Chiral stationary phase of poria acid polysaccharide and application

Abstract

The present application relates to the technical field of liquid chromatography column chiral stationary phase, in particular to a poria acid polysaccharide chiral stationary phase and application. The poria acid polysaccharide and its derivatives have spectral and high-efficiency chiral resolution capacity, and can realize effective resolution of chiral compounds in normal phase chromatography mode, so as to further obtain single configuration drugs, realize universal application of single configuration drugs, provide a new sugar-based stationary phase on the basis of traditional sugar-based stationary phases such as cellulose, amylose and beta-cyclodextrin, and enrich the range of chiral selectors. The chiral high-performance liquid chromatography column has the characteristics of fast separation speed, good resolution effect, good stability and repeated use.

Description

Technical Field

[0001] This invention relates to the field of chiral stationary phase technology in liquid chromatography, specifically to chiral stationary phases based on Poria cocos acidic polysaccharides and their applications. Background Technology

[0002] Chirality is the most common phenomenon in nature and a fundamental property of chiral substances. Specifically, it manifests as a mirror image difference between a substance and its enantiomers, much like the difference between the left and right hands. The stereospecific recognition and enantiomer identification of chiral molecules are fundamental phenomena in life sciences: the chirality of the constituent substances of living systems (nucleic acids, amino acids, proteins, etc.) has varying degrees of influence on bioactive compounds that participate in the interactions of life activities. The sensitivity of biological systems to stereochemistry is reflected in the activity differences between a pair of enantiomers. This discovery also indicates a huge demand for single optical isomer drugs in clinical treatment.

[0003] Most existing chiral resolution studies are primarily analytical, with only a small number of pure optical isomer drugs being prepared through chemical synthesis. Chiral resolution of natural compounds has become a reliable source of pure optical drugs. Matrine, matrine alcohol, paclitaxel, silymarin, and luteolin have all shown good pharmacological activity in cell and animal experiments, but detailed studies on the pharmacological effects of their different optical isomers have not yet been conducted. The bottleneck lies in the extreme difficulty of preparing pure optical compounds, a landmark achievement.

[0004] In chiral resolution studies, polysaccharide derivative chiral stationary phases are the most widely used, exhibiting the best resolution among existing commercial columns. Amylose and cellulose derivatives are widely used for column packing. Cyclodextrins have attracted considerable research attention due to their ability to function as both chiral mobile phase additives and CSPs in chiral HPLC separations. Most commercially available chiral columns are prepared by coating or chemically immobilizing polysaccharides such as cellulose and amylose onto silica gel. Cellulose tris(3-chloro-4-methylphenylcarbamate) effectively resolves five β-adrenergic blockers, including bisoprolol and carvedilol. Cellulose 6-benzoic acid-2,3-phenylcarbamate and cellulose tris(3,5-dimethylphenylcarbamate) can both resolve flavanones and atenolol, but cellulose 6-benzoic acid-2,3-phenylcarbamate shows better resolution. Chiral separation of flavanones and benzoin was achieved. Amylose tris(3-chloro-5-methylphenylcarbamate) showed excellent resolution for various flavanones. Giovanni et al. covalently immobilized amylose tris(3-chloro-5-methylphenylcarbamate) on aminopropylalkylated (APS) silica to prepare a chiral stationary phase. This stationary phase was used for chiral analysis of racemic mixtures of flavanones and pentobarbital under capillary electrochromatography (CSP), demonstrating resolution behavior for acidic, neutral, and basic analytes. β-cyclodextrin and its derivatives, as chiral stationary phases for high-performance liquid chromatography (HPLC), successfully resolved seven flavanones and nine triazole enantiomers. The widespread application of glycosyl chiral stationary phases such as β-cyclodextrin, cellulose, and amylose reveals the enormous chiral resolution potential of glycosyl stationary phases.

