Acylated hydroxypropyl-beta-cyclodextrin derivatization chiral stationary phase as well as preparation method and application thereof

Abstract

The invention discloses an acylated hydroxypropyl-beta-cyclodextrin derivatization chiral stationary phase as well as a preparation method and application thereof. The preparation method comprises the following steps: sequentially reacting hydroxypropyl-beta-cyclodextrin with a 3-isocyanate propyltriethoxysilane coupling agent and an anhydride derivatization reagent by utilizing a one-pot method, and then reacting with activated silica gel microspheres to prepare a target material; the acylated hydroxypropyl-beta-cyclodextrin derivative chiral stationary phase prepared by the invention has good chiral resolution capability compared with an aryl carboxylic acid compound with relatively large steric hindrance, and is suitable for reversed-phase high performance liquid chromatography conditions, and the stability of a chromatographic column is good.

Description

Technical Field

[0001] The present invention relates to an acylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase, a preparation method thereof and an application thereof, belonging to the technical fields of composite materials and high performance liquid chromatography chiral separation. Background Art

[0002] High performance liquid chromatography (HPLC) has now become one of the most widely used techniques in the field of chiral separation. A variety of chiral stationary phases (CSPs) have been widely studied, including mainly cellulose, starch, cyclodextrin (CD) and macrocyclic antibiotics, etc. Compared with typical polysaccharides and macrocyclic antibiotics, the complementary enantiomeric recognition ability of CD is one of the important aspects attracting research interest. CSPs based on CD have attracted wide attention and achieved remarkable and exciting results in experiments. Due to its hydrophilic outer surface and hydrophobic inner cavity, β-CD can encapsulate guest molecules in its hydrophobic cavity through hydrogen bonding and hydrophobic interaction, thus forming inclusion complexes with different stabilities to achieve enantiomeric separation. However, due to the limited binding sites and binding strength of natural β-CD, its chiral separation efficiency is not ideal. Therefore, researchers have developed multifunctional β-CD derivatized CSPs, and such stationary phases exhibit excellent chiral selectivity for a wide range of compounds through various interaction mechanisms.

[0003] Specifically, among these derivatized CSPs, substituted phenylcarbamate β-CD CSPs are the main representatives. They utilize the hydrophobic cavity to promote molecular recognition through inclusion; meanwhile, aromatic groups can be introduced to further enhance chiral selectivity through π-π stacking. In addition, these CSPs have polar hydrogen bond donor or acceptor sites due to the presence of carbamate groups, which can facilitate dipole-dipole interaction and hydrogen bond binding. The large volume of the non-polar aromatic ring also introduces steric hindrance effects and van der Waals forces, further improving chiral selectivity.

[0004] Although a large number of existing articles report that the chiral stationary phase modified by cyclodextrin can greatly improve its chiral resolution ability, compared with the wide usability and specificity of polysaccharide stationary phases, the existing reported preparation methods are difficult, the routes are complex, and the problems such as general resolution ability are still the difficulties for its difficult promotion. For example, although the chiral resolution ability of the commercial hydroxypropyl-β-cyclodextrin chiral stationary phase has been improved, it cannot achieve rapid and complete resolution, and there is no specific resolution exclusive object. Therefore, for the above reasons, a specific derivatization method is reported herein to modify hydroxypropyl-β-cyclodextrin, and a simple synthesis method with streamlined synthesis steps and independent of diazotizing reagents is designed, which is of great significance for improving the separation performance of hydroxypropyl-β-cyclodextrin chromatographic columns. Summary of the Invention

[0005] The object of the present invention is to provide an acylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase, a preparation method thereof and an application thereof. The present invention uses a one-pot method to sequentially react hydroxypropyl-β-cyclodextrin with a silane coupling agent and a derivatizing reagent, and then reacts with silica microspheres to obtain the target material.

[0006] The acylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase of the present invention has good chromatographic column stability and excellent chiral separation ability.

