Zwitterionic mixed-mode chromatographic stationary phase as well as preparation method and application thereof

Abstract

The invention discloses a zwitter-ion mixed-mode chromatographic stationary phase as well as a preparation method and application thereof. The polymer is mainly composed of a bottom tertiary amine (alkaline) group and a top negative ion (acidic) group, and hydrophobic groups are distributed in the polymer. Different groups can be modified on the surface of the silica gel through a direct bonding or indirect assembling method, and the process is simple, controllable and repeatable. The zwitter-ion stationary phase is novel in composition, stable in structure and adjustable in surface property, has zwitter-ion polar groups and non-polar groups such as alkyl / aryl, can provide multiple interactions at the same time, and is suitable for separating different types of substances in multiple chromatographic separation modes such as hydrophilicity, reverse phase and ion exchange.

Description

Technical Field

[0001] The present application relates to a zwitterion mixed mode chromatographic stationary phase and a preparation method and application thereof, belonging to the field of liquid chromatography. Background Art

[0002] As an effective separation and analysis method, high performance liquid chromatography has been widely used in the fields of chemical industry, environment, food, medicine, etc. The chromatographic column (chromatographic filler) is the core of the chromatographic separation system to achieve effective separation of different substances. The composition structure (matrix) and surface properties (stationary phase) of the filler directly determine its separation performance; therefore, the ideal chromatographic separation effect can be achieved by selecting a suitable matrix and stationary phase.

[0003] At present, reversed-phase chromatography stationary phase is the most popular in HPLC separation. It is mainly composed of non-polar groups (alkane or aromatic groups, such as C18 and phenyl, etc.), and is used to separate weakly polar or non-polar substances; while hydrophilic chromatography with polar groups (amino, hydroxyl, amide and zwitterion, etc.) as the stationary phase complements the reversed-phase chromatography selectivity, and can be used to separate highly polar or ionic substances that are difficult to retain by reversed-phase chromatography. Traditional reversed-phase and hydrophilic chromatography are usually stationary phases of a single nature, so the single retention mechanism (interaction) makes their separation ability relatively limited. Mixed-mode chromatography stationary phases selectively combine polar and non-polar groups so that they can provide multiple interactions at the same time, thus showing versatility: the properties of the stationary phase can be selectively used to separate different types of substances by adjusting the composition of the mobile phase.

[0004] The arrangement and combination of different polar groups can obtain mixed mode chromatographic stationary phases with different separation capabilities. Zwitterionic groups usually show good hydrophilicity and ion exchange capacity due to their special internal salt structure, and are generally used as hydrophilic chromatographic stationary phases (Guo Y. Biomed. Chromatogr, 2022, 36 (4): e5332.). Typical strong anionic and cationic groups-sulfobetaine (ZIC-HILIC) and phosphorylcholine (ZIC-cHILIC) zwitterionic stationary phases have been commercialized and promoted for use and show good separation selectivity. In addition, amino acids are used as zwitterionic groups as a whole to develop hydrophilic chromatographic stationary phases (CN102614847B), and new weak anionic and cationic groups-tertiary amines and carboxyl groups are also used in hydrophilic chromatography with adjustable zwitterionic ratios (CN104785230B).

[0005] At present, there are a few reports on zwitterionic mixed-mode chromatographic stationary phases, but there is still no commercial filler available (CN107106931A, CN115245816B, CN111389383B). Different types of stationary phase compositions show different separation performances, but it is still difficult to meet complex separation requirements. Moreover, these zwitterionic stationary phases are mostly distributed on the silica gel surface with monosilane as the medium, and the hydrolysis stability faces challenges in complex separation situations.

[0006] In summary, new zwitterionic mixed-mode chromatographic stationary phases with good stability and multifunctional separation performance are still the demand and goal of the development of HPLC. Summary of the invention

[0007] To this end, the object of the present invention is to provide a novel zwitterionic mixed-mode chromatographic stationary phase and a preparation method thereof, wherein the zwitterionic mixed-mode chromatographic stationary phase has both zwitterionic polar groups and non-polar groups, can provide multiple interactions simultaneously and has stable performance, and the preparation process is simple, controllable and repeatable.

