阅读说明：本技术 吡啶二甲酰胺基桥联双β-环糊精固定相及其制备方法和应用 (Pyridine dicarboxamide bridged bis-beta-cyclodextrin stationary phase and preparation method and application thereof ) 是由 李来生 钟慧 曾庆丽 张天赐 双亚洲 李良 于 2021-10-11 设计创作，主要内容包括：本发明公开了一种吡啶二甲酰胺基桥联双β-环糊精固定相及其制备方法和应用,该材料以吡啶-2,3-二甲酰胺基桥联双β-环糊精、3-异氰酸基丙基三乙氧基硅烷和SBA-15为原料制备得到,制备方法过程简单,条件温和、成本较低；制得的吡啶二甲酰胺基桥联双β-环糊精固定相是一类多模式固定相,具有广泛的分离对象,在液相色谱中能高效快速地拆分一系列酸性、碱性、中性及两性的手性药物或农药。(The invention discloses a pyridine dicarboxamide bridged bis-beta-cyclodextrin stationary phase and a preparation method and application thereof, the material is prepared by taking pyridine-2, 3-dicarboxamide bridged bis-beta-cyclodextrin, 3-isocyanatopropyltriethoxysilane and SBA-15 as raw materials, the preparation method has simple process, mild conditions and lower cost; the prepared pyridine dicarboxamide bridged bis beta-cyclodextrin stationary phase is a multi-mode stationary phase, has wide separation objects, and can efficiently and quickly separate a series of acidic, alkaline, neutral and amphoteric chiral drugs or pesticides in liquid chromatography.)

1. A preparation method of a pyridine dicarboxamide bridged bis beta-cyclodextrin stationary phase is characterized by comprising the following steps:

uniformly mixing pyridine-2, 3-dimethylamido bridged bis beta-cyclodextrin, DMF (dimethyl formamide) and 3-isocyanatopropyltriethoxysilane, and reacting for 1.5-2.5h at the temperature of 60-70 ℃ in a nitrogen atmosphere;

step two, adding SBA-15 silica gel, heating to 115-125 ℃, stirring for reacting for 16-24h, cooling to room temperature, filtering, washing filter residues by sequentially adopting DMF, methanol and acetone until washing liquid is clear, and drying to obtain a pyridine dicarboxamide bridged bis beta-cyclodextrin stationary phase; the proportion of pyridine-2, 3-dicarboxamido bridged bis beta-cyclodextrin, DMF, 3-isocyanatopropyltriethoxysilane and SBA-15 silica gel is (0.8-1.6) g: 40mL of: (0.2-0.4) mL: 4g of the total weight of the mixture;

the pyridine-2, 3-dicarboxamido bridged bis-beta-cyclodextrin is prepared by the following steps:

mono-6-amino-beta-cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and DMF as (6.5-7.5) g: (0.2-0.3) g: 0.86 g: 0.54 g: uniformly mixing the components in a ratio of 30mL, stirring the mixture to react for 32 to 40 hours, filtering the mixture, and performing DCC: acetone ═ 0.86 g: adding the filtrate into acetone according to the proportion of 500mL, precipitating, filtering, dissolving the filter residue with water, purifying by a sephadex fast column, adding acetone to precipitate, filtering, and drying to obtain the pyridine-2, 3-dicarboxamide bridged bis-beta-cyclodextrin.

2. The process according to claim 1, wherein the ratio of mono-6-amino- β -cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and DMF is 7.0 g: 0.25 g: 0.86 g: 0.54 g: 30 mL.

3. The method of claim 1, wherein the ratio of pyridine-2, 3-dicarboxamido-bridged bis β -cyclodextrin, DMF, 3-isocyanatopropyltriethoxysilane, and SBA-15 silica gel is 1.2 g: 40mL of: 0.3 mL: 4.0 g.

4. The preparation method according to claim 1, wherein the stirring reaction time is 36 hours in the preparation of pyridine-2, 3-dicarboxamido-bridged bis β -cyclodextrin.

5. The method according to claim 1, wherein the reaction is carried out for 2 hours in the first step under a nitrogen atmosphere at 65 ℃.

6. The process according to claim 1, wherein in the second step, the temperature is raised to 120 ℃ and the reaction is stirred for 20 hours.

7. The method according to claim 1, wherein in the second step, the drying is carried out under vacuum at 60 ℃; when preparing the pyridine-2, 3-dicarboxamido bridged bis beta-cyclodextrin, the drying condition is vacuum drying at 55 ℃.

8. A pyridine dicarboxamide-bridged bis β -cyclodextrin stationary phase prepared by the method of any one of claims 1 to 7.

9. The use of the pyridine dicarboxamide-bridged bis β -cyclodextrin stationary phase of claim 8 in the field of chiral enantiomeric separation.

10. The use according to claim 9, wherein the chiral enantiomer comprises a chiral drug which is a chiral triazole pesticide, a flavanone drug, a dansyl amino acid, or a beta-blocker drug.

Technical Field

The invention belongs to the field of chiral separation materials, and particularly relates to a pyridine dicarboxamide bridged bis-beta-cyclodextrin stationary phase, and a preparation method and application thereof.

Background

With the progress of stereochemical research, the difference of the enantiomer functions of chiral compounds increasingly draws the wide attention of international society. The space structure of the chiral drug can be better matched with chiral life substances (such as protein, nucleic acid, biological enzyme, active polysaccharide and the like), and the chiral drug has the advantages of definite action target, high drug effect, good curative effect and the like, and is widely applied to clinical treatment. However, only one of the levorotatory and dextrorotatory chiral drugs has high activity, and the other has low activity and may cause toxic and side effects. For example, L-dopa is used for treating Parkinson diseases, while D-dopa does not have the function and even causes granulocytopenia, and the safety of chiral drugs and pesticides has become a scientific problem to be solved urgently in the chemical industry, pharmaceutical industry, clinical medicine and social development. Therefore, the development of novel chiral separation materials and the establishment of an analysis method of the content of the drug enantiomer have important significance for guaranteeing the medication safety and food safety of people. Currently, High Performance Liquid Chromatography (HPLC) has been developed as one of the most common means for resolution and preparation of chiral substances, due to its numerous advantages, where chiral separation materials play an important role in HPLC resolution.

