A liquid chromatography packing for polar compound separation analysis and its preparation method and application

Abstract

The application discloses a liquid chromatography filler for polar compound separation analysis and a preparation method and application thereof, and the liquid chromatography filler is a hydrogel formed by polyvinyl alcohol, borax and tannic acid on the surface of silica gel microspheres. The liquid chromatography filler is obtained by modifying a layer of hydrogel formed by polyvinyl alcohol, borax and tannic acid on the surface of silica gel in a physical coating mode, and has the advantages of simple preparation path and convenient operation. Due to the hydrophilic property of the hydrogel and the existence of multiple action sites, the chromatography filler can be used in the separation of various polar compounds such as amino acids, nucleosides / bases / alkaloids and the like, and has excellent chromatography separation performance.

Description

Technical Field

[0001] This invention relates to a liquid chromatography packing material, specifically to a liquid chromatography packing material for the separation and analysis of polar compounds, its preparation method, and its application. Background Technology

[0002] The separation and analysis of polar compounds play a vital role in biological life and are of great significance in various fields such as pharmaceutical science, life science, food chemistry, and environmental science. However, these compounds face different challenges in the separation and analysis of reversed-phase chromatography (RPLC) and normal-phase chromatography (NPLC), such as limited retention capacity and selectivity, poor solubility in the mobile phase, and poor chromatographic reproducibility. Hydrophilic interaction liquid chromatography (HILIC) effectively solves the separation problem of polar molecules in RPLC and NPLC. It has excellent retention capacity for polar compounds, and the presence of water in the mobile phase ensures optimal sample solubility. The stationary phase is the foundation and core of high-performance liquid chromatography (HPLC), determining the retention mechanism, separation selectivity, and separation efficiency. Therefore, the development of novel HILIC chromatographic packing materials is of great significance for the separation and analysis of polar compounds.

[0003] Hydrogels are novel hydrophilic polymers with a three-dimensional network structure. They have attracted considerable attention due to their versatility in preparation, biocompatibility, tunable pore size, and excellent adhesion, and are widely used in energy, catalysis, wearable devices, and other fields. However, research on their application as chromatographic packing materials in high-performance liquid chromatography (HPLC) is limited. Utilizing the excellent adhesion of hydrogels, and by screening the types and amounts of hydrogel monomers to combine them with silica microspheres to prepare novel HPLC packing materials for the separation of polar compounds, is a promising research area and development direction. Summary of the Invention

[0004] The purpose of this invention is to provide a liquid chromatography packing material for the separation and analysis of polar compounds, its preparation method and application, which can be used for the separation of various polar compounds (such as amino acids, nucleosides / bases / alkaloids, etc.) and has excellent chromatographic separation performance.

[0005] To achieve the above objectives, the present invention provides a liquid chromatography packing material for the separation and analysis of polar compounds, wherein the liquid chromatography packing material is a hydrogel formed on the surface of silica microspheres by polyvinyl alcohol, borax and tannic acid.

[0006] Preferably, the mass ratio of polyvinyl alcohol, borax and tannic acid is (1.5-3):(0.3-0.5):1.

[0007] Preferably, the mass ratio of the silica microspheres to polyvinyl alcohol is (5-20):1.

[0008] Preferably, the molecular weight of the polyvinyl alcohol is 65,000 to 70,000.

[0009] More preferably, the molecular weight of the polyvinyl alcohol is 65,000 to 67,000.

[0010] Preferably, the size of the silica microspheres is 5 μm.

[0011] Another object of the present invention is to provide a method for preparing the aforementioned liquid chromatography packing material, the method comprising:

[0012] (1) Add polyvinyl alcohol to water and stir at 85-90°C to obtain a polyvinyl alcohol solution;

[0013] (2) Add the silica microspheres to the polyvinyl alcohol solution and stir at room temperature to obtain suspension A;

[0014] (3) Add the crosslinking agent borax to water, stir at room temperature, then add tannic acid, and continue stirring at room temperature to obtain solution B;

[0015] (4) Add the solution B to the suspension A, heat to 95°C, and reflux under stirring;

[0016] (5) After the reaction is complete, heat to 115°C to evaporate water, obtain solid product, wash and dry to obtain the liquid chromatography packing material.

[0017] Preferably, in step (1), the mixture is stirred at 85-90°C for 2-3 hours at a stirring speed of 150-250 rpm.

[0018] Preferably, in step (2), the mixture is stirred at room temperature for 0.5 to 1 hour; in step (3), the mixture is stirred at room temperature for 2 to 3 hours, then tannic acid is added, and the mixture is stirred at room temperature for another 0.5 to 1 hour.

[0019] Preferably, in step (4), the stirring is carried out for 2 to 4 hours at a stirring speed of 150 to 250 rpm.

