Preparation and application of nanocellulose bonded secondary amine molecular cage silica gel chiral stationary phase

Abstract

This invention discloses a nanocellulose-bonded secondary amine molecular cage silica chiral stationary phase, its preparation method, and its application, belonging to the field of chiral separation materials technology. The stationary phase uses 5mm silica spheres as a substrate, covalently bonding a secondary amine organic molecular cage RCC3-R based on (1R,2R)-cyclohexanediamine via a silanization reaction. Nanocellulose-tris(3,5-dimethylphenylcarbamate) (NCDMPC) is then bonded to its surface using a solvent evaporation method combined with isocyanate crosslinking technology, forming a multi-level synergistic recognition system that combines the three-dimensional cavity recognition capability of the molecular cage with the chirality of the nanocellulose helical conformation. The successful preparation of the stationary phase was verified by elemental analysis, infrared spectroscopy, and thermogravimetric analysis. The synergistic effect of hydrogen bonding, π-π stacking, and stereoconfinement significantly improves chiral separation performance. In normal-phase high-performance liquid chromatography (n-hexane / isopropanol) mode, 10 chiral compounds were successfully separated, with metalaxyl, 1-phenyl-1-propanol, 1-naphthylethanol, and p-methylphenylethanol achieving resolutions ≥1.5. In reversed-phase mode (methanol / water), 5 chiral compounds were separated, with metalaxyl achieving a resolution as high as 2.26. The nanocellulose-bonded secondary amine molecular cage silica chiral stationary phase exhibits good chiral recognition and separation performance, providing a theoretical basis for the study of the structure-property relationship of chiral separation materials.

Description

Technical Field

[0001] This invention relates to the field of chiral separation materials technology, specifically to a nanocellulose-bonded secondary amine molecular cage silica chiral stationary phase, its preparation method, and its application. Background Technology

[0002] Chirality is widespread in nature, and chiral resolution is a key technology in medicinal chemistry, biomedicine, and materials science. Different enantiomers of the same chiral compound can differ significantly in drug efficacy, toxicity, and metabolic pathways in vivo. Therefore, efficient separation of chiral molecules is crucial for drug development and quality control. High-performance liquid chromatography (HPLC) is a commonly used method for chiral resolution due to its speed, sensitivity, and wide applicability. The core of HPLC is the stationary phase. While traditional chiral stationary phases (such as cellulose, cyclodextrin, and proteins) are widely used, they still face problems such as poor stability, insufficient selectivity, and high cost. For example, existing cellulose-based stationary phases are prone to swelling and detachment under reversed-phase conditions; cyclodextrin stationary phases have fixed pore sizes, making them difficult to adapt to large molecule drugs. In recent years, the cross-integration of supramolecular stereostructures and nanomaterials has provided new ideas for the development of novel chiral separation materials. Porous organic molecular cages (POCs) are a class of novel porous materials with three-dimensional spatial structures, high specific surface areas, high porosity, and tunable chemical structures, and have been widely used in biology, gas storage, catalysis, drug delivery, and separation analysis. In separation analysis, secondary amine molecular cages (RCC3-R) exhibit excellent chiral selectivity due to their three-dimensional cavity structure and precise chiral recognition sites; however, single molecular cages suffer from low stationary phase loading and slow mass transfer. Nanocellulose, on the other hand, is an excellent medium for preparing chiral stationary phases due to its good orderliness, high specific surface area, abundant hydroxyl groups that are easy to functionalize. Its highly ordered helical spatial arrangement endows the inherent chiral centers in the three-dimensional conformation with excellent chiral selectivity. This invention utilizes nanocellulose to provide a stable framework and additional chiral sites, while molecular cages provide a three-dimensional cavity structure; the synergy of these two enhances stability and expands the recognition dimensions. Surface functionalization of RCC3-R was achieved by covalently bonding it to a silica substrate via silane coupling reaction. Subsequently, a nano-cellulose composite coating was constructed at the functionalized interface using solvent evaporation, creating a composite stationary phase with a multi-level chiral recognition interface. This invention leverages the synergistic effect of three-dimensional molecular cage recognition and the conformational chirality of cellulose derivatives to achieve highly efficient chromatographic separation of drug enantiomers, providing a new platform for high-throughput and highly selective separation of chiral drugs and demonstrating broad application prospects. Summary of the Invention

