A bicyclic bonded chromatographic stationary phase and its preparation method and application

Abstract

The present invention relates to a liquid chromatography stationary phase, which is characterized in that the bonding phase contains a bicyclic structure of thiazole and pyridine, and the structural formula is as follows: #imgabs0# wherein Silica Gel is silica gel, and n=0-10. The present invention also provides a method for preparing the above-mentioned chromatographic stationary phase, and a novel bicyclic chromatographic stationary phase is prepared by a mercapto-epoxy ring-opening reaction. The separation material preparation method provided by the present invention is simple and efficient, and the stationary phase contains a bicyclic structure of thiazole and pyridine, which can provide a variety of forces, and has a variety of functional groups such as ether bonds, hydroxyl groups and thioether bonds, which can provide different separation selectivities, and 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 double heterocyclic structure chromatographic stationary phase containing thiazole and pyridine in a bonding phase. Technical Background

[0002] Liquid chromatography is a separation and analysis technology that leaves the chromatographic column in different orders according to the different interactions between different substances in the species being measured and the stationary phase. It is widely used. Due to its advantages such as high resolution, high sensitivity, fast speed, repeated use of chromatographic columns, and easy collection of effluent components, it is widely used in various fields such as biochemistry, food analysis, medical research, environmental analysis, inorganic analysis, etc., and has become the most promising method for solving biochemical analysis problems.

[0003] The selection of liquid chromatography stationary phase plays an important role in the separation of samples, and sometimes even a decisive role. Different types of chromatographic modes select stationary phases of different structures. For example, octadecyl, pentafluorophenyl, etc. are commonly used as stationary phases for reversed-phase chromatography, and amino, diol, etc. are commonly used as stationary phases for normal-phase chromatography. Currently, the commonly used types of stationary phases cannot meet the separation requirements of all samples, so it is necessary to develop new structural stationary phases to improve separation selectivity. Chromatographic stationary phases containing heterocyclic structures contain oxygen, nitrogen, and sulfur atoms in their structures, and have multiple forces such as hydrogen bonding, electrostatic effects, and dipole-dipole effects. They can provide different separation selectivities and can be used in multiple action modes, such as reversed-phase chromatography mode, normal-phase chromatography mode, supercritical fluid chromatography mode, etc., and are widely used in the separation and analysis of different types of compounds. Lurie et al. reported that the use of 2-aminopyridine stationary phase in supercritical fluid mode can achieve baseline separation of 22 synthetic cannabinoids, with high resolution for positional isomers and diastereomers of synthetic cannabinoids [Lurie. SI et al, J. Chromatogr. A, 2016, 1440, 201-211]. West et al. reported the use of cinchona alkaloid stationary phase containing quinoline structure to separate and analyze 161 non-chiral compounds and 66 pairs of enantiomers in supercritical fluid mode, proving that sulfonic acid group and quinoline structure have an important influence on enantioselectivity [West. C. et al, J. Chromatogr. A, 2020, 1612, 460689-460701]. In order to further broaden the range of heterocyclic structure chromatographic stationary phases, the present invention designs a new type of chromatographic stationary phase with a double heterocyclic structure, which has not been reported as a liquid chromatography stationary phase and can improve the separation selectivity between different samples. Summary of the invention

[0004] The purpose of the present invention is to provide a liquid chromatography stationary phase and a preparation method thereof. The bonded phase contains a bicyclic structure of thiazole and pyridine, 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 10 to 40 wt%, heated under reflux and stirred for 1 to 48 hours, filtered, washed with water until pH = 6 to 7, and the resulting solid is dried in a drying oven at 100 to 160 ° C for 8 to 24 hours to obtain acidified silica gel;

[0010] The amount of strong acid solution used is 5-20mL per gram of silica gel;

[0011] b. Synthesis of epoxy intermediate: Use sodium bicarbonate to prepare an aqueous solution with pH = 8.5, add epoxy silane coupling agent and silica gel to the sodium bicarbonate aqueous solution, react at 20-130°C for 1-48 hours, filter, wash with water, methanol, water, 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 epoxy 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 sodium bicarbonate aqueous solution used is 5-15 mL.

[0013] c. Mercapto-epoxy ring-opening reaction: under nitrogen or argon protection, add 2-mercapto-4-(4-pyridyl)thiazole and epoxy intermediate in an organic solvent, react at 40-100°C for 4-48 hours, filter, wash with N,N-dimethylformamide, acetone, N,N-dimethylformamide, acetone 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] Based on each gram of silica gel, the amount of 2-mercapto-4-(4-pyridyl)thiazole used is 1-20 mmol, and the amount of the organic solvent 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 modify the bicyclic structure of thiazole and pyridine onto the surface of silica gel to prepare a novel liquid chromatography stationary phase. The stationary phase structure contains thiazole and pyridine, which has good hydrogen bonding, π-π interaction and electrostatic interaction. At the same time, the stationary phase structure also contains groups such as hydroxyl and thioether bonds, which can provide additional dipole-dipole and other forces.

