Preparation and application of general secondary amine molecular cage-nanocellulose composite chiral stationary phase for positive and negative phases

Abstract

The invention discloses a general secondary amine molecular cage-nanocellulose composite chiral stationary phase for positive and negative phases as well as a preparation method and application of the general secondary amine molecular cage-nanocellulose composite chiral stationary phase. A secondary amine molecular cage (RCC3-R) modified silica gel is used as a substrate, a nano cellulose derivative (NCDMPC) is loaded through a coating method to prepare two chiral stationary phases (CSP-1 and CSP-2), and aminopropyl silica gel is used as a substrate to prepare a control chiral stationary phase (CSP-3). By utilizing the three-dimensional cavity screening effect of RCC3-R and the synergistic effect of an NCDMPC chiral spiral skeleton, the common use of normal phase and reversed phase chromatography modes is realized, and the chiral recognition and separation performance is superior to that of CSP-3. 15 chiral compounds can be split in a normal-phase mode, 12 chiral compounds can be split in a reverse-phase mode, excellent splitting performance on alcohols, amines and chiral drugs is shown, a novel material can be provided for efficient separation of complex systems such as chiral drugs and pesticides, and the application prospect is wide.

Description

Technical Field

[0001] This invention belongs to the field of high performance liquid chromatography (HPLC) separation technology, specifically relating to a secondary amine molecular cage-nanocellulose composite chiral stationary phase that is suitable for both normal-phase and reverse-phase chiral separation. This HPLC chiral stationary phase is mainly used for the separation of chiral compounds. Background Technology

[0002] Chiral separation plays a crucial role in fields such as medicine, environment, and food. High-performance liquid chromatography (HPLC) is currently the mainstream chiral separation technique, and its separation effect is highly dependent on the performance of the chiral stationary phase. Traditional chiral stationary phases, such as polysaccharides and cyclodextrins, have drawbacks such as low separation efficiency, poor selectivity, and limited applicable chromatographic modes, making it difficult to meet the separation requirements of complex chiral systems.

[0003] To address the aforementioned issues, researchers are dedicated to developing novel multifunctional chiral stationary phases. Among these, porous organic cages (POCs), as a class of crystalline porous materials with well-defined cavity structures, have become a research hotspot in the field of chiral separation due to their precisely tunable molecular cavity size, abundant host-guest recognition sites, and good structural stability. Secondary amine molecular cages (RCC3-R), a typical representative of POCs, are prepared from trimesaldehyde and cyclohexanediamine via a Schiff base-reduction reaction. They possess high symmetry and suitable cavity size, enabling specific enantiomer recognition through size exclusion effects and non-covalent interactions (hydrogen bonds, π-π stacking, hydrophobic interactions, etc.). This provides an ideal framework material for constructing high-performance chiral stationary phases, and patents already exist for their use in gas chromatography columns (patent CN201911325921.4) and liquid chromatography columns (patent CN202111589677.X).

[0004] Meanwhile, cellulose nanoparticles, as natural and renewable biomaterials, possess advantages such as large specific surface area, abundant surface hydroxyl groups, and good biocompatibility. Their derivatives can be chemically modified to introduce chiral recognition sites, resulting in higher chiral loading and more exposed action sites compared to traditional cellulose materials. This demonstrates potential application value in chiral separation, and a patent already exists for its use in liquid chromatography columns (patent CN201610321675.5). However, chiral stationary phases prepared solely using cellulose nanoparticle derivatives as the active component still suffer from a limited chiral recognition mechanism and restricted applicability, hindering the full realization of their performance advantages. While there have been attempts to prepare chiral stationary phases by combining porous materials with cellulose, there are no reports of synergistically combining RCC3-R with cellulose nanoparticle derivatives and systematically verifying the advantages of secondary amine molecular cage silica gel (RCC3@SiO2) substrates using aminopropyl silica gel (a commonly used substrate) as a control. Furthermore, most existing composite stationary phases fail to achieve universal compatibility between normal / reverse phase modes, and the improvement in separation performance is limited, making it difficult to meet the high-efficiency separation requirements of complex chiral systems. Therefore, developing a composite chiral stationary phase with RCC3@SiO2 as the core substrate and loaded with nanocellulose derivatives, constructing multiple chiral recognition sites through the synergistic effect of the two, achieving universality of normal / inverse phase modes, and clarifying its performance advantages compared with traditional aminopropyl silica substrates is of great significance for breaking through the technical bottleneck of traditional chiral stationary phases, expanding the application scenarios of chiral separation materials, and providing a new solution for chiral separation technology. Summary of the Invention

