A mixed-mode liquid chromatography packing material, its preparation method and application

Abstract

This invention provides a mixed-mode liquid chromatography packing material, its preparation method, and its application. The method includes: dissolving monomers N-isopropylacrylamide, 1-vinyl-3-octylimidazolium bromide ionic liquid, and N,N-methylenebisacrylamide in anhydrous ethanol to obtain a mixed solution; adding silica microspheres to the mixed solution, sonicating, magnetically stirring, and then allowing the resulting mixture to stand at room temperature; adding the initiator 2,2'-azobisisobutyronitrile to the stood mixture, placing it in an oil bath, purging the system with N2 to remove air, and mechanically stirring the reaction; after the reaction is complete, heating to evaporate the ethanol, soaking the obtained solid product in deionized water, centrifuging and washing with deionized water and ethanol, and drying to finally obtain a mixed-mode liquid chromatography packing material based on an ionic liquid hybrid hydrogel. The method provided by this invention has a simple preparation path and is convenient to operate.

Description

Technical Field

[0001] This invention relates to the field of high performance liquid chromatography separation and analysis technology, and in particular to a mixed-mode liquid chromatography packing material, its preparation method and application. Background Technology

[0002] The core of chromatography is the chromatographic packing material. Currently, most chromatographic packing materials are single-mode packing materials (such as reversed-phase, hydrophilic, and ion-exchange chromatographic packing materials that use hydrophobic, polar, and ionic groups as functional groups, respectively). Their separation mechanisms are singular, which limits their ability to analyze complex, multi-component samples and fails to meet the practical needs of separating complex samples. Mixed-mode chromatographic packing materials, as an alternative or supplement to single-mode packing materials, have become a research hotspot in the field of chromatography. Unlike chromatography based on a single retention mode, mixed-mode chromatography uses multiple mechanisms synergistically to separate complex samples, achieving separation effects that single-mode chromatography cannot. Currently, the types of mixed-mode packing materials are still very limited; therefore, the development of novel mixed-mode chromatographic packing materials is of great significance for achieving efficient separation of complex samples. Summary of the Invention

[0003] This invention provides a mixed-mode liquid chromatography packing material, its preparation method, and its application.

[0004] A method for preparing a mixed-mode liquid chromatography packing material, comprising the following steps:

[0005] (1) Dissolve the hydrogel monomer N-isopropylacrylamide, 1-vinyl-3-octylimidazolium bromide ionic liquid and crosslinking agent N,N-methylenebisacrylamide in anhydrous ethanol to obtain a mixed solution;

[0006] (2) Add silica microspheres to the mixed solution, sonicate, stir magnetically, and then let the resulting mixture stand at room temperature for 10-12 hours;

[0007] (3) Add the initiator 2,2'-azobisisobutyronitrile to the mixture after it has been standing, place it in an oil bath, introduce N2 to remove the air from the system, and react at 65-75°C for 4-6 hours with mechanical stirring;

[0008] (4) After the reaction is completed, heat to 80°C to evaporate ethanol to obtain a solid product. Soak the solid product in deionized water, then wash it by centrifugation with deionized water and ethanol in sequence, and finally dry it in a vacuum drying oven to obtain a mixed-mode liquid chromatography packing material based on ionic liquid hybrid hydrogel.

[0009] Furthermore, in the preparation method of the mixed-mode liquid chromatography packing material as described above, in step (1), the mass ratio of N-isopropylacrylamide to 1-vinyl-3-octylimidazolium bromide ionic liquid is 1:1-1:1.6;

[0010] The mass ratio of the N,N-methylenebisacrylamide to the 1-vinyl-3-octylimidazolium bromide ionic liquid is 1:6 to 1:10.

[0011] Furthermore, in the preparation method of the mixed-mode liquid chromatography packing material as described above, the mass ratio of the 1-vinyl-3-octylimidazolium bromide ionic liquid to the silica microspheres is 1:5-1:10.

