Modified polyvinylsilsesquioxane microspheres, and methods of making and using the same

Abstract

This invention provides an amino-modified polyvinylsilsesquioxane microsphere. The vinyl groups on the surface of the polyvinylsilsesquioxane silica microsphere undergo a mercapto-olefin click addition reaction, and the basic functional groups are then immobilized on the organosilicon microsphere surface via thioether bonds. This invention also provides a method for preparing the modified polyvinylsilsesquioxane microspheres. Furthermore, this invention provides the application of the modified polyvinylsilsesquioxane microspheres in chromatographic separation. This invention uses polyvinylsilsesquioxane microspheres as the amino-based microsphere matrix, resulting in uniform particle size, a simple synthesis method, and easy scale-up. The direct amino modification of the organosilicon microsphere surface via click addition avoids the low stability of conventional amino-based silica gels.

Description

Technical Field

[0001] This invention belongs to the field of chromatographic stationary phases, specifically relating to a modified polyvinylsilsesquioxane microsphere, its preparation method, and its application. Background Technology

[0002] Pharmaceutical analysis is a class of qualitative and quantitative analyses of drugs, and is a crucial scientific and technological field related to drug quality control and new drug development. Chromatography is the most important analytical method in drug research, characterized by its superior separation capabilities and advantages such as high sensitivity, high specificity, high efficiency, and high speed, making it the most powerful tool for analyzing complex compounds. Among these methods, high-performance liquid chromatography (HPLC) is the most widely used and has become an indispensable tool in the analysis of active pharmaceutical ingredients (APIs) and their formulations. The chromatographic column packing material, as the core of HPLC, is a key factor limiting the analytical efficiency of chromatographic separation techniques. Therefore, the design, preparation, and optimization of chromatographic packing materials are crucial for the development of HPLC.

[0003] Chromatographic packing materials need to possess good physicochemical stability, regular and uniform spherical shape, and suitable pore structure. Currently, liquid chromatography packing materials are mainly divided into inorganic silica gel matrices, organic polymer matrices, and organic-inorganic hybrid matrices, with commonly used stationary phase pore sizes ranging from 6 to 50 nm. Silica gel matrices have advantages such as high mechanical strength, good thermal stability, controllable specific surface area and pore structure, abundant silanol content, and ease of chemical modification. However, their preparation process is complex, their pH tolerance range is narrow, silica gel is easily corroded by alkalis, has poor chemical stability, and the high reactivity of residual silanols can easily cause strong adsorption and tailing of basic drugs, greatly reducing their efficiency in drug analysis, especially in the separation and analysis of basic drugs.

[0004] Organic polymer matrices are suitable for a wide range of mobile phases, have high chemical stability across the entire pH range, and are easy to modify. However, they have poor pressure resistance, are prone to swelling, and the volume of the packing material can easily change depending on the proportion of organic solvent in the mobile phase. Small molecule mass transfer is slow, and compared with inorganic silica gel particles of the same particle size, the column efficiency and separation efficiency are much lower.

[0005] Organosilicon resins are organic / inorganic hybrid matrices with a unique network structure that combines the excellent properties of both organic and inorganic materials. The introduction of organic groups enhances the alkali corrosion resistance of the inorganic framework and can alter the physicochemical properties of the material, thereby giving it better stability, activity, and selectivity. Organic / inorganic hybrid matrix chromatographic packing materials significantly broaden the pH tolerance range of chromatographic columns and mitigate the tailing problem caused by alkaline compounds. Talanta 81 (2010) 824–830 (Non-Patent Literature 1) discloses a method for preparing organic / inorganic hybrid silica gel copolymerized from 1,4-bis-triethoxysilylbenzene and tetraethoxysilane; the higher the organosilicon content, the better the alkali resistance of the chromatographic column. Journal of Chromatography A, 1247 (2012) 63–70 (Non-Patent Literature 2) discloses a method for chemically bonding C18 groups directly onto the phenyl groups on the surface of phenyl-bridged hybrid silica gel, resulting in better alkali resistance of the column compared to the method of directly bonding with silane coupling agents.