[0003] This invention aims to provide a chiral stationary phase co-modified with secondary amine molecular cages and nanocellulose in silica gel. By synergistically combining the three-dimensional recognition of the secondary amine molecular cages with the conformational chirality of nanocellulose, it solves the problems of poor stability, insufficient selectivity, and narrow applicability of traditional chiral stationary phases. Furthermore, it provides a preparation method and applications to meet the high-efficiency separation requirements in fields such as chiral drugs. To achieve the above objectives, the technical solution of this invention is as follows: This invention relates to the preparation of a chiral silica gel stationary phase with nanocellulose-bonded secondary amine molecular cages and its chromatographic evaluation of chiral drugs under two high-performance liquid chromatography (HPLC) separation modes. First, nanocellulose-tris(3,5-dimethylphenylcarbamate) was coated multiple times onto the surface of RCC3@SiO2 using an volatile solvent. Then, 4,4'-diphenylmethyl diisocyanate was added to initiate the reaction. After the reaction was complete, the mixture was washed and dried to obtain the final chiral silica gel stationary phase with nanocellulose-bonded secondary amine molecular cages. The chromatographic performance of this stationary phase was evaluated in both normal-phase and reversed-phase chromatographic separation modes for various chiral compounds, including alcohols, acids, and amines. The results demonstrated that this chiral stationary phase exhibits excellent chiral separation performance. This technology shows broad application prospects in chiral drug separation and provides an innovative approach for the development of novel chromatographic packing materials.

[0004] The preparation and chiral resolution properties of RCC3-R bonded to nanocellulose silica chiral stationary phase are characterized by the following steps: (1) Preparation process of nanocellulose-bonded secondary amine molecular cage silica chiral stationary phase. Nanocellulose-tris(3,5-dimethylphenylcarbamate) was placed in a beaker, dried tetrahydrofuran was added, and the mixture was dissolved by ultrasonication and filtered through filter paper. Nanocellulose was coated onto the surface of CNP-1 multiple times as a volatile solvent and dried under vacuum at 60°C for 6 h. A mixed solution of dried toluene and chloroform was added to the coated silica gel, along with 55 mg of 4,4'-diphenylmethyl diisocyanate. The mixture was refluxed at 80°C for 6 h, followed by the addition of 3,5-dimethylphenyl isocyanate and refluxed for 16 h. After the reaction was completed, the product was washed with toluene, acetone, methanol / dichloromethane (1:1), and anhydrous ethanol, respectively. After vacuum drying, the composite chiral stationary phase CSP-1 was obtained. Using the same method, the above steps were repeated with CNP-2 as the substrate to obtain the composite chiral stationary phase CSP-2.

[0005] In step (1) of this invention, the ratio of nanocellulose-tris(3,5-dimethylphenylcarbamate), molecular cage silica gel, 4,4'-diphenylmethyl diisocyanate, toluene, chloroform, and derivatizing reagent is 0.25-1 g: 2-5 g: 0.05-0.2 g: 50-100 mL: 50-100 mL: 2-4 mL. Depending on the reaction ratio, a high-performance liquid chromatography (HPLC) stationary phase with a loading of 5%-30% of nanocellulose-bonded molecular cages can be prepared. This invention allows for adjustment of the loading amount based on the amount of stationary phase, thereby improving column efficiency.

