Modified silicon dioxide microsphere for high performance liquid chromatography and preparation method thereof

Abstract

The invention discloses modified silicon dioxide microspheres for high performance liquid chromatography and a preparation method of the modified silicon dioxide microspheres, and relates to the technical field of chromatographic packing. When the modified silicon dioxide microspheres for the high performance liquid chromatography are prepared, firstly, (perfluorohexyl) benzene reacts with isophthaloyl dichloride to prepare a perfluorohexyl benzophenone compound; the preparation method comprises the following steps: reacting a perfluorohexyl benzophenone compound with hydrazine hydrate to prepare a perfluorohexyl hydrazone compound; carrying out catalytic decomposition on the perfluorohexyl hydrazone compound to prepare a hydrophobic modified diazo compound; preparing monodispersed non-porous silicon dioxide microspheres by using a sol-gel method; modifying the non-porous silicon dioxide microspheres by an etching template method to prepare mesoporous core-shell silicon dioxide microspheres; and reacting the mesoporous core-shell silicon dioxide microspheres with a hydrophobic modified diazo compound to prepare the modified silicon dioxide microspheres for high performance liquid chromatography. The prepared modified silicon dioxide microspheres for high performance liquid chromatography have the advantages of monodisperse particle size, large specific surface area, small back pressure and high column efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of chromatographic fillers, in particular to modified silica microspheres for high performance liquid chromatography and a preparation method thereof. Background Art

[0002] As a separation technology and method, high performance liquid chromatography has become increasingly mature in recent years. Due to the advantages of high efficiency, speed and easy operation in the separation and analysis process, its application range is becoming increasingly wide, such as pesticide residue analysis, food safety testing, production process monitoring and protein separation and purification.

[0003] As the site and tool for separation, the chromatographic column is the core component of the entire HPLC system. Its performance directly determines the quality of the separation, hence the column's often referred to as the heart of HPLC. The development of chromatographic packings determines the development of chromatographic columns, so the preparation and optimization of chromatographic packings remain at the core of chromatographic technology. With the rapid development of biological and life sciences, new and complex samples have placed higher demands on separation and analysis. Rapid and efficient separation methods have always been the goal pursued by chromatographers.

[0004] In recent years, superficially porous core-shell packings have captured the attention of chromatographers with their unique structure and superior separation performance. Their unique non-porous core and mesoporous shell structure not only ensures high mechanical strength but also significantly shortens the diffusion path for solutes, significantly reducing separation time. The development of rapid separation technology for core-shell packings has ushered in a new era of rapid analysis in liquid chromatography. Compared to non-porous silica packings of the same particle size, core-shell packings offer higher sample loading capacity and lower column pressure. They also offer faster separation speeds and superior results compared to conventional 5μm fully porous packings. The methods for preparing such core-shell microspheres include self-assembly, template, template dissolution-induced redeposition, and polymerization-induced redeposition. However, the self-assembly method is cumbersome and generally requires 10 to 15 cycles to reach the desired particle size, resulting in low yield and high cost. The template and template dissolution-induced redeposition methods share the same problem, namely, the pore size of the surface shell is small, less than 5 to 8 nm, and usually requires secondary pore expansion before it can be used for the separation and analysis of macromolecules. Although the pore size prepared by the polymerization-induced redeposition method is larger, the micropores formed are parallel to the surface of the microspheres, which increases the diffusion path and reduces the column efficiency. Secondly, the particle size distribution of the microspheres prepared by this scheme is wide, and further particle size classification is required before they can be used for chromatographic analysis.

[0005] To improve separation efficiency and performance, silica microspheres are typically surface-modified with various functional groups, including octadecyl, octyl, phenyl, amino, and cyano groups. However, the conventional silica modification process is complex and time-consuming, typically involving multiple steps such as silica pretreatment, introduction of a coupling agent, and embedding of the target modification agent. Furthermore, the silane coupling agents commonly used in the modification process are often toxic and water-sensitive, requiring strict use under anhydrous conditions. These factors, in turn, can cause environmental pollution and significantly limit their application. Summary of the Invention

[0006] The purpose of the present invention is to provide a modified silica microsphere for high performance liquid chromatography and a preparation method thereof, so as to solve the problems existing in the prior art.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions: A modified silica microsphere for high performance liquid chromatography, wherein the modified silica microsphere for high performance liquid chromatography is prepared by reacting mesoporous core-shell silica microspheres with a hydrophobically modified diazo compound; The mesoporous core-shell silica microspheres are prepared by modifying non-porous silica microspheres using an etching template method; The non-porous silica microspheres are prepared by a sol-gel method; The hydrophobically modified diazo compound is prepared by catalytically decomposing a perfluorohexylhydrazone compound; The perfluorohexylhydrazone compound is prepared by reacting a perfluorohexylbenzophenone compound with hydrazine hydrate; The perfluorohexyl benzophenone compound is prepared by reacting (perfluorohexyl)benzene with isophthaloyl chloride.

[0008] As an optimization, the etching template method is carried out using sodium carbonate as an etchant, and octadecyltrimethylammonium chloride and tri(dodecyl)methylammonium chloride as template agents.

[0009] As an optimization, the catalytic decomposition is carried out by adding manganese dioxide, anhydrous sodium sulfate and potassium hydroxide.