[0005] The purpose of this invention is to provide a universally applicable secondary amine molecular cage-nanocellulose composite chiral stationary phase, its preparation method, and its application, which solves the problems of limited applicability and separation performance of existing chiral stationary phases. At the same time, by comparing it with the aminopropyl silica gel control stationary phase, the advantages of the RCC3@SiO2 substrate are highlighted.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: This invention relates to the preparation of a secondary amine molecular cage-nanocellulose composite chiral stationary phase and its chromatographic evaluation of chiral drugs under two separation modes in high-performance liquid chromatography (HPLC). First, nanocellulose-tris(3,5-dimethylphenylcarbamate) (NCDMPC) was coated multiple times onto the surface of RCC3@SiO2 using an volatile solvent, followed by drying to obtain the final RCC3-NCDMPC@SiO2 chiral stationary phase. Subsequently, the chromatographic performance of various chiral compounds, including alcohols, acids, and amines, was evaluated under normal-phase and reversed-phase chromatographic separation modes. The results confirmed that the RCC3@SiO2-based chiral stationary phase exhibits excellent chiral resolution performance, superior to that of chiral stationary phases based on aminopropyl silica gel, and demonstrates universal adaptability for both normal and reverse-phase chromatography. This technology shows broad application prospects in chiral drug separation and provides an innovative approach for the development of novel chromatographic packing materials.

[0007] Preparation and chiral resolution properties of a secondary amine molecular cage-nanocellulose composite chiral stationary phase, characterized by the following steps in the preparation method: (1) Preparation process of secondary amine molecular cage-nanocellulose composite chiral stationary phase. Nanocellulose-tris(3,5-dimethylphenylcarbamate) was placed in a beaker, dried tetrahydrofuran was added, ultrasonically dissolved, and filtered with filter paper; the filtered NCDMPC solution was coated onto the surface of RCC3@SiO2 in multiple batches by evaporating solvent, and dried under vacuum at 60℃ for 6 h to obtain RCC3-NCDMPC@SiO2 chiral stationary phases (CSP-1 and CSP-2); the NCDMPC solution was coated onto the surface of aminopropyl silica gel using the same method to obtain the control stationary phase CSP-3, which was then packed into a chromatographic column to obtain a high performance liquid chromatography column that can be used for normal-phase and reverse-phase chiral separation.

[0008] In step (1) of this invention, the ratio of NCDMPC, RCC3@SiO2, and tetrahydrofuran is 0.45-0.5 g: 2.55-2.65 g: 180-200 mL. By adjusting the above material ratio, RCC3@SiO2 substrate materials with different RCC3-R bonding amounts can be precisely prepared, thereby obtaining a performance-tunable RCC3-NCDMPC@SiO2 high-performance liquid chromatography chiral stationary phase.