[0007] The technical solution of the present invention is as follows:

[0008] A preparation method of an acylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase, comprising:

[0009] Dissolve hydroxypropyl-β-cyclodextrin in anhydrous N,N-dimethylformamide, add 3-isocyanatopropyltriethoxysilane coupling agent, stir and react at 80-100°C for 6-12 h, then add acid anhydride, stir and react at 90-120°C for 12-48 h, then add activated silica microspheres, continue to stir and react at 100-120°C for 24-36 h, then cool to room temperature, filter, wash the solid product, and dry to obtain the acylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase (pale yellow powder);

[0010] Wherein, the acid anhydride is selected from any one of acetic anhydride, propionic anhydride, and butyric anhydride;

[0011] The molar ratio of hydroxypropyl-β-cyclodextrin to acid anhydride is 1:10-1:30;

[0012] The feeding ratio of hydroxypropyl-β-cyclodextrin, 3-isocyanatopropyltriethoxysilane coupling agent, and activated silica microspheres is 1.8-4.6 g:0.365-1.50 mL:2-4 g;

[0013] The feeding ratio of hydroxypropyl-β-cyclodextrin to anhydrous N,N-dimethylformamide is 1.8-4.6 g, 30-50 mL;

[0014] The solid product is washed with acetone, methanol, and water respectively, and dried in a vacuum drying oven at 50-70°C for 6-24 h.

[0015] Specifically, the activation method of the silica microspheres is as follows:

[0016] Place the silica microspheres in an acid solution, stir at room temperature for 12-48 h, then reflux for 6-12 h, wash with deionized water until neutral, and obtain activated silica microspheres (SiO2) through freeze-drying and vacuum drying;

[0017] The acid solution is a hydrochloric acid solution or a nitric acid solution with a volume fraction of 5-18%;

[0018] The activated silica microspheres have a particle size of 5 - 8 μm and a pore diameter of 100 nm.

[0019] Specifically, the preparation method of hydroxypropyl - β - cyclodextrin is as follows:

[0020] Add 30 - 60 g of β - cyclodextrin into 20 - 60 mL of 13 - 22 wt% sodium hydroxide solution, heat it to 60 - 78 °C at a rate of 2 - 5 °C / min until all β - cyclodextrin is dissolved. Transfer the obtained β - cyclodextrin solution to an ice - water bath, cool it to 2 - 5 °C at a rate of 2 - 4 °C / min, and keep it for 20 - 30 min. Then, while stirring, dropwise add 20 - 120 mL of propylene oxide (0.280 - 1.682 mol) at a dropping rate of 130 - 200 g / h. After the dropping is completed, raise the temperature to room temperature at a rate of 2 °C / min, and continue stirring and reacting for 24 - 48 h. After post - treatment, hydroxypropyl - β - cyclodextrin is obtained;

[0021] The specific post - treatment method is: after the reaction is completed, neutralize the reaction solution with 30 - 38 wt% hydrochloric acid, then obtain a syrupy substance by vacuum distillation. Add 150 - 300 mL of 90 - 98% volume - fraction ethanol - aqueous solution and stir for 30 - 45 min to precipitate the NaCl generated by the neutralization reaction. Then filter out the precipitated NaCl with a Buchner funnel, wash the filter cake with pure ethanol (20 mL * 3), combine the filtrates and conduct vacuum distillation. Dissolve the concentrate in 50 - 100 mL of deionized water (to form a yellow transparent solution), and dialyze it with a dialysis bag with a 500 MW cut - off point against running water for 12 - 24 h (to remove residual trace NaCl and small - molecule impurities generated during the reaction process). Finally, freeze - dry to obtain hydroxypropyl - β - cyclodextrin;

[0022] The degree of substitution of the obtained hydroxypropyl - β - cyclodextrin is: 5 - 18; the yield is: 43.46 - 58.74%.

[0023] Specifically, the preparation method of the acylated hydroxypropyl - β - cyclodextrin derivative chiral stationary phase is as follows:

[0024] Accurately weigh 1.8 - 4.6 g of hydroxypropyl - β - cyclodextrin and dissolve it in 30 - 50 mL of dry anhydrous N,N - dimethylformamide. Add 0.365 - 1.50 mL of 3 - isocyanatopropyltriethoxysilane coupling agent and mix well. Place it in an oil bath at 80 - 100 °C and stir for 6 - 12 h. Then add a certain amount of anhydride (1.32 - 6.30 mL) to it, and place it in an oil bath at 90 - 120 °C for reaction for 12 - 48 h. Finally, add 2 - 4 g of activated silica microspheres and continue to react in an oil bath at 100 - 120 °C for 24 - 36 h. After the reaction is completed, filter, and wash the solid product with acetone, methanol, and water respectively. Finally, place it in a vacuum drying oven to dry for 6 - 24 h to obtain a pale yellow powdery solid, which is the acylated hydroxypropyl - β - cyclodextrin - derivatized chiral stationary phase.

