Multi-mode liquid chromatography filler of interpenetrating network hydrogel modified aminated silica gel as well as preparation method and application of multi-mode liquid chromatography filler

Abstract

The invention discloses a multi-mode liquid chromatography filler of interpenetrating network hydrogel modified aminated silica gel as well as a preparation method and application of the multi-mode liquid chromatography filler, and relates to the technical field of high performance liquid chromatography separation and analysis. A layer of interpenetrating network hydrogel formed by carboxymethyl chitosan and poly (N-isopropylacrylamide) is modified on the surface of aminated silica gel through a chemical polymerization technology to obtain the hydrogel. The filler provided by the invention can be used for separation and analysis of compounds with different polarities, and an interpenetrating network structure formed by two monomers enables a hydrogel modification layer to have higher mechanical strength; moreover, due to different acting sites, multi-mode (hydrophilic chromatography mode, hydrophobic chromatography mode and ion chromatography mode) separation analysis of compounds with different polarities can be realized. The preparation method provided by the invention is simple and convenient, and the prepared filler has remarkable application potential in the field of chromatographic separation and analysis.

Description

Technical Field

[0001] The present invention relates to the technical field of high performance liquid chromatography separation and analysis, and in particular to a multimodal liquid chromatography filler of an interpenetrating network hydrogel-modified amino silica gel, a preparation method thereof, and an application thereof. Background Art

[0002] To address the limited analytical range and water solubility limitations of single-retention mode chromatography, multimodal chromatography utilizes multiple mechanisms, such as hydrophobic interactions, ion exchange, and hydrophilic interactions, to synergistically separate diverse samples. This embodies the integrated utilization of the separation capabilities of each separation mode, significantly improving separation selectivity and achieving separation results not achievable by a single chromatographic mode. In recent years, with the growing demand for precise sample separations, multimodal chromatography has garnered increasing attention, and a variety of novel multimodal stationary phases have been successfully designed, prepared, and applied. Driven by the rapid advancement of liquid chromatography technology, the preparation of tunable chromatographic stationary phases has become a key research direction, and the development of multimodal stationary phases continues to flourish. However, at present, multimodal chromatography has not yet been widely applied in practical analytical processes. The selection of different functional ligands and how to enhance the synergistic effects of multiple interactions require further exploration and analysis. In particular, the development of multimodal stationary phases with enhanced selectivity for samples with diverse structural types and varying polarities remains a significant area of ​​research.

[0003] As a new type of polymer, hydrogels have attracted close attention since their introduction due to their diverse preparation methods, biocompatibility, and intelligent responsiveness. In particular, intelligent hydrogels exhibit different hydrophilicity, shrinkage and swelling properties, and volume variability under stimulus sources. Furthermore, they offer a wide variety of response types and regulatory methods, are highly adaptable, and have broad application prospects in the field of separation and analysis. However, the high swelling and high water absorption of traditional hydrogels, their poor mechanical stability, and the non-specific adsorption of the materials themselves limit their application as functional groups in multimodal chromatographic stationary phases. In order to improve the above properties of traditional hydrogels, there is a tendency to introduce new polymers, macrocyclic molecules, or new ionic liquids into the hydrogels. However, the inevitable introduction of errors due to the cumbersome experimental steps is not conducive to the precise regulation of hydrogel properties. In comparison, interpenetrating network hydrogels can effectively circumvent the above problems.

[0004] Interpenetrating network hydrogels are polymer networks formed by the entanglement of two or more independently cross-linked polymer chains. In an interpenetrating network hydrogel, each polymer chain is relatively independent, yet interdependent to form a whole. This interpenetrating network technology not only improves the performance of traditional hydrogels, but also enables the hydrogel to simultaneously possess the excellent properties of two polymers, such as temperature responsiveness, mechanical properties, and biocompatibility. Therefore, based on the differences in the chemical composition and structure of different polar compounds, the targeted design of interpenetrating network hydrogels is of great significance for achieving efficient and rapid separation and analysis of polar compounds with diverse structural types. Summary of the Invention

[0005] The purpose of the present invention is to provide a multimodal liquid chromatography filler of interpenetrating network hydrogel-modified amino silica gel that can be used for separation and analysis of compounds of different polarities, and can be applied in the field of chromatographic separation and analysis.