[0020] Preferably, in step (5), the water and ethanol are used for centrifugal washing in sequence; the vacuum drying temperature is 65-85℃ and the drying time is 10-12h.

[0021] Another object of the present invention is to provide the application of the described liquid chromatography packing material in the separation or analysis of polar compounds.

[0022] Preferably, the polar compound is selected from amino acids, nucleoside bases, alkaloids, or organic acid compounds.

[0023] The liquid chromatography packing material for the separation and analysis of polar compounds of the present invention, its preparation method, and its application have the following advantages:

[0024] (1) The liquid chromatography packing material of the present invention is obtained by physically coating a layer of hydrogel formed by polyvinyl alcohol, borax and tannic acid on the surface of silica gel. Polyvinyl alcohol is rich in hydroxyl groups, borax is a crosslinking agent to enhance the mechanical strength of the polymer, and the addition of tannic acid can give the hydrogel adhesive properties, which is beneficial to physical coating onto the surface of silica gel. In addition, the presence of benzene rings can provide π-π interaction sites. Therefore, the prepared hydrogel structure is rich in hydroxyl groups and benzene rings can provide a variety of interaction sites, which is beneficial to the separation of polar compounds. The preparation route is simple and the operation is convenient.

[0025] (2) Due to the hydrophilic properties of the hydrogel and the presence of multiple action sites, the chromatographic packing material of the present invention can be used for the separation of a variety of polar compounds (such as amino acids, nucleosides / bases / alkaloids, etc.) and has excellent chromatographic separation performance. Attached Figure Description

[0026] Figure 1 SEM images of the liquid chromatography packing material prepared in Example 1 of this invention and bare silica gel; wherein, a is bare silica gel; b is the liquid chromatography packing material of Example 1.

[0027] Figure 2 The image shows the FT-IR spectrum of the liquid chromatography packing material prepared in Example 1 of this invention and bare silica gel.

[0028] Figure 3 Thermogravimetric analysis (TGA) of the liquid chromatography packing material prepared in Example 1 of this invention and bare silica gel.

[0029] Figure 4 The images show the separation of amino acids on the liquid chromatography packing material prepared in Example 1 of this invention and the comparative amino column.

[0030] Figure 5 The liquid chromatograms showing the separation of nucleosides / bases on the liquid chromatography packing material prepared in Example 1 of this invention and the comparative amino column are shown.

[0031] Figure 6 The images show liquid chromatograms of alkaloids on the liquid chromatography packing material prepared in Example 1 of this invention and on the comparative amino column.

[0032] Figure 7 The images show liquid chromatograms of organic acids on the liquid chromatography packing material prepared in Example 1 of this invention and on the comparative amino column. Detailed Implementation

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Example 1

[0035] A liquid chromatography packing material for the separation and analysis of polar compounds, the preparation method of which includes the following steps:

[0036] (1) Add 0.3g of polyvinyl alcohol (molecular weight ~67000) to a round-bottom flask containing 40mL of deionized water, place it in an oil bath, and keep it at 85℃ for 2h under mechanical stirring at 250rpm to obtain a polyvinyl alcohol solution.

[0037] (2) Add 3g of silica microspheres (purchased from Suzhou Nanomicro Technology Co., Ltd., silica type UniSil 5-100, silica microsphere size 5μm) to the above polyvinyl alcohol solution and stir at room temperature for 0.5h to obtain suspension A;

[0038] (3) Add 0.05g of crosslinking agent borax to a round-bottom flask containing 10mL of deionized water, stir magnetically for 2h at room temperature, then add 0.1g of tannic acid, stir magnetically for 30min to obtain solution B;

[0039] (4) Add the obtained solution B to the suspension A, heat to 95°C, reflux and condense, and mechanically stir at 250 rpm for 3 hours.

[0040] (5) After the reaction is complete, heat to 115°C to evaporate the deionized water to obtain a solid product. Then, wash the solid product by centrifugation with deionized water and ethanol three times each. Place the solid product in a vacuum drying oven and dry at 80°C for 10 hours to obtain a hydrogel-modified silica gel chromatography packing.

[0041] Example 2

[0042] A liquid chromatography packing material for the separation and analysis of polar compounds, the preparation method of which includes the following steps:

[0043] (1) Add 0.15g of polyvinyl alcohol (molecular weight ~67000) to a round-bottom flask containing 40mL of deionized water, place it in an oil bath, and keep it at 90℃ for 2h under mechanical stirring at 150rpm to obtain a polyvinyl alcohol solution.