[0025] Pack the acylated hydroxypropyl - β - cyclodextrin - derivatized chiral stationary phase prepared in this application into the column tube of high - performance liquid chromatography, and it can be used for the separation of enantiomers.

[0026] The enantiomers preferably include aryl carboxylic acid compounds with large steric hindrance, such as: 2 - (4 - chlorophenyl) - 3 - methylbutyric acid, 2,3 - diphenylpropionic acid, α - cyclopentylmandelic acid, α - isopropylphenylacetic acid, voriconazole, paroctol, etc.

[0027] Use the slurry method to pack the acylated hydroxypropyl - β - cyclodextrin - derivatized chiral stationary phase into the column tube of high - performance liquid chromatography. The specific packing method is as follows: Take 3 - 5 g of the acylated hydroxypropyl - β - cyclodextrin - derivatized chiral stationary phase, add 20 - 50 mL of methanol, ultrasonically disperse for 30 - 60 min, load the obtained chiral stationary phase slurry into a slurry tank, use methanol as the displacement liquid, and press it into the stainless - steel column tube through a pneumatic pump. The specifications of the chromatographic column are 100*4.6 mm (I.D.), and the packing pressure is 35 - 60 MPa.

[0028] The advantages of the present invention are mainly reflected in:

[0029] (1) The process of the present invention is simple, the reaction conditions are mild, and the reaction efficiency is high, which is conducive to the rapid and efficient synthesis of cyclodextrin - functionalized silica microspheres. And the raw materials are cheap and easily available, which is conducive to realizing industrialized mass production.

[0030] (2) In the chiral stationary phase prepared in the present invention, the acyl groups on the edge of cyclodextrin expand the cavity of the cyclodextrin stationary phase, increasing the hydrophobic interaction, π - π stacking interaction, and hydrogen bond and other interaction forces between the stationary phase and solute molecules. And its narrow chiral cavity makes it more conducive to forming inclusion compounds with various compounds with obvious steric hindrance in the reversed - phase mode. It is a chiral stationary phase with specific separation specificity. Description of the Drawings

[0031] Figure 1 : Schematic diagram for the synthesis of acylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase.

[0032] Figure 2 : Infrared spectroscopy (IR) diagram of acylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase; (a) is acetyl hydroxypropyl-β-cyclodextrin chiral stationary phase; (b) is propionyl hydroxypropyl-β-cyclodextrin chiral stationary phase; (c) is butyryl hydroxypropyl-β-cyclodextrin chiral stationary phase; (d) is hydroxypropyl-β-cyclodextrin; (e) is hydroxypropyl-β-cyclodextrin chiral stationary phase; (f) is silica microspheres.

[0033] Figure 3 : Thermogravimetric (TGA) curve of acylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase; (a) is hydroxypropyl-β-cyclodextrin chiral stationary phase derivatized with acetic anhydride; (b) is hydroxypropyl-β-cyclodextrin chiral stationary phase derivatized with propionic anhydride; (c) is hydroxypropyl-β-cyclodextrin chiral stationary phase derivatized with butyric anhydride; (d) is hydroxypropyl-β-cyclodextrin chiral stationary phase; (e) is silica microspheres.

[0034] Figure 4 : Chromatographic separation diagram of the racemate of 2-(4-chlorophenyl)-3-methylbutyric acid.

[0035] Figure 5 : Chromatographic separation diagram of the racemate of 2,3-diphenylpropionic acid.

[0036] Figure 6 : Chromatographic separation diagram of the racemate of α-cyclopentylmandelic acid.

[0037] Figure 7 : Chromatographic separation diagram of the racemate of α-isopropylphenylacetic acid.

[0038] Figure 8 : Chromatographic separation diagram of the racemate of voriconazole.

[0039] Figure 9 : Chromatographic separation diagram of the racemate of paroxetine. Detailed implementation manners

[0040] The present invention will be further described below through specific embodiments, but the protection scope of the present invention is not limited thereto.

[0041] In the following embodiments,

[0042] Silica microspheres were purchased from Qingdao Haisu New Material Technology Co., Ltd., with a particle size of 5 μm; β-cyclodextrin was purchased from Shanghai Titan Technology Co., Ltd., with a purity of 99%.

[0043] The high-performance liquid chromatography is the Shimadzu analytical LC-20A high-performance liquid chromatograph (Shimadzu, Japan), which consists of an SPD-20A UV detector, a dual LC-20ATvp liquid pump, a CTO-10ASvp column oven, a LabSolutions chromatographic workstation, a 20 μL injection loop, etc.