Cyclodextrin has a special molecular structure of 'internal hydrophobicity and external hydrophilicity', and is easy to form an inclusion compound with guest molecules matched with the cavity size. The levorotatory body and the dextrorotatory body have different space structures, so that the levorotatory body and the dextrorotatory body are different in stability of an inclusion compound formed by cyclodextrin, and are separated by a cyclodextrin stationary phase. The cyclodextrin stationary phase has stable and durable structure, can realize chiral separation in various modes, is non-toxic and cheap, and has higher practical application value. Especially, a plurality of action sites can be introduced through derivatization of a cyclodextrin port, so that the chiral selectivity of the stationary phase is improved, the chiral selectivity mainly comprises alkylation, hydroxypropylation, benzoylation, phenylcarbamoylation, naphthylethylcarbamoylation, triazolization and the like, and a part of the derivatized cyclodextrin stationary phase is commercialized and plays an important role in chiral separation. However, the potential of the cyclodextrin stationary phase is difficult to further excavate only through derivatization, on one hand, derivatization reaction is time-consuming and labor-consuming, on the other hand, derivatization of a port can reduce hydrogen bond action of hydroxyl, and also can cause crowding of the port, and in addition, a single cyclodextrin cavity is small in size, so that the advantages of cyclodextrin inclusion effect are not favorably exerted.

Disclosure of Invention

Because a single beta-cyclodextrin cavity in the derivatized beta-cyclodextrin is small, the derivatized beta-cyclodextrin only can encapsulate a substrate with the volume of one naphthalene ring at most, and the separation of chiral substances with large volume is poor. The invention aims to solve the problem of the performance deficiency of the derivatization cyclodextrin stationary phase, and provides a pyridine dicarboxamide bridged bis-beta-cyclodextrin stationary phase and a preparation method thereof, the method is simple to operate and high in yield, and the prepared PyCDP can well separate triazole, flavanone and other chiral substances under different modes such as reversed phase chromatography, polar organic and the like, and is expected to solve the problem that the single-cyclodextrin derivatization stationary phase is difficult to separate a large-volume enantiomer to a certain extent. The following technical scheme is adopted specifically:

uniformly mixing pyridine-2, 3-dimethylamido bridged bis beta-cyclodextrin, DMF (dimethyl formamide) and 3-isocyanatopropyltriethoxysilane, and reacting for 1.5-2.5h at the temperature of 60-70 ℃ in a nitrogen atmosphere;

step two, adding SBA-15 silica gel, heating to 115-125 ℃, stirring for reacting for 16-24h, cooling to room temperature, filtering, washing filter residues by sequentially adopting DMF, methanol and acetone until washing liquid is clear, and drying to obtain a pyridine dicarboxamide bridged bis beta-cyclodextrin stationary phase; the proportion of pyridine-2, 3-dicarboxamido bridged bis beta-cyclodextrin, DMF, 3-isocyanatopropyltriethoxysilane and SBA-15 silica gel is (0.8-1.6) g: 40mL of: (0.2-0.4) mL: 4g of the total weight of the mixture;

the pyridine-2, 3-dicarboxamido bridged bis-beta-cyclodextrin is prepared by the following steps:

mono-6-amino- β -cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and DMF (N, N-dimethylformamide) as (6.5-7.5) g: (0.2-0.3) g: 0.86 g: 0.54 g: uniformly mixing the components in a ratio of 30mL, stirring the mixture to react for 32 to 40 hours, filtering the mixture, and performing DCC: acetone ═ 0.86 g: adding the filtrate into acetone according to the proportion of 500mL, precipitating, filtering, dissolving the filter residue with water, purifying by a sephadex fast column, adding acetone to precipitate, filtering, and drying to obtain the pyridine-2, 3-dicarboxamide bridged bis-beta-cyclodextrin.

Based on the phase action principle of pi-acid and pi-base such as Armstrong, the invention introduces pi-basic pyridine aryl as a bridge group, pyridine-2, 3-dicarboxylic acid and amino-beta-cyclodextrin are used as raw materials, under the existence of common dehydrating agents DCC (dicyclohexylcarbodiimide) and HOBT (hydroxybenzotriazole), pyridine dicarboxamide bridged bis-beta-cyclodextrin is synthesized conveniently by normal temperature condensation reaction, and is bonded to the surface of silica gel through a coupling agent to prepare a novel bridged cyclodextrin stationary phase (PyCDP for short). In the PyCDP obtained by the invention, two cyclodextrin cavities and a pyridine bridge group form a 'clamp-type' structure, and can be used for cooperatively encapsulating substrates with larger volume which cannot be encapsulated by the cyclodextrin cavities. The amido bond on the bridge group is not only a hydrogen bond donor, but also a hydrogen bond acceptor, and can play the role of the hydrogen bond of the stationary phase to a greater extent. Meanwhile, the introduced pyridyl can provide a pi-pi stacking function, and the interaction forces jointly act on substrate molecules, so that the chiral separation capability of the PyCDP is improved.

The stationary phase and the ligand are characterized by infrared, mass spectrum, element analysis and the like, and the successful synthesis of the pyridine dicarboxamide bridged bis beta-cyclodextrin ligand and the successful bonding on the silica gel matrix are proved. Experiments prove that the novel stationary phase obtained by the invention has stronger chiral separation capability and shows good separation performance on triazole pesticides, dansyl amino acids, flavanones and the like in an HPLC (high performance liquid chromatography) reversed phase mode; meanwhile, various beta-receptor retarder chiral compounds can be effectively resolved in a polar organic mode, and the separation capability is obviously superior to that of a common single cyclodextrin stationary phase.

The silica gel matrix used in the invention is SBA-15, the particle size distribution is 2.5-4.5 mu m, the pore diameter is about 20nm, and the average specific surface area is 400m²The larger specific surface area of the porous material is beneficial to improving the bonding amount, and the ordered pore structure can also accelerate solute mass transfer.