[0006] In order to achieve the above object, the present invention provides a method for preparing a multimodal liquid chromatography filler of an interpenetrating network hydrogel-modified amino-silica gel, comprising the following steps: S1: Take silica gel and completely disperse it in anhydrous ethanol, stir it evenly to form a suspension, then add the modifier and heat it in an inert atmosphere to react; S2: After the reaction is completed, the silica gel is washed with anhydrous ethanol by centrifugation and dried in a vacuum drying oven to obtain amino-modified silica gel; S3: Dissolve the hydrogel monomer in deionized water, stir evenly, add the crosslinker, and magnetically stir for 12-14 hours to obtain the hydrogel prepolymer solution; S4: adding the amino-modified silica gel obtained in S2 to the hydrogel prepolymer solution, ultrasonically treating the solution to obtain a suspension A, and allowing the suspension to stand; S5: Take the initiator and add it to the suspension A after standing under magnetic stirring, place it in a hot bath, and stir it after condensing and refluxing under an inert atmosphere to react; S6: After the reaction is completed, the solid product is dried under magnetic stirring to obtain a solid product, which is then soaked in deionized water, washed by centrifugation, and dried to obtain a multimodal liquid chromatography filler prepared by modifying the interpenetrating network hydrogel onto the amino-modified silica gel surface.

[0007] Preferably, the ratio of silica gel to modifier in the above S1 is 1 g:1-2 mL; the modifier is selected from any one of 3-aminopropyltriethoxysilane, N-(2-aminoethyl)-3-aminopropyltriethoxysilane, γ-aminopropyltrimethoxysilane, 2-aminoethyltriethoxysilane, and 3-aminopropyltrimethoxysilane; and the reaction conditions are: 150-300 rpm at 25-45 ° C for 3-5 h.

[0008] A more preferred preparation method, wherein the ratio of silica gel to modifier is 3g:4mL; the modifier is selected from 3-aminopropyltriethoxysilane.

[0009] Preferably, the centrifugal washing in S2 is performed 4-6 times; and the drying condition is 85-95° C. for 22-26 h.

[0010] Preferably, the hydrogel monomer in S3 is selected from carboxymethyl chitosan and N-isopropyl acrylamide, and the mass ratio thereof is 1:1, and the mass ratio of the hydrogel monomer to the silica gel in S1 is 1:15; the crosslinking agent is selected from a 25% aqueous solution of glutaraldehyde and N,N'-methylenebisacrylamide, and the ratio of the crosslinking agent to the hydrogel monomer is 2 mL / 1 g and 1 g / 10 g, respectively.

[0011] Preferably, the ultrasonication in S4 is for 30 min; and the standing is for 24-36 h at room temperature.

[0012] Preferably, the initiator in S5 is selected from any one of ammonium persulfate, azobisisobutyronitrile, hydrogen peroxide, and dichloride peroxide, and the ratio of the initiator to the silica gel is 1g:60g; the reaction conditions are: 150-300 rpm and 55-65°C for 4-6 h.

[0013] Preferably, the soaking in S6 is for 12-14 hours; the centrifugal washing is performed 4-6 times; and the drying is performed under vacuum at 85-95°C.

[0014] The present invention also provides a multimodal liquid chromatography filler prepared by the above preparation method. The prepared multimodal liquid chromatography filler can be used to prepare a liquid chromatography column. The liquid chromatography filler or liquid chromatography column can be used in the field of chromatographic separation.

[0015] The filler or chromatographic column provided by the present invention can be used for the separation and analysis of compounds of different polarities, including the separation of compounds such as nucleosides / bases, sulfonamide compounds, polycyclic aromatic hydrocarbons, aniline compounds, and anions, and can realize multi-mode (hydrophilic chromatography mode, hydrophobic chromatography mode, and ion chromatography mode) separation and analysis of compounds of different polarities.

[0016] The present invention has the following advantages: The present invention adopts chemical polymerization technology to obtain a multimodal liquid chromatography filler modified with an interpenetrating network hydrogel prepared from carboxymethyl chitosan and poly (N-isopropyl acrylamide) onto the surface of amino-modified silica gel. The preparation method is simple and can achieve efficient modification of the hydrogel on the silica gel surface.

[0017] Because the prepared hydrogel has multiple properties (such as hydrophilicity, hydrophobicity, ion exchange, etc.) and a three-dimensional network structure, the multimodal liquid chromatography filler can be used for the separation and analysis of compounds of different polarities and achieves chromatographic separation performance that is difficult to achieve with a single separation mode. It has good separation selectivity for nucleosides / bases, sulfonamide compounds, polycyclic aromatic hydrocarbons, aniline compounds, anions, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 These are SEM images of the multimodal liquid chromatography filler and bare silica gel prepared in Example 1, wherein (a) is bare silica gel and (b) is the prepared filler.