[0044] Example 1:

[0045] A preparation method of an acylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase is as follows:

[0046] (1) Place the silica microspheres (2.3 g) in a hydrochloric acid solution with a volume concentration of 8% and stir at room temperature for activation for 28 h. Then heat it under reflux for 12 h, wash it with deionized water until neutral, and obtain activated silica microspheres (SiO2) after freeze-drying and vacuum drying.

[0047] (2) Take 30 g of β-cyclodextrin and add it to a three-necked flask containing 30 mL of 14% sodium hydroxide solution. Then heat the obtained β-cyclodextrin solution to 68 °C at a rate of 5 °C / min until completely dissolved. Then transfer it to an ice-water bath and cool it to 5 °C at a rate of 3 °C / min and keep it for 30 min. Then gradually and evenly dropwise add propylene oxide (20 mL, 0.280 mol) while stirring, and the dropping rate is 130 g / h. After the dropping is completed, set the programmed temperature increase to 2 °C / min and adjust it to room temperature conditions and continue stirring and reacting for 24 h. After the reaction is completed, neutralize the greatly excessive sodium hydroxide with 30% concentrated hydrochloric acid. The reaction solution is subjected to solvent removal by vacuum distillation to obtain a residue similar to syrup. Then add 150 mL of 90% ethanol-water and stir for up to 45 min to precipitate the contained NaCl. Then use a Buchner funnel to filter out the precipitated NaCl, and wash the hydroxypropyl-β-cyclodextrin adsorbed on the surface of NaCl with pure ethanol (20 mL * 3). Combine the filtrates and perform vacuum distillation to obtain a pale yellow thick syrup. Transfer it to a round-bottom flask, then add 50 mL of deionized water to dissolve it into a yellow transparent solution, and dialyze it with a dialysis bag with a 500 MW cut-off point under running water for 24 h to remove the residual trace NaCl and small molecule impurities generated during the reaction process. Finally, obtain hydroxypropyl-β-cyclodextrin after freeze-drying. (Degree of substitution: 5.06; Yield: 43.46%)

[0048] (3) 1.8 g of the hydroxypropyl-β-cyclodextrin prepared in step (2) was dissolved in 30 mL of dry anhydrous N,N-dimethylformamide, 0.365 mL of 3-isocyanatepropyltriethoxysilane coupling agent was added and mixed, and the mixture was placed in an 80°C oil bath and stirred for 12 h. Then, a certain amount of acetic anhydride (1.32 mL) was added thereto, and the mixture was placed in an oil bath at 120°C and reacted for 48 h. Finally, 2 g of the silica gel microspheres activated in step (1) were added, and the mixture was continued to react in an oil bath at 120°C for 24 h. After the reaction was completed, the mixture was filtered, and the solid product was washed with acetone, methanol, and water respectively, and finally placed in a vacuum drying oven and dried for 24 h to obtain a light yellow powdery solid, which was the acetylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase.

[0049] (4) The acetylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase prepared in step (3) was filled into a high performance liquid chromatography column using a homogenization method. 3 g of the acetylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase prepared in step (3) was taken and 20 mL of methanol was added thereto. The chiral stationary phase was ultrasonically dispersed for 30 min. The resulting chiral stationary phase homogenate was placed in a homogenization tank and, with methanol as a displacement fluid, was pressed into a stainless steel column tube by a pneumatic pump. The specifications of the chromatographic column were 100*4.6 mm (ID). The filling pressure was 60 MPa.

[0050] The product in Example 1 was characterized by infrared, elemental analysis, and thermogravimetric analysis, and the results were analyzed as follows:

[0051] In such Figure 2 In the FT-IR spectrum shown in (a), 3440 cm -1 The peaks attributed to the hydroxyl groups of hydroxypropyl-β-cyclodextrin and the residual hydroxyl groups on the silanol silicon surface are 2956 and 2714 cm -1 1624 cm-1 is attributed to the stretching vibrations of the methyl and methylene groups of hydroxypropyl-β-cyclodextrin and the methyl and methylene groups of the introduced acyl groups. -1 Corresponding to the stretching vibration absorption peak of carbonyl C=O; at 1100cm -1 The absorption peak attributed to the stretching vibration of COC is 855 cm -1 It is the stretching vibration of the ether bond on cyclodextrin, 627cm -1 is the CH bending vibration of cyclodextrin. These characteristic peaks indicate that hydroxypropyl-β-cyclodextrin is successfully bonded to the surface of silica microspheres.

