Bonded silica gel liquid chromatography stationary phase as well as preparation method, preparation device and application thereof

Abstract

The invention discloses a bonded silica gel liquid chromatography stationary phase and a preparation method, a preparation device and application thereof, a silanization reagent and silica gel are used as raw materials, supercritical carbon dioxide is used as a solvent, in a high-pressure preparation device, active groups of the silanization reagent react with silicon hydroxyl on the surface of the silica gel, and the bonded silica gel liquid chromatography stationary phase is obtained. And bonding the silanization reagent on the surface of the silica gel to form a bonding layer on the surface of the silica gel so as to obtain the bonded silica gel. Compared with a traditional process, efficient bonding and end capping of the surface of the silica gel are achieved by optimizing silanization reagent selection, reaction conditions and a step-by-step washing process, the chemical stability, separation performance and batch consistency of the stationary phase are remarkably improved, and the prepared bonded silica gel stationary phase has better environmental friendliness, uniformity and stability; meanwhile, the method has the advantages of being environmentally friendly, high in reaction efficiency, high in product purity, high in process controllability and the like, and a brand new solution is provided for green manufacturing of the high-performance chromatographic stationary phase.

Description

Technical Field

[0001] The present invention relates to the technical field of chromatographic stationary phase preparation, and more specifically to a bonded silica gel liquid chromatography stationary phase and a preparation method, a preparation device and applications thereof. Background Art

[0002] In liquid chromatography, silica-based bonded stationary phases are a core material for chromatographic separations due to their high surface area, mechanical strength, and chemical stability. Traditional methods for preparing bonded silica stationary phases primarily use organic solvents such as toluene and xylene as reaction media. Silane reagents react with silanol (Si-OH) groups on the silica surface to introduce functional groups such as alkyl, phenyl, or amino groups. However, these processes have significant drawbacks: 1. Traditional organic solvents are highly toxic and volatile, posing a significant risk to environmental pollution and health during production. Global chromatographic packing production consumes tens of thousands of tons of organic solvents annually, of which only approximately 60% is recyclable, with the remainder requiring hazardous waste disposal. 2. Silica gel contains numerous microporous structures, resulting in low mass transfer efficiency in liquid-phase reactions. The surface tension of organic solvents can easily clog the capillaries within the silica micropores, hindering the diffusion of the silane reagent deep into the pores and resulting in uneven surface modification. 3. Post-processing is complex and energy-intensive. After the liquid-phase reaction, multiple washes with organic solvents are required to remove unreacted reagents and byproducts, leading to a lengthy process and the need for solvent recovery or hazardous waste disposal.

[0003] In recent years, supercritical carbon dioxide has emerged as a green solvent in materials synthesis. Its combination of high gas diffusivity and strong liquid solubility, coupled with the ability to dynamically control solvent polarity through pressure and temperature adjustments, makes it a viable alternative to traditional solvents in chemical bonding reactions. However, its systematic application in the preparation of high-performance chromatographic stationary phases has yet to be seen. Summary of the Invention

[0004] The purpose of the present invention is to overcome the above-mentioned defects of the prior art. The present invention innovatively provides a bonded silica gel liquid chromatography stationary phase based on supercritical carbon dioxide and its preparation method, preparation device and application. Compared with the traditional process, the present invention optimizes the selection of silanization reagents, reaction conditions and step-by-step washing process to achieve efficient bonding and end-capping of the silica gel surface, significantly improving the chemical stability, separation performance and batch consistency of the stationary phase. The prepared bonded silica gel stationary phase has better environmental protection, uniformity and stability; at the same time, the method has the advantages of being green and environmentally friendly, high reaction efficiency, high product purity, strong process controllability, etc., providing a new solution for the green manufacturing of high-performance chromatographic stationary phases.

[0005] To achieve the above object, the technical solution of the present invention is as follows: A method for preparing a bonded silica gel liquid chromatography stationary phase, the method using a silanization agent and silica gel as raw materials and supercritical carbon dioxide as solvent. In a high-pressure preparation device, the active groups of the silanization agent react with the silanol groups on the surface of the silica gel to bond the silanization agent to the silica gel surface, thereby forming a bonding layer on the silica gel surface to obtain the bonded silica gel. The high-pressure preparation device comprises a CO2 cylinder 1, a pressure gauge 2, a cooling circulation pump 3, a pressure pump 5, a high-pressure reaction device 6, and a separation tank 9 connected in sequence. A valve 4 is provided on the connecting pipeline between the cooling circulation pump 3 and the pressure pump 5. The high-pressure reaction device 6 comprises an autoclave 61, an autoclave heater 62 arranged on the outer wall of the autoclave 61, a temperature gauge 7 arranged on the top of the outer wall of the autoclave 61, and a grid 8 arranged above the interior of the main body of the autoclave 61.

