A silica gel chromatographic column, a filler and a preparation method and application thereof
By introducing quaternary ammonium groups into the surface of silica gel, aminopropylsilane was introduced to prepare silica gel spheres with amino and quaternary ammonium groups. This solved the problems of unstable peak elution, high noise, and short lifespan of aminopropyl-bonded silica gel columns in the determination of betaine, and achieved efficient separation of betaine.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- XIAMEN INST FOR FOOD & DRUG QUALITY INSPECTION (XIAMEN PORT DRUG INSPECTION INST OF THE PEOPLES REPUBLIC OF CHINA)
- Filing Date
- 2026-03-20
- Publication Date
- 2026-05-29
AI Technical Summary
Existing aminopropyl-bonded silica gel columns have problems such as unstable peak elution time, high baseline noise, inability to separate betaine from impurity peaks, and short column life when determining betaine.
By introducing quaternary ammonium groups with charge-shielding effects by bonding aminopropylsilane to the surface of silica gel, the non-specific adsorption between silanol groups and betaine is shielded, thus preparing silica gel spheres with surface-bonded amino and quaternary ammonium groups.
It achieves excellent peak shape, high column efficiency and stable peak area, and solves the problems of high baseline noise, low column efficiency and short life of traditional amino columns, making it suitable for the efficient separation of betaine.
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Figure CN122098529A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of liquid chromatography column production, and particularly to a silica gel chromatography column, packing material, preparation method and application thereof. Background Technology
[0002] Betaine, also known as trimethylglycine, is a binary molecule containing three hydrophobic methyl groups (-CH3) and hydrophilic carboxyl groups (-COOH), belonging to the quaternary ammonium base class. Betaine is found in the roots, stems, and leaves of mammals, poultry, and plants. Due to its physiological functions such as regulating osmotic pressure, improving growth performance and immunity, improving cardiovascular health, and possessing anti-inflammatory and antioxidant properties, it is widely used in medicine, food, feed, and cosmetics. For the determination of betaine in wolfberry, both the 2020 and 2025 editions of the *Pharmacopoeia of the People's Republic of China* use an amino-bonded silica gel column.
[0003] The most commonly used amino columns currently employ an aminopropyl-bonded stationary phase. Due to the high reactivity of the primary amine groups in this phase, they readily undergo nucleophilic addition reactions with aldehyde groups to form Schiff bases, altering the properties of the analyte and the bonded phase. Furthermore, this stationary phase is unstable and prone to leaching, easily leading to irreversible dead adsorption when applied to acidic substances, significantly reducing its lifespan. When using an aminopropyl-bonded silica gel column to determine betaine, the following problems are frequently encountered: (1) unstable elution time; (2) high baseline noise; (3) inability to distinguish betaine from impurity peaks; and (4) excessively short column lifespan. Summary of the Invention
[0004] In view of the above problems, this application provides a novel amino-bonded silica gel chromatographic column packing. This packing introduces quaternary ammonium groups with charge-shielding effects into the aminopropylsilane bonded to the silica gel surface, which can shield silanol groups to a certain extent, overcoming the "non-specific adsorption" between silanol groups and betaine. Therefore, when used for betaine separation, it exhibits excellent peak shape, high column efficiency, and stable peak area.
[0005] The first aspect of this application provides a silica gel chromatography column packing material, wherein the packing material is silica gel spheres with amino and quaternary ammonium groups bonded to their surface, and its structural formula is shown in Formula I:
[0006]
[0007] Formula I.
[0008] Unlike existing technologies, the above technical solution provides a silica gel chromatography column packing material, which consists of silica gel spheres with amino and quaternary ammonium groups bonded to their surface. This packing material introduces quaternary ammonium groups with charge-shielding effects into the aminopropylsilane bonded to the silica gel surface, which can shield silanol groups to a certain extent, overcoming the "non-specific adsorption" between silanol groups and betaine. Therefore, when used for betaine separation, it exhibits excellent peak shape, high column efficiency, and stable peak area.