[0012] Furthermore, in the preparation method of the mixed-mode liquid chromatography packing material as described above, the ultrasonic dispersion time in step (2) is 20-40 min, and the magnetic stirring time is 0.5-1 h.

[0013] Further, in the preparation method of the mixed-mode liquid chromatography packing material as described above, the mass ratio of the 2,2'-azobisisobutyronitrile in step (3) to the 1-vinyl-3-octylimidazolium bromide ionic liquid in step (1) is 1:6-1:10;

[0014] The mechanical stirring speed is 150-250 rpm.

[0015] Furthermore, in the preparation method of the mixed-mode liquid chromatography packing material as described above, in step (4), the solid product is soaked in deionized water for 10-12 hours; the number of centrifugal washings with deionized water and ethanol is 3-6 times; the vacuum drying temperature is 70℃-85℃; and the drying time is 10-16 hours.

[0016] Furthermore, in the preparation method of the mixed-mode liquid chromatography packing material as described above, the particle size of the silica microspheres is 5 μm.

[0017] A mixed-mode liquid chromatography packing material prepared by the method described above.

[0018] The above-described mixed-mode liquid chromatography packing material is used in the detection and separation of hydrophilic compounds, anions, and alkylbenzenes.

[0019] The present invention has the following advantages over the prior art:

[0020] 1. This invention uses physical coating technology to introduce ionic liquid hybrid hydrogels onto the surface of silica microspheres to obtain liquid chromatography packing material based on the ionic liquid hybrid hydrogel mixing mode. The preparation path is simple and the operation is convenient.

[0021] 2. Due to the presence of hydrophilic amide groups, cations, and hydrophobic C8 long chains in the ionic liquid hybrid hydrogel structure, the packing material of this invention exhibits typical mixed-mode chromatographic packing material, which can be used for various separation modes such as hydrophilic / hydrophobic / ion exchange, and has good separation selectivity for nucleosides / bases, antibiotics, organic acid compounds, alkylbenzenes, and anions. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 A schematic diagram of the separation of nucleoside bases using an ionic liquid hybrid hydrogel modified silica gel column in a mobile phase of acetonitrile / 100mM ammonium acetate (90 / 10, v / v).

[0024] Figure 2 A schematic diagram of the separation of antibiotics using an ionic liquid hybrid hydrogel modified silica gel column under the condition of mobile phase: acetonitrile / 100mM ammonium acetate (70 / 30, v / v);

[0025] Figure 3 A schematic diagram of the separation of organic acids using an ionic liquid hybrid hydrogel modified silica gel column in a mobile phase of acetonitrile / 100mM ammonium acetate (86 / 14, v / v).

[0026] Figure 4 A schematic diagram of the separation of alkylbenzenes using an ionic liquid hybrid hydrogel-modified silica column under the condition of acetonitrile / water (26 / 84, v / v) as the mobile phase;

[0027] Figure 5 A schematic diagram of the separation of anions using an ionic liquid hybrid hydrogel-modified silica column in a mobile phase of acetonitrile / 20mM potassium chloride (88 / 12, v / v). Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0029] Hydrogels are hydrophilic but water-insoluble polymer networks that swell to equilibrium in water while maintaining their shape and three-dimensional network structure. As a novel hydrophilic polymer, hydrogels have attracted close attention since their inception 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 still relatively limited. Ionic liquids possess advantages such as stability, high conductivity, and functional designability. Adding them as additives to mobile phases or modifying silica gel surfaces can achieve good separation results. Incorporating ionic liquids into the hydrogel network structure can combine the advantages of both. Modifying these liquids into silica gel microspheres to prepare novel mixed-mode chromatographic packing materials and applying them to the separation of different compounds is a research area and development direction worth exploring and attempting.

[0030] The present invention is further illustrated below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0031] High performance liquid chromatograph, Hitachi (Japan); ionic liquid hybrid hydrogel modified silica column (150mm×4.6mm), Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences.

