Modified silica microspheres for high performance liquid chromatography and method for preparing the same

Abstract

The application discloses modified silica microspheres for high performance liquid chromatography and a preparation method thereof, and relates to the technical field of chromatographic packing materials.In the preparation of the modified silica microspheres for high performance liquid chromatography, (perfluorohexyl) benzene is reacted with isophthaloyl chloride to obtain a perfluorohexyl benzophenone compound; the perfluorohexyl benzophenone compound is reacted with hydrazine hydrate to obtain a perfluorohexyl hydrazone compound; the perfluorohexyl hydrazone compound is catalytically decomposed to obtain a hydrophobic modified diazo compound; monodisperse non-porous silica microspheres are prepared by a sol-gel method; the non-porous silica microspheres are modified into mesoporous core-shell silica microspheres by an etching template method; and the mesoporous core-shell silica microspheres are reacted with the hydrophobic modified diazo compound to obtain the modified silica microspheres for high performance liquid chromatography.The modified silica microspheres for high performance liquid chromatography prepared by the application have the advantages of monodisperse particle size, large specific surface area, small back pressure and high column efficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chromatographic packing, in particular to modified silica microspheres for high performance liquid chromatography and a preparation method thereof. BACKGROUND

[0002] As a separation technology and method, high performance liquid chromatography (HPLC) has been developing maturely in recent years. Due to the advantages of high efficiency, rapidness and easy operation in the separation and analysis process, the application range of HPLC is becoming more and more extensive, such as pesticide residue analysis, food safety detection, production process monitoring and protein separation and purification.

[0003] As the place and tool of separation, the chromatographic column is the core part of the whole HPLC system, and its performance directly determines the separation effect. Therefore, the chromatographic column is called the heart of HPLC. The development of chromatographic packing determines the development of chromatographic column, so the preparation and optimization of chromatographic packing are always the core of the development of chromatographic technology. With the rapid development of biology and life science, various new samples and complex samples have put forward higher requirements for separation and analysis, and fast and efficient separation method has always been the goal pursued by chromatographic workers.

[0004] In recent years, the surface porous core-shell packing has gradually attracted the research enthusiasm of chromatographic workers due to its special structure and superior separation performance. The special non-porous core and mesoporous shell structure not only ensures high mechanical strength, but also greatly shortens the diffusion path of solutes, which can significantly shorten the time required for separation process. The development of core-shell packing fast separation technology makes liquid chromatography technology enter a new era of rapid analysis. Compared with the non-porous silica gel packing with the same particle size, the core-shell packing has large sample loading capacity and low column pressure. Compared with the conventional 5 μm full-porous packing, the separation speed is fast and the separation effect is good. The methods for preparing such core-shell microspheres include self-assembly method, template method, template dissolution induced re-deposition method and polymerization induced re-deposition method. However, the self-assembly method has a complicated preparation process, generally needs to be cycled for 10-15 times to reach the required particle size, has low yield and high cost. The template method and the template dissolution induced re-deposition method also have the same problem, that is, the pore size of the surface shell layer is smaller than 5-8 nm, and usually needs to be expanded twice to be used for separation and analysis of macromolecules. Although the pore size prepared by the polymerization induced re-deposition method is larger, the micropores formed are parallel to the surface of the microspheres, which increases the diffusion path and reduces the column efficiency, and the particle size distribution of the microspheres prepared by this method is wide, which needs to be further classified in particle size before being applied to chromatographic analysis.

[0005] In order to improve the separation efficiency and separation performance, the surface of the silica microspheres is usually modified by various functional groups. Commonly used modified functional groups include octadecyl, octyl, phenyl, amino, cyano and the like. However, the conventional modification process of silica is complex and time-consuming, and usually includes multiple steps such as pretreatment of silica, introduction of coupling agent and embedding of target modification reagent. In addition, the commonly used silane coupling agent in the modification process is often toxic and sensitive to water, and needs to be used under strict water-free conditions. These factors will cause environmental pollution and greatly limit its application. SUMMARY

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

[0007] In order to solve the above technical problems, the present application provides the following technical solutions:

[0008] A modified silica microsphere for high performance liquid chromatography, which is prepared by reacting mesoporous core-shell silica microspheres with a hydrophobic modified diazo compound;

[0009] The mesoporous core-shell silica microspheres are prepared by modifying non-porous silica microspheres by an etching template method;

[0010] The non-porous silica microspheres are prepared by a sol-gel method;

[0011] The hydrophobic modified diazo compound is prepared by catalytic decomposition of a perfluorohexyl thiohydroxamic compound;

[0012] The perfluorohexyl thiohydroxamic compound is prepared by reacting a perfluorohexyl benzophenone compound with hydrazine hydrate;

[0013] The perfluorohexyl benzophenone compound is prepared by reacting (perfluorohexyl) benzene with isophthaloyl chloride.