[0009] A second aspect of this application provides a method for preparing silica gel chromatography column packing material, comprising the following steps:
[0010] Activation of silica spheres: The surface of porous silica spheres is activated with hydrochloric acid to obtain activated silica spheres with alcohol groups; the porous silica spheres have a particle size of 5 μm and a pore size of 11-12 nm;
[0011] Amine formation: The activated silica spheres undergo a bonding reaction with 3-aminopropyltriethoxysilane to obtain amino silica spheres;
[0012] Epoxy group generation: The amino silica gel spheres undergo a bonding reaction with 3-glycidyl etheroxypropyltrimethoxysilane to obtain silica gel spheres with amino and epoxy groups bonded to their surfaces;
[0013] Quaternary ammonium group formation: In the silica spheres with surface-bonded amino and epoxy groups, the epoxy groups undergo a ring-opening reaction with trimethylamine to obtain silica spheres with surface-bonded amino and quaternary ammonium groups.
[0014] Furthermore, in the silica ball activation step, the porous silica balls are ultrasonically dispersed with 6 mol / L hydrochloric acid, stirred at 105-110℃ for 24 h, washed with water and dried to obtain activated silica balls with alcohol groups.
[0015] Furthermore, in the amino formation step, the activated silica gel spheres are reacted with 3-aminopropyltriethoxysilane at 105-110°C for 24 h; washed with anhydrous ethanol and dried under vacuum at 100°C for 12 h to obtain amino silica gel spheres.
[0016] Furthermore, in the epoxy group generation step, the amino silica gel balls are reacted with 3-glycidyl etheroxypropyltrimethoxysilane at 60°C for 24 h, washed with anhydrous ethanol, and dried under vacuum at 100°C for 12 h to obtain silica gel balls with amino and epoxy groups bonded to their surfaces.
[0017] Furthermore, in the step of generating the quaternary ammonium group, silica spheres with surface-bonded amino and epoxy groups are reacted with trimethylamine at 100°C for 2 h, washed with anhydrous ethanol, and vacuum dried at 100°C for 12 h to obtain silica spheres with surface-bonded amino and quaternary ammonium groups.
[0018] Furthermore, in the steps of amino group formation, epoxy group formation, and quaternary ammonium group formation, the reaction is carried out under nitrogen protection conditions.
[0019] Furthermore, in the steps of amino formation and epoxy formation, the reaction is carried out in toluene solvent.
[0020] Furthermore, in the quaternary ammonium group generation step, the reaction is carried out in an N,N-dimethylformamide solvent.
[0021] A third aspect of this application also provides a silica gel chromatography column, wherein the chromatography column is packed with the silica gel chromatography column packing material described in the first aspect of this application.
[0022] Furthermore, the chromatographic column was 250 mm long and had an inner diameter of 4.6 mm. The packing conditions were as follows: packing pressure 3000-7000 psi, homogenizer: acetonitrile and isopropanol, material-to-liquid ratio 1:10, and finally replaced with methanol for 30 min.
[0023] Unlike existing technologies, the chromatographic column provided in this application, when used for the determination of betaine content in wolfberry according to the requirements of the 2025 edition of the Pharmacopoeia of the People's Republic of China, can completely solve the problems of high baseline noise, low column efficiency, low resolution, and short lifespan of traditional amino columns. Furthermore, the preparation method of this silica gel chromatographic packing material has advantages such as good stability, high reproducibility, simple preparation method, and ease of large-scale production.
[0024] The fourth aspect of this application provides the application of the silica gel chromatographic column for detecting the betaine content in wolfberry.
[0025] Furthermore, acetonitrile-water (85:15) was used as the mobile phase; the detection wavelength was 195 nm, the column temperature was 30℃, and the flow rate was 1.0 min / mL. The targets for separation were betaine and betaine from wolfberry. The sample preparation method was in accordance with the 2025 edition of the Pharmacopoeia of the People's Republic of China.
