A mercaptopyridine 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 bonded phase contains a mercaptopyridine structure, and the structural formula thereof is as follows: 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 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 mercaptopyridine structure is contained in the stationary phase, which can provide a variety of forces, and at the same time, a variety of functional groups such as ether bonds and hydroxyl groups 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 chromatographic stationary phase containing mercaptopyridine structure 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 for reversed-phase chromatographic stationary phases, and amino, diol, etc. are commonly used for normal-phase chromatographic stationary phases. 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 pyridine structures contain nitrogen atoms and conjugated structures, and have multiple forces such as hydrogen bonding, electrostatic effects, and π-π effects. They can provide different separation selectivity from conventional chromatographic stationary phases, and can be used in multiple action modes, such as reversed-phase chromatography mode, normal-phase chromatography mode, supercritical fluid chromatography (SFC) mode, etc. They have been widely used in the separation and analysis of different types of compounds and have gradually attracted attention in recent years. 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]. The Viridis 2-EP chromatographic column containing pyridine structure developed by Waters can be widely used in non-chiral SFC separation, with good retention characteristics, peak shape and selectivity. In order to further improve the separation selectivity of pyridine bonded chromatographic stationary phases for different samples, it is still necessary to develop new chromatographic stationary phases. 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 mercaptopyridine 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 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 a pH of 7.5-9.5, 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;

[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 the protection of nitrogen or argon, add 2-mercaptopyridine and epoxy intermediate in an organic solvent, react at 40-100°C for 4-48 hours, filter, wash with N,N-dimethylformamide, methanol, N,N-dimethylformamide, methanol 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;

[0014] Based on each gram of silica gel, the amount of 2-mercaptopyridine 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 mercaptopyridine structure onto the silica gel surface to prepare a novel liquid chromatography stationary phase. The stationary phase structure contains thioether bonds and pyridine, and has good hydrogen bonding, π-π and electrostatic effects. At the same time, the stationary phase structure also contains hydroxyl groups and ether 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 with reference to 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 35 wt% hydrochloric acid solution, heat under reflux with stirring for 5 hours, filter, wash with water to pH = 6.5, and dry the obtained solid in a drying oven at 160°C for 24 hours to obtain acidified silica gel.

[0024] To a 250 mL flask, add 10 g of acidified silica gel, 10 mL of 3-(2,3-epoxypropoxy)propyltrimethoxysilane and 100 mL of sodium bicarbonate aqueous solution (pH = 8.5), 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 a 3-(2,3-epoxypropoxy)propyl intermediate.

[0025] Under nitrogen protection, 10 g of 3-(2,3-epoxypropoxy)propyl intermediate, 5.3 g of 2-mercaptopyridine (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, methanol, N,N-dimethylformamide and methanol in sequence, and the obtained solid was vacuum dried in a drying oven at 80°C for 16 hours to obtain a chromatographic stationary phase 1, with a thioether bond amount of 0.70 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, 5.3 g of 2-mercaptopyridine (molar number is 48 mmol) and 100 mL of dimethyl sulfoxide were added to a 250 mL flask, reacted at 70 ° C for 24 hours, filtered, washed with N, N-dimethylformamide, methanol, N, N-dimethylformamide 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, the thioether bond bonding amount was 0.56 mmol, and 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] Sample Type Iridoids Sesquiterpenes Sesquiterpenes Diterpenes Triterpenes Sample name Gentiopicroside β-elemene Atractylodesone Andrographolide Ursolic acid Retention time (min) 1.135 7.230 7.137 3.887 8.235

[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, 5-70% 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] Sample Type Aromatic aldehydes Iridoids Phenolic acid Flavonoids Alkaloids Sample name Protocatechuic aldehyde Gardenia glycoside Salvianolic acid B Rutin Evodiamine Retention time (min) 2.184 1.188 7.481 5.085 0.706

[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: methanol;

[0070] Elution: 0-5 min, 1%-5% B; 5-7 min, 5%-10% B; 7-10 min, 10%-20% B;

[0071] 10-15min, 20%-35% B;

[0072] Flow rate: 3.0 mL / min;

[0073] Column temperature: 30°C;

[0074] Detection: DAD (190nm-400nm) & UV (280nm);

[0075] Injection: 1 μL.

[0076] Example 7

[0077] Under nitrogen protection, 10 g of microspherical silica gel dried at 160°C for 16 hours, 6 mL of mercaptopropyltrimethoxysilane and 60 mL of xylene were added to a 100 mL flask, reacted at 110°C for 16 hours, filtered, washed with methanol, methanol water, methanol and tetrahydrofuran in sequence, and the obtained solid was dried in a drying oven at 80°C for 16 hours to obtain a mercapto stationary phase with the following structure:

[0078]

[0079] Example 8

[0080] The chromatographic stationary phase 1 obtained in Example 1 and the thiol stationary phase obtained in Example 7 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 the pyridine ring. The test results are shown in the table below. The chromatographic conditions are:

[0081] Chromatographic column: 4.6×50mm;

[0082] Solvent: A: acetonitrile, B: 100 mM ammonium formate (pH = 3.2), C: water;

[0083] Elution: A:B=50:50;

[0084] Flow rate: 1.0 mL / min;

[0085] Column temperature: 30°C;

[0086] Detection: DAD (190nm-400nm) & UV (254nm);

[0087] Injection: 1 μL.

[0088]

[0089]

Claims

1. A liquid chromatography stationary phase, characterized in that The bonded phase contains mercaptopyridine structure, and 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 0.1-1.4 mmol of thioether bonds.

3. The chromatographic stationary phase according to claim 2, characterized in that: Each gram of silica gel contains 0.1-0.9 mmol of thioether bonds.

4. A method for preparing the stationary phase according to any one of claims 1 to 3, characterized in that: The steps include: a. 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; b. Synthesis of epoxy intermediate: Use sodium bicarbonate to prepare an aqueous solution with a pH of 7.5-9.5, 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; c. Mercapto-epoxy ring-opening reaction: under the protection of nitrogen and / or argon, add 2-mercaptopyridine and epoxy intermediate in an organic solvent, react at 40-100°C for 4-48 hours, filter, wash with N,N-dimethylformamide, methanol, N,N-dimethylformamide, methanol 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; The strong acid used in step a is one or more of hydrochloric acid, nitric acid and sulfuric acid; 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.

5. 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.

6. 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-mercaptopyridine 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.

7. A stationary phase according to any one of claims 1 to 3 used for chromatographic separation of aromatic aldehyde compounds, alkaloid compounds, phenolic acid compounds, terpenoid compounds and flavonoid compounds.

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