Preparation method of anion organic hybrid monolithic column and chiral drug separation method of anion organic hybrid monolithic column in capillary electrophoresis

Abstract

The invention provides a preparation method of an anion organic hybrid monolithic column and a chiral drug separation method of the anion organic hybrid monolithic column in capillary electrophoresis. Specifically, the invention relates to a chromatographic column which takes hexanediol dimethyl acrylate (EDMA), bonded anion beta-cyclodextrin: sulfonated-beta-cyclodextrin and glutamic acid-beta-cyclodextrin (S-beta-CD and GMA-beta-CD) as raw materials and is used for capillary electrophoresis chiral drug analysis. The preparation method comprises the following steps: firstly preparing S-beta-CD and Glu-beta-CD framework materials, preparing an electrolyte, and finally carrying out condensation polymerization to prepare the organic hybrid monolithic column. The method is simple and rapid to operate, good in repeatability and small in environmental pollution, and the chiral drug separation efficiency including analysis time and drug separation degree can be remarkably improved. According to the technical scheme, 13 chiral drugs can be effectively separated.

Description

Technical Field

[0001] This invention belongs to the field of synthesis and analysis technology, and specifically relates to two anionic sulfonated β-cyclodextrin backbone materials, glutamic acid β-cyclodextrin backbone materials and their organic hybrid monolithic column preparation methods, and capillary electrophoretic analysis methods for chiral drugs. Background Technology

[0002] Chirality is widespread in nature; basic substances in living organisms such as proteins, polysaccharides, and nucleic acids are essentially chiral. Chirality refers to the asymmetry of molecular structure, specifically manifested as the enantiomer relationship of compounds. Chiral drugs consist of enantiomers, which exhibit different therapeutic effects and toxicological properties in vivo. Typically, one enantiomer of a racemic drug exhibits good activity in vivo, while the other may be less effective, ineffective, or even have side effects. Therefore, there is a demand in the pharmaceutical market for single-enantiomer drugs. Clearly, the study of enantiomer separation is an important research direction in fields such as chemical pharmaceuticals, biosciences, and food science. It greatly contributes to the understanding of drug activity, toxicity, and chiral recognition processes in vivo.

[0003] Capillary electrochromatography (CEC) combines the high resolution of capillary electrophoresis (CE) with the high specificity of high-performance liquid chromatography (HPLC), offering advantages such as high efficiency, speed, and simplicity. Monolithic columns are characterized by simple preparation, high column permeability, and fast chromatographic separation. In the field of modern electrochromatography, monolithic columns have become a popular new type of chromatographic column, hailed as the fourth-generation separation medium.

[0004] In capillary electrochromatography (CEC), the effectiveness of chiral separation depends on selecting the appropriate chiral recognition agent for various chiral drugs. Among these, β-cyclodextrin, a cyclodextrin-based chiral selector, is the most extensively studied and widely used. It has a moderate cavity size, the most stable structure among the three natural cyclodextrins, provides more hydrophobic cavities, and can derive hydroxyl groups. However, due to its bonding ability and water solubility, natural cyclodextrins have limitations in chiral separation applications. By introducing derivatized groups onto the cyclodextrin cavity, cyclodextrins can not only form a more stable bond with the stationary phase in terms of cavity size and shape, but also effectively improve the chiral recognition ability of the cyclodextrin stationary phase and alter the hydrophilic properties of cyclodextrins. Studies have shown that charged cyclodextrins have higher solubility in water and improved separation compared to uncharged cyclodextrins. Based on this, this study explored the preparation of two anionic charged cyclodextrin monolithic columns and their application in chiral drug separation. No similar studies have been published to date. Summary of the Invention

[0005] To address the above technical problems, in a first aspect, the present invention proposes a method for preparing an organic hybrid monolithic column, the method comprising the following steps:

[0006] Preparation steps of S-β-cyclodextrin monolithic column:

[0007] The specific steps include: 0.5 mL of electrolyte solution, 20 mg of sulfonated β-cyclodextrin backbone material, 20 μL of deionized water, 20 μL of dimethyl sulfoxide (DMSO), and 1.0 mg of azobisisobutyronitrile (AIBN) are vortexed and ultrasonically degassed for 5 min before use. The above-prepared solution is then siphoned into a pretreated quartz capillary tube, both ends are sealed with rubber stoppers, and heated in a water bath at a constant temperature (40 °C) for 19 hours to obtain an S-β-CD organosilicon hybrid single-column capillary column.