[0005] Poria cocos acidic polysaccharide is a linear glucan linked by β-1,3-glycosidic bonds. It is the main component of dried Poria cocos sclerotia, and its content in Poria cocos can be as high as 80%. Due to its extremely poor water solubility, there is very little research on it. Current research mainly focuses on its pharmacological activity and structural modification, hoping to discover more potential activities of this compound through structural modification. However, since it is a glucan like cellulose and amylose, there are no reports on the chiral resolution of Poria cocos acidic polysaccharide. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a chiral stationary phase based on Poria cocos acidic polysaccharides and its application. Poria cocos acidic polysaccharides and their derivatives are coated onto a chromatographic matrix as chiral selectors to serve as the chiral stationary phase of a chromatographic column, exhibiting excellent chiral separation performance.

[0007] In order to achieve the above-mentioned objectives, in a first aspect, the present invention provides a chiral stationary phase of Poria cocos acidic polysaccharide, comprising a chromatographic matrix and a chiral selector coated on the chromatographic matrix;

[0008] The chiral selector is selected from Poria cocos acidic polysaccharide and Poria cocos acidic polysaccharide derivatives.

[0009] Furthermore, the structure of the poria cocos acidic polysaccharide is shown in the following formula:

[0010]

[0011] In a specific embodiment, the acidic polysaccharide of Poria cocos was prepared by the following method: the dried Poria cocos residue after water extraction and alcohol extraction was taken, and NaOH solution was added and stirred for 1-2 hours. After the reaction was completed, the residue was filtered, the filtrate was neutralized with hydrochloric acid, centrifuged, and the precipitate was collected, washed and dried to obtain the product.

[0012] In a preferred embodiment, the dried Poria cocos acidic polysaccharide is dissolved in N,N-dimethylformamide at 60°C for 20-30 hours, and then anhydrous lithium chloride is added and stirred continuously for 2-4 hours to obtain a chiral selector.

[0013] Furthermore, the poria cocos acidic polysaccharide derivative is obtained by modifying poria cocos acidic polysaccharide with aromatic polyisocyanate / aliphatic polyisocyanate.

[0014] Furthermore, the poria cocos acidic polysaccharide derivative is prepared by the following method: poria cocos acidic polysaccharide is swollen in anhydrous pyridine, then aromatic polyisocyanate is added, the temperature is raised to 80-100℃, and the reaction is carried out under nitrogen protection for 24 hours. After the reaction is completed, the product is obtained by filtration, washing, and vacuum drying.

[0015] In a preferred embodiment, the aromatic polyisocyanate-modified Poria cocos acid polysaccharide obtained by vacuum drying is dissolved in N,N-dimethylformamide at 60°C to obtain a chiral selector.

[0016] Furthermore, the aromatic polyisocyanate is 3,5-dimethylphenyl isocyanate.

[0017] Furthermore, the poria cocos acidic polysaccharide derivative is prepared by the following method: poria cocos acidic polysaccharide is swollen in dimethyl sulfoxide, then aliphatic polyisocyanate is added, the temperature is raised to 80-100℃, and the reaction is carried out under nitrogen protection for 24 hours. After the reaction is completed, the product is filtered, washed, and vacuum dried to obtain the product.

[0018] In a preferred embodiment, the aliphatic polyisocyanate-modified Poria cocos acid polysaccharide obtained by vacuum drying is dissolved in N,N-dimethylformamide at 60°C to obtain a chiral selector.

[0019] Furthermore, the aliphatic polyisocyanate is 1,6-hexamethylenediisocyanate.

[0020] Secondly, this invention provides the application of a chiral stationary phase of Poria cocos acidic polysaccharides as a liquid chromatography column for chiral separation.

[0021] It has been discovered that the poria cocos acidic polysaccharide and its derivatives of the present invention can preferentially bind one enantiomer relative to another enantiomer when contacted with a mixture of enantiomers. Therefore, the poria cocos acidic polysaccharide and its derivatives of the present invention can be used in a resolution method.