[0019] Figure 2 FT-IR images of the multimodal liquid chromatography filler prepared in Example 1, amino silica gel, and bare silica gel, where the horizontal axis is the wave number (Wavenumber (cm -1 )); The vertical axis is intensity.

[0020] Figure 3 This is a thermogravimetric diagram of the multimodal liquid chromatography filler prepared in Example 1, amino-treated silica gel, and bare silica gel, where the abscissa represents temperature (°C); the ordinate represents the remaining mass percentage of the sample (TG (%)).

[0021] Figure 4 Schematic diagram of the prepared multimodal liquid chromatography column for the separation of nucleosides / bases using a mobile phase of acetonitrile / 50 mM ammonium acetate (90 / 10, v / v), where peak 1 is thymine; peak 2 is 5-methyluridine; peak 3 is adenosine; peak 4 is cytosine; peak 5 is cytosine nucleoside; and peak 6 is guanosine.

[0022] Figure 5 Schematic diagram of the separation of sulfonamide compounds using the prepared multimodal liquid chromatography column with acetonitrile / water (92 / 8, v / v) as the mobile phase; Peak 1 is sulfadimethoxine sodium; Peak 2 is sulfaguanidine; Peak 3 is sulfamethoxazole; Peak 4 is sulfathiazole; Peak 5 is sulfamethoxazole; and Peak 6 is sulfisoxazole.

[0023] Figure 6 Schematic diagram of the prepared multimodal liquid chromatography column for separating polycyclic aromatic hydrocarbons using acetonitrile / water (20 / 80, v / v) as the mobile phase; Peak 1 is naphthalene; Peak 2 is acenaphthene; Peak 3 is phenanthrene; Peak 4 is fluoranthene; and Peak 5 is benzo(a)anthracene.

[0024] Figure 7Schematic diagram of the separation of aniline compounds using the prepared multimodal liquid chromatography column with a mobile phase of acetonitrile / water (2 / 98, v / v); Peak 1 is aniline; Peak 2 is 3-chloroaniline; Peak 3 is 2,6-diisopropylaniline; and Peak 4 is N,N'-diethylaniline.

[0025] Figure 8 Schematic diagram of the separation of anions using the prepared multimodal liquid chromatography column with a mobile phase of acetonitrile / 20 mM sodium chloride (87 / 13, v / v); Peak 1 is SCN - Peak 2 is NO3 - Peak 3 is NO2 - Peak 4 is Br - . DETAILED DESCRIPTION

[0026] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0027] Note: Unless otherwise noted, the experimental methods in the following examples are conventional methods, performed according to the techniques and conditions described in literature in the field or according to product specifications. Materials and reagents used in the following examples, unless otherwise noted, are commercially available.

[0028] Example 1 Preparation of Multimodal Liquid Chromatography Filler The preparation of multimodal liquid chromatography filler of interpenetrating network hydrogel modified with amino-coated silica gel comprises the following steps: (1) Weigh 3.0 g of silica gel and disperse it in anhydrous ethanol. The amount of ethanol used should be at least enough to stir the silica gel evenly to form a suspension. Then add 4 mL of the modifier 3-aminopropyltriethoxysilane. Transfer to an oil bath / water bath, introduce N2 for 20 min to expel the air in the system, and react at 30 °C for 4 h under magnetic stirring at 150 rpm. Other modifiers can be selected, such as N-(2-aminoethyl)-3-aminopropyltriethoxysilane; γ-aminopropyltrimethoxysilane; 2-aminoethyltriethoxysilane; 3-aminopropyltrimethoxysilane, etc., which can convert the hydroxyl modification on the silica gel surface into amino groups. The ratio of silica gel to modifier is 1 g:1-2 mL, and the preferred ratio is 3 g:4 mL. The N2 atmosphere can also be other inert gases, such as helium, argon, etc.

[0029] (2) After the reaction is completed, the mixture is washed with anhydrous ethanol by centrifugation 5 times and dried in a vacuum drying oven at 85-95 °C for 22-26 h to obtain amino-modified amino silica gel.