[0008] Organic hybrid monolithic columns were prepared.

[0009] Secondly, the present invention also discloses an organic hybrid monolithic column prepared by the method for preparing the organic hybrid monolithic column proposed in the present invention.

[0010] Thirdly, the present invention also proposes a capillary electrophoresis method for separating chiral drugs using an organic hybrid monolithic column. The stationary phase of the capillary electrophoresis method is an organic hybrid monolithic column prepared by the method disclosed in the present invention. The mobile phase of the capillary electrophoresis method has a pH of 5.3-5.5 and includes a Tris buffer solution with a concentration range of 18-22 mM, wherein the Tris buffer solution contains 30-40% acetonitrile by mass. Preferably, the Tris buffer solution has a concentration of 20 mM and contains 30% acetonitrile by mass. The electrophoresis voltage of the capillary electrophoresis method is 12-18 kV, preferably 15 kV. More preferably, the chiral drug includes one or more chiral drugs selected from antihistamines, antihypertensive drugs, amino acids, azoles, and pesticides.

[0011] The organic hybrid monolithic column prepared according to the method of the present invention has the following technical effects: the organic hybrid monolithic column has good thermal stability and can be used as a stationary phase in capillary chromatography; it is beneficial for enantiomeric separation and identification and the separation effect is significant. Attached Figure Description

[0012] Figure 1 1H NMR spectra of S-β-CD and S-β-CD framework materials (dissolved in D2O)

[0013] Figure 2 1H NMR spectra of Glu-β-CD and Glu-β-CD framework materials (dissolved in DMSO-d6)

[0014] Figure 3 Infrared spectra of S-β-CD and S-β-CD framework materials (KBr pellets)

[0015] Figure 4 Infrared spectra of Glu-β-CD and Glu-β-CD framework materials (KBr pellets)

[0016] Figure 5 SEM images of the interior of S-β-CD (A,C) and Glu-β-CD (B,D) columns.

[0017] Figure 6 TGA plot of S-β-CD and Glu-β-CD organic hybrid silica monolithic column

[0018] Figure 7 Results of the effect of acetonitrile content (a) and pH change (b) on EOF in S-β-CD and Glu-β-CD organosilica hybrid monolithic columns.

[0019] Figure 8 The effect of acetonitrile concentration variation on imidazolium (A) and econazole (B) when using S-β-CD organic hybrid silica monolithic column; The effect of acetonitrile concentration variation on imidazolium (A) and econazole (B) when using Glu-β-CD organic hybrid silica monolithic column;

[0020] Figure 9 The effect of pH changes on imidazolium (A) and econazole (B) when using an S-β-CD organic hybrid silica monolithic column; The effect of pH changes on imidazolium (A) and econazole (B) when using a Glu-β-CD organic hybrid silica monolithic column;

[0021] Figure 10 When using an S-β-CD organic hybrid silica monolithic column, the optimal mobile phase conditions for the separation chromatograms of antihistamines, antihypertensive drugs, amino acids, azoles, and pesticides are as follows: 20 mM Tris-phosphate, voltage 15 kV; 20% acetonitrile, pH 5.5 for promethazine, methyl scopolamine, propranolol, lysine, imazalil ethanol, bifonazole, tebuconazole, miconazole, tebuconazole, and thioconazole; 40% acetonitrile, pH 5.5 for imazalil and econazole; and 20% acetonitrile, pH 4.5 for flutriafol.

