Captopril-modified weak cation exchange chromatography stationary phases, their preparation and applications

Abstract

The present invention relates to a liquid chromatography stationary phase, characterized in that the drug molecule captopril is used to modify the silica gel surface, the bonded phase contains a carboxyl group, a thioether bond and an amide group, and the structural formula is as follows: wherein Silica Gel is silica gel, and n=0-10. The present invention also provides a preparation method of the above-mentioned liquid chromatography stationary phase, and captopril is modified to the silica gel surface by a thiol-olefin click reaction to prepare a novel weak cation exchange chromatography stationary phase. The separation material preparation method provided by the present invention is simple and efficient, and a variety of functional groups can provide different separation selectivities, which can be widely used in the separation and analysis of various compounds.

Description

Technical Field

[0001] The invention relates to a liquid chromatography stationary phase, in particular to a weak cation exchange chromatography stationary phase modified on the surface of silica gel by using drug molecule captopril. Technical Background

[0002] Cation exchange chromatography uses strong cations containing sulfur ions or weak cation exchangers containing carboxyl functional groups to bind to positively charged compounds through electrostatic interaction. The binding between the charged molecules and the stationary phase is reversible. By changing the pH of the eluent or eluting with a buffer solution with gradually increasing ionic strength, the compounds bound to the ion exchanger can be exchanged with the ions in the eluent and eluted into the solution. Currently, the use of chromatographic stationary phases containing a single cation exchange group cannot meet the separation requirements of all compounds, and new functional groups need to be introduced to further enrich the force of the stationary phase to improve separation selectivity.

[0003] Bai et al. developed a novel aminocaproic acid-modified weak cation exchange / hydrophobic bifunctional mixed mode chromatographic stationary phase, which can achieve rapid separation of multiple proteins [Bai, Q. et al, Chin. J. Chromatogr. 2016, 34, 1228-1233]. Sze et al. reported a polyaspartic acid weak cation exchange chromatographic column for online mass spectrometry analysis of proteomes [Sze, SK et al, J. Proteome Res. 2018, 17, 2390-2400]. However, there is currently no preparation technology for a weak cation exchange stationary phase modified with the multifunctional drug molecule captopril. Summary of the invention

[0004] The purpose of the present invention is to provide a weak cation exchange liquid chromatography stationary phase and a preparation method thereof. The bonded phase contains the drug molecule captopril structure, and the preparation method is simple and has wide applicability.

[0005] 1. The technical solution of the present invention is: a liquid chromatography stationary phase, characterized in that the structure is:

[0006]

[0007] Wherein Silica Gel is silica gel, wherein Silica Gel is silica gel, and n=0-10.

[0008] The present invention also provides a method for preparing the above-mentioned stationary phase, which is characterized by comprising the following steps:

[0009] a. Silica gel pretreatment: silica gel is added to a strong acid solution with a concentration of 1 to 40 wt%, heated under reflux and stirred for 1 to 48 hours, filtered, washed with water until pH = 6 to 7, and the obtained solid is dried in a drying oven at 100 to 160 ° C for 8 to 24 hours to obtain acidified silica gel; the obtained dried acidified silica gel is placed in an atmosphere with a humidity of 10% to 80% for 12 to 72 hours to make the silica gel absorb water and increase its weight by 1% to 10% of the mass of the dry silica gel to obtain hydrated silica gel;

[0010] The amount of strong acid solution used is 2-20 mL per gram of silica gel;

[0011] b. Synthesis of alkenyl intermediates: under the protection of nitrogen or argon, add alkenyl silane coupling agent and hydrated silica gel in an organic solvent, react at 20-100°C for 1-48 hours, filter, wash with methanol, tetrahydrofuran and methanol in sequence, and vacuum dry the obtained solid in a drying oven at 40-80°C for 8-24 hours to obtain an alkenyl intermediate;

[0012] Based on each gram of silica gel, the amount of the silane coupling agent used is 1-10 mmol, and the amount of the organic solvent used is 5-15 mL.

