PFP chromatographic filler, preparation method thereof and application of PFP chromatographic filler in liquid chromatography

Abstract

The invention belongs to the field of chromatographic fillers, and provides a PFP chromatographic filler and a preparation method and application thereof in liquid chromatogram, and the preparation method comprises the following steps: uniformly mixing NH2 (at) SiO2 and pentafluorobenzaldehyde in an organic solvent, and reacting in the presence of glacial acetic acid to obtain PFP (at) SiO2. The PFP chromatographic packing prepared by the invention can be used for liquid chromatography separation of small molecules such as various drugs, and has the advantages of room-temperature synthesis, low column pressure and wide flow velocity coverage range.

Description

Technical Field

[0001] The present invention belongs to the technical field of chromatographic fillers, and specifically relates to a preparation method of a PFP chromatographic filler and a PFP chromatographic filler obtained based on the preparation method. The present invention also provides an application of loading the PFP chromatographic filler into a chromatographic column for liquid chromatographic separation and analysis. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Liquid chromatography is an important separation and analysis tool, and is widely used in the fields of pharmaceutical analysis, environmental analysis, industrial analysis, biological analysis, etc. The filler in the chromatographic column generates differential forces with different sample molecules through modified functional groups, so that the sample molecules can achieve differential separation. Therefore, the chromatographic filler is the key to separation. How to design chromatographic fillers with different functional groups, develop low-energy consumption and low-cost chromatographic filler synthesis methods, and meet the actual application requirements of laws, regulations, industry standards, etc., are technical problems that technicians in this field have always paid attention to.

[0004] 1,2,3,4,5-pentafluorophenyl (PFP) substituents can provide hydrophobic, π-π, dipole and special FF effects provided by fluorine atoms, and have been used to prepare PFP chromatographic fillers for separating polar molecules, isomers, etc. Many well-known companies, such as Phenomenex, Waters, and Yuexu Technology, have commercialized PFP columns for sale. The preparation of liquid chromatography fillers usually involves bonding functional groups to the surface of silica microspheres, a common carrier of liquid chromatography fillers, through chemical reactions to enable them to perform separation functions. Different bonding reaction routes have different reaction efficiencies (such as the inability to 100% produce residual groups due to the predetermined reaction), different connection methods between the functional group and the silica surface (such as connecting groups, spatial distance, etc.), the introduction of other chemical groups such as spacer arms, and even the use of other composite materials (such as covalent organic framework materials, carbon dots, etc.) to connect silica carriers and functional groups. All of these factors can affect the chromatographic separation performance of the functional group to a certain extent. In addition, the use of silica carriers with different particle sizes (such as 3 microns, 5 microns, etc.) and different chromatographic column tube sizes also affects the column pressure and flow rate range of the chromatographic column operation, which in turn affects the selection of suitable instruments (such as atmospheric pressure chromatograph, ultra-high pressure chromatograph, etc.) and analytical methods.