[0006] The nanocellulose-bonded secondary amine molecular cage silica chiral stationary phase prepared in this invention contains multiple secondary amines, benzene rings, chiral cyclohexanediamine, and nanocellulose molecules with a helical spatial arrangement. This allows for hydrophobic, hydrophilic, hydrogen bond, π-π, dipole-dipole, and ion exchange interactions between the stationary phase and chiral compounds, enhancing the chiral recognition ability between the stationary phase and the chiral compounds. Consequently, the stationary phase obtained in this invention exhibits excellent chiral separation performance, achieving the separation of chiral compounds in both normal-phase and reversed-phase chromatography modes, and holds promise as a novel material for application in the field of HPLC separation. Attached Figure Description

[0007] Figure 1 This is a schematic diagram illustrating the preparation of the nanocellulose-bonded secondary amine molecular cage silica chiral stationary phase in Example 1; Figure 2 The image shows a scanning electron microscope (SEM) image of the nanocellulose-bonded secondary amine molecular cage silica chiral stationary phase with loadings of 18.2% and 26.4% obtained in Example 1. Figure 3 The infrared spectra of the nanocellulose-bonded secondary amine molecular cage silica chiral stationary phases with loadings of 18.2% and 26.4% obtained in Example 1 are shown. Figure 4 Thermogravimetric analysis and nitrogen adsorption diagrams are shown for the nanocellulose-bonded secondary amine molecular cage silica chiral stationary phases with loadings of 18.2% and 26.4% obtained in Example 1. Figure 5 The following are the separation chromatograms of chiral compounds (1) propranolol hydrochloride, (2) metalaxyl, (3) quizalofop-p-ethyl, (4) 1-phenyl-1-propanol, (5) praziquantel, (6) benzoin, (7) benzoin methyl ether, (8) trans-benzoyl peroxide, (9) 1-naphthylethanol, and (10) p-methylphenylethanol in the normal phase chromatography separation mode of Example 2. Figure 6The following are the resolution chromatograms of chiral compounds (1) 6-hydroxyflavanone, (2) flavanone, (3) 6-methoxyflavanone, (4) metalaxyl, and (5) ranolazine in reversed-phase chromatography separation mode in Example 1. Figure 7 Example 3 shows the chromatogram of the effect of changes in alcohol content in the hexane / isopropanol mobile phase on chiral separation efficiency under normal phase chromatography separation mode. Figure 8 This is a chromatogram from Example 4, showing the effect of four alcohol modifiers (ethanol, n-propanol, isopropanol, and n-butanol) on the separation efficiency of the stationary phase in a normal-phase chromatographic separation mode in the n-hexane system. Figure 9 This is a chromatogram from Example 5, showing the separation efficiency of different mobile phase additives for chiral compounds under normal phase chromatography. Figure 10 This is a chromatogram from Example 6, showing the separation efficiency of the mobile phase at different flow rates for chiral compounds in normal phase chromatography. Detailed Implementation

[0008] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art. The examples are only used to better illustrate the present invention and are not intended to limit the present invention. Example

[0009] Preparation of a nanocellulose-bonded secondary amine molecular cage silica chiral stationary phase, the preparation process is as follows: Figure 1 ; First, secondary amine molecular cage functionalized silica intermediates were prepared: using porous silica with a particle size of 5 μm as a substrate, secondary amine organic molecular cages RCC3-R, constructed based on (1R,2R)-cyclohexanediamine, were covalently bonded to its surface via a silane coupling reaction. The mass ratio of RCC3-R to silica was controlled at 0.05~0.1:1. After washing and drying, two intermediates with different loadings were obtained, denoted as CNP-1 (RCC3-R loading of 8.2%) and CNP-2 (RCC3-R loading of 10.8%), respectively. 0.4850 g of nanocellulose was placed in a beaker, and 180 mL of dried tetrahydrofuran was added. The mixture was dissolved by ultrasonication and filtered through filter paper. The nanocellulose was coated three times onto the surface of 2.7038 g of RCC3@SiO2 (CNP-1 and CNP-2) using an evaporating solvent method, and then vacuum dried at 60 °C for 6 h. 3 g of coated silica gel was added to a mixture of 50 mL of dry toluene and 50 mL of chloroform, along with 55 mg of 4,4'-diphenylmethyl diisocyanate. The mixture was refluxed at 80 °C for 6 h, followed by the addition of 2 mL of 3,5-dimethylphenyl isocyanate and refluxed for 16 h. After the reaction was complete, the product was washed with toluene, acetone, methanol / dichloromethane (1:1), and anhydrous ethanol, respectively, and dried under vacuum to obtain the final chiral stationary phases CSP-1 (prepared from CNP-1, with a total organic loading of 18.2%) and CSP-2 (prepared from CNP-2, with a total organic loading of 26.4%).