[0010] A method for preparing modified silica microspheres for high performance liquid chromatography comprises the following steps: (1) In an ice-water bath, 0.5-0.6 parts of (perfluorohexyl)benzene, 0.3-0.35 parts of anhydrous aluminum chloride, and 25-30 parts of dichloromethane were mixed uniformly by mass, and stirred at 200-300 r / min for 20-30 min. A 1 wt% dichloromethane solution of isophthaloyl chloride and (perfluorohexyl)benzene was added dropwise at a rate of 20-30 drops / min, with a molar ratio of isophthaloyl chloride to (perfluorohexyl)benzene of 1:2. After the addition was completed, the mixture was stirred at 300-400 r / min for 11-12 h at room temperature, and washed with dilute hydrochloric acid solution and pure water for 2-3 times, respectively. After purification, a perfluorohexyl benzophenone compound was obtained. (2) By weight, 1-1.2 parts of perfluorohexyl benzophenone compound and 20-25 parts of anhydrous ethanol were mixed evenly, stirred at room temperature at 200-300 r / min for 20-30 min, 0.7-0.8 parts of hydrazine hydrate and 0.16-0.2 parts of acetic acid were added, and the mixture was refluxed at 80-85°C and 300-400 r / min for 90-96 h. The ethanol was removed by rotary evaporation, 20 parts of dichloromethane was added to redissolve the mixture, and the mixture was washed three times with saturated brine, the aqueous layer was separated, and the organic layer was dried over anhydrous magnesium sulfate. After filtering, the mixture was rotary evaporated to obtain a perfluorohexyl hydrazone compound; (3) By weight, 0.4-0.5 parts of perfluorohexylbenzophenone hydrazone compound and 20-30 parts of dichloromethane were mixed evenly, stirred at 200-300 r / min at room temperature for 20-30 min, 0.15-0.2 parts of manganese dioxide, 0.1-0.12 parts of anhydrous sodium sulfate, and 0.08-0.1 parts of potassium hydroxide were added, and the mixture was stirred at 200-300 r / min at room temperature for 6-7 h in the dark. The mixture was filtered and rotary evaporated in the dark to obtain a hydrophobically modified diazo compound; (4) By weight, 0.015-0.02 parts of potassium chloride, 6-7 parts of pure water, 65-70 parts of anhydrous ethanol, and 3-4 parts of 28 wt% ammonia water were mixed evenly, stirred at 300-400 r / min for 30-40 min at room temperature, and 38-40 parts of 10 wt% ethyl orthosilicate ethanol solution were added dropwise at a rate of 0.15-0.2 ml / min. After the addition was completed, the reaction was continued for 2-3 h, and the mixture was washed with anhydrous ethanol for 3-4 times by centrifugation, and vacuum dried at 50-60 ° C for 8-10 h to obtain non-porous silica microspheres; (5) According to the mass fraction, 1.1~1.3 parts of octadecyltrimethylammonium chloride, 0.9~1.07 parts of tri(dodecyl)methylammonium chloride and 180~200 parts of pure water were mixed evenly, stirred at 200~300 r / min for 20~30 min at room temperature, 2~2.5 parts of non-porous silica microspheres were added, ultrasonically dispersed for 25~30 min, and 0.14~0 16 parts of sodium carbonate and 10-12 parts of 28wt% ammonia water were refluxed at 85-90°C and 300-400 rpm for 20-24 hours. After cooling, the mixture was centrifuged at 3000-4000 rpm for 6-8 minutes, the liquid was removed, and the mixture was washed with anhydrous ethanol and pure water 2-3 times each by centrifugation. The mixture was vacuum dried at 50-60°C for 8-10 hours and calcined at 600-650°C for 10-12 hours to obtain mesoporous core-shell silica microspheres. (6) Under light-proof conditions, the hydrophobically modified diazo compound was dissolved in dichloromethane at a concentration of 0.1 g / ml, and mesoporous core-shell silica microspheres were added at a material-liquid ratio of 1:(25-30) g / ml. The mixture was ultrasonicated for 5-6 min, allowed to stand for 8-10 min, and the liquid was removed by centrifugation. The solid was vacuum-dried at 50-60 °C for 6-8 h, heated at 120-130 °C for 30-40 min, and ultrasonically cleaned with acetone, dichloromethane, and ethanol in sequence. The modified silica microspheres for high performance liquid chromatography were obtained.

[0011] As an optimization, the reaction process of the perfluorohexyl benzophenone compound in step (1) is as follows: .

[0012] As an optimization, the reaction process of the perfluorohexylhydrazone compound in step (2) is as follows: .

[0013] As an optimization, the reaction process of the hydrophobically modified diazo compound in step (3) is as follows: .

[0014] As an optimization, the molar ratio of octadecyltrimethylammonium chloride to tri(dodecyl)methylammonium chloride in step (5) is 1:2.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The invention prepares modified silica microspheres for high performance liquid chromatography. The invention comprises the following steps: first, (perfluorohexyl)benzene is reacted with isophthaloyl chloride to obtain a perfluorohexylbenzophenone compound; the perfluorohexylbenzophenone compound is reacted with hydrazine hydrate to obtain a perfluorohexylhydrazone compound; the perfluorohexylhydrazone compound is catalytically decomposed to obtain a hydrophobically modified diazo compound; monodispersed non-porous silica microspheres are obtained by a sol-gel method; the non-porous silica microspheres are modified by an etching template method to obtain mesoporous core-shell silica microspheres; and the mesoporous core-shell silica microspheres are reacted with the hydrophobically modified diazo compound to obtain the modified silica microspheres for high performance liquid chromatography.

[0016] First, (perfluorohexyl)benzene and isophthaloyl chloride undergo a Friedel-Crafts alkylation reaction in the presence of anhydrous aluminum chloride to form a benzophenone compound with three benzene rings and a perfluorocarbon chain. The carbonyl group is then converted into a hydrazone group by reacting with hydrazine hydrate. Finally, the compound is catalytically decomposed into a diazo group under the action of manganese dioxide, anhydrous sodium sulfate, and potassium hydroxide. The diazo group can be adsorbed on the surface of most matrix materials through hydrogen bonds and electrostatic effects, and can generate a carbene group with excellent reactivity under heating conditions. It undergoes a carbene insertion reaction with the active hydrogen on the matrix material to form a covalent bond. Compared with silane coupling agents, this modification method is convenient and fast, has a simple reaction, and has excellent modification results.

