 1 mg/mL 50 nm SiO2 and 20 mg/mL LSZ. (b): Corresponding microscope image after mixing 1 mg/mL 100 nm SiO2 and 20 mg/mL LSZ; Figure S5. UV spectra of silica with different particle sizes; Figure S6. UV spectra of lysozyme at different concentrations; Figure S7. CD spectra of lysozyme in the presence and absence of SNP.

**Author Contributions:** Conceptualization, Y.Z. (Yuxiao Zhang), X.Y., F.H. and X.W. (Xiaoqiang Wang); methodology, Y.Z. (Yuxiao Zhang), X.Y., F.H. and X.W. (Xiaoqiang Wang); investigation, Y.Z. (Yuxiao Zhang), X.J., X.W. (Xia Wu), Y.M., S.L., K.L. and G.Z., Y.Z. (Yuyu Zhou); writing—original draft preparation, Y.Z. (Yuxiao Zhang); writing—review and editing, X.Y., F.H. and X.W. (Xiaoqiang Wang) All authors have read and agreed to the published version of the manuscript.

**Funding:** The work was supported by the National Natural Science Foundation of China (22178387).

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Enhancing the Water Solubility of 9-Fluorenone Using Cyclodextrin Inclusions: A Green Approach for the Environmental Remediation of OPAHs**

**Yue Niu 1, Ling Zhou 1,\*, Huiqi Wang 1, Jiayu Dai 1, Ying Bao 1,2, Baohong Hou 1,2 and Qiuxiang Yin 1,2,\***

**Abstract:** Oxygenated polycyclic aromatic hydrocarbons (OPAHs) are toxic and carcinogenic compounds widely present in the natural environment, posing a serious threat to the environment and human health. However, the removal of OPAHs is mainly hindered by their low water solubility. Cyclodextrins (CDs) are frequently used to form inclusion complexes (ICs) with hydrophobic molecules to improve their solubility. In this study, we investigated the solubility enhancement ability of different CDs on 9-fluorenone, a common OPAH, through phase solubility experiments. We successfully prepared three solid ICs of 9-fluorenone with β-, hydroxypropyl-β-(HP-β-) and sulfobutylether-β-CD (SBE-β-CD) using the cooling crystallization method for the first time and characterized them via powder X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, etc. Molecular dynamics simulations were employed to investigate the binding modes and stable configurations of the ICs in the liquid phase and to explore the factors affecting their solubility enhancement ability. The results showed that all the CDs had a solubility enhancement effect on 9-fluorenone, with SBE-β-CD displaying the strongest effect, increasing the solubility of 9-fluorenone by 146 times. HP-β-CD, β-CD, α-CD, and γ-CD followed in decreasing order. Moreover, 9-fluorenone formed a ratio of 1:1 ICs to CDs. In addition, the interaction energy between SBE-β-CD and 9-fluorenone was the lowest among the CDs, which further validated the results of the phase solubility experiments from a theoretical perspective. Overall, this study provides a green method for the removal of 9-fluorenone pollutants in the environment and is expected to be applied to the removal and environmental remediation of other OPAHs.

**Keywords:** cyclodextrin inclusion complex; OPAHs; 9-fluorenone; solubilization; molecular dynamics simulation

#### **1. Introduction**

Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent and toxic organic pollutants that are commonly present in the environment [1]. Oxygenated polycyclic aromatic hydrocarbons (OPAHs) are derivatives of PAHs that contain at least one carbonyl oxygen on the aromatic ring and frequently coexist with PAHs in soils, air, and surface water [2]. OPAHs are primarily released into the environment through the incomplete combustion of solid fuels and the transformation of PAHs [3–5]. OPAHs are known for their high stability and resistance to degradation, and studies have found that OPAHs may exhibit stronger cytotoxicity and developmental toxicity than their parent PAHs [6,7]. OPAHs in the soil can accumulate in agricultural plants and be consumed by humans, while OPAHs in the atmosphere can be adsorbed onto particles and inhaled by humans [8,9]. These pollutants can cause allergies and even lead to cancer [10,11]. Therefore, the efficient

**Citation:** Niu, Y.; Zhou, L.; Wang, H.; Dai, J.; Bao, Y.; Hou, B.; Yin, Q. Enhancing the Water Solubility of 9-Fluorenone Using Cyclodextrin Inclusions: A Green Approach for the Environmental Remediation of OPAHs. *Crystals* **2023**, *13*, 775. https://doi.org/10.3390/ cryst13050775

Academic Editor: Waldemar Maniukiewicz

Received: 17 April 2023 Revised: 28 April 2023 Accepted: 4 May 2023 Published: 6 May 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

removal of PAHs and OPAHs from soil and air is an important environmental issue and a necessary step for sustainable development.