[0008] According to one aspect of the present application, a zwitterionic mixed-mode chromatographic stationary phase is provided, wherein the zwitterionic mixed-mode chromatographic stationary phase has a structure shown in Formula I:

[0009]

[0010] Formula I;

[0011] The Silica on the left is silica gel, including pure silica or impure silica, with a uniform spherical structure and a particle size distribution of D 90 / D 10 The particle size is 1-100 μm, the pore size is 6-600 nm, and the specific surface area is 10-1000 m 2 / g;

[0012] The structural formula on the right is the ligand;

[0013] Wherein, N is a nitrogen atom;

[0014] Z is a negatively charged group selected from a carboxyl group, a sulfonic acid group or a phosphonic acid group;

[0015] R 1 , R 2 , R 3 , R 4 , R 5 and R 6 Each is independently a linking group or a free group;

[0016] The linking group is an oxygen atom;

[0017] The free radical is selected from at least one of a methoxy group, an ethoxy group, a halogen atom, a hydroxyl group, an alkyl group and an aryl group;

[0018] R 1 , R 2 , R 3 , R 4 , R 5 and R 6 In the case of three adjacent groups (R 1 / R 2 / R 3 and R 4 / R 5 / R 6 ) has at least one linking group, when there are multiple linking groups, they are not the same linking group, the linking group connects the ligand to the silica gel, or the linking group connects the Si in the ligand to each other;

[0019] L 1 , L 2 , L 3 and L 4 Each independently selected from at least one of an alkyl group or an aryl group having 0 to 30 carbon atoms;

[0020] X and Y are each independently selected from at least one of an oxygen atom, a sulfur atom, a hydroxyl group, an amino group, an ester group, an amide group or a sulfonyl group;

[0021] n and m are each independently selected from integers between 0 and 30, including 0.

[0022] According to another aspect of the present application, a method for preparing the above-mentioned zwitterionic mixed-mode chromatographic stationary phase is provided, comprising the following steps:

[0023] Washing or vacuum drying the silica gel to obtain activated silica gel;

[0024] The reaction solvent, activated silica gel, disilyl secondary amine ligand and negatively charged group reagent are mixed, reacted, washed with the reaction solvent, methanol, pure water and methanol in sequence, and dried to obtain the zwitterionic mixed mode chromatographic stationary phase.

[0025] Optionally, the following steps are included:

[0026] Washing or vacuum drying the silica gel to obtain activated silica gel;

[0027] A reaction solvent, a disilyl secondary amine ligand and a negatively charged group reagent are mixed to assemble a zwitterionic silane target ligand;

[0028] The reaction solvent, activated silica gel and zwitterionic silane target ligand are mixed, reacted, washed with reaction solvent, methanol, pure water and methanol in sequence, and dried to obtain the zwitterionic mixed mode chromatographic stationary phase.

[0029] Optionally, the following steps are included:

[0030] Washing or vacuum drying the silica gel to obtain activated silica gel;

[0031] The reaction solvent, the disilyl secondary amine ligand and the activated silica gel are mixed, reacted, washed with the reaction solvent, methanol, pure water and methanol in sequence, and dried to obtain modified silica gel;

[0032] The reaction solvent, modified silica gel and negatively charged group reagent are mixed, reacted, washed with the reaction solvent, methanol, pure water and methanol in sequence, and dried to obtain the zwitterion mixed mode chromatographic stationary phase.

[0033] Washing the silica gel and then vacuum drying it or directly vacuum drying it can activate the hydroxyl groups on the surface of the silica gel and remove moisture adsorbed on the surface of the silica gel and impurities in the pores.

[0034] The washing treatment is to add silica gel into hydrochloric acid, nitric acid, hydrofluoric acid aqueous solution or pure water, stand at room temperature or heat and stir under reflux for 2 to 72 hours, then filter and rinse with pure water until neutral.

[0035] The vacuum drying treatment is to vacuum dry the silica gel at 60-180° C. for 2-72 hours until constant weight is reached.