[0017] 2. The liquid chromatography stationary phase provided by the present invention has good separation selectivity for compounds of different polarities 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, and the reaction conditions are mild, 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 6. 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 to a 250 mL flask, add to 100 mL of 25 wt% hydrochloric acid solution, heat under reflux with stirring for 5 hours, filter, wash with water to pH = 6.7, and dry the obtained solid in a drying oven at 160°C for 24 hours to obtain acidified silica gel.

[0024] Add 10 g of silica gel, 10 mL of 3-(2,3-epoxypropoxy)propyltrimethoxysilane and 100 mL of sodium bicarbonate aqueous solution (pH = 8.5) into a 250 mL flask, react at 30 ° C for 24 hours, filter, wash with water, methanol, water, and methanol in sequence, and dry the obtained solid in a drying oven at 80 ° C under vacuum for 24 hours to obtain 3-(2,3-epoxypropoxy)propyl intermediate.

[0025] Under nitrogen protection, 10 g of 3-(2,3-epoxypropoxy)propyl intermediate, 5.2 g of 2-mercapto-4-(4-pyridyl)thiazole (molar number is 48 mmol) and 100 mL of N,N-dimethylformamide were added to a 250 mL flask, reacted at 70°C for 24 hours, filtered, washed with N,N-dimethylformamide, acetone, N,N-dimethylformamide and acetone in sequence, and the obtained solid was vacuum dried in a drying oven at 80°C for 16 hours to obtain chromatographic stationary phase 1, with a thiazole bonding amount of 0.84 mmol. The structure was confirmed by infrared spectroscopy and nuclear magnetic carbon spectroscopy as follows:

[0026]

[0027] Example 2

[0028] Add 10 g of silica gel to a 250 mL flask, add to 100 mL of 30 wt% nitric acid solution, heat under reflux with stirring for 2 hours, filter, wash with water to pH = 6-7, and dry the obtained solid in a drying oven at 160°C for 24 hours to obtain acidified silica gel.

[0029] Add 10 g of silica gel, 18 mL of 3-(2,3-epoxypropoxy)heptyltrimethoxysilane and 100 mL of sodium bicarbonate aqueous solution into a 250 mL flask, react at 60 ° C for 24 hours, filter, wash with water, methanol, water and methanol in sequence, and dry the obtained solid in a drying oven at 80 ° C under vacuum for 24 hours to obtain 3-(2,3-epoxypropoxy)heptyl intermediate.

[0030] Under nitrogen protection, 10 g of 3-(2,3-epoxypropoxy)heptyl intermediate, 5.2 g of 2-mercapto-4-(4-pyridyl)thiazole (molar number is 48 mmol) and 100 mL of N,N-dimethylformamide were added to a 250 mL flask, reacted at 70°C for 24 hours, filtered, washed with N,N-dimethylformamide, acetone, N,N-dimethylformamide and acetone in sequence, and the obtained solid was vacuum dried in a drying oven at 80°C for 24 hours to obtain chromatographic stationary phase 2, with a thiazole bonding amount of 0.76 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 non-polar compounds in liquid chromatography mode. The stationary phase had good selectivity for polar compounds. Figure 1 As shown, the chromatographic conditions are:

[0034] Chromatographic column: 4.6×50mm;

[0035] Sample: mixed standard (uracil 1 mg / mL, nitrobenzene 1 mg / mL, naphthalene 2 mg / mL, fluorene 2.2 mg / mL);

[0036] Solvent: A: acetonitrile, D: water;

[0037] Elution: A:D = 45:55;

[0038] Flow rate: 1.5 mL / min;

[0039] Column temperature: 30°C;

[0040] Detection: PDA (190nm-400nm) & UV (254nm);

[0041] Injection: 1.0 μL.

[0042] Example 4

[0043] 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 different types of terpenoid compounds in liquid chromatography mode, and had good separation selectivity.

[0044] The test results are shown in the table below. The chromatographic conditions are:

[0045] Chromatographic column: 4.6×50mm;

[0046] Samples: see the table below (all concentrations are 0.40 mg / mL);

[0047] Solvent: A: acetonitrile, B: 0.1% formic acid in water;

[0048] Elution: 0-10-15 min, 10-80% A-90% A;

[0049] Flow rate: 1.0 mL / min;

[0050] Column temperature: 30°C;

[0051] Detection: DAD (190nm-400nm) & UV (240nm);

[0052] Injection: 10 μL.

