A high-efficiency chiral separation chromatographic column and preparation method thereof

Abstract

The present invention belongs to the technical field of polymer materials and chromatographic separation, and discloses a high-efficiency chiral separation chromatographic column and a preparation method thereof. The preparation method is as follows: dissolving cyclodextrin or its derivatives in an alkaline aqueous solution, then adding allyl epoxy-terminated polyether, heating to 30-80°C and stirring for etherification reaction, separating and purifying the product to obtain an allyl polyether-substituted cyclodextrin compound, then mixing it with an acrylate monomer, a cross-linking monomer, a porogenic solvent and an initiator, ultrasonically dissolving and degassing it, and then pouring it into a chromatographic column, heating it in a water bath to 40-80°C for polymerization reaction, and washing and removing unreacted monomers and porogenic solvents after the reaction is completed to obtain an high-efficiency chiral separation chromatographic column. The present invention adopts free radical copolymerization of an allyl polyether-substituted cyclodextrin compound with an acrylate monomer and a cross-linking monomer to prepare a monolithic chromatographic column solid phase filler, the preparation method is simple and controllable, and the obtained chromatographic column has the advantage of high separation efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of polymer materials and chromatographic separation, and particularly relates to a high-efficiency chiral separation chromatographic column and a preparation method thereof. Background Art

[0002] Chromatographic separation is an analytical technique based on the difference in the distribution coefficient of a substance between the stationary phase and the mobile phase. Its basic principle is to use the difference in the distribution behavior of each component in the mixture in different phases to achieve separation through the relative movement of the two phases. During the chromatographic separation process, one phase is fixed and is called the stationary phase; the other phase continuously flows through the stationary phase and is called the mobile phase. The mixture is introduced into the chromatographic column, which is filled with the stationary phase, and then the mobile phase is used to move the mixture. Different substances have different distribution coefficients in the stationary phase and the mobile phase, causing them to move along the stationary phase at different speeds, ultimately achieving the effect of separation. The properties of the stationary phase play a key role in the separation effect.

[0003] According to the preparation method of the stationary phase, chromatographic columns are mainly divided into packed columns and monolithic columns. The preparation of monolithic columns is based on the principle of in-situ polymerization or curing, and the mixture of monomers, cross-linking agents, initiators, etc. is directly polymerized in the column tube to form a continuous porous structure. Compared with traditional packed columns, monolithic columns have the advantages of good permeability, fast mass transfer rate, and low column pressure, so they can be used for efficient separation of target compounds.

[0004] Chirality is one of the basic properties of nature. From microscopic particles to macroscopic substances, chirality is ubiquitous. For example, most proteins and nucleic acids that make up living organisms are chiral, so chirality research plays an important role in pharmacology, analytical chemistry, life sciences and other fields. The focus of chirality research is mainly on the separation of chiral drugs. Among common drugs, a considerable number exist in the form of racemates. Generally, one of the enantiomers exerts its pharmacological effect, while the other enantiomer usually has no pharmacological effect or even offsets some of the effects. Therefore, exploring technologies and methods that can effectively separate racemates is a major challenge in the development of chiral drugs.

[0005] Cyclodextrin is a chiral compound that has the ability to recognize and select organic molecules and has been successfully applied to various chromatography and electrophoresis methods. At the same time, cyclodextrin can effectively increase the solubility and dissolution rate of some poorly water-soluble drugs in water, and has good affinity for drug molecules, so it can be used for the efficient separation of chiral drugs. Patent CN1603817 A discloses a method for preparing a high-performance liquid chromatography organic phase monolithic column bonded with cyclodextrin compounds, which uses cyclodextrin or its derivatives to react with the epoxy groups on the monolithic column matrix of glycidyl methacrylate-ethylene dimethacrylate polymer under the catalysis of boron trifluoride (BF3), which can be used for small molecule sample separation and analysis, chiral compound separation, and biomacromolecule separation. However, it is necessary to prepare a monolithic matrix column first, and then use a cyclodextrin derivatization solution for reaction bonding, which has the problem of low bonding efficiency. At the same time, cyclodextrin has a large steric hindrance due to its three-dimensional ring structure, which also leads to low bonding efficiency, and the separation efficiency of the prepared chromatographic column is not high. In addition, it directly uses cyclodextrin, hydroxyethyl cyclodextrin, and hydroxypropyl cyclodextrin to react with the epoxy groups on the polymer monolithic column matrix. The bonded cyclodextrin molecules cannot be effectively extended, and the binding efficiency with the to-be-separated substances in the mobile phase is low, which also leads to low separation efficiency. Summary of the invention

[0006] In view of the shortcomings and deficiencies of the above prior art, the primary purpose of the present invention is to provide a method for preparing an efficient chiral separation chromatographic column.

