β-Cyclodextrin Stationary Phase Using Tetrafluoroterephthalonitrile as Spacer Arm for Separation of Phenolic Compounds

β-Cyclodextrin Stationary Phase Using Tetrafluoroterephthalonitrile as Spacer Arm for Separation of Phenolic Compounds | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article β-Cyclodextrin Stationary Phase Using Tetrafluoroterephthalonitrile as Spacer Arm for Separation of Phenolic Compounds Li Jiang, Le Duan, Yan Teng, Ineza Urujeni Gisèle, Geyuan Li, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7475722/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In the field of chromatographic separation, high-performance liquid chromatography (HPLC) is widely used in the analysis of complex samples due to its high separation efficiency, fast analysis speed and wide application range. The core goal of the approach was to develop a more efficient chromatographic stationary phase. Although cyclodextrin chromatographic stationary phase has been reported previously, there are various challenges that prevent it from becoming the mainstream chromatographic stationary phase, such as low bonding amount, complicated synthesis process and poor separation effect. In this paper, a novel β-cyclodextrin chromatographic stationary phase was prepared with polysubstituted tetrafluoroterephthalonitrile (TFTPN) as a spacer arm for the separation of phenolic compounds. The chromatographic results showed that it had excellent chromatographic performance, and the mechanism of action was explained by computer simulation. This study provides new insights for the separation and analysis of structural analogues. tetrafluoroterephthalonitrile chromatographic stationary phase β-cyclodextrin column efficiency hydrophobic cavities Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction High-performance liquid chromatography (HPLC) is one of the most effective techniques to separate structural analogues due to its advantages of rapidity, high performance, wide separation range, and more improved fittings[ 1 ]. For high-performance liquid chromatography, separation of structural analogues can be achieved directly by using stationary phase, mobile phase additives, or indirectly by pre-column derivatization[ 2 – 4 ]. Cyclodextrins have many chiral centers[ 5 ] and hydrophobic cavities[ 6 ], which have high recognition ability for structural analogues, so they are often used in the preparation of chromatographic stationary phases[ 7 – 9 ]. Common commercial cyclodextrin columns and their analytical application are systematically summarized in the table S1 . Although cyclodextrin has been introduced into the preparation of HPLC stationary phase, it still has some problems such as low bonding amount, slow recognition speed and limited recognition ability. These factors restrict cyclodextrin chromatographic stationary phase from becoming mainstream and commercialized chromatographic stationary phase. In this paper, β-cyclodextrin is bonded to the surface of SiO2 microspheres carrier using tetrafluoroterephthalonitrile (TFTPN) as a spacer arm to obtain β-cyclodextrin chromatographic stationary phase for the separation of structurally similar phenol, 1-naphthol, and 2-naphthol mixtures. Taking advantage of the high reaction activity of TFTPN[ 10 ] and the ability to combine multiple β-cyclodextrin molecules[ 11 – 14 ], the problem of low cyclodextrin bonding amount prevalent in traditional cyclodextrin chromatographic stationary phases[ 15 ] has been solved. Besides, this novel cyclodextrin chromatographic stationary phase has abundant pore structure, which allows rapid recognition. Moreover, the novel β-cyclodextrin chromatographic stationary phase can provide various interaction sites such as π-π interaction, hydrogen bonding and cyclodextrin hydrophobic cavities, improving the selectivity for phenolic structural analogues. We investigated the influence of mobile phase composition, proportion, pH value, injection volume and other factors on the separation of phenolic compounds with similar structure, and evaluated the column efficiency, resolution, repeatability and the life span of β-cyclodextrin chromatographic column. In this study, the original traditional cyclodextrin stationary phase was improved, providing more options in the field of high-performance liquid chromatography. 2 Materials and Instruments 2.1 Materials All analytical grade chemical reagents were purchased from Shanghai Aladdin Biochemical Technology Co., LTD (China). They were N, N-dimethylformamide, β-cyclodextrins, potassium carbonate, tetrahydrofuran, ammonia, phenol, α- naphthol, and 2-naphthol. Tetrafluorophenonitrile (> 99%), tetraethyl silicate (reagent grade, 98%), 3-aminopropyl triethoxysilane (98%) were also purchased from here. SiO 2 microspheres was purchased from Tianjin Beilux. Methanol was obtained from Shanghai Xingke High purity Solvent Co., LTD. Acetonitrile was supplied by Shanghai Hutest Laboratory Equipment Co., LTD. 2.2 Instruments High performance liquid chromatography (LC-20AB), vacuum drying oven (DZF-6021), FT-IR spectrometer (TTIR-8400s) were purchased from Shimadzu, Japan. Elemental analyzer (PE 2400 series Ⅱ) was supplied by Beijing Jing ke Rui da technology Co., LTD. Heat collecting type magnetic (DF-101S) was obtained from Nanjing jiameilun scientific instrument Co., LTD. NC ultrasonic cleaner (DIGITAL PRO+) was supplied by Lifecode. Transmission electron microscope (Tecnai 12) was purchased from Philips. Laboratory pH meter (ST2100) was purchased from Ohaus Instrument Co., LTD. 3 Methods 3.1 Preparation of β -cyclodextrin stationary phase The preparation process of cyclodextrin chromatographic immobilization is mainly shown in Fig. 1 , and the synthesis process includes the following steps: (1) Acidification of SiO 2 microspheres[ 16 ]; (2) Preparation of aminated SiO 2 microspheres[ 17 ]; (3) Preparation of cyclodextrin stationary phases by the "one-step method"; (4) Preparation of cyclodextrin stationary phases by the "two-step method". 3.1.1 Acidification of SiO 2 microspheres Spherical silica gel (10 g) was weighed and placed into a three-necked flask. A condenser was installed, and 10% aqueous hydrochloric acid solutions (100 mL) were added. The reaction mixture was refluxed at 105℃ for 12 h under mechanical stirring. After that, the solution was repeatedly washed with deionized water until the filtrate was neutral and dried under vacuum at 90℃ for 12 h. 3.1.2 Synthesis of SiO 2 @NH 2 For the preparation of SiO 2 @NH 2, acidified SiO 2 microspheres (10 g) were weighed and evenly dispersed into water (60 mL). Ethanol (300 mL), ammonia (1.5 mL) and γ-aminopropyltriethoxysilane (20 mL) were then added respectively. The reaction mixture was stirred at room temperature for 6 h. The obtained product was washed with absolute ethanol and distilled water sequentially, and dried under vacuum at 60℃ to obtain aminated SiO 2 microspheres. 3.1.3 Synthesis of SiO 2 @NH 2 @TFTPN@β-CD-1 For the preparation of SiO 2 @NH 2 @TFTPN@β-CD-1, amino-modified SiO 2 microspheres (10 g), tetrafluoroterephthalonitrile (4 g), β-cyclodextrin (8 g) and potassium carbonate (9 g) were weighed and evenly dispersed into a mixed solvent (THF/DMF, v/v = 9/1). Nitrogen was passed into the system for 3 min, heated and stirred at 85 ℃ for 48 h. The supernatant was removed by centrifugation. The remaining products were washed sequentially with water, DMF and dichloromethane, then they were dried under vacuum at 60℃ to obtain β-cyclodextrin stationary phase SiO 2 @NH 2 @TFTPN@β-CD-1. 