[0014] As an optimization, the etching template method uses sodium carbonate as an etchant and octadecyltrimethylammonium chloride and tridodecylmethylammonium chloride as co-template agents.

[0015] As an optimization, the catalytic decomposition is carried out by adding manganese dioxide, anhydrous sodium sulfate and potassium hydroxide.

[0016] A preparation method of a modified silica microsphere for high performance liquid chromatography, comprising the following preparation steps:

[0017] (1) according to mass fraction, 0.5~0.6 parts of (perfluorohexyl) benzene, 0.3~0.35 parts of anhydrous aluminum chloride, 25~30 parts of dichloromethane are mixed uniformly, stirred at 200~300 r / min for 20~30 min, 1wt% isophthaloyl chloride dichloromethane solution is added at 20~30 drops / min, after the addition is completed, stirring at 300~400 r / min for 11~12 h at room temperature, washed with dilute hydrochloric acid solution and pure water for 2~3 times, and the perfluorohexyl benzophenone compound is prepared after purification;

[0018] (2) according to mass fraction, 1~1.2 parts of perfluorohexyl benzophenone compound, 20~25 parts of anhydrous ethanol are mixed uniformly, stirred at 200~300 r / min for 20~30 min at room temperature, 0.7~0.8 parts of hydrazine hydrate, 0.16~0.2 parts of acetic acid are added, refluxed at 80~85℃, 300~400 r / min for 90~96 h, the ethanol is removed by rotary evaporation, 20 parts of dichloromethane is added to redissolve, washed with saturated brine for 3 times, the water layer is removed by liquid separation, the organic layer is dried with anhydrous magnesium sulfate, filtered and rotary evaporated to prepare the perfluorohexyl hydrazone compound;

[0019] (3) according to mass fraction, 0.4~0.5 parts of perfluorohexyl benzophenone hydrazone compound, 20~30 parts of dichloromethane are mixed uniformly, stirred at 200~300 r / min for 20~30 min at room temperature, 0.15~0.2 parts of manganese dioxide, 0.1~0.12 parts of anhydrous sodium sulfate, 0.08~0.1 parts of potassium hydroxide are added, stirred at 200~300 r / min for 6~7 h at room temperature in the dark, filtered, rotary evaporated in the dark to prepare the hydrophobic modified diazonium compound;

[0020] (4) according to mass fraction, 0.015~0.02 parts of potassium chloride, 6~7 parts of pure water, 65~70 parts of anhydrous ethanol, 3~4 parts of 28wt% ammonia water are mixed uniformly, stirred at 300~400 r / min for 30~40 min at room temperature, 38~40 parts of 10wt% ethyl silicate ethanol solution is added at a speed of 0.15~0.2 ml / min, after the addition is completed, the reaction is continued for 2~3 h, washed with anhydrous ethanol by centrifugation for 3~4 times, dried at 50~60℃ under vacuum for 8~10 h to prepare the non-porous silica microspheres;

[0021] (5) 1.1-1.3 parts of octadecyl trimethyl ammonium chloride, 0.9-1.07 parts of tridodecylmethylammonium chloride and 180-200 parts of pure water are mixed uniformly, stirred at 200-300 r / min for 20-30 min at room temperature, 2-2.5 parts of non-porous silica microspheres are added, ultrasonic dispersion is carried out for 25-30 min, 0.14-0.16 parts of sodium carbonate and 10-12 parts of 28 wt% ammonia water are added at 300-400 r / min at room temperature, reflux reaction is carried out at 85-90°C for 20-24 h, after cooling, centrifugation is carried out at 3000-4000 rpm for 6-8 min, the liquid is removed, and the solid is washed with anhydrous ethanol and pure water for 2-3 times, vacuum drying is carried out at 50-60°C for 8-10 h, and calcination is carried out at 600-650°C for 10-12 h, to obtain the mesoporous core-shell silica microspheres;

[0022] (6) The hydrophobic modified diazo compound is dissolved in dichloromethane at a concentration of 0.1 g / ml under light-proof conditions, the mesoporous core-shell silica microspheres are added at a material-liquid ratio of 1:(25-30) g / ml, ultrasonic dispersion is carried out for 5-6 min, standing is carried out for 8-10 min, the liquid is removed by centrifugation, the solid is vacuum dried at 50-60°C for 6-8 h, and heating is carried out at 120-130°C for 30-40 min, and the modified silica microspheres for high-performance liquid chromatography are obtained by sequentially carrying out ultrasonic cleaning with acetone, dichloromethane and ethanol and vacuum drying at 50-60°C for 6-8 h.