[0013] c. Thiol-olefin click reaction: under nitrogen or argon protection, add captopril, free radical initiator and olefin intermediate to a mixed solution of organic solvent / water (v / v=1-7 / 3-9), react at 40-80°C for 8-48 hours, filter, wash with water, methanol, water and methanol in sequence, and dry the obtained solid in a drying oven at 40-80°C under vacuum for 8-24 hours to obtain a chromatographic stationary phase;

[0014] The amount of captopril used is 1-10 mmol per gram of silica gel, the amount of the free radical initiator used is 1-10 mmol per gram of silica gel, and the amount of the organic solvent / water (v / v=1-7 / 3-9) mixed solution used is 5-20 mL.

[0015] The present invention has the following advantages:

[0016] 1. The stationary phase structure is novel. The present invention proposes for the first time to use the drug molecule captopril as a bonding phase to modify the silica gel surface to prepare a novel weak cation exchange chromatography stationary phase. The stationary phase structure contains carboxylic acid groups, and the stationary phase surface can be negatively charged by regulating the pH of the mobile phase, which has an electrostatic effect. At the same time, the stationary phase structure also contains amide groups and thioether bonds, which have hydrogen bonding and dipole-dipole forces.

[0017] 2. The captopril chromatographic stationary phase provided by the present invention has good separation selectivity for most compounds containing polar groups and can be widely used in the separation and analysis of various samples.

[0018] 3. The preparation process of the liquid chromatography stationary phase provided by the present invention is simple and reliable, which is conducive to industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the chromatogram of Example 3;

[0020] Figure 2 This is the chromatogram of Example 4. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with examples, which are intended only to illustrate the present invention, but not to limit the present invention.

[0022] Example 1

[0023] Add 10 g of silica gel into a 250 mL flask, add into 100 mL of 20 wt% hydrochloric acid solution, heat under reflux with stirring for 2 hours, filter, wash with water until pH = 6.2, dry the obtained solid in a drying oven at 160° C. for 24 hours to obtain acidified silica gel; place the obtained dry acidified silica gel in an air atmosphere with a humidity of 50% for 12 hours to make the silica gel absorb water and increase its weight by 3% of the mass of the dry silica gel to obtain hydrated silica gel.

[0024] Under nitrogen protection, 10 g of hydrated silica gel, 6 mL of vinyltrichlorosilane and 60 mL of xylene were added to a 100 mL flask, and the mixture was reacted at 30 °C for 3 hours. The mixture was filtered and washed with methanol, tetrahydrofuran and methanol in sequence. The obtained solid was vacuum dried in a drying oven at 80 °C for 24 hours to obtain a vinyl intermediate.

[0025] Under nitrogen protection, 10 g of vinyl intermediate, 8.7 g of captopril (molar number is 40 mmol), 5.5 g of azobisisobutylimidazoline hydrochloride and 100 mL of methanol / water (v / v=7 / 3) mixed solution were added to a 250 mL flask, reacted at 60° C. for 48 hours, filtered, washed with water, methanol, water and methanol in sequence, and the obtained solid was vacuum dried in a drying oven at 80° C. for 24 hours to obtain a chromatographic stationary phase 1, with a carboxyl bonding amount of 0.66 mmol. The structure was confirmed by infrared spectroscopy and nuclear magnetic resonance carbon spectroscopy as follows:

[0026]

[0027] Example 2

[0028] Add 10 g of silica gel into a 250 mL flask, add into 100 mL of 30 wt% hydrochloric acid solution, heat under reflux with stirring for 2 hours, filter, wash with water until pH = 6-7, dry the obtained solid in a drying oven at 160° C. for 24 hours to obtain acidified silica gel; place the obtained dry acidified silica gel in an atmosphere of 30% dryness for 12 hours to make the silica gel absorb water and increase its weight by 2.1% of the mass of the dry silica gel to obtain hydrated silica gel.