Although OPAHs have better water solubility compared to their parent PAHs, their low water solubility is still a major challenge in removing them from contaminated soils [12,13]. However, recent studies have suggested that cyclodextrins (CDs) and their derivatives hold great promise as agents for improving the solubility of these persistent organic pollutants [14–16]. CDs are cyclic molecules with a hydrophobic interior cavity and a hydrophilic exterior, which allows them to form inclusion complexes (ICs) with hydrophobic molecules, thus enhancing their solubility [17]. Natural CDs such as α-, β-, and γ-CD have been shown to form such complexes with organic molecules, as have modified CD derivatives such as hydroxypropyl-β-(HP-β-) and sulfobutylether-β-CD(SBEβ-CD) [18,19]. These modified derivatives exhibit significantly improved water solubility compared to natural CDs, thus providing a more effective means of increasing the solubility of insoluble molecules [20].

In recent years, numerous studies have demonstrated that the preparation of CD ICs can lead to the effective removal of PAHs from the environment. T. Badr and K. Hanna et al. [14] studied the solubilization and extraction effects of CDs on the organic pollutants naphthalene and phenanthrene in soil and confirmed the formation of PAH ICs. Inga Tijunelyte et al. [16] investigated the ability to encapsulate two PAHs, naphthalene and fluoranthene, in CDs' cavities. Fuat Topuz et al. [21] described a novel concept of PAH removal from aqueous solutions using CD-functionalized mesostructured silica nanoparticles and pristine mesostructured silica nanoparticles. However, there is currently a dearth of research on the use of CD ICs to remove OPAHs from the environment. Meanwhile, the solubilization mechanism of cyclodextrins for pollutants has received relatively little attention in research. Therefore, further investigation is highly necessary. Molecular dynamics (MD) simulations have emerged as a powerful tool for investigating the mechanism of CD ICs' formation, which allows for more intuitive information on how the ICs are bound at the molecular level [22]. Various methods have been employed for the preparation of ICs, including mechanochemical methods, cooling crystallization, ultrasonication, and freeze drying. Cooling crystallization has gained considerable attention due to its low energy consumption and the high purity of the resulting ICs. Recent advances in the field have led to the development of novel techniques such as supercritical anti-solvent and supercritical assisted atomization, which have also been explored for the preparation of ICs [23,24].

9-Fluorenone (Figure 1A), also known as diphenylene ketone, is a common smallmolecule OPAH and a frequently used fine chemical raw material [7]. 9-Fluorenone in the environment is usually derived from the combustion of solid fuels or from the oxidation of its parent compound, fluorene [25,26]. Found in the environment, it typically exhibits direct mutagenicity and stronger toxicity and persistence, often being widely detected in soils, groundwater, and the atmosphere [27–29]. Due to its poor solubility and limited bioavailability, it appears to be difficult to effectively remove from the environment [30]. Therefore, the preparation of ICs of 9-fluorenone with CDs can significantly enhance its solubility in water, thereby effectively removing the pollutants from the soil. Additionally, for 9-fluorenone that is already present in the effluent, it can be precipitated out through the formation of solid ICs, which leads to environmental restoration.

The purpose of this study was to investigate the solubilization effects of different CDs on 9-fluorenone. We prepared and characterized 9-fluorenone/CD ICs and elucidated the structural details of the complexes in aqueous solution through molecular dynamics simulations, identifying the optimal host molecule for improving the water solubility of 9-fluorenone. This information provides valuable support for the removal of 9-fluorenone from the environment and also offers a green method for the removal of other OPAHs.

**Figure 1.** Molecular structure of (**A**) 9-fluorenone, (**B**) α-CD, (**C**) β-CD, (**D**) γ-CD, (**E**) (2 hydroxypropyl)-β-CD, (**F**) sulfobutylether-β-CD.