[0052] The elemental analysis (Table 1) shows that the acetylated hydroxypropyl-β-cyclodextrin chiral stationary phase has an elemental distribution of carbon, nitrogen, oxygen, and silicon. Compared with the hydroxypropyl-β-cyclodextrin chiral stationary phase, the carbon, nitrogen, and silicon contents increase, and the oxygen content decreases, which indicates that acetic anhydride is successfully bonded to the surface of the hydroxypropyl-β-cyclodextrin chiral stationary phase.

[0053] Furthermore, the acetylated hydroxypropyl-β-cyclodextrin chiral stationary phase prepared in Example 1 was characterized by thermogravimetric analysis. As Figure 3 shown in (a) of , the thermogravimetric curve shows that the thermogravimetric loss of the acetylated hydroxypropyl-β-cyclodextrin chiral stationary phase is significantly higher than that of the hydroxypropyl-β-cyclodextrin chiral stationary phase and silica microspheres, which is attributed to the thermal decomposition of acetic anhydride and hydroxypropyl-β-cyclodextrin, further proving the successful preparation of the acetylated hydroxypropyl-β-cyclodextrin chiral stationary phase.

[0054] Table 1 Elemental analysis results of acetylated hydroxypropyl-β-cyclodextrin chiral stationary phase, hydroxypropyl-β-cyclodextrin chiral stationary phase, and silica

[0055]

[0056] Preparation of mobile phase and sample: Triethylamine acetate buffer solutions with pH = 3.2 and 5.5 were prepared by mixing acetic acid and water at a volume ratio of 5:1000, and the pH was adjusted with triethylamine. Acetonitrile and triethylamine acetate buffer solution were sonicated for 30 min with an ultrasonic instrument before use.

[0057] The sample was prepared into 1 mg / mL with acetonitrile:water = 8:2 (v / v), and the sample loading amount was 20 μL.

[0058] Example 2:

[0059] The reaction steps and conditions were the same as those in Example 1, and the derivatization reagent used was acetic anhydride with a dosage of 2.64 mL. Finally, an acetylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase was obtained.

[0060] Example 3:

[0061] The reaction steps and conditions were the same as those in Example 1, and the derivatization reagent used was acetic anhydride with a dosage of 3.96 mL. Finally, an acetylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase was obtained.

[0062] Example 4:

[0063] The reaction steps and conditions were the same as those in Example 1, and the derivatization reagent used was propionic anhydride with a dosage of 1.72 mL. Finally, a propionylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase was obtained.

[0064] The product in Example 4 was characterized by infrared, elemental analysis, and thermogravimetric analysis, and the results are analyzed as follows:

[0065] In (b) of the FT-IR spectrum as shown in Figure 2 , 3440 cm -1 ​Attributed to the residual hydroxyl peaks of the hydroxyl groups of hydroxypropyl-β-cyclodextrin and the silanol groups on the silicon surface, 2956, 2714 cm -1 Attributed to the stretching vibrations of the methyl and methylene groups of hydroxypropyl-β-cyclodextrin and the methyl and methylene groups on the introduced acyl groups. 1624 cm -1 Corresponding to the stretching vibration absorption peak of the carbonyl C=O; at 1100 cm -1 Attributed to the stretching vibration absorption peak of C-O-C, 855 cm -1 Is the stretching vibration of the ether bond on the cyclodextrin, 627 cm -1 Is the C-H bending vibration of the cyclodextrin. These characteristic peaks all indicate that hydroxypropyl-β-cyclodextrin has been successfully bonded to the surface of the silica microspheres.

[0066] In the elemental analysis (Table 2), the chiral stationary phase of propionylated hydroxypropyl-β-cyclodextrin has the elemental distribution of carbon, nitrogen, oxygen, and silicon. Compared with the chiral stationary phase of hydroxypropyl-β-cyclodextrin, the increase in carbon and nitrogen contents and the decrease in oxygen and silicon contents indicate that propionic anhydride has been successfully bonded to the surface of the chiral stationary phase of hydroxypropyl-β-cyclodextrin.

[0067] Furthermore, the propionylated hydroxypropyl-β-cyclodextrin chiral stationary phase prepared in Example 4 was characterized by thermogravimetric analysis. As Figure 3 Shown in (b) of, the thermogravimetric curve shows that the thermogravimetric loss of the propionylated hydroxypropyl-β-cyclodextrin chiral stationary phase is significantly higher than that of the hydroxypropyl-β-cyclodextrin chiral stationary phase and the silica microspheres, which is attributed to the thermal decomposition of propionic anhydride and hydroxypropyl-β-cyclodextrin, further proving the successful preparation of the propionylated hydroxypropyl-β-cyclodextrin chiral stationary phase.

