CN111019067B

CN111019067B - Organic-inorganic hybrid porous monolithic material, preparation and application

Info

Publication number: CN111019067B
Application number: CN201811172346.4A
Authority: CN
Inventors: 欧俊杰; 王妍; 马淑娟; 叶明亮
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Dalian Institute of Chemical Physics of CAS
Priority date: 2018-10-09
Filing date: 2018-10-09
Publication date: 2021-04-02
Anticipated expiration: 2038-10-09
Also published as: CN111019067A

Abstract

The invention relates to a rapid preparation method of an organic-inorganic hybrid porous monolithic material based on Thiol-ene click polymerization (Thiol-ene click polymerization). The preparation method specifically comprises the steps of uniformly mixing polyhedral oligomeric silsesquioxane (POSS) (functional monomer) containing multi-mercapto functional groups, organic compounds (functional monomer) containing three double bonds, organic compounds (functional monomer) containing one double bond, a photoinitiator, a pore-forming agent and the like, and then carrying out mercapto-alkene click polymerization reaction at a certain temperature to prepare the organic-inorganic hybrid porous integral material with special functions. The preparation method has the advantages of mild condition, rapidness, controllability and the like. The prepared hybrid monolithic material can be applied to chromatographic separation, and realizes high-efficiency separation and analysis of small molecular compounds and complex biological samples.

Description

Organic-inorganic hybrid porous monolithic material, preparation and application

Technical Field

The invention relates to a method for quickly preparing an organic-inorganic hybrid porous monolithic material based on Thiol-ene click polymerization (Thiol-ene click polymerization). The organic-inorganic hybrid porous monolithic material with special functions is prepared by mixing and uniformly stirring a compound (functional monomer) containing multi-mercapto functional groups, an organic compound (functional monomer) containing three double bonds, an organic compound (functional monomer) containing one double bond, a photoinitiator, a pore-forming agent and the like, and then carrying out mercapto-methacrylate click polymerization reaction at a certain temperature.

Background

Capillary liquid chromatography (cLC) is one of the most important techniques in microanalysis and plays an important role in the field of separation science. Compared with the traditional liquid chromatography, the capillary liquid chromatography not only improves the effectiveness and chromatographic separation selectivity of the chromatographic column, but also reduces the waste of a mobile phase, a stationary phase and a sample. Capillary columns are the core of capillary liquid chromatography and can be generally divided into three types, including capillary packed columns, capillary monolith columns and capillary open columns. Suitable functional monomers are readily polymerized in situ within the capillary, which avoids the complicated preparation of packed columns. Because the preparation of the packed column requires the burning of the plug and the packing of the packing. In addition, the peak capacity of the monolithic column is much higher than that of the open-tube capillary column. The monolithic material has a series of advantages of easy preparation, high mass transfer rate, good permeability, stable performance and the like, is widely applied to the field of chromatographic separation and can be used for preparing a whole body.

Monolithic columns are prepared by a variety of methods, such as sol-gel and free radical polymerization. Although the sol-gel method has good versatility, the preparation process is relatively complicated. Free radical polymerization is relatively simple and easy to operate, but the reaction is difficult to control because of the very fast chain growth rate. Click chemistry was first proposed in 2001 by Sharpless who obtained the nobel prize for chemistry (document 1.Kolb, h.c., Finn, m.g., Sharpless, k.b., Click chemistry: reverse chemical function from a good reactions, angew.chem.int.ed.2001,40, 2004-2021.). Originally, it was widely used in organic synthesis, bioconjugation and surface modification, and now it has been extended to the synthesis of novel polymers. The click chemical reaction has the characteristics of easiness in control, mild reaction conditions, insensitivity to oxygen and the like, and can be used for preparing the monolithic column. Thiol-based click polymerization offers the advantages of classical click chemistry, and in particular avoids some side effects in biological applications, such as the CuAAC reaction, which is most typical of click chemistry, leads to residual copper, a heavy metal, in the product. Photoinitiated mercapto-ene click polymerization is a stepwise polymerization reaction that is not sensitive to oxygen in the air, greatly simplifying the overall manufacturing process (document 2.Chan, j.w., Shin, j., Hoyle, c.e., Bowman, c.n., Lowe, a.b., Synthesis, clinical-ene "polymerization, and physical properties of network derivative from novel functional industries, macromolecules 2010,43, 4937-4942).

According to the sulfydryl-alkene click polymerization reaction, a proper compound is selected as a functional monomer, and the hybrid monolithic column with the three-dimensional framework structure can be prepared. The prepared organic-inorganic hybrid whole body can be applied to chromatographic separation and analysis.