[0006] Amino-functionalized silica gel is one of the most representative stationary phases in carbohydrate analysis columns. It mainly relies on the strong hydrogen bonding between the amino groups in the stationary phase and polyhydroxy compounds such as sugars and sugar alcohols, as well as polar compounds such as nucleosides, bases, and saponins, to achieve the separation of carbohydrates.

[0007] Talanta, 2003, 59(6): 1173-1188, disclosed the reasons for the short lifespan of silica-based amino-bonded chromatographic columns. Besides the common problem of column bleed in silica-based columns, the amino functional groups themselves undergo an autocatalytic reaction, which exacerbates the hydrolysis of Si-O-Si on the silica surface, leading to the loss of the bonded phase. Therefore, using more stable chromatographic packing matrix and more stable bonded stationary phase is the way to improve the stability and lifespan of amino chromatographic columns.

[0008] Polysilsesquioxane is a high-performance organosilicon resin material with advantages such as high mechanical strength, good chemical stability, and high content of organic functional groups. It is used as a chromatographic packing material, offering advantages such as simple preparation process, high column efficiency, and good compatibility with existing chromatographic instruments, showing broad application prospects. However, the microspheres prepared by existing methods have relatively small pore sizes. A method for preparing porous monodisperse mercaptopropyl-functionalized polymethylsilsesquioxane microspheres by hydrolytic co-condensation of methyltrimethoxysilane and mercaptopropyltrimethoxysilane, and further functionalizing them, is disclosed in *Ion Exchange and Adsorption*, 2018, 34 (02), 168-177 (Non-Patent Literature 4), with the microsphere pore size range of 7-8 nm; and in *Analytical Laboratory*, 2018, 37 (01). Non-patent literature 5 (79-83) discloses a method for preparing monodisperse porous vinyl-functionalized polymethylsilsesquioxane microspheres by hydrolysis and co-condensation of methyltrimethoxysilane and vinyltrimethoxysilane, wherein the pore size of the obtained microspheres is in the range of 3-4 nm; Patent application number 201610380069.0 discloses a method for preparing monodisperse porous polysilsesquioxane microspheres by hydrolysis and condensation of at least one of methyltrimethoxysilane, vinyltrimethoxysilane, mercaptopropyltrimethoxysilane and chloropropyltrimethoxysilane followed by ammonia pore formation, wherein the pore size of the porous polyvinyl / polymethylsilsesquioxane microspheres is only 4.3 nm. Summary of the Invention

[0009] The first objective of this invention is to provide a modified polyvinylsilsesquioxane microsphere with a high theoretical plate number that can be used as a stationary phase in liquid chromatography.

[0010] A second objective of this invention is to provide a method for preparing the modified polyvinylsilsesquioxane microspheres.

[0011] A third objective of this invention is to provide an application of the modified polyvinylsilsesquioxane microspheres.

[0012] This invention is achieved through the following technical solution: A modified polyvinylsilsesquioxane microsphere, wherein the vinyl groups on the surface of the polyvinylsilsesquioxane silica microsphere are bonded together after an addition reaction. or .

[0013] The method for preparing the modified polyvinylsilsesquioxane microspheres includes the following steps: S1 involves mixing vinyltrimethoxysilane and water, then adding acid to hydrolyze the vinyltrimethoxysilane. S2 is then alkali is added to carry out an alkaline thermal reaction to create pores, resulting in polyvinyl silsesquioxane microspheres; S3 is obtained by adding polyvinyl silsesquioxane microspheres to an initiator and compound 1 in an organic solvent; Compound 1 is or .

[0014] The hydrolysis temperature is 5-40℃; The hydrolysis time is 0.1-4 hours; The weight ratio of the vinyltrimethoxysilane to water is 1:0.1-20; The acid is one of formic acid, glacial acetic acid, phosphoric acid, or hydrochloric acid; The amount of acid used is 0.00005-0.005% of the water volume.

[0015] The alkali is one of sodium carbonate, triethylamine, ammonia, or pyridine; The amount of alkali used is 0.001-0.1% of the water volume.

