SiO2@pMPC chromatographic stationary phase and its preparation method and application

Abstract

The present invention discloses a SiO2@pMPC chromatographic stationary phase, its preparation method, and application, belonging to the technical field of hydrophilic chromatographic stationary phases. The preparation method comprises the following steps: acidifying silica gel to obtain activated silica gel; subjecting the activated silica gel to a silane coupling reaction with a silanizing agent under a nitrogen atmosphere to obtain amino-modified silica gel; modifying the surface of the silica gel with an initiator to obtain bromosilica gel with bromine on the surface; mixing the bromosilica gel with bromine on the surface with a catalyst, a catalyst ligand, and a functional monomer; and subjecting the mixture to a surface-initiated atom transfer radical polymerization reaction at 20 to 75°C to obtain the SiO2@pMPC chromatographic stationary phase. The SiO2@pMPC chromatographic stationary phase synthesized by this method greatly improves the surface monomer grafting rate, has excellent chemical stability, and exhibits excellent chromatographic performance in actual separation and analysis applications, thus having great promotional value.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrophilic chromatography stationary phases, and more particularly to a SiO2@pMPC chromatography stationary phase and a preparation method and application thereof. Background Art

[0002] With the development of chromatographic technology, high performance liquid chromatography (HPLC) has been widely used in important fields such as food and medicine, environmental safety, metabolomics, etc. Drugs and their metabolites are usually polar, but their traditional reverse phase and normal phase chromatography modes are difficult to meet the analytical requirements for polar compounds. Conventional reverse phase chromatography uses non-polar stationary phase and polar mobile phase conditions to achieve the separation of target analytes, and the retention of hydrophilic high polar substances is very weak or substantially unretained. Normal phase chromatography based on a combination of polar stationary phase and non-polar mobile phase conditions is expected to overcome such problems, but hydrophilic polar compounds have low solubility under normal phase chromatography mobile phase conditions [Rizzi A, Katz E, Eksteen R, et al. Handbook of HPLC. CRC Press, 1998: 1-223], which brings great challenges to its analysis. Against this backdrop, hydrophilic chromatography (HILIC) emerged [Alpert A J. Journal of Chromatography A, 1990, 499: 177-196]. By combining a polar stationary phase with a less polar aqueous mobile phase, HILIC can simultaneously address the shortcomings of both normal and reversed-phase chromatography in separating water-soluble polar compounds.

[0003] Among currently commercialized HILIC stationary phases, zwitterionic stationary phases are increasingly popular in the market due to their unique structure of coexisting positive and negative charges, resulting in excellent hydrophilicity. Most commercial zwitterionic stationary phases are based on silica gel and are easily soluble in alkaline environments. Currently, the pH tolerance of commonly used zwitterionic chromatography stationary phases is only 3 to 8. Zwitterionic chromatography stationary phases based on organic polymer matrices can tolerate a pH range of 2 to 10, but their mechanical strength is lower than that of silica gel matrices and they are prone to swelling.

[0004] Surface-initiated atomic radical polymerization (SI-ATRP) is a highly controllable surface modification method that can graft polymer chains with controlled molecular weight and narrow molecular weight distribution onto the surface of materials [Nagase K, Kobayashi J, Kikuchi A, et al. Langmuir, 2008, 24(2): 511-517]. Polymer coatings with various morphologies have been successfully synthesized on the surface of silica supports by SI-ATRP [Mu B, Wang T, Liu P. Industrial & Engineering Chemistry Research, 2007, 46(10): 3069-3072]. More and more scholars are using SI-ATRP as a new tool to develop stationary phases for grafting polymerization onto the surface of silica microspheres.