3.1.4 Synthesis of SiO 2 @NH 2 @TFTPN@β-CD-2 For the preparation of SiO 2 @NH 2 @TFTPN@β-CD-2, amino-modified SiO 2 microspheres (10 g), tetrafluoroterephthalonitrile (4 g) and potassium carbonate (9 g) were weighed and evenly dispersed into a mixed solvent (THF/DMF, v/v = 9/1). Nitrogen was passed into the system for 3 min, heated and stirred at 85℃ for 48 h. At the end of the reaction, the supernatant was removed by centrifugation. The remaining products were washed sequentially with water, tetrahydrofuran and dichloromethane, then they were dried under vacuum at 60℃ to obtain solid SiO 2 @NH 2 @TFTPN. SiO 2 @NH 2 @TFTPN (10 g), β-cyclodextrin (8 g) and potassium carbonate (9 g) were weighed and evenly dispersed into the mixed solvent (THF/DMF, v/v = 9/1). Nitrogen was instilled into the system for 3 min, heated at 85℃ for 48 h. At the end of the reaction, the supernatant was removed by centrifugation. The remaining products were washed successively with water, DMF and dichloromethane, then they were dried under vacuum at 60℃ to obtain β-cyclodextrin stationary phase SiO 2 @NH 2 @TFTPN@β-CD-2. 3.2 Characterization of Materials In order to confirm the synthesized β-cyclodextrin stationary phase, infrared spectroscopy (FTIR), elemental analyzer and scanning electron microscope were used respectively to characterize them. 3.3 Adsorption experiment 3 mg of adsorbent materials (SiO 2 microspheres, SiO 2 @NH 2 @TFTPN@β-CD-1, SiO 2 @NH 2 @TFTPN@β-CD-2) were added to 3 mL of 0.1 mmol/L sample solution (1-naphthol, phenol) and dispersed evenly, then placed in a constant-temperature oscillator at 25°C for shaking. The absorbance of the supernatant was measured at specific time intervals, and the adsorption capacity was calculated based on the concentration change before and after adsorption. \(\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\text{Q=}\frac{\left({\text{C}}_{\text{0}}\text{-}{\text{C}}_{\text{t}}\right)\text{V}}{\text{m}}\) …….…………………Formula-1 where C 0 and Ct (mg L − 1 ) are the residual concentration of solute in solution after adsorption at time t = 0 and t (min), m (mg) is the mass of the adsorbent, V (L) is the volume of the adsorption system. 3.4 Chromatographic column packing The column was packed using the classical slurry packing techniques[ 18 ]. Approximately 3 g of each of the above prepared stationary phase is weighed and dispersed into an appropriate solvent by sonication. A stainless steel column (150 mm × 4.6 mm) is packed with the prepared solution at 5000 psi(34.5MPa) pressure for 30 minutes using methanol as the substitute. Then the chromatographic column is washed with methanol until the baseline is stable. 3.5 Chromatographic evaluation Phenol, 1-naphthol and 2-naphthol are dissolved with methanol to prepare single sample solutions and mixed sample solutions at a concentration of 100 µg/mL, respectively. The mobile phases are acetonitrile /acetic acid-ammonium acetate buffer solution and methanol/ammonium acetate-acetic acid buffer solution. Both the mobile phase and sample solution are filtered through 0.22 µm organic filter membrane before use and degassed by sonication. The detection wavelength is 280 nm and the detection temperature is 30°C。 In order to investigate the separation effect, stability and repeatability of the novel β-cyclodextrin column, we carried out chromatographic separation on the mixed solution of phenol, α-naphthol and 2-naphthol, mainly focusing on the ratio of mobile phase, composition, pH value, sample volume and repeated sample injection. For the calculation of chromatographic separation results, resolution R is calculated as \(\:\text{R=1.18×}\frac{{\text{t}}_{\text{2}}\text{-}{\text{t}}_{\text{1}}}{{\text{W}}_{\text{h}\text{1}}\text{+}{\text{W}}_{\text{h}\text{2}}}\) ………………………………Formula-2 Where t is the retention time of the sample and W is the full width at half maximum (FWHM). 3.6 Schrödinger Molecular Docking All host molecules and guest molecules involved in docking in this paper are taken from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) or are self-drawn by Chemdraw, molecular docking simulations were performed using Schrödinger software. Rotatable single bonds, nonpolar hydrogens, etc. in the guest molecular structure are not modified according to the program default values. Receptor Grid Generation optimizes receptor molecules to produce receptor point files, ligand molecules are optimized by ligprep, and the molecular docking process is performed in cube lattice, which is 25 Å long, wide, and high, with the remaining parameters referenced to default values. 4 Results and Discussion 4.1 Characterization of β -cyclodextrin chromatographic stationary phase 4.1.1 Electron microscopic characterization Morphology of the materials was observed and analyzed through SEM. As SEM images show in Fig. 2 , the size of the SiO 2 microspheres was about 4 ~ 7 µm. The chromatographic stationary phase obtained via one-step reaction did not change significantly in particle size, indicating that the reaction conditions were mild; the overall structure of the SiO 2 microspheres was not destroyed, but a thin layer of functional groups was grafted on the surface of the silicon sphere with uniform size and regular shape, which was extremely important for obtaining excellent column efficiency and resolution of the HPLC column. However, the products synthesized by the two-step method contained spherical particles and fragments, indicating that the morphology was not uniform and not suitable for chromatographic stationary phase. 4.1.2 FI-IR spectroscopy The SiO 2 microspheres grafted with different functional groups were verified by IR. As shown in Fig. 3 , the absorption peak at 2235 cm − 1 was the stretching vibration of -C ≡ N on TFTPN. And the peak at 1467cm − 1 was the absorption peak of benzene ring skeleton. These results indicated that TFTPN had been modified on the surface of SiO 2 microspheres. The absorption peak at 3387 cm − 1 was ascribed to stretching vibration of -OH. The infrared characteristic absorption peak of 2929 cm − 1 was ascribed to stretching vibration of aliphatic C-H. These results proved that β-cyclodextrin was successfully bonded on the surface of SiO 2 microspheres. 4.1.3 Elemental analysis The rationality and success of the synthesis route of β-cyclodextrin stationary phase was verified by EA. Data are shown in Table S2. SiO 2 @NH 2 was the synthetic substrate for SiO 2 @NH 2 @TFTPN@β-CD-1 and SiO 2 @NH 2 @TFTPN@β-CD-2. After the introduction of TFTPN and β-cyclodextrin groups, the percentage composition of carbon increased significantly, while the percentage composition of hydrogen decreased slightly. These results indicated that β-cyclodextrin was successfully bonded on the surface of SiO 2 microspheres with TFTPN as the spacer arm. Furthermore, the percentage compositions of carbon and nitrogen in SiO 2 @NH 2 @TFTPN@β-CD-1 were similar to those in SiO 2 @NH 2 @TFTPN@β-CD-2, which were much greater than those in SiO 2 @NH 2 . These results demonstrated that the cyclodextrin chromatographic stationary phases prepared via one-step method and two-step method both had a high cyclodextrin bonding amount of cyclodextrin. 4.2 Adsorption performance To investigate the adsorption rate and capacity of three materials for phenolic structural analogues, the adsorption capacity of three materials for 1-naphthol in 120 min was measured. As shown in Figure S1 , the adsorption equilibrium was reached within 3 min. The adsorption capacity of 1-naphthol by SiO 2 microspheres was 27 mg/g at the adsorption equilibrium. When modified with β-cyclodextrin, the adsorption capacity of SiO 2 @NH 2 @TFTPN@β-CD-1 and SiO 2 @NH 2 @TFTPN@β-CD-2 was significantly increased to 45 mg/g and 52 mg/g, respectively. Besides, the adsorption of 1-naphthol by two kinds of cyclodextrin modified SiO 2 microspheres was equivalent, which was much greater than that by SiO 2 microspheres. Therefore, it could be speculated that β-cyclodextrin had been successfully bonded on the surface of SiO 2 microspheres. These experimental results were consistent with the results of elemental analysis. 