[0032] Example 1

[0033] A method for preparing liquid chromatography packing material based on ionic liquid hybrid hydrogel mixing mode includes the following steps:

[0034] (1) Dissolve the hydrogel monomer N-isopropylacrylamide (0.3g), 1-vinyl-3-octylimidazolium bromide (0.5g), ionic liquid, and crosslinking agent N,N-methylenebisacrylamide (0.05g) in 40ml of anhydrous ethanol;

[0035] (2) Add 2.5g of silica microspheres to the above mixed solution, sonicate for 20min, stir magnetically for 0.5h, and then let the mixture stand at room temperature for 12h. The silica microspheres were purchased from Suzhou Nanomicro Technology Co., Ltd., 5μm, 400m 2 / g;

[0036] (3) Add 0.05g of initiator 2,2'-azobisisobutyronitrile to the above mixture, place it in an oil bath, introduce N2 to remove air from the system, and heat to 70℃ for 5h under mechanical stirring (250rpm);

[0037] (4) After the reaction is complete, heat to 80°C to evaporate ethanol to obtain solid product A. Add deionized water and soak for 12 hours. Then, wash with deionized water and ethanol three times by centrifugation and place in a vacuum drying oven at 80°C for 12 hours to obtain liquid chromatography packing material based on ionic liquid hybrid hydrogel mixed mode.

[0038] Example 2:

[0039] The difference between this embodiment and Embodiment 1 is that: the mass ratio of N-isopropylacrylamide to 1-vinyl-3-octylimidazolium bromide ionic liquid is 1:1; the mass ratio of N,N-methylenebisacrylamide to 1-vinyl-3-octylimidazolium bromide ionic liquid is 1:6; and the mass ratio of 1-vinyl-3-octylimidazolium bromide ionic liquid to silica microspheres is 1:10.

[0040] In step (2), the ultrasonic dispersion time is 40 min and the magnetic stirring time is 1 h;

[0041] The mass ratio of the 2,2'-azobisisobutyronitrile to the 1-vinyl-3-octylimidazolium bromide ionic liquid is 1:6; the mechanical stirring speed is 150 rpm;

[0042] The solid product was soaked in deionized water for 10 hours; it was washed by centrifugation with deionized water and ethanol 6 times; the vacuum drying temperature was 70℃; and the drying time was 16 hours.

[0043] Example 3:

[0044] The difference between this embodiment and Embodiment 1 is that: the mass ratio of N-isopropylacrylamide to 1-vinyl-3-octylimidazolium bromide ionic liquid is 1:1.2; the mass ratio of N,N-methylenebisacrylamide to 1-vinyl-3-octylimidazolium bromide ionic liquid is 1:8; and the mass ratio of 1-vinyl-3-octylimidazolium bromide ionic liquid to silica microspheres is 1:7.

[0045] In step (2), the ultrasonic dispersion time is 30 min and the magnetic stirring time is 0.8 h;

[0046] The mass ratio of the 2,2'-azobisisobutyronitrile to the 1-vinyl-3-octylimidazolium bromide ionic liquid is 1:7; the mechanical stirring speed is 200 rpm;

[0047] The solid product was soaked in deionized water for 11 hours; it was washed by centrifugation with deionized water and ethanol 5 times; the vacuum drying temperature was 85℃; and the drying time was 13 hours.

[0048] Example 4: Isolation of nucleoside / base compounds

[0049] Preparation of ionic liquid hybrid hydrogel-modified silica gel chromatographic column: The ionic liquid hybrid hydrogel mixed-mode liquid chromatography packing material prepared in Example 1 was packed into a 150×4.6mm stainless steel liquid chromatography column using a high-pressure homogenization method for the separation and analysis of nucleosides / bases. During packing, both the dispersion and displacement solvent were methanol, and the pressure was 40 MPa.

[0050] Chromatographic separation of nucleosides / bases: Chromatographic analysis conditions were as follows: mobile phase: acetonitrile / 100mM ammonium acetate (90 / 10, v / v); flow rate: 1.0 mL / min; UV detector: 254 nm; injection volume: 20 μL.