[0029] Under nitrogen protection, 10 g of hydrated silica gel, 8 mL of butenyltrichlorosilane and 60 mL of toluene were added to a 100 mL flask, and the mixture was reacted at 60 ° C for 3 hours. The mixture was filtered and washed with methanol, tetrahydrofuran and methanol in sequence. The obtained solid was vacuum dried in a drying oven at 80 ° C for 24 hours to obtain a butenyl intermediate.

[0030] Under nitrogen protection, 10 g of the butenyl intermediate, 8.7 g of captopril (molar number is 40 mmol), 5.5 g of azobisisobutylimidazoline hydrochloride and 100 mL of ethanol / water (v / v=7 / 3) mixed solution were added to a 250 mL flask, reacted at 60° C. for 48 hours, filtered, washed with water, methanol, water and methanol in sequence, and the obtained solid was vacuum dried in a drying oven at 80° C. for 24 hours to obtain a chromatographic stationary phase 2, with a carboxyl bonding amount of 0.46 mmol. The structure was confirmed by infrared spectroscopy and nuclear magnetic carbon spectroscopy as follows:

[0031]

[0032] Example 3

[0033] The chromatographic stationary phase 1 obtained in Example 1 was used to fill a 4.6×50 mm chromatographic column for separation and analysis of polar compounds. The stationary phase had good selectivity for polar compounds. Figure 1 As shown, the chromatographic conditions are:

[0034] Chromatographic column: 4.6×50mm;

[0035] Solvent: A: acetonitrile, D: 100 mM ammonium formate (pH = 3.2);

[0036] Elution: A:D = 85:15;

[0037] Flow rate: 1.0 mL / min;

[0038] Column temperature: 30°C;

[0039] Detection: PDA (190nm-400nm) & UV (254nm);

[0040] Injection: 1.0 μL.

[0041] Example 4

[0042] The chromatographic stationary phase 1 obtained in Example 1 was used to fill a 4.6×50 mm chromatographic column for separation and analysis of alkaloids. The stationary phase had good separation selectivity for different types of alkaloids. Figure 2 As shown, the chromatographic conditions are:

[0043] Chromatographic column: 4.6×50mm;

[0044] Solvent: A: acetonitrile, B: 100 mM ammonium formate (pH = 3.2), C: water;

[0045] Elution: 0-10min-15min-20min, 5%-60%A-90%A-90%A-10%B;

[0046] Flow rate: 1.0 mL / min;

[0047] Column temperature: 30°C;

[0048] Detection: DAD (190nm-400nm) & UV (254nm);

[0049] Injection: 1 μL.

[0050] Example 5

[0051] Under nitrogen protection, add 10 g of microspherical silica gel dried at 160°C for 16 hours, 6 mL of aminopropyltrimethoxysilane and 60 mL of xylene into a 100 mL flask, react at 110°C for 16 hours, filter, wash with methanol, methanol water, methanol and tetrahydrofuran in sequence, and dry the obtained solid in a drying oven at 80°C for 16 hours to obtain amino silica gel.

[0052] Add 10g amino silica gel, 3g 4-dimethylaminopyridine, 2.4g succinic anhydride and 100mL N,N-dimethylformamide to a 250mL flask and react at 40°C for 24 hours. Filter and wash with methanol, 50mM sodium acetate solution, water and methanol in sequence. Dry the obtained solid in a drying oven at 80°C for 16 hours to obtain a carboxylic acid stationary phase with the following structure:

[0053]

[0054] Example 6

[0055] The chromatographic stationary phase 1 obtained in Example 1 and the carboxyl stationary phase obtained in Example 5 were used to fill 4.6×50 mm chromatographic columns for separation and analysis of alkaloids. The chromatographic stationary phase 1 has better separation selectivity for different types of alkaloids due to its captopril structure. The test results are shown in the table below. The chromatographic conditions are:

[0056] Chromatographic column: 4.6×50mm;

[0057] Solvent: A: acetonitrile, B: 100 mM ammonium formate (pH = 3.2), C: water;

[0058] Elution: A:B=50:50;

[0059] Flow rate: 1.0 mL / min;

[0060] Column temperature: 30°C;

[0061] Detection: DAD (190nm-400nm) & UV (254nm);

[0062] Injection: 1 μL.