Disclosure of Invention

The invention provides a method for preparing a porous hybrid monolithic material based on a mercapto-alkene click chemical reaction. The organic-inorganic hybrid monolithic material is prepared by uniformly mixing polyhedral oligomeric silsesquioxane containing eight sulfydryl groups, two acrylate monomers, a pore-forming agent and a photoinitiator through ultrasound, exposing in an ultraviolet crosslinking instrument, and performing polymerization reaction initiated by light.

The technical scheme adopted by the invention is as follows:

the method comprises the steps of dissolving polyhedral oligomeric silsesquioxane containing eight sulfydryl groups, functional monomers containing three double bonds, functional monomers containing one double bond and a photoinitiator modified by mercaptopropyl trimethoxy silane in a pore-forming solvent, ultrasonically mixing the solution to obtain a uniform and transparent solution, preparing an organic-inorganic porous hybrid integral material by utilizing a sulfydryl-alkene click chemical reaction, and preparing the organic-inorganic porous hybrid integral material with different properties by adjusting the monomer concentration and the pore-forming agent ratio according to different requirements.

The hybrid porous monolithic material prepared by the invention can be applied to chromatographic analysis, is particularly suitable for capillary liquid chromatography, and can realize the separation and identification of small molecules and complex biological samples. The separation objects are five kinds of benzene series, amine compounds, phenolic compounds and complex biological samples such as bovine serum protein enzymolysis liquid and murine liver protein enzymolysis liquid. The results show that five benzene series compounds, eight phenol compounds and five amine compounds respectively achieve baseline separation in a reversed phase chromatography mode, and meanwhile, in the LC-MS, the material can realize the separation of peptide fragments in a complex sample and the identification of proteins.

The invention has the following beneficial effects and advantages:

the method adopts the photoinitiated sulfydryl-ene click polymerization reaction (Thiol-ene click polymerization), and the click chemical reaction has the characteristics of easy control, mild reaction condition, insensitivity to oxygen and the like, so that the preparation process of the integral material is simpler and the preparation time is shortened. The formation of the hybrid porous monolithic material only needs exposure reaction in an ultraviolet crosslinking instrument, and the aperture, the pore structure and the permeability of the hybrid porous monolithic material can be regulated and controlled by changing the concentration of the added crosslinking agent and the functional monomer and changing the composition or the content of the pore-forming solvent.

The hybrid monolithic material prepared by the invention has a uniform three-dimensional network structure and is suitable for chromatographic separation and analysis. Water contact angle experiments show that the hybrid porous monolithic material has strong hydrophobicity, and liquid chromatography investigation results show that a neutral compound shows a typical reversed-phase retention mechanism on the material. It can be seen that the organic-inorganic hybrid monolithic column prepared by the experiment shows stronger hydrophobicity and higher column efficiency.

The hybrid porous monolithic material prepared by the invention has the advantages of good permeability, good biocompatibility, good thermal stability, strong universality and the like, and can be used for preparing various monolithic materials for chromatographic separation by adopting other functional monomers with sulfydryl or double bonds as precursors.

Drawings

Fig. 1 is a schematic diagram of the preparation of organic-inorganic hybrid monolithic materials based on thiol-ene click polymerization.

FIG. 2 is a scanning electron micrograph of the capillary hybrid monolithic column of example 1 (a is 1000 times and b is 5000 times).

FIG. 3 is a thermogravimetric analysis of the porous hybrid monolith of example 1.

FIG. 4 is a diagram of capillary liquid chromatography separation of benzene series in capillary hybrid monolithic column of example 1.

FIG. 5 is a graph showing the effect of flow rate of the capillary hybridization monolith column on the height of the tray in example 1.

FIG. 6 is a graph of the effect of acetonitrile content on analyte retention factor (example 1).

FIG. 7 is a diagram showing capillary liquid chromatography separation of phenolic compounds and amine compounds on a capillary hybridized monolithic column in example 1 (a is a phenolic compound, and b is an amine compound).

FIG. 8 is a cLC-MS/MS separation chart of BSA enzymatic hydrolysate and mouse liver proteolytic hydrolysate in capillary hybrid monolithic column of example 1 (a is BSA enzymatic hydrolysate, b is mouse liver proteolytic hydrolysate).

FIG. 9 is a diagram of capillary liquid chromatography separation of benzene series in capillary hybrid monolithic column of example 2.