[0017] Secondly, by improving the sol-gel method, monodisperse non-porous silica microspheres with a particle size of 2.5~3μm were prepared. The conventional sol-gel method can only prepare microspheres with a particle size of less than 200nm. By controlling the amount of potassium chloride added as the electrolyte, the particle size of the prepared microspheres can be significantly increased to the micron level, making it meet the filler particle size requirements of high-performance liquid chromatography; then, sodium carbonate was used as an etchant, and octadecyltrimethylammonium chloride and tri(dodecyl)methylammonium chloride were used as templates to prepare mesoporous core-shell silica microspheres. The prepared mesoporous core-shell silica microspheres have a surface mesoporous and an internal solid core-shell structure. Small silica fragments are separated from the silica microspheres under the etching action of sodium carbonate, while octadecyltrimethylammonium chloride and tri(dodecyl)methylammonium chloride as templates. Ammonium chloride forms micelles that are adsorbed on the surface of the silica microspheres through electrostatic force, and the silica fragments separated by etching are captured and coated by the micelles adsorbed on the surface of the silica microspheres, thereby being directionally deposited on the surface of the silica microspheres again. As etching and re-deposition continue, the structure of the surface of the non-porous silica microspheres gradually transforms into a hybrid structure containing silica and micelles. After calcination to remove the octadecyltrimethylammonium chloride and tri(dodecyl)methylammonium chloride serving as templates, a shell layer with a radial pore structure is formed on the surface of the silica microspheres. At the same time, the micelles formed by the specific molar ratio of octadecyltrimethylammonium chloride and tri(dodecyl)methylammonium chloride have the largest volume, and can form larger mesopores after calcination, thereby effectively improving the separation efficiency of macromolecular substances.

[0018] Finally, the hydrophobically modified diazo compound with a diazo group is adsorbed on the surface and mesoporous wall of the mesoporous core-shell silica microspheres through electrostatic and hydrogen bonding. Then, under the action of high temperature, the diazo group is converted into a highly reactive carbene group, which then reacts with the hydroxyl groups on the surface of the mesoporous core-shell silica microspheres through a carbene insertion reaction, thereby introducing perfluoroalkyl chains and benzene rings into the surface of the silica microspheres, completing the hydrophobic modification and improving its separation efficiency and column efficiency. DETAILED DESCRIPTION

[0019] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0020] The concentration of the dilute hydrochloric acid solution used in all the following examples and comparative examples is 0.1 mol / L.

[0021] Example 1: A method for preparing modified silica microspheres for high performance liquid chromatography, comprising the following steps: (1) In an ice-water bath, 0.5 parts of (perfluorohexyl)benzene, 0.3 parts of anhydrous aluminum chloride, and 25 parts of dichloromethane were mixed uniformly by mass and stirred at 200 r / min for 30 min. A 1 wt% dichloromethane solution of isophthaloyl chloride was added dropwise at a rate of 20 drops / min, with a molar ratio of isophthaloyl chloride to (perfluorohexyl)benzene of 1:2. After the addition was completed, the mixture was stirred at 300 r / min for 12 h at room temperature. The mixture was washed twice with dilute hydrochloric acid solution and purified with water, respectively, to obtain a perfluorohexyl benzophenone compound. (2) By weight, 1 part of perfluorohexyl benzophenone compound and 20 parts of anhydrous ethanol were mixed evenly, stirred at room temperature at 200 r / min for 30 min, 0.7 parts of hydrazine hydrate and 0.16 parts of acetic acid were added, and the mixture was refluxed at 80°C and 300 r / min for 96 h. The ethanol was removed by rotary evaporation, 20 parts of dichloromethane was added to redissolve the mixture, and the mixture was washed three times with saturated brine. The aqueous layer was separated and removed, and the organic layer was dried over anhydrous magnesium sulfate. After filtering, the mixture was rotary evaporated to obtain a perfluorohexyl hydrazone compound; (3) 0.4 parts of perfluorohexylbenzophenone hydrazone compound and 20 parts of dichloromethane were mixed uniformly by weight, stirred at 200 r / min at room temperature for 30 min, 0.15 parts of manganese dioxide, 0.1 parts of anhydrous sodium sulfate, and 0.08 parts of potassium hydroxide were added, and the mixture was stirred at 200 r / min at room temperature for 7 h in the dark. The mixture was filtered and rotary evaporated in the dark to obtain a hydrophobically modified diazo compound; (4) By weight, 0.015 parts of potassium chloride, 6 parts of pure water, 65 parts of anhydrous ethanol, and 3 parts of 28 wt% ammonia water were mixed evenly, stirred at 300 r / min for 40 min at room temperature, and 38 parts of 10 wt% ethyl orthosilicate ethanol solution were added dropwise at a rate of 0.15 ml / min. After the addition was completed, the reaction was continued for 2 h, and the mixture was washed three times by centrifugation with anhydrous ethanol, and vacuum dried at 50 ° C for 10 h to obtain non-porous silica microspheres; (5) By weight, 1.1 parts of octadecyltrimethylammonium chloride, 0.9 parts of tridodecylmethylammonium chloride, and 180 parts of pure water were mixed evenly, stirred at 200 r / min at room temperature for 30 min, 2 parts of non-porous silica microspheres were added, and ultrasonic dispersion was carried out for 25 min. At room temperature, 0.14 parts of sodium carbonate and 10 parts of 28 wt% ammonia water were added at 300 r / min. The mixture was refluxed at 85 ° C and 300 r / min for 24 h. After cooling, the mixture was centrifuged at 3000 rpm for 8 min, the liquid was removed, and the mixture was washed twice with anhydrous ethanol and pure water respectively. The mixture was vacuum dried at 50 ° C for 10 h and calcined at 600 ° C for 12 h to obtain mesoporous core-shell silica microspheres. (6) Under light-shielding conditions, the hydrophobically modified diazo compound was dissolved in dichloromethane at a concentration of 0.1 g / ml, and mesoporous core-shell silica microspheres were added at a material-liquid ratio of 1:25 g / ml. The mixture was ultrasonicated for 5 min, allowed to stand for 10 min, and the liquid was removed by centrifugation. The solid was vacuum-dried at 50 °C for 8 h, heated at 120 °C for 40 min, ultrasonically cleaned with acetone, dichloromethane, and ethanol in sequence, and vacuum-dried at 50 °C for 8 h to obtain modified silica microspheres for high performance liquid chromatography.