[0005] The methods for preparing PFP chromatographic fillers disclosed in the prior art are: (1) Lin Zi'an's team at Fuzhou University synthesized a fluorinated spherical covalent organic framework as a liquid chromatography stationary phase for high-resolution separation of halogen-containing compounds and hydrophobic compounds. The prepared fluorinated COF spherical material has a small particle size. Although a small-sized column tube is used, there is still a problem of high column pressure (Analytical Chemistry, 2022, 94, 18067-18073). (2) Chen Lingxin's team at the Yantai Institute of Coastal Research, Chinese Academy of Sciences, used 1H, 1H, 2H, 2H-perfluorooctyltriethoxysilane to modify silica microspheres; the prepared liquid chromatography column showed excellent separation performance in separating highly polar compounds (such as ginsenosides, sulfonamides, nucleosides and benzoylurea pesticides). The synthetic route of this scheme is quite simple, but the types of fluorinated silanization reagents used are limited and the source is single, which limits the synthetic method to a certain extent. (Microchemical Journal, 2022, 181, 107670). (3) The Schmitz team of the University of Duisburg-Essen used pentafluorobenzoic acid monomers to generate peptide bonds at the connection point through the condensation reaction of amino and carboxyl groups, thereby obtaining the PFP stationary phase. The initial step of this synthetic route requires the use of toluene as a solvent and heating and reflux. The reaction of amino and carboxyl groups requires a metered-grade catalyst, which is used in large quantities. This chromatographic column is used to separate the differences between biphenyl isomers, nucleoside molecules and capsaicinoid molecules. Although it can be operated under low flow rates (less than 0.3 mL / min), detailed column efficiency and column pressure data are not listed, and a 2.1×150 mm column tube is used, resulting in an injection volume of no more than 5 microliters (usually the liquid chromatography injection volume is 10-20 microliters). (Journal of Chromatography A, 2024, 1717, 464688). (4) Jinan Huadao New Materials Technology Co., Ltd. uses fluorinated unsaturated olefin monomers and divinylbenzene crosslinkers as raw materials and adopts a relatively complex precipitation polymerization method to synthesize fluorinated polymer microspheres for chromatographic stationary phases. It can separate fluorinated compounds under lower column pressure conditions, but lacks more detailed application data to determine the separation object (CN 115340622A). (5) Chen Lingxin's team has also developed a fluorinated embedded carbon dot stationary phase, which has perfect chromatographic performance in separating pesticides, nucleosides, sulfonamides, alkaloids and alkylbenzenes (Analytical Chemistry, 2024, 96, 16590-16598).(6) The inventor's research group introduced PFP groups into covalent organic framework materials (COF) and modified fluorinated COF onto the surface of silica microspheres to prepare fluorinated COF fillers for separating drugs such as alkaloids, phenolic acids and antiviral drugs. Although the column pressure of this scheme is low, the separation performance such as column efficiency does not meet the requirements of the pharmacopoeia for drug separation. (Microchemical Journal, 2025, 208, 112513). Among them, the prior art (5) and the prior art (6) (numbered the same as the number used when listing the synthesis method) respectively use carbon dots and COF to connect PFP groups and silica carriers, and the raw materials used are complex. In the aforementioned synthesis methods, the prior art (1) and (6) use room temperature synthesis methods, and the rest involve reflux, heating and other operations during the preparation process. Based on the above analysis, the prior art still lacks a room temperature synthesis method that can regulate the separation performance of PFP chromatographic fillers to meet the actual application requirements, so as to efficiently and conveniently complete the synthesis of PFP fillers. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a method for synthesizing PFP chromatographic filler at room temperature, and PFP chromatographic filler prepared by the method, which is used for liquid chromatography separation of various drugs and other small molecules, and the operating parameters of the chromatographic column are evaluated in detail and given.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides a method for preparing a PFP chromatographic filler, comprising:

[0009] NH2@SiO2 and pentafluorobenzaldehyde are uniformly mixed in an organic solvent and reacted in the presence of glacial acetic acid to obtain PFP@SiO2.

[0010] Different monomers have different prices, and the resulting connecting groups are also different, which affects the final chromatographic performance. In order to achieve the separation of antiviral drugs, phenolic acid drugs, and flavonoid drugs, the present invention has selected pentafluorobenzaldehyde monomers after systematic research and large-scale experimental exploration, and generates a C=N double bond at the connection point through the condensation reaction of the amino group and the aldehyde group. Therefore, in some embodiments, the mass volume ratio of the NH2@SiO2, pentafluorobenzaldehyde and glacial acetic acid is 2g:2-3mL:0.01mL.

[0011] In some embodiments, the reaction temperature is room temperature;

[0012] In some embodiments, the reaction time is 48 hours or more.