Detailed Description

Example 1

Preparing a POSS-SH functional monomer: firstly, 15mL of mercaptopropyl trimethoxy silane (MPTS) is dissolved in 360mL of methanol, 30mL of concentrated hydrochloric acid (37%) is dropwise added into the solution, and the mixture is refluxed and reacted in an oil bath at 90 ℃ for 24 hours; after the reaction is finished, cooling the reaction solution in an ice bath (-4 ℃) to obtain a crude product, washing the crude product for five times by using methanol, and removing residual mercaptopropyltrimethoxysilane; then, the obtained crude product was dissolved with dichloromethane, water was added to the solution for extraction, the dichloromethane layer was collected, water was added for extraction, which was repeated three times, finally dichloromethane was collected, and anhydrous sodium sulfate was added thereto and dried overnight (12 hours); and finally, carrying out reduced pressure distillation on the obtained solution to obtain the product POSS-SH.

1) Adding POSS-SH 63.6mg as a functional monomer into an empty centrifugal tube;

2) adding 145 mu L of diethylene glycol diethyl ether into the centrifugal tube in the step 1) as a pore-foaming agent;

3) adding 157 mu L of n-propanol into the centrifugal tube in the step 2) as a pore-foaming agent;

4) adding 19.4 mu L of 1,2, 4-triethylcyclohexane serving as a functional monomer into the centrifugal tube in the step 3);

5) adding 35.3 mu L of isophytol serving as a functional monomer into the centrifugal tube in the step 4);

6) adding 0.2mg of 2, 2-dimethoxy-phenylacetophenone into the centrifuge tube obtained in the step 5) to serve as a photoinitiator;

7) performing ultrasonic treatment on the centrifugal tube in the step 6) at room temperature for 3min to completely dissolve the centrifugal tube to form a uniform and transparent solution;

8) introducing 1 μ L of the prepolymer solution obtained in step 7) into a 75 μm (inner diameter) capillary tube which had been subjected to 3- (methacryloyloxy) propyltrimethoxysilane activation treatment in advance, using a syringe, then sealing both ends of the capillary tube with silica gel, and then sealing the centrifuge tube containing the remaining mixed solution.

9) Placing the reactor sealed with the mixed solution in the step 8) in an ultraviolet crosslinking instrument with the wavelength of 365nm, and reacting for 10min until the mixed solution in the centrifuge tube reacts to become a white solid;

10) washing the product obtained in the step 9) reactor for at least 3 times by using ethanol, and flushing out the pore-foaming agent and the unreacted or un-combined substances to obtain the hybrid porous monolithic material.

11) And (3) washing the capillary with ethanol, and washing out the pore-foaming agent and some substances which do not participate in the reaction to prepare the capillary hybrid monolithic column. Scanning electron micrographs of the capillary hybrid monolithic column are shown in FIG. 2(a, b); the thermogravimetric analysis of the porous hybrid monolith is shown in FIG. 3; the capillary liquid chromatography separation diagram is shown in FIG. 4; van Deemter of capillary hybridization monolith column is shown in FIG. 5; the effect of acetonitrile content on retention factor is shown in FIG. 6.

FIG. 3 is a thermogravimetric analysis of a porous hybrid monolith. The weight loss range of the whole material is 285-600 ℃, which shows that the material has good thermal stability.

FIG. 4 is a diagram of capillary liquid chromatography separation of benzene series in capillary hybrid monolithic column. The chromatographic conditions were capillary column (19.4 cm. times.75 μm i.d.), mobile phase acetonitrile/water (70/30, v/v) and flow rate 240 μ L/min (before splitting). Peaks in the chromatogram are (1) thiourea, (2) benzene, (3) toluene, (4) ethylbenzene, (5) propylbenzene and (6) butylbenzene in sequence. The appearance of peaks from weak hydrophobicity to strong hydrophobicity is a typical reversed-phase chromatographic retention mechanism.

FIG. 5 is a graph showing the effect of flow rate of a capillary tube hybridization monolith column on the height of a tray. Analytes were (. tangle-solidup.) benzene, (. smallcircle.) toluene, (●) ethylbenzene, (□) propylbenzene and (■) butylbenzene; the chromatographic conditions were capillary column (19.4 cm. times.75 μm i.d.), mobile phase acetonitrile/water (70/30, v/v) and flow rate 20-220 μ L/min (before splitting).

FIG. 6 is a graph of the effect of acetonitrile content on retention factor. The chromatographic conditions were capillary column (19.4 cm. times.75 μm i.d.), mobile phase acetonitrile/water (40/60-70/30, v/v), flow rate 240 μ L/min (before splitting).