[0009] The secondary amine molecular cage-nanocellulose composite chiral stationary phase prepared in this invention contains multiple secondary amines, benzene rings, chiral cyclohexanediamine, and nanocellulose molecules with a helical spatial arrangement. These components enable hydrophobic, hydrophilic, hydrogen bond, π-π, dipole-dipole, and ion exchange interactions between the stationary phase and chiral compounds, significantly enhancing the chiral recognition ability between the stationary phase and the chiral compounds. This results in a stationary phase with excellent chiral resolution performance, capable of separating 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

[0010] Figure 1 This is a schematic diagram illustrating the preparation of the secondary amine molecular cage-nanocellulose composite chiral stationary phase in Example 1; Figure 2 The images shown are scanning electron microscope images of the stationary phases used in Example 1 of this invention, where (a) SiO2, (b) CSP-1, (c) CSP-2, and (d) CSP-3. Figure 3 The infrared spectrum of the stationary phase prepared in Example 1 of the present invention includes SiO2, CSP-1, CSP-2 and CSP-3; Figure 4 Thermogravimetric analysis (a) and nitrogen adsorption (b) of the stationary phase in Example 1 are shown. Figure 5 The following are the separation chromatograms of CSP-1 and CSP-2 in normal phase chromatography separation mode in Example 2: (a) phenylethanol, (b) p-methylphenylethanol, (c) 1-phenyl-1-propanol, (d) 1-naphthylethanol, (e) benzoin, (f) flavanone, (g) 6-methoxyflavanone, (h) trans-oxobenzoic acid, and (i) metalaxyl. Figure 6 The following is a chromatogram of the CSP-3 in Example 2, in normal phase chromatographic separation mode, showing (a) phenylethanol, (b) p-methylphenylethanol, (c) 1-phenyl-1-propanol, (d) 1-naphthylethanol, (e) benzoin, (f) flavanone, (g) 6-methoxyflavanone, (h) trans-oxobenzoic acid, and (i) metalaxyl. Figure 7 The following are the resolution chromatograms of CSP-1 and CSP-2 in Example 2 under reversed-phase chromatography separation mode: (a) p-methylphenylethanol, (b) 1-phenyl-1-propanol, (c) 1-naphthylethanol, (d) flavanone, (e) 6-hydroxyflavanone, (f) 6-methoxyflavanone, (g) 2-hydroxyflavanone, (h) trans-benzoyl oxonide, (i) benzoyl methyl ether, and (j) triazolone. Figure 8The chromatograms of CSP-1 and CSP-2 in Example 3, separated in normal phase chromatographic mode, are as follows: (a) tetraimidazole hydrochloride, (b) quinolone, (c) ranolazine, (d) metoprolol, (e) triadimefon, (f) tebuconazole, (g) metalaxyl, and (h) quizalofop-p-ethyl. Figure 9 This is the chromatogram of CSP-1 and CSP-2 used in Example 4 to examine their stability in reversed-phase chromatography. Figure 10 This is a chromatogram of the chiral stationary phase of the present invention in Example 4, after multiple switching between normal / reversed phase chromatographic modes, demonstrating its separation efficiency for chiral compounds. Detailed Implementation

[0011] 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 embodiments are only used to better illustrate the present invention and are not intended to limit the present invention.

[0012] Example 1 Preparation of secondary amine molecular cage-cellulose nanocomposite chiral stationary phase, the preparation process is as follows: Figure 1 ; 0.450 g of nanocellulose was placed in a beaker, 180 mL of dried tetrahydrofuran was added, and the mixture was dissolved by ultrasonication and filtered through filter paper. The nanocellulose solution was coated onto the surfaces of two RCC3@SiO2 in multiple applications as an evaporating solvent. After vacuum drying at 60 °C for 6 h, chiral composite stationary phases (CSP-1 and CSP-2) with secondary amine molecular cages and nanocellulose having RCC3-R loadings of 8% and 12% were obtained. The control stationary phase CSP-3 was prepared by coating NCDMPC solution onto the surface of aminopropyl silica gel using the same method.