[0023] As optimization, the reaction process of the perfluorohexyl benzophenone compound in step (1) is as follows:

[0024] .

[0025] As optimization, the reaction process of the perfluorohexyl benzophenone compound in step (1) is as follows:

[0026] .

[0027] As optimization, the reaction process of the perfluorohexyl benzophenone compound in step (1) is as follows:

[0028] .

[0029] As optimization, the molar ratio of octadecyl trimethyl ammonium chloride and tridodecylmethylammonium chloride in step (5) is 1:2.

[0030] Compared with the prior art, the present application has the following beneficial effects:

[0031] The application is used for preparing modified silica microspheres for high performance liquid chromatography, wherein (perfluorohexyl) benzene is reacted with isophthaloyl chloride to prepare perfluorohexyl benzophenone compound; the perfluorohexyl benzophenone compound is reacted with hydrazine hydrate to prepare perfluorohexyl hydrazone compound; the perfluorohexyl hydrazone compound is catalytically decomposed to prepare hydrophobic modified diazo compound; monodisperse non-porous silica microspheres are prepared by sol-gel method; the non-porous silica microspheres are modified into mesoporous core-shell silica microspheres by etching template method; the mesoporous core-shell silica microspheres are reacted with the hydrophobic modified diazo compound to prepare modified silica microspheres for high performance liquid chromatography.

[0032] Firstly, (perfluorohexyl) benzene is subjected to Friedel-Crafts alkylation reaction in the presence of anhydrous aluminum chloride, thereby forming benzophenone compound with three benzene rings and perfluorocarbon chain, then reacted with hydrazine hydrate to convert carbonyl into hydrazone group, and finally catalytically decomposed into 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 substrate materials through hydrogen bond and electrostatic interaction, and can produce carbene group with excellent reactivity under heating condition, and react with active hydrogen on the substrate material through carbene insertion reaction to form covalent bond connection, the modification method is convenient, fast, simple and excellent compared with silane coupling agent.

[0033] Secondly, by improving the sol-gel method, monodisperse non-porous silica microspheres with a particle size of 2.5-3 μm are prepared. The conventional sol-gel method can only prepare microspheres with a particle size of less than 200 nm, and by controlling the amount of added electrolyte potassium chloride, the particle size of the prepared microspheres can be significantly increased to the micron level, meeting the particle size requirements of high performance liquid chromatography fillers; then, using sodium carbonate as an etchant and octadecyltrimethylammonium chloride and tridodecylmethylammonium chloride as co-templates, mesoporous core-shell silica microspheres are prepared. The prepared mesoporous core-shell silica microspheres have a surface mesoporous and solid core-shell structure. Under the etching action of sodium carbonate, small silica fragments are separated out, and octadecyltrimethylammonium chloride and tridodecylmethylammonium chloride, as templates, form micelles that are adsorbed on the surface of the silica microspheres by electrostatic force. The silica fragments separated by etching are captured and coated by the micelles adsorbed on the surface of the silica microspheres, and thus are again deposited on the surface of the silica microspheres. With the continuous etching and re-deposition, the structure on the surface of the non-porous silica microspheres gradually changes to a hybrid structure containing silica and micelles. After calcination to remove octadecyltrimethylammonium chloride and tridodecylmethylammonium chloride 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 a specific molar ratio of octadecyltrimethylammonium chloride and tridodecylmethylammonium chloride have the largest volume, and can form larger mesoporous pore diameters after calcination, effectively improving the separation efficiency of macromolecular substances.

[0034] 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 by electrostatic and hydrogen bonding. Then, under the action of high temperature, the diazo group is converted into a carbene group with high reactivity, which then reacts with the hydroxyl groups on the surface of the mesoporous core-shell silica microspheres through a carbene insertion reaction, thereby introducing a perfluoroalkyl chain and a benzene ring onto the surface of the silica microspheres, completing the hydrophobic modification of the silica microspheres and improving their separation efficiency and column efficiency. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of the present application.

[0036] The concentration of the dilute hydrochloric acid solution used in all the following examples and comparative examples is 0.1 mol / L.

