Double-layer porous SiO2-coated SiO2 core-shell microsphere based on double-phase method and preparation method and application of double-layer porous SiO2-coated SiO2 core-shell microsphere

Abstract

The invention belongs to the technical field of chromatographic packing, and particularly relates to a double-layer hole type SiO2 (at) SiO2 core-shell microsphere based on a double-phase method and a preparation method and application of the double-layer hole type SiO2 (at) SiO2 core-shell microsphere. According to the core-shell microsphere, micron-sized non-porous SiO2 serves as a core, a mesoporous SiO2 framework with pore channels perpendicular to the surface of the non-porous SiO2 core serves as a porous shell layer, the porous shell layer sequentially comprises a first ordered mesoporous shell layer and a second ordered mesoporous shell layer which are different in pore diameter from inside to outside, and the pore channels of the two shell layers are completely consistent in direction and are communicated with each other. According to the invention, a porous shell structure with dual or multiple pore size distribution is constructed on the surface of a SiO2 core, and by integrating more than two pore sizes with obvious size difference, exclusive mass transfer paths are provided for solute molecules with different sizes, exclusive mass transfer channels can be provided for small-molecular compounds and macromolecular substances at the same time, and the pore size difference is greatly reduced. Therefore, the selectivity and column efficiency of chromatographic separation are remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of chromatographic fillers, and in particular relates to double-layer porous SiO2@SiO2 core-shell microspheres based on a two-phase method, a preparation method and an application thereof. Background Art

[0002] Core-shell stationary phases, composed of a nonporous SiO2 core and a porous SiO2 shell, have been widely used in chromatographic separation and quantitative analysis due to their excellent rapid separation performance. Existing technologies have developed a variety of synthesis methods, including polymerization-induced colloidal coacervation, layer-by-layer self-assembly, one-pot, dual-template, template-induced redeposition, and two-phase methods. These methods can be used to prepare SiO2@SiO2 core-shell microspheres with two typical pore structures: stacked pore-type core-shell microspheres formed by the accumulation of nano-SiO2 particles, and radial pore-type core-shell microspheres with pores perpendicular to the surface of the nonporous SiO2 core. However, both types of core-shell microspheres are limited by a single pore size.

[0003] In practical chromatographic separation applications, core-shell stationary phases with a single pore size require a pre-determined determination of the molecular size of the substance being separated in order to select a chromatographic column with a matching pore size. For unknown samples with complex molecular weight distributions, tedious sample pretreatment and molecular weight fractionation are often required, which not only increases analysis costs but can also damage the column due to pore clogging. Summary of the Invention

[0004] In order to solve the limitations of the single pore size core-shell stationary phase, the present invention provides a double-layer porous SiO2@SiO2 core-shell microsphere based on a two-phase method, as well as its preparation method and application. The core-shell microsphere constructs an ordered mesoporous shell structure with a dual pore size distribution on the surface of the SiO2 core, including a first ordered mesoporous shell and a second ordered mesoporous shell arranged in sequence from the inside to the outside; wherein the pore size of the first ordered mesoporous shell is relatively small, between 7 nm and 12 nm, and the pore size of the second ordered mesoporous shell is relatively large, about 20 nm to 40 nm. The present invention innovatively integrates two pore structures with significant size differences to provide exclusive mass transfer paths for solute molecules of different sizes, thereby providing exclusive mass transfer paths for solute molecules of different sizes, and can simultaneously provide exclusive mass transfer channels for small molecule compounds and large molecular substances, thereby significantly improving the selectivity and column efficiency of chromatographic separation, and can directly separate and analyze mixtures of unknown molecular weight, thereby simplifying the operating process and reducing analysis costs.

[0005] The present invention solves the above technical problems through the following technical solutions.

[0006] The first object of the present invention is to provide a double-layer porous SiO2@SiO2 core-shell microsphere based on a two-phase method, wherein the core-shell microsphere has a micron-sized non-porous SiO2 as a core and a radial mesoporous SiO2 shell as a shell; the mesoporous shell includes a first ordered mesoporous shell and a second ordered mesoporous shell from the inside to the outside, wherein the pore size of the first ordered mesoporous shell is 7 nm to 12 nm, and the pore size of the second ordered mesoporous shell is 20 nm to 40 nm, and the pore channels of the first ordered mesoporous shell and the second ordered mesoporous shell are radially arranged perpendicular to the surface of the micron-sized non-porous SiO2 core and are interconnected.

[0007] Furthermore, the thickness of the first ordered mesoporous shell layer and the second ordered mesoporous shell layer are both 100 nm to 200 nm.

[0008] Furthermore, the particle size of the micron-sized non-porous SiO2 is 2 μm to 3 μm.

[0009] The second object of the present invention is to provide a method for preparing the above-mentioned double-layer porous SiO2@SiO2 core-shell microspheres based on the two-phase method, comprising the following steps: S1. Micron-sized non-porous SiO2 microspheres were prepared using the improved Stöber method.

[0010] S2. Add surfactant and urea to the micron-sized non-porous SiO2 microspheres and disperse them evenly to form a first reaction liquid. Cyclohexane is used as the organic phase, tetraethyl orthosilicate is used as the silicon source, octadecylamine is used as the pore expander and charge inducer, and isopropyl alcohol is used as the cosolvent to form a second reaction liquid. The first reaction liquid is refluxed at 70°C, and then the second reaction liquid is added to carry out a reflux reaction to deposit a first ordered mesoporous shell precursor on the surface of the micron-sized non-porous SiO2 microspheres to form a SiO2@SiO2 core-shell microsphere precursor.

[0011] S3. Under the same reaction conditions for preparing SiO2@SiO2 core-shell microsphere precursors, the cyclohexane organic phase is replaced with a toluene organic phase, and a reflux reaction is performed to deposit a second layer of mesoporous shell precursor on the surface of the first ordered mesoporous shell precursor to form double-layer porous SiO2@SiO2 core-shell microspheres.