[0013] Figure 2 The following are the scanning electron microscope (SEM) images of the stationary phases: (a) bare silicon spheres, (b) CSP-1, (c) CSP-2, and (d) CSP-3. Analysis by scanning electron microscope (SEM) shows that the bare silicon spheres are smooth spherical particles, while the stationary phase spheres of CSP-1, CSP-2, and CSP-3 have rough surfaces and obvious deposits on their surfaces, indicating that NCDMPC was successfully coated onto the RCC3@SiO2 surface. Figure 3 The infrared spectra of the stationary phases are shown, specifically those of SiO2, CSP-1, CSP-2, and CSP-3. Infrared spectral analysis reveals that SiO2 exhibits a broad absorption peak in the 3400-3500 cm⁻¹ range, corresponding to the OH stretching vibration, and a characteristic peak of the Si-O-Si stretching vibration near 1057 cm⁻¹, indicating a typical inorganic silicon-oxygen framework structure. Meanwhile, the peak at 2928 cm⁻¹...-1 and 2841cm -1 The peaks at 1713 cm⁻¹ are characteristic absorption peaks of methyl and methylene groups in CSP-1 and CSP-2. The infrared spectra of CSP-1, CSP-2 and CSP-3 show a C=O stretching vibration peak at 1713 cm⁻¹ and an absorption peak of -NH group at 3399 cm⁻¹, confirming the existence of the urethane ester structure (-NH-CO-O-), indicating that NCDMPC has been successfully coated on RCC3@SiO2 and aminopropyl silica gel.

[0014] Figure 4 Thermogravimetric analysis and nitrogen adsorption diagrams of the stationary phases are shown in (a) and (b) are thermogravimetric analysis diagrams of SiO2, CSP-1 and CSP-2. Thermogravimetric analysis experimental results ( Figure 4 a) This indicates that the RCC3-NCDMPC@SiO2 chiral stationary phase is a thermally stable material; the weight loss at 30-100℃ is the thermal loss due to the physical adsorption of water by SiO2, while the weight loss at 200-800℃ mainly originates from the thermal decomposition of NCDMPC and RCC3-R. The weight loss rate of CSP-2 (23.56%) is higher than that of CSP-1 (20.82%), consistent with the higher CC3-R bonding amount of CSP-2. Furthermore, neither stationary phase shows significant weight loss below 200℃, indicating good thermal stability under conventional chromatographic operating temperatures; N2 adsorption / desorption isotherms ( Figure 4 b) The results show that the BET surface area of ​​SiO2, CSP-1, and CSP-2 is 253.96 m². 2 / g, 142.04m 2 / g and 122.37m 2 / g, the isothermal adsorption curves of RCC3-NCDMPC@SiO2 all conform to the type IV isotherm.

[0015] Example 2 The separation effect of CSP-1, CSP-2, and CSP-3 obtained in Example 1 on chiral compounds is shown in the chromatograms of the separation. Figure 5 , Figure 6 ; Figure 5 This is a chromatogram showing the separation of chiral compounds (a) phenethyl alcohol, (b) p-methylphenethyl alcohol, (c) 1-phenyl-1-propanol, (d) 1-naphthylethanol, (e) benzoin, (f) flavanone, (g) 6-methoxyflavanone, (h) trans-benzoyl peroxide, and (i) metalaxyl under normal-phase chromatographic conditions. Mobile phase: n-hexane / isopropanol (90 / 10); flow rate: 1 mL / min; detection wavelength: 254 nm; temperature: 25 °C; column pressure: 980 psi (a), 1050 psi (b); from Figure 5It can be demonstrated that the complex interaction between the RCC3-NCDMPC@SiO2 chiral stationary phase and the chiral compounds enables the stationary phase to have a strong separation ability for these nine chiral compounds under normal phase chromatography conditions. CSP-1 is superior to CSP-2 in terms of retention capacity, resolution and versatility. This difference is due to the fact that the amount of RCC3-R bonding regulates the polarity-hydrophobicity balance of the stationary phase surface, thereby changing the dominant force of the host-guest interaction. CSP-1 has moderate surface hydrophobicity and high polarity matching with the normal phase mobile phase, which is conducive to the recognition of weakly polar molecules, providing a new strategy for the efficient separation of complex chiral compounds. Figure 6 This is the CSP-3 resolution chromatogram for nine chiral compounds, compared to... Figure 5 CSP-3 was less effective than CSP-1 and CSP-2 in separating chiral compounds. Under normal phase chromatography, RCC3-R and NCDMPC had a synergistic enhancement effect, which improved the enantiomeric separation efficiency.