4.3 Chromatographic evaluation 4.3.1 Column efficiency Since the results of elemental analysis, IR and kinetic adsorption experiments showed that the bonding amount of β-cyclodextrin was similar between SiO 2 @NH 2 @TFTPN@β-CD-2 prepared by “two-step method” and SiO 2 @NH 2 @TFTPN@β-CD-1 prepared by one-step method. Considering that the experimental process of “one-step method” was simple, timesaving and easy to operate, while microspheres obtained by “two-step method” were broken and had uneven morphology. Therefore, SiO 2 @NH 2 @TFTPN@β-CD-1 prepared by one-step method was used in the subsequent studies. The column efficiency was evaluated by naphthalene. The chromatograms are shown in Figure S2, and the theoretical plate number of naphthalene was detected as 26266/m. The asymmetry factor was 0.943. 4.3.2 Effect of mobile phase proportion on separation efficiency of three phenolic structural analogues Under reversed phase chromatography conditions, we investigated the proportion of mobile phase on the separation results of phenolic structural analogues. The results are shown in Fig. 4 . As the water content increased, the elution ability of mobile phase decreased and the retention time of phenolic structural analogues increased. These results indicate that hydrophobic interactions between β-cyclodextrin and three phenolic structural analogues dominated. As shown in Table S3, β-cyclodextrin stationary phase shows good resolution over phenol, 1-naphthol, and 2-naphthol structural analogues at a mobile phase proportion of 50:50 (v/v), with short retention times, complete peak appearance within 15 min and baseline separation. The above experimental results show that baseline separation of phenolic compounds could be achieved by adjusting the proportion of mobile phase on the novel β-cyclodextrin column. At the same time, three kinds of phenols were compared between β-cyclodextrin column and C18 column, and the results are shown in Figure S3. The results show that β-cyclodextrin column and C18 column have roughly the same column efficiency and considerable resolution, especially the peak sequence, which shows that the two stationary phases have different separation selectivity. 4.3.3 The effect of pH values on the separation of three phenolic structural analogues To further investigate the separation performance of β-cyclodextrin chromatographic stationary phase, we investigated the effect of pH of mobile phase on the separation of β-cyclodextrin chromatographic column[ 19 ], and the results are shown in Fig. 5 and Table S4. The retention time of the three phenolic structural analogues on the stationary phase shortened with the decreasing of pH. When pH was less than 4, all three phenolic structural analogues were completely separated. Because the three phenolic structural analogues existed mainly in molecular form in acidic mobile phase and could better interact with active groups on stationary phase. 4.3.4 The effect of mobile phase composition on resolution of phenolic structural analogues The composition of mobile phase effected the separation of phenolic structural analogues[ 20 ]. The results are shown in Fig. 6 and Table S5. Compared with the mobile phase using methanol and water, the retention time of phenolic structural analogues was shorter, the resolution was significantly better and the peak shape had better symmetry in the mobile phase using acetonitrile and water. The column efficiency of separating phenolic compounds in the acetonitrile / water system (20926/m) was significantly higher than that in the methanol / water system (9706/m). Therefore, compared with methanol system, the separation efficiency of three phenolic structural analogues was better and the column efficiency was higher in acetonitrile system. The results indicated that the mobile phase had a significant impact on the separation results. 4.3.5 Effect of sample volume on separation efficiency of β-cyclodextrin column The injection volume affected the separation performance of the chromatographic column[ 21 ]. As shown in Fig. 7 , as the injection volume increased from 3 µL to 20 µL, the retention time of the three phenolic structural analogues slightly decreased. The baseline separation could still be achieved. A good linear relationship was maintained between the peak area and the injection volume. The results indicated that the β-cyclodextrin chromatographic column had the potential for quantitative analysis. 4.3.6 β-Cyclodextrin Column Repeatability Study The repeatability of a chromatographic column is an essential parameter for its application prospect. As shown in Figure S4, the elution time of the analyte showed no significant change after repeated injections on a β-cyclodextrin column that had been used for 10 months for 40 times. The calculated RSD values for the retention time of phenol, 1-naphthol and 2- naphthol in the 1st, 10th, 20th, 30th and 40th injections were less than 0.7%. The values for the retention time of peak area were less than 2.5%. The resolution of 1- naphthol and 2- naphthol was as high as 1.95. The results showed that β-cyclodextrin column had good repeatability in HPLC separation. No abnormal increase of column pressure, collapse of column packing, bifurcation or serious tailing of chromatographic peak occurred, which indicated that β-cyclodextrin chromatographic column had a long lifespan and promising market prospect. 4.4 Molecular docking simulation Molecular docking of β-CD and cyclodextrin stationary phases to phenolic structural analogues was performed to investigate the separation of β-cyclodextrin stationary phase on phenolic structural analogues. The docking results are shown in FIGURE 8. None of the phenolic structural analogues; phenol, 1-naphthol, and 2-naphthol was found to be separated from the β-cyclodextrin cavity, which could indicate that these three phenolic structural analogues could form stable inclusion complexes with β-cyclodextrin. Stable hydrogen bonds were formed between the hydrogen atoms on the hydroxyl groups of the three phenolic structural analogues and the oxygen atoms at the small mouth end of β-cyclodextrin[ 22 ]. In addition, β-cyclodextrin was hydrophobic inside. Hydrophobic aromatic structure entered β-cyclodextrin hydrophobic cavity. Hydrogen bond and hydrophobic interaction reduced the energy of the complex[ 23 – 25 ], which maintained the stability of this structure. As seen in Figure S5, the docking energy of cyclodextrin stationary phase to phenolic structural analogues was significantly lower than that of β-CD due to the introduction of TFTPN, indicating that the interaction between cyclodextrin stationary phase and the three phenolic structural analogues was significantly stronger than that of β-CD. The docking energies of phenols with cyclodextrin stationary phase were: phenol (-4.623 kJ/mol), 1-naphthol (-5.490 kJ/mol) and 2-naphthol (-5.846 kJ/mol), and the retention of phenol was less than that of 1-naphthol and 2-naphthol, which was consistent with the molecular docking results. However, the retention of 1-naphthol was instead greater than that of 2-naphthol, which was contrary to molecular docking results. It might be due to mobile phase effects[ 26 ], as the nature of the mobile phase affected the selectivity and retention time of β-cyclodextrin separation as a stationary phase, which had also been recently reported for regioisomers of xylene separated as a stationary phase by MIL-53[ 27 ]. 