[0012] S4. calcining the double-layer porous SiO2@SiO2 core-shell microsphere precursor to obtain double-layer porous SiO2@SiO2 core-shell microspheres.

[0013] Furthermore, the mass ratio of micron-sized non-porous SiO2 microspheres, surfactant and urea is 2:1~2:0.2~3, and the surfactant is hexadecyltrimethylammonium bromide; the mass-volume ratio of octadecylamine and cyclohexane is 0.1 g~1.8 g:30 mL~120 mL, the mass-volume ratio of octadecylamine and tetraethyl orthosilicate is 0.1 g~1.8 g:0.1 mL~3 mL, and the volume ratio of tetraethyl orthosilicate and isopropyl alcohol is 0.1~3:0.1~6.

[0014] Furthermore, the volume ratio of the first reaction liquid to the second reaction liquid is 1:0.1-2, and the reflux reaction time is 1 h-24 h.

[0015] Furthermore, the amounts of toluene and cyclohexane used are the same.

[0016] Furthermore, the calcination temperature is 500°C to 650°C, and the calcination time is 5 h to 20 h.

[0017] The third object of the present invention is to provide the use of the above-mentioned double-layer stacked porous SiO2@SiO2 core-shell microspheres as filler in chromatographic separation.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The double-layer porous SiO2@SiO2 core-shell microspheres provided by the present invention construct a porous shell structure with a double pore size distribution on the surface of the SiO2 core, wherein micron-level non-porous SiO2 is used as the core, and then a first ordered mesoporous shell layer and a second ordered mesoporous shell layer are sequentially constructed on the surface of the micron-level non-porous SiO2 core. The pore size distribution of the first ordered mesoporous shell layer is between 7 nm and 12 nm, and the pore channel is perpendicular to the core surface, with a high degree of order, mainly providing an action site for the separation of small molecule compounds. The second ordered mesoporous shell layer is constructed with the first ordered mesoporous shell layer with a smaller pore size as the base, and its pore size is 20 nm to 40 nm. The pore channel direction is consistent with that of the first ordered mesoporous shell layer and they are interconnected. The unique dual-pore channel provides exclusive mass transfer pathways for analytes of different sizes, and can provide suitable mass transfer channels for both small molecule compounds and macromolecules, thereby significantly improving the selectivity and column efficiency of chromatographic separation, while achieving efficient and rapid separation of small molecules and biomacromolecules. The differentiated shell pore structure enables the core-shell stationary phase to directly achieve efficient, rapid, and low-cost separation without the need for size-fractionation pretreatment of samples with unknown molecular weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the structure of the double-layer porous SiO2@SiO2 core-shell microspheres of the present invention.

[0020] Figure 2This is a flow chart of the preparation process of double-layer porous SiO2@SiO2 core-shell microspheres of the present invention.

[0021] Figure 3 This is a morphology and structure diagram of micron-sized non-porous SiO2 microspheres according to Example 1 of the present invention. Figure 3 a is a scanning electron microscopy at a size of 5 μm, and b is a scanning electron microscopy at a size of 1 μm.

[0022] Figure 4 This is a morphological structure diagram of the first ordered mesoporous shell layer in Example 1 of the present invention. Figure 4 a is a scanning electron microscopy at a size of 2.5 μm, b is a scanning electron microscopy at a size of 0.5 μm, c is a transmission electron microscopy at a size of 1 μm, d is a transmission electron microscopy at a size of 0.5 μm, e is a nitrogen adsorption-desorption curve, and f is a pore size distribution diagram.

[0023] Figure 5 This is a morphology and structure diagram of the double-porous SiO2@SiO2 core-shell microspheres in Example 1 of the present invention. Figure 5 a is a scanning electron microscopy at a size of 2.5 μm, b is a scanning electron microscopy at a size of 1 μm, c is a transmission electron microscopy at a size of 2 μm, d is a transmission electron microscopy at a size of 0.5 μm, e is a nitrogen adsorption-desorption curve, and f is a pore size distribution diagram.

[0024] Figure 6 This is a morphological structure diagram of the second ordered mesoporous layer in Example 1 of the present invention. Figure 6 a is the scanning electron micrograph at 2.5 μm size, b is the scanning electron micrograph at 0.5 μm size, c is the nitrogen adsorption-desorption curve, and d is the pore size distribution diagram.

[0025] Figure 7 This is a column pressure-flow rate curve of the double-pore SiO2@SiO2-C18 core-shell column of the present invention.

[0026] Figure 8 This is a graph showing the reduced plate height-reduced flow rate curve of the double-pore SiO2@SiO2-C18 core-shell column for naphthalene.

[0027] Figure 9 Figure 1 is a chromatographic separation diagram of alkylbenzenes and aromatic alcohol homologues by the dual-pore SiO2@SiO2-C18 core-shell column of the present invention. a is a chromatogram of six alkylbenzene homologues, and b is a chromatogram of five aromatic alcohol homologues.

[0028] Figure 10 This is the chromatographic separation diagram of the dual-pore SiO2@SiO2-C18 core-shell column of the present invention for Bovine serum albumin hydrolysate and Ovalbumin hydrolysate. Figure 10a is the chromatographic separation diagram of Bovine serum albumin enzymatic hydrolysate, and b is the chromatographic separation diagram of Ovalbumin enzymatic hydrolysate.