[0022] Figure 11 The optimal separation chromatograms for antihistamines, antihypertensive drugs, amino acids, azoles, and pesticides when using a Glu-β-CD organic hybrid silica monolithic column.

[0023] Mobile phase conditions: 20 mM Tris-phosphate, voltage 15 kV, promethazine, methyl scopolamine, propranolol, lysine, tebuconazole, miconazole, tebuconazole, econazole, and thioconazole in 10% ACN, pH 5.5; imazalil in 40% acetonitrile; imazalil ethanol in 0% acetonitrile, pH 5.5; bifonazole in 20% acetonitrile, pH 5.5. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, what is described is only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention. First, the relevant technical fields involved in the present invention will be explained and described.

[0025] Capillary electrophoresis (CE) is based on the principle of separating charged molecules in a narrow capillary under the influence of an electric field. It can obtain results quickly and efficiently, and in many cases outperforms other techniques such as liquid chromatography (LC).

[0026] CEC columns are classified into three types: packed columns, monolithic columns, and open tubular columns, which are important components of CEC. Specifically, monolithic columns are popular in the field of chiral separation in CEC due to their good selectivity, large surface area, and strong adsorption capacity. Based on the properties of the monolithic material, they can be divided into three main categories: (1) organic polymer-based monolithic columns, (2) silica-based monolithic columns, and (3) organic-silica hybrid monolithic columns. Compared with the first two types of monolithic columns, organic-silica hybrid monolithic columns are superior in terms of good pH stability and mechanical stability.

[0027] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the present invention.

[0028] It should be noted that the buffer solution C4H 11 NO3 is also known as Tris buffer, and ethyl methacrylate (EDMA) has the molecular formula C60-32 ... 12 H 18 O4.

[0029] Thermogravimetric analysis (TG or TGA) is a thermal analysis technique that measures the relationship between the mass of a sample and temperature change under programmed temperature control. It is used to study the thermal stability and composition of materials.

[0030] Example 1-1 Preparation of Sulfonated β-Cyclodextrin Monolithic Column

[0031] 1) Take sulfonated β-cyclodextrin and the following amounts of raw materials:

[0032] Sulfonated β-cyclodextrin (S-β-CD) 20±1mg

[0033] EDMA (dimethacrylate) 21±4 μL

[0034] Anhydrous dimethyl sulfoxide (DMSO) 200±10μL

[0035] The above-mentioned amounts of sulfonated β-cyclodextrin (S-β-CD), dimethacrylate (EDMA), and anhydrous dimethyl sulfoxide (DMSO) were placed in a container and stirred in a 100°C water bath for 30 minutes to obtain a sulfonated β-cyclodextrin monolithic column framework material.

[0036] 2) Mix glacial acetic acid (0.01M, 1.0mL) and 1,4-butanediol (20μL) and stir magnetically in an ice bath for 2 hours to obtain an electrolyte solution.

[0037] 3) Take 0.5 mL of the product from step 2), 20 mg of the product from step 1), 20 μL of deionized water, 20 μL of dimethyl sulfoxide (DMSO), and 1.0 mg of azobisisobutyronitrile (AIBN), vortex mix, and sonicate to degas for 5 min before use. Siphon the above-prepared solution into a pretreated quartz capillary tube, seal both ends with rubber stoppers, and heat in a water bath at a constant temperature (40℃) for 19 hours to obtain an S-β-CD organosilicon hybrid single-column capillary column.

[0038] Example 1-2 Preparation of Glu-β-CD Hybrid Single-Column Capillary Column

[0039] 1) Take glutamic acid-β-cyclodextrin and use the following amounts of raw materials:

[0040] Glutamic acid-β-cyclodextrin (Glu-β-CD) 60±1mg

[0041] EDMA (dimethacrylate) 21±4 μL

[0042] Anhydrous dimethyl sulfoxide (DMSO) 200±10μL

[0043] The above-mentioned amounts of glutamic acid-β-cyclodextrin (Glu-β-CD), dimethacrylate (EDMA), and anhydrous dimethyl sulfoxide (DMSO) were placed in a container and stirred in a 100°C water bath for 30 minutes to obtain sulfonated-β-cyclodextrin monolithic column framework material.