[0022] The average particle size of the prepared modified silica microspheres for high performance liquid chromatography was 2.74 μm, and the polydispersity index was 4.27%.

[0023] Example 2: A method for preparing modified silica microspheres for high performance liquid chromatography, comprising the following steps: (1) In an ice-water bath, 0.55 parts of (perfluorohexyl)benzene, 0.32 parts of anhydrous aluminum chloride, and 28 parts of dichloromethane were mixed uniformly, stirred at 250 r / min for 25 min, and a 1 wt% dichloromethane solution of isophthaloyl chloride was added dropwise at a rate of 25 drops / min, with a molar ratio of isophthaloyl chloride to (perfluorohexyl)benzene of 1:2. After the addition was completed, the mixture was stirred at 350 r / min for 11.5 h at room temperature, washed twice with dilute hydrochloric acid solution and purified with water, and purified to obtain a perfluorohexyl benzophenone compound; (2) By weight, 1.1 parts of perfluorohexyl benzophenone compound and 22 parts of anhydrous ethanol were mixed evenly, stirred at room temperature at 250 r / min for 25 min, 0.75 parts of hydrazine hydrate and 0.18 parts of acetic acid were added, and the mixture was refluxed at 80°C and 350 r / min for 93 h. The ethanol was removed by rotary evaporation, 20 parts of dichloromethane was added to redissolve the mixture, and the mixture was washed three times with saturated brine. The aqueous layer was separated and removed, and the organic layer was dried over anhydrous magnesium sulfate. After filtering, the mixture was rotary evaporated to obtain a perfluorohexyl hydrazone compound; (3) By weight, 0.45 parts of perfluorohexylbenzophenone hydrazone compound and 25 parts of dichloromethane were mixed evenly, stirred at 250 r / min at room temperature for 25 min, 0.18 parts of manganese dioxide, 0.11 parts of anhydrous sodium sulfate, and 0.09 parts of potassium hydroxide were added, and the mixture was stirred at 250 r / min at room temperature for 6.5 h in the dark. The mixture was filtered and rotary evaporated in the dark to obtain a hydrophobically modified diazo compound; (4) By weight, 0.018 parts of potassium chloride, 6.5 parts of pure water, 68 parts of anhydrous ethanol, and 3.5 parts of 28 wt% ammonia water were mixed evenly, stirred at 350 r / min at room temperature for 35 min, and 39 parts of 10 wt% ethyl orthosilicate ethanol solution were added dropwise at a rate of 0.18 ml / min. After the addition was completed, the reaction was continued for 2.5 h, washed three times with anhydrous ethanol by centrifugation, and dried in vacuum at 55 °C for 9 h to obtain non-porous silica microspheres; (5) By weight, 1.2 parts of octadecyltrimethylammonium chloride, 0.987 parts of tridodecylmethylammonium chloride, and 190 parts of pure water were mixed evenly, stirred at 250 r / min at room temperature for 25 min, 2.2 parts of non-porous silica microspheres were added, and ultrasonic dispersion was carried out for 28 min. At room temperature, 0.15 parts of sodium carbonate and 11 parts of 28 wt% ammonia water were added at 350 r / min. The mixture was refluxed at 88 ° C and 350 r / min for 22 h. After cooling, the mixture was centrifuged at 3500 rpm for 7 min, the liquid was removed, and the mixture was washed twice with anhydrous ethanol and pure water respectively, dried in a vacuum at 55 ° C for 9 h, and calcined at 625 ° C for 11 h to obtain mesoporous core-shell silica microspheres. (6) Under light-shielding conditions, the hydrophobically modified diazo compound was dissolved in dichloromethane at a concentration of 0.1 g / ml, and mesoporous core-shell silica microspheres were added at a material-liquid ratio of 1:28 g / ml. The mixture was ultrasonicated for 5.5 min, allowed to stand for 9 min, and the liquid was removed by centrifugation. The solid was vacuum-dried at 55 °C for 7 h, heated at 125 °C for 35 min, ultrasonically cleaned with acetone, dichloromethane, and ethanol in sequence, and vacuum-dried at 55 °C for 7 h to obtain modified silica microspheres for high performance liquid chromatography.

[0024] The average particle size of the prepared modified silica microspheres for high performance liquid chromatography was 2.83 μm, and the polydispersity index was 3.94%.