[0006] Optionally, the critical temperature of the supercritical carbon dioxide is 31.1° C. and the critical pressure is 7.38 MPa.

[0007] Optionally, the silanization reagent consists of a bonding reagent and a capping reagent, which are used for chemical bonding and surface capping of silica gel respectively.

[0008] Optionally, the bonding reagent includes at least one of alkylsilanes, phenylsilanes, methoxysilanes, ethoxysilanes and aminosilane compounds; preferably, the bonding reagent includes at least one of octadecyltrichlorosilane, octadecyldimethylchlorosilane, n-octyltrichlorosilane, chlorodimethyloctylsilane, phenyltrichlorosilane, phenyltrimethoxysilane, diphenyldichlorosilane and 3-aminopropyltrimethoxysilane.

[0009] Optionally, the capping reagent is used to block unreacted silanol groups on the surface of the silica gel, and includes at least one of hexamethyldisilazane (HMDS), trimethylchlorosilane (TMCS) and dimethyldichlorosilane.

[0010] Optionally, the mass volume ratio of the bonding reagent, the capping reagent and the silica gel is 1: (0.05~0.5): (0.01~0.1).

[0011] Optionally, the step of preparing the bonded silica gel liquid chromatography stationary phase is carried out in the high-pressure preparation device, comprising the following steps: S1, pretreating silica gel to obtain pretreated silica gel; S2, wrapping the pretreated silica gel with a metal mesh to obtain wrapped silica gel; S3, supercritical CO2 bonding: the bonding reagent is added to the bottom of the autoclave 61, and the wrapped silica gel is placed on the grid 8, the valve 4 is opened, the liquid CO2 stored in the cylinder is cooled to 0°C to -6°C by the cooling circulation pump 3, and injected into the autoclave 61 through the pressure pump 5. When the pressure in the autoclave 61 reaches 10MPa to 30MPa, the pressure pump 5 and the valve 4 are closed, and the autoclave 61 is heated to react so that the active groups in the bonding reagent condense with the silanol groups in the silica gel to form a bonding layer. After the reaction, the CO2 is released to relieve the pressure; S4, one supercritical washing: the coated silica gel obtained in step S3 is placed at the bottom of the autoclave 61, the valve 4 is opened, the liquid CO2 stored in the cylinder is cooled to 0°C to -6°C by the cooling circulation pump 3, and injected into the autoclave 61 through the pressure pump 5. When the pressure in the autoclave 61 reaches 25 MPa, the pressure pump 5 and the valve 4 are closed, and the autoclave 61 is heated to 40°C and maintained for 30 minutes. The CO2 is then released, and the released CO2 carries the unreacted bonding reagent and small molecule by-products to the separation tank 9. This washing process is repeated until the quality of the metal screen coated with silica gel remains unchanged, ensuring that the residue is completely removed; S5, supercritical CO2 end-capping: the end-capping reagent is added to the bottom of the autoclave 61, the wrapped silica gel obtained in step S4 is placed on the grid 8, the valve 4 is opened, the liquid CO2 stored in the cylinder is cooled to 0°C to -6°C by the cooling circulation pump 3, and injected into the autoclave 61 through the pressure pump 5. When the pressure in the autoclave 61 reaches 10MPa to 30MPa, the pressure pump 5 and the valve 4 are closed, and the autoclave 61 is heated to perform the end-capping reaction to further block the unbonded silanol groups. After the reaction is completed, the CO2 is released to relieve the pressure; S6, secondary supercritical washing: Place the wrapped silica gel obtained in step S5 at the bottom of the autoclave 61, open the valve 4, cool the liquid CO2 stored in the cylinder to 0°C~-6°C through the cooling circulation pump 3, and inject it into the autoclave 61 through the pressure pump 5. When the pressure in the autoclave 61 reaches 25MPa, close the pressure pump 5 and the valve 4, and heat the autoclave 61 to 40°C and maintain it for 30 minutes. Then release CO2, and the released CO2 carries the unreacted end-capping reagent and small molecule by-products to the separation tank 9. Repeat this washing process until the mass of the metal screen coated with silica gel remains unchanged, thereby obtaining the bonded silica gel liquid chromatography stationary phase.