[0016] The alkaline solution used in the alkaline-thermal reaction includes one of KOH solution, NaOH solution, ammonia water, and Ba(OH)2 solution; The concentration of the alkaline solution is 0.01-0.5 mol / L; The temperature of the alkaline thermal reaction is 40-120℃; The organic solvent used in the alkaline thermal reaction includes ethanol; The alkaline-thermal reaction takes 0.5-8 hours; The volume ratio of the organic solvent ethanol to the alkaline solution is 1:1-10.

[0017] The organic solvents used in S3 include ethanol; The mass ratio of polyvinyl silsesquioxane microspheres to organic solvent in S3 is 1:1-50; The addition reaction temperature is 40-120℃.

[0018] The initiator is one of ABVN, AIBN, DTBP, and BPO; The mass ratio of the polyvinyl silsesquioxane microspheres, the initiator, and compound 1 is 1:0.05-1:0.5-5. The modified polyvinyl silsesquioxane microspheres have a particle size of 0.5-12 μm; The modified polyvinylsilsesquioxane microspheres have a pore size of 3-45 nm.

[0019] The method for preparing the modified polyvinyl silsesquioxane microspheres further includes the steps of washing the modified polyvinyl silsesquioxane microspheres alternately with ethanol and water until the filtrate is neutral, and then drying them under reduced pressure at 60°C and baking them.

[0020] The modified polyvinylsilsesquioxane microspheres are used in the chromatographic separation of nucleoside base compounds; or Applications in the chromatographic separation of carbohydrates; or Application in the chromatographic separation of sugar alcohols; Applications in the chromatographic separation of purine compounds; or It is used for the chromatographic separation of pyrimidine substances.

[0021] The nucleoside compounds include thymidine, methyluridine, adenosine, uridine, inosine, cytidine, or guanosine.

[0022] The purine substances include adenine, theophylline, or theobromine; The pyrimidine substances include cytosine, uracil, or orotic acid; The sugars include rhamnose, fructose, glucose, sucrose, maltose, or raffinose; The sugar alcohols include glycerol, erythritol, xylitol, glucosyl alcohol, maltitol, or inositol.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: The modified polyvinylsilsesquioxane microspheres provided by this invention use polyvinylsilsesquioxane as a matrix, which has better stability than silica gel, uniform particle size, and is easy to synthesize.

[0024] The modified polyvinyl silsesquioxane microspheres provided by this invention are surface functionalized through double bonds on the surface of organosilicon spheres. Compared with conventional methods of modification using silane coupling agents (through Si-O-Si-R), the bonded phase has better stability.

[0025] The modified polyvinylsilsesquioxane microspheres provided by this invention are an amino-modified chromatographic packing material. They are widely used in sugar separation and hydrophilic separation, but a drawback is their short lifespan due to self-corrosion. This invention uses a more stable organosilicon resin and combines it with click chemistry to modify the surface, improving the stability of the separation medium. Thioethers can not only connect amine-containing organic groups to the structural units on the material surface, but the presence of both amine groups and thioethers during separation causes different interaction forces between the microspheres and the substances being separated, resulting in separation. The combined presence of both can increase the theoretical plate number of the chromatographic column. Column efficiency is mainly related to the particle size, particle size distribution, pore size, and pore structure of the packing particles. The organosilicon microspheres provided by this invention have uniform particle size, large pore size, and concentrated pore size distribution, thus exhibiting excellent column efficiency.