[0025] Example 3: A method for preparing modified silica microspheres for high performance liquid chromatography, comprising the following steps: (1) In an ice-water bath, 0.6 parts of (perfluorohexyl)benzene, 0.35 parts of anhydrous aluminum chloride, and 30 parts of dichloromethane were mixed uniformly by mass, stirred at 300 r / min for 20 min, and a 1 wt% dichloromethane solution of isophthaloyl chloride was added dropwise at a rate of 30 drops / min according to a molar ratio of isophthaloyl chloride to (perfluorohexyl)benzene of 1:2. After the addition was completed, the mixture was stirred at 400 r / min for 11 h at room temperature, washed three times with dilute hydrochloric acid solution and purified with water, and purified to obtain a perfluorohexyl benzophenone compound; (2) By weight, 1.2 parts of perfluorohexyl benzophenone compound and 25 parts of anhydrous ethanol were mixed evenly, stirred at room temperature at 300 r / min for 20 min, 0.8 parts of hydrazine hydrate and 0.2 parts of acetic acid were added, and the mixture was refluxed at 85°C and 400 r / min for 90 h. The ethanol was removed by rotary evaporation, 20 parts of dichloromethane was added to redissolve the mixture, and the mixture was washed three times with saturated brine. The aqueous layer was separated and removed, and the organic layer was dried over anhydrous magnesium sulfate. After filtering, the mixture was rotary evaporated to obtain a perfluorohexyl hydrazone compound; (3) By weight, 0.5 parts of perfluorohexylbenzophenone hydrazone compound and 30 parts of dichloromethane were mixed evenly, stirred at 300 r / min at room temperature for 20 min, 0.2 parts of manganese dioxide, 0.12 parts of anhydrous sodium sulfate, and 0.1 parts of potassium hydroxide were added, and the mixture was stirred at 300 r / min at room temperature for 6 h in the dark. The mixture was filtered and rotary evaporated in the dark to obtain a hydrophobically modified diazo compound; (4) 0.02 parts of potassium chloride, 7 parts of pure water, 70 parts of anhydrous ethanol, and 4 parts of 28 wt% ammonia water were mixed uniformly by weight, stirred at 400 r / min for 30 min at room temperature, and 40 parts of 10 wt% ethyl orthosilicate ethanol solution were added dropwise at a rate of 0.2 ml / min. After the addition was completed, the reaction was continued for 3 h, and the mixture was washed with anhydrous ethanol by centrifugation 4 times, and vacuum dried at 60 ° C for 8 h to obtain non-porous silica microspheres; (5) 1.3 parts of octadecyltrimethylammonium chloride, 1.07 parts of tridodecylmethylammonium chloride and 200 parts of pure water were mixed uniformly by mass, stirred at 300 r / min for 20 min at room temperature, 2.5 parts of non-porous silica microspheres were added, and ultrasonic dispersion was carried out for 30 min. 0.16 parts of sodium carbonate and 12 parts of 28 wt% ammonia water were added at 400 r / min at room temperature, and refluxed at 90 ° C and 400 r / min for 20 h. After cooling, centrifuged at 4000 rpm for 6 min, the liquid was removed, and the mixture was washed with anhydrous ethanol and pure water for 3 times each. The mixture was vacuum dried at 60 ° C for 8 h and calcined at 650 ° C for 10 h to obtain mesoporous core-shell silica microspheres. (6) Under light-shielding conditions, the hydrophobically modified diazo compound was dissolved in dichloromethane at a concentration of 0.1 g / ml, and mesoporous core-shell silica microspheres were added at a material-liquid ratio of 1:30 g / ml. The mixture was ultrasonicated for 6 min, allowed to stand for 8 min, and the liquid was removed by centrifugation. The solid was vacuum-dried at 60 °C for 6 h, heated at 130 °C for 30 min, ultrasonically cleaned with acetone, dichloromethane, and ethanol in sequence, and vacuum-dried at 60 °C for 6 h to obtain modified silica microspheres for high performance liquid chromatography.

[0026] The average particle size of the prepared modified silica microspheres for high performance liquid chromatography was 2.78 μm, and the polydispersity index was 4.32%.

[0027] Comparative Example 1: The difference between the preparation method of modified silica microspheres for high performance liquid chromatography in Comparative Example 1 and Example 2 is that steps (1), (2) and (3) are not performed, and step (6) is modified as follows: by mass, 2 parts of mesoporous core-shell silica microspheres are soaked in 1 mol / L hydrochloric acid solution for 6 h, washed with pure water until neutral, vacuum dried at 60 ° C for 12 h, dispersed in 50 parts of anhydrous toluene, ultrasonically dispersed for 30 min, and 2 parts of perfluorododecyltrichlorosilane, 0. 6 parts of triethylamine were refluxed at 95°C and 300 rpm for 24 hours, washed three times by centrifugation with anhydrous toluene, methanol, and dichloromethane, dried in vacuo at 60°C for 12 hours, and then dispersed in 50 parts of anhydrous toluene. 1 part of trimethylchlorosilane was added, and the mixture was refluxed at 95°C and 300 rpm for 24 hours. The mixture was washed three times by centrifugation with anhydrous toluene and dichloromethane, and dried in vacuo at 60°C for 12 hours to produce modified silica microspheres for high performance liquid chromatography. The remaining steps were the same as in Example 2.

[0028] The average particle size of the prepared modified silica microspheres for high performance liquid chromatography is 2.85 μm, and the polydispersity index is 3.97%.

[0029] Comparative Example 2: The difference between the preparation method of modified silica microspheres for high performance liquid chromatography in Comparative Example 2 and Example 2 lies in the difference in step (5). Step (5) is modified as follows: 1.8 parts of tridodecylmethylammonium chloride and 190 parts of pure water are mixed uniformly by mass, stirred at 250 r / min for 25 min at room temperature, 2.2 parts of non-porous silica microspheres are added, ultrasonically dispersed for 28 min, 0.15 parts of sodium carbonate and 11 parts of 28 wt% ammonia water are added at 350 r / min at room temperature, refluxed at 88°C and 350 r / min for 22 h, cooled and centrifuged at 3500 rpm for 7 min, the liquid is removed, and the mixture is centrifuged twice with anhydrous ethanol and pure water, dried under vacuum at 55°C for 9 h, and calcined at 625°C for 11 h to obtain mesoporous core-shell silica microspheres. The remaining steps are the same as in Example 2.

[0030] The average particle size of the prepared modified silica microspheres for high performance liquid chromatography was 2.86 μm, and the polydispersity index was 4.01%.