[0029] Figure 11 This is the chromatographic separation diagram of 6 mixed proteins using the dual-pore SiO2@SiO2-C18 core-shell column of the present invention. DETAILED DESCRIPTION

[0030] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention, in conjunction with the accompanying drawings. Obviously, the described embodiments are only a portion of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0031] It should be noted that the professional terms used in the present invention are only used to describe specific embodiments and do not limit the scope of protection. The reference of the terms to specific components should not be understood as a distinction standard. Those skilled in the art know that the same component may have different naming methods. The components of the present invention are distinguished based on their functional characteristics rather than differences in names. As mentioned throughout the specification and claims, "including" is an open-ended expression and should be understood as "including but not limited to".

[0032] The present invention provides a dual-porous SiO2@SiO2 core-shell microsphere based on a dual-phase method, such as Figure 1 As shown, the core-shell microspheres include, from the inside to the outside, a micron-sized non-porous SiO2 core, a first ordered mesoporous shell layer and a second ordered mesoporous shell layer; wherein, the pore size of the first ordered mesoporous shell layer is 7 nm to 12 nm, and the pore size of the second ordered mesoporous shell layer is 20 nm to 40 nm; the pore channels of the first ordered mesoporous shell layer and the pore channels of the second ordered mesopore layer are radially arranged perpendicular to the surface of the micron-sized non-porous SiO2 core and are interconnected.

[0033] It should be noted that, in response to the problem that existing single-pore core-shell stationary phases are difficult to directly separate mixtures with complex size distributions, the present invention innovatively designs a dual-pore SiO2@SiO2 core-shell microsphere stationary phase. This stationary phase uses micron-scale non-porous SiO2 as the core, and sequentially constructs an ordered mesoporous shell structure with a dual pore size distribution on its surface. Among them, the pore size distribution of the first ordered mesoporous shell is between 7 nm and 12 nm, and the pores are perpendicular to the core surface, with a high degree of order, mainly providing action sites for the separation of small molecule compounds; the second ordered mesoporous shell is constructed with the first ordered mesoporous shell with a smaller pore size as the base, and its pore size is 20 nm to 40 nm. The pore direction is consistent with that of the first ordered mesoporous shell and they are interconnected. The unique dual-pore channel provides exclusive mass transfer pathways for analytes of different sizes, and can provide suitable mass transfer channels for both small-molecule compounds and large-molecule substances, thereby significantly improving the selectivity and column efficiency of chromatographic separation. The differentiated shell pore structure enables the core-shell stationary phase to directly achieve efficient, rapid, and low-cost separation without the need for size-fractionation pretreatment of samples with unknown molecular weight.

[0034] In addition, the double pores of the first ordered mesoporous shell and the second ordered mesoporous shell are completely separated in space, and the independently distributed structural characteristics can ensure that the solute molecules can diffuse from the surface of the core-shell microspheres to the interior of the pores through a relatively uniform mass transfer path, thereby effectively suppressing the chromatographic peak broadening phenomenon.

[0035] In some embodiments, the thickness of the first ordered mesoporous shell layer and the second ordered mesoporous shell layer are both 100 nm to 200 nm.

[0036] It should be noted that the key advantage of core-shell fillers over fully porous fillers lies in their shorter solute diffusion paths, making shell thickness a decisive factor influencing column efficiency and separation speed. In the present embodiment, although the typical thickness of the first ordered mesoporous shell is only 140 nm, the actual shell thickness can be controlled and adjusted within a range of 0.1 nm to 200 nm by precisely controlling the reaction time and the amount of ethyl orthosilicate added, thereby meeting the specific requirements for separation speed and efficiency in different analytical scenarios.

[0037] In some embodiments, the particle size of the micron-sized non-porous SiO2 is 2 μm to 3 μm.

[0038] It should be noted that the column efficiency of a chromatographic column is inversely proportional to the particle size of the filler, making the development of chromatographic stationary phases with smaller particle sizes and ease of packing a research focus in the field of chromatography. However, reducing the particle size will lead to increased operating pressure and may exceed the instrument limit. When the particle size of the core-shell filler is less than 2 μm, its packing uniformity will face great challenges, and the column bed tightness and uniformity will significantly deteriorate with decreasing particle size. In addition, reducing the particle size will also aggravate the uneven distribution of the mobile phase in the column cross-section and the temperature gradient effect, causing chromatographic peak broadening. Therefore, current research on the particle size of core-shell microspheres mainly focuses on sub-3 micron and sub-2 micron specifications. The present invention uses 2 μm to 3 μm non-porous SiO2 as the core, preferably sub-3 μm SiO2@SiO2 core-shell microspheres prepared with a particle size of about 2 μm, which have both excellent column efficiency and low column pressure characteristics.

[0039] The second object of the present invention is to provide a method for preparing the above-mentioned dual-porous SiO2@SiO2 core-shell microspheres based on the two-phase method, such as Figure 2 As shown, the following steps are included: S1. Micron-sized non-porous SiO2 microspheres were prepared using the improved Stöber method.

[0040] S2. Add surfactant and urea to the micron-sized non-porous SiO2 microspheres and disperse them evenly to form a first reaction liquid. Use cyclohexane as the organic phase, tetraethyl orthosilicate as the silicon source, octadecylamine as the pore expander and charge inducer, and isopropyl alcohol as the cosolvent to mix and form a second reaction liquid. Reflux the first reaction liquid at 70°C, then add the second reaction liquid and perform a reflux reaction to deposit a first ordered mesoporous layer on the surface of the micron-sized non-porous SiO2 microspheres to form a SiO2@SiO2 core-shell microsphere precursor.

[0041] S3. Under the same reaction conditions for preparing SiO2@SiO2 core-shell microsphere precursor, the cyclohexane organic phase is replaced with a toluene organic phase, and a reflux reaction is performed to deposit a second ordered mesoporous shell layer on the surface of the first ordered mesoporous layer to form a double-layer porous SiO2@SiO2 core-shell microsphere precursor.

[0042] S4. calcining the double-porous SiO2@SiO2 core-shell microsphere precursor to obtain double-porous SiO2@SiO2 core-shell microspheres.

