A copolymer nanobrush type chromatographic stationary phase and its preparation method and application

Abstract

The present invention provides a copolymer nanobrush-type chromatographic stationary phase, its preparation method, and application. The copolymer nanobrush-type chromatographic stationary phase comprises styrene-maleic acid as a copolymer grafted onto the surface of spherical silica to form a nanobrush-like structure. The material of the present invention has excellent solubilization ability and separation potential, enabling efficient chromatographic separation and analysis of complex phospholipid samples. The material preparation method provided by the present invention features a simple process, mild reaction conditions, good preparation reproducibility and stability, and is readily applicable.

Description

Technical Field

[0001] The present invention relates to a chromatographic stationary phase material and a preparation method thereof, in particular to a copolymer nano brush type chromatographic stationary phase and a preparation method and application thereof. Background Art

[0002] Surface-modified copolymer brushes are currently a popular surface modification technology that can be used to change the inherent surface physical and chemical properties of materials. By designing the conformation and structure of the copolymer brush chain, the performance of the material, such as surface hydrophilicity, friction, and anti-fouling ability, can be changed, thereby achieving diversified applications. Copolymer brushes can be modified onto the surfaces of a variety of materials, such as carbon nanotubes, planar substrates, and nanoparticles. Among them, copolymer brushes grafted onto nanoparticles are widely used, such as gold nanoparticles, spherical silica, and nanoliposomes, which can achieve applications such as protein adsorption, drug delivery, and macromolecular separation and analysis. Currently, there are relatively few reports on the preparation and application of copolymer nanobrush-type stationary phases at home and abroad, especially those designed based on the chain structure of copolymer nanobrush according to the properties of the target object.

[0003] In recent years, styrene-maleic acid copolymers have been widely used in the biological field as membrane protein extractants. Styrene-maleic acid copolymers can be embedded in membrane lipids, dissolving biological membranes by forming nanodiscs, demonstrating excellent solubilization and extraction capabilities. Phospholipids are the primary components of membrane lipids, so styrene-maleic acid copolymers also have a good affinity for phospholipids. Nie Yangyang et al. proposed a modified styrene-maleic anhydride copolymer chromatographic stationary phase for phospholipid separation and analysis (Nie Yangyang, 2023). However, its separation performance is limited, and it cannot effectively separate structurally complex phospholipid samples or amide compounds.

[0004] Based on the above background, it is of great significance to prepare a new copolymer nanobrush stationary phase to meet various separation applications. Summary of the Invention

[0005] The purpose of the present invention is to provide a copolymer nano-brush type chromatographic stationary phase and its preparation method and application, so as to achieve efficient separation and analysis of target substances, especially phospholipids.

[0006] The object of the present invention is achieved like this:

[0007] A copolymer nanobrush type chromatographic stationary phase, wherein the copolymer nanobrush type chromatographic stationary phase is a styrene-maleic acid copolymer grafted onto the surface of spherical silica to form a nanobrush shape, and its structure is:

[0008] Preferably, the particle size of the spherical silica is 1.7-10 μm.

[0009] The preparation method of the copolymer nanobrush type chromatographic stationary phase comprises the following steps:

[0010] (1) Spherical silica is suspended in a solvent, (3-epoxyethylmethoxypropyl)trimethoxysilane is quickly added, and triethylamine as a catalyst is added. After mixing, the mixture is reacted at 90-95°C under nitrogen protection for 18-24 hours. The mass ratio of (3-epoxyethylmethoxypropyl)trimethoxysilane to spherical silica is (4-5):5, and the mass ratio of triethylamine to spherical silica is (1-2):100. After the reaction is completed, the mixture is cooled to room temperature, washed by centrifugation with detergent for 2-5 times, and vacuum dried to constant weight to obtain epoxy-functionalized silica.