[0013] The reaction solvent affects the reaction efficiency. The present invention uses methanol as a solvent to carry out the reaction at room temperature, which has lower energy consumption and the toxicity of methanol is much lower than that of toluene. In some embodiments, the organic solvent is methanol;

[0014] In some embodiments, the volume ratio of methanol to pentafluorobenzaldehyde is 15-20:1.

[0015] In some embodiments, after the reaction is completed, PFP@SiO2 is washed with methanol to remove residual reaction reagents and then dried.

[0016] The second aspect of the present invention provides a PFP chromatographic filler prepared by the above method.

[0017] The third aspect of the present invention provides a PFP chromatographic column filled with the above-mentioned PFP chromatographic filler. Different column sizes and filling pressures result in different chromatographic column pressures. The present invention has the advantage of low column pressure and can adapt to more liquid phase instruments.

[0018] In some embodiments, a 4.6*50 mm column tube is used, and the injection volume is 1-20 μL. Different column tubes have different effects, especially injection volume, and the present invention has a wider injection volume range.

[0019] A fourth aspect of the present invention provides a method for packing a PFP chromatographic column, comprising:

[0020] Dispersing the above-mentioned PFP chromatographic filler into the mixed solution to obtain a dispersion of the filler;

[0021] Methanol is used as a displacement liquid to pump the dispersion of the filler into an empty chromatographic column tube at a preset pressure, and then the sieve plate and the column tube are installed to obtain a PFP chromatographic column.

[0022] In some embodiments, the mass volume ratio of the PFP chromatographic filler to the mixed solution is 0.8 g: 50-60 mL;

[0023] In some embodiments, the mixed solution consists of methanol and isopropanol, and the volume ratio of the two is 9:1-1.5;

[0024] In some embodiments, the preset pressure is 45-50 MPa.

[0025] The fourth aspect of the present invention provides the use of the above-mentioned PFP chromatographic filler and the above-mentioned PFP chromatographic column in the detection of antiviral drugs, phenolic acid drugs, and flavonoid drugs.

[0026] Beneficial effects of the present invention

[0027] (1) The present invention uses pentafluorobenzaldehyde monomers to generate a C=N double bond at the connection point through a condensation reaction between an amino group and an aldehyde group, thereby realizing the room temperature synthesis of PFP chromatographic filler. At the same time, the present invention has the advantages of low column pressure and a wide flow rate coverage range. When detecting antiviral drugs, phenolic acid drugs, and flavonoid drugs, the column efficiency meets the pharmacopoeia separation requirements.

[0028] (2) The method of the present invention is simple, practical and easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0030] Figure 1 The structure of the PFP chromatographic filler (A) and the scanning electron microscope energy spectrum analysis result (B);

[0031] Figure 2 It is the column pressure test result of PFP chromatographic column;

[0032] Figure 3 The effect of mobile phase ratio on the chromatographic separation of antiviral drugs;

[0033] Figure 4 The chromatogram of antiviral drug separation when the mobile phase is acetonitrile: 0.1% formic acid aqueous solution = 3:97;

[0034] Figure 5 The effect of flow rate on the chromatographic separation of antiviral drugs;

[0035] Figure 6 The effect of column temperature on the chromatographic separation of antiviral drugs;

[0036] Figure 7 The effect of injection volume on the chromatographic separation of antiviral drugs;

[0037] Figure 8 The influence of sample solvent on the chromatographic separation of antiviral drugs;

[0038] Fig. 9 The effect of mobile phase ratio on the chromatographic separation of phenolic acid drug molecules;

[0039] Fig.10 The chromatogram of separation of phenolic acid drug molecules when the mobile phase is acetonitrile: 0.1% formic acid aqueous solution = 3:97;

[0040] Fig.11 The effect of flow rate on the chromatographic separation of phenolic acid drug molecules;

[0041] Fig.12The effect of column temperature on the chromatographic separation of phenolic acid drug molecules;

[0042] Fig.13 The effect of injection volume on the chromatographic separation of phenolic acid drug molecules;