[0036] The disilyl secondary amine ligand has a structure shown in Formula II:

[0037]

[0038] Formula II;

[0039] Where R 1 , R 2 , R 3 , R 4 , R 5 and R 6 Each is independently a leaving group or an inert group;

[0040] The leaving group is selected from at least one of a methoxy group, an ethoxy group or a halogen atom;

[0041] The inert group is selected from at least one of an alkyl group or an aryl group;

[0042] R 1 , R 2 , R 3 , R 4 , R5 and R 6 In the case of three adjacent groups (R 1 / R 2 / R 3 and R 4 / R 5 / R 6 ) has at least one leaving group.

[0043] The negatively charged group reagent is selected from sultone or a compound having a structure shown in Formula III:

[0044]

[0045] Formula III;

[0046] Wherein, A is selected from one of a halogen atom, an epoxy group or a double bond group.

[0047] The reaction solvent is selected from at least one of toluene, xylene, ethanol, acetonitrile, tetrahydrofuran, dimethylformamide and dichloromethane.

[0048] The ratio of the activated silica gel to the reaction solvent is 1 g: 5 to 50 mL;

[0049] The ratio of the activated silica gel to the disilyl secondary amine ligand is 1g: 0.1-10mmol;

[0050] The molar ratio of the disilyl secondary amine ligand to the negatively charged group reagent is 1:1-2.

[0051] The assembly and reaction are performed under an inert gas atmosphere, and the inert gas atmosphere is selected from a nitrogen atmosphere and / or an argon atmosphere.

[0052] The reaction temperature is from room temperature to the boiling point of the reaction solvent;

[0053] The reaction time is 2 to 72 hours;

[0054] The drying is vacuum drying;

[0055] The drying temperature is 60 to 180°C;

[0056] The drying time is 2 to 72 hours.

[0057] According to another aspect of the present application, there is provided an application of the above-mentioned zwitterionic mixed-mode chromatographic stationary phase for use in liquid chromatography.

[0058] The beneficial effects of this application include:

[0059] The zwitterionic mixed-mode chromatographic stationary phase has a novel composition, stable structure, and adjustable surface properties. It has both tertiary amine and negative ion groups, and the stationary phase can be selectively designed by adjusting the mobile phase composition or reagent type, thereby adjusting the surface properties and separation performance of the stationary phase. In addition, the tertiary amine group is connected to the silica gel surface through disilane, which can greatly increase the hydrolysis stability of the stationary phase and extend its service life.

[0060] Zwitterionic mixed-mode chromatographic stationary phases are multifunctional. They have both zwitterionic polar groups and non-polar groups such as alkyl / aryl groups. They can provide multiple interactions simultaneously and selectively exhibit different degrees of hydrophilicity, hydrophobicity and ion exchange properties. Therefore, they can separate different types of substances under the same conditions, and can also selectively separate the same type of substances under different conditions, thereby achieving efficient utilization of resources, that is, one column can be used for multiple purposes in chromatographic separations.

[0061] The preparation process of zwitterionic mixed-mode chromatographic stationary phase is simple, controllable and repeatable. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Schematic diagram of the stationary phase structure.

[0063] Figure 2 This is a schematic diagram of the stationary phase structure prepared in Example 2.

[0064] Figure 3 The chromatogram is a hydrophilic mode separation.

[0065] Figure 4 The chromatogram is for the reverse phase separation mode. DETAILED DESCRIPTION

[0066] The embodiments may help to understand the present invention, but the present invention is not limited to the embodiments.

[0067] The silica gel described in the examples was prepared by the laboratory, and its particle size and distribution were measured by Mastersizer 3000 combined with JSM-7800F field emission scanning electron microscope; its pore structure characteristics were measured by Autosorb-iQ2 physical adsorption instrument. The application of zwitterionic mixed mode chromatographic stationary phase in the examples was evaluated by Agilent 1260 InfinityⅡ liquid chromatograph.

[0068] Example 1

[0069] (1) Take 30 g of silica gel (5 μm, 300 m 2 / g, 8nm) was vacuum dried at 120°C for 72h to obtain activated silica gel, which was placed in a desiccator for later use.