[0005] Phosphorylcholine (PC) groups are terminal hydrophilic groups on the outer bilayer of cell membranes, possessing a zwitterionic structure and equal positive and negative charges. MPC, the simplest compound containing a PC group, has been widely used in chromatography and exhibits excellent chromatographic performance. However, existing MPC-modified silica microsphere chromatographic stationary phases synthesized using thiol-ene click chemistry methods exhibit low levels of surface-grafted monomers [Xiong C, Yuan J, Wang Z, et al. Journal of Chromatography A, 2018, 1546:56-65]. Therefore, there is a need for a more efficient method for synthesizing organic / inorganic hybrid HILIC stationary phases with MPC monomers grafted onto the surface of silica microspheres. Summary of the Invention

[0006] Against this background, the primary objective of the present invention is to provide a method for preparing a SiO2@pMPC chromatographic stationary phase (an organic / inorganic hybrid HILIC stationary phase composed of silica microspheres grafted with MPC monomers). This method, based on the SI-ATRP method, aims to improve the pH tolerance of the chromatographic stationary phase while maintaining the high mechanical strength of the silica matrix. The successful preparation of this SiO2@pMPC chromatographic stationary phase not only enriches the variety of zwitterion separation materials in this category but also provides a reference for the development of such chromatographic fillers.

[0007] The second object of the present invention is to provide a SiO2@pMPC chromatographic stationary phase (organic / inorganic hybrid HILIC stationary phase with MPC monomer grafted onto the surface of silica microspheres) prepared by the above method.

[0008] The third object of the present invention is to provide the separation application of the above-mentioned SiO2@pMPC chromatographic stationary phase (organic / inorganic hybrid HILIC stationary phase with MPC monomer grafted onto the surface of silica microspheres) in the hydrophilic chromatography mode.

[0009] The primary purpose of the present invention can be achieved by the following technical solutions:

[0010] A method for preparing a SiO2@pMPC chromatographic stationary phase comprises the following steps:

[0011] (1) Preparation of activated silica gel: Silica gel was acidified with a concentrated hydrochloric acid / methanol mixed solution in an equal volume ratio of 1 g:50-200 mL to obtain activated silica gel;

[0012] (2) Surface modification of amino-modified silica gel: Using toluene as solvent, under nitrogen atmosphere, the activated silica gel in S1 was subjected to silane coupling reaction with a silanization reagent at a mass volume ratio of 1 g: 1-3 mL, and then dried to obtain amino-modified silica gel;

[0013] (3) Synthesis of brominated silica spheres: Using anhydrous tetrahydrofuran as solvent, the amino-modified silica gel in S2, triethylamine and 2-bromo-isobutyryl bromide were coupled in a mass volume ratio of 1 g: 0.3-1: 0.1-2.0 mL, and vacuum dried to obtain brominated silica gel with bromine modified on the surface;

[0014] (4) Synthesis of SiO2@pMPC chromatographic stationary phase: Under a protective gas atmosphere, phospholipid functional monomers, brominated silica gel in S3, catalyst: 2,2-bipyridine, and cuprous chloride are reacted by atom transfer radical polymerization in a molar ratio of 50-200:1:1-4:2-8, and vacuum dried to obtain SiO2@pMPC chromatographic stationary phase.

[0015] Preferably, the acidification treatment temperature in step (1) is 70-110° C., the reaction time is 4-12 h, and the activated silica gel obtained after the reaction is placed at 80-120° C. and vacuum dried for 12 h.

[0016] Preferably, the silanization agent in step (2) is aminopropyltriethoxysilane.

[0017] Preferably, in step (2), the silane coupling reaction temperature is 20-200° C., and the continuous stirring time is 2-48 h; the drying temperature is 80-120° C., and the drying time is 12 h.

[0018] Preferably, the coupling reaction temperature in step (3) is 0°C, the coupling reaction time is 0.5-2h, and then the reaction is transferred to room temperature for 12-24h; the vacuum drying temperature is 60-120°C, and the vacuum drying time is 12h.

[0019] Preferably, in step (4), the reaction temperature is 20-75° C., and the reaction time is 12-24 h; the vacuum drying temperature is 60-120° C., and the vacuum drying time is 12 h.