[0026] The modified polyvinyl silsesquioxane microspheres provided by this invention, due to the presence of thioether bonds, can enhance selectivity through coordination. The sulfur atoms in the thioether bonds can coordinate with certain metal ions or polar analytes, thereby increasing the affinity and selectivity of the stationary phase for specific compounds (such as transition metals and nitrogen-containing compounds). The thioether bonds are poorly hydrophilic, thus influencing retention behavior through hydrophobic interactions. The carbon chain portion of the thioether bonds exhibits hydrophobicity, facilitating van der Waals interactions with nonpolar or weakly polar analytes, regulating the distribution of components between the stationary and mobile phases, and thus affecting retention values. The Lewis basicity and flexibility of the thioether bonds facilitate π-π or dipole-dipole interactions with analytes, enhancing selective recognition of aromatic or polar molecules. Simultaneously, due to the presence of amine and guanidine groups, the modified polyvinyl silsesquioxane microspheres can enhance the retention of polar compounds through hydrogen bonding. Secondly, the polarity of amine and guanidine groups allows for direct dipole-dipole interactions with polar analytes, achieving selective retention of compounds such as sugars and amino acids. In an acidic mobile phase, amine groups can be protonated to form primary ammonium ions, thereby exerting a weak anion exchange effect and retaining negatively charged analytes (such as organic acids or nucleotides) through electrostatic attraction. Amine groups can also participate in π-π interactions (when the stationary phase contains aromatic structures) or dipole-dipole interactions, further enhancing the selectivity for analytes with specific functional groups. Under the combined π-π or dipole-dipole interactions of both, the modified polyvinyl silsesquioxane microspheres provided by this invention exhibit higher chromatographic separation efficiency and better performance for amphoteric organic compounds. Simultaneously, both thioether bonds and amine groups possess lone pairs of electrons; therefore, the modified polyvinyl silsesquioxane microspheres can chelate with specific groups to regulate the distribution of components between the stationary and mobile phases, improving separation efficiency. In certain configurations, such as when the guanidinium group and aromatic ring are stacked in parallel or in a T-shape, the guanidinium group, in addition to the electrostatically dominant cation-π interaction, also exhibits superimposed π-π interactions, collectively enhancing binding stability and regulating the distribution of components between the stationary and mobile phases. Although the imidazole ring of the guanidinium group is not usually directly conjugated, if the two are tightly positioned by the peptide chain and in a coplanar conformation, they can form an electronically coupled microenvironment through hydrogen bonds or salt bridges, indirectly promoting local π-electron interactions, thereby further enhancing binding stability and regulating the distribution of components between the stationary and mobile phases.

[0027] The modified polyvinylsilsesquioxane microspheres provided by this invention exhibit structural stability, resulting in a long stationary phase lifetime. This is because the high bond energy of the thioether bonds makes them less prone to breakage under conventional chromatographic operating conditions, helping to maintain the three-dimensional network structure of the stationary phase and improving its mechanical strength and durability.

[0028] This invention employs a hydrolysis-condensation method to prepare modified polyvinylsilsesquioxane microspheres. This method is simple, easily scaled up, allows for tunable pore structure, and exhibits good reproducibility and easily controllable pore structure. Attached Figure Description

[0029] Figure 1 Electron micrograph of monodisperse porous polyvinyl silsesquioxane microspheres prepared in Example 1; Figure 2 The pore size distribution curves of the microspheres prepared in Examples 1, 2, and 3 are shown. Figure 3 Pore ​​size distribution curves of microspheres prepared in Examples 1, 7, 8, and 9; Figure 4 Pore ​​size distribution curves of microspheres prepared in Examples 1 and 10; Figure 5 The pore size distribution curves of the microspheres prepared in Examples 3 and 11 are shown. Figure 6 For Example 10, microspheres were prepared and packed into a column for alkali resistance testing. Figure 7 The liquid chromatogram for separating 13 nucleoside base analytes after microsphere packing in Example 10; Figure 8 The liquid chromatogram for separating 13 nucleoside base analytes after packing microspheres into a column in Example 11 is shown below. The peaks in the figure represent the following substances: 1-theophylline, 2-theobromine, 3-uracil, 4-thymidine, 5-adenine, 6-5-methyluridine, 7-adenosine, 8-uridine, 9-cytosine, 10-inosine, 11-cytidine, 12-orotic acid, and 13-guanosine.

[0030] Figure 9 The liquid chromatogram of 12 components of steviol glycosides prepared by microsphere packing in Example 10; Figure 10 The liquid chromatogram for separating six sugars after packing microspheres into a column in Example 11 is shown below. The peaks in the figure represent the following substances: 1-rhamnose, 2-fructose, 3-glucose, 4-sucrose, 5-maltose, and 6-raffinose.