[0031] Comparative Example 3: The difference between the preparation method of modified silica microspheres for high performance liquid chromatography in Comparative Example 3 and Example 2 lies in the difference in step (5). Step (5) is modified as follows: 1.8 parts of octadecyltrimethylammonium chloride and 190 parts of pure water are mixed uniformly by mass, stirred at 250 r / min for 25 min at room temperature, 2.2 parts of non-porous silica microspheres are added, and ultrasonic dispersion is carried out for 28 min. At room temperature, 0.15 parts of sodium carbonate and 11 parts of 28 wt% ammonia water are added at 350 r / min, and the mixture is refluxed at 88°C and 350 r / min for 22 h. After cooling, the mixture is centrifuged at 3500 rpm for 7 min, the liquid is removed, and the mixture is centrifuged twice with anhydrous ethanol and pure water, dried under vacuum at 55°C for 9 h, and calcined at 625°C for 11 h to obtain mesoporous core-shell silica microspheres. The remaining steps are the same as those in Example 2.

[0032] The average particle size of the prepared modified silica microspheres for high performance liquid chromatography was 2.88 μm, and the polydispersity index was 4.11%.

[0033] Comparative Example 4: The difference between the preparation method of modified silica microspheres for high performance liquid chromatography in Comparative Example 4 and Example 2 lies in the difference in step (5). Step (5) is modified as follows: 1.2 parts of octadecyltrimethylammonium chloride, 1.38 parts of tri(dodecyl)methylammonium chloride, and 190 parts of pure water are mixed uniformly by mass, stirred at 250 r / min for 25 min at room temperature, 2.2 parts of non-porous silica microspheres are added, and ultrasonic dispersion is carried out for 28 min. At room temperature, 0.15 parts of sodium carbonate and 11 parts of 28 wt% ammonia water are added at 350 r / min, and the mixture is refluxed at 88°C and 350 r / min for 22 h. After cooling, the mixture is centrifuged at 3500 rpm for 7 min, the liquid is removed, and the mixture is centrifuged twice with anhydrous ethanol and pure water, dried under vacuum at 55°C for 9 h, and calcined at 625°C for 11 h to obtain mesoporous core-shell silica microspheres. The remaining steps are the same as those in Example 2.

[0034] The average particle size of the prepared modified silica microspheres for high performance liquid chromatography was 2.85 μm, and the polydispersity index was 4.02%.

[0035] Comparative Example 5: The difference between the preparation method of modified silica microspheres for high performance liquid chromatography in Comparative Example 5 and Example 2 lies in the difference in step (5). Step (5) is modified as follows: 1.2 parts of octadecyltrimethylammonium chloride, 0.6 parts of tri(dodecyl)methylammonium chloride, and 190 parts of pure water are mixed uniformly by mass, stirred at 250 r / min for 25 min at room temperature, 2.2 parts of non-porous silica microspheres are added, and ultrasonic dispersion is carried out for 28 min. At room temperature, 0.15 parts of sodium carbonate and 11 parts of 28 wt% ammonia water are added at 350 r / min, and the mixture is refluxed at 88°C and 350 r / min for 22 h. After cooling, the mixture is centrifuged at 3500 rpm for 7 min, the liquid is removed, and the mixture is centrifuged twice with anhydrous ethanol and pure water, dried under vacuum at 55°C for 9 h, and calcined at 625°C for 11 h to obtain mesoporous core-shell silica microspheres. The remaining steps are the same as those in Example 2.

[0036] The average particle size of the prepared modified silica microspheres for high performance liquid chromatography was 2.86 μm, and the polydispersity index was 4.07%.

[0037] Comparative Example 6: The preparation method of modified silica microspheres for high performance liquid chromatography in Comparative Example 6 differs from that in Example 2 in that step (5) is not performed, and step (6) is modified as follows: under light-shielding conditions, the hydrophobically modified diazo compound is dissolved in dichloromethane at a concentration of 0.1 g / ml, non-porous silica microspheres are added at a material-liquid ratio of 1:28 g / ml, ultrasonically treated for 5.5 min, allowed to stand for 9 min, centrifuged to remove the liquid, and the solid is vacuum-dried at 55°C for 7 h, heated at 125°C for 35 min, ultrasonically cleaned with acetone, dichloromethane, and ethanol in sequence, and vacuum-dried at 55°C for 7 h to prepare modified silica microspheres for high performance liquid chromatography. The remaining steps are the same as in Example 2.

[0038] The average particle size of the prepared modified silica microspheres for high performance liquid chromatography was 2.73 μm, and the polydispersity index was 3.88%.

[0039] Test Example 1: Basic performance test: The specific surface area and average pore size of the prepared modified silica microspheres for high performance liquid chromatography were tested using a BETA201B specific surface and pore size analyzer. The specific surface area and average pore size of the modified silica microspheres for high performance liquid chromatography were calculated using the BET method and the BJH method. Each group was tested in parallel 5 times, and the average value was recorded.

[0040] The results are shown in Table 1.

[0041] Table 1 ; From the comparison of the experimental data of Examples 1 to 3 and Comparative Examples 1 to 6 in Table 1, it can be found that the modified silica microspheres for high performance liquid chromatography prepared in the present invention have a higher specific surface area, a larger pore size and a narrower pore size distribution.

[0042] By comparing the data in the table, the data of Comparative Examples 2 and 3 show that the use of octadecyltrimethylammonium chloride and tri(dodecyl)methylammonium chloride as templates effectively expands the pore size of the surface mesoporous shell. Octadecyltrimethylammonium chloride and tri(dodecyl)methylammonium chloride synergistically form micelles with larger volumes, thereby leaving a larger pore size after calcination to remove the template. Although the specific surface area is reduced, the larger pore size allows it to be used in the separation of large molecules, expanding its application range.

[0043] By comparing the data in the table, the data of Comparative Examples 4 and 5 show that octadecyltrimethylammonium chloride and tri(dodecyl)methylammonium chloride can exert the best effect at a specific molar ratio and provide the highest pore enlargement effect. Whether increasing or decreasing the molar ratio will lead to a decrease in effect. Although the specific surface area is reduced, the larger pore size allows it to be used in the separation of large molecules, expanding the scope of application.