[0063]

Claims

1. A captopril-modified weak cation exchange chromatography stationary phase, characterized in that: The drug molecule Captopril is used to modify the silica surface, and the bonded phase contains carboxyl, thioether and amide groups; its structural formula is as follows: , wherein Silica Gel is silica gel, and n= 0-10.

2. The chromatographic stationary phase according to claim 1, characterized in that: Each gram of silica gel contains 0.1-2.4 mmol of carboxyl groups.

3. The chromatographic stationary phase according to claim 1, characterized in that: Each gram of silica gel contains 0.3-1.3 mmol of carboxyl groups.

4. A method for preparing the stationary phase according to claim 1, 2 or 3, characterized in that: The steps include: a. Silica gel pretreatment: Add silica gel to a strong acid solution with a concentration of 1-40 wt%, heat reflux and stir for 1-48 hours, filter, wash with water until pH=6-7, and dry the obtained solid in a drying oven at 100-160°C for 8-24 hours to obtain acidified silica gel; the obtained dried acidified silica gel is placed in an atmosphere with a humidity of 10%-80% for 12-72 hours to make the silica gel absorb water and increase its weight by 1%-10% of the mass of the dry silica gel to obtain hydrated silica gel; b. Synthesis of alkenyl intermediates: under the protection of nitrogen and / or argon, add alkenyl silane coupling agent and hydrated silica gel to an organic solvent, react at 20-100°C for 1-48 hours, filter, wash with methanol, tetrahydrofuran and methanol in sequence, and vacuum dry the obtained solid in a drying oven at 40-80°C for 8-24 hours to obtain an alkenyl intermediate; c. Thiol-olefin click reaction: Under the protection of nitrogen and / or argon, captopril, a free radical initiator and an olefin intermediate are added to a mixed solution of an organic solvent / water volume ratio of 1-7 / 3-9, and the mixture is reacted at 40-80°C for 8-48 hours. The mixture is filtered and washed with water, methanol, water and methanol in sequence. The obtained solid is vacuum dried in a drying oven at 40-80°C for 8-24 hours to obtain a chromatographic stationary phase.

5. The preparation method according to claim 4, characterized in that: The strong acid used in step a is one of hydrochloric acid, nitric acid and sulfuric acid.

6. The preparation method according to claim 4, characterized in that: The silane coupling agent used in step b has the following structure: , wherein X is one or more of chlorine, methoxy or ethoxy, and n= 0-10.

7. The preparation method according to claim 4, characterized in that: The organic solvent used in step b is one or more of dichloromethane, toluene, xylene, isooctane and N,N-dimethylformamide.

8. The preparation method according to claim 4, characterized in that: The organic solvent used in step c is one or more of methanol, ethanol, isopropanol, dimethyl sulfoxide and N,N-dimethylformamide.

9. The preparation method according to claim 4, characterized in that: The free radical initiator used in step c is one or more of azobisisobutyronitrile, dimethyl azobisisobutyrate, azobisisobutylamidine hydrochloride, azobisisobutylimidazoline hydrochloride, azobiscyanovaleric acid, and azobisisopropylimidazoline.

10. The preparation method according to claim 4, characterized in that: The amount of the strong acid solution used in step a is 2-20 mL per gram of silica gel; The amount of the silane coupling agent used in step b is 1-10 mmol per gram of silica gel; The amount of the organic solvent used in step b is 5-15 mL per gram of silica gel; The amount of captopril used in step c is 1-10 mmol per gram of silica gel; The amount of the free radical initiator used in step c is 1-10 mmol per gram of silica gel; The amount of the mixed solution with the organic solvent / water volume ratio of 1-7 / 3-9 used in step c is 5-20 mL.

11. Use of the stationary phase according to claim 1, 2 or 3 for chromatographic separation of polar compounds and alkaloid compounds.

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