[0012] Optionally, in step S1, the pretreatment step includes: vacuum drying the silica gel at 80°C to 200°C for 5h to 24h to remove physically adsorbed water, and transferring it to a dry inert atmosphere (nitrogen or argon) environment after cooling for standby use to avoid moisture absorption.

[0013] Optionally, the particle size of the silica gel is 3 μm to 10 μm, and the specific surface area of ​​the silica gel is 200 m 2 / g~500m 2 / g, and the pore size of the silica gel is 5nm~20nm.

[0014] Optionally, in step S2, the pore size of the metal mesh is 2.6 μm, and the pore size of the metal mesh is smaller than the particle size of the silica gel.

[0015] Optionally, in step S3, the reaction temperature is 50° C. to 130° C., and the reaction time is 0.5 h to 10 h.

[0016] Optionally, in step S4, the reaction temperature is 40° C. and the reaction time is 0.5 h.

[0017] Optionally, in step S5, the reaction temperature is 50° C. to 130° C., and the reaction time is 0.5 h to 10 h.

[0018] Optionally, in step S6, the reaction temperature is 40° C. and the reaction time is 0.5 h.

[0019] The present invention also discloses a high-pressure preparation device for a bonded silica gel liquid chromatography stationary phase, which is suitable for the above-mentioned preparation method; the high-pressure preparation device includes a CO2 cylinder 1, a pressure gauge 2, a cooling circulation pump 3, a pressure pump 5, a high-pressure reaction device 6 and a separation tank 9 connected in sequence; wherein, a valve 4 is provided on the connecting pipeline between the cooling circulation pump 3 and the pressure pump 5; the high-pressure reaction device 6 includes an autoclave 61, an autoclave heater 62 arranged on the outer wall of the autoclave 61, a temperature gauge 7 arranged on the top of the outer wall of the autoclave 61, and a grid 8 arranged above the inside of the main body of the autoclave 61.

[0020] The invention also discloses a bonded silica gel liquid chromatography stationary phase prepared by the above preparation method.

[0021] The invention also discloses an application of the bonded silica gel liquid chromatography stationary phase prepared by the above preparation method in chromatographic separation.

[0022] The implementation of the present invention will have the following beneficial effects: (1) The present invention provides a bonded silica gel liquid chromatography stationary phase based on supercritical carbon dioxide, and its preparation method, preparation device and application. In a high-pressure reaction device, silica gel and a silanization agent are placed together in a supercritical CO2 environment; when the temperature exceeds 31.1°C and the pressure is higher than 7.38 MPa, CO2 enters a supercritical state. At this time, it has both the high diffusivity of gas and the strong solubility of liquid, which can fully dissolve the silanization agent and carry it into the pore structure of silica gel. During the dynamic mass transfer process, the active groups of the silanization agent react with the silanol groups on the surface of silica gel to form a stable Si-O-Si covalent bond, thereby constructing a uniform organic functional layer on the surface of silica gel. Compared with the traditional liquid phase method, this method replaces toxic organic solvents such as toluene and xylene, avoids the blockage of pores caused by capillary forces, improves the bonding rate of silica gel surface, and has excellent application prospects.

[0023] (2) The present invention uses supercritical CO2 to replace organic solvents throughout the process, and no harmful waste is discharged, which meets the requirements of green chemistry.

[0024] (3) The high diffusivity and low surface tension of the supercritical CO2 of the present invention promote the penetration of reagents into the silica gel pores, thereby improving bonding uniformity.

[0025] (4) The present invention can adjust the bonding density and end-capping degree by adjusting the temperature, pressure and reaction time to adapt to different chromatographic separation requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of a high-pressure preparation device for the bonded silica gel liquid chromatography stationary phase according to Example 1 of the present invention.

[0027] Among them, 1. CO2 cylinder, 2. pressure gauge, 3. cooling circulation pump, 4. valve, 5. pressure pump, 6. high-pressure reaction device, 61. autoclave, 62. autoclave heater, 7. thermometer, 8. grid, 9. separation tank.

[0028] Figure 2 Graphs showing the separation performance of Example 2 and Comparative Example 1. A represents Example 2, and B represents Comparative Example 1. DETAILED DESCRIPTION

[0029] The present invention will be further described below with reference to specific examples, but the present invention is not limited thereto in any way.