[0022] Furthermore, the chiral chromatographic column coated with Poria cocos acidic polysaccharide was applied to the chiral separation of benzoin, 1-(1-naphthyl)-ethanol, 1,1'-bi-2-naphthol, bifurfural, and Trogel base compounds; the chiral chromatographic column coated with Poria cocos acidic polysaccharide modified with 3,5-dimethylphenyl isocyanate was applied to the chiral separation of ketoprofen, flurbiprofen, ofloxacin, praziquantel, and other chiral compounds; and the chiral chromatographic column coated with Poria cocos acidic polysaccharide modified with 1,6-hexamethylenediisocyanate was applied to the chiral separation of warfarin sodium, propranolol hydrochloride, sulpiride, amlodipine, and other chiral compounds.

[0023] Thirdly, the present invention provides a method for separating mixtures of enantiomers, comprising the following steps:

[0024] S1. Pass the composition containing the enantiomer mixture through a chromatographic column containing the above-described poria cocos acidic polysaccharide chiral stationary phase;

[0025] S2. Contact the composition containing the enantiomer mixture with the chiral selector described above, so that the components in the composition reach equilibrium on the chromatographic column;

[0026] S3. Enantiomer mixtures are separated by utilizing the different retention times of each component on the chromatographic column.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The Poria cocos acid polysaccharide and its derivatives of the present invention have broad-spectrum and efficient chiral resolution capabilities, and can achieve effective separation of chiral compounds in normal phase chromatography mode. In order to further obtain single-configuration drugs and realize the widespread application of single-configuration drugs, the novel glycosyl stationary phase provided on the basis of traditional glycosyl stationary phases such as cellulose, amyl starch, and β-cyclodextrin enriches the range of chiral selectors.

[0029] (2) The acidic polysaccharide of Poria cocos of the present invention is derived from the waste residue after water extraction and alcohol extraction of Poria cocos sclerotium. Its extraction and preparation process is simple and easy to implement. It is an economical, green and sustainable new chiral material.

[0030] (3) The high performance liquid chromatography column of the present invention has the characteristics of fast separation speed, good separation effect, good stability and reusability. Attached Figure Description

[0031] Figure 1 Flowchart for the preparation of chiral chromatographic columns for Poria cocos acidic polysaccharides and their derivatives;

[0032] Figure 2 SEM images of the stationary phase of Poria cocos acidic polysaccharide and its derivatives;

[0033] Among them, 2A is a polysaccharide-coated stationary phase with a scale of 10,000 times; 2B is a 3,5-dimethylphenyl isocyanate-derived polysaccharide-coated stationary phase with a scale of 10,000 times; 2D is a 1,6-hexamethylenediisocyanate-derived polysaccharide-coated stationary phase with a scale of 10,000 times; 2a is a polysaccharide-coated stationary phase with a scale of 30,000 times; 2b is a 3,5-dimethylphenyl isocyanate-derived polysaccharide-coated stationary phase with a scale of 20,000 times; and 2d is a 1,6-hexamethylenediisocyanate-derived polysaccharide-coated stationary phase with a scale of 15,000 times.

[0034] Figure 3 FT-IR chromatograms of each step in the synthesis of Poria cocos acidic polysaccharide and its derivatives using a stationary phase;

[0035] Figure 4 The chromatogram for the separation of chiral compounds from acidic polysaccharides in Poria cocos;

[0036] Figure 5 The image shows a chiral resolution chromatogram of poria cocos acidic polysaccharide derivatized with 3,5-dimethylphenyl isocyanate.

[0037] Figure 6 The chromatogram is a chiral resolution chromatogram of 1,6-hexamethylenediisocyanate-derived Poria cocos acidic polysaccharide.

[0038] Figure 7 To improve the stability of chiral chromatographic columns coated with poria cocos acidic polysaccharides;

[0039] Figure 8 Reproducibility of chiral chromatographic columns for 3,5-dimethylphenyl isocyanate-derived Poria cocos acidic polysaccharides;

[0040] Figure 9 To improve the stability of chromatographic columns coated with 1,6-hexamethylene diisocyanate-derived Poria cocos acidic polysaccharide. Detailed Implementation

[0041] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, the test materials used in the following embodiments were purchased from conventional biochemical reagent stores. Unless otherwise stated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0042] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0043] The inventors developed a chiral resolution stationary phase using Poria cocos, a unique variety of Yunnan medicinal herbs, as a substrate. The stationary phase exhibits excellent resolution performance and enantioselectivity for various types of chiral compounds, and possesses broad-spectrum and efficient chiral resolution capabilities. It is an indispensable front-end research for realizing the widespread application of single optical isomer drugs.