#### **2. Materials and Methods**

#### *2.1. Materials*

9-Fluorenone was purchased from Tianjin Heowns Biochem LLC (purity 99%, Tianjin, China). α-CD (purity 99%, average MW 973 g/mol), β-CD (purity > 98%, average MW 1135 g/mol), γ-CD (purity > 98%, average MW 1297 g/mol), (2-hydroxypropyl)-β-CD (purity > 98%, average MW 1542 g/mol), and sulfobutylether-β-CD (purity > 98%, average MW 1451 g/mol) were purchased from Tianjin Hiensi Biochemical Technology Co. (Tianjin, China). Distilled–deionized water was formed in the laboratory using a NANOPURE system from BARNSTEAD (Thermo Scientific Co., Tianjin, China). All chemicals were used as received. Ethanol of analytical grade was from Tianjin Kewei Chemical Technique Co., Ltd. (Tianjin, China).

#### *2.2. Phase Solubility Experiments*

#### 2.2.1. Standard Curve Construction

9-Fluorenone was accurately weighed (5 mg) and dissolved in anhydrous ethanol to prepare a 0.1 mg/mL stock solution. Aliquots of the stock solution (2.0, 4.0, 6.0, 8.0, and 10.0 mL) were transferred into 25 mL volumetric flasks and diluted with anhydrous ethanol to the mark, with anhydrous ethanol as the reference blank. The solutions were shaken well and subjected to full-spectrum scanning analysis using a UV–visible spectrophotometer (UV-3600, Shimadzu, Kyoto, Japan). The absorption peak of 9-fluorenone appeared at 298 nm, while β-CD did not show any absorption peak at this wavelength. A standard curve was constructed by plotting the absorbance as the ordinate against the mass concentration (ρ) as the abscissa.

#### 2.2.2. Solubility Curve Construction

Solutions of various CDs with concentrations ranging from 0 to 10 mM (10 mL) were prepared. Excess 9-fluorenone was added to the CD solutions, and the resulting solutions were stirred at 300 rpm and 25 ◦C for 48 h. After reaching the solid–liquid equilibrium, the supernatants were filtered using a 0.45 μm aqueous syringe filter, diluted to a certain degree, and the absorbance values of the samples at 298 nm were measured using a UV– visible spectrophotometer. The solubility of 9-fluorenone in the different CD concentrations was calculated from the corresponding standard curve, and the solubility of fluorenone was plotted as the ordinate against the CD concentration as the abscissa to construct the solubility curve.

#### 2.2.3. Calculation of Apparent Stability Constants

The binding constants for each CD were calculated using the Higuchi–Connor method [31,32]. According to the Higuchi–Connor formula for the binding constant *K*:

$$K = \frac{Slope}{S\_0 \times (1 - Slope)}$$

where *Slope* represents the slope of the phase solubility curve and *S*0 represents the solubility of the guest molecule in water.

Meanwhile, the solubilization efficiency (*Se*) is calculated using the following equation:

$$S\_c = \frac{S\_{CD}}{S\_0}$$

where *SCD* is the apparent solubility of 9-fluorenone at a fixed concentration of 100 mM CD.

#### *2.3. Preparation of 9-Fluorenone/CD ICs*

#### 2.3.1. Solid Samples

For the different CDs, saturated solutions (200mL) were prepared at 50 ◦C. 9-Fluorenone (1:1 molar ratio of host to guest) was dissolved in 5 mL of ethanol (as a co-solvent) and added to the prepared aqueous solution of CDs. The mixed solution was mechanically stirred at 300 rpm for 6 h while avoiding exposure to light. Afterward, the solution was cooled down to 5 ◦C at a rate of 8 ◦C/h, resulting in a pale-yellow precipitate. The precipitate was washed with a small amount of ethanol, filtered, and dried for 12 h in a vacuum-drying oven at 40 ◦C.

#### 2.3.2. Physical Mixture

CDs and 9-fluorenone (at a ratio of 1:1) were lightly ground manually using a mortar and pestle for 10 min to obtain a homogeneous mixture.