[0044] 2) Mix glacial acetic acid (0.01M, 1.0mL) and 1,4-butanediol (20μL) and stir magnetically in an ice bath for 2 hours to obtain the electrolyte product.

[0045] 3) Take 0.5 mL of the product from step 2), 20 mg of the product from step 1), 20 μL of deionized water, 20 μL of dimethyl sulfoxide (DMSO), and 1.0 mg of azobisisobutyronitrile (AIBN), vortex mix, and sonicate to degas for 5 min before use. Siphon the above-prepared solution into a pretreated quartz capillary tube, seal both ends with rubber stoppers, and heat in a water bath at a constant temperature (40℃) for 19 hours to obtain a Glu-β-CD organosilicon hybrid single-column capillary column.

[0046] Example 2: A method for separating chiral drugs

[0047] Column: S-β-cyclodextrin-organosilicon hybrid monolithic column;

[0048] Mobile phase: NaH2PO4 (20mM) with 20-30% acetonitrile added;

[0049] The gas was degassed by ultrasound and then filtered through a filter membrane with a pore size of 0.22 μm.

[0050] pH: 4.5–5.5;

[0051] Detection wavelength: 220nm;

[0052] Column temperature: 25℃;

[0053] Separation voltage: 15kV.

[0054] Example 3: A method for separating chiral drugs

[0055] Column: Glu-β-cyclodextrin organosilicon hybrid monolithic column;

[0056] Mobile phase: Tris-H3PO4 (20 mM) with 0–40% acetonitrile added;

[0057] The gas was degassed by ultrasound and then filtered through a filter membrane with a pore size of 0.22 μm.

[0058] pH: 5.5–6.5;

[0059] Detection wavelength: 220nm;

[0060] Column temperature: 25℃;

[0061] Separation voltage: 15kV

[0062] Experiment Example 1: Structure Confirmation

[0063] 3.3.1 1 H-NMR spectrum

[0064] The methacrylate group signal at 5.60–6.2 ppm and the methyl proton resonance at 1.72–2.12 ppm in the NMR spectrum indicate that a methacrylate group was successfully attached to the S-β-CD molecule (Figure S2AB). The same is true for Glu-β-CD (Figure S3AB).

[0065] The synthesized compounds were further identified using IR spectroscopy. Compared to the IR spectrum of S-β-CD (Fig. S4a), the IR spectrum of S-β-CD (Fig. S4b) showed characteristic peaks at 1714.0 cm⁻¹ (C=O) and 1619.8 cm⁻¹ (C=C), indicating that S-β-CD was successfully modified onto the capillary surface. The same was true for Glu-β-CD (Fig. S5ab).

[0066] based on 1 H-NMR and FT-IR spectral evidence show that S-β-CD and Glu-β-CD have been successfully immobilized on the capillary wall.

[0067] Experiment Example 2: Scanning Electron Microscopy

[0068] The S-β-CD and Glu-β-CD organic hybrid monolithic columns were prepared, and their internal morphology and structure were observed using electron microscopy (SEM). The SEM images of the inner surface are shown below. Figure 1 As shown (S-β-CD is) Figure 1 A5,000×, C 10,000×; Glu-β-CD is Figure 1 B 5,000×, D 10,000×). From Figure 6 As can be seen, the monolithic column prepared in this application, composed of sulfonated-β-cyclodextrin and glutamic acid-β-cyclodextrin organosilicon hybrids, exhibits amorphous silica polymers on its inner surface, forming a high-density microporous structure. This microporous structure is uniformly distributed, with relatively large pore sizes, and possesses good permeability characteristics. Furthermore, the capillary surface modified with Glu-β-CD displays an even more pronounced porous morphology. This enhanced porosity is believed to provide more chiral recognition sites, thereby improving the efficiency and selectivity of the monolithic column in enantioselective separation applications.