[0007] Another object of the present invention is to provide a high-efficiency chiral separation chromatographic column prepared by the above method.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A method for preparing a high-efficiency chiral separation chromatographic column comprises the following preparation steps:

[0010] (1) dissolving cyclodextrin or its derivative in an alkaline aqueous solution, then adding allyl epoxy-terminated polyether, heating to 30-80° C. and stirring for etherification reaction, and separating and purifying the product to obtain an allyl polyether-substituted cyclodextrin compound;

[0011] (2) The allyl polyether-substituted cyclodextrin compound is mixed with an acrylate monomer, a cross-linking monomer, a porogenic solvent and an initiator, dissolved by ultrasonication, degassed and then poured into a chromatographic column, heated in a water bath to 40 to 80° C. for polymerization reaction, and after the reaction is completed, the unreacted monomer and the porogenic solvent are washed away to obtain a high-efficiency chiral separation chromatographic column.

[0012] Furthermore, the cyclodextrin or its derivative in step (1) is β-cyclodextrin, hydroxyethyl-β-cyclodextrin or hydroxypropyl-β-cyclodextrin.

[0013] Furthermore, the alkaline aqueous solution in step (1) is a NaOH aqueous solution with a mass concentration of 5% to 15%.

[0014] Furthermore, the molecular weight of the allyl epoxy terminated polyether in step (1) is 400 to 1000.

[0015] The present invention uses allyl epoxy-terminated polyether to carry out etherification reaction on cyclodextrin compounds, which can cut off the hydrogen bonding of hydroxyl groups between cyclodextrin molecules and improve the solubility of subsequent reactions; on the other hand, the connecting arm of appropriate length can reduce the reaction steric hindrance of cyclodextrin molecules and improve the copolymerization effect. In addition, it can promote the extension of polymerized cyclodextrin molecules in the mobile phase, improve the bonding efficiency with the mixture to be separated, and effectively improve the separation efficiency.

[0016] Furthermore, in step (1), the molar ratio of the cyclodextrin or its derivative to the allyl epoxy-terminated polyether is 1:1-2.

[0017] The present invention can adjust the degree of substitution of the etherification reaction by controlling the addition amount of cyclodextrin or its derivatives and allyl epoxy-terminated polyether, so as to achieve a suitable crosslinking density and separation effect. If the addition amount of allyl epoxy-terminated polyether is too low, its degree of substitution is reduced, the solubility is poor, it is not easy to dissolve in the subsequent polymerization reaction process, and the terminal allyl content of the subsequent polymerization reaction with the acrylate monomer is too low, the polymerization effect is poor, and the separation effect is poor; if the addition amount of allyl epoxy-terminated polyether is too high, it will lead to excessive crosslinking and embedding of the cyclodextrin compound substituted by the allyl polyether, and the cyclodextrin compound cannot effectively extend to combine and interact with the mixture to be separated in the mobile phase, and the separation effect is poor.

[0018] Furthermore, the etherification reaction time in step (1) is 1 to 4 hours.

[0019] Furthermore, the separation and purification method in step (1) is as follows: after the etherification reaction is completed, acid is added for neutralization, the reaction solution is concentrated and then ethanol is added for precipitation, the salt is removed by filtration, and the filtrate is taken for removal of the solvent by vacuum distillation to obtain an allyl polyether-substituted cyclodextrin compound.

[0020] Furthermore, in step (2), the acrylate monomer is butyl methacrylate (BMA) or dimethylaminoethyl methacrylate (DMAEMA); and the crosslinking monomer is methylenebisacrylamide (MBA) or ethylene glycol dimethacrylate (EGDMA).

[0021] Furthermore, the porogenic solvent in step (2) is a mixed solvent of at least one solvent selected from N,N-dimethylformamide, N,N-dimethylacetamide and dimethyl sulfoxide and isopropanol in a volume ratio of 100:20 to 40.

[0022] Furthermore, the initiator in step (2) is azobisisobutyronitrile (AIBN).