[0070] (2) 30 g of activated silica gel was added to 300 mL of anhydrous toluene and stirred evenly, then 30.74 g of bis(3-trimethoxysilyl)propylamine was added thereto, heated under reflux for 48 h in a nitrogen environment, cooled to room temperature, filtered, and washed with toluene, methanol, pure water and methanol in sequence, and finally dried under vacuum at 120°C for 24 h to obtain secondary amine bonded silica gel. 10 g of secondary amine bonded silica gel was dispersed in 100 mL of anhydrous dimethylformamide, then 7.54 g of 10-bromodecanoic acid and 6.07 g of triethylamine were added thereto, heated at 60°C for 72 h in a nitrogen environment, cooled to room temperature, filtered, and washed with dimethylformamide, methanol, pure water and methanol in sequence, and finally dried under vacuum at 60°C for 12 h to obtain a zwitterionic mixed mode chromatographic stationary phase.

[0071] Example 2

[0072] (1) Take 10 g of silica gel (5 μm, 300 m 2 / g, 8nm) was added into 100mL 10wt.% hydrochloric acid solution and stirred evenly, heated to reflux for 12h, cooled to room temperature, filtered and washed to neutrality, and then vacuum dried at 180°C for 12h to obtain activated silica gel, which was placed in a desiccator for use.

[0073] (2) 10 g of activated silica gel was added to 100 mL of anhydrous xylene and stirred evenly, then 8.20 g of bis(3-trimethoxysilyl)propylamine was added thereto, heated under reflux for 24 h in a nitrogen environment, cooled to room temperature, filtered, and washed with xylene, methanol, pure water and methanol in sequence, and finally dried under vacuum at 120°C for 12 h to obtain secondary amine-bonded silica gel. 3 g of secondary amine-bonded silica gel was dispersed in 50 mL of anhydrous acetonitrile, then 1.75 g of 2-(4-bromomethylphenyl)propionic acid was added thereto, heated under reflux for 12 h in a nitrogen environment, cooled to room temperature, filtered, and washed with acetonitrile, methanol, pure water and methanol in sequence, and finally dried under vacuum at 60°C for 12 h to obtain a zwitterionic mixed mode chromatographic stationary phase.

[0074] Example 3

[0075] (1) Take 10 g of silica gel (5 μm, 300 m 2 / g, 8nm) was added into 100mL of 0.02wt.% hydrofluoric acid solution and stirred evenly, heated under reflux for 24h, cooled to room temperature, filtered and washed to neutrality, and then vacuum dried at 180°C for 12h to obtain activated silica gel, which was placed in a desiccator for use.

[0076] (2) 10 g of activated silica gel was added to 100 mL of anhydrous xylene and stirred evenly, then 4.10 g of bis(3-trimethoxysilyl)propylamine was added thereto, heated under reflux for 24 h in a nitrogen environment, cooled to room temperature, filtered, and washed with xylene, methanol, pure water and methanol in sequence, and finally dried under vacuum at 60°C for 12 h to obtain secondary amine-bonded silica gel. 3 g of secondary amine-bonded silica gel was dispersed in 50 mL of anhydrous acetonitrile, then 0.55 g of 3-bromopropionic acid was added thereto, heated under reflux for 24 h in a nitrogen environment, cooled to room temperature, filtered, and washed with acetonitrile, methanol, pure water and methanol in sequence, and finally dried under vacuum at 60°C for 6 h to obtain a zwitterionic mixed mode chromatographic stationary phase.

[0077] Example 4

[0078] (1) Take 10 g of silica gel (5 μm, 300 m 2 / g, 8nm) was added into 100mL 10wt.% nitric acid solution and stirred evenly, heated to reflux for 12h, cooled to room temperature, filtered and washed to neutrality, and then vacuum dried at 180°C for 24h to obtain activated silica gel, which was placed in a desiccator for use.