[0020] Preferably, the specific method for pre-treating cuprous chloride in step (4) is: repeatedly washing cuprous chloride with 0.1 mol / L dilute acetic acid solution until the green solid turns white, and then repeatedly washing the solution with methanol until it becomes neutral (4-6 times).

[0021] The second object of the present invention can be achieved by the following technical solutions:

[0022] A SiO2@pMPC chromatographic stationary phase is prepared by the above method.

[0023] Preferably, the structural formula of the SiO2@pMPC chromatographic stationary phase is as follows:

[0024]

[0025] The third object of the present invention can be achieved by the following technical solutions:

[0026] A SiO2@pMPC chromatographic stationary phase (an organic / inorganic hybrid HILIC stationary phase with MPC monomer grafted onto the surface of silica microspheres) is used for separation in hydrophilic chromatography mode.

[0027] Compared with the prior art, the present invention has the following technical advantages and beneficial effects:

[0028] (1) The SiO2@pMPC chromatographic stationary phase of the present invention has good separation performance for common hydrophilic compounds;

[0029] (2) The SiO2@pMPC chromatographic stationary phase of the present invention has good stability, long service life, and can maintain good separation performance under harsh acid and alkaline conditions;

[0030] (3) In the preparation process of the SiO2@pMPC chromatographic stationary phase described in the present invention, cuprous chloride is used to replace the original cuprous bromide in the reaction, which improves the reaction performance of SI-ATRP and further increases the density of the grafted polymer on the silica gel surface. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Comparison of the separation effects of SiO2@pMPC chromatographic stationary phases synthesized with different catalytic systems;

[0032] Figure 2 This is the electron microscopy characterization of SiO2@pMPC chromatographic stationary phase;

[0033] Figure 3 This is the full spectrum of X-ray electron energy elemental analysis of SiO2@pMPC chromatographic stationary phase;

[0034] Figure 4 This is the characteristic spectrum of phosphorus element analyzed by X-ray electron energy of SiO2@pMPC chromatographic stationary phase;

[0035] Figure 5 This is the chromatographic separation diagram of urea and allantoin separated by SiO2@pMPC chromatographic stationary phase as described in Example 2;

[0036] Figure 6 This is the chromatographic separation diagram of phenol compounds separated by SiO2@pMPC chromatographic stationary phase as described in Example 2;

[0037] Figure 7 This is a graph showing the continuous injection and long-term stability test of the SiO2@pMPC chromatographic stationary phase described in Example 2;

[0038] Figure 8 This is a test chart of the pH tolerance (acid-base) stability of the SiO2@pMPC chromatographic stationary phase described in Example 2. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below with reference to specific examples, but the embodiments of the present invention are not limited thereto. The materials used in the examples of the present invention can all be purchased commercially.

[0040] Example 1

[0041] The preparation method of SiO2@pMPC chromatographic stationary phase comprises the following steps:

[0042] (1) Preparation of activated silica gel: Weigh 1 g of spherical silica gel (particle size 5 μm, pore size 10 nm, specific surface area 450 m 2 / g) was placed in a 250mL three-necked flask, 50mL of methanol solution and 50mL of concentrated hydrochloric acid solution were added in sequence, heated to 70℃ and refluxed for 4h, and after the reaction, repeatedly washed with pure water, and dried at 120℃ for 12h to obtain activated silica gel;

[0043] (2) Surface modification of amino-modified silica gel: The dried activated silica gel was placed in a 250 mL three-necked flask, and 100 mL of anhydrous toluene was added. After the container was sealed, 1.5 mL of APTES was added via a syringe after heating the system to 110 °C and then heated under reflux for 12 h. After the reaction, the silica gel was washed with toluene, pure water, and acetone in sequence, and dried at 120 °C for 12 h to obtain amino-modified silica gel.