[0023] Furthermore, in step (2), the mass ratio of the allyl polyether-substituted cyclodextrin compound, the acrylate monomer, the crosslinking monomer, the porogenic solvent and the initiator is 10-20:30-50:20-40:60-100:0.2-1.

[0024] Furthermore, the flushing in step (2) refers to connecting a high-pressure pump to flush with methanol.

[0025] A high-efficiency chiral separation chromatographic column is prepared by the method.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention adopts free radical copolymerization of allyl polyether-substituted cyclodextrin compounds with acrylate monomers and cross-linking monomers to prepare the integral chromatographic column solid phase filler, which has the advantages of high cyclodextrin molecular grafting efficiency and easy structure control, and the obtained chromatographic column has the advantage of high separation efficiency.

[0028] (2) The present invention uses an allyl epoxy-terminated polyether of suitable molecular weight to carry out an etherification substitution reaction on the cyclodextrin compound in advance, which is beneficial to improving the compatibility of the subsequent copolymerization reaction and enhancing the copolymerization effect; at the same time, the introduction of a polyether chain of suitable length can promote the extension of the polymerized cyclodextrin molecules in the mobile phase, improve the binding efficiency with the mixture to be separated, and effectively improve the separation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a chromatogram of the chromatographic column obtained in Example 1 used to separate the sample with structure I and its corresponding isomers.

[0030] Figure 2 and Figure 3 The chromatograms of the chromatographic columns obtained in Example 2 and Comparative Example 1 used to separate the sample with structure II and its corresponding isomers are shown respectively.

[0031] Figure 4 and Figure 5 The chromatograms of the chromatographic columns obtained in Example 2 and Comparative Example 2 used to separate the sample with structure III and its corresponding isomers are shown respectively.

[0032] Figure 6 and Figure 7 The chromatograms of the chromatographic columns obtained in Example 2 and Comparative Example 3 used to separate the sample with structure IV and its corresponding isomers are shown respectively. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0034] Example 1

[0035] A method for preparing a high-efficiency chiral separation chromatographic column comprises the following preparation steps:

[0036] (1) 113.5 g of β-cyclodextrin was heated and dissolved in 1 L of a 15% NaOH aqueous solution, and then 160 g of an allyl epoxy-terminated polyether with an average molecular weight of 1000 (Liaoning Kelong Fine Chemical Co., Ltd. KL-91B) was added, and the temperature was raised to 50° C. and stirred for etherification reaction for 4 h. After the etherification reaction was completed, hydrochloric acid was added for neutralization. The reaction solution was concentrated to a volume of about 400 ml, and then 800 ml of ethanol was added and stirred until no precipitation was precipitated. The salt was removed by filtration, and the filtrate was taken and the solvent was removed by vacuum distillation to obtain an allyl polyether-substituted cyclodextrin compound.

[0037] (2) The allyl polyether-substituted cyclodextrin compound was mixed with acrylate monomer BMA, cross-linking monomer MBA, porogenic solvent (a mixed solvent of N,N-dimethylformamide and isopropanol in a volume ratio of 100:25) and initiator AIBN in a mass ratio of 20:40:30:75:0.5, dissolved by ultrasonication, degassed and poured into a 150*4.6 mm chromatographic column, sealed at both ends, heated in a water bath to 60°C for polymerization reaction for 2 hours, and after the reaction was completed, one end was connected to a high-pressure pump to flush with methanol to remove unreacted monomers and porogenic solvent, thereby obtaining a high-efficiency chiral separation chromatographic column.

[0038] The chromatogram of the chromatographic column obtained in this example for separating the sample of formula I and its corresponding isomers is shown in FIG. Figure 1 As shown (mobile phase: acetonitrile: 0.1% trifluoroacetic acid = 60:40 (v / v), 1 ml / min, 25°C).

[0039]

[0040] pass Figure 1 The results show that the chromatographic column of the present invention can achieve good chiral separation.