[0031] Figure 11 The image shows the liquid chromatogram of the separation of six sugar alcohols after microspheres were prepared and packed into a column in Example 11. The peaks in the image represent the following substances: 1-glycerol, 2-erythritol, 3-xylitol, 4-glucol, 5-maltitol, and 6-inositol. Detailed Implementation

[0032] Example 1 Step (1) In a 250mL round-bottom beaker, add 20.0g vinyltrimethoxysilane, 100mL pure water and 0.4μL glacial acetic acid, and stir and hydrolyze for 1h in a 15℃ water bath until the solution changes from a turbid emulsion to a clear and transparent solution. Step (2) Add 12 μL of ammonia water to (1), continue stirring until homogeneous, then stop stirring. The solution gradually changes from clear and transparent to milky white and is left to stand for 18 h. Step (3) After discarding the supernatant of the product from (2), filter it and wash it several times with anhydrous ethanol. Transfer the filter cake to a 250 mL round-bottom flask, add 50 mL of anhydrous ethanol, stir to disperse evenly, transfer to an oil bath, heat to 70ºC, add 150 mL of 0.1 mol / L NaOH solution, and heat for 2 h. Step (4) The product from (3) was filtered and washed alternately with anhydrous ethanol and pure water until the filtrate was neutral. The filter cake was transferred to a beaker and dried under reduced pressure at 60ºC for 6 hours. After drying, polyvinyl silsesquioxane microspheres were obtained. The microspheres have a particle size of 5 μm, and their specific surface area was measured to be 437 m² using a specific surface area analyzer. 2 / g, pore volume 0.92cm 3 / g, with an average pore size of 6.91nm.

[0033] Example 2 In step (1) of Example 1, 20.0g vinyltrimethoxysilane, 100mL pure water, and 0.4μL glacial acetic acid were replaced with 40.0g vinyltrimethoxysilane, 200mL pure water, and 0.8μL glacial acetic acid. In step (2), 12μL ammonia water was replaced with 24μL ammonia water. In step (3), the amounts of ethanol and NaOH solution added were replaced with 100mL and 300mL, respectively. All other steps were performed as in Example 1. The microsphere particle size was 5μm, and the specific surface area was measured to be 391m² using a surface area analyzer. 2 / g, pore volume 0.85cm 3 / g, with an average pore size of 6.79nm.

[0034] Example 3 In step (1) of Example 1, 20.0g vinyltrimethoxysilane, 100mL pure water, and 0.4μL glacial acetic acid were replaced with 80.0g vinyltrimethoxysilane, 400mL pure water, and 1.6μL glacial acetic acid. In step (2), 12μL ammonia water was replaced with 48μL ammonia water. In step (3), the amounts of ethanol and NaOH solution added were replaced with 200mL and 600mL, respectively. All other steps were performed as in Example 1. The microsphere particle size was 5μm, and the specific surface area was measured to be 400m² using a surface area analyzer. 2 / g, pore volume 0.95cm3 / g, with an average pore size of 7.34nm.

[0035] Example 4 In step (2) of Example 1, 12 μL of ammonia water was replaced with 15 μL of ammonia water, and all other steps were performed as in Example 1. The microsphere particle size was 3 μm, and the specific surface area was measured to be 393 m² using a specific surface area analyzer. 2 / g, pore volume 0.98cm 3 / g, with an average pore size of 8.03nm.

[0036] Example 5 In step (2) of Example 1, 12 μL of ammonia water was replaced with 9 μL of ammonia water, and all other steps were performed as in Example 1. The microsphere particle size was 7 μm, and the specific surface area was measured to be 367 m² using a specific surface area analyzer. 2 / g, pore volume 0.68cm 3 / g, with an average pore size of 5.93nm.

[0037] Example 6 In step (1) of Example 1, 20.0 g of vinyltrimethoxysilane was replaced with 30.0 g of vinyltrimethoxysilane, and in step (2), 12 μL of ammonia water was replaced with 8 μL of ammonia water. All other steps were performed as in Example 1. The microsphere particle size was 10 μm, and the specific surface area was measured to be 254 m² using a specific surface area analyzer. 2 / g, pore volume is 0.34cm 3 / g, with an average pore size of 4.84nm.