[0044] Example 1: Preparation of chiral selectors

[0045] (1) Chiral selector based on Poria cocos acidic polysaccharide: The dried residue after water and alcohol extraction was extracted with 70 times the amount of 0.5 mol / L NaOH and stirred for 1 h. After filtration using a Büchner funnel, the filtrate was neutralized with 20% hydrochloric acid and allowed to stand overnight. The suspension was centrifuged at 4000 r / min, the supernatant was discarded, and the gel-like precipitate was repeatedly washed with distilled water to desalt and then freeze-dried to obtain Poria cocos acidic polysaccharide. 0.5 g of Poria cocos acidic polysaccharide was weighed into a 50 mL round-bottom flask, and 20 mL of N,N-dimethylformamide was added and dissolved at 90 °C for 24 h. Then, 0.1 g of anhydrous lithium chloride was added and stirred continuously for 2.5 h to obtain the final product.

[0046] (2) Chiral selector for derivatizing Poria cocos acidic polysaccharide based on 3,5-dimethylphenyl isocyanate: Weigh 1.0 g of lyophilized Poria cocos acidic polysaccharide and vacuum dry it at 90 °C for 2 h. Add 40 mL of anhydrous pyridine to swell it for 2.5 h. Add 3.8 mL of 3,5-dimethylphenyl isocyanate to the nitrogen-protected system and reflux at 90 °C for 24 h. After cooling to room temperature, precipitate it with 1 L of methanol, filter it, and wash it with methanol and n-hexane until there is no pyridine odor. Vacuum dry it at 60 °C for 5 h. Weigh 0.5 g of the above product into a 50 mL round-bottom flask, add 20 mL of N,N-dimethylformamide, and dissolve it at 60 °C to obtain the final product.

[0047] (3) Chiral selector for derivatizing Poria cocos acidic polysaccharide based on 1,6-hexamethylene diisocyanate: Weigh 1.0 g of lyophilized Poria cocos acidic polysaccharide, vacuum dry at 90 °C for 2 h, add 40 mL of dimethyl sulfoxide to swell for 2.5 h, add 3.8 mL of 1,6-hexamethylene diisocyanate to a nitrogen-protected system, reflux at 90 °C for 24 h, cool to room temperature, precipitate with 1 L of methanol, filter and wash with methanol and n-hexane until no pyridine odor remains, vacuum dry at 60 °C for 5 h. Weigh 0.5 g of the above product into a 50 mL round-bottom flask, add 20 mL of N,N-dimethylformamide and dissolve at 60 °C to obtain the final product.

[0048] Example 2: Preparation of Chiral Stationary Phase

[0049] Weigh 10g of silica gel (5μm) into a 250mL round-bottom flask, add 100mL of 10% dilute hydrochloric acid, heat at 90℃ for 8h, filter with a sintered glass funnel, wash with distilled water until neutral, and dry the filter residue under reduced pressure at 180℃ for 5h. Take 8g of the above spherical silica gel and transfer it to a 250mL two-necked flask, slowly add 80mL of anhydrous benzene, 1.2mL of anhydrous pyridine, and 1.6mL of 3-aminopropyltriethoxysilane, reflux at 80℃ for 24h under nitrogen protection, cool and filter, then wash with appropriate amounts of methanol, acetone, and n-hexane respectively, and dry under vacuum at 60℃ for 5h to obtain the final product. The three chiral selectors prepared in Example 1 were applied in small, repeated applications (3-6 drops each time) to the surface of 2.5 g of 3-aminopropyltriethoxysilane-modified silica gel. Once the silica gel showed signs of dampness, the coating process was stopped, and the gel was vacuum-dried at 60°C for 30 min. The chiral selectors were then added again, and the coating process was repeated. After the final coating, the gel was vacuum-dried at 60°C for 5 h to remove the solvent, yielding the chiral stationary phase.