[0030] Example 1 The high-pressure preparation apparatus for a bonded silica gel liquid chromatography stationary phase of this embodiment is applicable to the preparation method of any of the following embodiments. The high-pressure preparation apparatus comprises a CO2 cylinder 1, a pressure gauge 2, a cooling circulation pump 3, a pressure pump 5, a high-pressure reaction apparatus 6, and a separator 9, connected in sequence. A valve 4 is provided on the connecting pipe between the cooling circulation pump 3 and the pressure pump 5. The high-pressure reaction apparatus 6 comprises an autoclave 61, an autoclave heater 62 disposed on the outer wall of the autoclave 61, a thermometer 7 disposed on the top of the outer wall of the autoclave 61, and a grid 8 disposed above the interior of the main body of the autoclave 61.

[0031] Example 2: Preparation of octadecyl bonded silica gel stationary phase The preparation method of the octadecyl bonded silica gel stationary phase of this embodiment is carried out in the high-pressure preparation apparatus of Example 1, comprising the following steps: S1, 20g of porous spherical silica gel (particle size 5μm, specific surface area 300m 2 / g, pore size of 10nm) was placed in a vacuum drying oven and vacuum dried at 150°C for 12h to remove physically adsorbed water. After cooling to room temperature, the silica gel was transferred to a sealed container in a nitrogen glove box for standby use to obtain pretreated silica gel.

[0032] S2. Wrap the pretreated silica gel with a 5000-mesh stainless steel sieve to obtain wrapped silica gel.

[0033] S3. Add 5 mL of octadecyltrichlorosilane to the bottom of the autoclave 61, and place the wrapped silica gel on the grid 8. Open the valve 4, cool the liquid CO2 stored in the cylinder to -6°C through the cooling circulation pump 3, and inject it into the autoclave 61 through the pressure pump 5. When the pressure in the autoclave 61 reaches 20 MPa, close the pressure pump 5 and valve 4, and heat the autoclave 61 to 110°C for reaction for 2 hours. After the reaction, release the CO2 to relieve the pressure.

[0034] S4. Place the wrapped silica gel obtained in step S3 at the bottom of the autoclave 61, open valve 4, cool the liquid CO2 stored in the cylinder to -6°C through the cooling circulation pump 3, and inject it into the autoclave 61 through the pressure pump 5. When the pressure in the autoclave 61 reaches 25 MPa, close the pressure pump 5 and valve 4, and heat the autoclave 61 to 40°C at the same time, maintain for 30 minutes, and then release CO2. The released CO2 carries unreacted octadecyltrichlorosilane and small molecular by-products to the separation tank 9. Repeat the washing 3 times until the quality of the screen remains unchanged to ensure complete removal of residues.

[0035] S5. Add 3 mL of hexamethyldisilazane to the bottom of the autoclave 61, place the wrapped silica gel obtained in step S4 on the grid 8, open the valve 4, cool the liquid CO2 stored in the cylinder to -6°C through the cooling circulation pump 3, and inject it into the autoclave 61 through the pressure pump 5. When the pressure in the autoclave 61 reaches 20 MPa, close the pressure pump 5 and the valve 4, and heat the autoclave 61 at 120°C for a capping reaction for 5 hours to further block the unbonded silanol groups. After the reaction is completed, release the CO2 to relieve the pressure.

[0036] S6. Repeat step S4 and wash three times to obtain the final product - octadecyl bonded silica gel stationary phase.

[0037] Example 3: Preparation of phenylamino bifunctional bonded silica gel stationary phase The preparation method of the phenylamino bifunctional bonded silica gel stationary phase of this embodiment is carried out in the high-pressure preparation apparatus of Example 1, comprising the following steps: S1, 10g of porous spherical silica gel (particle size 3μm, specific surface area 500m 2 / g, pore size 7nm) was placed in a vacuum drying oven and vacuum dried at 80°C for 24h. After cooling to room temperature, the silica gel was transferred to a sealed container in a nitrogen glove box for standby use to obtain pretreated silica gel.

[0038] S2. Wrap the pretreated silica gel with a 5000-mesh stainless steel sieve to obtain wrapped silica gel.

[0039] S3. Add 2 mL of a mixed solution of phenyltrimethoxysilane and 3 mL of 3-aminopropyltrimethoxysilane to the bottom of the autoclave 61 and perform the treatment according to step S3 in Example 2, wherein the reaction temperature is 130° C., the pressure is maintained at 15 MPa, the reaction time is 8 h, and CO2 is released to relieve pressure after the reaction.