[0051] The HPLC chromatogram for the separation of nucleosides / bases is shown below. Figure 1 The analytes were: 1. thymine, 2. thymidine, 3. uridine, 4. adenosine, 5. 6-chlorouracil, 6. inosine, 7. cytosine, and 8. guanosine. Figure 1 It can be seen that the liquid chromatography packing material based on the ionic liquid hybrid hydrogel hybrid mode has good separation selectivity for nucleosides / bases.

[0052] Example 5

[0053] Preparation of ionic liquid hybrid hydrogel-modified silica gel chromatographic column: The ionic liquid hybrid hydrogel mixed-mode liquid chromatography packing material prepared in Example 1 was packed into a 150×4.6mm stainless steel liquid chromatography column using a high-pressure homogenization method for the separation and analysis of antibiotic compounds. During packing, both the dispersion and displacement solvent were methanol, and the pressure was 40 MPa.

[0054] Chromatographic separation of antibiotics: The chromatographic analysis conditions were as follows: mobile phase: acetonitrile / 100mM ammonium acetate (70 / 30, v / v); flow rate: 1.0 mL / min; UV detector: 254 nm; injection volume: 20 μL. The liquid chromatogram of the antibiotics is shown below. Figure 2 The analytes were: 1. Ornidazole, 2. Metronidazole, 3. Cefuroxime sodium, 4. Cephalexin, and 5. Cefpirome sulfate. Figure 2 It can be seen that the liquid chromatography packing material based on the ionic liquid hybrid hydrogel hybrid mode has good separation selectivity for antibiotics.

[0055] Example 6: Isolation of Organic Acids

[0056] Preparation of ionic liquid hybrid hydrogel-modified silica gel chromatographic column: The ionic liquid hybrid hydrogel mixed-mode liquid chromatography packing material prepared in Example 1 was packed into a 150×4.6mm stainless steel liquid chromatography column using a high-pressure homogenization method for the separation and analysis of antibiotic compounds. During packing, both the dispersion and displacement solvent were methanol, and the pressure was 40 MPa.

[0057] Chromatographic separation of organic acid compounds: The chromatographic conditions were as follows: mobile phase: acetonitrile / 100 mM ammonium acetate (86 / 14, v / v); flow rate: 1.0 mL / min; UV detector: 254 nm; injection volume: 20 μL. The liquid chromatogram of antibiotics is shown below. Figure 3 The analytes were: 1. 3,5-dinitrobenzic acid; 2. salicylic acid; 3. p-nitrobenzoic acid; 4. α-naphthaleneacetic acid; 5. benzoic acid; 6. cinnamic acid; 7. p-hydroxybenzoic acid; and 8. nicotinic acid. Figure 3 It can be seen that the liquid chromatography packing material based on the ionic liquid hybrid hydrogel hybrid mode has good separation selectivity for organic acids.

[0058] Example 7: Separation of Alkylbenzene

[0059] Preparation of ionic liquid hybrid hydrogel-modified silica gel chromatographic column: The ionic liquid hybrid hydrogel mixed-mode liquid chromatography packing material prepared in Example 1 was packed into a 150×4.6mm stainless steel liquid chromatography column using a high-pressure homogenization method for the separation and analysis of antibiotic compounds. During packing, both the dispersion and displacement solvent were methanol, and the pressure was 40 MPa.

[0060] Chromatographic separation of alkylbenzene compounds: The chromatographic conditions were as follows: mobile phase: acetonitrile / water (26 / 74, v / v); flow rate: 0.8 mL / min; UV detector: 207 nm; injection volume: 20 μL. The liquid chromatogram of alkylbenzenes is shown below. Figure 4 The analytes were: 1. Benzene, 2. Toluene, 3. Ethylbenzene, 4. Propylbenzene, 5. Butylbenzene, and 6. Pentylbenzene. Figure 4 It can be seen that the liquid chromatography packing material based on the ionic liquid hybrid hydrogel hybrid mode has good separation selectivity for alkylbenzenes.