[0026] The above description of the invention is merely an overview of the technical solution of this application. In order to enable those skilled in the art to better understand the technical solution of this application and to implement it based on the textual description, and to make the above-mentioned objectives and other objectives, features and advantages of this application easier to understand, the following description is provided in conjunction with the specific embodiments and accompanying drawings of this application. Attached Figure Description
[0027] The accompanying drawings are only used to illustrate the principles, implementation methods, applications, features, and effects of specific embodiments of this application and other related content, and should not be considered as limitations on this application.
[0028] In the accompanying drawings of the instruction manual:
[0029] Figure 1 This is a schematic diagram of a method for preparing silica gel chromatography packing material;
[0030] Figure 2 To prepare chromatographic columns for separating betaine content chromatograms;
[0031] Figure 3 To prepare chromatographic packing material for separating betaine content in wolfberry;
[0032] Figure 4 To prepare chromatographic packing material for the separation and analysis of reproducible chromatograms of betaine content in wolfberry. Detailed Implementation
[0033] To illustrate the possible application scenarios, technical principles, implementable specific solutions, and achievable objectives and effects of this application in detail, the following description, in conjunction with the listed specific embodiments and accompanying drawings, provides a detailed explanation. The embodiments described herein are merely illustrative of the technical solutions of this application and are therefore intended to limit the scope of protection of this application.
[0034] In this document, the term "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The term "embodiment" appearing in various places throughout the specification does not necessarily refer to the same embodiment, nor does it specifically limit its independence or connection with other embodiments. In principle, in this application, as long as there are no technical contradictions or conflicts, the technical features mentioned in each embodiment can be combined in any way to form corresponding implementable technical solutions.
[0035] Unless otherwise defined, the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the use of related terms herein is merely for the purpose of describing particular embodiments and is not intended to limit this application.
[0036] In the description of this application, the term "and / or" is used to describe the logical relationship between objects, indicating that three relationships can exist. For example, A and / or B means: A exists, B exists, and A and B exist simultaneously. Additionally, the character " / " in this document generally indicates that the preceding and following objects have an "or" logical relationship.
[0037] In this application, terms such as “first” and “second” are used only to distinguish one entity or operation from another, and do not necessarily require or imply any actual quantity, hierarchy or order relationship between these entities or operations.
[0038] Unless otherwise specified, the use of terms such as “comprising,” “including,” “having,” or other similar expressions in this application is intended to cover non-exclusive inclusion, which does not exclude the presence of additional elements in a process, method, or product that includes the stated elements, such that a process, method, or product that includes a list of elements may include not only those defined elements but also other elements not expressly listed, or elements inherent to such a process, method, or product.
[0039] As understood in the Examination Guidelines, in this application, expressions such as "greater than," "less than," and "exceeding" are understood to exclude the stated number; expressions such as "above," "below," and "within" are understood to include the stated number. Furthermore, in the description of the embodiments in this application, "multiple" means two or more (including two), and similar expressions related to "multiple" are also understood in this way, such as "multiple groups" and "multiple times," unless otherwise explicitly specified.
[0040] The first aspect of this application provides a silica gel chromatography column packing material, wherein the packing material is silica gel spheres with amino and quaternary ammonium groups bonded to their surface, and its structural formula is shown in Formula I:
[0041]
[0042] Formula I.
[0043] Unlike existing technologies, the above technical solution provides a silica gel chromatography column packing material, which consists of silica gel spheres with amino and quaternary ammonium groups bonded to their surface. This packing material introduces quaternary ammonium groups with charge-shielding effects into the aminopropylsilane bonded to the silica gel surface, which can shield silanol groups to a certain extent, overcoming the "non-specific adsorption" between silanol groups and betaine. Therefore, when used for betaine separation, it exhibits excellent peak shape, high column efficiency, and stable peak area.