[0079] (2) Add 10 g of activated silica gel to 100 mL of anhydrous xylene and stir evenly, then add 4.10 g of bis(3-trimethoxysilyl)propylamine, heat and reflux for 24 h under nitrogen, cool to room temperature, filter and wash with xylene, methanol, pure water and methanol in turn, and finally dry under vacuum at 60 ° C for 12 h to obtain secondary amine bonded silica gel. Take 3 g of secondary amine bonded silica gel and disperse it in 50 mL of anhydrous xylene, then add 0.44 g of propane sultone, heat and reflux for 24 h under nitrogen, cool to room temperature, filter and wash with xylene, methanol, pure water and methanol in turn, and finally dry under vacuum at 60 ° C for 6 h to obtain a zwitterionic mixed mode chromatographic stationary phase.

[0080] Example 5

[0081] (1) Take 10 g of silica gel (5 μm, 300 m 2 / g, 8nm) was vacuum dried at 180°C for 24h to obtain activated silica gel, which was placed in a desiccator for later use.

[0082] (2) 3.07 g of bis[3-(triethoxysilyl)propyl]amine was added to 50 mL of anhydrous acetonitrile and stirred evenly, and then 1.75 g of 2-(4-bromomethylphenyl)propionic acid was added thereto. The mixture was heated to reflux for 12 h under a nitrogen environment. After cooling to room temperature, 3 g of activated silica gel was added thereto. The mixture was heated to reflux for 48 h under a nitrogen environment. After cooling to room temperature, the mixture was filtered and washed with acetonitrile, methanol, pure water and methanol in sequence. Finally, the mixture was dried under vacuum at 60°C for 12 h to obtain a zwitterionic mixed mode chromatographic stationary phase.

[0083] Example 6

[0084] (1) Take 10 g of silica gel (5 μm, 300 m 2 / g, 8nm) was vacuum dried at 120°C for 72h to obtain activated silica gel, which was placed in a desiccator for later use.

[0085] (2) 10.22 g of bis[3-(triethoxysilyl)propyl]amine was added to 100 mL of anhydrous toluene and stirred evenly, and then 6.03 g of 10-bromodecanoic acid was added thereto. The mixture was heated to reflux for 24 h under a nitrogen environment. After cooling to room temperature, 10 g of activated silica gel was added thereto. The mixture was heated to reflux for 72 h under a nitrogen environment. After cooling to room temperature, the mixture was filtered and washed with toluene, methanol, pure water and methanol in sequence. Finally, the mixture was dried under vacuum at 60°C for 12 h to obtain a zwitterionic mixed mode chromatographic stationary phase.

[0086] Example 7

[0087] The zwitterionic mixed mode stationary phase prepared in Example 2 (its stationary phase structure schematic diagram is shown in Figure 2 ) was used as an example to evaluate in the hydrophilic mode. The chromatographic evaluation conditions were: chromatographic column specifications: 150×4.6mm id; detector: UV-visible detector 254nm; mobile phase: acetonitrile / water = 90 / 10v / v; flow rate: 1mL / min; column temperature: 30℃; injection volume: 1μL. The chromatographic evaluation results are shown in Figure 3 :Six hydrophilic nucleosides were successfully separated. The results showed that the stationary phase has good hydrophilicity and can be used in the hydrophilic mode to selectively separate polar compounds.

[0088] Example 8

[0089] The zwitterionic mixed mode stationary phase prepared in Example 2 was used as an example for evaluation in reverse phase mode. The chromatographic evaluation conditions were: chromatographic column specifications: 150×4.6 mm id; detector: UV-visible light detector 254 nm; mobile phase: acetonitrile / water = 30 / 70 v / v; flow rate: 1 mL / min; column temperature: 30°C; injection volume: 1 μL. The chromatographic evaluation results are shown in Figure 4 :Six alkylbenzenes were successfully separated, and the results showed that the stationary phase has good hydrophobicity and can be used in reversed-phase mode to selectively separate non-polar compounds. Therefore, the stationary phase has mixed-mode separation capabilities and exhibits multifunctionality.