[0040] S4. Place the wrapped silica gel obtained in step S3 at the bottom of the autoclave 61 and treat it according to step S4 in Example 2. Then release CO2. The released CO2 carries unreacted phenyltrimethoxysilane, 3-aminopropyltrimethoxysilane and small molecule by-products to the separation tank 9. Repeat the washing three times until the quality of the screen remains unchanged to ensure complete removal of the residue.

[0041] S5. Add 2 mL of TMCS to the bottom of the autoclave 61 and process according to step S5 in Example 2, wherein the reaction temperature is 80° C., the reaction pressure is 15 MPa, and the reaction time is 3 h to perform the end-capping reaction to further block the unbonded silanol groups. After the reaction is completed, release CO2 to relieve the pressure.

[0042] S6. Repeat step S4 and wash three times to obtain the final product - phenylamino bifunctional bonded silica gel stationary phase.

[0043] Example 4: Preparation of octyl bonded silica gel stationary phase The preparation method of the octyl bonded silica gel stationary phase of this embodiment is carried out in the high-pressure preparation apparatus of Example 1, comprising the following steps: S1, 15g of porous spherical silica gel (particle size 10μm, specific surface area 270m 2 / g, pore size of 12 nm) was placed in a vacuum drying oven and vacuum dried at 100°C for 15 h. After cooling to room temperature, the silica gel was transferred to a sealed container in a nitrogen glove box for standby use to obtain pretreated silica gel.

[0044] S2. Wrap the pretreated silica gel with a 5000-mesh stainless steel sieve to obtain wrapped silica gel.

[0045] S3. Add 4 mL of n-octyltrichlorosilane to the bottom of the autoclave 61 and perform the treatment according to step S3 in Example 2, wherein the reaction temperature is 60° C., the pressure is maintained at 25 MPa, the reaction time is 8 h, and CO2 is released to relieve pressure after the reaction.

[0046] S4. Place the wrapped silica gel obtained in step S3 at the bottom of the autoclave 61 and treat it according to step S4 in Example 2. Then release CO2. The released CO2 carries unreacted n-octyltrichlorosilane and small molecular by-products to the separation tank 9. Repeat the washing three times until the quality of the screen remains unchanged to ensure complete removal of the residue.

[0047] S5. Add 2 mL of trimethylchlorosilane to the bottom of the autoclave 61 and process according to step S5 in Example 2, wherein the reaction temperature is 80° C., the reaction pressure is 30 MPa, and the reaction time is 0.5 h to perform the end-capping reaction to further block the unbonded silanol groups. After the reaction is completed, release CO2 to relieve the pressure.

[0048] S6. Repeat step S4 and wash three times to obtain the final product - octyl bonded silica gel stationary phase.

[0049] Example 5: Preparation of phenyl bonded silica gel stationary phase The preparation method of the phenyl bonded silica gel stationary phase of this embodiment is carried out in the high-pressure preparation apparatus of Example 1, comprising the following steps: S1, 25g of porous spherical silica gel (particle size 8μm, specific surface area 400m 2 / g, pore size of 10nm) was placed in a vacuum drying oven and vacuum dried at 200°C for 5h. After cooling to room temperature, the silica gel was transferred to a sealed container in a nitrogen glove box for standby use to obtain pretreated silica gel.

[0050] S2. Wrap the pretreated silica gel with a 5000-mesh stainless steel sieve to obtain wrapped silica gel.

[0051] S3. Add 8 mL of phenyltrichlorosilane to the bottom of the autoclave 61 and perform the treatment according to step S3 in Example 2, wherein the reaction temperature is 70° C., the pressure is maintained at 20 MPa, the reaction time is 10 h, and CO2 is released to relieve pressure after the reaction.

[0052] S4. Place the wrapped silica gel obtained in step S3 at the bottom of the autoclave 61 and treat it according to step S4 in Example 2. Then release CO2. The released CO2 carries unreacted phenyltrichlorosilane and small molecular by-products to the separation tank 9. Repeat the washing three times until the quality of the screen remains unchanged to ensure complete removal of the residue.