In some preferred embodiments, the ratio of mono-6-amino- β -cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and DM F is 7.0 g: 0.25 g: 0.86 g: 0.54 g: 30 mL.

In some preferred embodiments, the ratio of pyridine-2, 3-dicarboxamido-bridged bis β -cyclodextrin, DMF, 3-isocyanatopropyltriethoxysilane, and SBA-15 silica gel is 1.2 g: 40mL of: 0.3 mL: 4.0 g.

In some preferred embodiments, the reaction is stirred for 36 hours in the preparation of pyridine-2, 3-dicarboxamido-bridged bis β -cyclodextrin.

In some preferred embodiments, in step one, the reaction is carried out for 2 hours under a nitrogen atmosphere at 65 ℃.

In some preferred embodiments, in step two, the temperature is raised to 120 ℃ and the reaction is stirred for 20 h.

In some preferred embodiments, in step two, the drying conditions are vacuum drying at 60 ℃; when preparing the pyridine-2, 3-dicarboxamido bridged bis beta-cyclodextrin, the drying condition is vacuum drying at 55 ℃.

The pyridine dicarboxamide bridged bis-beta-cyclodextrin stationary phase prepared by the preparation method can be used as a chromatographic stationary phase for separating chiral enantiomers, such as chiral drugs, including chiral triazole pesticides, flavanone drugs, dansyl amino acid or beta-receptor blocker drugs.

The invention has the beneficial effects that:

(1) the stationary phase prepared by the invention is a multimode stationary phase, and is simultaneously suitable for reversed phase and polar organic modes of high performance liquid chromatography; meanwhile, the PyCDP has good chromatographic performance, good chromatographic separation reproducibility, stable chromatographic performance, higher chiral separation capability in a wider temperature range, strong acid resistance and high-concentration organic solvent resistance and higher practical application value.

(2) The preparation method has the advantages of simple process, convenient operation, good reproducibility, high yield and the like.

(3) The PyCDP prepared by the invention has wide separation objects, can efficiently and quickly separate a series of acidic, alkaline, neutral and amphoteric chiral drugs or pesticides in liquid chromatography, comprises triazole pesticides, flavanone drugs, dansylated amino acids, beta-receptor blocker drugs and the like, and has good separation performance on weakly acidic dansylated glutamic acid and dansylated aspartic acid.

Drawings

FIG. 1 is a schematic diagram showing the structure of a pyridine-2, 3-dimethylamide-bridged bis β -cyclodextrin stationary phase in example 1;

FIG. 2 is a schematic diagram showing the chemical structure of each standard;

FIG. 3 shows chiral chromatograms obtained by resolving individual standards.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments and the accompanying drawings to fully understand the objects, aspects and effects of the present invention.

In the following examples, the silica gel matrix used in the present invention was SBA-15, the particle size distribution was 2.5 to 4.5. mu.m, the pore diameter was about 20nm, and the average specific surface area was 400m²/g。

Example 1:

a preparation method of a pyridine-2, 3-dicarboxamide bridged bis beta-cyclodextrin stationary phase comprises the following steps:

(1) adding mono-6-amino-beta-cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and anhydrous DMF according to the proportion of 6.5g, 0.25g, 0.86g, 0.54g and 30mL, stirring for dissolving, and reacting for 36h under magnetic stirring at room temperature. The unreacted solid was filtered, and the filtrate was poured into 500mL of acetone (in an amount of 0.86g of DCC, the same applies below), and after precipitation, the crude solid was filtered to obtain a crude product. Dissolving the solid with a small amount of water, purifying by a sephadex (C-25) rapid column, collecting filtrate, concentrating, adding acetone to separate out a precipitate, performing suction filtration, and drying the solid at 55 ℃ in vacuum to obtain pyridine-2, 3-dicarboxamide bridged bis beta-cyclodextrin (PyCD) serving as a chiral ligand;

(2) PyCD, anhydrous DMF, 3-isocyanatopropyltriethoxysilane 1.2g, 40mL, 0.30mL are added according to the proportion, stirred evenly, and reacted for 2.0h under the protection of nitrogen and magnetic stirring at 65 ℃. Then 4.0g of SBA-15 silica gel was added and the reaction was stirred rapidly at 120 ℃ for 20 h. And (3) cooling to room temperature, filtering, repeatedly washing the solid with DMF (dimethyl formamide), methanol and acetone in sequence until the washing liquid is clear, and vacuum-drying the solid at 60 ℃ to obtain PyCDP. The structure of the device is schematically shown in figure 1.

The bonding amount of the PyCDP obtained was calculated from the carbon content in the elemental analysis results, and the measured data are shown in table 1.

TABLE 1

Example 2:

a preparation method of a pyridine-2, 3-dicarboxamide bridged bis beta-cyclodextrin stationary phase comprises the following steps:

(1) adding mono-6-amino-beta-cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and anhydrous DMF according to the proportion of 7.0g, 0.25g, 0.86g, 0.54g and 30mL, stirring for dissolving, and reacting for 36h under magnetic stirring at room temperature. The unreacted solid was filtered, and the filtrate was poured into 500mL of acetone (in an amount of 0.86g of DCC, the same applies below), and after precipitation, the crude solid was filtered to obtain a crude product. Dissolving the solid with a small amount of water, purifying by a sephadex (C-25) rapid column, collecting filtrate, concentrating, adding acetone to precipitate, filtering, and drying the solid at 55 ℃ in vacuum to obtain PyCD used as a chiral ligand;

(2) PyCD, anhydrous DMF, 3-isocyanatopropyltriethoxysilane 1.2g, 40mL, 0.30mL are added according to the proportion, stirred evenly, and reacted for 2.0h under the protection of nitrogen and magnetic stirring at 65 ℃. Then 4.0g of SBA-15 silica gel was added and the reaction was stirred rapidly at 120 ℃ for 20 h. And (3) cooling to room temperature, filtering, repeatedly washing the solid with DMF (dimethyl formamide), methanol and acetone in sequence until the washing liquid is clear, and vacuum-drying the solid at 60 ℃ to obtain PyCDP.