[0068] Table 2 Elemental analysis results of the propionylated hydroxypropyl-β-cyclodextrin chiral stationary phase, hydroxypropyl-β-cyclodextrin chiral stationary phase, and silica

[0069]

[0070] Preparation of the mobile phase and the sample: Triethylamine acetate buffer solutions with pH = 3.2 and 5.5 were prepared by mixing acetic acid and water at a volume ratio of 5:1000, and the pH was adjusted with triethylamine. Acetonitrile and triethylamine acetate buffer were sonicated for 30 min with an ultrasonic instrument before use.

[0071] The sample was prepared into 1 mg / mL with acetonitrile:water = 8:2 (v / v), and the sample loading amount was 20 μL.

[0072] Example 5:

[0073] The reaction steps and conditions were the same as those in Example 1, and the derivatization reagent used was propionic anhydride, with a dosage of 3.44 mL. Finally, a propionylated hydroxypropyl-β-cyclodextrin chiral stationary phase was obtained.

[0074] Example 6:

[0075] The reaction steps and conditions are the same as those in Example 1. The derivatization reagent used is propionic anhydride, and its dosage is 5.16 mL. Finally, a propionylated hydroxypropyl-β-cyclodextrin chiral stationary phase is obtained.

[0076] Example 7:

[0077] The reaction steps and conditions are the same as those in Example 1. The derivatization reagent used is butyric anhydride, and its dosage is 2.10 mL. Finally, a butyrylated hydroxypropyl-β-cyclodextrin chiral stationary phase is obtained.

[0078] The product in Example 7 was characterized by infrared, elemental analysis, and thermogravimetric analysis. The results are analyzed as follows:

[0079] In (c) of the FT-IR spectrum as shown in Figure 2 , 3440 cm -1 is attributed to the hydroxyl groups of hydroxypropyl-β-cyclodextrin and the residual hydroxyl groups on the surface of the silanol group silicon, and 2956, 2714 cm -1 are attributed to the stretching vibrations of the methyl and methylene groups of hydroxypropyl-β-cyclodextrin and the methyl and methylene groups on the introduced acyl group. 1624 cm -1 corresponds to the stretching vibration absorption peak of the carbonyl C=O; at 1100 cm -1 is attributed to the stretching vibration absorption peak of C-O-C, and 855 cm -1 is the stretching vibration of the ether bond on the cyclodextrin, and 627 cm -1 is the C-H bending vibration of the cyclodextrin. These characteristic peaks all indicate that hydroxypropyl-β-cyclodextrin has been successfully bonded to the surface of the silica microspheres.

[0080] In the elemental analysis (Table 3), the butyrylated hydroxypropyl-β-cyclodextrin chiral stationary phase has the elemental distribution of carbon, nitrogen, oxygen, and silicon. The increase in the carbon, nitrogen, and silicon contents and the decrease in the oxygen content compared to the hydroxypropyl-β-cyclodextrin chiral stationary phase indicate that butyric anhydride has been successfully bonded to the surface of the hydroxypropyl-β-cyclodextrin chiral stationary phase.

[0081] Furthermore, the butyrylated hydroxypropyl-β-cyclodextrin chiral stationary phase prepared in Example 7 was characterized by thermogravimetric analysis. As shown in (c) of Figure 3 , the thermogravimetric curve shows that the thermogravimetric loss of the butyrylated hydroxypropyl-β-cyclodextrin chiral stationary phase is significantly higher than that of the hydroxypropyl-β-cyclodextrin chiral stationary phase and the silica microspheres, which is attributed to the thermal decomposition of butyric anhydride and hydroxypropyl-β-cyclodextrin, further proving the successful preparation of the butyrylated hydroxypropyl-β-cyclodextrin chiral stationary phase.

[0082] Table 3 Elemental analysis results of butyrylated hydroxypropyl-β-cyclodextrin chiral stationary phase, hydroxypropyl-β-cyclodextrin chiral stationary phase, and silica

[0083]

[0084] Preparation of mobile phase and sample: Triethylamine acetate buffer solutions with pH = 3.2 and 5.5 were prepared with acetic acid and water at a volume ratio of 5:1000, and the pH was adjusted with triethylamine. Acetonitrile and triethylamine acetate buffer solution were ultrasonically treated for 30 min with an ultrasonic instrument before use.