[0038] Example 7 In step (3) of Example 1, the amounts of ethanol and NaOH solution added were replaced with 150 mL and 50 mL, respectively. In step (3), the 2-hour heating treatment was replaced with a 1-hour heating treatment. All other steps were performed as in Example 1. The microsphere particle size was 5 μm, and the specific surface area was measured to be 480 m² using a surface area analyzer. 2 / g, pore volume 0.50cm 3 / g, with an average pore size of 3.39nm.

[0039] Example 8 In step (3) of Example 1, the heat treatment of 2 hours was replaced with heat treatment of 6 hours, and all other steps were performed as in Example 1. The microsphere particle size was 5 μm, and the specific surface area was measured to be 255 m² using a specific surface area analyzer. 2 / g, pore volume 0.85cm 3 / g, with an average pore size of 11.39nm.

[0040] Example 9 In step (3) of Example 1, the amount of ethanol and NaOH solution added was replaced with 100 mL and 100 mL respectively. In step (3), the heating treatment for 2 hours was replaced with heating treatment for 14 hours. All other steps were performed as in Example 1. The microsphere particle size was 5 μm, and the specific surface area was measured to be 170 m² using a specific surface area analyzer. 2 / g, pore volume 0.52cm 3 / g, with an average pore size of 12.77nm.

[0041] The particle size and pore structure data for the above cases are listed in the table below: Comparative analysis of the microsphere particle size and specific surface area of ​​the microspheres obtained in Examples 1, 2, and 3 shows that the polyvinyl silsesquioxane microspheres prepared by the present invention have good repeatability. Comparative analysis of the specific surface area of ​​the microspheres obtained in Examples 1, 4, 5, and 6 shows that the particle size of the polyvinyl silsesquioxane microspheres prepared by the present invention is easy to control. Comparative analysis of the specific surface area of ​​the microspheres obtained in Examples 1, 7, 8, and 9 shows that the pore structure of the polyvinyl silsesquioxane microspheres prepared by the present invention is tunable.

[0042] Example 10 Take 5g of the microspheres prepared in Example 1 and disperse them in 50mL of anhydrous ethanol. Add 0.5g of AIBN and stir until homogeneous. Add 15g of... Dissolved in 50 mL of 80 °C hot water, cooled to room temperature and added to the ethanol solution of dispersed microspheres, stirred evenly, purged with nitrogen and heated to 65 °C for 24 h, the product was washed with 80 °C hot water and ethanol and then dried to obtain the target product. The microspheres have a particle size of 5 μm, and their specific surface area was measured to be 255 m² using a specific surface area analyzer. 2 / g, pore volume 0.49cm 3 / g, with an average pore size of 7.21nm; Elemental analysis revealed that the amount of amino bonds was 4.35 mmol / g.

[0043] Example 11 Take 5g of the microspheres prepared in Example 3 and disperse them in 100 mL of anhydrous ethanol. Add 0.5g of AIBN and 15g of... After thorough stirring, the mixture was purged with nitrogen and then heated to 65℃ for 24 h. The product was washed with hot water at 80℃, then washed with ethanol, and finally dried to obtain the target product. The microspheres had a particle size of 5 μm, and the specific surface area was measured to be 252 m². 2 / g, pore volume 0.62cm 3The average pore size was 7.05 nm. Elemental analysis revealed that the amino group bonding amount was 3.99 mmol / g.

[0044] Comparing the surface area analysis data and elemental analysis data of the polyvinyl silsesquioxane microspheres obtained in Examples 1, 10, 3, and 11, it can be seen that the polyvinyl silsesquioxane microspheres prepared in this invention have good modifiability.

[0045] Example 12 The microspheres prepared in Example 10 were packed into a 150 × 4.6 id mm stainless steel column using a homogenization method. Acetonitrile and triethylamine were used as the mobile phase. After running at high pH for 600 column volumes, the performance did not change significantly. The changes are as follows: Figure 6 As shown, the amino-modified polyvinyl silsesquioxane microspheres prepared in this invention exhibit good stability under alkaline conditions.