[0044] A second aspect of this application provides a method for preparing silica gel chromatography column packing material, comprising the following steps:
[0045] Activation of silica spheres: The surface of porous silica spheres is activated with hydrochloric acid to obtain activated silica spheres with alcohol groups; the porous silica spheres have a particle size of 5 μm and a pore size of 11-12 nm;
[0046] Amine formation: The activated silica spheres undergo a bonding reaction with 3-aminopropyltriethoxysilane to obtain amino silica spheres;
[0047] Epoxy group generation: The amino silica gel spheres undergo a bonding reaction with 3-glycidyl etheroxypropyltrimethoxysilane to obtain silica gel spheres with amino and epoxy groups bonded to their surface;
[0048] Quaternary ammonium group formation: In the silica spheres with surface-bonded amino and epoxy groups, the epoxy groups undergo a ring-opening reaction with trimethylamine to obtain silica spheres with surface-bonded amino and quaternary ammonium groups.
[0049] Furthermore, in the silica ball activation step, the porous silica balls are ultrasonically dispersed with 6 mol / L hydrochloric acid, stirred at 105-110℃ for 24 h, washed with water and dried to obtain activated silica balls with alcohol groups.
[0050] Furthermore, in the amino formation step, the activated silica gel spheres are reacted with 3-aminopropyltriethoxysilane at 105-110°C for 24 h; washed with anhydrous ethanol and dried under vacuum at 100°C for 12 h to obtain amino silica gel spheres.
[0051] Furthermore, in the epoxy group generation step, the amino silica gel balls are reacted with 3-glycidyl etheroxypropyltrimethoxysilane at 60°C for 24 h, washed with anhydrous ethanol, and dried under vacuum at 100°C for 12 h to obtain silica gel balls with amino and epoxy groups bonded to their surfaces.
[0052] Furthermore, in the step of generating the quaternary ammonium group, silica spheres with surface-bonded amino and epoxy groups are reacted with trimethylamine at 100°C for 2 h, washed with anhydrous ethanol, and vacuum dried at 100°C for 12 h to obtain silica spheres with surface-bonded amino and quaternary ammonium groups.
[0053] Furthermore, in the steps of amino group formation, epoxy group formation, and quaternary ammonium group formation, the reaction is carried out under nitrogen protection conditions.
[0054] Furthermore, in the steps of amino formation and epoxy formation, the reaction is carried out in toluene solvent.
[0055] Furthermore, in the quaternary ammonium group generation step, the reaction is carried out in an N,N-dimethylformamide solvent.
[0056] Furthermore, in the silica ball activation step, the porous silica balls are ultrasonically dispersed with 6 mol / L hydrochloric acid, stirred at 105-110℃ for 24 h, washed with water until pH=7.0, and dried at 150℃ to obtain activated silica balls with alcohol groups.
[0057] Furthermore, in the amino formation step, the activated silica spheres are ultrasonically suspended in toluene, then 3-aminopropyltriethoxysilane is added and reacted at 110°C under nitrogen protection for 24 h. After filtration and washing with anhydrous ethanol, the silica spheres are finally dried under vacuum at 100°C for 12 h to obtain amino silica spheres.
[0058] Furthermore, in the epoxy group generation step, the amino silica gel spheres are ultrasonically suspended in toluene, 3-glycidyl etheroxypropyltrimethoxysilane is added, and the reaction is carried out at 60°C under nitrogen protection for 24 h. Then, the spheres are filtered and washed with anhydrous ethanol, and finally dried under vacuum at 100°C for 12 h to obtain silica gel spheres with amino and epoxy groups bonded to their surfaces.
[0059] Furthermore, in the step of generating quaternary ammonium groups, silica spheres with surface-bonded amino and epoxy groups are ultrasonically suspended with N,N-dimethylformamide, and then trimethylamine is added and reacted at 100°C under nitrogen protection for 2 h. After that, they are washed by filtration with anhydrous ethanol and finally dried under vacuum at 100°C for 12 h to obtain silica spheres with surface-bonded amino and quaternary ammonium groups.
[0060] A third aspect of this application also provides a silica gel chromatography column, wherein the chromatography column is packed with the silica gel chromatography column packing material described in the first aspect of this application.
[0061] Furthermore, the chromatographic column was 250 mm long and had an inner diameter of 4.6 mm. The packing conditions were as follows: packing pressure 3000-7000 psi, homogenizer: acetonitrile and isopropanol, material-to-liquid ratio 1:10, and finally replaced with methanol for 30 min.