[0037] Example 1:

[0038] A preparation method of modified silica microspheres for high performance liquid chromatography, the preparation method of modified silica microspheres for high performance liquid chromatography comprises the following preparation steps:

[0039] (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, stirred at 200 r / min for 30 min, 1wt% isophthaloyl chloride in dichloromethane was added dropwise at a rate of 20 drops / min, after the dropwise addition was completed, the reaction was stirred at room temperature at 300 r / min for 12 h, and then washed with dilute hydrochloric acid solution and pure water for 2 times respectively, and the (perfluorohexyl) benzophenone compound was prepared after purification;

[0040] (2) 1 part of the (perfluorohexyl) benzophenone compound and 20 parts of anhydrous ethanol were mixed uniformly, stirred at 200 r / min for 30 min at room temperature, 0.7 parts of hydrazine hydrate and 0.16 parts of acetic acid were added, and the reaction was carried out at 80℃ under reflux at 300 r / min for 96 h, then the ethanol was removed by rotary evaporation, 20 parts of dichloromethane was added for redissolution, washed with saturated brine for 3 times, the water layer was removed by liquid separation, the organic layer was dried with anhydrous magnesium sulfate, and then filtered and rotary evaporated to prepare the (perfluorohexyl) hydrazone compound;

[0041] (3) 0.4 parts of the (perfluorohexyl) benzophenone hydrazone compound and 20 parts of dichloromethane were mixed uniformly, stirred at 200 r / min for 30 min at room temperature, 0.15 parts of manganese dioxide, 0.1 parts of anhydrous sodium sulfate and 0.08 parts of potassium hydroxide were added, and the reaction was carried out at room temperature under light shielding and stirring at 200 r / min for 7 h, then the reaction mixture was filtered and rotary evaporated under light shielding to prepare the hydrophobic modified diazonium compound;

[0042] (4) 0.015 parts of potassium chloride, 6 parts of pure water, 65 parts of anhydrous ethanol, and 3 parts of 28wt% ammonia water were mixed uniformly, stirred at 300 r / min for 40 min at room temperature, 38 parts of 10wt% tetraethyl orthosilicate ethanol solution was added dropwise at a rate of 0.15 ml / min, after the dropwise addition was completed, the reaction was continued for 2 h, then washed with anhydrous ethanol by centrifugation for 3 times, and dried at 50℃ under vacuum for 10 h to prepare the non-porous silica microspheres;

[0043] (5) 1.1 parts of octadecyl trimethyl ammonium chloride, 0.9 parts of tridodecyl methyl ammonium chloride, 180 parts of pure water were mixed uniformly, stirred at 200 r / min for 30 min at room temperature, 2 parts of non-porous silica microspheres were added, ultrasonic dispersion was performed for 25 min, 0.14 parts of sodium carbonate, 10 parts of 28 wt% ammonia water were added at 300 r / min at room temperature, reflux reaction was performed at 85℃ for 24 h, after cooling, centrifugation was performed at 3000 rpm for 8 min, the liquid was removed, washed with anhydrous ethanol and pure water for 2 times, vacuum drying was performed at 50℃ for 10 h, calcination was performed at 600℃ for 12 h, and mesoporous core-shell silica microspheres were prepared;

[0044] (6) Under light-proof condition, the hydrophobic modified diazo compound was dissolved in dichloromethane at a concentration of 0.1 g / ml, the mesoporous core-shell silica microspheres were added at a material-liquid ratio of 1:25 g / ml, ultrasonic dispersion was performed for 5 min, standing was performed for 10 min, the liquid was removed by centrifugation, the solid was vacuum dried at 50℃ for 8 h, heated at 120℃ for 40 min, sequentially washed with acetone, dichloromethane and ethanol by ultrasonic dispersion, vacuum dried at 50℃ for 8 h, and modified silica microspheres for high performance liquid chromatography were prepared.

[0045] 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%.

[0046] Example 2:

[0047] A preparation method of modified silica microspheres for high performance liquid chromatography, the preparation method of the modified silica microspheres for high performance liquid chromatography comprises the following preparation steps:

[0048] (1) 0.55 parts of (perfluorohexyl) benzene, 0.32 parts of anhydrous aluminum chloride, 28 parts of dichloromethane were mixed uniformly in an ice water bath, stirred at 250 r / min for 25 min, 1 wt% isophthaloyl chloride dichloromethane solution was added dropwise at a molar ratio of isophthaloyl chloride to (perfluorohexyl) benzene of 1:2 at 25 drops / min, after the dropwise addition was completed, stirring was performed at 350 r / min for 11.5 h at room temperature, washed with dilute hydrochloric acid solution and pure water for 2 times respectively, and a perfluorohexyl benzophenone compound was prepared after purification;

[0049] (2) 1.1 parts of perfluorohexyl benzophenone compound, 22 parts of anhydrous ethanol were mixed uniformly, stirred at 250 r / min for 25 min at room temperature, 0.75 parts of hydrazine hydrate, 0.18 parts of acetic acid were added, reflux reaction was performed at 80℃ at 350 r / min for 93 h, ethanol was removed by rotary evaporation, redissolved with 20 parts of dichloromethane, washed with saturated brine for 3 times, removed the water layer by liquid separation, dried the organic layer with anhydrous magnesium sulfate, filtered, and rotary evaporated to prepare a perfluorohexyl hydrazine compound.