[0041] Example 2

[0042] A method for preparing a high-efficiency chiral separation chromatographic column comprises the following preparation steps:

[0043] (1) 144.3 g of hydroxyethyl-β-cyclodextrin was heated and dissolved in 0.8 L of 10% NaOH aqueous solution, and then 70 g of allyl epoxy-terminated polyether with an average molecular weight of 500 (Liaoning Kelong Fine Chemical Co., Ltd. KL-11B) was added, and the temperature was raised to 60° C. and stirred for etherification reaction for 3 h. After the etherification reaction was completed, hydrochloric acid was added for neutralization. The reaction solution was concentrated to a volume of about 350 ml, and then 700 ml of ethanol was added and stirred until no precipitation was precipitated. The salt was removed by filtration, and the filtrate was taken and the solvent was removed by vacuum distillation to obtain an allyl polyether-substituted cyclodextrin compound.

[0044] (2) The allyl polyether-substituted cyclodextrin compound was mixed with the acrylate monomer BMA, the cross-linking monomer EGDMA, the porogenic solvent (a mixed solvent of N,N-dimethylacetamide and isopropanol in a volume ratio of 100:30) and the initiator AIBN in a mass ratio of 15:50:20:60:0.5, and after ultrasonic dissolution and degassing, the mixture was poured into a 150*4.6 mm chromatographic column, sealed at both ends, heated to 70°C in a water bath for polymerization reaction for 2 hours, and after the reaction was completed, one end was connected to a high-pressure pump to flush with methanol to remove the unreacted monomers and the porogenic solvent, thereby obtaining a high-efficiency chiral separation chromatographic column.

[0045] Example 3

[0046] A method for preparing a high-efficiency chiral separation chromatographic column comprises the following preparation steps:

[0047] (1) 154.2 g of hydroxypropyl-β-cyclodextrin was heated and dissolved in 0.6 L of 5% NaOH aqueous solution, and then 50 g of allyl epoxy-terminated polyether (Liaoning Kelong Fine Chemical Co., Ltd. KL-11) with an average molecular weight of 450 was added, and the temperature was raised to 70° C. and stirred for etherification reaction for 2 h. After the etherification reaction was completed, hydrochloric acid was added for neutralization. The reaction solution was concentrated to a volume of about 300 ml, and then 600 ml of ethanol was added and stirred until no precipitation was precipitated. The salt was removed by filtration, and the filtrate was taken and the solvent was removed by vacuum distillation to obtain an allyl polyether-substituted cyclodextrin compound.

[0048] (2) The allyl polyether-substituted cyclodextrin compound was mixed with the acrylate monomer DMAEMA, the cross-linking monomer EGDMA, the porogenic solvent (a mixed solvent of dimethyl sulfoxide and isopropanol in a volume ratio of 100:40) and the initiator AIBN in a mass ratio of 10:30:40:100:0.5, ultrasonically dissolved and degassed, and then poured into a 150*4.6 mm chromatographic column. Both ends were sealed and heated to 80°C in a water bath for polymerization reaction for 1 hour. After the reaction was completed, one end was connected to a high-pressure pump to flush with methanol to remove unreacted monomers and porogenic solvent, thereby obtaining a high-efficiency chiral separation chromatographic column.

[0049] Comparative Example 1

[0050] A method for preparing a high-efficiency chiral separation chromatographic column, compared with Example 2, using an equal molar amount of glycidyl methacrylate (GMA) to replace the allyl epoxy-terminated polyether with an average molecular weight of 500, and the rest is the same.

[0051] The chromatograms of the chromatographic columns obtained in Example 2 and Comparative Example 1 for separating the sample of formula II structure and its corresponding isomers are shown as follows: Figure 2 and Figure 3 As shown (mobile phase: acetonitrile: 0.1% phosphoric acid = 80:20 (v / v), 1 ml / min, 25°C).

[0052]

[0053] It can be seen from the comparison results of this comparative example and Example 2 that the connecting arm for etherification reaction with the cyclodextrin compound is too short, and the resulting chromatographic column cannot effectively separate the corresponding isomers. The reason may be that the steric hindrance of the copolymerization of the substituted cyclodextrin compound with the acrylate monomer and the cross-linking monomer obtained by the too short connecting arm is large, resulting in a low copolymerization grafting rate of the cyclodextrin compound and a reduced chiral recognition ability of the resulting polymer chromatographic column.

[0054] Comparative Example 2

[0055] A method for preparing a high-efficiency chiral separation chromatographic column. Compared with Example 2, the amount of KL-11B added is reduced to 40 g, and the rest is the same.

[0056] The chromatograms of the chromatographic columns obtained in Example 2 and Comparative Example 2 for separating the sample of formula III and its corresponding isomers are shown respectively as follows: Figure 4 and Figure 5 As shown (mobile phase: acetonitrile: 0.1% trifluoroacetic acid = 60:40 (v / v), 1 ml / min, 25°C).