[0062] Unlike existing technologies, the chromatographic column provided in this application, when used for the determination of betaine content in wolfberry according to the requirements of the 2025 edition of the Pharmacopoeia of the People's Republic of China, can completely solve the problems of high baseline noise, low column efficiency, low resolution, and short lifespan of traditional amino columns. Furthermore, the preparation method of this silica gel chromatographic packing material has advantages such as good stability, high reproducibility, simple preparation method, and ease of large-scale production.
[0063] The fourth aspect of this application provides the application of the silica gel chromatographic column for detecting the betaine content in wolfberry.
[0064] Furthermore, acetonitrile-water (85:15) was used as the mobile phase; the detection wavelength was 195 nm, the column temperature was 30℃, and the flow rate was 1.0 min / mL. The targets for separation were betaine and betaine from wolfberry. The sample preparation method was in accordance with the 2025 edition of the Pharmacopoeia of the People's Republic of China.
[0065] Example 1 Preparation of packing material and chromatographic column
[0066] The preparation process of the filler is as follows: Figure 1 As shown, it includes:
[0067] Activation of silica spheres: 5g of porous silica spheres were ultrasonically dispersed with 6 mol / L hydrochloric acid, stirred at 105-110℃ for 24h, washed with pure water until pH=7.0, and dried at 150℃ for 12h to obtain activated silica spheres with alcohol groups.
[0068] Amine formation: 3g of activated silica gel balls were added to toluene solvent, followed by 4.68 mL of 3-aminopropyltriethoxysilane (20 mmol / L). The mixture was sonicated until uniformly dispersed and reacted at 110℃ under nitrogen protection for 24 h. The mixture was then washed sequentially with water and anhydrous ethanol and dried under vacuum at 100℃ for 12 h to obtain amino silica gel balls.
[0069] Epoxy group formation: 3g of amino silica gel balls were added to toluene solvent, and 0.69 mL / L of 3-glycidyl etheroxypropyltrimethoxysilane was added. The reaction was carried out at 60℃ (3 mmol / L) under nitrogen protection for 24 h. The balls were washed with water and anhydrous ethanol in sequence, and dried under vacuum at 100℃ for 12 h to obtain silica gel balls with amino and epoxy groups bonded to the surface.
[0070] In the quaternary ammonium group generation step, 3g of silica spheres with surface-bonded amino and epoxy groups were added to N,N-dimethylformamide solvent, and then 1.29 mL of trimethylamine (6 mmol / L) was added. The mixture was reacted at 100℃ under nitrogen protection for 2 h. The spheres were washed sequentially with water and anhydrous ethanol, and then dried under vacuum at 100℃ for 12 h to obtain silica spheres with surface-bonded amino and quaternary ammonium groups. The filler preparation was completed.
[0071] Preparation of silica gel chromatographic column: Silica gel beads with amino and quaternary ammonium groups bonded to their surfaces were packed into the column to obtain the chromatographic column. The column length was 250 mm and the inner diameter was 4.6 mm. The packing conditions were as follows: packing pressure 3000-7000 psi; homogenizer: acetonitrile and isopropanol; material-to-liquid ratio 1:10; and finally, methanol was used to replace the liquid for 30 min.
[0072] Example 2: Determination and Analysis of Betaine Content in Lycium barbarum
[0073] 1. The sample preparation method for betaine and betaine in wolfberry was in accordance with the 2025 edition of the Pharmacopoeia of the People's Republic of China, using acetonitrile-water (85:15) as the mobile phase; the detection wavelength was 195 nm, the column temperature was 30℃, and the flow rate was 1.0 min / mL.
[0074] 2. Separation results are as follows Figure 2 , Figure 3 As shown
[0075] 3. Stability test: The effect of the wolfberry sample after 200 injections into the silica gel column is shown in the figure below. Figure 4 ,Depend on Figure 4 It can be seen that after 200 needle tests, the peak shape of the chromatographic packing material was good and the column efficiency did not decrease significantly.