[0069] Experimental Example 3 Thermogravimetric Analysis

[0070] TGA curves of the two newly prepared columns are as follows: Figure 7 As shown, the chiral stationary phases showed almost no decomposition at any temperature, indicating their high thermal and chemical stability. Under nitrogen atmosphere and a heating rate of 20 °C / min, SCD and Glu-CD exhibited weight loss rates of approximately 1.65% and 1.47%, respectively, upon increasing from room temperature to 800 °C. The data confirm the thermal stability of both single columns.

[0071] Experiment Example 4: Electroosmotic Flow Experiment

[0072] Electroosmotic flow (EOF) plays a driving role in enantiomeric separation in capillary electrochromatography (CEC). It is affected by applied voltage, organic solvent, buffer solution, and temperature. In this experiment, the effects of acetonitrile (ACN) content (0–30%) and different pH values ​​(4.5–6.5) on EOF were investigated. Figure 8 As shown, the EOF value decreases with increasing ACN content; conversely, for S-β-CD and Glu-β-CD silica hybrid single columns, an increasing trend is observed with increasing pH. Therefore, changes in ACN content and pH are significant for CEC enantiomeric separation efficiency.

[0073] Experimental Example 5: Chromatographic Evaluation of S-β-CD and Glu-β-CD Hybrid Silica Columns

[0074] Enantiomeric separation of 13 analytes was tested in a CEC using two types of capillary tubes to compare the recognition capabilities of S-β-CD and Glu-β-CD in a single column. The 13 analytes included: promethazine, methylhyoscyamine, propranolol, lysine, imazalil ethanol, bifonazole, tebuconazole, miconazole, methazine, imazalil, econazole, flutriafol, and thioconazole.

[0075] In addition, the effects of mobile phase pH and acetonitrile content on the separation efficiency were investigated, and the results are shown below:

[0076] The concentration of acetonitrile can affect the ionic strength of the electrophoresis medium, and thus the EOF (exchange factor). As the acetonitrile content increases, the EOF decreases, which in turn promotes longer retention times and improves separation efficiency. Simultaneously, as the viscosity coefficient of the electrolyte increases, drug enrichment, viscosity, and dielectric constant also increase. Ultimately, separation efficiency and analytical sensitivity are affected.

[0077] On an S-β-CD column: The separation efficiency of 30 analytes on a preparative column was investigated using a mobile phase of 20 mM Tris-H3PO4 (pH = 5.5) with ACN content (10-30%). For example, phenazine ethanol was separated on an S-β-CD column using a mobile phase containing 20% ​​ACN. Figure 9 A). When the acetonitrile concentration is below 10%, the interaction between the molecule and the chiral stationary phase is insufficient. When the ACN content is set above 20%, almost all drugs are effectively separated, but the retention time is longer and the peak shape is poorer. The same situation was observed in the separation of econazole enantiomers. Figure 9 B). Considering elution time and resolution, 20% ACN was selected for enantiomeric separation on the S-β-CD monolithic column. Except for econazole and imidacloprid ethanol (20% acetonitrile), 40% ACN was set as the optimal condition for all others.

[0078] On Glu-β-CD columns: The ACN content on Glu-β-CD single columns was optimized using the same method, and the results showed that most drugs (a total of 4 pairs: promethazine, bifonazole, tebuconazole, and imidacloprid) were well separated using 20 mM Tris HCl containing 10% ACN as the mobile phase. Enantiomers of econazole could be baseline separated using the optimized 40% ACN content, such as... Figure 9 As shown in C and D. Imazalil ethanol was used without the addition of ACN. Therefore, enantiomeric separation was performed on a Glu-β-CD organic hybrid single column with 0%, 10%, and 40% ACN.