#### *2.4. Characterization*

Crystal shapes were observed using an Olympus BX51 polarized light microscope (PLM, Olympus, Tokyo, Japan). Powder X-ray diffraction (PXRD) was performed with a D/MAX-2500 (Rigaku, Tokyo, Japan) using Cu-Kα radiation (0.15405 nm) in the 2θ between 2◦ and 40◦ with a scanning rate of 8◦/min. The applied voltage and current were 40 kV and 100 mA, respectively. Differential scanning calorimetry (DSC) curves were measured with a DSC 1/500 (Mettler Toledo, Co., Zurich, Switzerland) under the protection of nitrogen, and the temperature range was from 25 to 300 ◦C at a heating rate of 10 ◦C/min. Fourier transform infrared spectroscopy (FTIR) spectra were collected via the KBr tablet method using a Nexus Fourier transform infrared spectrometer (FTIR, Thermo Fisher, Waltham, MA, USA), focusing on the wavelength range of 4000 to 400 cm−1 at a resolution of 4 cm−1 under ambient conditions. The crystal morphology of the raw materials, physical mixture, and ICs were observed using scanning electron microscopy (SEM, TM3000, Hitachi, Tokyo, Japan).

#### *2.5. Molecular Dynamics (MD) Simulation*

MD simulations were carried out using GROMACS [33] to investigate the IC conformations in the solution. All molecular topology and structure files were obtained using Automated Topology Builder (ATB) [34]. The structural modifications of HP-β-CD and SBE-β-CD were obtained based on the β-CD framework acquired from ATB and further refined using Guessview6.0. The resulting structures were submitted to ATB for topology and molecular structure file generation via computation. Molecular modeling was performed using the open-source software Packmol [35]. The GROMOS96 force field was employed. The default steps and protocols of the MD were selected to optimize the system with an equilibrium of 100ps, and the production run was carried out for 10ns (NPT ensemble)

in triplicate with a step of 2 fs at constant temperature (323K). The energies and guest dispositions were registered every 1000 steps. VMD [36] software was utilized to visualize the simulation results, ensuring that the outcomes were easily interpretable.

#### **3. Results and Discussion**

#### *3.1. Phase Solubility Study*

A standard curve equation was obtained by fitting the absorbance values of 9-fluorenone with the mass concentration, which is depicted in Figure 2A. The equation was A = 13.6875 m + 0.015, with a high fitting accuracy of R2 = 0.99986. The solubility curves of the various CDs at 25 ◦C are presented in Figure 2B, and the fitting curves and apparent stability constants K of the ICs were calculated and are listed in Table 1 and Table S1. The results indicated that increasing the concentration of CDs could linearly enhance the solubility of 9-fluorenone, and the enhancing capacity order was SBE-β- > HP-β- > β- > α- > γ-CD. Based on the Higuchi–Connors classification [37], the solubility curves belong to the AL type, which indicates that the stoichiometric ratio of 9-fluorenone and CDs in the liquid state was 1:1. The highest K value was detected for 9-fluorenone/SBE-β-CD (1528 M−1), followed by 9-fluorenone/HP-β-CD (799 M−1) and 9-fluorenone/β-CD (346 M−1). Notably, γ-CD showed almost no significant solubility-enhancing effect on 9-fluorenone, possibly due to insufficient intermolecular interaction forces between the γ-CD and 9-fluorenone molecules, resulting in weak inclusion effects. The solubility of 9-fluorenone was significantly increased by approximately 17-fold, 37-fold, 82-fold, 146-fold, and 2-fold, respectively, after forming complexes with α-, β-, HP-β-, SBE-β-, and γ-CD, as shown in Table 1. These findings suggest that natural CDs and their derivatives can significantly enhance the solubility of hydrophobic guest molecules, especially SBE-β-CD, which exhibits a more significant improvement in the stability and solubility of 9-fluorenone than natural CDs.

**Figure 2.** (**A**) Standard UV curve of 9-fluorenone. (**B**) The phase solubility curve of 9-fluorenone/CDs.

**Table 1.** Apparent stability constants (K, M−1) and solubilization efficiency (Se) for the 9 fluorenone/CD ICs.

*3.2. Characterization of 9-Fluorenone/CD ICs*

Solid ICs of 9-fluorenone with β-CD, HP-β-CD, and SBE-β-CD were prepared using the cooling crystallization method. However, the solids of 9-fluorenone with α-CD and γ-CD obtained using the same method were determined to be physical mixtures after analysis, which may be due to their weaker binding ability in the liquid state. Figure S1 shows photographs of all the prepared ICs. As 9-fluorenone has a yellow crystalline form, the liquid ICs exhibit varying degrees of yellow coloration depending on their solubility. The solid ICs obtained are also pale-yellow powders.