[0011] (2) Add epoxy functionalized silica to the solvent and fully disperse it, add S-thiobenzoyl acetic acid, mix thoroughly, and heat under reflux for 12-18 h under nitrogen protection. The reaction temperature is 90-100 ° C, and the mass ratio of S-thiobenzoyl acetic acid to epoxy functionalized silica is 1:1. After the reaction, cool to room temperature, wash with detergent by centrifugation 4-6 times, and vacuum dry to constant weight to obtain chain transfer functionalized silica.

[0012] (3) Add maleic anhydride and styrene to the chain transfer functionalized silica, add the catalyst azobisisobutyronitrile, and finally add the solvent, ultrasonicate until mixed, and magnetically stir at 60-65°C for 12-24 h. The molar ratio of maleic anhydride to styrene is 1:1, and the mass ratio of the total mass of maleic anhydride and styrene to the chain transfer functionalized silica is (4-5):5. After the reaction is completed, cool to room temperature, wash with detergent 20-30 times, and vacuum dry to constant weight to obtain maleic anhydride-styrene copolymer modified silica.

[0013] (4) Ethylene glycol isooctyl ether is added to the maleic anhydride-styrene copolymer modified silica, followed by the catalyst 4-dimethylaminopyridine, and finally the solvent. The mixture is ultrasonically mixed and magnetically stirred at 60-65°C for 12-24 h under nitrogen protection. The mass ratio of ethylene glycol isooctyl ether to maleic anhydride-styrene copolymer modified silica is (2-5):10; the amount of 4-dimethylaminopyridine added is 0.5-1% of the molar mass of ethylene glycol isooctyl ether; after the reaction is completed, the mixture is cooled to room temperature, washed with detergent 4-6 times, and vacuum dried to constant weight to obtain a copolymer nanobrush type chromatographic stationary phase.

[0014] Furthermore, the solvent is anhydrous toluene, 4-methyl-2-pentanone or tetrahydrofuran.

[0015] Furthermore, the detergent is methanol, petroleum ether or tetrahydrofuran.

[0016] The present invention also provides application of the copolymer nano-brush type chromatographic stationary phase in liquid chromatography separation and analysis.

[0017] When applied, the copolymer nano-brush type chromatographic stationary phase is filled into a stainless steel chromatographic column, which can realize efficient separation and analysis of hydrophilic amides, different phospholipid types and phosphatidylcholine.

[0018] Beneficial effects of the present invention:

[0019] The present invention provides a copolymer nanobrush type chromatographic stationary phase, which has good solubilization ability and separation potential and can realize efficient chromatographic separation and analysis of complex phospholipid samples.

[0020] The preparation method of the copolymer nano-brush type chromatographic stationary phase provided by the present invention has a simple process, mild reaction conditions, good preparation reproducibility and stability, and is easy to promote and apply. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The infrared spectra of spherical silica, epoxy functionalized silica, chain transfer functionalized silica, maleic anhydride-styrene copolymer modified silica and the copolymer nano-brush type chromatographic stationary phase of the present invention are shown.

[0022] Figure 2 It is a thermogravimetric analysis graph of spherical silica, epoxy functionalized silica, chain transfer functionalized silica, maleic anhydride-styrene copolymer modified silica and the copolymer nanobrush type chromatographic stationary phase of the present invention.

[0023] Figure 3 It is a transmission electron micrograph of spherical silica (A) and the copolymer nanobrush type chromatographic stationary phase (B) of the present invention.

[0024] Figure 4 This is the separation chromatogram of four amide substances.

[0025] Figure 5 This is a chromatogram showing the separation of three different types of phospholipids.

[0026] Figure 6 This is a chromatogram showing the separation of four phosphatidylcholine molecules. DETAILED DESCRIPTION

[0027] Example 1

[0028] (1) Preparation of epoxy functionalized silica:

[0029] Weigh spherical silica and resuspend it in anhydrous toluene. Quickly add (3-epoxyethylmethoxypropyl)trimethoxysilane (90% by weight of the spherical silica) and triethylamine (1% by weight of the spherical silica) as a catalyst. Mix thoroughly and react at 90°C under nitrogen for 24 hours. After cooling to room temperature, wash the mixture five times with methanol by centrifugation and vacuum dry to constant weight to obtain epoxy-functionalized silica.