[0050] The three stationary phases were characterized using scanning electron microscopy (SEM), such as... Figure 2 As shown, the three stationary phase particles are uniform and coated with a chiral selector, making them suitable for column packing.

[0051] The content ratios of C, H, O, and N elements in activated silica gel, 3-aminopropyltriethoxysilane-modified silica gel, and the three chiral stationary phases were determined using an elemental analyzer (results are shown in Table 1).

[0052] Table 1. Elemental analysis results of each step of the reaction in the stationary phase.

[0053]

[0054] As shown in the table above, compared with activated silica gel, the C, H, and N content of 3-aminopropyltriethoxysilane silica gel is significantly increased, indicating that the synthesis of 3-aminopropyltriethoxysilane silica gel is successful and the silanol effect is successfully masked. The coating of polysaccharides increases the proportion of C, H, and O elements in the stationary phase. After coating with 3,5-dimethylphenyl isocyanate-derived polysaccharides, in addition to the increase in the proportion of C, H, and O, the N element also increases. After coating with 1,6-hexamethylene diisocyanate-derived polysaccharides, the increase in N and O elements is more significant compared with that of 3,5-dimethylphenyl isocyanate-derived polysaccharides, indicating that the derivatization of 1,6-hexamethylene diisocyanate is successful.

[0055] To further investigate the success of bonding in each step of the stationary phase process, FT-IR characterization was performed on the stationary phases consisting of activated silica gel, 3-aminopropyltriethoxysilane-modified silica gel, polysaccharide coating, 3,5-dimethylphenyl isocyanate-derived polysaccharide coating, and 1,6-hexamethylene diisocyanate-derived polysaccharide coating. The results are as follows: Figure 3 As shown: activated silica gel, 3-aminopropyltriethoxysilane-modified silica gel, and three stationary phases were incubated at 3450 cm⁻¹. -1 The broad absorption peaks at 1092 and 789 cm⁻¹ are caused by the stretching vibrations of the silanol groups on the silica gel surface and the introduced NH stretching vibrations. -1 The strong absorption peaks are related to the overlapping absorption bands of Si-O, CN, and CO, at 2930 and 2861 cm⁻¹. -1 The absorption peak at 1553 cm⁻¹ is the CH stretching vibration peak; the absorption peak at 1553 cm⁻¹ is the 3,5-dimethylphenyl isocyanate-derived polysaccharide stationary phase. -1 Characteristic absorptions of the benzene ring were observed, and the stationary phases coated with 3,5-dimethylphenyl isocyanate-derived polysaccharides and 1,6-hexamethylene diisocyanate-derived polysaccharides reached 1745 cm⁻¹. -1 Characteristic absorption peaks of ester groups appeared at the infrared spectrum, indicating that all three chiral stationary phases based on Poria cocos acidic polysaccharides were successfully prepared.

[0056] Example 3: Preparation of chromatographic column

[0057] Weigh 1.2 g of each of the three stationary phases prepared in Example 2, add an appropriate amount of n-hexane / isopropanol (9:1, v / v) and pass through a 250-mesh sieve to make the particle size uniform. Use 200 mL of n-hexane / isopropanol (9:1, v / v) as the displacement solvent, pack the column at 40 MPa for 2 min, then slowly reduce the pressure to 15 to 20 MPa and stabilize for 30 min. After stabilizing at normal pressure for 30 min, a coated chiral chromatographic column is obtained. Elute the column using n-hexane / isopropanol (9:1, v / v) as the mobile phase at a flow rate of 0.1 mL / min on a high performance liquid chromatograph. After the baseline stabilizes, the chiral chromatographic column of Poria cocos acidic polysaccharide and its chiral chromatographic column derived from 3,5-dimethylphenyl isocyanate and 1,6-hexamethylenediisocyanate are obtained.