[0016] Figure 7 The stationary phase of this invention, under reversed-phase chromatographic conditions, comprises chiral compounds (a) p-methylphenylethanol, (b) 1-phenyl-1-propanol, (c) 1-naphthylethanol, (d) flavanones, (e) 6-hydroxyflavanones, (f) 6-methoxyflavanones, (g) 2-hydroxyflavanones, (h) trans-benzoyl oxadiene, (i) benzoyl methyl ether, and (j) triazolones; mobile phase: methanol / water (80 / 20, V / V); flow rate: 1.0 mL / min; detection wavelength: 254 nm; temperature: 25 °C; column pressure: 1905 psi (a), 2030 psi (b); from Figure 7 It can be seen that CSP-1 is superior to CSP-2 in normal phase chromatography; in reversed phase chromatography, CSP-2, by balancing the RCC3-R bond amount and structural compatibility, exhibits better separation performance than CSP-1. This is because CSP-2, with its increased RCC3-R density and more polar sites, interacts more gently with the reversed phase mobile phase, demonstrating a more stable and broader separation advantage for polar molecules. Example 3 The separation performance of chiral drugs was investigated using CSP-1 and CSP-2 obtained in Example 1. The separation effect is shown in [Figure 1]. Figure 8 ; Figure 8The chromatographic conditions and the separated compounds were as follows: mobile phase: n-hexane / isopropanol (95:5, v / v) (ab, df, h), methanol / water (95:5, v / v) (c, g); flow rate: 1.0 mL / min; detection wavelength: 254 nm. nm; Analytes: (a) tetraimidazole hydrochloride, (b) quinolones, (c) ranolazine, (d) metoprolol, (e) triadimefon, (f) tebuconazole, (g) metalaxyl, (h) quizalofop-p-ethyl; Eight chiral drugs were separated to varying degrees on CSP-1 and CSP-2. CSP-1 showed significantly better separation of metoprolol, triadimefon, tebuconazole, and quizalofop-p-ethyl in normal phase mode than CSP-2, with quizalofop-p-ethyl achieving a resolution of 3.08 on CSP-1. CSP-2, however, showed better separation of quinolones in normal phase mode. The resolution of quinolones on CSP-2 (Rs=1.61) was significantly higher than that on CSP-1 (Rs=1.05). The results indicate that CSP-1 and CSP-2 have their own advantages and disadvantages in chiral separation, and selecting a suitable stationary phase and chromatographic mode is crucial for the separation of specific chiral compounds.

[0017] Example 4 The stability of the chromatographic columns was investigated using CSP-1 and CSP-2 obtained in Example 1, and the separation results are shown in [see figure]. Figure 9 ; Figure 9 Chromatographic conditions: Mobile phase: methanol / water (80 / 20, v / v); flow rate: 1.0 mL / min; detection wavelength: 254 nm; temperature: 25℃; analytes: (a) metalaxyl, (b) 1-phenyl-1-propanol. As shown in Example 6, the intraday relative standard deviation (RSD) was calculated by injecting samples five times consecutively on the same day, and the interday RSD was calculated by injecting samples once a day for five consecutive days. The results showed that the intraday RSDs of CSP-1 and CSP-2 for metalaxyl and 1-phenyl-1-propanol were both less than 0.40%, and the interday RSDs were both less than 0.50%, indicating that the stationary phase had good repeatability.