[0030] (2) Preparation of chain transfer functionalized silica:

[0031] 4-Methyl-2-pentanone was added as a solvent to the epoxy-functionalized silica to fully disperse it. S-thiobenzoyl acetic acid was then added in a mass ratio of 1:1 to the product of step (1). After thorough mixing by ultrasonication, the mixture was heated under reflux at 90°C for 12 h in a nitrogen atmosphere. After the reaction was completed, the mixture was cooled to room temperature, washed with petroleum ether by centrifugation 6 times, and vacuum dried to constant weight to obtain chain transfer-functionalized silica.

[0032] (3) Preparation of maleic anhydride-styrene copolymer modified silica:

[0033] Maleic anhydride and styrene were added to the chain transfer functionalized silica in a molar ratio of 1:1, and the total mass of maleic anhydride and styrene was 80% of the mass of the reactants obtained in step (2). Azobisisobutyronitrile was then added as a catalyst in an amount of 0.5% of the mass of maleic anhydride and styrene. Finally, tetrahydrofuran was added as a solvent, and the mixture was ultrasonically mixed. The mixture was stirred under magnetic stirring at 65°C for 12 h. After the reaction, the mixture was cooled to room temperature, washed 20 times with tetrahydrofuran, and vacuum dried to constant weight to obtain maleic anhydride-styrene copolymer modified silica.

[0034] (4) Preparation of copolymer nanobrush chromatographic stationary phase:

[0035] To the maleic anhydride-styrene copolymer modified silica, ethylene glycol isooctyl ether was added in an amount equal to 50% of the mass of the reactants obtained in step (3), followed by the addition of catalyst 4-dimethylaminopyridine in an amount equal to 1% of the molar mass of ethylene glycol isooctyl ether, and finally tetrahydrofuran was added as a solvent. The mixture was ultrasonically mixed and reacted under a nitrogen atmosphere at 65°C with magnetic stirring for 24 h. After the reaction, the mixture was cooled to room temperature, washed with tetrahydrofuran six times, and vacuum dried to constant weight to obtain a copolymer nanobrush chromatographic stationary phase.

[0036] The product was characterized and the results are shown in Figure 1-3 .

[0037] Figure 1The infrared spectra of spherical silica, epoxy functionalized silica, chain transfer functionalized silica, maleic anhydride-styrene copolymer modified silica and the copolymer nanobrush type chromatographic stationary phase of the present invention are shown in FIG. Figure 1 As shown in the infrared spectrum of spherical silica at 980 cm -1 There is a characteristic peak of stretching vibration of Si-OH bond on the surface of silica sphere at 980 cm -1 The peak at 1740 cm-1 disappears, indicating that (3-epoxyethylmethoxypropyl)trimethoxysilane has been successfully bonded to the surface of spherical silica. -1 The carbonyl C=O bond characteristic peak appears at 1780 cm -1 The carbonyl C=O bond characteristic peak of maleic anhydride appears at 1780 cm -1 The carbonyl peak at 1740 cm -1 The carbonyl peak at 1477 nm increased, indicating that ethylene glycol isooctyl ether successfully reacted with maleic acid. Infrared spectroscopy showed that the copolymer nanobrush type chromatographic stationary phase was successfully prepared.

[0038] Figure 2 Thermogravimetric analysis (TGA) of spherical silica, epoxy-functionalized silica, chain-transfer-functionalized silica, maleic anhydride-styrene copolymer-modified silica, and the copolymer nanobrush-type chromatographic stationary phase of the present invention are shown. Compared to spherical silica, epoxy-functionalized silica, chain-transfer-functionalized silica, maleic anhydride-styrene copolymer-modified silica, and the copolymer nanobrush-type chromatographic stationary phase all exhibit higher mass loss, further demonstrating the successful preparation of the copolymer nanobrush-type chromatographic stationary phase synthesized by the present invention.