#### 3.2.1. Powder X-ray Diffraction

The PXRD patterns of 9-fluorenone, the CDs, physical mixtures, and ICs are presented in Figure 3. The diffraction peaks observed in the physical mixtures of all three CDs represent an overlay of the 9-fluorenone and CD spectra. The XRD pattern of the 9 fluorenone/β-CD IC shows new distinct diffraction peaks at 2θ = 4.9◦, 7.1◦, 7.5◦, 10.0◦, 14.0◦, and 21.5◦, which indicate that a new crystalline structure was formed. The disappearance of some of the characteristic peaks of β-CD and 9-fluorenone in the IC pattern suggests that the two compounds underwent a molecular rearrangement. The XRD pattern of 9-fluorenone/HP-β-CD IC shows two broad peaks at 2θ = 9.5◦ and 18.1◦, which differ from those observed in HP-β-CD and the physical mixtures. The disappearance of the crystalline peak corresponding to 9-fluorenone in the X-ray diffraction pattern indicates the formation of an inclusion complex between 9-fluorenone and HP-β-CD. The complexation process leads to the amorphization of the guest molecule upon encapsulation within the CD cavity [38]. The amorphous state observed in the XRD pattern of 9-fluorenone/SBE-β-CD IC may indicate that 9-fluorenone is either present on the surface of SBE-β-CD or is present in a disordered state within the cavity of SBE-β-CD. Overall, the XRD results confirm the formation of solid ICs and provide evidence of the structural changes that occurred during complexation.

#### 3.2.2. Fourier Transform Infrared Spectroscopy

Figure 4 shows the FTIR spectra of 9-fluorenone, the CDs, their physical mixtures, and the ICs. The FTIR spectrum of 9-fluorenone displays characteristic peaks at 667 cm−1, 725 cm−1, 758 cm−1, 915 cm−1, 1086 cm−1, 1297 cm−1, 1450 cm−1, 1592 cm−1, and 1710 cm−1. The peaks observed at 1450 cm−1 and 1592 cm−1 correspond to the stretching and bending vibrations of the benzene ring skeleton, respectively. The peak observed at 1710 cm−1 is attributed to the stretching vibration of the C=O bond [39]. The peaks observed at 667 cm−1 and 725 cm−1 are attributed to the bending vibrations of the =C-H bond [40]. A significant and broad absorption band is observed at 3280 cm−1 in the spectrum of β-CD, which is attributed to the stretching vibration of the O-H bond of the hydroxyl group. A small characteristic peak is observed at 2925 cm−1, which is attributed to the asymmetric stretching vibration of -CH2. The spectrum of HP- exhibits a characteristic peak of -CH3 stretching vibration at 2930 and 2837 cm−1. In the spectrum of SBE-CD, the peak observed at 1153 cm−1 is attributed to the stretching vibration of the S=O bond [41]. Distinct differences in characteristic peaks were observed upon comparing the spectra of the ICs and physical mixtures composed of compounds with the same dose ratio, indicating the successful preparation of ICs. In the spectrum of β-IC, the presence of C=O (1710 cm−1) and C=C (1592 cm−1) indicates that the basic skeleton of the 9-fluorenone structure has not changed. A comparison of the infrared spectra of the IC and prednisolone did not reveal any new peaks, indicating the absence of new chemical bonds. The presence of an -OH peak in the region of 3600 to 3100 cm−1 in the IC spectrum is due to the abundance of hydroxyl groups in β-CD. Many typical, characteristic peaks of 9-fluorenone are masked by the characteristic peaks of β-CD, indicating that 9-fluorenone is encapsulated within the cavity of β-CD. The -OH absorption band is observed to shift from 3290 to 3261 cm−1, a redshift phenomenon, indicating the formation of new hydrogen bonds between 9-fluorenone and β-CD [42].

Similarly, the -OH absorption band of HP-β-IC shifted from 3347 to 3310 cm−1, and that of SBE-β-IC shifted from 3372 to 3353 cm−1. This also indicates the formation of hydrogen bonds between 9-fluorenone and CDs. The intensity of the peak at 1710 cm−1 in the IC spectrum is also weak compared to that in the spectrum of 9-fluorenone alone, suggesting that the C=O bond in 9-fluorenone is involved in the inclusion complexation with CDs. The formation of hydrogen bonds between CDs and 9-fluorenone molecules can improve the solubility and stability of 9-fluorenone in aqueous solution by reducing the intermolecular attractive forces between 9-fluorenone molecules and enhancing the intermolecular interaction forces between 9-fluorenone and water molecules. Overall, the FTIR spectra provide strong evidence for the successful formation of ICs and the involvement of hydrogen bonding in the inclusion process, confirming the results of the solubility studies.