[0044] By comparing the data in the table, the data of Comparative Example 6 show that the surface mesoporous shell was successfully prepared. The presence of the surface mesoporous shell not only provides a large number of mesopores with larger pore sizes, but also greatly increases the specific surface area. The large specific surface area and large-pore mesopores have a good promoting effect on improving the column efficiency of high-performance liquid chromatography.

[0045] Test Example 2: Chromatographic column filling: The prepared modified silica microspheres for high performance liquid chromatography were loaded into a 75 mm × 4.6 mm ID stainless steel chromatographic column using a chromatographic column packing machine at a column filling pressure of 25 MPa using a mixture of isopropanol and methanol in a volume ratio of 1:1 as the pressurizing liquid. The loaded chromatographic column was connected to the high-pressure infusion pump of the high performance liquid chromatograph, and methanol was used as the mobile phase. The column was equilibrated at a flow rate of 0.1 ml / min for approximately 3 h until the UV detection baseline was flat.

[0046] Column pressure test: Use a mixture of acetonitrile and pure water with a volume ratio of 80:20 as the mobile phase and a flow rate of 0.8 ml / min to measure the pressure of the chromatographic column. Each group of parallel tests shall be repeated 5 times and the average value shall be recorded.

[0047] Separation performance test: The packed column was tested on an Acquity UPLC H-class high-performance liquid chromatograph connected to a UV detector for analysis. The mobile phase consisted of a mixture of acetonitrile and pure water in a 75:25 volume ratio, with a flow rate of 0.2 ml / min, a detection wavelength of 254 nm, a column temperature of 25°C, and an injection volume of 0.1 μL. Samples included uracil, nitrobenzene, toluene, and naphthalene. The maximum retention time was recorded.

[0048] The results are shown in Table 2.

[0049] Table 2 ; From the comparison of the experimental data of Examples 1 to 3 and Comparative Examples 1 to 6 in Table 2, it can be found that the modified silica microspheres for high performance liquid chromatography prepared in the present invention have lower column pressure and faster separation speed.

[0050] By comparing the data in the table, the data of Comparative Example 1 show that the use of hydrophobically modified diazo compounds to modify mesoporous core-shell silica microspheres has a better modification effect than the traditional use of silane coupling agents, and can effectively improve the separation speed.

[0051] By comparing the data in the table, the data of Comparative Examples 2 and 3 show that the use of octadecyltrimethylammonium chloride and tri(dodecyl)methylammonium chloride as templates effectively expands the pore size of the surface mesoporous shell, and the larger pore size effectively reduces the column pressure, increases the life of the chromatographic column, and increases the separation speed, resulting in better separation effect.

[0052] By comparing the data in the table, the data of Comparative Examples 4 and 5 show that octadecyltrimethylammonium chloride and tri(dodecyl)methylammonium chloride can exert the best effect at a specific molar ratio and provide the highest pore enlargement effect. Whether increasing or decreasing the molar ratio will lead to a decrease in the effect, and the largest mesopore diameter brings lower column pressure and faster separation speed.

[0053] By comparing the data in the table, the data of Comparative Example 6 show that the surface mesoporous shell was successfully prepared. The presence of the surface mesoporous shell not only provides a large number of mesopores with larger pore sizes, but also greatly increases the specific surface area. The large specific surface area and large-pore mesopores have a good improvement effect on improving the column efficiency and reducing the column pressure of high-performance liquid chromatography.

[0054] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A modified silica microsphere for high performance liquid chromatography, characterized in that: The modified silica microspheres for high performance liquid chromatography are prepared by reacting mesoporous core-shell silica microspheres with a hydrophobically modified diazo compound; The mesoporous core-shell silica microspheres are prepared by modifying non-porous silica microspheres using an etching template method; The non-porous silica microspheres are prepared by a sol-gel method; The hydrophobically modified diazo compound is prepared by catalytically decomposing a perfluorohexylhydrazone compound; The perfluorohexylhydrazone compound is prepared by reacting a perfluorohexylbenzophenone compound with hydrazine hydrate; The perfluorohexyl benzophenone compound is prepared by reacting (perfluorohexyl)benzene with isophthaloyl chloride.

2. The modified silica microspheres for high performance liquid chromatography according to claim 1, characterized in that: The etching template method is carried out using sodium carbonate as an etchant and octadecyltrimethylammonium chloride and tri(dodecyl)methylammonium chloride as template agents.

3. The modified silica microspheres for high performance liquid chromatography according to claim 1, characterized in that: The catalytic decomposition is carried out by adding manganese dioxide, anhydrous sodium sulfate and potassium hydroxide.

4. A method for preparing modified silica microspheres for high performance liquid chromatography, characterized in that: The method comprises the following preparation steps: (1) In an ice-water bath, (perfluorohexyl)benzene, anhydrous aluminum chloride, and dichloromethane are mixed evenly, stirred, and a dichloromethane solution of isophthaloyl dichloride is added dropwise. After the addition is complete, the mixture is stirred for reaction at room temperature, washed, and purified to obtain a perfluorohexyl benzophenone compound; (2) The perfluorohexyl benzophenone compound and anhydrous ethanol are mixed evenly, stirred at room temperature, hydrazine hydrate and acetic acid are added, refluxed, rotary evaporated, dichloromethane is added to dissolve again, washed, separated, dried, filtered and rotary evaporated to obtain a perfluorohexyl hydrazone compound; (3) Mix the perfluorohexylbenzophenone hydrazone compound and dichloromethane evenly, stir at room temperature, add manganese dioxide, anhydrous sodium sulfate, and potassium hydroxide, stir and react at room temperature in the dark, filter, and evaporate in the dark to obtain a hydrophobically modified diazo compound; (4) Mix potassium chloride, pure water, anhydrous ethanol, and ammonia water evenly, stir at room temperature, add ethyl orthosilicate ethanol solution dropwise, continue the reaction after the addition is complete, wash, and dry to obtain non-porous silica microspheres; (5) Octadecyltrimethylammonium chloride, tri(dodecyl)methylammonium chloride, and pure water were mixed evenly, stirred at room temperature, non-porous silica microspheres were added, ultrasonically dispersed, sodium carbonate and ammonia water were added, refluxed, cooled, centrifuged, washed, dried, and calcined to obtain mesoporous core-shell silica microspheres; (6) The hydrophobically modified diazo compound is dissolved in dichloromethane, and mesoporous core-shell silica microspheres are added, ultrasonicated, allowed to stand, centrifuged, dried, heated for reaction, ultrasonically cleaned, and dried to prepare modified silica microspheres for high performance liquid chromatography.