[0039] Figure 3 These are transmission electron micrographs of spherical silica (A) and the copolymer nanobrush-type chromatographic stationary phase (B) synthesized in the present invention. Compared with the smooth surface of spherical silica (A), the copolymer nanobrush-type chromatographic stationary phase synthesized in the present invention shows a typical core-shell structure, further indicating that the copolymer nanobrush-type chromatographic stationary phase synthesized in the present invention has been successfully prepared.

[0040] Example 2

[0041] (1) Preparation of epoxy functionalized silica:

[0042] Weigh spherical silica and resuspend it in anhydrous toluene. Quickly add (3-epoxyethylmethoxypropyl)trimethoxysilane (100% by weight of the spherical silica) and triethylamine (2% by weight of the spherical silica) as a catalyst. Mix thoroughly and react at 95°C under nitrogen for 24 hours. After cooling to room temperature, wash the mixture five times with methanol by centrifugation and then vacuum dry to constant weight to obtain epoxy-functionalized silica.

[0043] (2) Preparation of chain transfer functionalized silica:

[0044] Add 4-methyl-2-pentanone as a solvent to the reactant obtained in step (1) to fully disperse it. Then add S-thiobenzoyl acetic acid in a mass ratio of 1:1 to the product of step a. After ultrasonication until fully mixed, heat and reflux at 100°C under a nitrogen atmosphere for 18 hours. After the reaction is completed, cool to room temperature, wash with petroleum ether by centrifugation six times, and vacuum dry to constant weight to obtain chain transfer functionalized silica.

[0045] (3) Preparation of maleic anhydride-styrene copolymer modified silica:

[0046] Maleic anhydride and styrene were added to the reactants obtained in step (2) in a molar ratio of 1:1, and the total mass of maleic anhydride and styrene was 85% of the mass of the reactants obtained in step (2). Azobisisobutyronitrile was then added as a catalyst in an amount of 0.5% of the mass of the maleic anhydride and styrene monomers. Finally, tetrahydrofuran was added as a solvent, and the mixture was ultrasonically mixed. The mixture was stirred under magnetic stirring at 65°C for 24 hours. After the reaction was completed, the mixture was cooled to room temperature, washed with tetrahydrofuran 30 times, and vacuum dried to constant weight to obtain maleic anhydride-styrene copolymer modified silica.

[0047] (4) Preparation of copolymer nanobrush chromatographic stationary phase:

[0048] To the reactant obtained in step (3), add ethylene glycol isooctyl ether in an amount equal to 50% of the mass of the reactant obtained in step (3), then add the catalyst 4-dimethylaminopyridine in an amount equal to 0.5% of the molar mass of ethylene glycol isooctyl ether, and finally add tetrahydrofuran as a solvent. Ultrasonic mixing is carried out, and the mixture is reacted at 65°C with magnetic stirring for 24 hours under nitrogen protection. After the reaction is completed, the mixture is cooled to room temperature, washed with tetrahydrofuran six times, and vacuum dried to constant weight to obtain a copolymer nanobrush chromatographic stationary phase.

[0049] Example 3

[0050] (1) Preparation of epoxy functionalized silica:

[0051] Weigh spherical silica and resuspend it in anhydrous toluene. Quickly add (3-epoxyethylmethoxypropyl)trimethoxysilane at a concentration of 80% by weight of the spherical silica. Add triethylamine as a catalyst at a concentration of 2% by weight of the spherical silica. Mix thoroughly and react at 95°C under nitrogen for 20 hours. After the reaction is complete, cool to room temperature, wash the mixture five times with methanol, and then vacuum dry to constant weight to obtain epoxy-functionalized silica.