[0043] It should be noted that the present invention adopts a two-phase method as a synthesis technology, with micron-scale non-porous SiO2 as the core, and deposits the first ordered mesoporous shell layer and the second ordered mesoporous shell layer in sequence by precisely controlling the synthesis conditions, and ensures that the pore orientations of the two shell layers are consistent and interconnected. Specifically: when preparing the first ordered mesoporous shell layer, cyclohexane is used as the organic phase to regulate the pore size; then, without destroying the pore structure of the first layer, the organic phase is replaced with toluene, and the second ordered mesoporous shell layer is directly constructed on the surface of the first ordered mesoporous shell layer; finally, the organic porogen in the pore channel is removed by calcination, and a two-pore core-shell structure with an inner pore size of 7 to 12 nm and an outer pore size of 20 nm to 40 nm is obtained. The unique two-pore channel provides exclusive mass transfer paths for analytes of different sizes, and can provide suitable mass transfer channels for small molecule compounds and large molecular substances at the same time, thereby significantly improving the selectivity and column efficiency of chromatographic separation.

[0044] In some embodiments, the mass ratio of micron-sized non-porous SiO2 microspheres, surfactant and urea is 2:1~2:0.2~3, and the surfactant is hexadecyltrimethylammonium bromide; the mass volume ratio of octadecylamine and cyclohexane is 0.1 g~1.8 g:30 mL~120 mL, the mass volume ratio of octadecylamine and tetraethyl orthosilicate is 0.1 g~1.8 g:0.1 mL~3 mL, and the volume ratio of tetraethyl orthosilicate and isopropyl alcohol is 0.1 mL~3 mL:0.1 mL~6 mL.

[0045] In some embodiments, the volume ratio of the first reaction liquid to the second reaction liquid is 1:0.1-2, and the reflux reaction time is 1 h to 24 h.

[0046] In some embodiments, the amounts of toluene and cyclohexane are the same. It should be noted that the amounts of toluene and cyclohexane can be adjusted as needed and do not have to be used in a strict 1:1 ratio.

[0047] In some embodiments, the calcination temperature is 500° C. to 650° C., and the calcination time is 5 h to 20 h.

[0048] The third object of the present invention is to provide a double-layer radial pore SiO2@SiO2 core-shell microsphere as a filler in chromatographic separation.

[0049] The following is further described through specific examples.

[0050] Example 1 A dual-porous SiO2@SiO2 core-shell microsphere with a micron-sized, nonporous SiO2 core and a porous shell consisting of a first ordered mesoporous shell and a second ordered mesoporous shell, from the inside out. The first ordered mesoporous shell has a smaller pore size, with pores perpendicular to the surface of the nonporous SiO2 core. The second ordered mesoporous shell is tightly connected to the first ordered mesoporous shell, but has a larger pore size and similarly radially arranged pores.

[0051] The preparation method of the double-layer radial pore SiO2@SiO2 core-shell microspheres comprises the following steps: S1. Preparation of micron-sized non-porous SiO2 microspheres: The cores of micron-sized non-porous SiO2 microspheres were prepared by the improved Stöber method, specifically: 500 mL of anhydrous ethanol was added to a 2000 mL three-necked flask, the temperature was raised to 30°C and stirred at 110 rpm, followed by the addition of 18 mL of KCl and 38 mL of ammonia water; a mixed solution of 80 mL of ethyl orthosilicate and 220 mL of ethanol was slowly dripped into the three-necked flask through a peristaltic pump, and the reaction was terminated after the particle size of the SiO2 core grew to the target size; after the reaction, the ethanol mixed solution containing the SiO2 core was centrifuged and washed to remove incompletely hydrolyzed silicate oligomers, and finally the SiO2 core was washed with deionized water until neutral, and dried in an oven at 60°C to obtain micron-sized non-porous SiO2 microspheres.

[0052] S2. Preparation of the first ordered mesoporous shell: 2 g of the nonporous SiO2 microspheres obtained in S1 were weighed and placed in a 250 mL three-necked flask. 2 g of hexadecyltrimethylammonium bromide (as a surfactant to assist the diffusion of TEOS-containing organic phase micelles into the aqueous phase), 1.2 g of urea (as a catalyst), and 60 mL of deionized water were added sequentially. Solution A was obtained by ultrasonic dispersion. Separately, 1.2 g of octadecylamine was dissolved in 60 mL of cyclohexane, and 1.4 mL of tetraethyl orthosilicate and 1.84 mL of isopropanol were added. Solution B was sonicated until clear. Solution A was placed in a 70°C oil bath, stirred at 140 rpm, and refluxed using a serpentine condenser. Solution B was then rapidly added to Solution A. After 12 h of reaction, the first ordered mesoporous layer was deposited on the surface of the nonporous SiO2 core, resulting in a SiO2@SiO2 core-shell microsphere precursor containing a mesoporous shell.

[0053] S3. Preparation of the Second Ordered Mesoporous Shell: The cyclohexane solution in the upper layer of the S2 reaction system was removed to obtain Solution A. 1.2 g of octadecylamine was dissolved in 60 mL of toluene, and 1.4 mL of ethyl orthosilicate and 1.84 mL of isopropanol were added. The mixture was sonicated until clear to obtain Solution B. Solution A was again placed in a 70°C oil bath, stirred at 130 rpm, and refluxed using a serpentine condenser. Solution B was then rapidly added to Solution A. After a reaction time of 12 h, a second ordered mesoporous layer was deposited on the surface of the SiO2@SiO2 core-shell microsphere precursor. After completion of the reaction, the product was collected by centrifugation at 2000 rpm, washed with deionized water and anhydrous ethanol, and dried in a 60°C forced air drying oven. Finally, the product was calcined in a muffle furnace at 600°C for 10 h to obtain the dual-porous SiO2@SiO2 core-shell microspheres.