[0061] Example 8 Separation of anions

[0062] Preparation of ionic liquid hybrid hydrogel-modified silica gel chromatographic column: The ionic liquid hybrid hydrogel mixed-mode liquid chromatography packing material prepared in Example 1 was packed into a 150×4.6mm stainless steel liquid chromatography column using a high-pressure homogenization method for the separation and analysis of antibiotic compounds. During packing, both the dispersion and displacement solvent were methanol, and the pressure was 40 MPa.

[0063] Chromatographic separation of alkylbenzene compounds: The chromatographic conditions were as follows: mobile phase: acetonitrile / 20 mM potassium chloride (26 / 74, v / v); flow rate: 1 mL / min; UV detector: 210 nm; injection volume: 20 μL. The liquid chromatogram of alkylbenzenes is shown below. Figure 5 The analytes included were: 1. SCN - 2.I -3.NO3 - 4.NO2 - ,5.Br - .from Figure 5 It can be seen that the liquid chromatography packing material based on the ionic liquid hybrid hydrogel hybrid mode has good separation selectivity for anions.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a mixed-mode liquid chromatography packing material, characterized in that, Includes the following steps: (1) Dissolve the hydrogel monomer N-isopropylacrylamide, 1-vinyl-3-octylimidazolium bromide ionic liquid and crosslinking agent N,N-methylenebisacrylamide in anhydrous ethanol to obtain a mixed solution; (2) Add silica microspheres to the mixed solution, sonicate, stir magnetically, and then let the resulting mixture stand at room temperature for 10-12 hours; (3) Add the initiator 2,2'-azobisisobutyronitrile to the mixture after it has been standing, place it in an oil bath, introduce N2 to remove the air from the system, and react at 65-75°C for 4-6 hours with mechanical stirring; (4) After the reaction is completed, heat to 80°C to evaporate ethanol to obtain a solid product. Soak the solid product in deionized water, then wash it by centrifugation with deionized water and ethanol in sequence, and finally dry it in a vacuum drying oven to obtain a mixed-mode liquid chromatography packing material based on ionic liquid hybrid hydrogel.

2. The method for preparing the mixed-mode liquid chromatography packing material as described in claim 1, characterized in that: In step (1), the mass ratio of N-isopropylacrylamide to 1-vinyl-3-octylimidazolium bromide ionic liquid is 1:1 to 1:1.6; The mass ratio of the N,N-methylenebisacrylamide to the 1-vinyl-3-octylimidazolium bromide ionic liquid is 1:6 to 1:

10.

3. The method for preparing the mixed-mode liquid chromatography packing material as described in claim 1, characterized in that: The mass ratio of the 1-vinyl-3-octylimidazolium bromide ionic liquid to the silica microspheres is 1:5 to 1:

10.

4. The method for preparing the mixed-mode liquid chromatography packing material as described in claim 1, characterized in that: In step (2), the ultrasonic dispersion time is 20-40 min and the magnetic stirring time is 0.5-1 h.

5. The method for preparing the mixed-mode liquid chromatography packing material as described in claim 1, characterized in that: The mass ratio of the 2,2'-azobisisobutyronitrile in step (3) to the 1-vinyl-3-octylimidazolium bromide ionic liquid in step (1) is 1:6-1:10; The mechanical stirring speed is 150-250 rpm.

6. The method for preparing the mixed-mode liquid chromatography packing material as described in claim 1, characterized in that: In step (4), the solid product is soaked in deionized water for 10-12 hours; the deionized water and ethanol are centrifuged and washed 3-6 times; the vacuum drying temperature is 70℃-85℃; and the drying time is 10-16 hours.

7. The method for preparing the mixed-mode liquid chromatography packing material as described in claim 1, characterized in that: The silica microspheres have a particle size of 5 μm.

8. A mixed-mode liquid chromatography packing material prepared by the method of any one of claims 1-7.

9. The application of the mixed-mode liquid chromatography packing material as described in claim 8 in the detection and separation of hydrophilic compounds, anions, and alkylbenzenes.

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