**Figure 3.** Powder X-ray diffraction pattern of 9-fluorenone, CDs, physical mixtures, and ICs.

**Figure 4.** FTIR spectra of 9-fluorenone, CDs, physical mixtures, and ICs.

3.2.3. Differential Scanning Calorimetry

The DSC thermal spectra of 9-fluorenone, CDs, physical mixtures, and ICs are presented in Figure 5 (For detailed thermal analysis data see Table S2). The DSC curve of 9-fluorenone exhibits an endothermic peak at approximately 86 ◦C due to its melting. The endothermic peak of β-CD corresponds to the release of water from its cavity at approximately 65 ◦C. In the DSC curve of the β-CD IC sample, the melting peak of 9-fluorenone vanishes due to the formation of the IC, and the endothermic peak represents the evaporation of water in the IC, resulting in an increase in the dehydration temperature to approximately 75 ◦C [43]. In the DSC curve of HP-β-CD, two endothermic peaks emerge, corresponding to the evaporation of water and the melting temperature of the CD, respectively. In HP-β-CD IC, the dehydration temperature increases to approximately 70 ◦C, while the melting peak of HP-β-CD disappears, which might indicate that the melting point of the IC rose to above 300 ◦C. A similar situation occurs in SBE-β-CD IC, except that the dehydration temperature is lower than that of SBE-β-CD. The DSC spectra do not reveal the presence of 9-fluorenone, providing additional evidence that 9-fluorenone was fully incorporated into the IC. These findings demonstrate that the thermodynamic behavior of 9-fluorenone can be altered by combining it with different types of CDs.

**Figure 5.** DSC thermogram of 9-fluorenone, CDs, physical mixtures, and ICs.

3.2.4. Scanning Electron Microscopy and Polarized Light Microscopy

The SEM images provide valuable information about the morphology and particle size of the materials. Figure 6A demonstrates that pure 9-fluorenone exists as blockshaped crystals, whereas the morphology of the CDs varies depending on the type of CD. Specifically, β-CD appears as a particulate structure with a relatively small particle size, while HP-β-CD and SBE-β-CD exhibit spherical particulate structures. Both 9-fluorenone and CDs are present alone in the physical mixtures, according to Figure 6B, and they do not combine to produce a new solid form. The SEM images of β-CD IC show that its crystal morphology is one of massive, aggregated particles with a more uniform particle size distribution. The SEM images of HP-β-IC and SBE-β-IC show that they are both massive structures with a large particle size. Additionally, their morphology is completely different from the original, spherical, amorphous structure of CDs. Overall, these SEM images demonstrate the differences in morphology and structure between the ICs and their corresponding raw materials.

**Figure 6.** SEM images of (**A**) raw materials, (**B**) physical mixtures, and (**C**) ICs.

3.2.5. Polarized Light Microscopy

The crystal morphology of 9-fluorenone/β-CD IC in the liquid and solid states was observed using optical microscopy (Figure 7). It can clearly be seen that the IC is a rectangular, sheet-like crystal in the liquid state. When solid inclusion occurs, the flake crystals agglomerate together to form a granular product due to the small particle size, which is consistent with the SEM observation.

**Figure 7.** Light micrographs of 9-fluorenone/β-CD IC: (**A**) in the liquid state and (**B**) in the solid state (a drop of cedar oil for better observation).

#### *3.3. Molecular Dynamics Simulations*

To investigate the binding mode of 9-fluorenone with the CDs in solution, MD simulations were performed. A water box of 4 × 4 nanometers was established, and the 9-fluorenone and CD molecules were placed inside it. Two initial position settings were chosen for each CD system to study the binding mode of the guest and host molecules (Figure 8). The final structures of different CD systems are presented in Figure 9. During the simulation process, the 9-fluorenone molecule could easily enter the CD cavity of α-, β-, and SBE-β-CD, regardless of the initial position settings. However, for HP-β-CD and

γ-CD, 9-fluorenone could only enter the CD cavity from a specific direction. In the case of HP-β-CD, the substitution of the hydroxyl group at the CD's primary hydroxyl end affects the entry of the guest molecule; hence, it can only enter the CD molecule through the secondary hydroxyl end. Meanwhile, γ-CD's cavity size is significantly larger than that of the 9-fluorenone molecule, which prevents it from stably encapsulating the guest molecule (Figure S2). This explains the poorer solubilization effect of γ-CD. These findings provide insights into the binding mechanisms of CDs with guest molecules in solution.