FIG. 7 shows the capillary liquid chromatography separation of phenolic compounds and amine compounds on a capillary hybridized monolithic column (a is phenolic compound, b is amine compound). Peaks in the chromatogram are (a) (1) phloroglucinol, (2) o-phenylphenol, (3) phenol, (4) p-cresol, (5) o-cresol, (6)2, 6-dimethylphenol, (7) naphthol, and (8)2, 4-dichlorophenol in sequence; (b) p-phenylenediamine (1), barbiturate (2), 4-methylenedianiline (3), 2-aminofluorene (4) and 1-naphthylamine (5). The mobile phase was acetonitrile/water (30/70, v/v); the flow rate was 170. mu.L/min (before splitting). Under the reverse phase chromatography mode, the eight phenolic compounds and the five amine compounds respectively achieve baseline separation and symmetrical peak shapes.

FIG. 8 is a cLC-MS/MS separation chart of bovine serum albumin enzymatic hydrolysate and murine liver protein enzymatic hydrolysate in a capillary hybridization monolithic column (a is bovine serum albumin enzymatic hydrolysate, and b is murine liver protein enzymatic hydrolysate). Chromatographic conditions were capillary column (25.9cm × 100 μm i.d.); the mobile phase A is 0.1% formic acid water solution, and the phase B is 0.1% formic acid acetonitrile solution; mobile phase gradient (a) 0-5% B (0-2min), 5-35% B (2-32min), 35-80% B (32-62min), 80% B (62-92min), (B) 0-5% B (0-2min), 5-35% B (2-95min), 35-80% B (95-103min), 80% B (103-133 min); the flow rate was 70. mu.L/min (before splitting). The result shows that 108 non-redundant peptide fragments are identified by analyzing the bovine serum albumin enzymatic hydrolysate, and the protein coverage rate is more than 85 percent; the highest 1087 proteins and 2905 peptides were identified by the analysis of the liver proteolysis solution.

As can be seen from the examples and the accompanying drawings, the method has the advantages of simple preparation process and short time consumption, and the prepared organic-inorganic hybrid monolithic column has a three-dimensional reticular framework structure, good thermal stability, proper permeability and uniform pore appearance. The micro-molecular compound is prepared into a whole and used in capillary liquid chromatography, so that a good separation effect is obtained, the column efficiency of separating the micro-molecular compound is high, and a satisfactory separation and analysis result is obtained when the micro-molecular compound is applied to separation of a complex biological sample. More importantly, various different monolithic columns can be prepared by replacing any functional monomer in the reaction system and can be widely applied to chromatographic separation.

Example 2

1) Adding POSS-SH 51.0mg as a functional monomer into an empty centrifugal tube;

2) adding 90 mu L of diethylene glycol diethyl ether into the centrifuge tube in the step 1) to serve as a pore-foaming agent;

3) adding 149 mu L of n-propanol into the centrifugal tube in the step 2) to serve as a pore-foaming agent;

11) And (3) washing the capillary with ethanol, and washing out the pore-foaming agent and some substances which do not participate in the reaction to prepare the capillary hybrid monolithic column. The capillary liquid chromatography separation diagram is shown in FIG. 9.

FIG. 9 is a diagram of capillary liquid chromatography separation of benzene series in capillary hybrid monolithic column. The chromatographic conditions were capillary column (20.7 cm. times.75 μm i.d.), mobile phase acetonitrile/water (70/30, v/v) and flow rate 120. mu.L/min (before splitting). Peaks in the chromatogram are (1) thiourea, (2) benzene, (3) toluene, (4) ethylbenzene, (5) propylbenzene and (6) butylbenzene in sequence. The five benzene series did not reach baseline separation due to the column being too loose.

Claims

1. A preparation method of an organic-inorganic hybrid porous monolithic material is characterized by comprising the following steps:

firstly, mixing and ultrasonically dissolving a polyhedral oligomeric silsesquioxane reagent with multi-sulfydryl, a functional monomer with three double bonds, a functional monomer with one double bond, a pore-forming agent and a photoinitiator, and carrying out sulfydryl-alkene click chemical reaction under the irradiation of an ultraviolet lamp to prepare an organic-inorganic porous hybrid integral material in one step;

the functional monomer containing three double bonds is 1,2, 4-trivinylcyclohexane, and the functional monomer containing one double bond is isophytol.