5 Conclusion In the field of chromatographic separation, it is essential to develop more efficient chromatographic stationary phases. In response to the shortcomings of the traditional cyclodextrin stationary phase, a novel β-cyclodextrin stationary phase with porous structure and fast adsorption kinetics is successfully prepared using TFTPN as spacer arm for the first time. Utilizing the multi-substitution characteristics of TFTPN, it has the advantages of higher reactivity and the ability to combine multiple β-cyclodextrin molecules, resulting in the increase of the bonding amount of β-cyclodextrin. Furthermore, the introduction of TFTPN enhances the hydrogen bonding and π-π interaction between the stationary phase and the target compound. TFTPN cooperates with the inclusion of the cyclodextrin hydrophobic cavity, enhancing the selectivity of β-cyclodextrin stationary phase on phenolic structural analogues. The effects of mobile phase ratio, pH value and injection volume on the separation of phenolic compounds with similar structure were investigated. The results showed that the three phenolic compounds could realize baseline separation on β-cyclodextrin column. At the same time, the repeatability of β-cyclodextrin chromatographic column was evaluated. During ten months of usage, the relative standard deviations of retention time of β-cyclodextrin chromatographic column for three phenolic compounds were less than 0.7%, and the relative standard deviations of peak areas were lower than 2.5%. The results indicated the chromatographic column had a good lifetime. Furthermore, its excellent separation ability and column efficiency also showed that the β-cyclodextrin chromatographic stationary phase had excellent industrialization potential. Declarations Competing interests The authors declare no competing interests. Author Contribution Li Jiang, Le Duan and Yan Teng wrote the main manuscript text and Ineza Urujeni Gisèle, Geyuan Li prepared figures and tables. All authors reviewed the manuscript. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 81402899). References M. Attimarad, K.N. Venugopala, A.B. Nair, N. Sreeharsha, P.K. 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Li,(2015) Investigation on interaction between Ligupurpuroside A and pepsin by spectroscopic and docking methods, Spectrochimica Acta Part a-Molecular and Biomolecular Spectroscopy, 135 256–263. http://dx.doi.org/10.1016/j.saa.2014.06.087 M.A. Moreira, J.C. Santos, A.F.P. Ferreira, J.M. Loureiro, A.E. Rodrigues,(2011) Influence of the Eluent in the MIL-53(Al) Selectivity for Xylene Isomers Separation, Industrial & Engineering Chemistry Research, 50 7688–7695. http://dx.doi.org/10.1021/ie200206n K.J. Hartlieb, J.M. Holcroft, P.Z. Moghadam, N.A. Vermeulen, M.M. Algaradah, M.S. Nassar, Y.Y. Botros, R.Q. Snurr, J.F. Stoddart,(2016) CD-MOF: A Versatile Separation Medium, Journal of the American Chemical Society, 138 2292–2301. http://dx.doi.org/10.1021/jacs.5b12860 Additional Declarations No competing interests reported. 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16:14:45","extension":"xml","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":91036,"visible":true,"origin":"","legend":"","description":"","filename":"7cdbc9b192004306a265c98a186fd65a1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7475722/v1/af932571dd8baf9c75c14a56.xml"},{"id":92014499,"identity":"a0c3e22e-490f-489d-8233-45e8f069bf23","added_by":"auto","created_at":"2025-09-23 16:14:45","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":98772,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7475722/v1/174ab1ba3b98b335c05acbe6.html"},{"id":92014479,"identity":"4bebcd9f-0571-47a0-8c9b-bcdcc395b255","added_by":"auto","created_at":"2025-09-23 16:14:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":122583,"visible":true,"origin":"","legend":"\u003cp\u003eTwo methods of preparing cyclodextrin stationary phases (A: one-step method, B: two-step method)\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7475722/v1/c180cc79d33f47c2636350bd.png"},{"id":92014485,"identity":"ae220c3a-6cfd-4949-87c5-26af2ca55cf8","added_by":"auto","created_at":"2025-09-23 16:14:45","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":299999,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of the three materials: A1 and A2：SiO\u003csub\u003e2\u003c/sub\u003e microspheres；B1 and B2：SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e@TFTPN@β-CD-1；C1 and C2：SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e@TFTPN@β-CD-2（Scale bars: A1-C1: 100 μm，A2-C2: 5 μm）.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7475722/v1/faf8fb6a65fd049475d7c629.jpeg"},{"id":92014480,"identity":"a11a0206-a298-4028-ab45-7256306f7705","added_by":"auto","created_at":"2025-09-23 16:14:45","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":165057,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR spectrums of cyclodextrin stationary phase\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7475722/v1/04ffce5135840f0ccc369b5b.jpeg"},{"id":92015732,"identity":"abb4ffa6-84f9-413a-b210-ede4e051b935","added_by":"auto","created_at":"2025-09-23 16:30:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":63339,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Separation of three phenolic structural analogues by β-Cyclodextrin stationary phase; (B) The effect of the proportion of mobile phase on the resolution of phenolic structural\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7475722/v1/954922542fc7314c40a0148f.png"},{"id":92015001,"identity":"019aea70-3c35-4023-b4db-3d46023a8375","added_by":"auto","created_at":"2025-09-23 16:22:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":40765,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Separation of phenolic compounds under different pH conditions, (B) Effect of pH value on retention time of phenolic compounds. Chromatographic conditions: mobile phase: methanol/0.02mol/L aqueous ammonium acetate solution; sample concentration: 100 μL/mL; injection volume: 10 μL; UV detection wavelength: 280 nm; column temperature: 30 ºC; flow rate: 0.7 min\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7475722/v1/1ce8fd12e548e6af42ce9651.png"},{"id":92014483,"identity":"28fde63a-aca4-493f-8e6f-7aaff730915e","added_by":"auto","created_at":"2025-09-23 16:14:45","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":57236,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of mobile phase type on the separation effect of phenolic compounds. Chromatographic conditions: mobile phase: (a) methanol / 0.02mol/L ammonium acetate at pH 4.0 (70 / 30, v/v), (b) acetonitrile / 0.02mol/L ammonium acetate at pH 4.0 (50 / 50, v/v); sample concentration: 100 μL/mL; injection volume: 10 μL; UV detection wavelength: 280 nm; column temperature: 30 ºC; flow rate: 0.7 min\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7475722/v1/4801dbe3390f84af2fb82fdc.jpeg"},{"id":92017107,"identity":"0ed6a497-bfa4-4ad4-a694-91002f8ed03d","added_by":"auto","created_at":"2025-09-23 16:46:45","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":363751,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Influence of injection amount on the separation effect of phenolic compounds with similar structure; (B) Influence of injection amount on retention time and peak area; Chromatographic conditions: mobile phase: methanol / 0.02 mol/L ammonium acetate at pH 4.0 (70:30, v/v); sample concentration: 100 μg/mL; injection volume: 5 μL; UV detection wavelength: 280 nm; column temperature: 30 ºC; flow rate: 0.7 mL min\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7475722/v1/2c10bfb0bb05a6e3b9e69b93.jpeg"},{"id":94474710,"identity":"dab8a1eb-4335-4b82-940f-5b9426c13ca9","added_by":"auto","created_at":"2025-10-27 15:49:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2052114,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7475722/v1/aaeaddcb-900d-4902-b722-88f64a4a4fff.pdf"},{"id":92016495,"identity":"81fe615d-beeb-479f-86e7-8b634dabb693","added_by":"auto","created_at":"2025-09-23 16:38:45","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":676101,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7475722/v1/f1ba63a9629da43bf175e925.