[0050] (3) 0.45 parts of perfluorohexyl benzophenone hydrazone compound, 25 parts of dichloromethane were mixed uniformly, stirred at 250 r / min for 25 min at room temperature, 0.18 parts of manganese dioxide, 0.11 parts of anhydrous sodium sulfate, 0.09 parts of potassium hydroxide were added, stirred at 250 r / min for 6.5 h at room temperature in the dark, filtered, and rotary evaporated in the dark to prepare a hydrophobic modified diazonium compound;

[0051] (4) 0.018 parts of potassium chloride, 6.5 parts of pure water, 68 parts of anhydrous ethanol, 3.5 parts of 28 wt% ammonia water were mixed uniformly, stirred at 350 r / min for 35 min at room temperature, 39 parts of 10 wt% ethyl silicate ethanol solution was added dropwise at a speed of 0.18 ml / min, and after the dropwise addition was completed, the reaction was continued for 2.5 h, washed with anhydrous ethanol by centrifugation for 3 times, and dried at 55°C under vacuum for 9 h to prepare non-porous silica microspheres;

[0052] (5) 1.2 parts of octadecyl trimethyl ammonium chloride, 0.987 parts of tridodecylmethyl ammonium chloride, 190 parts of pure water were mixed uniformly, stirred at 250 r / min for 25 min at room temperature, 2.2 parts of non-porous silica microspheres were added, ultrasonic dispersed for 28 min, 0.15 parts of sodium carbonate, 11 parts of 28 wt% ammonia water were added at 350 r / min at room temperature, and the reaction was carried out at 88°C under reflux for 22 h, after cooling, centrifuged at 3500 rpm for 7 min, the liquid was removed, washed with anhydrous ethanol and pure water for 2 times by centrifugation, dried at 55°C under vacuum for 9 h, and calcined at 625°C for 11 h to prepare mesoporous core-shell silica microspheres;

[0053] (6) The hydrophobic modified diazonium compound was dissolved in dichloromethane at a concentration of 0.1 g / ml under dark conditions, the mesoporous core-shell silica microspheres were added at a material-liquid ratio of 1:28 g / ml, ultrasonic dispersed for 5.5 min, stood for 9 min, the liquid was removed by centrifugation, the solid was dried at 55°C under vacuum for 7 h, heated at 125°C for 35 min, and sequentially washed with acetone, dichloromethane, and ethanol by ultrasonic, and dried at 55°C under vacuum for 7 h to prepare modified silica microspheres for high performance liquid chromatography.

[0054] 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%.

[0055] Example 3:

[0056] A preparation method of modified silica microspheres for high performance liquid chromatography, the preparation method of modified silica microspheres for high performance liquid chromatography comprises the following preparation steps:

[0057] (1) According to the mass fraction, 0.6 parts of (perfluorohexyl) benzene, 0.35 parts of anhydrous aluminum chloride, 30 parts of dichloromethane were mixed uniformly in an ice water bath, stirred at 300 r / min for 20 min, 1wt% isophthaloyl chloride dichloromethane solution was added at a rate of 30 drops / min, after the addition was completed, it was stirred at room temperature at 400 r / min for 11 h, washed with dilute hydrochloric acid solution and pure water for 3 times respectively, and the perfluorohexyl benzophenone compound was prepared after purification;

[0058] (2) According to the mass fraction, 1.2 parts of perfluorohexyl benzophenone compound, 25 parts of anhydrous ethanol were mixed uniformly, 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, refluxed at 85℃ at 400 r / min for 90 h, the ethanol was removed by rotary evaporation, 20 parts of dichloromethane was added for redissolution, washed with saturated brine for 3 times, the water layer was removed by liquid-liquid separation, the organic layer was dried with anhydrous magnesium sulfate, filtered and rotary evaporated to prepare the perfluorohexyl hydrazone compound;

[0059] (3) According to the mass fraction, 0.5 parts of perfluorohexyl benzophenone hydrazone compound, 30 parts of dichloromethane were mixed uniformly, stirred at room temperature at 300 r / min 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, stirred at room temperature at 300 r / min for 6 h in the dark, filtered, and rotary evaporated in the dark to prepare the hydrophobic modified diazonium compound;

[0060] (4) According to the mass fraction, 0.02 parts of potassium chloride, 7 parts of pure water, 70 parts of anhydrous ethanol and 4 parts of 28wt% ammonia water were mixed uniformly, stirred at room temperature at 400 r / min for 30 min, 40 parts of 10wt% ethyl silicate ethanol solution was added at a rate of 0.2 ml / min, after the addition was completed, it was continuously reacted for 3 h, washed with anhydrous ethanol by centrifugation for 4 times, and dried at 60℃ under vacuum for 8 h to prepare the non-porous silica microspheres;