[0010] Figure 2 The following are the scanning electron microscope (SEM) images of the stationary phases: (a) is the bare silicon sphere, (b) is CSP-1, and (c) is CSP-2. According to the analysis by scanning electron microscope (SEM), the bare silicon sphere is a smooth spherical particle, while the surface of the stationary phase spheres of CSP-1 and CSP-2 is rough, and there are obvious adhering substances on the surface of the spheres. Figure 3 The infrared spectra of the stationary phases are shown below: bare silicon spheres, CNP-1 / CNP-2, and CSP-1 / CSP-2 Fourier transform infrared (FT-IR) spectra. The results show that the peak at 1060 cm⁻¹ in the bare silicon spheres is clearly due to the asymmetric stretching vibration of Si-O-Si, and the peak at 3655 cm⁻¹ is the stretching vibration peak of OH. The presence of these peaks indicates the presence of a silicon-oxygen framework and surface hydroxyl groups in the compound. Furthermore, the peaks at 3342 cm⁻¹ and 1617 cm⁻¹ in the CNP and CSP series samples are the stretching vibration peak of NH and the characteristic peak of the bending vibration of the aromatic ring C=C (or NH), respectively, indicating successful bonding of the RCC3 molecular cage in the compound. By comparing the infrared spectra of CNP and CSP, the peak at 1726 cm⁻¹ in the CSP series samples... -1The characteristic peaks at the point can prove that carbonyl functional groups were successfully introduced into the sample surface, indicating that the molecular cage and nanofibers are bonded together, and the intensity change of the 1060 cm⁻¹ peak of the silicon-oxygen framework further supports the existence of surface modification. Figure 4 Figure 1 shows the thermogravimetric analysis (TGA) and nitrogen adsorption (TGA) curves of the prepared stationary phases. Figure 2(a) shows the TGA curves of bare silicon spheres, CNP-1 / CNP-2, and CSP-1 / CSP-2. The thermal stability of the prepared stationary phases was evaluated by TGA under nitrogen from 25 °C to 800 °C. As shown in the figure, the thermal stability of the stationary phases at 100 °C is... The weight loss at ℃ is due to the evaporation of water adsorbed on the surface silica gel; a significant second-stage weight loss is observed in the 250-450℃ range, which is attributed to the decomposition of the RCC3 molecular cages on the silica gel surface, with CNP-1 and CNP-2 loadings of 8.2% and 10.8%, respectively; a third weight loss occurs in the 300-500℃ range, corresponding to the thermal degradation of the nanocellulose derivatives. Based on the comprehensive analysis of the total weight loss rate, the loadings of CSP-1 and CSP-2 reach 18.2% and 26.4%, respectively, which are significantly higher than those of the CNP series, confirming that molecular cages and nanocellulose derivatives are simultaneously bonded to their surfaces; Figure (b) is the nitrogen adsorption (BET) characterization diagram of bare silica spheres and CSP-1 / CSP-2. It can be seen that all materials exhibit typical type IV isothermal characteristics, with significant adsorption-desorption hysteresis curves in the relative pressure range of 0.45-0.9, confirming the presence of capillary condensation in the system with a mesoporous structure. Specifically, the original silica gel exhibits typical monolayer adsorption characteristics in the low-pressure region (P / P0=0.05-0.3), with a BET specific surface area of ​​241 m² / g, a pore volume of 0.54 cm³ / g, and an average pore size of 8.6 nm. After functionalization modification, the specific surface area of ​​CSP-1 is significantly reduced to 111 m² / g, the pore volume is reduced to 0.18 cm³ / g, and the pore size is narrowed to 5.6 nm. Similarly, CSP-2 has a specific surface area of ​​121 m² / g, a pore volume of 0.17 cm³ / g, and an average pore size of 5.3 nm. Example