[0044] (2) Add 3g of silica microspheres (purchased from Suzhou Nanomicro Technology Co., Ltd., silica type UniSil 5-100, silica microsphere size 5μm) to the above polyvinyl alcohol solution and stir at room temperature for 1h to obtain suspension A;

[0045] (3) Add 0.05g of crosslinking agent borax to a round-bottom flask containing 10mL of deionized water, stir magnetically at room temperature for 3h, then add 0.1g of tannic acid, and continue to stir magnetically at room temperature for 30min to obtain solution B;

[0046] (4) Add the obtained solution B to the suspension A, heat to 95°C, reflux and condense, and mechanically stir at 200 rpm for 3 hours.

[0047] (5) After the reaction is complete, heat to 115°C to evaporate the deionized water to obtain a solid product. Then, wash the solid product by centrifugation with deionized water and ethanol three times each. Place the solid product in a vacuum drying oven and dry at 65°C for 12 hours to obtain a hydrogel-modified silica gel chromatography packing.

[0048] Example 3

[0049] A liquid chromatography packing material for the separation and analysis of polar compounds, the preparation method of which includes the following steps:

[0050] (1) Add 0.3g of polyvinyl alcohol (molecular weight ~67000) to a round-bottom flask containing 40mL of deionized water, place it in an oil bath, and keep it at 85℃ for 3h under mechanical stirring at 250rpm to obtain a polyvinyl alcohol solution.

[0051] (2) Add 3g of silica microspheres (purchased from Suzhou Nanomicro Technology Co., Ltd., silica type UniSil 5-100, silica microsphere size 5μm) to the above polyvinyl alcohol solution and stir at room temperature for 0.5h to obtain suspension A;

[0052] (3) Add 0.03g of crosslinking agent borax to a round-bottom flask containing 10mL of deionized water, stir magnetically at room temperature for 2.5h, then add 0.1g of tannic acid, and continue stirring magnetically at room temperature for 30min to obtain solution B;

[0053] (4) Add the obtained solution B to the suspension A, heat to 95°C, reflux and condense, and mechanically stir at 150 rpm for 4 hours.

[0054] (5) After the reaction is complete, heat to 115°C to evaporate the deionized water to obtain a solid product. Then, wash the solid product by centrifugation with deionized water and ethanol three times each. Place the solid product in a vacuum drying oven and dry at 85°C for 12 hours to obtain a hydrogel-modified silica gel chromatography packing.

[0055] Example 4: Application in amino acid separation

[0056] Preparation of a liquid chromatography column for the separation and analysis of polar compounds: The liquid chromatography packing material prepared in Example 1 for the separation and analysis of polar compounds 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 amino acid compounds. During the packing process, both the dispersion and the displacement solvent were methanol, and the pressure was 40 MPa.

[0057] Amino acids were separated using an amino column (NH2) in liquid chromatography as a reference.

[0058] Chromatographic separation of amino acids:

[0059] 1. The chromatographic analysis conditions are as follows:

[0060] The mobile phase was acetonitrile / 150mM ammonium acetate (72 / 28, v / v); the flow rate was 1.0 mL / min; the evaporative light detector was set at T = 90℃, the gas flow rate was 2.5 L / min, and the injection volume was 20 μL.

[0061] 2. The analyte is a mixture of several amino acids.

[0062] The liquid chromatogram for the separation of amino acids is shown below. Figure 4 Among them, 1 is tryptophan, 2 is phenylalanine; 3 is isoleucine, 4 is cysteine, 5 is threonine, 6 is asparagine, 7 is glutamic acid, and 8 is histidine. Figure 4 As can be seen from A and B, the liquid chromatography packing material of the present invention for the separation and analysis of polar compounds has excellent separation selectivity for amino acids.

[0063] Example 5: Application in nucleoside / base separation

[0064] Preparation of a liquid chromatography column for the separation and analysis of polar compounds: The liquid chromatography packing material prepared in Example 1 for the separation and analysis of polar compounds 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. During the packing process, both the dispersion and the displacement solvent were methanol, and the pressure was 40 MPa.

[0065] Nucleosides / bases were separated using an amino column (NH2) in liquid chromatography as a reference.

[0066] Chromatographic separation of nucleosides / bases:

[0067] 1. The chromatographic analysis conditions are as follows:

[0068] The mobile phase was acetonitrile / 100mM ammonium acetate (88 / 12, v / v); the flow rate was 1.0 mL / min; the UV detector was 254 nm; and the injection volume was 20 μL.

[0069] 2. The analyte is a mixture of several nucleosides / bases.

[0070] The HPLC chromatogram for the separation of nucleosides / bases is shown below. Figure 5 Among them, 1 is thymine, 2 is thymidine, 3 is 6-chlorouracil, 4 is uridine, 5 is adenosine, 6 is inosine, 7 is cytosine, and 8 is cytidine. Figure 5 As can be seen from A and B, the liquid chromatography packing material of the present invention for the separation and analysis of polar compounds has excellent separation selectivity for nucleosides / bases.