[0061] (5) According to the mass fraction, 1.3 parts of octadecyl trimethyl ammonium chloride, 1.07 parts of tridodecylmethyl ammonium chloride and 200 parts of pure water were mixed uniformly, stirred at room temperature at 300 r / min for 20 min, 2.5 parts of non-porous silica microspheres were added, ultrasonic dispersed for 30 min, 0.16 parts of sodium carbonate and 12 parts of 28wt% ammonia water were added at room temperature at 400 r / min, refluxed at 90℃ at 400 r / min for 20 h, centrifuged at 4000 rpm for 6 min after cooling to remove the liquid, washed with anhydrous ethanol and pure water for 3 times by centrifugation, dried at 60℃ under vacuum for 8 h, and calcined at 650℃ for 10 h to prepare the mesoporous core-shell silica microspheres;

[0062] (6) Under the light-avoiding condition, the hydrophobic modified diazo compound was dissolved in dichloromethane at a concentration of 0.1 g / ml, and the mesoporous core-shell silica microspheres were added at a material-liquid ratio of 1:30 g / ml, ultrasonic treatment was performed for 6 min, and then the liquid was removed by centrifugation, the solid was vacuum dried at 60°C for 6 h, heated at 130°C for 30 min, sequentially washed with acetone, dichloromethane and ethanol by ultrasonic treatment, and vacuum dried at 60°C for 6 h, to obtain the modified silica microspheres for high performance liquid chromatography.

[0063] 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%.

[0064] Comparative Example 1

[0065] The preparation method of the modified silica microspheres for high performance liquid chromatography in Comparative Example 1 was different from that in Example 2 in that steps (1), (2) and (3) were not performed, and step (6) was modified as follows: 2 parts of mesoporous core-shell silica microspheres were 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, ultrasonic dispersed for 30 min, added with 2 parts of perfluorododecyltrichlorosilane and 0.6 parts of triethylamine, refluxed at 95°C and 300 r / min for 24 h, sequentially washed with anhydrous toluene, methanol and dichloromethane by centrifugation for 3 times, vacuum dried at 60°C for 12 h, dispersed in 50 parts of anhydrous toluene again, added with 1 part of trimethylchlorosilane, refluxed at 95°C and 300 r / min for 24 h, sequentially washed with anhydrous toluene and dichloromethane by centrifugation for 3 times, and vacuum dried at 60°C for 12 h, to obtain the modified silica microspheres for high performance liquid chromatography. The remaining steps were the same as those in Example 2.

[0066] The average particle size of the prepared modified silica microspheres for high performance liquid chromatography was 2.85 μm, and the polydispersity index was 3.97%.

[0067] Comparative Example 2

[0068] The preparation method of the modified silica microspheres for high performance liquid chromatography of Comparative Example 2 is different from that of Example 2 in that step (5) is modified as follows: 1.8 parts of tridodecylmethylammonium chloride, 190 parts of pure water are uniformly mixed, stirred at 250 r / min for 25 min at room temperature, 2.2 parts of non-porous silica microspheres are added, ultrasonic dispersion is performed for 28 min, 0.15 parts of sodium carbonate, 11 parts of 28 wt% ammonia water are added at 350 r / min at room temperature, reflux reaction is performed at 88°C at 350 r / min for 22 h, after cooling, centrifugation is performed at 3500 rpm for 7 min, the liquid is removed, washing is performed twice with anhydrous ethanol and pure water by centrifugation, vacuum drying is performed at 55°C for 9 h, and calcination is performed at 625°C for 11 h to prepare mesoporous core-shell silica microspheres. The remaining steps are the same as those of Example 2.

[0069] The average particle size of the prepared modified silica microspheres for high performance liquid chromatography is 2.86 μm, and the polydispersity index is 4.01%.

[0070] Comparative Example 3:

[0071] The preparation method of the modified silica microspheres for high performance liquid chromatography of Comparative Example 3 is different from that of Example 2 in that step (5) is modified as follows: 1.8 parts of octadecyltrimethylammonium chloride, 190 parts of pure water are uniformly mixed, stirred at 250 r / min for 25 min at room temperature, 2.2 parts of non-porous silica microspheres are added, ultrasonic dispersion is performed for 28 min, 0.15 parts of sodium carbonate, 11 parts of 28 wt% ammonia water are added at 350 r / min at room temperature, reflux reaction is performed at 88°C at 350 r / min for 22 h, after cooling, centrifugation is performed at 3500 rpm for 7 min, the liquid is removed, washing is performed twice with anhydrous ethanol and pure water by centrifugation, vacuum drying is performed at 55°C for 9 h, and calcination is performed at 625°C for 11 h to prepare mesoporous core-shell silica microspheres. The remaining steps are the same as those of Example 2.