[0058] Example 4: Chromatographic Separation Effect Test

[0059] The chromatographic column was used to investigate its performance in separating chiral samples. The chromatographic conditions were as follows: the mobile phase was hexane / different concentrations of trifluoroacetic acid, ethylenediamine, and triethylamine isopropanol (see Table 2 for details); the absorption wavelength was 254 nm; the injection volume was 10 μL; and the column temperature was room temperature.

[0060] Preparation of chiral test solutions: Take appropriate amounts of racemic compound and chiral drug and dilute them with hexane / isopropanol (9:1, v / v) as solvent to prepare stock solutions. Store all solutions at 4°C for later use.

[0061] Table 2

[0062]

[0063]

[0064]

[0065] The resolution results of some racemic compounds for each chromatographic column are shown in Table 3:

[0066] Table 3

[0067]

[0068]

[0069] From Table 3 and Figure 4-6As can be seen, baseline separation with a resolution (Rs) exceeding 1.5 was achieved for 10 chiral compounds, including some racemic compounds such as 1,2-diphenylethylenediamine (Rs = 9.81), ketoprofen (Rs = 6.59), benzoin (Rs = 5.09), dihydroflavonoids (Rs = 3.42), flurbiprofen (Rs = 3.06), 1-(1-naphthyl)ethanol (Rs = 2.97), praziquantel (Rs = 2.81), ofloxacin (Rs = 2.77), and amlodipine (Rs = 1.98); among them, such as Figure 4 As shown, the chiral chromatographic column for Poria cocos acidic polysaccharides achieved baseline separation of 1-(1-naphthyl)ethanol, 1,1'-bi-2-naphthol, bifurfural, and benzoin compounds, and showed signs of resolution for Trogel bases. This preliminarily proves that Poria cocos acidic polysaccharides are chiral selective biomolecules; Figure 5 As shown, the 3,5-dimethylphenyl isocyanate-derived polysaccharide chiral column exhibits significantly superior chiral separation performance compared to the Poria cocos acidic polysaccharide chiral column, achieving separation of 17 compounds, including alcohols, ketones, amines, and carboxylic acids. In NP-HPLC mode, it demonstrates superior chiral separation performance compared to the Poria cocos acidic polysaccharide chiral column, exhibiting advantages in both the variety and number of separated compounds. Figure 6 As shown, the 1,6-hexamethylene diisocyanate-derived polysaccharide chiral column exhibited chiral separation behavior for sulpiride and warfarin sodium, while these two racemic mixtures were not separated on the Poria cocos acidic polysaccharide chiral column or the 3,5-dimethylphenyl isocyanate-derived polysaccharide chiral column. This indicates that the 1,6-hexamethylene diisocyanate-derived polysaccharide chiral column also demonstrates certain advantages. However, overall, the 3,5-dimethylphenyl isocyanate-derived polysaccharide chiral column has better chiral recognition ability, separating more compounds in both number and variety than both the Poria cocos acidic polysaccharide chiral column and the 1,6-hexamethylene diisocyanate-derived polysaccharide chiral column, demonstrating its broad-spectrum chiral recognition. Furthermore, it exhibits higher resolution for multiple drugs, which is more advantageous for the preparation of single optical isomers. From a production perspective, the 3,5-dimethylphenyl isocyanate-derived polysaccharide chiral column is more conducive to the production of drugs with single optical properties, making it more significant for clinical applications.

[0070] Example 5 examines the resolution of a 3,5-dimethylphenyl isocyanate-derived chiral column.

[0071] The chromatographic separation performance of chiral columns for 3,5-dimethylphenyl isocyanate-derived polysaccharides and commercially available columns Chirapak OD-H and Chirapak AD-H was investigated. The chromatographic conditions were as follows: absorption wavelength: 254 nm; injection volume: 10 μL; column temperature: room temperature. The separation results of some racemic compounds for each column are shown in Table 4.