[0054] Example 2 A dual-porous SiO2@SiO2 core-shell microsphere with a micron-sized, nonporous SiO2 core and a porous shell consisting of a first ordered mesoporous shell and a second ordered mesoporous shell, from the inside out. The first ordered mesoporous shell has a smaller pore size, with pores perpendicular to the surface of the nonporous SiO2 core. The second ordered mesoporous shell is tightly connected to the first ordered mesoporous shell, but has a larger pore size and similarly radially arranged pores.

[0055] The preparation method of the double-layer radial pore SiO2@SiO2 core-shell microspheres comprises the following steps: S1. Preparation of micron-sized non-porous SiO2 microspheres: The cores of micron-sized non-porous SiO2 microspheres were prepared by the improved Stöber method, specifically: 500 mL of anhydrous ethanol was added to a 2000 mL three-necked flask, the temperature was raised to 30°C and stirred at 110 rpm, followed by the addition of 18 mL of KCl and 38 mL of ammonia water; a mixed solution of 80 mL of ethyl orthosilicate and 220 mL of ethanol was slowly dripped into the three-necked flask through a peristaltic pump, and the reaction was terminated after the particle size of the SiO2 core grew to the target size; after the reaction, the ethanol mixed solution containing the SiO2 core was centrifuged and washed to remove incompletely hydrolyzed silicate oligomers, and finally the SiO2 core was washed with deionized water until neutral, and dried in an oven at 60°C to obtain micron-sized non-porous SiO2 microspheres.

[0056] S2. Preparation of the first ordered mesoporous shell: 2 g of the nonporous SiO2 microspheres obtained in S1 were weighed and placed in a 250 mL three-necked flask. 1.5 g of hexadecyltrimethylammonium bromide (as a surfactant to assist the diffusion of TEOS-containing organic phase micelles into the aqueous phase), 0.2 g of urea (as a catalyst), and 60 mL of deionized water were added sequentially. Solution A was obtained by ultrasonic dispersion. Separately, 0.1 g of octadecylamine was dissolved in 30 mL of cyclohexane, and 0.5 mL of tetraethyl orthosilicate and 0.5 mL of isopropanol were added. Solution B was sonicated until clear. Solution A was placed in a 70°C oil bath, stirred at 140 rpm, and refluxed using a serpentine condenser. Solution B was then rapidly added to Solution A. After 12 h of reaction, the first ordered mesoporous layer was deposited on the surface of the nonporous SiO2 core, resulting in a SiO2@SiO2 core-shell microsphere precursor containing a mesoporous shell.

[0057] S3. Preparation of the Second Ordered Mesoporous Shell: The cyclohexane solution in the upper layer of the S2 reaction system was removed to obtain Solution A. 1.2 g of octadecylamine was dissolved in 60 mL of toluene, and 1.4 mL of ethyl orthosilicate and 1.84 mL of isopropanol were added. The mixture was sonicated until clear to obtain Solution B. Solution A was again placed in a 70°C oil bath, stirred at 130 rpm, and refluxed using a serpentine condenser. Solution B was then rapidly added to Solution A. After a reaction time of 12 h, a second ordered mesoporous layer was deposited on the surface of the SiO2@SiO2 core-shell microsphere precursor. After completion of the reaction, the product was collected by centrifugation at 2000 rpm, washed with deionized water and anhydrous ethanol, and dried in a 60°C forced air drying oven. Finally, the product was calcined in a muffle furnace at 600°C for 10 h to obtain the dual-porous SiO2@SiO2 core-shell microspheres.

[0058] Example 3 A dual-porous SiO2@SiO2 core-shell microsphere with a micron-sized, nonporous SiO2 core and a porous shell consisting of a first ordered mesoporous shell and a second ordered mesoporous shell, from the inside out. The first ordered mesoporous shell has a smaller pore size, with pores perpendicular to the surface of the nonporous SiO2 core. The second ordered mesoporous shell is tightly connected to the first ordered mesoporous shell, but has a larger pore size and similarly radially arranged pores.

[0059] The preparation method of the double-layer radial pore SiO2@SiO2 core-shell microspheres comprises the following steps: S1. Preparation of micron-sized non-porous SiO2 microspheres: The cores of micron-sized non-porous SiO2 microspheres were prepared by the improved Stöber method, specifically: 500 mL of anhydrous ethanol was added to a 2000 mL three-necked flask, the temperature was raised to 30°C and stirred at 110 rpm, followed by the addition of 18 mL of KCl and 38 mL of ammonia water; a mixed solution of 80 mL of ethyl orthosilicate and 220 mL of ethanol was slowly dripped into the three-necked flask through a peristaltic pump, and the reaction was terminated after the particle size of the SiO2 core grew to the target size; after the reaction, the ethanol mixed solution containing the SiO2 core was centrifuged and washed to remove incompletely hydrolyzed silicate oligomers, and finally the SiO2 core was washed with deionized water until neutral, and dried in an oven at 60°C to obtain micron-sized non-porous SiO2 microspheres.

[0060] S2. Preparation of the first ordered mesoporous shell: 2 g of the nonporous SiO2 microspheres obtained in S1 were weighed and placed in a 250 mL three-necked flask. 1 g of hexadecyltrimethylammonium bromide (as a surfactant to assist the diffusion of the organic phase micelles containing TEOS into the aqueous phase), 3 g of urea (as a catalyst), and 80 mL of deionized water were added sequentially. Solution A was obtained by ultrasonic dispersion. Separately, 1.8 g of octadecylamine was dissolved in 120 mL of cyclohexane, 3 mL of tetraethyl orthosilicate and 6 mL of isopropanol were added, and solution B was obtained by ultrasonic treatment until clear. Solution A was placed in a 70°C oil bath, stirred at 140 rpm, and refluxed using a serpentine condenser. Solution B was then rapidly added to Solution A. After 12 h of reaction, the first ordered mesoporous layer was deposited on the surface of the nonporous SiO2 core, resulting in a SiO2@SiO2 core-shell microsphere precursor containing a mesoporous shell.