The bonding amount of the PyCDP obtained was calculated from the carbon content in the elemental analysis results, and the measured data are shown in table 2.

TABLE 2

Example 3:

a preparation method of a pyridine-2, 3-dicarboxamide bridged bis beta-cyclodextrin stationary phase comprises the following steps:

(1) adding mono-6-amino-beta-cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and anhydrous DMF according to the proportion of 7.5g, 0.25g, 0.86g, 0.54g and 30mL, stirring for dissolving, and reacting for 36h under magnetic stirring at room temperature. The unreacted solid was filtered, and the filtrate was poured into 500mL of acetone (in an amount of 0.86g of DCC, the same applies below), and after precipitation, the crude solid was filtered to obtain a crude product. Dissolving the solid with a small amount of water, purifying by a sephadex (C-25) rapid column, collecting filtrate, concentrating, adding acetone to precipitate, filtering, and drying the solid at 55 ℃ in vacuum to obtain PyCD used as a chiral ligand;

(2) PyCD, anhydrous DMF, 3-isocyanatopropyltriethoxysilane 1.2g, 40mL, 0.30mL are added according to the proportion, stirred evenly, and reacted for 2.0h under the protection of nitrogen and magnetic stirring at 65 ℃. Then 4.0g of SBA-15 silica gel was added and the reaction was stirred rapidly at 120 ℃ for 20 h. And (3) cooling to room temperature, filtering, repeatedly washing the solid with DMF (dimethyl formamide), methanol and acetone in sequence until the washing liquid is clear, and vacuum-drying the solid at 60 ℃ to obtain PyCDP.

The bonding amount of the PyCDP obtained was calculated from the carbon content in the elemental analysis results, and the measured data are shown in table 3.

TABLE 3

Example 4:

a preparation method of a pyridine-2, 3-dicarboxamide bridged bis beta-cyclodextrin stationary phase comprises the following steps:

(1) adding mono-6-amino-beta-cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and anhydrous DMF according to the proportion of 7.0g, 0.20g, 0.86g, 0.54g and 30mL, stirring for dissolving, and reacting for 36h under magnetic stirring at room temperature. The unreacted solid was filtered, and the filtrate was poured into 500mL of acetone (in an amount of 0.86g of DCC, the same applies below), and after precipitation, the crude solid was filtered to obtain a crude product. Dissolving the solid with a small amount of water, purifying by a sephadex (C-25) rapid column, collecting filtrate, concentrating, adding acetone to precipitate, filtering, and drying the solid at 55 ℃ in vacuum to obtain PyCD used as a chiral ligand;

(2) PyCD, anhydrous DMF, 3-isocyanatopropyltriethoxysilane 1.2g, 40mL, 0.30mL are added according to the proportion, stirred evenly, and reacted for 2.0h under the protection of nitrogen and magnetic stirring at 65 ℃. Then 4.0g of SBA-15 silica gel was added and the reaction was stirred rapidly at 120 ℃ for 20 h. And (3) cooling to room temperature, filtering, repeatedly washing the solid with DMF (dimethyl formamide), methanol and acetone in sequence until the washing liquid is clear, and vacuum-drying the solid at 60 ℃ to obtain PyCDP.

The bonding amount of the PyCDP obtained was calculated from the carbon content in the elemental analysis results, and the measured data are shown in table 4.

TABLE 4

Example 5:

a preparation method of a pyridine-2, 3-dicarboxamide bridged bis beta-cyclodextrin stationary phase comprises the following steps:

(1) adding mono-6-amino-beta-cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and anhydrous DMF according to the proportion of 7.0g, 0.30g, 0.86g, 0.54g and 30mL, stirring for dissolving, and reacting for 36h under magnetic stirring at room temperature. The unreacted solid was filtered, and the filtrate was poured into 500mL of acetone (in an amount of 0.86g of DCC, the same applies below), and after precipitation, the crude solid was filtered to obtain a crude product. Dissolving the solid with a small amount of water, purifying by a sephadex (C-25) rapid column, collecting filtrate, concentrating, adding acetone to precipitate, filtering, and drying the solid at 55 ℃ in vacuum to obtain PyCD used as a chiral ligand;

(2) PyCD, anhydrous DMF, 3-isocyanatopropyltriethoxysilane 1.2g, 40mL, 0.30mL are added according to the proportion, stirred evenly, and reacted for 2.0h under the protection of nitrogen and magnetic stirring at 65 ℃. Then 4.0g of SBA-15 silica gel was added and the reaction was stirred rapidly at 120 ℃ for 20 h. And (3) cooling to room temperature, filtering, repeatedly washing the solid with DMF (dimethyl formamide), methanol and acetone in sequence until the washing liquid is clear, and vacuum-drying the solid at 60 ℃ to obtain PyCDP.

The bonding amount of the resulting PyCDP was calculated from the carbon content in the elemental analysis results, and the measured data are shown in table 5.

TABLE 5

Example 6:

a preparation method of a pyridine-2, 3-dicarboxamide bridged bis beta-cyclodextrin stationary phase comprises the following steps:

(1) adding mono-6-amino-beta-cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and anhydrous DMF according to the proportion of 7.0g, 0.25g, 0.86g, 0.54g and 30mL, stirring for dissolving, and reacting for 32h under magnetic stirring at room temperature. The unreacted solid was filtered, and the filtrate was poured into 500mL of acetone (in an amount of 0.86g of DCC, the same applies below), and after precipitation, the crude solid was filtered to obtain a crude product. Dissolving the solid with a small amount of water, purifying by a sephadex (C-25) rapid column, collecting filtrate, concentrating, adding acetone to precipitate, filtering, and drying the solid at 55 ℃ in vacuum to obtain PyCD used as a chiral ligand;

(2) PyCD, anhydrous DMF, 3-isocyanatopropyltriethoxysilane 1.2g, 40mL, 0.30mL are added according to the proportion, stirred evenly, and reacted for 2.0h under the protection of nitrogen and magnetic stirring at 65 ℃. Then 4.0g of SBA-15 silica gel was added and the reaction was stirred rapidly at 120 ℃ for 20 h. And (3) cooling to room temperature, filtering, repeatedly washing the solid with DMF (dimethyl formamide), methanol and acetone in sequence until the washing liquid is clear, and vacuum-drying the solid at 60 ℃ to obtain PyCDP.