5. The method for preparing modified silica microspheres for high performance liquid chromatography according to claim 4, wherein: The perfluorohexyl benzophenone compound in step (1) is calculated by weight. In an ice-water bath, 0.5-0.6 parts of (perfluorohexyl) benzene, 0.3-0.35 parts of anhydrous aluminum chloride, and 25-30 parts of dichloromethane are mixed uniformly, stirred at 200-300 r / min for 20-30 min, and a 1wt% dichloromethane solution of isophthaloyl chloride and (perfluorohexyl) benzene is added dropwise at a molar ratio of 1:2 at 20-30 drops / min. After the addition is completed, the mixture is stirred at 300-400 r / min for 11-12 h at room temperature, washed with dilute hydrochloric acid solution and pure water 2-3 times, and purified to obtain the product.

6. The method for preparing modified silica microspheres for high performance liquid chromatography according to claim 4, wherein: The perfluorohexylhydrazone compound in step (2) is prepared by uniformly mixing 1 to 1.2 parts of a perfluorohexyl benzophenone compound and 20 to 25 parts of anhydrous ethanol, stirring at room temperature at 200 to 300 r / min for 20 to 30 minutes, adding 0.7 to 0.8 parts of hydrazine hydrate and 0.16 to 0.2 parts of acetic acid, and reflux reaction at 80 to 85°C and 300 to 400 r / min for 90 to 96 hours, removing the ethanol by rotary evaporation, adding 20 parts of dichloromethane to redissolve, washing with saturated brine three times, separating and removing the water layer, drying the organic layer with anhydrous magnesium sulfate, filtering and rotary evaporation to obtain the product.

7. The method for preparing modified silica microspheres for high performance liquid chromatography according to claim 4, wherein: The hydrophobically modified diazo compound in step (3) is prepared by uniformly mixing 0.4-0.5 parts of perfluorohexyl benzophenone hydrazone compound and 20-30 parts of dichloromethane, stirring at 200-300 r / min for 20-30 min at room temperature, adding 0.15-0.2 parts of manganese dioxide, 0.1-0.12 parts of anhydrous sodium sulfate, and 0.08-0.1 parts of potassium hydroxide, and reacting at room temperature in the dark at 200-300 r / min for 6-7 h, filtering, and rotary evaporation in the dark to obtain the hydrophobically modified diazo compound.

8. The method for preparing modified silica microspheres for high performance liquid chromatography according to claim 4, wherein: The non-porous silica microspheres in step (4) are prepared by uniformly mixing 0.015-0.02 parts of potassium chloride, 6-7 parts of pure water, 65-70 parts of anhydrous ethanol, and 3-4 parts of 28 wt% ammonia water, stirring at 300-400 r / min for 30-40 min at room temperature, and adding dropwise 38-40 parts of 10 wt% ethyl orthosilicate ethanol solution at a rate of 0.15-0.2 ml / min. After the addition is completed, the reaction is continued for 2-3 h, the mixture is centrifugally washed with anhydrous ethanol for 3-4 times, and vacuum dried at 50-60 ° C for 8-10 h.

9. The method for preparing modified silica microspheres for high performance liquid chromatography according to claim 4, wherein: The mesoporous core-shell silica microspheres in step (5) are prepared by mixing 1.1-1.3 parts of octadecyltrimethylammonium chloride, 0.9-1.07 parts of tridodecylmethylammonium chloride and 180-200 parts of pure water, stirring at 200-300 r / min for 20-30 min at room temperature, adding 2-2.5 parts of non-porous silica microspheres, ultrasonically dispersing for 25-30 min, and stirring at 300-400 r / min at room temperature. 0.14-0.16 parts of sodium carbonate and 10-12 parts of 28wt% ammonia water are added at r / min, and reflux reaction is carried out at 85-90°C and 300-400 r / min for 20-24 hours. After cooling, the mixture is centrifuged at 3000-4000 rpm for 6-8 minutes, the liquid is removed, and the mixture is centrifuged with anhydrous ethanol and pure water for 2-3 times respectively. The mixture is vacuum dried at 50-60°C for 8-10 hours, and calcined at 600-650°C for 10-12 hours to obtain the product.

10. The method for preparing modified silica microspheres for high performance liquid chromatography according to claim 4, wherein: The modified silica microspheres for high performance liquid chromatography in step (6) are prepared by dissolving a hydrophobically modified diazo compound in dichloromethane at a concentration of 0.1 g / ml under light-proof conditions, adding mesoporous core-shell silica microspheres at a material-liquid ratio of 1:(25-30) g / ml, ultrasonicating for 5-6 minutes, standing for 8-10 minutes, centrifuging to remove the liquid, and vacuum drying the solid at 50-60°C for 6-8 hours, heating at 120-130°C for 30-40 minutes, ultrasonically cleaning with acetone, dichloromethane, and ethanol in sequence, and vacuum drying at 50-60°C for 6-8 hours.