[0044] (3) Synthesis of bromosilica spheres: The dried amino-coated silica gel was placed in a 100 mL three-necked flask, 30 mL of anhydrous THF was added and the mixture was placed at 0°C, followed by 2.3 mL of TEA and 1.5 mL of BIBB (added dropwise using a separatory funnel). The mixture was reacted at 0°C for 1.5 h and then transferred to room temperature for 24 h. After the reaction, the mixture was washed with THF, pure water, and acetone in sequence and dried at 80°C for 12 h to obtain bromosilica spheres.

[0045] (4) Synthesis of SiO2@pMPC chromatographic stationary phase: Add the dried brominated silica gel into a 100 mL high-pressure sealed reaction bottle, and then add 12 mL of ultra-dry methanol, 2.4 g of MPC monomer, 15 mg of pretreated cuprous chloride, 21 mg of 2-2-bipyridine, and 10 mg of sodium chloride in sequence. Then, repeatedly evacuate and fill with nitrogen 5-6 times. Place the sealed container at 55°C for 24 h, then let it stand for 12 h to quench the reaction. Repeatedly wash with 0.1 mol / L EDTA solution, distilled water, ethanol, and acetone in sequence, and then dry at 60°C for 12 h to obtain the SiO2@pMPC chromatographic stationary phase.

[0046] Column efficiency test: N 尿嘧啶 =38224N / m.

[0047] Example 2

[0048] The difference from Example 1 is the synthesis of SiO2@pMPC in step (4): the dried bromosilica gel is added to a 100 mL high-pressure sealed reaction bottle, followed by the addition of 12 mL of ultra-dry methanol, 2.4 g of MPC monomer, 15 mg of pretreated cuprous bromide, 21 mg of 2-2-bipyridine, and 10 mg of sodium chloride. The reaction is then repeated by vacuuming and nitrogen filling for 5-6 times. The sealed container is placed at 55°C for 24 hours, then allowed to stand for 12 hours to quench the reaction, and then washed repeatedly with 0.1 mol / L EDTA solution, distilled water, ethanol, and acetone, followed by drying at 60°C for 12 hours to obtain the SiO2@pMPC chromatogram. Column efficiency test: N 尿嘧啶 =19423N / m.

[0049] Phospholipid functional monomers, brominated silica gel in S3, catalyst: 2,2-bipyridine, cuprous chloride in a mass volume ratio of 50-200:1:1-4:2-8 were subjected to atom transfer radical polymerization reaction and vacuum dried.

[0050] Example 3

[0051] The difference from Example 2 lies in the synthesis of SiO2@pMPC in step (4): the dried bromosilica gel was added to a 100 mL high-pressure sealed reaction bottle, followed by the addition of 12 mL of ultra-dry methanol, 2.4 g of MPC monomer, 20 mg of pretreated cuprous chloride, 28 mg of 2-2-bipyridine, and 10 mg of sodium chloride. The mixture was then repeatedly evacuated and filled with nitrogen 5-6 times. The sealed container was placed at 55°C for reaction for 24 h, then allowed to stand for 12 h to quench the reaction, and washed repeatedly with 0.1 mol / L EDTA solution, distilled water, ethanol, and acetone, followed by drying at 60°C for 12 h to obtain the SiO2@pMPC chromatographic stationary phase.

[0052] Column efficiency test: N 尿嘧啶 =9863N / m.

[0053] Example 4

[0054] The difference from Example 2 lies in the synthesis of SiO2@pMPC in step (4): the dried bromosilica gel was added to a 100 mL high-pressure sealed reaction bottle, followed by the addition of 12 mL of ultra-dry methanol, 2.4 g of MPC monomer, 10 mg of pretreated cuprous chloride, 14 mg of 2-2-bipyridine, and 10 mg of sodium chloride. The mixture was then repeatedly evacuated and filled with nitrogen 5-6 times. The sealed container was placed at 55°C for 24 h, then allowed to stand for 12 h to quench the reaction. The mixture was then washed repeatedly with 0.1 mol / L EDTA solution, distilled water, ethanol, and acetone, and then dried at 60°C for 12 h to obtain the SiO2@pMPC chromatographic stationary phase.

[0055] Column efficiency test: N 尿嘧啶 =12655N / m.