The bonding amount of the PyCDP obtained was calculated from the carbon content in the elemental analysis results, and the measured data are shown in table 6.

TABLE 6

Example 7:

a preparation method of a pyridine-2, 3-dicarboxamide bridged bis beta-cyclodextrin stationary phase comprises the following steps:

(1) adding mono-6-amino-beta-cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and anhydrous DMF according to the proportion of 7.0g, 0.25g, 0.86g, 0.54g and 30mL, stirring for dissolving, and reacting for 36h under magnetic stirring at room temperature. The unreacted solid was filtered, and the filtrate was poured into 500mL of acetone (in an amount of 0.86g of DCC, the same applies below), and after precipitation, the crude solid was filtered to obtain a crude product. Dissolving the solid with a small amount of water, purifying by a sephadex (C-25) rapid column, collecting filtrate, concentrating, adding acetone to precipitate, filtering, and drying the solid at 55 ℃ in vacuum to obtain PyCD used as a chiral ligand;

(2) PyCD, anhydrous DMF, 3-isocyanatopropyltriethoxysilane 1.2g, 40mL, 0.30mL are added according to the proportion, stirred evenly, and reacted for 2.0h under the protection of nitrogen and magnetic stirring at 65 ℃. Then 4.0g of SBA-15 silica gel was added and the reaction was stirred rapidly at 120 ℃ for 20 h. And (3) cooling to room temperature, filtering, repeatedly washing the solid with DMF (dimethyl formamide), methanol and acetone in sequence until the washing liquid is clear, and vacuum-drying the solid at 60 ℃ to obtain PyCDP.

The bonding amount of the PyCDP obtained was calculated from the carbon content in the elemental analysis results, and the measured data are shown in table 7.

TABLE 7

Example 8:

a preparation method of a pyridine-2, 3-dicarboxamide bridged bis beta-cyclodextrin stationary phase comprises the following steps:

(1) adding mono-6-amino-beta-cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and anhydrous DMF according to the proportion of 7.0g, 0.25g, 0.86g, 0.54g and 30mL, stirring for dissolving, and reacting for 32h under magnetic stirring at room temperature. The unreacted solid was filtered, and the filtrate was poured into 500mL of acetone (in an amount of 0.86g of DCC, the same applies below), and after precipitation, the crude solid was filtered to obtain a crude product. Dissolving the solid with a small amount of water, purifying by a sephadex (C-25) rapid column, collecting filtrate, concentrating, adding acetone to precipitate, filtering, and drying the solid at 55 ℃ in vacuum to obtain PyCD used as a chiral ligand;

(2) PyCD, anhydrous DMF and 3-isocyanatopropyltriethoxysilane are added according to the proportion of 0.8g, 40mL and 0.30mL, stirred evenly and reacted for 2.0h at 65 ℃ by magnetic stirring under the protection of nitrogen. Then 4.0g of SBA-15 silica gel was added and the reaction was stirred rapidly at 120 ℃ for 20 h. And (3) cooling to room temperature, filtering, repeatedly washing the solid with DMF (dimethyl formamide), methanol and acetone in sequence until the washing liquid is clear, and vacuum-drying the solid at 60 ℃ to obtain PyCDP.

The bonding amount of the PyCDP obtained was calculated from the carbon content in the elemental analysis results, and the measured data are shown in table 8.

TABLE 8

Example 9:

a preparation method of a pyridine-2, 3-dicarboxamide bridged bis beta-cyclodextrin stationary phase comprises the following steps:

(1) adding mono-6-amino-beta-cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and anhydrous DMF according to the proportion of 7.0g, 0.25g, 0.86g, 0.54g and 30mL, stirring for dissolving, and reacting for 32h under magnetic stirring at room temperature. The unreacted solid was filtered, and the filtrate was poured into 500mL of acetone (in an amount of 0.86g of DCC, the same applies below), and after precipitation, the crude solid was filtered to obtain a crude product. Dissolving the solid with a small amount of water, purifying by a sephadex (C-25) rapid column, collecting filtrate, concentrating, adding acetone to precipitate, filtering, and drying the solid at 55 ℃ in vacuum to obtain PyCD used as a chiral ligand;

(2) PyCD, anhydrous DMF and 3-isocyanatopropyltriethoxysilane are added according to the proportion of 1.6g, 40mL and 0.30mL, stirred evenly and reacted for 2.0h at 65 ℃ under the protection of nitrogen and magnetic stirring. Then 4.0g of SBA-15 silica gel was added and the reaction was stirred rapidly at 120 ℃ for 20 h. And (3) cooling to room temperature, filtering, repeatedly washing the solid with DMF (dimethyl formamide), methanol and acetone in sequence until the washing liquid is clear, and drying the solid in vacuum at 60 ℃ to obtain the PyCDP.

The bonding amount of the PyCDP obtained was calculated from the carbon content in the elemental analysis results, and the measured data are shown in table 9.

TABLE 9

Example 10:

a preparation method of a pyridine-2, 3-dicarboxamide bridged bis beta-cyclodextrin stationary phase comprises the following steps:

(1) adding mono-6-amino-beta-cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and anhydrous DMF according to the proportion of 7.0g, 0.25g, 0.86g, 0.54g and 30mL, stirring for dissolving, and reacting for 32h under magnetic stirring at room temperature. The unreacted solid was filtered, and the filtrate was poured into 500mL of acetone (in an amount of 0.86g of DCC, the same applies below), and after precipitation, the crude solid was filtered to obtain a crude product. Dissolving the solid with a small amount of water, purifying by a sephadex (C-25) rapid column, collecting filtrate, concentrating, adding acetone to precipitate, filtering, and drying the solid at 55 ℃ in vacuum to obtain PyCD used as a chiral ligand;

(2) PyCD, anhydrous DMF and 3-isocyanatopropyltriethoxysilane are added according to the proportion of 1.2g, 40mL and 0.20mL, stirred evenly and reacted for 2.0h at 65 ℃ under the protection of nitrogen and magnetic stirring. Then 4.0g of SBA-15 silica gel was added and the reaction was stirred rapidly at 120 ℃ for 20 h. And (3) cooling to room temperature, filtering, repeatedly washing the solid with DMF (dimethyl formamide), methanol and acetone in sequence until the washing liquid is clear, and drying the solid in vacuum at 60 ℃ to obtain the PyCDP.