[0053] S5. Add 4 mL of hexamethyldisilazane to the bottom of the autoclave 61 and process according to step S5 in Example 2, wherein the reaction temperature is 50° C., the reaction pressure is 15 MPa, and the reaction time is 2 h to perform the end-capping reaction to further block the unbonded silanol groups. After the reaction is completed, release CO2 to relieve the pressure.

[0054] S6. Repeat step S4 and wash three times to obtain the final product - phenyl bonded silica gel stationary phase.

[0055] Example 6: Preparation of octadecyl bonded silica gel stationary phase The preparation method of the octadecyl bonded silica gel stationary phase of this embodiment is carried out in the high-pressure preparation apparatus of Example 1, comprising the following steps: S1, 15g of porous spherical silica gel (particle size 5μm, specific surface area 300m 2 / g, pore size of 10 nm) was placed in a vacuum drying oven and vacuum dried at 130°C for 10 h. After cooling to room temperature, the silica gel was transferred to a sealed container in a nitrogen glove box for standby use to obtain pretreated silica gel.

[0056] S2. Wrap the pretreated silica gel with a 5000-mesh stainless steel sieve to obtain wrapped silica gel.

[0057] S3. Add 2 mL of a mixture of octadecyltrichlorosilane and 2 mL of octadecyldimethylchlorosilane to the bottom of the autoclave 61 and perform treatment according to step S3 in Example 2, wherein the reaction temperature is 50° C., the pressure is maintained at 30 MPa, the reaction time is 9 h, and CO2 is released after the reaction to relieve pressure.

[0058] S4. Place the wrapped silica gel obtained in step S3 at the bottom of the autoclave 61 and treat it according to step S4 in Example 2. Then release CO2. The released CO2 carries unreacted octadecyltrichlorosilane, octadecyldimethylchlorosilane and small molecular by-products to the separation tank 9. Repeat the washing three times until the quality of the screen remains unchanged to ensure complete removal of the residue.

[0059] S5. Add 3 mL of trimethylchlorosilane to the bottom of the autoclave 61 and process according to step S5 in Example 2, wherein the reaction temperature is 100° C., the reaction pressure is 20 MPa, and the reaction time is 2 h to perform the end-capping reaction to further block the unbonded silanol groups. After the reaction is completed, release CO2 to relieve the pressure.

[0060] S6. Repeat step S4 and wash three times to obtain the final product - octadecyl bonded silica gel stationary phase.

[0061] Example 7: Preparation of octyl bonded silica gel stationary phase The preparation method of the octyl bonded silica gel stationary phase of this embodiment is carried out in the high-pressure preparation apparatus of Example 1, comprising the following steps: S1, 30g of porous spherical silica gel (particle size 3μm, specific surface area 500m 2 / g, pore size 7nm) was placed in a vacuum drying oven and vacuum dried at 180°C for 6h. After cooling to room temperature, the silica gel was transferred to a sealed container in a nitrogen glove box for standby use to obtain pretreated silica gel.

[0062] S2. Wrap the pretreated silica gel with a 5000-mesh stainless steel sieve to obtain wrapped silica gel.

[0063] S3. Add 5 mL of a mixture of n-octyltrichlorosilane and 5 mL of chlorodimethyloctylsilane to the bottom of the autoclave 61 and perform the treatment according to step S3 in Example 2, wherein the reaction temperature is 80° C., the pressure is maintained at 15 MPa, the reaction time is 6 h, and CO2 is released to relieve the pressure after the reaction.

[0064] S4. Place the wrapped silica gel obtained in step S3 at the bottom of the autoclave 61 and treat it according to step S4 in Example 2. Then release CO2. The released CO2 carries unreacted n-octyltrichlorosilane, chlorodimethyloctylsilane and small molecule by-products to the separation tank 9. Repeat the washing three times until the quality of the screen remains unchanged to ensure complete removal of the residue.

[0065] S5. Add 8 mL of trimethylchlorosilane to the bottom of the autoclave 61 and process according to step S5 in Example 2, wherein the reaction temperature is 60° C., the reaction pressure is 10 MPa, and the reaction time is 6 h to perform the end-capping reaction to further block the unbonded silanol groups. After the reaction is completed, release CO2 to relieve the pressure.

[0066] S6. Repeat step S4 and wash three times to obtain the final product - octyl bonded silica gel stationary phase.

[0067] Comparative Example 1 This comparative example adopts the traditional liquid phase method to prepare the octadecyl bonded silica gel stationary phase, and the preparation method comprises: S1, 20g of porous spherical silica gel (particle size 5μm, specific surface area 300m 2 / g, pore size of 10nm) was pretreated with the same processing steps as in Example 2 to obtain pretreated silica gel.