[0061] S3. Preparation of the Second Ordered Mesoporous Shell: The cyclohexane solution in the upper layer of the S2 reaction system was removed to obtain Solution A. 1.2 g of octadecylamine was dissolved in 60 mL of toluene, and 1.4 mL of ethyl orthosilicate and 1.84 mL of isopropanol were added. The mixture was sonicated until clear to obtain Solution B. Solution A was again placed in a 70°C oil bath, stirred at 130 rpm, and refluxed using a serpentine condenser. Solution B was then rapidly added to Solution A. After a reaction time of 12 h, a second ordered mesoporous layer was deposited on the surface of the SiO2@SiO2 core-shell microsphere precursor. After completion of the reaction, the product was collected by centrifugation at 2000 rpm, washed with deionized water and anhydrous ethanol, and dried in a 60°C forced air drying oven. Finally, the product was calcined in a muffle furnace at 650°C for 6 h to obtain dual-porous SiO2@SiO2 core-shell microspheres.

[0062] Since the dual-porous SiO2@SiO2 core-shell microspheres prepared in Examples 1 to 3 have similar structures and basically the same performance, the dual-porous SiO2@SiO2 core-shell microspheres prepared in Example 1 were taken as an example to test the structure and morphology of the dual-porous SiO2@SiO2 core-shell microspheres prepared in Example 1. The results are as follows: Figure 3 This is a morphology and structure diagram of micron-sized non-porous SiO2 microspheres according to Example 1 of the present invention. Figure 3 In the figure, a is a scanning electron microscope at a size of 5 μm, and b is a scanning electron microscope at a size of 1 μm. Figure 3 As shown in the figure, the particle size of the micron-sized non-porous SiO2 microspheres is about 3 μm and the particle size is uniform.

[0063] The SiO2@SiO2 core-shell microsphere precursor was calcined in a muffle furnace at 600 °C for 10 h to form the first ordered mesoporous shell on the micron-sized non-porous SiO2 microspheres. Figure 4 This is a morphological structure diagram of the first ordered mesoporous shell layer in Example 1 of the present invention. Figure 4 a is a scanning electron micrograph at 2.5 μm, b is a scanning electron micrograph at 0.5 μm, c is a transmission electron micrograph at 1 μm, d is a transmission electron micrograph at 0.5 μm, e is a nitrogen adsorption-desorption curve, and f is a pore size distribution diagram. Figure 4 As shown in the figure, the first ordered mesoporous shell was constructed on the micron-sized non-porous SiO2 microspheres. The thickness of the first ordered mesoporous shell was 140 nm and the pore diameter was 8.96 nm.

[0064] Figure 5 This is a morphology and structure diagram of the double-porous SiO2@SiO2 core-shell microspheres in Example 1 of the present invention. Figure 5 a is a scanning electron micrograph at 2.5 μm, b is a scanning electron micrograph at 1 μm, c is a transmission electron micrograph at 2 μm, d is a transmission electron micrograph at 0.5 μm, e is a nitrogen adsorption-desorption curve, and f is a pore size distribution diagram. Figure 5 As shown, the dual-porous SiO2@SiO2 core-shell microspheres successfully encapsulate two mesoporous shells of varying pore sizes on the SiO2 core surface. Pore size distribution measurements show that the average pore size increases from 8.96 nm to 9.65 nm, but the expected bimodal distribution is not observed. This phenomenon may be attributed to the structural similarity of the pores in the two fibrous shells, making it difficult for the instrument to effectively distinguish between the two types of pores.

[0065] To accurately determine the pore size of the second ordered mesoporous shell, a second ordered mesoporous shell was directly prepared on the prepared micron-sized nonporous SiO2 microspheres. 2 g of the nonporous SiO2 microspheres obtained in step S1 of Example 1 were weighed and placed in a 250 mL three-necked flask. 2 g of hexadecyltrimethylammonium bromide, 1.2 g of urea, and 60 mL of deionized water were added, followed by ultrasonic dispersion to obtain Solution A. Separately, 1.2 g of octadecylamine was dissolved in 60 mL of toluene, followed by 1.4 mL of ethyl orthosilicate and 1.84 mL of isopropanol. The mixture was ultrasonically treated until clear to obtain Solution B. Solution A was placed in a 70°C oil bath, stirred at 130 rpm, and refluxed using a serpentine condenser. Solution B was then rapidly added to Solution A. After a reaction time of 12 h, the precursor of the second ordered mesoporous shell was deposited on the surface of the nonporous SiO2 core. Finally, the precursor was calcined in a muffle furnace at 600°C for 10 h to obtain the second ordered mesoporous shell.

[0066] Figure 6 This is a morphological structure diagram of the second ordered mesoporous layer in Example 1 of the present invention. Figure 6 a is the scanning electron micrograph at 2.5 μm size, b is the scanning electron micrograph at 0.5 μm size, c is the nitrogen adsorption-desorption curve, and d is the pore size distribution diagram. Figure 6 As shown, the morphology of the mesoporous shell is highly similar to that of the double-layer core-shell microspheres. The pore size distribution curve shows an average pore diameter of 31.87 nm, confirming that the pore size of the second ordered mesoporous shell is approximately 30 nm, providing a reliable reference for pore size analysis of double-layer mesoporous structures.