[0056] Example 5

[0057] The difference from Example 2 lies in the synthesis of SiO2@pMPC in step (4): the dried bromosilica gel was added to a 100 mL high-pressure sealed reaction bottle, followed by the addition of 12 mL of ultra-dry methanol, 1.2 g of MPC monomer, 20 mg of pretreated cuprous chloride, 28 mg of 2-2-bipyridine, and 10 mg of sodium chloride. The mixture was then repeatedly evacuated and filled with nitrogen 5-6 times. The sealed container was placed at 55°C for reaction for 24 h, then allowed to stand for 12 h to quench the reaction, and washed repeatedly with 0.1 mol / L EDTA solution, distilled water, ethanol, and acetone, followed by drying at 60°C for 12 h to obtain the SiO2@pMPC chromatographic stationary phase.

[0058] Column efficiency test: N 尿嘧啶 =9426N / m.

[0059] Example 6

[0060] The difference from Example 2 lies in the synthesis of SiO2@pMPC in step (4): the dried bromosilica gel was added to a 100 mL high-pressure sealed reaction bottle, followed by the addition of 12 mL of ultra-dry methanol, 0.6 g of MPC monomer, 20 mg of pretreated cuprous chloride, 28 mg of 2-2-bipyridine, and 10 mg of sodium chloride. The mixture was then repeatedly evacuated and filled with nitrogen 5-6 times. The sealed container was placed at 55°C for reaction for 24 h, then allowed to stand for 12 h to quench the reaction, and washed repeatedly with 0.1 mol / L EDTA solution, distilled water, ethanol, and acetone, followed by drying at 60°C for 12 h to obtain the SiO2@pMPC chromatographic stationary phase.

[0061] Column efficiency test: N 尿嘧啶 =4531N / m.

[0062] Test Example 1

[0063] The stationary phases obtained in Example 1 and Example 2 were respectively loaded into 0.1×150 mm capillary columns and used for the separation and analysis of phenol, thiourea, and uracil. Figure 1 As shown in the figure, the SiO2@pMPC chromatographic stationary phase synthesized by the cuprous chloride catalytic system described in Example 1 exhibits better chromatographic performance.

[0064] Chromatographic column: 0.1×150mm;

[0065] Mobile phase: A: 5% H2O B: 95% can;

[0066] Flow rate: 0.003 mL / min;

[0067] Detection wavelength: 254nm.

[0068] Test Example 2

[0069] The SiO2@pMPC chromatographic stationary phase obtained in Example 2 was packed into a 0.1×150 mm capillary column for separation and analysis of urea and allantoin. Figure 4 As shown, urea and allantoin were well separated on the SiO2@pMPC chromatographic stationary phase;

[0070] Chromatographic column: 0.1×150mm;

[0071] Mobile phase: A: 5% H2O B: 95% ACN;

[0072] Flow rate: 0.003 mL / min;

[0073] Detection wavelength: 190nm.

[0074] Test Example 3

[0075] The SiO2@pMPC chromatographic stationary phase obtained in Example 2 was packed into a 0.1×150 mm capillary column for the analysis of phenolic compounds. Figure 5 As shown, the four phenolic compounds were well separated;

[0076] Chromatographic column: 0.1×150mm;

[0077] Mobile phase: A: 5% H2O B: 95% ACN 10mM AF pH 10.0;

[0078] Flow rate: 0.003 mL / min;

[0079] Detection wavelength: 270nm.

[0080] Test Example 4

[0081] The SiO2@pMPC chromatographic stationary phase obtained in Example 2 was packed into a 0.1×150 mm capillary column for continuous injection and long-term stability experiments. Figure 6 As shown in the figure, the column showed good stability after 500 consecutive injections and a usage time of up to three months.

[0082] Test Example 5

[0083] The SiO2@pMPC chromatographic stationary phase obtained in Example 2 was packed into a 0.1×150 mm capillary column for stability experiments under extreme pH conditions. Figure 7 As shown in the figure, the column has good stability at pH 2 to 10.