[0071] Example 6: Application in Alkaloid Separation

[0072] Preparation of a liquid chromatography column for the separation and analysis of polar compounds: The liquid chromatography packing material prepared in Example 1 for the separation and analysis of polar compounds 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 alkaloid compounds. During the packing process, both the dispersion and the displacement solvent were methanol, and the pressure was 40 MPa.

[0073] Alkaloids were separated by using an amino column (NH2) in liquid chromatography as a reference.

[0074] Chromatographic separation of alkaloids:

[0075] 1. The chromatographic analysis conditions are as follows:

[0076] The mobile phase was acetonitrile / 150mM ammonium acetate (90 / 10, v / v); the flow rate was 1.0 mL / min; the UV detector was 254 nm; and the injection volume was 20 μL.

[0077] 2. The analyte is a mixture of several alkaloids.

[0078] The liquid chromatogram of alkaloids is shown below. Figure 6 Among them, 1 is corydaline, 2 is caffeine, 3 is colchicine, 4 is theobromine, 5 is berberine, and 6 is tetrandrine. Figure 6 As can be seen from A and B, the liquid chromatography packing material of the present invention for the separation and analysis of polar compounds has excellent separation selectivity for alkaloids.

[0079] Example 7: Application in the separation of organic acids

[0080] Preparation of a liquid chromatography column for the separation and analysis of polar compounds: The liquid chromatography packing material prepared in Example 1 for the separation and analysis of polar compounds 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 organic acid compounds. During the packing process, both the dispersion and the displacement solvent were methanol, and the pressure was 40 MPa.

[0081] Organic acids were separated using an amino column (NH2) in liquid chromatography as a reference.

[0082] Chromatographic separation of organic acid compounds:

[0083] 1. The chromatographic analysis conditions are as follows:

[0084] The mobile phase was acetonitrile / 100mM ammonium acetate (89 / 11, v / v); the flow rate was 1.0 mL / min; the UV detector was 254 nm; and the injection volume was 20 μL.

[0085] 2. The analyte is a mixture of several organic acids.

[0086] The liquid chromatograms of organic acids are shown below. Figure 7 Wherein, 1 is 3,5-dinitrobenzoic acid, 2 is p-nitrobenzoic acid, 3 is m-methylbenzoic acid, 4 is benzoic acid, 5 is p-hydroxybenzoic acid, and 6 is nicotinic acid. Figure 7 As can be seen from A and B, the liquid chromatography packing material of the present invention for the separation and analysis of polar compounds has excellent separation selectivity for organic acids.

[0087] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A liquid chromatography packing material for the separation and analysis of polar compounds, characterized in that, The liquid chromatography packing material is a hydrogel formed on the surface of silica microspheres by polyvinyl alcohol, borax and tannic acid. The mass ratio of polyvinyl alcohol, borax, and tannic acid is (1.5~3):(0.3~0.5):1; The mass ratio of the silica microspheres to polyvinyl alcohol is (5~20):

1.

2. The method for preparing the liquid chromatography packing material as described in claim 1, characterized in that, The method includes: (1) Add polyvinyl alcohol to water and stir at 85~90 ℃ to obtain a polyvinyl alcohol solution; (2) Add the silica microspheres to the polyvinyl alcohol solution and stir at room temperature to obtain suspension A; (3) Add the crosslinking agent borax to water, stir at room temperature, then add tannic acid, and continue stirring at room temperature to obtain solution B; (4) Add the solution B to the suspension A, heat to 95°C, reflux and stir; (5) After the reaction is complete, heat to 115 °C to evaporate water, obtain solid product, wash and dry to obtain the liquid chromatography packing material.

3. The preparation method according to claim 2, characterized in that, In step (1), the mixture is stirred at 85~90 ℃ for 2~3 h at a stirring speed of 150~250 rpm.

4. The preparation method according to claim 2, characterized in that, In step (2), the mixture is stirred at room temperature for 0.5 to 1 h; in step (3), the mixture is stirred at room temperature for 2 to 3 h, then tannic acid is added, and the mixture is stirred at room temperature for another 0.5 to 1 h.

5. The preparation method according to claim 2, characterized in that, In step (4), the mixture is stirred for 2 to 4 hours at a speed of 150 to 250 rpm.

6. The preparation method according to claim 2, characterized in that, In step (5), the water and ethanol are used for centrifugation and washing in sequence; the vacuum drying temperature is 65~85 ℃ and the drying time is 10~12 h.

7. The application of the liquid chromatography packing material as described in claim 1 in the separation or analysis of polar compounds.

8. The application according to claim 7, characterized in that, The polar compound is selected from amino acids, nucleoside bases, alkaloids, or organic acid compounds.

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