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"β-Cyclodextrin Stationary Phase Using Tetrafluoroterephthalonitrile as Spacer Arm for Separation of Phenolic Compounds","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eHigh-performance liquid chromatography (HPLC) is one of the most effective techniques to separate structural analogues due to its advantages of rapidity, high performance, wide separation range, and more improved fittings[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. For high-performance liquid chromatography, separation of structural analogues can be achieved directly by using stationary phase, mobile phase additives, or indirectly by pre-column derivatization[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Cyclodextrins have many chiral centers[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and hydrophobic cavities[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], which have high recognition ability for structural analogues, so they are often used in the preparation of chromatographic stationary phases[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Common commercial cyclodextrin columns and their analytical application are systematically summarized in the table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eAlthough cyclodextrin has been introduced into the preparation of HPLC stationary phase, it still has some problems such as low bonding amount, slow recognition speed and limited recognition ability. These factors restrict cyclodextrin chromatographic stationary phase from becoming mainstream and commercialized chromatographic stationary phase.\u003c/p\u003e\u003cp\u003eIn this paper, β-cyclodextrin is bonded to the surface of SiO2 microspheres carrier using tetrafluoroterephthalonitrile (TFTPN) as a spacer arm to obtain β-cyclodextrin chromatographic stationary phase for the separation of structurally similar phenol, 1-naphthol, and 2-naphthol mixtures. Taking advantage of the high reaction activity of TFTPN[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and the ability to combine multiple β-cyclodextrin molecules[\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], the problem of low cyclodextrin bonding amount prevalent in traditional cyclodextrin chromatographic stationary phases[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] has been solved. Besides, this novel cyclodextrin chromatographic stationary phase has abundant pore structure, which allows rapid recognition. Moreover, the novel β-cyclodextrin chromatographic stationary phase can provide various interaction sites such as π-π interaction, hydrogen bonding and cyclodextrin hydrophobic cavities, improving the selectivity for phenolic structural analogues. We investigated the influence of mobile phase composition, proportion, pH value, injection volume and other factors on the separation of phenolic compounds with similar structure, and evaluated the column efficiency, resolution, repeatability and the life span of β-cyclodextrin chromatographic column. In this study, the original traditional cyclodextrin stationary phase was improved, providing more options in the field of high-performance liquid chromatography.\u003c/p\u003e"},{"header":"2 Materials and Instruments","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials\u003c/h2\u003e\u003cp\u003eAll analytical grade chemical reagents were purchased from Shanghai Aladdin Biochemical Technology Co., LTD (China). They were N, N-dimethylformamide, β-cyclodextrins, potassium carbonate, tetrahydrofuran, ammonia, phenol, α- naphthol, and 2-naphthol. Tetrafluorophenonitrile (\u0026gt;\u0026thinsp;99%), tetraethyl silicate (reagent grade, 98%), 3-aminopropyl triethoxysilane (98%) were also purchased from here. SiO\u003csub\u003e2\u003c/sub\u003e microspheres was purchased from Tianjin Beilux. Methanol was obtained from Shanghai Xingke High purity Solvent Co., LTD. Acetonitrile was supplied by Shanghai Hutest Laboratory Equipment Co., LTD.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Instruments\u003c/h2\u003e\u003cp\u003eHigh performance liquid chromatography (LC-20AB), vacuum drying oven (DZF-6021), FT-IR spectrometer (TTIR-8400s) were purchased from Shimadzu, Japan. Elemental analyzer (PE 2400 series Ⅱ) was supplied by Beijing Jing ke Rui da technology Co., LTD. Heat collecting type magnetic (DF-101S) was obtained from Nanjing jiameilun scientific instrument Co., LTD. NC ultrasonic cleaner (DIGITAL PRO+) was supplied by Lifecode. Transmission electron microscope (Tecnai 12) was purchased from Philips. Laboratory pH meter (ST2100) was purchased from Ohaus Instrument Co., LTD.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Preparation of β -cyclodextrin stationary phase\u003c/h2\u003e\u003cp\u003eThe preparation process of cyclodextrin chromatographic immobilization is mainly shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, and the synthesis process includes the following steps:\u003c/p\u003e\u003cp\u003e(1) Acidification of SiO\u003csub\u003e2\u003c/sub\u003e microspheres[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e];\u003c/p\u003e\u003cp\u003e(2) Preparation of aminated SiO\u003csub\u003e2\u003c/sub\u003e microspheres[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e];\u003c/p\u003e\u003cp\u003e(3) Preparation of cyclodextrin stationary phases by the \"one-step method\";\u003c/p\u003e\u003cp\u003e(4) Preparation of cyclodextrin stationary phases by the \"two-step method\".\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e3.1.1 Acidification of SiO\u003csub\u003e2\u003c/sub\u003e microspheres\u003c/h2\u003e\u003cp\u003eSpherical silica gel (10 g) was weighed and placed into a three-necked flask. A condenser was installed, and 10% aqueous hydrochloric acid solutions (100 mL) were added. The reaction mixture was refluxed at 105℃ for 12 h under mechanical stirring. After that, the solution was repeatedly washed with deionized water until the filtrate was neutral and dried under vacuum at 90℃ for 12 h.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e3.1.2 Synthesis of SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e\u003cp\u003eFor the preparation of SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2,\u003c/sub\u003e acidified SiO\u003csub\u003e2\u003c/sub\u003e microspheres (10 g) were weighed and evenly dispersed into water (60 mL). Ethanol (300 mL), ammonia (1.5 mL) and γ-aminopropyltriethoxysilane (20 mL) were then added respectively. The reaction mixture was stirred at room temperature for 6 h. The obtained product was washed with absolute ethanol and distilled water sequentially, and dried under vacuum at 60℃ to obtain aminated SiO\u003csub\u003e2\u003c/sub\u003e microspheres.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e3.1.3 Synthesis of SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e@TFTPN@β-CD-1\u003c/h2\u003e\u003cp\u003eFor the preparation of SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e@TFTPN@β-CD-1, amino-modified SiO\u003csub\u003e2\u003c/sub\u003e microspheres (10 g), tetrafluoroterephthalonitrile (4 g), β-cyclodextrin (8 g) and potassium carbonate (9 g) were weighed and evenly dispersed into a mixed solvent (THF/DMF, v/v\u0026thinsp;=\u0026thinsp;9/1). Nitrogen was passed into the system for 3 min, heated and stirred at 85 ℃ for 48 h. The supernatant was removed by centrifugation. The remaining products were washed sequentially with water, DMF and dichloromethane, then they were dried under vacuum at 60℃ to obtain β-cyclodextrin stationary phase SiO\u003csub\u003e2\u003c/sub\u003e @NH\u003csub\u003e2\u003c/sub\u003e@TFTPN@β-CD-1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e3.1.4 Synthesis of SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e@TFTPN@β-CD-2\u003c/h2\u003e\u003cp\u003eFor the preparation of SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e@TFTPN@β-CD-2, amino-modified SiO\u003csub\u003e2\u003c/sub\u003e microspheres (10 g), tetrafluoroterephthalonitrile (4 g) and potassium carbonate (9 g) were weighed and evenly dispersed into a mixed solvent (THF/DMF, v/v\u0026thinsp;=\u0026thinsp;9/1). Nitrogen was passed into the system for 3 min, heated and stirred at 85℃ for 48 h. At the end of the reaction, the supernatant was removed by centrifugation. The remaining products were washed sequentially with water, tetrahydrofuran and dichloromethane, then they were dried under vacuum at 60℃ to obtain solid SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e@TFTPN.