[0011] The CSP-1(a) and CSP-2(b) obtained in Example 1 were used to test their separation effect on chiral compounds. The chromatograms of the separation are shown in [Figure 1]. Figure 5 ; Figure 5This is a chromatogram of the stationary phase of the present invention under normal phase chromatographic conditions for the separation of chiral compounds (1) propranolol hydrochloride, (2) metalaxyl, (3) quizalofop-p-ethyl, (4) 1-phenyl-1-propanol, (5) praziquantel, (6) benzoin, (7) benzoin methyl ether, (8) trans-benzoyl peroxide, (9) 1-naphthylethanol, and (10) p-methylphenylethanol; mobile phase: (1-4) hexane / isopropanol (80 / 20), (5-9) hexane / isopropanol (90 / 10), (10) hexane / isopropanol (95 / 5); flow rate: 1 mL / min; detection wavelength: 254 nm; temperature: 25 °C; column pressure: 980 psi(a), 1050 The results (psi(b)) demonstrate that the complex interactions between the nanocellulose-bonded secondary amine molecular cage silica chiral stationary phase and the chiral compounds result in strong resolution of these 10 chiral compounds under normal-phase chromatography. CSP-1 showed resolutions higher than 1.5 for four compounds (metalaxyl, 1-phenyl-1-propanol, 1-naphthylethanol, and p-methylphenylethanol), and higher than 0.85 for the remaining four compounds, superior to CSP-2. This may be due to the introduction of the RCC3-R structure enhancing the stereoselectivity of the chiral cavity of the stationary phase, while optimizing hydrogen bonding and π-π interactions, thus significantly improving chiral recognition. However, excessive loading may lead to pore blockage and reduced mass transfer efficiency. Notably, the resolution of metalaxyl on CSP-2 was significantly higher than that on CSP-1, possibly related to the high matching degree of metalaxyl on its unique RCC3-R structure. The experimental results indicate that CSP-1 outperforms CSP-2 in terms of retention capacity, resolution, and versatility, providing a new strategy for the efficient resolution of complex chiral compounds.

[0012] Figure 6 This is a chromatogram of the stationary phase of the present invention under reversed-phase chromatographic conditions for the separation of chiral compounds (1) 6-hydroxyflavanone, (2) flavanone, (3) 6-methoxyflavanone, (4) metalaxyl, and (5) ranolazine; mobile phase: (1) methanol / water (70 / 30), (4-5) methanol / water (80 / 20); flow rate: 1 mL / min; detection wavelength: 254 nm; temperature: 25℃; column pressure: 1905 psi (a), 2030 psi (b); from Figure 5 It can be seen that the resolution of flavanones on CSP-1 is significantly higher than that on CSP-2. The resolution of 6-methoxyflavanone on CSP-1 is 1.17, while the resolution on CSP-2 is only 0.71. Furthermore, ranolazine can still be effectively separated on CSP-1, while the resolution on CSP-2 is only 0.40, indicating that CSP-1 is more advantageous in the separation of compounds with complex structures. The resolution of metalaxyl on CSP-2 is 2.26, significantly higher than that on CSP-1, which may be related to the high matching degree of its unique RCC3-R structure with metalaxyl. Example