[0090] The above are only a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A zwitterionic mixed-mode chromatographic stationary phase, characterized in that: The zwitterionic mixed-mode chromatographic stationary phase has a structure shown in Formula I: Among them, the Silica on the left is silica gel, including pure silica or impure silica, with a uniform spherical structure and a particle size distribution of D 90 / D 10 The particle size is 1-100 μm, the pore size is 6-600 nm, and the specific surface area is 10-1000 m 2 / g; The structural formula on the right is the ligand; Wherein, N is a nitrogen atom; Z is a negatively charged group selected from a carboxyl group, a sulfonic acid group or a phosphonic acid group; R 1 , R 2 , R 3 , R 4 , R 5 and R 6 Each is independently a linking group or a free group; The linking group is an oxygen atom; The free radical is selected from at least one of a methoxy group, an ethoxy group, a halogen atom, a hydroxyl group, an alkyl group and an aryl group; R 1 , R 2 , R 3 , R 4 , R 5 and R 6 In the embodiment, there is at least one connecting group among the three adjacent groups on the same Si. When there are multiple connecting groups, they are not the same connecting group. The connecting group connects the ligand to the silica gel, or the connecting group connects the Si in the ligand to each other. L 1 , L 2 , L 3 and L 4 Each independently selected from at least one of an alkyl group or an aryl group having 0 to 30 carbon atoms; X and Y are each independently selected from at least one of an oxygen atom, a sulfur atom, a hydroxyl group, an amino group, an ester group, an amide group or a sulfonyl group; n and m are each independently selected from integers between 0 and 30, including 0.

2. A method for preparing the zwitterionic mixed-mode chromatographic stationary phase according to claim 1, characterized in that: The following steps are involved: Washing or vacuum drying the silica gel to obtain activated silica gel; The reaction solvent, activated silica gel, disilyl secondary amine ligand and negatively charged group reagent are mixed, reacted and dried to obtain the zwitterion mixed mode chromatographic stationary phase.

3. The preparation method according to claim 2, characterized in that: The following steps are involved: Washing or vacuum drying the silica gel to obtain activated silica gel; A reaction solvent, a disilyl secondary amine ligand and a negatively charged group reagent are mixed to assemble a zwitterionic silane target ligand; The reaction solvent, activated silica gel and zwitterionic silane target ligand are mixed, reacted and dried to obtain the zwitterionic mixed mode chromatographic stationary phase.

4. The preparation method according to claim 2, characterized in that: The following steps are involved: Washing or vacuum drying the silica gel to obtain activated silica gel; The reaction solvent, the disilyl secondary amine ligand and the activated silica gel are mixed, reacted, and dried to obtain modified silica gel; The reaction solvent, modified silica gel and negatively charged group reagent are mixed, reacted and dried to obtain the zwitterion mixed mode chromatographic stationary phase.

5. The preparation method according to claim 2, characterized in that: The disilyl secondary amine ligand has a structure shown in Formula II: wherein R1, R2, R3, R4, R5 and R6 are each independently a leaving group or an inert group; The leaving group is selected from at least one of a methoxy group, an ethoxy group or a halogen atom; The inert group is selected from at least one of an alkyl group or an aryl group; Among R1, R2, R3, R4, R5 and R6, there is at least one leaving group among the three adjacent groups on the same Si.

6. The preparation method according to claim 2, characterized in that: The negatively charged group reagent is selected from sultone or a compound having a structure shown in Formula III: Formula III; Wherein, A is selected from one of a halogen atom, an epoxy group or a double bond group.

7. The preparation method according to claim 2, characterized in that: The reaction solvent is selected from at least one of toluene, xylene, ethanol, acetonitrile, tetrahydrofuran, dimethylformamide and dichloromethane.

8. The preparation method according to claim 2, characterized in that: The ratio of the activated silica gel to the reaction solvent is 1 g: 5 to 50 mL; The ratio of the activated silica gel to the disilyl secondary amine ligand is 1g: 0.1-10mmol; The molar ratio of the disilyl secondary amine ligand to the negatively charged group reagent is 1:1-2.

9. The preparation method according to claim 2, characterized in that: The reaction time is 2 to 72 hours; The drying temperature is 60 to 180°C; The drying time is 2 to 72 hours.

10. A use of the zwitterionic mixed mode chromatographic stationary phase according to claim 1, characterized in that: Used in liquid chromatography.

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