\u003c/p\u003e\u003cp\u003eSiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e@TFTPN (10 g), β-cyclodextrin (8 g) and potassium carbonate (9 g) were weighed and evenly dispersed into the mixed solvent (THF/DMF, v/v\u0026thinsp;=\u0026thinsp;9/1). Nitrogen was instilled into the system for 3 min, heated at 85℃ for 48 h. At the end of the reaction, the supernatant was removed by centrifugation. The remaining products were washed successively with water, DMF and dichloromethane, then they were dried under vacuum at 60℃ to obtain β-cyclodextrin stationary phase SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e@TFTPN@β-CD-2.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Characterization of Materials\u003c/h2\u003e\u003cp\u003eIn order to confirm the synthesized β-cyclodextrin stationary phase, infrared spectroscopy (FTIR), elemental analyzer and scanning electron microscope were used respectively to characterize them.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Adsorption experiment\u003c/h2\u003e\u003cp\u003e3 mg of adsorbent materials (SiO\u003csub\u003e2\u003c/sub\u003e microspheres, SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e@TFTPN@β-CD-1, SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e@TFTPN@β-CD-2) were added to 3 mL of 0.1 mmol/L sample solution (1-naphthol, phenol) and dispersed evenly, then placed in a constant-temperature oscillator at 25\u0026deg;C for shaking. The absorbance of the supernatant was measured at specific time intervals, and the adsorption capacity was calculated based on the concentration change before and after adsorption.\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\text{Q=}\\frac{\\left({\\text{C}}_{\\text{0}}\\text{-}{\\text{C}}_{\\text{t}}\\right)\\text{V}}{\\text{m}}\\)\u003c/span\u003e\u003c/span\u003e\u0026hellip;\u0026hellip;.\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;Formula-1\u003c/p\u003e\u003cp\u003ewhere \u003cem\u003eC\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eCt\u003c/em\u003e (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) are the residual concentration of solute in solution after adsorption at time \u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0 and \u003cem\u003et\u003c/em\u003e (min), \u003cem\u003em\u003c/em\u003e (mg) is the mass of the adsorbent, \u003cem\u003eV\u003c/em\u003e (L) is the volume of the adsorption system.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Chromatographic column packing\u003c/h2\u003e\u003cp\u003eThe column was packed using the classical slurry packing techniques[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Approximately 3 g of each of the above prepared stationary phase is weighed and dispersed into an appropriate solvent by sonication. A stainless steel column (150 mm \u0026times; 4.6 mm) is packed with the prepared solution at 5000 psi(34.5MPa) pressure for 30 minutes using methanol as the substitute. Then the chromatographic column is washed with methanol until the baseline is stable.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Chromatographic evaluation\u003c/h2\u003e\u003cp\u003ePhenol, 1-naphthol and 2-naphthol are dissolved with methanol to prepare single sample solutions and mixed sample solutions at a concentration of 100 \u0026micro;g/mL, respectively. The mobile phases are acetonitrile /acetic acid-ammonium acetate buffer solution and methanol/ammonium acetate-acetic acid buffer solution. Both the mobile phase and sample solution are filtered through 0.22 \u0026micro;m organic filter membrane before use and degassed by sonication. The detection wavelength is 280 nm and the detection temperature is 30\u0026deg;C。\u003c/p\u003e\u003cp\u003eIn order to investigate the separation effect, stability and repeatability of the novel β-cyclodextrin column, we carried out chromatographic separation on the mixed solution of phenol, α-naphthol and 2-naphthol, mainly focusing on the ratio of mobile phase, composition, pH value, sample volume and repeated sample injection. For the calculation of chromatographic separation results, resolution \u003cem\u003eR\u003c/em\u003e is calculated as\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{R=1.18\u0026times;}\\frac{{\\text{t}}_{\\text{2}}\\text{-}{\\text{t}}_{\\text{1}}}{{\\text{W}}_{\\text{h}\\text{1}}\\text{+}{\\text{W}}_{\\text{h}\\text{2}}}\\)\u003c/span\u003e\u003c/span\u003e \u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;Formula-2\u003c/p\u003e\u003cp\u003eWhere \u003cem\u003et\u003c/em\u003e is the retention time of the sample and \u003cem\u003eW\u003c/em\u003e is the full width at half maximum (FWHM).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Schr\u0026ouml;dinger Molecular Docking\u003c/h2\u003e\u003cp\u003eAll host molecules and guest molecules involved in docking in this paper are taken from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) or are self-drawn by Chemdraw, molecular docking simulations were performed using Schr\u0026ouml;dinger software. Rotatable single bonds, nonpolar hydrogens, etc. in the guest molecular structure are not modified according to the program default values. Receptor Grid Generation optimizes receptor molecules to produce receptor point files, ligand molecules are optimized by ligprep, and the molecular docking process is performed in cube lattice, which is 25 \u0026Aring; long, wide, and high, with the remaining parameters referenced to default values.\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Results and Discussion","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Characterization of β -cyclodextrin chromatographic stationary phase\u003c/h2\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e\u003cb\u003e4.1.1 Electron microscopic characterization\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eMorphology of the materials was observed and analyzed through SEM. As SEM images show in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the size of the SiO\u003csub\u003e2\u003c/sub\u003e microspheres was about 4\u0026thinsp;~\u0026thinsp;7 \u0026micro;m. The chromatographic stationary phase obtained via one-step reaction did not change significantly in particle size, indicating that the reaction conditions were mild; the overall structure of the SiO\u003csub\u003e2\u003c/sub\u003e microspheres was not destroyed, but a thin layer of functional groups was grafted on the surface of the silicon sphere with uniform size and regular shape, which was extremely important for obtaining excellent column efficiency and resolution of the HPLC column. However, the products synthesized by the two-step method contained spherical particles and fragments, indicating that the morphology was not uniform and not suitable for chromatographic stationary phase.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e4.1.2 FI-IR spectroscopy\u003c/h2\u003e\u003cp\u003eThe SiO\u003csub\u003e2\u003c/sub\u003e microspheres grafted with different functional groups were verified by IR. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the absorption peak at 2235 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was the stretching vibration of -C\u0026thinsp;\u0026equiv;\u0026thinsp;N on TFTPN. And the peak at 1467cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was the absorption peak of benzene ring skeleton. These results indicated that TFTPN had been modified on the surface of SiO\u003csub\u003e2\u003c/sub\u003e microspheres. The absorption peak at 3387 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was ascribed to stretching vibration of -OH. The infrared characteristic absorption peak of 2929 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was ascribed to stretching vibration of aliphatic C-H. These results proved that β-cyclodextrin was successfully bonded on the surface of SiO\u003csub\u003e2\u003c/sub\u003e microspheres.