[0067] The bi-porous SiO2@SiO2 core-shell microspheres prepared in Example 1 were used as a chromatographic filler matrix for chromatographic separation testing, wherein the bi-porous SiO2@SiO2 core-shell microspheres were used as a stationary phase chromatographic filler. The preparation method included the following steps: 1 g of double-layer porous SiO2@SiO2 core-shell microspheres prepared in Example 1 was dispersed in 40 mL of 6 molL -1 The reaction mixture was stirred and refluxed in a 120°C oil bath for 6 h. After cooling, the mixture was filtered through a G4 funnel and repeatedly washed with deionized water until the filtrate was neutral. The resulting solid was dried in an oven at 125°C for 10 h. It was then ultrasonically dispersed with 1.0 mL of octadecyltrichlorosilane in 50 mL of anhydrous toluene and stirred at reflux for 24 h at 120°C to complete the C18 reverse-phase modification. After the reaction, the product was washed three times with toluene and anhydrous ethanol, respectively, and then dried at 60°C for 12 h. Finally, the product was tail-capped with trimethylchlorosilane to obtain a dual-porous SiO2@SiO2-C18 stationary phase.

[0068] 0.4 g of the bi-porous SiO2@SiO2-C18 stationary phase was ultrasonically dispersed in 30 mL of isopropanol for 5 min and filled into a 50 mm × 2.1 mm ID stainless steel column using an HPPS-20-400 column filling system at a pressure of 80 MPa using methanol and isopropanol (volume ratio of 1:1) as propellants for 40 min.

[0069] Permeability testing was performed using reverse phase chromatography using Waters' ACQUITY TM Ultra-high performance liquid chromatography (UPLC) was used, with ultrapure water as mobile phase A and chromatographically pure acetonitrile as mobile phase B, at a volume ratio of 20:80, at a flow rate of 0.1-1.0 mL·min -1 Perform pressure tests within a range of flow rates. Figure 7 The column pressure-flow rate curve of the double-pore SiO2@SiO2-C18 core-shell column of the present invention is shown in FIG. Figure 7 As shown in the figure, the column pressure of the core-shell column increases linearly with the increase of the flow rate of the mobile phase, and the linear correlation coefficient is R This phenomenon confirms that the chromatographic stationary phase of the core-shell column is uniformly packed and structurally stable.

[0070] Waters ACQUITY TM Ultra-high performance liquid chromatography (UPLC) was used. Mobile phase A was ultrapure water, and mobile phase B was chromatographically pure acetonitrile. The volume ratio of the two was 50:50. The detection wavelength was 254 nm, the injection volume was 1 μL, and the flow rate was 0.1 mL min -1 ~1.0 mL·min -1 Tests were performed within a range of flow rates. Figure 8 The graph of the reduced plate height-reduced flow rate of the double-layer porous SiO2@SiO2-C18 core-shell column for naphthalene is shown in FIG. Figure 8 As shown in the figure, when naphthalene is used as the test object for column efficiency analysis, the minimum equivalent plate height of the core-shell column for naphthalene is h min is 2.04.

[0071] Waters ACQUITY TM Ultra-high performance liquid chromatography (UPLC) was used in isocratic elution mode with ultrapure water as mobile phase A and chromatographic-grade acetonitrile as mobile phase B. The chromatographic conditions were set at a flow rate of 0.6 mL min -1 , detection wavelength of 254 nm, injection volume of 1 μL; a double-layer pore SiO2@SiO2-C18 core-shell column was used for the separation and analysis of alkylbenzene homologues and aromatic alcohol homologues. Figure 9Figure 1 is a diagram of the separation and analysis of alkylbenzenes and aromatic alcohol homologues by the dual-pore SiO2@SiO2-C18 core-shell column of the present invention. a is a diagram of the separation and analysis of 6 alkylbenzene homologues, and b is a diagram of the separation and analysis of 5 aromatic alcohol homologues. Figure 9 As shown in a, the volume ratio of acetonitrile and water in the mobile phase is 70:30. Figure 9 As shown in b, the volume ratio of acetonitrile and water in the mobile phase is 40:60, and the flow rate is 0.6 mL min -1 Detection wavelength: 254 nm; Figure 9 The chromatographic peaks are: 1-toluene, 2-ethylbenzene, 3-propylbenzene, 4-butylbenzene, 5-pentylbenzene, 6-hexylbenzene, 7-phenylethanol, 8-phenylpropanol, 9-phenylbutanol, 10-phenylpentanol, 11-phenylhexanol. Figure 9 As shown in Table 1, the core-shell reversed-phase column achieved baseline separation of six alkylbenzenes and five aromatic alcohol homologues within 2 min, with the theoretical plate number of p-hexylbenzene reaching 127,400·m -1 .

[0072] Table 1 Separation and analysis parameters of alkylbenzenes and aromatic alcohol homologues The peptide separation test was performed using Waters ACQUITY TM Ultra-high performance liquid chromatography (UPLC) was used. Mobile phase A was ultrapure water containing 0.1% trifluoroacetic acid, and mobile phase B was chromatographic grade acetonitrile containing 0.1% trifluoroacetic acid. A linear gradient elution program was used. The detection wavelength was 280 nm, the injection volume was 10 μL, and the flow rate was 0.6 mL min. -1 . Figure 10 This is a chromatographic separation analysis diagram of the dual-pore SiO2@SiO2-C18 core-shell column of the present invention for Bovine serum albumin hydrolysate and Ovalbumin hydrolysate. Figure 10 Figure a is the separation and analysis diagram of Bovineserum albumin enzymatic hydrolysate, and figure b is the separation and analysis diagram of Ovalbumin enzymatic hydrolysate. Figure 10 For the separation and analysis of Bovine serum albumin hydrolysate, a linear gradient elution program was used to increase the mobile phase B from 10% to 25% and 100% at 0 min, 10 min and 14 min, respectively. For the separation and analysis of Ovalbumin hydrolysate, a linear gradient elution program was used to increase the mobile phase B from 10% to 25% and 100% at 0 min, 10 min and 14 min, respectively. Figure 10 As shown in the figure, after identification by UV detector, the enzymatic hydrolysate of Bovine serum albumin can be separated into more than 30 components, and the enzymatic hydrolysate of Ovalbumin can be identified into nearly 40 components.