[0043] Fig.14 The effect of sample solvent on the chromatographic separation of phenolic acid drug molecules;

[0044] Fig.15 The effect of mobile phase ratio on the chromatographic separation of flavonoids;

[0045] Fig.16 The chromatogram of the separation of flavonoid drug molecules when the mobile phase is acetonitrile: 0.1% formic acid aqueous solution = 3:97;

[0046] Fig.17 The effect of flow rate on the chromatographic separation of flavonoids molecules;

[0047] Fig.18 The effect of column temperature on the chromatographic separation of flavonoids molecules;

[0048] Fig.19 The effect of injection volume on the chromatographic separation of flavonoids;

[0049] Fig. 20 The effect of sample solvent on the chromatographic separation of flavonoids;

[0050] Fig.21 is the chromatogram of deuterated benzoic acid;

[0051] Fig. 22 is a chromatogram of continuous operation;

[0052] Fig.23 This is the chromatogram of the actual sample Sanguisorba officinalis charcoal. DETAILED DESCRIPTION

[0053] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0054] The present invention will be further described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0055] Example 1

[0056] Preparation of PFP chromatographic filler (PFP@SiO2):

[0057] The synthesis of PFP@SiO2 includes three main steps: (1) Activation of silica microspheres: 20 g of silica microspheres (particle size 3 μm, pore size 10 nm) were added to 100 mL of 10% (v / v) hydrochloric acid solution, and the solution was stirred at room temperature for 48 h. The activated silica microspheres were then washed with water until the washing solution was neutral. The silica microspheres were then washed with methanol to remove residual moisture and dried at 65 °C to obtain activated silica microspheres. (2) Preparation of amino-modified silica microspheres (NH2@SiO2): 10 g of activated silica microspheres were added to 50 mL of a methanol solution of an aminosilylating agent ((3 -aminopropyl)trimethoxysilane, concentration 40% (v / v)), the solution was stirred at room temperature for 48 hours, and then NH2@SiO2 was washed with methanol to remove the residual reaction reagents, and dried at 65°C to obtain NH2@SiO2; (3) Preparation of PFP@SiO2: 2.0 g of NH2@SiO2 and 2 mL of pentafluorobenzaldehyde were added to 30 mL of methanol and mixed evenly, and then 10 μL of glacial acetic acid was added as a catalyst, and the reaction system was stirred at room temperature for 48 hours. After the reaction was completed, PFP@SiO2 was washed with methanol to remove the residual reaction reagents, and dried at 65°C to obtain PFP@SiO2, the structure of which is shown in FIG. Figure 1 As shown in A.

[0058] Example 2

[0059] PFP column filling (homogenization method):

[0060] 0.8 g of PFP@SiO2 filler was fully dispersed in 50 mL of a mixed solution (methanol: isopropanol = 9:1, v / v) to obtain a dispersion of the filler, and then methanol was used as a displacement liquid to pump the dispersion of the filler into an empty chromatographic column tube (4.6×50 mm) at a pressure of 45 MPa, and then the sieve plate and column tube were installed to obtain a PFP chromatographic column; the loaded PFP chromatographic column was eluted with methanol until the baseline at a detection wavelength of 220 nm was stable, and then the chromatographic column was filled with methanol and sealed for storage; before conducting any chromatographic experiment, the chromatographic column was equilibrated with an appropriate mobile phase.

[0061] Example 3

[0062] Characterization of chromatographic packing materials:

[0063] The prepared PFP@SiO2 filler contains fluorine element through scanning electron microscopy. Figure 1B, proves the feasibility of the synthetic route. The column pressure of the chromatographic column (the size of the test chromatographic column: 4.6×50mm) was tested at different flow rates using methanol and acetonitrile, the organic solvent mobile phases commonly used in liquid chromatography. The results are shown in Figure 2 , indicating that the column pressure of the chromatographic column filled with the prepared PFP filler is low, and it can be tested at a variety of common flow rates. It is suitable for a variety of liquid chromatography instruments such as low pressure, medium and high pressure, and has a wide range of applications.