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003e4.1.3 Elemental analysis\u003c/h2\u003e\u003cp\u003eThe rationality and success of the synthesis route of β-cyclodextrin stationary phase was verified by EA. Data are shown in Table S2. SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e was the synthetic substrate for SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e@TFTPN@β-CD-1 and SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e@TFTPN@β-CD-2. After the introduction of TFTPN and β-cyclodextrin groups, the percentage composition of carbon increased significantly, while the percentage composition of hydrogen decreased slightly. These results indicated that β-cyclodextrin was successfully bonded on the surface of SiO\u003csub\u003e2\u003c/sub\u003e microspheres with TFTPN as the spacer arm. Furthermore, the percentage compositions of carbon and nitrogen in SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e@TFTPN@β-CD-1 were similar to those in SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e@TFTPN@β-CD-2, which were much greater than those in SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e. These results demonstrated that the cyclodextrin chromatographic stationary phases prepared via one-step method and two-step method both had a high cyclodextrin bonding amount of cyclodextrin.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e4.2 Adsorption performance\u003c/h2\u003e\u003cp\u003eTo investigate the adsorption rate and capacity of three materials for phenolic structural analogues, the adsorption capacity of three materials for 1-naphthol in 120 min was measured. As shown in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, the adsorption equilibrium was reached within 3 min. The adsorption capacity of 1-naphthol by SiO\u003csub\u003e2\u003c/sub\u003e microspheres was 27 mg/g at the adsorption equilibrium. When modified with β-cyclodextrin, the adsorption capacity of SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e@TFTPN@β-CD-1 and SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e@TFTPN@β-CD-2 was significantly increased to 45 mg/g and 52 mg/g, respectively. Besides, the adsorption of 1-naphthol by two kinds of cyclodextrin modified SiO\u003csub\u003e2\u003c/sub\u003e microspheres was equivalent, which was much greater than that by SiO\u003csub\u003e2\u003c/sub\u003e microspheres. Therefore, it could be speculated that β-cyclodextrin had been successfully bonded on the surface of SiO\u003csub\u003e2\u003c/sub\u003e microspheres. These experimental results were consistent with the results of elemental analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Chromatographic evaluation\u003c/h2\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003e\u003cb\u003e4.3.1 Column efficiency\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eSince the results of elemental analysis, IR and kinetic adsorption experiments showed that the bonding amount of β-cyclodextrin was similar between SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e@TFTPN@β-CD-2 prepared by \u0026ldquo;two-step method\u0026rdquo; and SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e@TFTPN@β-CD-1 prepared by one-step method. Considering that the experimental process of \u0026ldquo;one-step method\u0026rdquo; was simple, timesaving and easy to operate, while microspheres obtained by \u0026ldquo;two-step method\u0026rdquo; were broken and had uneven morphology. Therefore, SiO\u003csub\u003e2\u003c/sub\u003e@NH\u003csub\u003e2\u003c/sub\u003e@TFTPN@β-CD-1 prepared by one-step method was used in the subsequent studies. The column efficiency was evaluated by naphthalene. The chromatograms are shown in Figure S2, and the theoretical plate number of naphthalene was detected as 26266/m. The asymmetry factor was 0.943.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\u003ch2\u003e4.3.2 Effect of mobile phase proportion on separation efficiency of three phenolic structural analogues\u003c/h2\u003e\u003cp\u003eUnder reversed phase chromatography conditions, we investigated the proportion of mobile phase on the separation results of phenolic structural analogues. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. As the water content increased, the elution ability of mobile phase decreased and the retention time of phenolic structural analogues increased. These results indicate that hydrophobic interactions between β-cyclodextrin and three phenolic structural analogues dominated. As shown in Table S3, β-cyclodextrin stationary phase shows good resolution over phenol, 1-naphthol, and 2-naphthol structural analogues at a mobile phase proportion of 50:50 (v/v), with short retention times, complete peak appearance within 15 min and baseline separation. The above experimental results show that baseline separation of phenolic compounds could be achieved by adjusting the proportion of mobile phase on the novel β-cyclodextrin column. At the same time, three kinds of phenols were compared between β-cyclodextrin column and C18 column, and the results are shown in Figure S3. The results show that β-cyclodextrin column and C18 column have roughly the same column efficiency and considerable resolution, especially the peak sequence, which shows that the two stationary phases have different separation selectivity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003e4.3.3 The effect of pH values on the separation of three phenolic structural analogues\u003c/h2\u003e\u003cp\u003eTo further investigate the separation performance of β-cyclodextrin chromatographic stationary phase, we investigated the effect of pH of mobile phase on the separation of β-cyclodextrin chromatographic column[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Table S4. The retention time of the three phenolic structural analogues on the stationary phase shortened with the decreasing of pH. When pH was less than 4, all three phenolic structural analogues were completely separated. Because the three phenolic structural analogues existed mainly in molecular form in acidic mobile phase and could better interact with active groups on stationary phase.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003e4.3.4 The effect of mobile phase composition on resolution of phenolic structural analogues\u003c/h2\u003e\u003cp\u003eThe composition of mobile phase effected the separation of phenolic structural analogues[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Table S5. Compared with the mobile phase using methanol and water, the retention time of phenolic structural analogues was shorter, the resolution was significantly better and the peak shape had better symmetry in the mobile phase using acetonitrile and water. The column efficiency of separating phenolic compounds in the acetonitrile / water system (20926/m) was significantly higher than that in the methanol / water system (9706/m). Therefore, compared with methanol system, the separation efficiency of three phenolic structural analogues was better and the column efficiency was higher in acetonitrile system. The results indicated that the mobile phase had a significant impact on the separation results.