[0079] pH can affect the dissociation ability of drugs, the degree of protonation of silanol groups, and the chemical stability of solutes. In polar media, the degree of drug dissociation increases, electrophoretic velocity increases, and the selectivity and sensitivity of separation are affected. Therefore, in this experiment, the separation efficiency of 13 analytes on an S-β-CD single column was studied at pH 4.5–6.5. As the pH increased, the migration time of the analytes decreased. In terms of separation results, all drugs were partially or completely separated at pH 5.5. Therefore, the pH of all analytes was fixed at 5.5. Here, imazalil (10A) and econazole (10B) were used as model analytes for demonstration.

[0080] Using a Glu-β-CD column, the effect of pH on enantiomeric separation (10C, D) was also optimized. Similar to S-β-CD, except for imazalil (pH 6.5), the optimal pH for all analytes was selected as 5.5.

[0081] The results of separation of 13 chiral samples using S-β-CD and Glu-β-CD monolithic columns under optimal conditions are shown in Table 1. According to the results, 10 chiral compounds were baseline separated on the S-β-CD column, while only 6 chiral compounds were effectively separated on the Glu-β-CD column. The results indicate that the prepared S-β-CD column has better enantiomeric separation capability. This may be because the chiral stationary phase containing sulfonic acid groups has higher enantiomeric separation performance than the chiral stationary phase containing glutamate groups. Chromatograms are shown below. Figure 11 middle.

[0082] Example 6: Repeatability and stability of S-β-CD and Glu-β-CD silica hybrid monolithic columns

[0083] Using econazole (analysis time) as a model drug, the stability and repeatability of the newly prepared column were investigated. Table 2 lists the intra-day (n=5), inter-day (n=3), inter-column (n=3), and inter-batch RSDs. All RSDs were below 5.0%. Furthermore, after 80 uses, the chromatographic performance remained largely unchanged. These results indicate that the column possesses excellent repeatability and stability.

[0085] Table 1. Chiral drug separation performance using S-β-CD and Glu-β-CD monolithic columns

[0086]

[0087] *There are two chiral carbons.

[0088] Table 2. Repeatability test results of S-β-CD organosilica hybrid monolithic column

[0089]

[0090] Experimental conditions: mobile phase: 20% ACN in 20mM Tris-H3PO4 buffer; voltage: +15.0kV;

[0091] The detection wavelength is 220nm.

[0092] RSD: Relative Standard Deviation

[0093] Appendix 1: Table 2 of Comparison Document 1

[0094] Table 2 shows the separation results of 12 drugs on a carboxymethyl-β-cyclodextrin-organosilicon hybrid monolithic column.

[0095] Chiral drugs <![CDATA[t1]]> <![CDATA[t2]]> α <![CDATA[R s ]]> <![CDATA[N1]]> <![CDATA[N2]]> atropine 5.47 8.50 7.98 20.63 117050 22028 Betamethasone 7.11 12.30 2.34 18.57 35424 14480 ofloxacin 6.02 10.92 7.96 16.29 15396 11462 Acephate 7.13 7.68 10.75 4.43 41453 79369 Beclomethasone propionate 6.46 10.28 1.46 21.23 179498 18924 Imidazole ethanol 6.48 6.76 16.88 2.39 110497 32448 Sertaconazole 6.16 6.58 18.47 3.22 174582 17654 Vitamin C 8.63 9.21 3.29 3.67 28355 112115 uniconazole 6.42 6.99 15.44 9.86 210484 224580 Sertraline hydrochloride 5.87 6.52 19.15 5.43 152881 20973 Promethazine hydrochloride 5.79 6.35 20.47 4.80 144350 22310 Ibuprofen 19.73 26.92 50.46 16.12 178668 18521

[0096] Appendix 1: Table 2 of Comparison Document 2

[0097] Table 2. Analytical results of 22 drugs from the α-Alk-β-CD organic hybrid monolithic column.