[0052] (2) Preparation of chain transfer functionalized silica:

[0053] 4-Methyl-2-pentanone was added as a solvent to the reactant obtained in step (1) to fully disperse it. S-thiobenzoyl acetic acid was then added in a mass ratio of 1:1 to the product of step (1). After thorough mixing by ultrasonication, the mixture was heated under reflux at 90°C for 15 h in a nitrogen atmosphere. After the reaction was completed, the mixture was cooled to room temperature, washed by centrifugation with petroleum ether six times, and vacuum dried to constant weight to obtain chain transfer functionalized silica.

[0054] (3) Preparation of maleic anhydride-styrene copolymer modified silica:

[0055] Maleic anhydride and styrene were added to the reactants obtained in step (2) in a molar ratio of 1:1, and the total mass of maleic anhydride and styrene was 100% of the mass of the reactants obtained in step (2). Azobisisobutyronitrile was then added as a catalyst in an amount of 1% of the mass of the maleic anhydride and styrene monomers. Finally, tetrahydrofuran was added as a solvent, and the mixture was ultrasonically mixed. The mixture was stirred magnetically at 65°C for 12 h. After the reaction was completed, the mixture was cooled to room temperature, washed with tetrahydrofuran 25 times, and vacuum dried to constant weight to obtain maleic anhydride-styrene copolymer modified silica.

[0056] (4) Preparation of copolymer nanobrush chromatographic stationary phase:

[0057] To the reactant obtained in step (3), ethylene glycol isooctyl ether was added in an amount equal to 20% of the mass of the reactant obtained in step (3), followed by the addition of a catalyst, 4-dimethylaminopyridine, in an amount equal to 1% of the molar mass of ethylene glycol isooctyl ether. Finally, tetrahydrofuran was added as a solvent, and ultrasonic mixing was performed. Under nitrogen protection, the mixture was stirred magnetically at 60°C for 24 hours. After the reaction, the mixture was cooled to room temperature, washed four times with tetrahydrofuran, and vacuum dried to constant weight to obtain a copolymer nanobrush chromatographic stationary phase.

[0058] Example 4

[0059] Chromatographic separation results of different amides in the packed column of the copolymer nanobrush type chromatographic stationary phase prepared in Example 1. Chromatographic conditions: chromatographic column (150×4.6 mm), mobile phase: acetonitrile / water (99 / 1, v / v), flow rate: 1.0 mL / min, wavelength: 214 nm. The order of peaks is: 1. thioacetamide, 2. iodoacetamide, 3. benzamide, 4. cinnamamide. The four hydrophilic polar substances achieved baseline separation, with the highest theoretical plate number reaching 65,700 N / m, showing good separation performance ( Figure 4 ).

[0060] Example 5

[0061] Chromatographic separation results of different types of phospholipids in the packed column of the copolymer nanobrush type chromatographic stationary phase prepared in Example 1. Chromatographic conditions: chromatographic column (150×4.6 mm), mobile phase conditions are AB (13:87, v / v), where phase A is water and phase B is methanol-acetonitrile (8:1, v / v), the detector is an evaporative light scattering detector, the gas flow rate is 2.0 L / min, and the drift tube temperature is 100°C. The order of peaks is: 1. dioleoylphosphatidylglycerol, 2. dimyristoylphosphatidylcholine, 3. 1-palmitoyl-2-oleoylphosphatidylethanolamine. The three different types of phospholipids achieved baseline separation, showing the good separation performance of the chromatographic column for phospholipid types ( Figure 5 ).

[0062] Example 6

[0063] Chromatographic separation results of different types of phosphatidylcholine molecules in the packed column of the copolymer nanobrush type chromatographic stationary phase prepared in Example 1. Chromatographic conditions: chromatographic column (150×4.6 mm), mobile phase conditions are AB (13:87, v / v), where phase A is water and phase B is methanol-acetonitrile (8:1, v / v), the detector is an evaporative light scattering detector, the gas flow rate is 2.0 L / min, and the drift tube temperature is 100°C. The order of peaks is: 1, hemolyzed phosphatidylcholine, 2, dimyristoyl phosphatidylcholine, 3, dipalmitoyl phosphatidylcholine, 4, dioleoyl phosphatidylcholine. The four phosphatidylcholine molecules achieved baseline separation, showing the good separation performance of the chromatographic column for phosphatidylcholine phospholipid molecules ( Figure 6 ).