[0084] Chromatographic column: 0.1×150mm;

[0085] Sample preparation: toluene, thiourea, uracil;

[0086] Mobile phase: acetonitrile / water (95 / 5, v / v);

[0087] Total flow rate: 0.003 mL / min;

[0088] Detection wavelength: 254nm.

[0089] Finally, it is emphasized that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments without inventive effort, or replace some of the technical features therein with equivalents. Therefore, any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing a SiO2@pMPC chromatographic stationary phase, characterized in that: The following steps are included: (1) Preparation of activated silica gel: Spherical silica gel was acidified with a concentrated hydrochloric acid / methanol solution in an equal volume ratio of 1 g: 50-200 mL to obtain activated silica gel; (2) Surface modification of amino-modified silica gel: Using toluene as solvent, under nitrogen atmosphere, the activated silica gel in step (1) is subjected to silane coupling reaction with a silanization reagent at a mass volume ratio of 1 g: 1-3 mL, and then dried to obtain amino-modified silica gel; the silanization reagent is aminopropyltriethoxysilane; (3) Synthesis of brominated silica spheres: Using anhydrous tetrahydrofuran as solvent, the amino-modified silica gel, triethylamine and 2-bromo-isobutyryl bromide in step (2) were coupled in a mass volume ratio of 1 g: 0.3~1 mL: 0.1~2.0 mL, and vacuum dried to obtain brominated silica gel with bromine modified on the surface; (4) Synthesis of SiO2@pMPC chromatographic stationary phase: Under a protective gas atmosphere, MPC monomer, the brominated silica gel in step (3), the catalyst: 2,2-bipyridine, and cuprous chloride were subjected to atom transfer radical polymerization in a molar ratio of 50-200:1:1-4:2-8, and vacuum dried to obtain SiO2@pMPC chromatographic stationary phase.

2. The method for preparing the SiO2@pMPC chromatographic stationary phase according to claim 1, characterized in that: In step (1), the acidification treatment temperature is 70-110°C, the reaction time is 4-12 h, and the activated silica gel obtained after the reaction is placed in a vacuum dryer at 80-120°C for 12 h.

3. The method for preparing the SiO2@pMPC chromatographic stationary phase according to claim 1, characterized in that: In step (2), the silane coupling reaction temperature is 20-200°C, and the continuous stirring time is 2-48 h; the drying temperature is 80-120°C, and the drying time is 12 h.

4. The method for preparing the SiO2@pMPC chromatographic stationary phase according to claim 1, characterized in that: The coupling reaction temperature in step (3) is 0°C, the coupling reaction time is 0.5~2 h, and then the reaction is transferred to room temperature for 12~24 h; the vacuum drying temperature is 60~120°C, and the vacuum drying time is 12 h.

5. The method for preparing the SiO2@pMPC chromatographic stationary phase according to claim 1, characterized in that: In step (4), the reaction temperature is 20-75°C, and the reaction time is 12-24 h; the vacuum drying temperature is 60-120°C, and the vacuum drying time is 12 h.

6. The method for preparing the SiO2@pMPC chromatographic stationary phase according to claim 1, characterized in that: In step (4), cuprous chloride is pretreated. The specific method of cuprous chloride pretreatment is: repeatedly washing cuprous chloride with 0.1 mol / L dilute acetic acid solution until the green solid turns white, and then repeatedly washing the solution with methanol until it becomes neutral.

7. A SiO2@pMPC chromatographic stationary phase, characterized in that: The SiO2@pMPC chromatographic stationary phase is prepared by the preparation method of any one of claims 1 to 6.

8. The SiO2@pMPC chromatographic stationary phase according to claim 7, characterized in that The structural formula of the SiO2@pMPC chromatographic stationary phase is as follows: 。 9. Use of the SiO2@pMPC chromatographic stationary phase according to claim 8 in a hydrophilic chromatography mode for separation.

Images