[0072] The average particle size of the prepared modified silica microspheres for high performance liquid chromatography is 2.88 μm, and the polydispersity index is 4.11%.

[0073] Comparative Example 4:

[0074] The preparation method of the modified silica microspheres for high performance liquid chromatography of Comparative Example 4 is different from that of Example 2 in that step (5) is modified as follows: 1.2 parts by mass of octadecyltrimethylammonium chloride, 1.38 parts by mass of tridodecylmethylammonium chloride, and 190 parts of pure water are uniformly mixed, stirred at 250 r / min for 25 min at room temperature, 2.2 parts of non-porous silica microspheres are added and 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, and reflux reaction is performed at 88°C for 22 h at 350 r / min, centrifuged at 3500 rpm for 7 min after cooling, the liquid is removed, washed with anhydrous ethanol and pure water for 2 times, dried at 55°C for 9 h in vacuum, and calcined at 625°C for 11 h to prepare mesoporous core-shell silica microspheres. The remaining steps are the same as those of Example 2.

[0075] The average particle size of the prepared modified silica microspheres for high performance liquid chromatography is 2.85 μm, and the polydispersity index is 4.02%.

[0076] Comparative Example 5:

[0077] The preparation method of the modified silica microspheres for high performance liquid chromatography of Comparative Example 5 is different from that of Example 2 in that step (5) is modified as follows: 1.2 parts by mass of octadecyltrimethylammonium chloride, 0.6 parts by mass of tridodecylmethylammonium chloride, and 190 parts of pure water are uniformly mixed, stirred at 250 r / min for 25 min at room temperature, 2.2 parts of non-porous silica microspheres are added and 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, and reflux reaction is performed at 88°C for 22 h at 350 r / min, centrifuged at 3500 rpm for 7 min after cooling, the liquid is removed, washed with anhydrous ethanol and pure water for 2 times, dried at 55°C for 9 h in vacuum, and calcined at 625°C for 11 h to prepare mesoporous core-shell silica microspheres. The remaining steps are the same as those of Example 2.

[0078] The average particle size of the prepared modified silica microspheres for high performance liquid chromatography is 2.86 μm, and the polydispersity index is 4.07%.

[0079] Comparative Example 6:

[0080] The preparation method of the modified silica microspheres for high performance liquid chromatography of Comparative Example 6 is different from that of Example 2 in that step (5) is not performed, and step (6) is modified as follows: the hydrophobic modified diazonium compound is dissolved in dichloromethane at a concentration of 0.1 g / ml under light shielding, the non-porous silica microspheres are added at a material-liquid ratio of 1:28 g / ml, ultrasonic treatment is performed for 5.5 min, standing is performed for 9 min, liquid is removed by centrifugation, the solid is dried at 55°C under vacuum for 7 h, heating is performed at 125°C for 35 min, and the solid is sequentially cleaned with acetone, dichloromethane and ethanol by ultrasonic treatment, and then dried at 55°C under vacuum for 7 h to obtain the modified silica microspheres for high performance liquid chromatography. The remaining steps are the same as those of Example 2.

[0081] The average particle size of the prepared modified silica microspheres for high performance liquid chromatography is 2.73 μm, and the polydispersity index is 3.88%.

[0082] Test Example 1

[0083] Basic performance test: The specific surface area and average pore size of the prepared modified silica microspheres for high performance liquid chromatography are tested by using a BETA201B specific surface area and pore size analyzer, and the specific surface area and average pore size of the modified silica microspheres for high performance liquid chromatography are calculated by using the BET method and the BJH method. Each group is tested in parallel for 5 times, and the average value is recorded.

[0084] The results are shown in Table 1.

[0085] Table 1

[0086] ;

[0087] From the experimental data of Examples 1 to 3 and Comparative Examples 1 to 6 in Table 1, it can be found that the prepared modified silica microspheres for high performance liquid chromatography have a higher specific surface area, a larger pore size and a narrow pore size distribution.

[0088] Through comparison of the data in the table, the data of Comparative Example 2 to Comparative Example 3 show that the use of octadecyltrimethylammonium chloride and tridodecylmethylammonium chloride as co-templates effectively expands the pore size of the surface mesoporous shell. The octadecyltrimethylammonium chloride and tridodecylmethylammonium chloride cooperatively form micelles with a larger volume, so that larger pore sizes are left after the templates are removed by calcination. Although the specific surface area is reduced, the larger pore size enables it to be applied in the separation of macromolecules, thereby expanding the application range.