[0064] Example 4

[0065] Chromatographic separation performance for antiviral drugs:

[0066] Column size: 4.6×50 mm; Test samples: 1, ganciclovir, 2, acyclovir, 3, favipiravir, Sample concentration: 50 μg / mL (prepare 2 mg / mL sample solution with methanol, then dilute with water, dilute with methanol, water, and acetonitrile respectively when testing solvent effect); Mobile phase: (A) acetonitrile, (B) 0.1% formic acid aqueous solution; Injection volume: 20 μL (when examining the effect of injection volume, the actual injection volume shall prevail); Column temperature: 30°C (when examining the effect of column temperature, the actual column temperature shall prevail); Flow rate: 0.3 mL / min (when examining the effect of flow rate, the actual flow rate shall prevail); Detection wavelength: 232 nm; Chromatographic performance under different mobile phase ratios is shown in Figure 3 (with acetonitrile ratio as the horizontal axis), wherein the chromatogram when the mobile phase is acetonitrile: 0.1% formic acid aqueous solution = 3:97 is shown in Figure 4 ; Chromatographic performance at different flow rates is shown in Figure 5 (Expressed by Van Deemter equation, HETP means theoretical plate number, other conditions are the same Figure 4 ), where the column efficiency is greater than 4500, meeting the requirements of the pharmacopoeia for chromatographic column efficiency (usually requiring chromatographic column efficiency to be greater than 2500-3000); the effect of different column temperatures on the chromatographic separation of antiviral drugs can be found in Figure 6 (Graphically expressed using Van't Hoff's law, other conditions are the same Figure 4 ), the test compounds can be well separated in the common column temperature range (25-40℃) with high column efficiency; the effect of different injection volumes on the chromatographic separation of antiviral drugs can be seen in Figure 7 , showing a linear relationship within the common injection volume range (1-20 μL), meeting the needs of different injection volumes; the solvent effect of different sample solvents (methanol, acetonitrile and water) on the chromatographic separation of antiviral drugs is shown in Figure 8,The results showed that methanol and water could be used directly for sample preparation and had no significant effect on chromatographic separation, while acetonitrile had no effect on the chromatographic retention of favipiravir and produced a significant solvent effect on ganciclovir and acyclovir.

[0067] Example 5

[0068] Chromatographic separation performance for phenolic acid drug molecules:

[0069] Column size: 4.6×50 mm; Test samples: 1. gallic acid, 2. ferulic acid, 3. isoferulic acid; Sample concentration: 50 μg / mL (prepare 2 mg / mL sample solution with methanol, then dilute with water; dilute with methanol, water, and acetonitrile respectively when testing solvent effect); Mobile phase: (A) acetonitrile, (B) 0.1% formic acid aqueous solution; Injection volume: 20 μL (the actual injection volume shall prevail when examining the effect of injection volume); Column temperature: 30°C (the actual column temperature shall prevail when examining the effect of column temperature); Flow rate: 0.3 mL / min (the actual flow rate shall prevail when examining the effect of flow rate); Detection wavelength: 280 nm; Chromatographic performance under different mobile phase ratios is shown in Fig. 9 (with acetonitrile ratio as the horizontal axis), wherein the chromatogram when the mobile phase is acetonitrile: 0.1% formic acid aqueous solution = 80:20 is shown in Fig.10 ; Chromatographic performance at different flow rates is shown in Fig.11 (Expressed by Van Deemter equation, HETP means theoretical plate number, other conditions are the same Fig.10 ), where the column efficiency is greater than 4500, meeting the requirements of the pharmacopoeia for chromatographic column efficiency (usually requiring chromatographic column efficiency to be greater than 2500-3000); the effect of different column temperatures on the chromatographic separation of phenolic acid drug molecules can be found in Fig.12 (Graphically expressed using Van't Hoff's law, other conditions are the same Fig.10 ), the test compounds can be well separated in the common column temperature range (25-40℃), and the column efficiency is high; the effect of different injection volumes on the chromatographic separation of phenolic acid drug molecules can be seen in Fig.13 , showing a linear relationship within the common injection volume range (1-20μL), meeting the needs of different injection volumes (other conditions are the same Fig.10 ); The solvent effects of different sample solvents (methanol, acetonitrile and water) on the chromatographic separation of phenolic acid drugs are shown in Fig.14 ,The results showed that methanol, water and acetonitrile could be directly used for sample preparation, ,with no significant effect on chromatographic separation.