[0030] (3) Weigh 0.2 g of hydrogel monomer carboxymethyl chitosan and 0.2 g of hydrogel monomer N-isopropyl acrylamide and dissolve them in deionized water until fully dissolved; stir evenly and add crosslinker (select 0.4 mL of glutaraldehyde aqueous solution (25%) and 0.02 g of N,N'-methylenebisacrylamide), and stir magnetically for 12-14 h to obtain hydrogel prepolymer solution.

[0031] (4) Weigh 3.0 g of amino-coated silica gel and add it to the above hydrogel prepolymer solution. Ultrasonicate for 30 min and let it stand for 24 h to obtain suspension A.

[0032] (5) Weigh 0.05 g of initiator ammonium persulfate and add it to suspension A under magnetic stirring to obtain solution B. The obtained solution B is placed in a hot bath, and inert gas N2 is introduced to expel air from the system for 20 min. The solution is condensed and refluxed, and the temperature is raised to 60 °C under magnetic stirring at 150 rpm for reaction for 5 h. The initiator can also be selected from azobisisobutyronitrile, hydrogen peroxide, dichlorine peroxide, etc.

[0033] (6) After the reaction, the temperature was raised to 115 °C under slow magnetic stirring to evaporate the deionized water to obtain a solid product, which was added to deionized water and soaked for 12 h. The solid product was then washed five times with deionized water by centrifugation. The solid product was placed in a vacuum drying oven at 90 °C and dried for 24 h. Finally, a multimodal liquid chromatography filler prepared by modifying the interpenetrating network hydrogel on the amino-modified silica gel surface was obtained.

[0034] See also Figure 1-3 ,in Figure 1 The SEM images of the prepared multimodal liquid chromatography filler and bare silica gel, where (a) is bare silica gel and (b) is the prepared filler; it can be seen that compared with the smooth surface of bare silica gel, the surface of the prepared filler is rough, proving that the hydrogel coating has been successfully modified on the silica gel surface. Figure 2 FT-IR images of the prepared multimodal liquid chromatography filler, amino silica gel, and bare silica gel. It can be seen that compared with bare silica gel, amino silica gel has a higher -1 There is no absorption at 804 cm -1 The intensity decreases at 1508 cm -1 The absorption peak at 958 cm corresponds to the stretching vibration of the -C-N- bond in PNIPAM, while the absorption peak at 958 cm -1 The absorption peak at 30 corresponds to the stretching vibration of the COC bond in CMC, which proves that the silica gel surface is successfully modified with amino groups and the hydrogel coating is successfully modified on the silica gel surface. Figure 3 The thermogravimetric comparison of the prepared multimodal liquid chromatography packing, amino-treated silica gel and bare silica gel shows that when the temperature gradually increases to 800°C, the weight losses of the multimodal liquid chromatography packing, amino-treated silica gel and bare silica gel are 16.3 wt%, 7.5 wt% and 3.3 wt%, respectively, proving that the silica gel surface is successfully modified with amino groups and the hydrogel coating is successfully modified on the silica gel surface.

[0035] Example 2 Separation of Nucleosides / Bases Preparation of a multimodal liquid chromatography column: The multimodal liquid chromatography packing prepared in Example 1 was packed into a 150 × 4.6 mm stainless steel liquid chromatography column using a high-pressure homogenization method for the separation and analysis of nucleosides / bases (including thymine, 5-methyluridine, adenosine, cytosine, cytosine nucleoside, and guanosine). During the loading process, both the dispersing liquid and the displacement liquid were methanol, and the pressure was 40 MPa.

[0036] Chromatographic separation of nucleosides / bases: Chromatographic analysis conditions were as follows: mobile phase: acetonitrile / 50 mM ammonium acetate (90 / 10, v / v); flow rate: 1.0 mL / min; UV detector: 254 nm; injection volume: 20 µL.

[0037] The liquid chromatography diagram of nucleoside / base separation is shown in Figure 4 As shown in the figure, the analytes are: 1. Thymine; 2. 5-methyluridine; 3. Adenosine; 4. Cytosine; 5. Cytosine; 6. Guanosine. The figure demonstrates the excellent separation selectivity of this multimodal LC packing for nucleosides / bases. The column efficiency for thymine is 3358.7. Furthermore, the resolution between thymine and 5-methyluridine is R = 1.911, demonstrating successful baseline separation.