[0072] Table 4

[0073]

[0074]

[0075] Example 6: Reproducibility and stability testing of the chromatographic column

[0076] To investigate the stability and repeatability of the three chiral chromatographic columns prepared in Example 3, benzoin and 1,1'-bi-2-naphthol were selected as analytes for testing. Chromatographic conditions were: absorption wavelength: 254 nm; injection volume: 10 μL; column temperature: room temperature. The resolution chromatograms of each chiral column are shown below. Figure 7-9 As shown, the chiral chromatographic columns coated with Poria cocos acidic polysaccharide, 3,5-dimethylphenyl isocyanate-derived Poria cocos acidic polysaccharide, and 1,6-hexamethylenediisocyanate-derived Poria cocos acidic polysaccharide all exhibited relatively stable chiral resolution performance. Among them, the Poria cocos acidic polysaccharide chiral column retained its chiral resolution performance for benzoin even after more than 600 injections, demonstrating good reproducibility. Furthermore, the 3,5-dimethylphenyl isocyanate-derived Poria cocos acidic polysaccharide column, prepared repeatedly using the same column preparation procedure, still showed good reproducibility and stability for 1,1'-bi-2-naphthol.

[0077] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Those skilled in the art, under the guidance of the present invention, can make various similar representations without departing from the spirit and claims of the present invention, and such modifications all fall within the protection scope of the present invention.