[0070] Example 6

[0071] Chromatographic separation performance of flavonoid drug molecules:

[0072] Column size: 4.6×50 mm; Test samples: 1, neohesperidin, 2, naringenin, 3, hesperidin, Sample concentration: 50 μg / mL (prepare 2 mg / mL sample solution with methanol, then dilute with water, dilute with methanol, water, and acetonitrile respectively when testing solvent effect); Mobile phase: (A) acetonitrile, (B) 0.1% formic acid aqueous solution; Injection volume: 20 μL (when examining the effect of injection volume, the actual injection volume shall prevail); Column temperature: 30°C (when examining the effect of column temperature, the actual column temperature shall prevail); Flow rate: 0.3 mL / min (when examining the effect of flow rate, the actual flow rate shall prevail); Detection wavelength: 280 nm; Chromatographic performance under different mobile phase ratios is shown in Fig.15 (with acetonitrile ratio as the horizontal axis), wherein the chromatogram when the mobile phase is acetonitrile: 0.1% formic acid aqueous solution = 80:20 is shown in Fig.16 ; Chromatographic performance at different flow rates is shown in Fig.17 (Expressed by Van Deemter equation, HETP means theoretical plate number, other conditions are the same Fig.16 ), where the column efficiency is greater than 4500, meeting the requirements of the pharmacopoeia for chromatographic column efficiency (usually requiring chromatographic column efficiency to be greater than 2500-3000); the effect of different column temperatures on the molecular chromatographic separation of flavonoid drugs can be found in Fig.18 (Graphically expressed using Van't Hoff's law, other conditions are the same Fig.16 ), the test compounds can be well separated in the common column temperature range (25-40℃), and the column efficiency is high; the effect of different injection volumes on the molecular chromatographic separation of flavonoid drugs can be seen in Fig.19 , showing a linear relationship within the common injection volume range (1-20μL), meeting the needs of different injection volumes (other conditions are the same Fig.16 ); the solvent effects of different sample solvents (methanol, acetonitrile and water) on the chromatographic separation of flavonoid drugs are shown in Fig. 20 The results showed that methanol and water could be used directly for sample preparation and had no significant effect on chromatographic separation, while acetonitrile produced a solvent effect on flavonoid molecules, causing the chromatographic peaks to deform.

[0073] Example 7

[0074] Chromatographic retention of deuterated reagents:

[0075] Column size: 4.6×50 mm; Test sample: deuterated benzoic acid, sample concentration: 50 μg / mL (prepare 2 mg / mL sample solution with methanol, then dilute with water); Mobile phase: (A) acetonitrile: (B) 0.1% formic acid aqueous solution = 45:55; Injection volume: 5 μL; Column temperature: 30°C; Flow rate: 0.3 mL / min; Detection wavelength: 220 nm; Results are as follows Fig.21 , indicating that the prepared chromatographic column has good retention capacity for deuterated reagents and can be used for the analysis of deuterated reagents.