[0038] Example 3 Separation of sulfonamide compounds Preparation of a multimodal liquid chromatography column: The multimodal liquid chromatography packing prepared in Example 1 was packed into a 150 × 4.6 mm stainless steel liquid chromatography column using a high-pressure homogenization method. This column was used for the separation and analysis of sulfonamide compounds (including sulfadimethoxine sodium, sulfaguanidine, sulfamethoxazole, sulfathiazole, sulfamethoxazole, and sulfisoxazole). During the loading process, both the dispersing liquid and the displacement liquid were methanol, and the pressure was 40 MPa.

[0039] Chromatographic separation of sulfonamides: Chromatographic analysis conditions were as follows: mobile phase: acetonitrile / water (92 / 8, v / v); flow rate: 1.0 mL / min; UV detector: 254 nm; injection volume: 20 µL.

[0040] The separation liquid chromatogram of sulfonamide compounds is shown in Figure 5The analytes are: 1. Sulfadimethoxine sodium; 2. Sulfamidine; 3. Sulfadimethoxazole; 4. Sulfathiazole; 5. Sulfadimethoxine; and 6. Sulfisoxazole. The figure shows that this multimodal LC packing material exhibits excellent separation selectivity for sulfonamides. The column efficiency for sulfathiazole is 2425.1. Furthermore, the resolution between sulfadimethoxine and sulfisoxazole is R = 2.199, demonstrating successful baseline separation.

[0041] Example 4 Separation of polycyclic aromatic hydrocarbons Preparation of a multimodal liquid chromatography column: The multimodal liquid chromatography packing prepared in Example 1 was packed into a 150 × 4.6 mm stainless steel liquid chromatography column using a high-pressure homogenization method for the separation and analysis of polycyclic aromatic hydrocarbons (PAHs), including naphthalene, acenaphthene, phenanthrene, fluoranthene, and benz(a)anthracene. During the loading process, both the dispersing and displacement fluids were methanol, and the pressure was 40 MPa.

[0042] Chromatographic separation of PAHs: Chromatographic analysis conditions were as follows: mobile phase: acetonitrile / water (20 / 80, v / v); flow rate: 1.0 mL / min; UV detector: 223 nm; injection volume: 20 µL.

[0043] The liquid chromatography of polycyclic aromatic hydrocarbons is shown in Figure 6 The analytes are: 1. Naphthalene; 2. Acenaphthene; 3. Phenanthrene; 4. Fluoranthene; 5. Benzo(a)anthracene. The figure shows that this multimodal LC packing material exhibits excellent separation selectivity for polycyclic aromatic hydrocarbons. The column efficiency for acenaphthene is 2701.2. Furthermore, the resolution between fluoranthene and benz(a)anthracene is R = 1.940, demonstrating successful baseline separation.

[0044] Example 5 Separation of Aniline Compounds Preparation of a multimodal liquid chromatography column: The multimodal liquid chromatography packing prepared in Example 1 was packed into a 150 × 4.6 mm stainless steel liquid chromatography column using a high-pressure homogenization method. This column was used for the separation and analysis of aniline compounds (including aniline, 3-chloroaniline, 2,6-diisopropylaniline, and N,N'-diethylaniline). During the loading process, both the dispersing liquid and the displacement liquid were methanol, and the pressure was 40 MPa.

[0045] Chromatographic separation of aniline compounds: Chromatographic analysis conditions are as follows: mobile phase: acetonitrile / water (2 / 98, v / v); flow rate: 1.0 mL / min; UV detector: 230 nm; injection volume: 20 µL.

[0046] The liquid chromatography of aniline compounds is shown in Figure 7The analytes are: 1. Aniline; 2. 3-chloroaniline; 3. 2,6-diisopropylaniline; and 4. N,N'-diethylaniline. The figure shows that this multimodal liquid chromatography packing material exhibits excellent separation selectivity for aniline compounds. The column efficiency for aniline is 2470.1. Furthermore, the resolution R of aniline and 3-chloroaniline is 1.614, demonstrating successful baseline separation.

[0047] Example 6 Separation of Anions Preparation of multi-mode liquid chromatography column: The multi-mode liquid chromatography filler prepared in Example 1 was packed into a 150×4.6 mm stainless steel liquid chromatography column using a high pressure homogenization method for anion (including SCN - 、NO3 - 、NO2 - Br - During the filling process, both the dispersion liquid and the displacement liquid were methanol, and the pressure was 40 MPa.

[0048] Chromatographic separation of anions: Chromatographic analysis conditions were as follows: mobile phase: acetonitrile / 20 mM sodium chloride (83 / 17, v / v); flow rate: 1.0 mL / min; UV detector: 210 nm; injection volume: 20 µL.