[0076] The silica gel chromatography column described in this application can provide polar interaction sites, thereby reducing the hydrolysis of amino groups and giving the amino-bonded silica gel chromatography packing material excellent stability.
[0077] According to the requirements of the 2025 edition of the Pharmacopoeia of the People's Republic of China, this application can completely solve the problems of high baseline noise, low column efficiency, low resolution, and short lifespan of traditional amino columns for the determination of betaine content in wolfberry. The preparation method of silica gel chromatography packing material has the advantages of good stability, high reproducibility, simple preparation method, and ease of large-scale production.
[0078] Finally, it should be noted that although the above embodiments have been described in the text and drawings of this application, this should not limit the scope of patent protection of this application. Any technical solutions that are based on the essential concept of this application and utilize the content described in the text and drawings of this application, resulting in equivalent structural or procedural substitutions or modifications, as well as the direct or indirect application of the technical solutions of the above embodiments to other related technical fields, are all included within the scope of patent protection of this application.
Claims
1. A silica gel chromatography column packing material, characterized in that, The filler is a silica gel ball with amino and quaternary ammonium groups bonded to its surface, and its structural formula is shown in Formula I: Formula I.
2. A method for preparing silica gel chromatography column packing material, characterized in that, Includes the following steps: Activation of silica spheres: The surface of porous silica spheres is activated with hydrochloric acid to obtain activated silica spheres with alcohol groups; the porous silica spheres have a particle size of 5 μm and a pore size of 11-12 nm; Amine formation: The activated silica spheres undergo a bonding reaction with 3-aminopropyltriethoxysilane to obtain amino silica spheres; Epoxy group generation: The amino silica gel spheres undergo a bonding reaction with 3-glycidyl etheroxypropyltrimethoxysilane to obtain silica gel spheres with amino and epoxy groups bonded to their surfaces; Quaternary ammonium group formation: In the silica spheres with surface-bonded amino and epoxy groups, the epoxy groups undergo a ring-opening reaction with trimethylamine to obtain silica spheres with surface-bonded amino and quaternary ammonium groups.
3. The preparation method according to claim 2, characterized in that, In the activated silica sphere step, porous silica spheres are ultrasonically dispersed with 6 mol / L hydrochloric acid, stirred at 105-110℃ for 24 h, washed with water and dried to obtain activated silica spheres with alcohol groups.
4. The preparation method according to claim 2, characterized in that, In the amino formation step, the activated silica gel spheres are reacted with 3-aminopropyltriethoxysilane at 105-110℃ for 24 h; washed with anhydrous ethanol and dried under vacuum at 100℃ for 12 h to obtain amino silica gel spheres.
5. The preparation method according to claim 2, characterized in that, In the epoxy group generation step, the amino silica gel balls are reacted with 3-glycidyl etheroxypropyltrimethoxysilane at 60°C for 24 h, washed with anhydrous ethanol, and dried under vacuum at 100°C for 12 h to obtain silica gel balls with amino and epoxy groups bonded to their surfaces.
6. The preparation method according to claim 2, characterized in that, In the step of generating quaternary ammonium groups, mixed silica spheres containing amino and epoxy groups are reacted with trimethylamine at 100°C for 2 h, washed with anhydrous ethanol, and vacuum dried at 100°C for 12 h to obtain silica spheres with amino and quaternary ammonium groups bonded to their surfaces.
7. The preparation method according to claims 2-6, characterized in that, In the steps of amino group formation, epoxy group formation, and quaternary ammonium group formation, the reaction is carried out under nitrogen protection.
8. The preparation method according to claims 2-6, characterized in that, In the steps of generating amino groups and epoxy groups, the reaction is carried out in toluene solvent; in the step of generating quaternary ammonium groups, the reaction is carried out in N,N-dimethylformamide solvent.
9. A silica gel chromatography column, characterized in that, The chromatographic column is packed with the silica gel chromatographic column packing material as described in claim 1.
10. The application of the silica gel chromatography column according to claim 9, characterized in that, The chromatographic column was used to detect the betaine content in wolfberries.