The actual measurement data of the bonding amount of PyCDP obtained by calculation based on the carbon content in the elemental analysis results are shown in table 10.

Watch 10

Example 11:

a preparation method of a pyridine-2, 3-dicarboxamide bridged bis beta-cyclodextrin stationary phase comprises the following steps:

(1) adding mono-6-amino-beta-cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and anhydrous DMF according to the proportion of 7.0g, 0.25g, 0.86g, 0.54g and 30mL, stirring for dissolving, and reacting for 32h under magnetic stirring at room temperature. The unreacted solid was filtered, and the filtrate was poured into 500mL of acetone (in an amount of 0.86g of DCC, the same applies below), and after precipitation, the crude solid was filtered to obtain a crude product. Dissolving the solid with a small amount of water, purifying by a sephadex (C-25) rapid column, collecting filtrate, concentrating, adding acetone to precipitate, filtering, and drying the solid at 55 ℃ in vacuum to obtain PyCD used as a chiral ligand;

(2) PyCD, anhydrous DMF, 3-isocyanatopropyltriethoxysilane 1.2g, 40mL, 0.40mL are added according to the proportion, stirred evenly, and reacted for 2.0h under the protection of nitrogen and magnetic stirring at 65 ℃. Then 4.0g of SBA-15 silica gel was added and the reaction was stirred rapidly at 120 ℃ for 20 h. And (3) cooling to room temperature, filtering, repeatedly washing the solid with DMF (dimethyl formamide), methanol and acetone in sequence until the washing liquid is clear, and vacuum-drying the solid at 60 ℃ to obtain PyCDP.

The bonding amount of the PyCDP obtained was calculated from the carbon content in the elemental analysis results, and the measured data are shown in table 11.

TABLE 11

Example 12:

a preparation method of a pyridine-2, 3-dicarboxamide bridged bis beta-cyclodextrin stationary phase comprises the following steps:

(1) adding mono-6-amino-beta-cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and anhydrous DMF according to the proportion of 7.0g, 0.25g, 0.86g, 0.54g and 30mL, stirring for dissolving, and reacting for 32h under magnetic stirring at room temperature. The unreacted solid was filtered, and the filtrate was poured into 500mL of acetone (in an amount of 0.86g of DCC, the same applies below), and after precipitation, the crude solid was filtered to obtain a crude product. Dissolving the solid with a small amount of water, purifying by a sephadex (C-25) rapid column, collecting filtrate, concentrating, adding acetone to precipitate, filtering, and drying the solid at 55 ℃ in vacuum to obtain PyCD used as a chiral ligand;

(2) PyCD, anhydrous DMF, 3-isocyanatopropyltriethoxysilane 1.2g, 40mL, 0.40mL are added according to the proportion, stirred evenly, and reacted for 2.0h under the protection of nitrogen and magnetic stirring at 60 ℃. Then 4.0g of SBA-15 silica gel was added and the reaction was stirred rapidly at 120 ℃ for 20 h. And (3) cooling to room temperature, filtering, repeatedly washing the solid with DMF (dimethyl formamide), methanol and acetone in sequence until the washing liquid is clear, and vacuum-drying the solid at 60 ℃ to obtain PyCDP.

The bonding amount of the PyCDP obtained was calculated from the carbon content in the elemental analysis results, and the measured data are shown in table 12.

TABLE 12

Example 13:

a preparation method of a pyridine-2, 3-dicarboxamide bridged bis beta-cyclodextrin stationary phase comprises the following steps:

(1) adding mono-6-amino-beta-cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and anhydrous DMF according to the proportion of 7.0g, 0.25g, 0.86g, 0.54g and 30mL, stirring for dissolving, and reacting for 32h under magnetic stirring at room temperature. The unreacted solid was filtered, and the filtrate was poured into 500mL of acetone (in an amount of 0.86g of DCC, the same applies below), and after precipitation, the crude solid was filtered to obtain a crude product. Dissolving the solid with a small amount of water, purifying by a sephadex (C-25) rapid column, collecting filtrate, concentrating, adding acetone to precipitate, filtering, and drying the solid at 55 ℃ in vacuum to obtain PyCD used as a chiral ligand;

(2) PyCD, anhydrous DMF, 3-isocyanatopropyltriethoxysilane 1.2g, 40mL, 0.40mL are added according to the proportion, stirred evenly, and reacted for 2.0h at 70 ℃ under the protection of nitrogen and magnetic stirring. Then 4.0g of SBA-15 silica gel was added and the reaction was stirred rapidly at 120 ℃ for 20 h. And (3) cooling to room temperature, filtering, repeatedly washing the solid with DMF (dimethyl formamide), methanol and acetone in sequence until the washing liquid is clear, and vacuum-drying the solid at 60 ℃ to obtain PyCDP.

The bonding amount of the PyCDP obtained was calculated from the carbon content in the elemental analysis results, and the measured data are shown in table 13.