[0085] The sample was prepared into 1 mg / mL with acetonitrile:water = 8:2 (v / v), and the sample injection volume was 20 μL.

[0086] Example 8:

[0087] The reaction steps and conditions were the same as those in Example 1, and the derivatization reagent used was butyric anhydride, with a dosage of 4.20 mL. Finally, a butyrylated hydroxypropyl-β-cyclodextrin chiral stationary phase was obtained.

[0088] Example 9:

[0089] The reaction steps and conditions were the same as those in Example 1, and the derivatization reagent used was butyric anhydride, with a dosage of 6.30 mL. Finally, a butyrylated hydroxypropyl-β-cyclodextrin chiral stationary phase was obtained.

[0090] Example 10:

[0091] The racemate of 2-(4-chlorophenyl)-3-methylbutyric acid was resolved using the acetylated hydroxypropyl-β-cyclodextrin chiral chromatographic column prepared in Example 1, and its chromatographic separation diagram is as Figure 4 shown. The chromatographic conditions used were: chromatographic column 10×4.6 mm (i.d.), mobile phase acetonitrile / buffer salt (triethylamine acetate buffer salt 0.5%, pH, 3.20) = 80 / 20 (v / v); flow rate 0.6 mL / min; UV detection wavelength 254 nm; column temperature 25 °C.

[0092] Example 11:

[0093] The racemate of 2,3-diphenylpropionic acid was resolved using the acetylated hydroxypropyl-β-cyclodextrin chiral chromatographic column prepared in Example 2, and its chromatographic separation diagram is as Figure 5 shown. The chromatographic conditions used were: chromatographic column 10×4.6 mm (i.d.), mobile phase acetonitrile / buffer salt (triethylamine acetate buffer salt 0.5%, pH, 3.20) = 80 / 20 (v / v); flow rate 0.6 mL / min; UV detection wavelength 254 nm; column temperature 25 °C.

[0094] Example 12:

[0095] The racemate of α-cyclopentylmandelic acid was resolved using the butyrylated hydroxypropyl-β-cyclodextrin chiral chromatographic column prepared in Example 7, and its chromatogram is as shown in Figure 6 Figure. The chromatographic conditions used were as follows: chromatographic column 10×4.6 mm (i.d.), mobile phase acetonitrile / buffer salt (triethylamine acetate buffer salt 0.5%, pH 3.20) = 80 / 20 (v / v); flow rate 0.6 mL / min; UV detection wavelength 254 nm; column temperature 25 °C.

[0096] Example 13:

[0097] The racemate of α-isopropylphenylacetic acid was separated using the propionylated hydroxypropyl-β-cyclodextrin chiral chromatographic column prepared in Example 5, and its chromatogram is as shown in Figure 7 Figure. The chromatographic conditions used were as follows: chromatographic column 10×4.6 mm (i.d.), mobile phase acetonitrile / buffer salt (triethylamine acetate buffer salt 0.5%, pH 3.20) = 80 / 20 (v / v); flow rate 0.6 mL / min; UV detection wavelength 254 nm; column temperature 25 °C.

[0098] Example 14:

[0099] The racemate of voriconazole was separated using the acetylated hydroxypropyl-β-cyclodextrin chiral chromatographic column prepared in Example 2, and its chromatogram is as shown in Figure 8 Figure. The chromatographic conditions used were as follows: chromatographic column 10×4.6 mm (i.d.), mobile phase acetonitrile / buffer salt (triethylamine acetate buffer salt 0.5%, pH 3.20) = 80 / 20 (v / v); flow rate 0.6 mL / min; UV detection wavelength 254 nm; column temperature 25 °C.

[0100] Example 15:

[0101] The racemate of paroxetine was separated using the acetylated hydroxypropyl-β-cyclodextrin chiral chromatographic column prepared in Example 3, and its chromatogram is as shown in Figure 9 Figure. The chromatographic conditions used were as follows: chromatographic column 10×4.6 mm (i.d.), mobile phase acetonitrile / buffer salt (triethylamine acetate buffer salt 0.5%, pH 5.50) = 95 / 5 (v / v); flow rate 0.6 mL / min; UV detection wavelength 254 nm; column temperature 25 °C.