[0098]

[0099]

[0100]

Claims

1. A method for preparing an organic hybrid monolithic column, characterized in that, The method includes the following steps: Steps for preparing sulfonated β-cyclodextrin (S-β-CD) framework materials; S-β-cyclodextrin 20±1mg EDMA (dimethacrylate) 21±4 μL Anhydrous dimethyl sulfoxide (DMSO) 200±10μL The Glu-β-cyclodextrin backbone material was prepared by the same method, except that the amount of Glu-β-cyclodextrin was 60±1 mg.

2. The method for preparing the organic hybrid monolithic column as described in claim 1, characterized in that, In the polycondensation reaction step, the amount of S-β-cyclodextrin backbone material used to prepare the organic hybrid monolithic column is 20±1 mg, and the amount of Glu-β-cyclodextrin backbone material is 60±1 mg.

3. A method for preparing the sulfonated-β-cyclodextrin, glutamic-β-cyclodextrin-organosilicon hybrid monolithic column according to claim 1, characterized in that, The preparation method includes the following steps: Preparation steps of monolithic column for sulfonated β-cyclodextrin (S-β-CD) and glutamic acid β-cyclodextrin (Glu-β-CD) Preparation of S-β-cyclodextrin and Glu-β-cyclodextrin backbone materials; Prepare a homogeneous electrolyte containing 1,4-butanediol as a porogen; S-β-cyclodextrin backbone material, DMSO and azobisisobutyronitrile were added to a homogeneous hydrolysate, mixed well, and injected into a capillary tube for polymerization. Preparation of Glu-β-cyclodextrin using a monolithic column method.

4. The method for preparing the organic hybrid monolithic column as described in claim 1, characterized in that, In the polycondensation reaction step, the amount of dimethacrylate (EDMA) used to prepare each unit of the organic hybrid monolithic column is 21 ± 4 μL.

5. The method for preparing the organic hybrid monolithic column as described in claim 3, characterized in that, The preferred polycondensation reaction step is a polycondensation reaction at 40 degrees Celsius for 19 hours.

6. The preparation method according to claim 3, characterized in that, The method for preparing sulfonated β-cyclodextrin backbone material includes: placing sulfonated β-cyclodextrin, dimethacrylate (EDMA) and anhydrous DMSO in a reaction vessel, and continuously stirring the reaction vessel in a water bath to obtain the sulfonated β-cyclodextrin backbone material. The glutamic acid-β-cyclodextrin backbone material was prepared by the same method.

7. The preparation method according to claim 3, characterized in that... Acetic acid and 1,4-butanediol were mixed and magnetically stirred in an ice bath to obtain an electrolyte solution.

8. The preparation method according to claim 7, characterized in that, The mixture consisted of glacial acetic acid (0.01 M, 1.0 mL), 1,4-butanediol (20 μL), and ethyl methacrylate (EDMA) (21 ± 4 μL). After mixing, the mixture was magnetically stirred in an ice bath for 2 hours.

9. An organic hybrid monolithic column, characterized in that, The organic hybrid monolithic column is prepared by any of the organic hybrid monolithic column preparation methods described in claims 1-8, and is used to separate chiral drugs in an organosilicon hybrid monolithic column.

10. A capillary electrophoresis method for separating chiral drugs using an organic hybrid monolithic column, characterized in that, The stationary phase of the capillary electrophoresis method is any of the organic hybrid monolithic columns described in claims 1-10, and the capillary electrophoresis method is as follows: The pH of the mobile phase should be between 4.5 and 6.

5. Mobile phase concentration: 20 mM Tris buffer containing 10–30% (S-β-CD) and 0–40% Glu-β-CD in acetonitrile; Electrophoresis voltage 15kV; The chiral drug includes one or more chiral drugs among antihistamines, antihypertensive drugs, amino acids, azoles, and pesticides.

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