2. The method for preparing the hybrid porous monolithic material according to claim 1, wherein:

the polyhedral oligomeric silsesquioxane reagent with multi-sulfydryl is an octa (3-mercaptopropyl) octa-polysilsesquioxane reagent; the pore-foaming agent is diethylene glycol diethyl ether and n-propanol; the photoinitiator is 2, 2-dimethoxy-phenyl acetophenone.

3. The method for preparing the hybrid porous monolithic material according to claim 1 or 2, characterized in that:

comprises the following steps of (a) carrying out,

1) preparing a multi-sulfydryl polyhedral oligomeric silsesquioxane reagent;

2) dissolving 50-70 mg of the multi-mercapto polyhedral oligomeric silsesquioxane reagent obtained in the step 1) in 0.09-0.16 mL of pore-foaming agent diethylene glycol diethyl ether in a centrifugal tube;

3) adding 0.14-0.17 mL of n-propanol into the solution obtained in the step 2) to serve as a pore-foaming agent;

4) adding 0.02-0.03 mL of 1,2, 4-trivinylcyclohexane as a functional monomer into the solution obtained in the step 3);

5) adding 0.03-0.04 mL of functional monomer isophytol into the solution obtained in the step 4);

6) adding 0.15-0.30 mg of photoinitiator 2, 2-dimethoxy-phenylacetophenone into the solution obtained in the step 5);

7) carrying out ultrasonic treatment on the solution obtained in the step 6) to enable the solution to completely form uniform and transparent pre-polymerization liquid;

8) placing the prepolymerization liquid obtained in the step 7) into an ultraviolet crosslinking instrument, and exposing the prepolymerization liquid by an ultraviolet lamp to obtain a white opaque solid;

9) washing the product obtained in the centrifugal tube in the step 8) with ethanol, and washing out the pore-foaming agent and the residual substances to obtain the hybrid porous monolithic material.

4. The production method according to claim 3, characterized in that:

preparation of Polymercapto polyhedral oligomeric silsesquioxane reagent: firstly, dissolving 15mL of mercaptopropyl trimethoxysilane in 358-362 mL of methanol, dropwise adding 28-32 mL of concentrated hydrochloric acid with the mass concentration of 36-38% into the solution, and carrying out reflux reaction in an oil bath at the temperature of 88-92 ℃ for 24-26 h; after the reaction is finished, cooling the reaction solution in an ice bath at the temperature of-4-4 ℃ to obtain a crude product, washing the crude product for three to five times by using methanol, and removing residual mercaptopropyl trimethoxysilane; then, dissolving the obtained crude product by using dichloromethane, adding water into the solution for extraction, collecting a dichloromethane layer, adding water for extraction, repeating for three to five times, finally collecting the dichloromethane layer, adding anhydrous sodium sulfate into the dichloromethane layer, and drying for 12 to 14 hours; and finally, carrying out reduced pressure distillation on the obtained solution to obtain the product, namely the multi-sulfydryl polyhedral oligomeric silsesquioxane reagent.

5. The production method according to claim 3, characterized in that: the functional monomer 1,2, 4-trivinylcyclohexane containing three double bonds is 0.02-0.03 mL, and the functional monomer isophytol containing one double bond is 0.03-0.04 mL; 50-70 mg of multi-sulfydryl polyhedral oligomeric silsesquioxane reagent; the pore-foaming agent is 0.23-0.34 mL; the photoinitiator is 0.15-0.30 mg of 2, 2-dimethoxy-phenyl acetophenone; the volume ratio of the diethylene glycol diethyl ether to the n-propanol in the pore-forming solvent is 1: 1.9-1.1: 1.

6. The production method according to claim 3, characterized in that: and 7) carrying out ultrasonic treatment for 2-4 min, and carrying out the photoinitiated reaction of the step 8) at room temperature for 7-11 min.

7. A porous hybrid monolithic material obtained by the preparation method of any one of claims 1 to 6.

8. Use of the porous hybrid monolithic material according to claim 7, wherein: the porous hybrid monolithic material is applied to chromatographic analysis as a chromatographic packing.

9. Use of the porous hybrid monolith according to claim 8, wherein: the porous hybrid monolithic material is used as a chromatographic packing for high-efficiency chromatographic separation of small molecular compounds and/or chromatographic analysis of complex biological samples.

10. Use according to claim 9, characterized in that: the small molecular compound is one or more of phenol and aniline compounds; the complex biological sample is one or more of bovine serum albumin enzymatic hydrolysate and rat liver protein enzymatic hydrolysate.