[0013] The effect of changes in alcohol content in the hexane / isopropanol mobile phase on chiral separation efficiency was investigated using the CSP-1 obtained in Example 1 under normal phase chromatography. The separation results are shown in [Figure 1]. Figure 7 ; Figure 7 The chromatographic conditions and separated compounds were as follows: mobile phase: n-hexane / isopropanol (95:5, 85 / 15, 75 / 25, v / v); flow rate: 1.0 mL / min; detection wavelength: 254 nm; analytes: (a) benzoin, (b) 1-naphthylethanol. As can be seen from Example 3, by adjusting the volume fraction of isopropanol, a significant correlation was found between the polarity of the solvent system and the solute retention behavior. As the isopropanol content gradually increased from 5% to 25%, the elution intensity of the mobile phase continuously increased, which significantly weakened the stereoselective interaction between the chiral compound and the chiral recognition site of the stationary phase. When the isopropanol addition was 5%, the system exhibited optimal separation performance. The experimental results confirm that by precisely controlling the mobile phase composition, dual optimization of chromatographic retention behavior and separation selectivity can be achieved. Example

[0014] The effects of four alcohol modifiers (ethanol, n-propanol, isopropanol, and n-butanol) on the separation efficiency of the stationary phase in a normal-phase chromatography system were investigated using the CSP-1 obtained in Example 1. The separation results are shown in [Figure 1]. Figure 8 ; Figure 8 Chromatographic conditions: Mobile phase: n-hexane / alcohol modifier (90 / 10); Flow rate: 1 mL / min; Detection wavelength: 254 nm; Temperature: 25℃; Analytes: (a) quizalofop-p-ethyl, (b) benzoin methyl ether.

[0015] As can be seen from Example 4, due to the differences in molecular structure and polarity, different alcohol modifiers for two chiral compounds will have specific interactions with the chiral recognition sites and enantiomers of the stationary phase, thereby significantly affecting the separation selectivity. Example

[0016] The effect of mobile phase additives on the resolution performance of chiral compounds was investigated using CSP-1 with the loading obtained in Example 1 in normal phase chromatography. The separation results are shown in [Figure 1]. Figure 9 ; Figure 9 Chromatographic conditions: Mobile phase: n-hexane / isopropanol (90 / 10), additives tetrahydrofuran (THF), acetone and chloroform (0%, 1%, 3%, 5%); flow rate 1 mL / min; detection wavelength: 254 nm; temperature: 25℃; analytes: (a) benzoin methyl ether, (b) quizalofop-p-ethyl.

[0017] As can be seen from Example 5, when the mobile phase additive is tetrahydrofuran at a concentration of 1%, the chiral separation effect of the two compounds is the best; for the chloroform additive, benzoin methyl ether has the best separation at a concentration of 3%, while quizalofop-P-ethyl has the best separation at 1% chloroform; for the acetone additive, both chiral compounds have poor separation, so the acetone additive is not suitable as a mobile phase component used in conjunction with this stationary phase. Example

[0018] The effect of different mobile phase flow rates on the separation performance of chiral compounds was investigated using the CSP-1 loading obtained in Example 1 in normal phase chromatography. The separation results are shown in [Figure 1]. Figure 10 ; Figure 10 Chromatographic conditions: Mobile phase: n-hexane / isopropanol (80 / 20, v / v); Flow rate: 0.3 mL / min, 0.5 mL / min, 0.8 mL / min, 1 mL / min; Detection wavelength: 254 nm; Temperature: 25 °C; Analytes: (a) benzoin, (b) 1-naphthylethanol.

[0019] As can be seen from Example 6, although a high degree of separation can be obtained when the flow rate is reduced to 0.3 mL / min, the excessively long analysis time leads to a significant increase in mobile phase consumption, which contradicts the concept of green chemistry. By balancing multiple dimensions such as separation efficiency, analysis speed, and environmental friendliness, 1.0 mL / min was finally determined as the optimal flow rate parameter for this chiral stationary phase system.