[0068] S2. Add the dried silica gel and 10 mL of octadecyltrichlorosilane to a 500 mL three-necked flask. Add 200 mL of anhydrous toluene and purge with nitrogen. Install a reflux condenser and heat to 110°C, the toluene reflux temperature, with continuous stirring for 24 hours. After the reaction is complete, stop heating and allow the mixture to cool naturally to room temperature.

[0069] S3. Transfer the reaction mixture to a fritted funnel and wash with toluene, methanol, and acetone three times (100 mL each) to remove unreacted octadecyltrichlorosilane and byproducts. Place the washed silica gel in a vacuum drying oven at 60°C for 12 hours.

[0070] S4. Add the dried silica gel and 6 mL of hexamethyldisilazane into a three-necked flask, add 150 mL of anhydrous toluene, heat to 110°C, stir and react for 8 hours to block the residual silanol groups.

[0071] S5. Repeat the washing process from step C (toluene → methanol → acetone three times, 100 ml each time) to remove unreacted hexamethyldisilazane and byproducts. The final product is vacuum-dried at 60°C for 12 hours to obtain the conventionally prepared octadecyl bonded silica stationary phase.

[0072] The effects of Examples 3 to 7 are the same as those of Example 2.

[0073] Test Case 1. The performance of the products obtained in Example 2 and Comparative Example 1 was compared, as shown in Table 1.

[0074] Table 1 Performance comparison between Example 2 and Comparative Example 1

[0075] From the analysis of Comparative Example 1 and Example 2, it can be seen that Example 2 of the present invention can significantly improve the bonding rate of hydroxyl groups on the silica gel surface.

[0076] 2. The chromatographic column was packed with the C18 bonded silica stationary phase of Example 2 and Comparative Example 1. The column efficiency test conditions were: mobile phase: water: acetonitrile: 100 mM ammonium formate (45:45:10, v / v / v), injection volume: 1.0 μL, test mixture: uracil, desipramine, amitriptyline, butyl parahydroxybenzoate. The column efficiency test results are shown in Table 2.

[0077] Table 2 Comparison of column efficiency performance between Example 2 and Comparative Example 1

[0078] By comparing the chromatographic separation data of Example 2 with that of Example 1, it can be seen that the chromatographic column prepared in Example 2 of the present invention exhibits a significant performance improvement in separating the above test mixture.

[0079] The above-described embodiments merely represent several implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for preparing a bonded silica gel liquid chromatography stationary phase, characterized in that: The preparation method uses a silanization agent and silica gel as raw materials and supercritical carbon dioxide as solvent. In a high-pressure preparation device, the active groups of the silanization agent react with the silanol groups on the surface of the silica gel to bond the silanization agent to the silica gel surface, thereby forming a bonding layer on the silica gel surface to obtain a bonded silica gel. The high-pressure preparation device includes a CO2 cylinder, a pressure gauge, a cooling circulation pump, a pressure pump, a high-pressure reaction device and a separation tank connected in sequence; wherein a valve is provided on the connecting pipe between the cooling circulation pump and the pressure pump; The high-pressure reaction device includes an autoclave, an autoclave heater arranged on the outer wall of the autoclave, a temperature gauge arranged on the top of the outer wall of the autoclave, and a grid arranged above the interior of the autoclave body.

2. The method for preparing a bonded silica gel liquid chromatography stationary phase according to claim 1, wherein The critical temperature of the supercritical carbon dioxide is 31.1°C and the critical pressure is 7.38MPa; The silanization reagent consists of a bonding reagent and a capping reagent; The bonding agent includes at least one of alkylsilanes, phenylsilanes, methoxysilanes, ethoxysilanes and aminosilane compounds; The end-capping agent includes at least one of hexamethyldisilazane, trimethylchlorosilane and dimethyldichlorosilane.

3. The method for preparing a bonded silica gel liquid chromatography stationary phase according to claim 2, wherein: The mass ratio of the silica gel, the bonding reagent and the capping reagent is 1: (0.05-0.5): (0.01-0.1).