Claims

1. A copolymer nanobrush type chromatographic stationary phase, characterized in that: The copolymer nanobrush type chromatographic stationary phase is a copolymer of styrene and maleic acid grafted onto the surface of spherical silica to form a nanobrush type, and its structure is: 。 2. The copolymer nanobrush type chromatographic stationary phase according to claim 1, characterized in that The particle size of the spherical silicon dioxide is 1.7-10 μm.

3. The method for preparing the copolymer nanobrush type chromatographic stationary phase according to claim 1 or 2, characterized in that: The following steps are involved: (1) Spherical silica is suspended in a solvent, (3-epoxyethylmethoxypropyl)trimethoxysilane is quickly added, and triethylamine as a catalyst is added. After mixing, the mixture is reacted at 90-95°C under nitrogen protection for 18-24 hours. The mass ratio of (3-epoxyethylmethoxypropyl)trimethoxysilane to spherical silica is (4-5):5, and the mass ratio of triethylamine to spherical silica is (1-2):

100. After the reaction is completed, the mixture is cooled to room temperature, washed by centrifugation with detergent for 2-5 times, and vacuum dried to constant weight to obtain epoxy-functionalized silica. (2) Add epoxy functionalized silica to the solvent and fully disperse it, add S-thiobenzoyl acetic acid, mix thoroughly, and heat under reflux for 12-18 h under nitrogen protection. The reaction temperature is 90-100 ° C, and the mass ratio of S-thiobenzoyl acetic acid to epoxy functionalized silica is 1:

1. After the reaction, cool to room temperature, wash with detergent by centrifugation 4-6 times, and vacuum dry to constant weight to obtain chain transfer functionalized silica. (3) Add maleic anhydride and styrene to the chain transfer functionalized silica, add the catalyst azobisisobutyronitrile, and finally add the solvent, ultrasonicate until mixed, and magnetically stir at 60-65°C for 12-24 h. The molar ratio of maleic anhydride to styrene is 1:1, and the mass ratio of the total mass of maleic anhydride and styrene to the chain transfer functionalized silica is (4-5):

5. After the reaction is completed, cool to room temperature, wash with detergent 20-30 times, and vacuum dry to constant weight to obtain maleic anhydride-styrene copolymer modified silica. (4) Ethylene glycol isooctyl ether is added to the maleic anhydride-styrene copolymer modified silica, followed by the catalyst 4-dimethylaminopyridine, and finally the solvent. The mixture is ultrasonically mixed and magnetically stirred at 60-65°C for 12-24 h under nitrogen protection. The mass ratio of ethylene glycol isooctyl ether to maleic anhydride-styrene copolymer modified silica is (2-5):10; the amount of 4-dimethylaminopyridine added is 0.5-1% of the molar mass of ethylene glycol isooctyl ether; after the reaction is completed, the mixture is cooled to room temperature, washed with detergent 4-6 times, and vacuum dried to constant weight to obtain a copolymer nanobrush type chromatographic stationary phase.

4. The preparation method according to claim 3, characterized in that The solvent is anhydrous toluene, 4-methyl-2-pentanone or tetrahydrofuran.

5. The preparation method according to claim 3, characterized in that The detergent is methanol, petroleum ether or tetrahydrofuran.

6. Use of the copolymer nanobrush type chromatographic stationary phase according to claim 1 or 2 in liquid chromatography separation and analysis.

7. Use of the copolymer nanobrush type chromatographic stationary phase according to claim 1 or 2 in the separation and analysis of hydrophilic amides, different phospholipid types or different phosphatidylcholines.

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