Claims

1. A preparation method of an acylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase, characterized in that, The described preparation method includes: Dissolve hydroxypropyl-β-cyclodextrin in anhydrous N,N-dimethylformamide, add 3-isocyanatopropyltriethoxysilane coupling agent, stir and react at 80-100 °C for 6-12 h, then add acid anhydride, stir and react at 90-120 °C for 12-48 h, subsequently add activated silica microspheres, continue to stir and react at 100-120 °C for 24-36 h, then cool to room temperature, filter, wash the solid product, and dry to obtain the acylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase; Wherein, The acid anhydride is selected from any one of acetic anhydride, propionic anhydride, and butyric anhydride.

2. The preparation method of the acylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase according to claim 1, wherein, The molar ratio of hydroxypropyl-β-cyclodextrin to acid anhydride is 1:10-1:

30.

3. The preparation method of the acylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase according to claim 1, wherein, The feeding ratio of hydroxypropyl-β-cyclodextrin, 3-isocyanatopropyltriethoxysilane coupling agent, and activated silica microspheres is 1.8-4.6 g: 0.365-1.50 mL: 2-4 g.

4. The preparation method of the acylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase according to claim 1, characterized in that, The feeding ratio of hydroxypropyl-β-cyclodextrin to anhydrous N,N-dimethylformamide is 1.8-4.6 g: 30-50 mL.

5. The preparation method of the acylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase according to claim 1, characterized in that, The solid product is washed with acetone, methanol, and water respectively, and placed in a vacuum drying oven at 50-70 °C for drying for 6-24 h.

6. The preparation method of the acylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase according to claim 1, wherein, The preparation method of the described activated silica microspheres is as follows: Place the silica microspheres in an acid solution, stir at room temperature for 12-48 h, then reflux for 6-12 h, wash with deionized water until neutral, and obtain the activated silica microspheres through freeze-drying and vacuum drying; The acid solution is hydrochloric acid solution or nitric acid solution, with a volume fraction of 5-18%; The activated silica microspheres have a particle size of 5-8 μm and a pore size of 100 nm.

7. The preparation method of the acylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase according to claim 1, characterized in that, The preparation method of the described hydroxypropyl-β-cyclodextrin is as follows: Add 30-60 g of β-cyclodextrin to 20-60 mL of 13-22 wt% sodium hydroxide solution, heat to 60-78 °C at a rate of 2-5 °C / min until the β-cyclodextrin is completely dissolved, transfer the obtained β-cyclodextrin solution to an ice-water bath, cool to 2-5 °C at a rate of 2-4 °C / min, maintain for 20-30 min, then dropwise add 20-120 mL of propylene oxide while stirring, with a dropping rate of 130-200 g / h. After the dropping is completed, heat to room temperature at a rate of 2 °C / min, continue to stir and react for 24-48 h, and then obtain hydroxypropyl-β-cyclodextrin through post-treatment; The post-treatment method is: after the reaction is completed, neutralize the reaction solution with 30-38 wt% hydrochloric acid, then obtain a syrupy substance through vacuum distillation, add 150-300 mL of 90-98% ethanol aqueous solution by volume fraction and stir for 30-45 min to precipitate the NaCl generated by the neutralization reaction, then filter out the precipitated NaCl with a Buchner funnel, wash the filter cake with pure ethanol, combine the filtrates for vacuum distillation, dissolve the concentrate in 50-100 mL of deionized water, dialyze with a dialysis bag with a 500 MW cut-off point under running water for 12-24 h, and finally freeze-dry to obtain hydroxypropyl-β-cyclodextrin.

8. The acylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase prepared by the preparation method according to any one of claims 1-7.

9. Use of the acylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase in the separation of enantiomers as described in claim 8.

10. The application according to claim 9, characterized in that, The acylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase is filled into the column tube of high performance liquid chromatography for the separation of enantiomers; The enantiomers are: 2-(4-chlorophenyl)-3-methylbutyric acid, 2,3-diphenylpropionic acid, α-cyclopentylmandelic acid, α-isopropylphenylacetic acid, voriconazole or paroctol; The acylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase is filled into the column tube of high performance liquid chromatography by the slurry method. The packing method is as follows: Take 3-5 g of the acylated hydroxypropyl-β-cyclodextrin derivatized chiral stationary phase, add 20-50 mL of methanol, ultrasonically disperse for 30-60 min, load the obtained chiral stationary phase slurry into the slurry tank, use methanol as the displacement liquid, and press it into the stainless steel column tube through a pneumatic pump; the specification of the chromatographic column is 100*4.6 mm, and the packing pressure is 35-60 MPa.

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