Watch 13

Example 14:

a preparation method of a pyridine-2, 3-dicarboxamide bridged bis beta-cyclodextrin stationary phase comprises the following steps:

(1) adding mono-6-amino-beta-cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and anhydrous DMF according to the proportion of 7.0g, 0.25g, 0.86g, 0.54g and 30mL, stirring for dissolving, and reacting for 32h under magnetic stirring at room temperature. The unreacted solid was filtered, and the filtrate was poured into 500mL of acetone (in an amount of 0.86g of DCC, the same applies below), and after precipitation, the crude solid was filtered to obtain a crude product. Dissolving the solid with a small amount of water, purifying by a sephadex (C-25) rapid column, collecting filtrate, concentrating, adding acetone to precipitate, filtering, and drying the solid at 55 ℃ in vacuum to obtain PyCD used as a chiral ligand;

(2) PyCD, anhydrous DMF, 3-isocyanatopropyltriethoxysilane 1.2g, 40mL, 0.40mL are added according to the proportion, stirred evenly, and reacted for 1.5h under the protection of nitrogen and magnetic stirring at 65 ℃. Then 4.0g of SBA-15 silica gel was added and the reaction was stirred rapidly at 120 ℃ for 20 h. And (3) cooling to room temperature, filtering, repeatedly washing the solid with DMF (dimethyl formamide), methanol and acetone in sequence until the washing liquid is clear, and vacuum-drying the solid at 60 ℃ to obtain PyCDP.

The bonding amount of the PyCDP obtained was calculated from the carbon content in the elemental analysis results, and the measured data are shown in table 14.

TABLE 14

Example 15:

a preparation method of a pyridine-2, 3-dicarboxamide bridged bis beta-cyclodextrin stationary phase comprises the following steps:

(1) adding mono-6-amino-beta-cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and anhydrous DMF according to the proportion of 7.0g, 0.25g, 0.86g, 0.54g and 30mL, stirring for dissolving, and reacting for 32h under magnetic stirring at room temperature. The unreacted solid was filtered, and the filtrate was poured into 500mL of acetone (in an amount of 0.86g of DCC, the same applies below), and after precipitation, the crude solid was filtered to obtain a crude product. Dissolving the solid with a small amount of water, purifying by a sephadex (C-25) rapid column, collecting filtrate, concentrating, adding acetone to precipitate, filtering, and drying the solid at 55 ℃ in vacuum to obtain PyCD used as a chiral ligand;

(2) PyCD, anhydrous DMF, 3-isocyanatopropyltriethoxysilane 1.2g, 40mL, 0.40mL are added according to the proportion, stirred evenly, and reacted for 2.5h under the protection of nitrogen and magnetic stirring at 65 ℃. Then 4.0g of SBA-15 silica gel was added and the reaction was stirred rapidly at 120 ℃ for 20 h. And (3) cooling to room temperature, filtering, repeatedly washing the solid with DMF (dimethyl formamide), methanol and acetone in sequence until the washing liquid is clear, and drying the solid in vacuum at 60 ℃ to obtain the PyCDP.

The bonding amount of the PyCDP obtained was calculated from the carbon content in the elemental analysis results, and the measured data are shown in table 15.

Watch 15

Example 16:

a preparation method of a pyridine-2, 3-dicarboxamide bridged bis beta-cyclodextrin stationary phase comprises the following steps:

(1) adding mono-6-amino-beta-cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and anhydrous DMF according to the proportion of 7.0g, 0.25g, 0.86g, 0.54g and 30mL, stirring for dissolving, and reacting for 32h under magnetic stirring at room temperature. The unreacted solid was filtered, and the filtrate was poured into 500mL of acetone (in an amount of 0.86g of DCC, the same applies below), and after precipitation, the crude solid was filtered to obtain a crude product. Dissolving the solid with a small amount of water, purifying by a sephadex (C-25) rapid column, collecting filtrate, concentrating, adding acetone to precipitate, filtering, and drying the solid at 55 ℃ in vacuum to obtain PyCD used as a chiral ligand;

(2) PyCD, anhydrous DMF, 3-isocyanatopropyltriethoxysilane 1.2g, 40mL, 0.40mL are added according to the proportion, stirred evenly, and reacted for 2.0h under the protection of nitrogen and magnetic stirring at 65 ℃. Then 4.0g of SBA-15 silica gel was added and the reaction was stirred rapidly at 115 ℃ for 20 h. And (3) cooling to room temperature, filtering, repeatedly washing the solid with DMF (dimethyl formamide), methanol and acetone in sequence until the washing liquid is clear, and vacuum-drying the solid at 60 ℃ to obtain PyCDP.

The bonding amount of the PyCDP obtained was calculated from the carbon content in the elemental analysis results, and the measured data are shown in table 16.

TABLE 16

Example 17:

a preparation method of a pyridine-2, 3-dicarboxamide bridged bis beta-cyclodextrin stationary phase comprises the following steps:

(1) adding mono-6-amino-beta-cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and anhydrous DMF according to the proportion of 7.0g, 0.25g, 0.86g, 0.54g and 30mL, stirring for dissolving, and reacting for 32h under magnetic stirring at room temperature. The unreacted solid was filtered, and the filtrate was poured into 500mL of acetone (in an amount of 0.86g of DCC, the same applies below), and after precipitation, the crude solid was filtered to obtain a crude product. Dissolving the solid with a small amount of water, purifying by a sephadex (C-25) rapid column, collecting filtrate, concentrating, adding acetone to precipitate, filtering, and drying the solid at 55 ℃ in vacuum to obtain PyCD used as a chiral ligand;

(2) PyCD, anhydrous DMF, 3-isocyanatopropyltriethoxysilane 1.2g, 40mL, 0.40mL are added according to the proportion, stirred evenly, and reacted for 2.0h under the protection of nitrogen and magnetic stirring at 65 ℃. Then 4.0g of SBA-15 silica gel was added and the reaction was stirred rapidly at 125 ℃ for 20 h. And (3) cooling to room temperature, filtering, repeatedly washing the solid with DMF (dimethyl formamide), methanol and acetone in sequence until the washing liquid is clear, and vacuum-drying the solid at 60 ℃ to obtain PyCDP.

The bonding amount of the PyCDP obtained was calculated from the carbon content in the elemental analysis results, and the measured data are shown in table 17.