[0018] Simultaneously, the CSP-2 stationary phase was selected to investigate the switching between forward and reverse modes, such as... Figure 10 As shown, after three normal / reverse phase mode switchings, CSP-2 still performed well in separating chiral compounds. After the first switch to reverse phase mode, the retention times of metalaxyl and 1-phenyl-1-propanol were extended in normal phase mode, but after the second switch to reverse phase mode, the retention times remained almost unchanged. For reverse phase mode, the normal / reverse phase mode switching had almost no effect on the separation of chiral compounds. This indicates that RCC3-NCDMPC@SiO2 successfully achieved both normal and reverse phase separation.

[0019] 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 content of the present invention's specification and drawings, 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 universally applicable secondary amine molecular cage-cellulose nanocomposite chiral stationary phase, characterized in that: Using RCC3@SiO2, a secondary amine molecular cage (RCC3-R) prepared by Schiff base reaction-reduction of pyromellitic trimethylolpropane and cyclohexanediamine, as a substrate, nanocellulose-tris(3,5-dimethylphenylcarbamate) (NCDMPC) is loaded onto the substrate. The composite chiral stationary phase contains secondary amine, benzene ring, chiral cyclohexanediamine, and helically arranged nanocellulose molecules, which can achieve chiral recognition through hydrophobic, hydrophilic, hydrogen bonding, π-π, dipole-dipole, and ion exchange interactions. It is also compatible with both normal-phase and reversed-phase chromatographic separation modes, and can effectively recognize chiral enantiomers in both hexane / isopropanol and methanol / water systems.

2. The composite chiral stationary phase according to claim 1, characterized in that: The RCC3-R is prepared by a Schiff base-reduction reaction of pyromellitic aldehyde and cyclohexanediamine. The ratio of NCDMPC, RCC3@SiO2, and tetrahydrofuran is 0.45-0.5g:2.55-2.65g:180-200mL. This ratio can be used to control the amount of RCC3-R bonded in the RCC3@SiO2 substrate, thereby obtaining an RCC3-NCDMPC@SiO2 high-performance liquid chromatography chiral stationary phase with adjustable chiral recognition and separation performance.

3. A method for preparing the composite chiral stationary phase according to claim 1, characterized in that, Includes the following steps: (1) Place NCDMPC in a beaker, add dried tetrahydrofuran, dissolve by sonication, and filter with filter paper to obtain NCDMPC filtrate; (2) The NCDMPC filtrate from step (1) was coated onto the RCC3@SiO2 surface multiple times as an evaporating solvent and dried under vacuum at 60°C for 6 hours to obtain nanocellulose-modified RCC3 molecular cage silica chiral stationary phases (referred to as CSP-1 and CSP-2 respectively). (3) Using the same operation as in steps (1)-(2), the NCDMPC filtrate was coated onto the surface of aminopropyl silica gel to prepare the control stationary phase (denoted as CSP-3). (4) The stationary phase obtained in step (2) or (3) is loaded into the chromatographic column to obtain a high performance liquid chromatography column that can be used for normal phase and reversed phase chiral separation.

4. The preparation method according to claim 3, characterized in that: In step (2), the RCC3-R loading of RCC3@SiO2 is 8%-12%. By adjusting the ratio of molecular cage to silica gel, the amount of RCC3-R bonding can be precisely controlled, thereby adjusting the chiral separation performance of the composite stationary phase.

5. An application of the composite chiral stationary phase according to claim 1, characterized in that: This is used for the separation of chiral compounds in high performance liquid chromatography, including alcohols (phenylethanol, p-methylphenylethanol, 1-phenyl-1-propanol, etc.), acids, amines, and chiral drugs (tetraimidazole hydrochloride, quinolones, metoprolol, triazolone, etc.), and is compatible with normal phase and reversed phase chromatography separation modes.

6. The application according to claim 5, characterized in that: The composite chiral stationary phase exhibits no significant weight loss below 200℃ and good thermal stability. During continuous injection, the intraday relative standard deviation (RSD) for metalaxyl and 1-phenyl-1-propanol is <0.40%, and the interday RSD is <0.50%. After three normal / reverse phase mode switchings, the resolution efficiency of chiral compounds does not decrease significantly.

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