[0073] Waters ACQUITY TM Ultra-high performance liquid chromatography (UPLC) was used. Mobile phase A was ultrapure water containing 0.1% trifluoroacetic acid, and mobile phase B was chromatographically pure acetonitrile containing 0.1% trifluoroacetic acid. The protein concentration was 5 mg / mL. -1 A linear gradient elution program was used to increase mobile phase B from 10% to 60% within 2 min, with a detection wavelength of 280 nm, an injection volume of 10 μL, and a flow rate of 0.6 mL / min. -1 . Figure 11 This is the chromatographic separation diagram of 6 mixed proteins using the dual-pore SiO2@SiO2-C18 core-shell column of the present invention. Figure 11 In the chromatographic peaks: 1 is Ribonuclease A, 2 is Insulin, 3 is Cytochrome C, 4 is Lysozyme, 5 is Bovine serum albumin, and 6 is Ovalbumin. Figure 10 As shown in the figure, the double-layer pore SiO2@SiO2-C18 core-shell column can achieve complete separation of 6 proteins within 2 minutes.

[0074] In summary, the dual-pore SiO2@SiO2-C18 core-shell column provided by the present invention can effectively achieve separation tasks that originally required two core-shell columns with different pore sizes. This feature significantly improves the efficiency and economy of chromatographic separation, and provides a new technical solution for the rapid separation and analysis of complex samples.

[0075] It should be noted that when numerical ranges are mentioned in the present invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints may be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes preferred embodiments. Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0076] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A dual-porous SiO2@SiO2 core-shell microsphere based on a two-phase method, characterized in that: The core-shell microspheres have micron-scale non-porous SiO2 as the core and two layers of mesoporous SiO2 skeletons with different pore sizes and pores perpendicular to the surface of the non-porous SiO2 core as the porous shell; the porous shell includes a first ordered mesoporous shell and a second ordered mesoporous shell from the inside to the outside, the pore size of the first ordered mesoporous shell is 7 nm to 12 nm, and the pore size of the second ordered mesopore is 20 nm to 40 nm, and the pores of the first ordered mesoporous shell and the second ordered mesopore are radially arranged, perpendicular to the surface of the micron-scale non-porous SiO2 core and interconnected.

2. The double-layer porous SiO2@SiO2 core-shell microspheres based on the two-phase method according to claim 1, characterized in that: The thickness of the first ordered mesoporous shell layer and the second ordered mesoporous shell layer are both 100 nm to 200 nm.

3. The double-layer porous SiO2@SiO2 core-shell microspheres based on the two-phase method according to claim 1, characterized in that: The particle size of the micron-sized non-porous SiO2 core is 2 μm to 3 μm.

4. A method for preparing double-layer porous SiO2@SiO2 core-shell microspheres based on a two-phase method according to any one of claims 1 to 3, characterized in that: The following steps are involved: Micron-sized non-porous SiO2 microspheres were prepared by the improved Stöber method; Surfactant and urea were added to micron-sized non-porous SiO2 microspheres and dispersed uniformly to form a first reaction solution. Cyclohexane was used as the organic phase, tetraethyl orthosilicate was used as the silicon source, octadecylamine was used as the pore-enlarging agent and charge-inducing agent, and isopropyl alcohol was used as the cosolvent to form a second reaction solution. After the first reaction solution was refluxed at 70°C, the second reaction solution was added and the reflux reaction was continued, thereby depositing a first ordered mesoporous shell layer on the surface of the micron-sized non-porous SiO2 microspheres and forming a SiO2@SiO2 core-shell microsphere precursor. Under the same reaction conditions as those for preparing the SiO2@SiO2 core-shell microsphere precursor, the cyclohexane organic phase was replaced with a toluene organic phase, and after reflux reaction, a second mesoporous shell layer was deposited on the surface of the first ordered mesoporous shell precursor to obtain a double-layer porous SiO2@SiO2 core-shell microsphere precursor; The double-layer porous SiO2@SiO2 core-shell microsphere precursor is calcined to obtain double-layer porous SiO2@SiO2 core-shell microspheres.

5. The method for preparing double-layer porous SiO2@SiO2 core-shell microspheres according to claim 4, characterized in that: The mass ratio of micron-sized non-porous SiO2 microspheres, surfactant and urea is 2:1~2:0.2~3, and the surfactant is hexadecyltrimethylammonium bromide; the mass-to-volume ratio of octadecylamine and cyclohexane is 0.1 g~1.8 g:30 mL~120 mL, the mass-to-volume ratio of octadecylamine and tetraethyl orthosilicate is 0.1 g~1.8 g:0.1 mL~3 mL, and the volume ratio of tetraethyl orthosilicate and isopropyl alcohol is 0.1~3:0.1~6.

6. The method for preparing double-layer porous SiO2@SiO2 core-shell microspheres based on a two-phase method according to claim 4, characterized in that: The volume ratio of the first reaction liquid to the second reaction liquid is 1:0.1-2, and the reflux reaction time is 1 h-24 h.

7. The method for preparing double-layer porous SiO2@SiO2 core-shell microspheres based on a two-phase method according to claim 4, characterized in that: The amounts of toluene and cyclohexane used were the same.

8. The method for preparing double-layer porous SiO2@SiO2 core-shell microspheres based on a two-phase method according to claim 4, characterized in that: The calcination temperature is 500 ℃ ~ 650 ℃, and the time is 5 h ~ 20 h.

9. Use of the double-layer porous SiO2@SiO2 core-shell microspheres according to any one of claims 1 to 3 as a filler in chromatographic separation.

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