[0076] Example 8

[0077] Stability test of chromatographic separation performance:

[0078] Column size: 4.6×50 mm; Test samples: 1. Gallic acid 2. Ferulic acid 3. Isoferulic acid; Sample concentration: 50 μg / mL (prepare 2 mg / mL sample solution with methanol and dilute with water); Mobile phase: (A) acetonitrile, (B) 0.1% formic acid aqueous solution, mobile phase ratio: acetonitrile: 0.1% formic acid aqueous solution = 80:20; Injection volume: 20 μL; Column temperature: 30°C; Flow rate: 0.3 mL / min; Detection wavelength: 280 nm; Number of runs: 6. See the chromatogram of continuous run. Fig. 22 The statistical results of retention time and peak area are shown in Table 1. The RSD value is small, which proves that the chromatographic column has good stability.

[0079] Table 1 RSD statistical results of retention time and peak area

[0080] Analytes Retention time RSD / % Peak area RSD / % gallic acid 0.2229 0.9341 Ferulic acid 0.5431 0.2905 Isoferulic acid 0.6051 1.4616

[0081] Example 9

[0082] Chromatographic separation performance for complex samples:

[0083] Chromatographic column size: 4.6×50mm; Test sample: Sanguisorba officinalis charcoal extract, sample preparation method: put 0.2g Sanguisorba officinalis charcoal sample powder into a 50mL centrifuge tube, add 25mL methanol to dissolve, shake well, seal, and extract in an ultrasonic bath for 1 hour. After extraction, cool to room temperature, weigh, add methanol to make up for weight loss, mix well, filter, collect the filtrate, and obtain the blood charcoal test solution, which is stored at 4°C for future use. Mobile phase: (A) acetonitrile, (B) 0.1% formic acid aqueous solution; gradient (calculated as the volume percentage change of A): 0-2min from 5% to 10%, 2-6min from 10% to 50%, 6-10min from 50% to 60%, 10.1-15min maintained at 5%; injection volume: 5μL; column temperature: 30°C; flow rate: 0.3mL / min; detection wavelength: 280nm; chromatogram see Fig.23 , proving that the column is capable of separating complex samples in gradient mode.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a PFP chromatographic filler, characterized in that: include: NH2@SiO2 and pentafluorobenzaldehyde are uniformly mixed in an organic solvent and reacted in the presence of glacial acetic acid to obtain PFP@SiO2.

2. The method for preparing the PFP chromatographic filler according to claim 1, characterized in that: The mass volume ratio of the NH2@SiO2, pentafluorobenzaldehyde and glacial acetic acid is 2g:2-3mL:0.01mL.

3. The method for preparing the PFP chromatographic filler according to claim 1, characterized in that: The reaction temperature is room temperature; Or, the reaction time is 48 hours or more.

4. The method for preparing the PFP chromatographic filler according to claim 1, characterized in that: The organic solvent is methanol; Alternatively, the volume ratio of the methanol to pentafluorobenzaldehyde is 15-20:

1.

5. The method for preparing the PFP chromatographic filler according to claim 1, characterized in that: After the reaction, PFP@SiO2 was washed with methanol to remove the residual reaction reagents and then dried.

6. PFP chromatographic filler prepared by the method described in any one of claims 1-5.

7. A PFP chromatographic column, characterized in that: The material is filled with the PFP chromatography filler according to claim 6.

8. A method for filling a PFP chromatographic column, characterized in that: include: Dispersing the PFP chromatographic filler according to claim 6 into the mixed solution to obtain a dispersion of the filler; Methanol is used as a displacement liquid to pump the dispersion of the filler into an empty chromatographic column tube at a preset pressure, and then the sieve plate and the column tube are installed to obtain a PFP chromatographic column.

9. The method for filling a PFP chromatographic column as claimed in claim 8, characterized in that: The mass volume ratio of the PFP chromatographic filler to the mixed solution is 0.8 g: 50-60 mL; Or, the mixed solution consists of methanol and isopropanol, and the volume ratio of the two is 9:1-1.5; Alternatively, the preset pressure is 45-50 MPa.

10. Use of the PFP chromatographic filler according to claim 6 and the PFP chromatographic column according to claim 7 in detecting antiviral drugs, phenolic acid drugs, and flavonoid drugs.

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