**Figure 8.** Two conformations of the initial position setting of 9-fluorenone toward the head (H) or tail (T) of different CDs.

**Figure 9.** The final structure of 9-fluorenone/CDs based on simulations at two initial positions. (For clearer viewing, the C, O, and H atoms of 9-fluorenone are shown in purple, red, and silver, respectively. The C and H atoms of the CDs are shown in white, the S atom in yellow, and the Na+ in blue).

Through MD simulations, we determined the stable configuration of the 9-fluorenone/ CD ICs by comparing the binding energies between the host and guest molecules (Figure 10). The 9-fluorenone molecule was found to be upright inside the cavity of α-, β-, and SBE-β-CD, whereas it was tilted in HP-β-CD. The ICs were stabilized by hydrogen bonds formed between the 9-fluorenone molecule and the O or H atoms inside the CD cavity. Figure 11

illustrates the simulation results of the lower interaction energy for each CD at different initial positions. The root-mean-square deviation (RMSD) of the system was calculated to estimate the equilibration of the system (Figure 11A), and it was observed that each system reached equilibrium before 500 ps of the MD simulation, indicating that the simulation time was sufficient for each system. The number of hydrogen bonds formed between the 9-fluorenone molecule and the CD molecule is illustrated in Figure 11B. During the binding process with α-, β-, and SBE-β-CDs, the 9-fluorenone molecule formed one or two hydrogen bonds, whereas with HP-β-CD, it formed three hydrogen bonds at certain times, which may be attributed to the orientation of the 9-fluorenone molecule inside the cavity. This is consistent with the red shift of the hydroxyl peaks in the FTIR spectrum. It was found that hydrogen bonds were not the absolute factor affecting the water solubility of the ICs but were important for the stable existence of the structure of the ICs. The interaction energy between the host and guest molecules at different initial positions is shown in Figure S3. Van der Waals interactions contribute far more to the total interaction of the IC system than electrostatic interactions [44]. The collective interaction energies between the host and guest molecules is presented in Figure 11C. Among the four CDs, the interaction energy between SBE-β-CD and the 9-fluorenone molecule is the lowest, with an average value of approximately 120 kJ/mol. This indicates that the complex structure of SBE-β-CD and 9-fluorenone is the most stable among the four CDs, which also explains why it has the strongest solubilization ability. Finally, the solvent-accessible surface area (SASA) of the 9-fluorenone molecule was analyzed during the simulation. The SASA of 9-fluorenone could be seen to decrease and then remain stable, indicating that the guest molecule was encapsulated, thereby reducing its area of contact with the solvent. After reaching the equilibrium state, the SASA of 9-fluorenone was almost identical after its inclusion in the different CDs, indicating that each type of CD can stably contain 9-fluorenone molecules within its cavity to an almost equal extent. In the MD experiments, we did not search for signs of the unstable formation of α-CD with 9-fluorenone in the liquid state; hence, the reason for its inability to form a solid IC may be related to the mechanism of supramolecular self-assembly during crystallization, and further studies are needed.

**Figure 10.** Stable conformation and hydrogen bonding (blue dotted line) of 9-fluorenone with CDs.

**Figure 11.** MD results: (**A**) RMSD of the system with the simulation time (ps); (**B**) the number of hydrogen bonds during the simulation; (**C**) total interaction energy between 9–fluorenone and CDs; (**D**) solvent-accessible and surface area (SASA) values of 9-fluorenone with the simulation time (ps).