TABLE 17

Example 18:

a preparation method of a pyridine-2, 3-dicarboxamide bridged bis beta-cyclodextrin stationary phase comprises the following steps:

(1) adding mono-6-amino-beta-cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and anhydrous DMF according to the proportion of 7.0g, 0.25g, 0.86g, 0.54g and 30mL, stirring for dissolving, and reacting for 32h under magnetic stirring at room temperature. The unreacted solid was filtered, and the filtrate was poured into 500mL of acetone (in an amount of 0.86g of DCC, the same applies below), and after precipitation, the crude solid was filtered to obtain a crude product. Dissolving the solid with a small amount of water, purifying by a sephadex (C-25) rapid column, collecting filtrate, concentrating, adding acetone to precipitate, filtering, and drying the solid at 55 ℃ in vacuum to obtain PyCD used as a chiral ligand;

(2) PyCD, anhydrous DMF, 3-isocyanatopropyltriethoxysilane 1.2g, 40mL, 0.40mL are added according to the proportion, stirred evenly, and reacted for 2.0h under the protection of nitrogen and magnetic stirring at 65 ℃. Then 4.0g of SBA-15 silica gel was added and the reaction was stirred rapidly at 120 ℃ for 16 h. And (3) cooling to room temperature, filtering, repeatedly washing the solid with DMF (dimethyl formamide), methanol and acetone in sequence until the washing liquid is clear, and vacuum-drying the solid at 60 ℃ to obtain PyCDP.

The bonding amount of the PyCDP obtained was calculated from the carbon content in the elemental analysis results, and the measured data are shown in table 18.

Watch 18

Example 19:

a preparation method of a pyridine-2, 3-dicarboxamide bridged bis beta-cyclodextrin stationary phase comprises the following steps:

(1) adding mono-6-amino-beta-cyclodextrin, pyridine-2, 3-dicarboxylic acid, DCC, HOBT and anhydrous DMF according to the proportion of 7.0g, 0.25g, 0.86g, 0.54g and 30mL, stirring for dissolving, and reacting for 32h under magnetic stirring at room temperature. The unreacted solid was filtered, and the filtrate was poured into 500mL of acetone (in an amount of 0.86g of DCC, the same applies below), and after precipitation, the crude solid was filtered to obtain a crude product. Dissolving the solid with a small amount of water, purifying by a sephadex (C-25) rapid column, collecting filtrate, concentrating, adding acetone to precipitate, filtering, and drying the solid at 55 ℃ in vacuum to obtain PyCD used as a chiral ligand;

(2) PyCD, anhydrous DMF, 3-isocyanatopropyltriethoxysilane 1.2g, 40mL, 0.40mL are added according to the proportion, stirred evenly, and reacted for 2.0h under the protection of nitrogen and magnetic stirring at 65 ℃. Then 4.0g of SBA-15 silica gel was added and the reaction was stirred rapidly at 120 ℃ for 24 h. And (3) cooling to room temperature, filtering, repeatedly washing the solid with DMF (dimethyl formamide), methanol and acetone in sequence until the washing liquid is clear, and vacuum-drying the solid at 60 ℃ to obtain PyCDP.

The bonding amount of the PyCDP obtained was calculated from the carbon content in the elemental analysis results, and the measured data are shown in table 19.

Watch 19

High performance liquid chromatography resolution experiments:

the PyCDP prepared in example 2 was used as the stationary phase of a high performance liquid chromatography column, and some chiral compounds were subjected to enantiomeric resolution; chiral chromatographic performance of PyCDP was evaluated. The prepared PyCDP was loaded on a stainless steel column (250 mm. times.4.6 mm) using a typical slurry packing technique with methanol as the packing solvent (packing pressure: 34.5MPa) for about 40 min. Before use, the new column is installed on a chromatograph, repeatedly washed by methanol and water alternately, finally washed by a mobile phase until a base line is stable, and then subjected to sample injection analysis. Setting the sample injection volume to be 5-10 mu L; the flow rate is set to be 0.8 mL/min; the separation of the hand substances is carried out at room temperature in the reversed phase mode, and the temperature in the polar organic mode is about 15 ℃; solvent peak as dead time.

The mobile phase consisted of simple water-methanol (acetonitrile) or 1% triethylammonium acetate or 1% formic acid (1% triethylammonium acetate means by adding 1% by volume of triethylamine to ultrapure water and adjusting the pH to a specific value using acetic acid; 1% formic acid means by adding 1% by volume of formic acid to ultrapure water). The mobile phase was filtered using a G4 sand core funnel and sonicated for 10min before use. The preparation method of the racemate standard substance comprises the following steps: the analyte is dissolved in a solution with a volume ratio of methanol to water of 1:1 to prepare a stock solution of 100-200 mg/L, and the stock solution is filtered by using a 0.22 mu m microporous membrane and subjected to ultrasound for 10min before use.

The split standards (structure shown in figure 2) are respectively: weakly acidic 2' -hydroxyflavanone (reverse phase mode, volume ratio of mobile phase water to acetonitrile 80: 20), dansylated glutamic acid (reverse phase mode, volume ratio of 1% triethylammonium acetate to methanol at pH 6.5 95: 5); weakly basic diniconazole (reverse phase mode, volume ratio of mobile phase water to acetonitrile 80: 20); neutral dansylated threonine (reverse phase mode, volume ratio of 1% triethylammonium acetate to methanol at pH 6.5 95: 5); naringin with larger volume (reversed phase mode, volume ratio of mobile phase water to acetonitrile is 92: 8); arotinolol (polar organic mode, volume ratio of mobile phase acetonitrile: methanol: triethylamine: acetic acid is 93: 7: 1.2: 0.8).

The results of the experiment are shown in FIG. 3, and it can be seen that the above standards were all separated by baseline. Obviously, the PyCDP can be used for separating enantiomers of partial chiral dansyl amino acid, flavanone drugs, beta-receptor blocker drugs and certain triazole chiral pesticides by high performance liquid chromatography, and has better separation effect.

The above description is only a preferred embodiment of the present invention, and the present invention is not limited to the above embodiment, and the present invention shall fall within the protection scope of the present invention as long as the technical effects of the present invention are achieved by the same means. The invention is capable of other modifications and variations in its technical solution and/or its implementation, within the scope of protection of the invention.