Claims

1. A chiral stationary phase of Poria cocos acidic polysaccharides, characterized in that: This includes the chromatographic matrix and the chiral selector coated on the chromatographic matrix; The chromatographic matrix refers to 3-aminopropyltriethoxysilane-modified silica gel. The chiral selector is selected from chiral selectors based on Poria cocos acidic polysaccharides, chiral selectors based on 3,5-dimethylphenyl isocyanate-derived Poria cocos acidic polysaccharides, and chiral selectors based on 1,6-hexamethylene diisocyanate-derived Poria cocos acidic polysaccharides. Chiral selector based on Poria cocos acidic polysaccharide: Take the dried residue after water extraction and alcohol extraction, add 70 times the amount of 0.5mol / L NaOH and stir for 1h. After filtration through a Büchner funnel, neutralize the filtrate with 20% hydrochloric acid, let it stand overnight, centrifuge the suspension at 4000r / min, discard the supernatant, wash the gel-like precipitate repeatedly with distilled water to desalt it, and freeze-dry it to obtain Poria cocos acidic polysaccharide. Weigh 0.5g of Poria cocos acidic polysaccharide into a 50mL round-bottom flask, add 20mL of N,N-dimethylformamide and dissolve it at 90℃ for 24h, then add 0.1g of anhydrous lithium chloride and stir continuously for 2.5h to obtain the final product. Chiral selector for 3,5-dimethylphenyl isocyanate-derived Poria cocos acidic polysaccharide: Weigh 1.0 g of lyophilized Poria cocos acidic polysaccharide and vacuum dry it at 90 °C for 2 h. Add 40 mL of anhydrous pyridine to swell it for 2.5 h. Add 3.8 mL of 3,5-dimethylphenyl isocyanate to the nitrogen-protected system and reflux at 90 °C for 24 h. After cooling to room temperature, precipitate it with 1 L of methanol, filter it, and wash it with methanol and n-hexane until there is no pyridine odor. Vacuum dry it at 60 °C for 5 h. Weigh 0.5 g of the product into a 50 mL round-bottom flask and add 20 mL of N,N-dimethylformamide to dissolve it at 60 °C to obtain the product. Chiral selector for 1,6-hexamethylene diisocyanate-derived Poria cocos acidic polysaccharide: Weigh 1.0 g of lyophilized Poria cocos acidic polysaccharide, vacuum dry at 90 °C for 2 h, add 40 mL of dimethyl sulfoxide to swell for 2.5 h, add 3.8 mL of 1,6-hexamethylene diisocyanate to a nitrogen-protected system, reflux at 90 °C for 24 h, cool to room temperature, precipitate with 1 L of methanol, filter and wash with methanol and n-hexane until no pyridine odor is detected, vacuum dry at 60 °C for 5 h, weigh 0.5 g of the product into a 50 mL round-bottom flask, add 20 mL of N,N-dimethylformamide and dissolve at 60 °C to obtain the product; The preparation method of the chiral stationary phase is as follows: Weigh 10g of silica gel with a particle size of 5μm into a 250mL round-bottom flask, add 100mL of 10% dilute hydrochloric acid, heat at 90℃ for 8h, filter with a sintered funnel, wash with distilled water until neutral, and dry the filter residue under reduced pressure at 180℃ for 5h. Take 8g of spherical silica gel and transfer it to a 250mL two-necked flask, slowly add 80mL of anhydrous benzene, 1.2mL of anhydrous pyridine, and 1.6mL of 3-aminopropyltriethoxysilane, reflux at 80℃ for 24h under nitrogen protection, cool and filter, then wash with appropriate amounts of methanol, acetone, and n-hexane respectively, and dry under vacuum at 60℃ for 5h to obtain 3. -Aminaminopropyltriethoxysilane-modified silica gel; Using a tapping method, chiral selectors based on Poria cocos acidic polysaccharides, chiral selectors based on 3,5-dimethylphenyl isocyanate-derived Poria cocos acidic polysaccharides, and chiral selectors based on 1,6-hexamethylenediisocyanate-derived Poria cocos acidic polysaccharides were applied in small, repeated amounts to the surface of 2.5 g of 3-aminopropyltriethoxysilane-modified silica gel, with 3-6 drops of coating solution applied each time. When the silica gel showed signs of dampness, coating was stopped, and the gel was vacuum-dried at 60°C for 30 min. The chiral selectors were then added again, and the coating process was repeated. After the final coating, the gel was vacuum-dried at 60°C for 5 h to remove the solvent, yielding the chiral stationary phase. The method for preparing chromatographic columns using the chiral stationary phases involves weighing 1.2 g of each of the three stationary phases, adding an appropriate amount of hexane and isopropanol in a volume ratio of 9:1, and passing the mixture through a 250-mesh sieve to ensure uniform particle size. Using 200 mL of hexane and isopropanol in a volume ratio of 9:1 as the displacement solvent, the column is packed at 40 MPa for 2 min, then the pressure is slowly reduced to 15-20 MPa and stabilized for 30 min. After stabilizing at ambient pressure for 30 min, a coated chiral chromatographic column is obtained. Elution is performed on a high-performance liquid chromatogram using hexane and isopropanol in a volume ratio of 9:1 as the mobile phase at a flow rate of 0.1 mL / min. Once the baseline stabilizes, the chiral chromatographic column of Poria cocos acidic polysaccharide and its chiral chromatographic columns derived from 3,5-dimethylphenyl isocyanate and 1,6-hexamethylenediisocyanate are obtained.

2. The application of the Poria cocos acidic polysaccharide chiral stationary phase as a liquid chromatography column in chiral separation according to claim 1.

3. The application according to claim 2, characterized in that: Applications of chiral chromatographic columns for Poria cocos acidic polysaccharides in the chiral separation of benzoin, 1-(1-naphthyl)-ethanol, bifurfural, and Trogel base compounds; application of chiral chromatographic columns for polysaccharides derived from 3,5-dimethylphenyl isocyanate in the chiral separation of ketoprofen, flurbiprofen, ofloxacin, and praziquantel; application of chiral chromatographic columns for polysaccharides derived from 1,6-hexamethylenediisocyanate in the chiral separation of warfarin sodium, propranolol hydrochloride, sulpiride, and amlodipine.

4. A method for separating mixtures of enantiomers, characterized in that, Includes the following steps: S1. Passing the composition containing the enantiomer mixture through a chromatographic column containing the chiral stationary phase of Poria cocos acidic polysaccharide according to claim 1; S2. Contact the composition containing the enantiomer mixture with the chiral selector according to claim 1, so that the components in the composition reach equilibrium on the chromatographic column; S3. Enantiomer mixtures are separated by utilizing the different retention times of each component on the chromatographic column.

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