[0057]

[0058] By comparing the results of this comparative example with those of Example 2, it can be seen that the addition amount of the allyl epoxy-terminated polyether is too low, and the separation effect of the obtained chromatographic column becomes poor.

[0059] Comparative Example 3

[0060] A method for preparing a high-efficiency chiral separation chromatographic column. Compared with Example 2, the amount of KL-11B added is increased to 125 g, and the rest is the same.

[0061] The chromatograms of the chromatographic columns obtained in Example 2 and Comparative Example 3 for separating the sample of Formula IV structure and its corresponding isomers are shown as follows: Figure 6 and Figure 7As shown (mobile phase: methanol: 20mM PH=4.0 ammonium acetate=70:30 (v / v), 1ml / min, 25°C).

[0062]

[0063] By comparing the results of this comparative example with those of Example 2, it can be seen that the addition amount of the allyl epoxy-terminated polyether is too high, and the separation effect of the obtained chromatographic column becomes poor.

[0064] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a high-efficiency chiral separation chromatographic column, characterized in that: The method comprises the following preparation steps: (1) dissolving cyclodextrin or its derivative in an alkaline aqueous solution, then adding allyl epoxy-terminated polyether, heating to 30-80° C. and stirring to carry out etherification reaction, and separating and purifying the product to obtain an allyl polyether-substituted cyclodextrin compound; (2) The allyl polyether-substituted cyclodextrin compound is mixed with an acrylate monomer, a cross-linking monomer, a porogenic solvent and an initiator, dissolved by ultrasonication, degassed and then poured into a chromatographic column, heated in a water bath to 40-80°C for polymerization reaction, and after the reaction is completed, the unreacted monomer and porogenic solvent are washed away to obtain a high-efficiency chiral separation chromatographic column; The molar ratio of the cyclodextrin or its derivative to the allyl epoxy-terminated polyether added in step (1) is 1:1-2.

2. The method for preparing a high-efficiency chiral separation chromatographic column according to claim 1, characterized in that: In step (1), the cyclodextrin or its derivative is β-cyclodextrin, hydroxyethyl-β-cyclodextrin or hydroxypropyl-β-cyclodextrin; and the alkaline aqueous solution is a NaOH aqueous solution with a mass concentration of 5% to 15%.

3. The method for preparing a high-efficiency chiral separation chromatographic column according to claim 1, characterized in that: The molecular weight of the allyl epoxy terminated polyether in step (1) is 400-1000.

4. The method for preparing a high-efficiency chiral separation chromatographic column according to claim 1, characterized in that: The time of the etherification reaction in step (1) is 1 to 4 hours; the separation and purification method is as follows: after the etherification reaction is completed, acid is added for neutralization, the reaction solution is concentrated and then ethanol is added for precipitation, the salt is removed by filtration, and the filtrate is taken and distilled under reduced pressure to remove the solvent to obtain an allyl polyether-substituted cyclodextrin compound.

5. The method for preparing a high-efficiency chiral separation chromatographic column according to claim 1, characterized in that: In step (2), the acrylic acid ester monomer is butyl methacrylate or dimethylaminoethyl methacrylate; and the cross-linking monomer is methylene bisacrylamide or ethylene glycol dimethacrylate.

6. The method for preparing a high-efficiency chiral separation chromatographic column according to claim 1, characterized in that: The porogenic solvent in step (2) is a mixed solvent of at least one solvent selected from N,N-dimethylformamide, N,N-dimethylacetamide and dimethyl sulfoxide and isopropanol in a volume ratio of 100:20-40; and the initiator is azobisisobutyronitrile.

7. The method for preparing a high-efficiency chiral separation chromatographic column according to claim 1, characterized in that: The mass ratio of the allyl polyether-substituted cyclodextrin compound, the acrylate monomer, the crosslinking monomer, the porogenic solvent and the initiator in step (2) is 10-20:30-50:20-40:60-100:0.2-1.

8. The method for preparing a high-efficiency chiral separation chromatographic column according to claim 1, characterized in that: The flushing described in step (2) refers to connecting a high-pressure pump to flush with methanol.

9. A high-efficiency chiral separation chromatographic column, characterized in that: It is prepared by the method according to any one of claims 1 to 8.

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