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003e4.3.5 Effect of sample volume on separation efficiency of β-cyclodextrin column\u003c/h2\u003e\u003cp\u003eThe injection volume affected the separation performance of the chromatographic column[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, as the injection volume increased from 3 \u0026micro;L to 20 \u0026micro;L, the retention time of the three phenolic structural analogues slightly decreased. The baseline separation could still be achieved. A good linear relationship was maintained between the peak area and the injection volume. The results indicated that the β-cyclodextrin chromatographic column had the potential for quantitative analysis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section3\"\u003e\u003ch2\u003e4.3.6 β-Cyclodextrin Column Repeatability Study\u003c/h2\u003e\u003cp\u003eThe repeatability of a chromatographic column is an essential parameter for its application prospect. As shown in Figure S4, the elution time of the analyte showed no significant change after repeated injections on a β-cyclodextrin column that had been used for 10 months for 40 times. The calculated RSD values for the retention time of phenol, 1-naphthol and 2- naphthol in the 1st, 10th, 20th, 30th and 40th injections were less than 0.7%. The values for the retention time of peak area were less than 2.5%. The resolution of 1- naphthol and 2- naphthol was as high as 1.95. The results showed that β-cyclodextrin column had good repeatability in HPLC separation. No abnormal increase of column pressure, collapse of column packing, bifurcation or serious tailing of chromatographic peak occurred, which indicated that β-cyclodextrin chromatographic column had a long lifespan and promising market prospect.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e4.4 Molecular docking simulation\u003c/h2\u003e\u003cp\u003eMolecular docking of β-CD and cyclodextrin stationary phases to phenolic structural analogues was performed to investigate the separation of β-cyclodextrin stationary phase on phenolic structural analogues. The docking results are shown in FIGURE 8. None of the phenolic structural analogues; phenol, 1-naphthol, and 2-naphthol was found to be separated from the β-cyclodextrin cavity, which could indicate that these three phenolic structural analogues could form stable inclusion complexes with β-cyclodextrin. Stable hydrogen bonds were formed between the hydrogen atoms on the hydroxyl groups of the three phenolic structural analogues and the oxygen atoms at the small mouth end of β-cyclodextrin[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In addition, β-cyclodextrin was hydrophobic inside. Hydrophobic aromatic structure entered β-cyclodextrin hydrophobic cavity. Hydrogen bond and hydrophobic interaction reduced the energy of the complex[\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], which maintained the stability of this structure.\u003c/p\u003e\u003cp\u003eAs seen in Figure S5, the docking energy of cyclodextrin stationary phase to phenolic structural analogues was significantly lower than that of β-CD due to the introduction of TFTPN, indicating that the interaction between cyclodextrin stationary phase and the three phenolic structural analogues was significantly stronger than that of β-CD. The docking energies of phenols with cyclodextrin stationary phase were: phenol (-4.623 kJ/mol), 1-naphthol (-5.490 kJ/mol) and 2-naphthol (-5.846 kJ/mol), and the retention of phenol was less than that of 1-naphthol and 2-naphthol, which was consistent with the molecular docking results. However, the retention of 1-naphthol was instead greater than that of 2-naphthol, which was contrary to molecular docking results. It might be due to mobile phase effects[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], as the nature of the mobile phase affected the selectivity and retention time of β-cyclodextrin separation as a stationary phase, which had also been recently reported for regioisomers of xylene separated as a stationary phase by MIL-53[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eIn the field of chromatographic separation, it is essential to develop more efficient chromatographic stationary phases. In response to the shortcomings of the traditional cyclodextrin stationary phase, a novel β-cyclodextrin stationary phase with porous structure and fast adsorption kinetics is successfully prepared using TFTPN as spacer arm for the first time. Utilizing the multi-substitution characteristics of TFTPN, it has the advantages of higher reactivity and the ability to combine multiple β-cyclodextrin molecules, resulting in the increase of the bonding amount of β-cyclodextrin. Furthermore, the introduction of TFTPN enhances the hydrogen bonding and π-π interaction between the stationary phase and the target compound. TFTPN cooperates with the inclusion of the cyclodextrin hydrophobic cavity, enhancing the selectivity of β-cyclodextrin stationary phase on phenolic structural analogues. The effects of mobile phase ratio, pH value and injection volume on the separation of phenolic compounds with similar structure were investigated. The results showed that the three phenolic compounds could realize baseline separation on β-cyclodextrin column. At the same time, the repeatability of β-cyclodextrin chromatographic column was evaluated. During ten months of usage, the relative standard deviations of retention time of β-cyclodextrin chromatographic column for three phenolic compounds were less than 0.7%, and the relative standard deviations of peak areas were lower than 2.5%. The results indicated the chromatographic column had a good lifetime. Furthermore, its excellent separation ability and column efficiency also showed that the β-cyclodextrin chromatographic stationary phase had excellent industrialization potential.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLi Jiang, Le Duan and Yan Teng wrote the main manuscript text and Ineza Urujeni Gis\u0026egrave;le, Geyuan Li prepared figures and tables. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grant No. 81402899).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eM. Attimarad, K.N. Venugopala, A.B. Nair, N. Sreeharsha, P.K. 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Stoddart,(2016) CD-MOF: A Versatile Separation Medium, Journal of the American Chemical Society, 138 2292\u0026ndash;2301.\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1021/jacs.5b12860\u003c/span\u003e\u003cspan address=\"10.1021/jacs.5b12860\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"tetrafluoroterephthalonitrile, chromatographic stationary phase, β-cyclodextrin, column efficiency, hydrophobic cavities","lastPublishedDoi":"10.21203/rs.3.rs-7475722/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7475722/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn the field of chromatographic separation, high-performance liquid chromatography (HPLC) is widely used in the analysis of complex samples due to its high separation efficiency, fast analysis speed and wide application range. The core goal of the approach was to develop a more efficient chromatographic stationary phase. Although cyclodextrin chromatographic stationary phase has been reported previously, there are various challenges that prevent it from becoming the mainstream chromatographic stationary phase, such as low bonding amount, complicated synthesis process and poor separation effect. In this paper, a novel β-cyclodextrin chromatographic stationary phase was prepared with polysubstituted tetrafluoroterephthalonitrile (TFTPN) as a spacer arm for the separation of phenolic compounds. The chromatographic results showed that it had excellent chromatographic performance, and the mechanism of action was explained by computer simulation. This study provides new insights for the separation and analysis of structural analogues.\u003c/p\u003e","manuscriptTitle":"β-Cyclodextrin Stationary Phase Using Tetrafluoroterephthalonitrile as Spacer Arm for Separation of Phenolic Compounds","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-23 16:14:40","doi":"10.21203/rs.3.rs-7475722/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ef31656e-47f5-42b6-8279-25e8218e89e8","owner":[],"postedDate":"September 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-27T14:36:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-23 16:14:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7475722","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7475722","identity":"rs-7475722","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}