#### **4. Conclusions**

In this study, we utilized phase solubility analysis to investigate the solubilization effects of different CDs on 9-fluorenone. Our results revealed significant differences in the solubilization efficiency of the different CDs towards 9-fluorenone. Specifically, SBE-β-CD exhibited the highest solubilization ability, with an apparent stability constant of 1527 M−1, followed by HP-β-CD, β-CD, and α-CD, while γ-CD showed the weakest solubilization efficacy. Additionally, it was confirmed that ICs were formed between 9-fluorenone and the CDs in a 1:1 ratio in the liquid phase. We successfully prepared three solid ICs of 9 fluorenone using the cooling crystallization method and characterized them using various techniques, including PXRD, FTIR, DSC, and SEM. MD simulation was employed to investigate the binding modes between 9-fluorenone and the CDs in the liquid phase. Our results revealed that the binding between SBE-β-CD and 9-fluorenone was the most stable, which was consistent with the findings of the phase solubility experiments. Hydrogen bonding and van der Waals interactions were found to play crucial roles in stabilizing the ICs. Overall, our study provides theoretical and experimental support for the design and preparation of ICs of OPAHs in the environment and offers a green and environmentally friendly method for their removal from soil. Furthermore, the successful production of solid ICs provides guidance for the removal of these pollutants from water.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/cryst13050775/s1, Figure S1: Photographs of the prepared 9-fluorenone/CD ICs in (A) the liquid state and (B) the solid state; Figure S2: Binding process of 9-fluorenone with γ-CD; Figure S3: The interaction energy of host and guest molecules of different conformations; Table S1: Data and fitted equations for 9-fluorenone at different CD concentrations; Table S2: Thermal analysis data.

**Author Contributions:** Conceptualization, Y.N.; methodology, Y.N.; validation, L.Z., H.W. and J.D.; formal analysis, Y.N.; data curation, H.W. and L.Z.; writing—original draft preparation, Y.N.; writing review and editing, Y.N., L.Z. and Q.Y. visualization, B.H. and Y.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors are grateful for the financial support of the Tianjin Municipal Natural Science Foundation (No. 21JCYBJC00600).

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

## *Article* **The Influence of Different Types of SiO2 Precursors and Ag Addition on the Structure of Selected Titania-Silica Gels**

**Anna Adamczyk**

Material Science and Ceramics Department, AGH University of Science and Technology, Al. Mickiewicza 30, 30-059 Kraków, Poland<EMAIL_ADDRESS>
**Abstract:** In this paper, samples of titania-silica system were obtained by the sol-gel method as bulk materials, using titanium propoxide Ti(C3H7O)4 to introduce titania and two precursors of SiO2: TEOS tetraethoxysilane Si(OC2H5)4 and DDS dimethyldiethoxysilane (CH3)2(C2H5O)2Si. To enhance antibacterial properties, Ag was added to gels of selected compositions. The main aim of the performed studies was to find the correlations between the structural changes and the applied precursor of silica. Simultaneously, the influence of different compositions of gels and the addition of Ag on the specimens' structure was investigated. To study the structure, two complementary methods, FTIR (Fourier Transform InfraRed) spectroscopy and X-ray diffraction, were applied. The analysis of the FTIR spectra and the XRD patterns made it possible to confirm the amorphous state of all dried gels and establish the presence of TiO2 polymorphs: anatase and rutile in all annealed samples. The addition of Ag atoms into the gels caused the crystallization of cristobalite phase in addition to titania polymorphs. The presence of crystalline Ag phase in the annealed gels allowed the calculation dimensions of Ag crystallites based on the Scherrer equation. The use of DDS as a silica precursor led to easier and faster crystallization of different TiO2 phases in the annealed samples and parallel increases in the depolymerization of silica lattice.

**Keywords:** TiO2-SiO2 system; sol-gel method; silica precursors; FTIR spectroscopy; X-ray diffraction

#### **1. Introduction**

Titania-silica materials are widely applied in many fields because of their unique properties and usually simple preparation. They exhibit high self-cleaning and deodorising abilities [1–5], and possess good mechanical properties as well as high thermal stability [6,7]. Materials of TiO2-SiO2 systems can be applied in the form of monoliths or as coatings. As TiO2-SiO2 coatings, they can be anticorrosive and/or antibacterial layers [8]. More importantly, such thin films can be used in the field of human health protection because of their bioactivity, biocompatibility and antibacterial as well as anticorrosive behaviours [9–11].

Studies of the structure of TiO2-SiO2 samples have shown that they mainly consist of TiO2 molecules dispersed in a silica matrix. When titania concentration is low, one can observe TiO2 microcrystals that grow and thus cause the depolymerization of silica lattice during annealing [3,12]. These small titania crystals can act as crystallisation nuclei and decrease the temperature of crystallization in all phases of a sample.