[0089] The data of the comparative example 4 to the comparative example 5 in the table show that octadecyl trimethyl ammonium chloride and tridodecyl methyl ammonium chloride can have the best effect at a special molar ratio, and provide the highest pore size expansion effect, and the effect will decrease whether the molar ratio is increased or the molar ratio is decreased, although the specific surface area is reduced, but the larger pore size enables it to be applied in the separation of macromolecules, and the application range is expanded.

[0090] The data of the comparative example 6 in the table show that the surface mesoporous shell is successfully prepared, and the existence of the surface mesoporous shell not only provides a large number of mesopores with large pore size, but also greatly increases the specific surface area, and the large specific surface area and the large-pore mesopore have a good promoting effect on the column efficiency of high performance liquid chromatography.

[0091] Test example 2:

[0092] Chromatographic column packing: the prepared modified silica microspheres for high performance liquid chromatography are packed into a 75mmx4.6mm I.D. stainless steel chromatographic column under a packing pressure of 25MPa by using isopropanol and methanol mixed solution with a volume ratio of 1:1 as a pressurized liquid, and the packed chromatographic column is connected with a high-pressure liquid pump of a high performance liquid chromatograph, and methanol is used as a mobile phase, and the chromatographic column is equilibrated at a flow rate of 0.1ml / min for about 3h until the ultraviolet detection baseline is flat.

[0093] Column pressure test: the mixed solution of acetonitrile and pure water with a volume ratio of 80:20 is used as a mobile phase, and the flow rate is 0.8ml / min, and the pressure of the chromatographic column is measured, and each group is tested in parallel for 5 times, and the average value is recorded.

[0094] Separation performance test: the packed chromatographic column is tested on an Acquity UPLC H-class high performance liquid chromatograph, and an ultraviolet detector is connected for analysis test, the mobile phase is a mixed solution of acetonitrile and pure water with a volume ratio of 75:25, the flow rate is 0.2ml / min, the detection wavelength is 254nm, the column temperature is 25℃, the sample amount is 0.1μL, and the sample is uracil, nitrobenzene, toluene and naphthalene. The maximum retention time is recorded.

[0095] The results are shown in table 2.

[0096] Table 2

[0097] ;

[0098] It can be found from the experimental data of the examples 1 to 3 and the comparative examples 1 to 6 in table 2 that the prepared modified silica microspheres for high performance liquid chromatography have low column pressure and fast separation speed.

[0099] By comparing the data in the table, the data of Comparative Example 1 shows that the modification of the mesoporous core-shell silica microspheres using the hydrophobic modified diazo compound has a better modification effect than the traditional use of silane coupling agent, and can effectively improve the separation speed.

[0100] By comparing the data in the table, the data of Comparative Example 2 to Comparative Example 3 shows that the use of octadecyl trimethyl ammonium chloride and tridodecyl methyl ammonium chloride as co-templates effectively expands the pore size of the surface mesoporous shell, and the larger pore size effectively reduces the column pressure, improves the column life, and improves the separation speed, and has a better separation effect.

[0101] By comparing the data in the table, the data of Comparative Example 4 to Comparative Example 5 shows that octadecyl trimethyl ammonium chloride and tridodecyl methyl ammonium chloride can have the best effect under a special molar ratio, providing the highest pore size expansion effect, and whether increasing or decreasing the molar ratio will result in a decrease in effect, and the largest mesoporous pore size brings lower column pressure and faster separation speed.

[0102] By comparing the data in the table, the data of Comparative Example 6 shows that the surface mesoporous shell is successfully prepared, and the presence of the surface mesoporous shell not only provides a large number of large-pore mesopores, but also greatly increases the specific surface area, and the large specific surface area and large-pore mesopores have a good improvement effect on improving column efficiency and reducing column pressure for high-performance liquid chromatography.

[0103] The above specific embodiments further illustrate the purpose, technical solutions and beneficial effects of the present application. It should be understood that the above description is only a specific embodiment of the present application and is not intended to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

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; The preparation of modified silica microspheres for high performance liquid chromatography 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) dissolving the hydrophobically modified diazo compound in dichloromethane, adding mesoporous core-shell silica microspheres, ultrasonicating, standing, centrifuging, drying, heating for reaction, ultrasonically cleaning, and drying to obtain modified silica microspheres for high performance liquid chromatography; The reaction process of the perfluorohexyl benzophenone compound is as follows: ； The reaction process of the perfluorohexylhydrazone compound is as follows: ； The reaction process of the hydrophobically modified diazo compound is as follows: 。 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 according to claim 1, 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.

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