[0046] Figure 6 The chromatographic column used was a 150×4.6 id mm stainless steel column packed with the microspheres prepared in Example 10. The liquid chromatography analysis conditions were as follows: ACN / 50mM triethylamine (pH=9.6) = 90 / 10 (v / v), flow rate 1.0 mL / min, detection wavelength 254 nm, analyte 5-methyluridine, changes in column pressure, tailing factor, column efficiency, and retention factor were recorded every 50 column volumes.

[0047] Example 13 The modified polyvinylsilsesquioxane microspheres prepared in Examples 10 and 11 were packed into 150 × 4.6 id mm stainless steel chromatographic columns using a homogenization method. Acetonitrile and ammonium formate solution were used as the mobile phase, and the separation of 13 nucleoside bases was recorded. Figure 7 , 8 As shown in the figure, the functionalized modified polyvinyl silsesquioxane microspheres exhibit good separation ability in hydrophilic liquid chromatography mode.

[0048] Figure 7 The chromatographic column used was a 150×4.6 id mm stainless steel column packed with the microspheres prepared in Example 10. The liquid chromatography analysis conditions were as follows: The mobile phase was ACN / 15 mM ammonium formate solution (pH=3.28), the flow rate was 1.0 mL / min, the detection wavelength was 254 nm, and the elution parameters were 0 min-0 min 95 / 5-85 / 15 (v / v); 20 min-40 min 85 / 15 (v / v).

[0049] Figure 8The chromatographic column used was a 150 × 4.6 id mm stainless steel column packed with the modified polyvinyl silsesquioxane microspheres prepared in Example 11. The liquid chromatography analysis conditions were as follows: The mobile phase was ACN / 15 mM ammonium formate solution (pH=3.28), the flow rate was 1.0 mL / min, the detection wavelength was 254 nm, and the elution parameters were 0 min-0 min 95 / 5-85 / 15 (v / v); 20 min-40 min 85 / 15 (v / v).

[0050] Example 14 The components of steviol glycosides were separated and analyzed using a 150×4.6 id mm stainless steel column packed with the modified polyvinyl silsesquioxane microspheres prepared in Example 10. The results are as follows: Figure 9 As shown, the functionalized modified polyvinyl silsesquioxane microspheres can achieve good separation of twelve components in steviol glycosides.

[0051] Figure 9 The chromatographic column used was a 150 × 4.6 id mm stainless steel column packed with the modified polyvinyl silsesquioxane microspheres prepared in Example 10. The liquid chromatography analysis conditions were as follows: The mobile phase was ACN / H2O, the flow rate was 1.0 mL / min, the detection wavelength was 254 nm, and the elution parameters were 0 min-20 min 80 / 20 (v / v); 20 min-40 min 80 / 20-60 / 40 (v / v); 40 min-60 min 60 / 40 (v / v).

[0052] Example 15 The microspheres prepared in Example 11 were packed into a 150 × 4.6 id mm stainless steel chromatographic column using a homogenization method. Acetonitrile and water were used as the mobile phase, and the separation of six sugars and six sugar alcohols was recorded. Figure 10 , 11 It can be seen that the functionalized modified polyvinyl silsesquioxane microspheres have good separation ability for sugars and sugar alcohols.

[0053] Figure 10 The chromatographic column used was a 150 × 4.6 id mm stainless steel column packed with the microspheres prepared in Example 11. The liquid chromatography analysis conditions were as follows: The mobile phase was ACN / H₂O = 75 / 25 (v / v), the column temperature was 40°C, the flow rate was 1.0 mL / min, and the detection wavelength was 254 nm. Elution parameters were: 0 min-20 min 70 / 30 (v / v); 20 min-40 min 70 / 30-50 / 50 (v / v); 40 min-60 min 50 / 50 (v / v).

[0054] Figure 11 The chromatographic column used was a 150 × 4.6 id mm stainless steel column packed with the modified polyvinyl silsesquioxane microspheres prepared in Example 11. The liquid chromatography analysis conditions were as follows: The mobile phase was ACN / H2O = 75 / 25 (v / v), the column temperature was 40℃, the flow rate was 1.0 mL / min, and the detection wavelength was 254 nm. Elution parameters were: 0 min-20 min 70 / 30 (v / v); 20 min-40 min 70 / 30-50 / 50 (v / v); 40 min-60 min 50 / 50 (v / v).