[0020] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the description and drawings of the present invention, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A nanocellulose-bonded secondary amine molecular cage silica chiral stationary phase, characterized in that, Using porous silica gel as a substrate, RCC3-R is covalently bonded to the surface via silane coupling reaction. The RCC3-R is further covalently linked to nanocellulose-tris(3,5-dimethylphenylcarbamate) (NCDMPC) via 4,4'-diphenylmethyl diisocyanate crosslinking reaction, forming a composite structure with a multi-level chiral recognition interface consisting of a secondary amine cavity, an aromatic benzene ring, a chiral cyclohexanediamine unit, and a helical nanocellulose chain.

2. A method for preparing the composite chiral stationary phase as described in claim 1, characterized in that, Includes the following steps: (1) Dissolve NCDMPC in tetrahydrofuran, sonicate to dissolve and filter to obtain NCDMPC solution; (2) The NCDMPC solution was applied to the RCC3@SiO2 surface in multiple applications and dried under vacuum at 60°C for 6 hours to obtain coated silicon spheres; (3) The coated silica gel was dispersed in a toluene-chloroform mixed solution, and 4,4'-diphenylmethyl diisocyanate was added. The mixture was refluxed at 80°C for 6 hours. Then, 3,5-dimethylphenyl isocyanate was added, and the mixture was refluxed for another 16 hours. (4) After the reaction is complete, the mixture is washed sequentially with toluene, acetone, methanol / dichloromethane (1:1) and anhydrous ethanol, and then dried under vacuum to obtain the composite chiral stationary phase. The RCC3@SiO2 is a functionalized silica gel obtained by covalently bonding RCC3-R to the surface of porous silica gel using a silane coupling agent, wherein RCC3-R is a secondary amine organic molecular cage based on (1R,2R)-cyclohexanediamine. The nanocellulose-tris(3,5-dimethylphenylcarbamate) is a chiral derivative obtained by reacting nanocellulose with 3,5-dimethylphenylisocyanate.

3. The preparation method according to claim 2, characterized in that, In step (1), the mass-to-volume ratio of NCDMPC to tetrahydrofuran is 0.25-1 g: 150-200 mL; in step (3), the mass ratio of NCDMPC to RCC3@SiO2 is 1:4 to 1:6, the volume ratio of toluene to chloroform is 1:1, and the amounts of 4,4'-diphenylmethyl diisocyanate and 3,5-dimethylphenyl isocyanate are 15-60 mg: 1.5-3 mL.

4. The preparation method according to claim 3, characterized in that, In the preparation of RCC3@SiO2, the mass ratio of RCC3-R to silica gel is 0.05~0.1:

1.

5. An application of the composite chiral stationary phase as described in claim 1, characterized in that, Used for the separation of chiral compounds in high performance liquid chromatography, it can be used in both normal and reversed phase chromatography modes.

6. The application as described in claim 5, characterized in that, The chiral compounds are selected from β-receptor blockers (such as propranolol hydrochloride), amide fungicides (such as metalaxyl and quizalofop-p-ethyl), flavonoid derivatives (such as flavanones and 6-hydroxyflavanones), aromatic secondary alcohols (such as 1-phenyl-1-propanol and 1-naphthylethanol), or chiral amine compounds (such as ranolazine). In normal-phase mode, ten chiral compounds can be separated: propranolol hydrochloride, metalaxyl, quizalofop-p-ethyl, 1-phenyl-1-propanol, praziquantel, benzoin, benzoin methyl ether, trans-benzoyl oxadiene, 1-naphthylethanol, and p-methylphenylethanol. In reverse-phase mode, five chiral compounds can be separated: 6-hydroxyflavanone, flavanone, 6-methoxyflavanone, metalaxyl, and ranolazine.

7. The application as described in claim 6, characterized in that, The reverse phase separation of the composite chiral stationary phase relative to metalaxyl is 2.26; the normal phase separation of metalaxyl, 1-phenyl-1-propanol, 1-naphthylethanol, and p-methylphenylethanol is ≥1.5.

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