[0049] The liquid chromatogram of anions is shown in Figure 8 Among them, the analytes are: 1. SCN - ; 2. NO3 - 3. NO2 - ;4.Br - From the figure, we can see that the multi-mode liquid chromatography filler has a good separation selectivity for anions. - The column efficiency is 2427.2. At the same time, NO2 - and Br - The resolution R = 2.268, successfully achieving baseline separation.

[0050] In summary, the present invention provides a method for preparing a multimodal liquid chromatography filler based on amino-coated silica gel modified with interpenetrating network hydrogels. The prepared filler can be used for the separation and analysis of compounds of varying polarity, including nucleosides / bases, sulfonamides, polycyclic aromatic hydrocarbons, anilines, and anions. It enables multimodal (hydrophilic, hydrophobic, and ion) separation and analysis of compounds of varying polarity. The preparation method provided by the present invention is simple to operate, and the prepared filler has significant development potential and application in the field of chromatographic separation and analysis.

[0051] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A method for preparing a multimodal liquid chromatography filler of an interpenetrating network hydrogel-modified amino-silica gel, characterized in that: The following steps are included: S1: Take silica gel and completely disperse it in anhydrous ethanol, stir it evenly to form a suspension, then add the modifier and heat it in an inert atmosphere to react; S2: After the reaction is completed, the silica gel is washed with anhydrous ethanol by centrifugation and dried in a vacuum drying oven to obtain amino-modified silica gel; S3: Dissolve the hydrogel monomer in deionized water, stir evenly, add the crosslinker, and magnetically stir for 12-14 hours to obtain the hydrogel prepolymer solution; S4: adding the amino-treated silica gel described in S2 to the hydrogel prepolymer solution, ultrasonically treating the solution to obtain a suspension A, and allowing the suspension to stand; S5: taking an initiator and adding it to the suspension A after standing under magnetic stirring, and then heating it in a hot bath. Under an inert atmosphere, the mixture is condensed and refluxed, and then stirred to react; S6: After the reaction is completed, the solid product is dried under magnetic stirring to obtain a solid product, which is then soaked in deionized water, washed by centrifugation, and dried to obtain a multimodal liquid chromatography filler prepared by modifying the interpenetrating network hydrogel onto the amino-modified silica gel surface.

2. The preparation method according to claim 1, characterized in that The ratio of silica gel to modifier in S1 is 1 g:1-2 mL; the modifier is selected from any one of 3-aminopropyltriethoxysilane, N-(2-aminoethyl)-3-aminopropyltriethoxysilane, γ-aminopropyltrimethoxysilane, 2-aminoethyltriethoxysilane, and 3-aminopropyltrimethoxysilane; the reaction conditions are: 150-300 rpm at 25-45 ° C for 3-5 h.

3. The preparation method according to claim 2, characterized in that The ratio of the silica gel to the modifier is 3 g:4 mL; the modifier is selected from 3-aminopropyltriethoxysilane.

4. The preparation method according to claim 1, characterized in that The centrifugal washing in S2 is performed 4-6 times; the drying condition is 85-95° C. for 22-26 hours.

5. The preparation method according to claim 1, characterized in that The hydrogel monomers in S3 are selected from carboxymethyl chitosan and N-isopropyl acrylamide, with a mass ratio of 1:1, and a mass ratio of 1:15 to the silica gel in S1; the crosslinking agent is selected from a 25% aqueous solution of glutaraldehyde and N,N'-methylenebisacrylamide, with a ratio of 2 mL / 1 g and 1 g / 10 g to each hydrogel monomer, respectively.

6. The preparation method according to claim 1, characterized in that The ultrasonication in S4 is for 30 min; and the standing is for 24-36 h at room temperature.

7. The preparation method according to claim 1, characterized in that The initiator in S5 is selected from any one of ammonium persulfate, azobisisobutyronitrile, hydrogen peroxide, and dichlorine peroxide, and the ratio of the initiator to the silica gel is 1g:60g; the reaction is carried out at 150-300 rpm and 55-65°C for 4-6 hours.

8. The preparation method according to claim 1, characterized in that The soaking in S6 is for 12-14 hours; the centrifugal washing is performed 4-6 times; and the drying is vacuum drying at 85-95°C.

9. A multimodal liquid chromatography filler prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the multimodal liquid chromatography filler according to claim 9 in a preparative liquid chromatography column.

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