[0053]

[0054] Example 5

[0055] 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 different types of traditional Chinese medicine samples in liquid chromatography mode, and had good separation selectivity. The test results are shown in the table below, and the chromatographic conditions are:

[0056] Chromatographic column: 4.6×50mm;

[0057] Sample: see the table below (all concentrations are 0.20 mg / mL);

[0058] Solvent: A: 0.1% phosphoric acid in acetonitrile, B: 0.1% phosphoric acid in water;

[0059] Elution: 0-10-15 min, 5-50% A-90% A;

[0060] Flow rate: 1.0 mL / min;

[0061] Column temperature: 30°C;

[0062] Detection: DAD (190nm-400nm) & UV (254nm);

[0063] Injection: 1 μL.

[0064]

[0065] Example 6

[0066] 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 different types of alkaloid samples under supercritical fluid chromatography mode, and had good separation selectivity. Figure 2 As shown, the chromatographic conditions are:

[0067] Chromatographic column: 4.6×50mm;

[0068] Sample: mixed standard (concentration of all samples was 1.0 mg / mL);

[0069] Solvent: A: CO2, B: 0.1% triethylamine / methanol;

[0070] Elution: A:B = 85%:15%;

[0071] Flow rate: 3.0 mL / min;

[0072] Column temperature: 30°C;

[0073] Detection: DAD (190nm-400nm) & UV (280nm);

[0074] Injection: 1 μL.

[0075] Example 7

[0076] Different types of chromatographic stationary phases were used to pack 4.6×50mm chromatographic columns for separation and analysis of acidic compounds. The test results are shown in the table below. The chromatographic conditions are:

[0077] Chromatographic column: 4.6×50mm;

[0078] Sample: mixed standard (caffeic acid 0.10 mg / mL, ferulic acid 0.05 mg / mL);

[0079] Solvent: A: acetonitrile, B: 0.1% formic acid in water;

[0080] Elution: gradient 0-10-15 min, 5%-30%-90% A;

[0081] Flow rate: 1.0 mL / min;

[0082] Column temperature: 30°C;

[0083] Detection wavelength: PDA (190nm-400nm) & UV (254nm);

[0084]

[0085]

Claims

1. A bicyclic heterocyclic chromatographic stationary phase, characterized in that The bonded phase contains a bicyclic structure of thiazole and pyridine, and its structural formula is as follows: Wherein Silica Gel is silica gel, n= 0-10.

2. The chromatographic stationary phase according to claim 1, characterized in that: Each gram of silica gel contains 0.3-1.5 mmol of thiazole groups.

3. The chromatographic stationary phase according to claim 2, characterized in that: Each gram of silica gel contains 0.3-1.0 mmol of thiazole groups.

4. A method for preparing the stationary phase according to claim 1 or 2, characterized in that: The steps include: Silica gel pretreatment: Add silica gel to a strong acid solution with a concentration of 10-40 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; Synthesis of epoxy intermediate: use sodium bicarbonate to prepare an aqueous solution with a pH of 8.5-9.5, add epoxy silane coupling agent and silica gel to the sodium bicarbonate aqueous solution, react at 20-130°C for 1-48 hours, filter, wash with water, methanol, water, 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 epoxy intermediate; Mercapto-epoxy ring-opening reaction: Under the protection of nitrogen and / or argon, add 2-mercapto-4-(4-pyridyl)thiazole and epoxy intermediate in an organic solvent, react at 40-100°C for 4-48 hours, filter, wash with N,N-dimethylformamide, acetone, N,N-dimethylformamide, and acetone in sequence, and vacuum dry the obtained solid 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 or more 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 c is one or more of dichloromethane, toluene, methanol, ethanol, isopropanol, dimethyl sulfoxide, and N,N-dimethylformamide.

8. The preparation method according to claim 4, characterized in that: The amount of the strong acid solution used in step a is 5-20 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 sodium bicarbonate aqueous solution used in step b is 5-15 mL per gram of silica gel; The amount of 2-mercapto-4-(4-pyridyl)thiazole used in step c is 1-20 mmol per gram of silica gel; The amount of the organic solvent used in step c is 5-20 mL per gram of silica gel.

9. Use of the stationary phase according to claim 1 or 2 for chromatographic separation of flavonoid glycosides, aromatic aldehydes, phenolic acids, terpenes and alkaloids.

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