4. The method for preparing a bonded silica gel liquid chromatography stationary phase according to claim 3, wherein: The step of preparing the bonded silica gel liquid chromatography stationary phase is carried out in the high-pressure preparation device, comprising the following steps: S1, pretreating silica gel to obtain pretreated silica gel; S2, wrapping the pretreated silica gel with a metal mesh to obtain wrapped silica gel; S3, adding the bonding reagent to the bottom of the autoclave, and placing the wrapped silica gel on the grid, opening the valve, cooling the liquid CO2 stored in the cylinder to 0°C~-6°C through the cooling circulation pump, and injecting it into the autoclave through the pressure pump. When the pressure in the autoclave reaches 10MPa~30MPa, the pressure pump and the valve are closed, and the autoclave is heated to react. After the reaction, CO2 is released to relieve pressure; S4. Placing the wrapped silica gel obtained in step S3 at the bottom of the autoclave, opening the valve, cooling the liquid CO2 stored in the cylinder to 0°C to -6°C via the cooling circulation pump, and injecting the liquid CO2 into the autoclave via the pressure pump. When the pressure in the autoclave reaches 25 MPa, closing the pressure pump and the valve, heating the autoclave to 40°C and maintaining it for 30 minutes, then releasing the CO2. The released CO2 carries the unreacted bonding reagent and small molecule byproducts to the separation tank, and repeating this washing process until the quality of the metal screen coated with silica gel remains unchanged. S5, adding the end-capping reagent to the bottom of the autoclave, placing the wrapped silica gel obtained in step S4 on the grid, opening the valve, cooling the liquid CO2 stored in the cylinder to 0°C~-6°C via the cooling circulation pump, and injecting it into the autoclave via the pressure pump. When the pressure in the autoclave reaches 10MPa~30MPa, the pressure pump and the valve are closed, and the autoclave is heated to react. After the reaction is completed, the CO2 is released to relieve the pressure; S6. Place the wrapped silica gel obtained in step S5 at the bottom of the autoclave, open the valve, cool the liquid CO2 stored in the cylinder to 0°C~-6°C via the cooling circulation pump, and inject it into the autoclave through the pressure pump. When the pressure in the autoclave reaches 25 MPa, close the pressure pump and the valve, and heat the autoclave to 40°C at the same time, maintain for 30 minutes, and then release CO2. The released CO2 carries the unreacted end-capping reagent and small molecule by-products to the separation tank. Repeat this washing process until the mass of the metal screen coated with silica gel remains unchanged, thereby obtaining the bonded silica gel liquid chromatography stationary phase.

5. The method for preparing a bonded silica gel liquid chromatography stationary phase according to claim 4, characterized in that: In step S1, the pretreatment step includes: vacuum drying the silica gel at 80° C. to 200° C. for 5 h to 24 h, and then transferring it to a dry inert atmosphere for standby use after cooling; The particle size of the silica gel is 3 μm to 10 μm, and the specific surface area of ​​the silica gel is 200 m 2 / g~500m 2 / g, and the pore size of the silica gel is 5nm~20nm.

6. The method for preparing a bonded silica gel liquid chromatography stationary phase according to claim 4, characterized in that: In step S2, the pore size of the metal mesh is 2.6 μm, and the pore size of the metal mesh is smaller than the particle size of the silica gel.

7. The method for preparing a bonded silica gel liquid chromatography stationary phase according to claim 4, characterized in that: In step S3, the reaction temperature is 50°C to 130°C, and the reaction time is 0.5h to 10h; In step S4, the reaction temperature is 40° C. and the reaction time is 0.5 h; In step S5, the reaction temperature is 50°C to 130°C, and the reaction time is 0.5h to 10h; In step S6, the reaction temperature is 40° C., and the reaction time is 0.5 h.

8. A high-pressure preparation device for bonded silica gel liquid chromatography stationary phase, characterized in that: Applicable to the preparation method according to any one of claims 1 to 7; The high-pressure preparation device includes a CO2 cylinder, a pressure gauge, a cooling circulation pump, a pressure pump, a high-pressure reaction device and a separation tank connected in sequence; wherein a valve is provided on the connecting pipe between the cooling circulation pump and the pressure pump; The high-pressure reaction device includes an autoclave, an autoclave heater arranged on the outer wall of the autoclave, a temperature gauge arranged on the top of the outer wall of the autoclave, and a grid arranged above the interior of the autoclave body.

9. A bonded silica gel liquid chromatography stationary phase prepared by the preparation method according to any one of claims 1 to 7.

10. Use of the bonded silica gel liquid chromatography stationary phase prepared by the preparation method according to any one of claims 1 to 7 in chromatographic separation.

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