Claims

1. A modified polyvinylsilsesquioxane microsphere, characterized in that: The vinyl groups on the surface of the polyvinylsilsesquioxane silica microspheres, after an addition reaction, are connected to... or .

2. The method for preparing modified polyvinylsilsesquioxane microspheres as described in claim 1, characterized in that: Includes the following steps: S1 involves mixing vinyltrimethoxysilane and water, then adding acid to hydrolyze the vinyltrimethoxysilane. S2 is then alkali is added to carry out an alkaline thermal reaction to create pores, resulting in polyvinyl silsesquioxane microspheres; S3 is obtained by adding polyvinyl silsesquioxane microspheres to an initiator and compound 1 in an organic solvent; The compound 1 is or .

3. The method for preparing modified polyvinylsilsesquioxane microspheres as described in claim 2, characterized in that: The hydrolysis temperature is 5-40℃; The hydrolysis time is 0.1-4 hours; The weight ratio of the vinyltrimethoxysilane to water is 1:0.1-20; The acid is one of formic acid, glacial acetic acid, phosphoric acid, or hydrochloric acid; The amount of acid used is 0.00005-0.005% of the water volume.

4. The method for preparing modified polyvinylsilsesquioxane microspheres as described in claim 2, characterized in that: The alkali is one of sodium carbonate, triethylamine, ammonia, or pyridine; The amount of alkali used is 0.001-0.1% of the water volume.

5. The method for preparing modified polyvinylsilsesquioxane microspheres as described in claim 2, characterized in that: The alkaline solution used in the alkaline-thermal reaction includes one of KOH solution, NaOH solution, ammonia water, and Ba(OH)2 solution; The concentration of the alkaline solution is 0.01-0.5 mol / L; The temperature of the alkaline thermal reaction is 40-120℃; The organic solvent used in the alkaline thermal reaction includes ethanol; The alkaline-thermal reaction takes 0.5-8 hours; The volume ratio of the organic solvent ethanol to the alkaline solution is 1:1-10.

6. The method for preparing modified polyvinylsilsesquioxane microspheres as described in claim 2, characterized in that: The organic solvents used in S3 include ethanol; The mass ratio of polyvinyl silsesquioxane microspheres to organic solvent in S3 is 1:1-50; The addition reaction temperature is 40-120℃.

7. The method for preparing modified polyvinylsilsesquioxane microspheres as described in claim 2, characterized in that: The initiator is one of ABVN, AIBN, DTBP, and BPO; The mass ratio of the polyvinyl silsesquioxane microspheres, the initiator, and compound 1 is 1:0.05-1:0.5-5. The modified polyvinyl silsesquioxane microspheres have a particle size of 0.5-12 μm; The modified polyvinylsilsesquioxane microspheres have a pore size of 3-45 nm.

8. The method for preparing modified polyvinylsilsesquioxane microspheres as described in claim 2, characterized in that: It also includes the steps of washing the modified polyvinyl silsesquioxane microspheres alternately with ethanol and water until the filtrate is neutral, and then drying them under reduced pressure at 60°C and baking them.

9. The application of the modified polyvinylsilsesquioxane microspheres as described in claim 1, characterized in that: Applied to the chromatographic separation of nucleoside bases; or Applications in the chromatographic separation of carbohydrates; or Application in the chromatographic separation of sugar alcohols; Applications in the chromatographic separation of purine compounds; or It is used for the chromatographic separation of pyrimidine substances.

10. The application of the modified polyvinylsilsesquioxane microspheres as described in claim 9, characterized in that: The nucleoside compounds include thymidine, methyluridine, adenosine, uridine, inosine, cytidine, or guanosine. The purine substances include adenine, theophylline, or theobromine; The pyrimidine substances include cytosine, uracil, or orotic acid; The sugars include rhamnose, fructose, glucose, sucrose, maltose, or raffinose; The sugar alcohols include glycerol, erythritol, xylitol, glucosyl alcohol, maltitol, or inositol.

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