CN103965419B - A kind of separation, the magnetic blotting method for producing polymer of purification Ractopamine - Google Patents

Abstract

The invention relates to a preparation method for separating and purifying ractopamine magnetic molecularly imprinted polymers, the purpose of which is to solve the problem of leakage of magnetic ferric iron tetroxide during template elution and practical application of existing magnetic ractopamine molecularly imprinted polymers . The steps of the method are as follows: (1) preparing magnetic ferric oxide nano-microspheres; (2) preparing ATPS-modified ferric oxide material (ATPS-Fe ₃ O ₄ ); (3) preparing magnetic Fe ₃ O ₄ functional monomer (MAC‑ATPS‑Fe ₃ O ₄ ); (4) Preparation of magnetic RAC molecularly imprinted polymers (Fe ₃ O ₄ @MIPs). The ractopamine magnetic molecularly imprinted polymer prepared by the present invention has stable performance, and there is no leakage of magnetic ferric iron tetroxide. The magnetic polymer has a relatively fast adsorption rate for ractopamine, meets the needs of rapid detection, and has rapid magnetic separation capabilities. Ideal for on-site sample handling.

Description

A preparation method of magnetically imprinted polymer for separation and purification of ractopamine

technical field

The invention relates to a preparation method of a core-shell type magnetic molecularly imprinted polymer, in particular to a preparation method for separating, purifying and enriching ractopamine magnetic nanometer molecularly imprinted polymer.

Background technique

Ractopamine (RAC) is a β ₂ -agonist, which can selectively stimulate the β ₂ -receptors of cell membrane smooth muscle, and is clinically used to prevent and treat congestive heart failure, bronchial asthma and muscular atrophy. RAC is a kind of "lean meat extract", which can show "nutrient redistribution effect", by changing the metabolic pathway of nutrients, it can promote animal muscle growth and animal protein deposition, especially the synthesis of protein in skeletal muscle. At the same time, it inhibits the synthesis and accumulation of fat, so as to improve the carcass quality and accelerate the growth rate. However, RAC remains in the blood for a long time and has serious toxic side effects on the body, which can cause muscle tremors, vomiting, nervousness, palpitations and other symptoms. my country and most EU countries strictly prohibit the application of RAC in livestock and poultry breeding.

Molecularly imprinted technology (MIT) is a technology for preparing polymers with specific selectivity for target molecules. Molecularly imprinted polymers (MIPs) prepared based on this technology have a complementary spatial structure and specific arrangement of binding sites for target molecules. It is the basis that MIPs have specific selectivity for target molecules. With its unique specific recognition, physical and chemical stability, and wide application, MIPs are well used in sample pretreatment, biosensors, biomimetic immunoassays, and chromatographic stationary phases.

At present, the preparation methods of molecularly imprinted polymers targeting RAC include gel-sol method (Wang S, LiuL, Fang G, et al.J Sep Sci,2009,32(9):1333-1339.), ontology Polymerization method (Zhigang Xu, YufeiHu, Yuling Hu, et al.Journal of Chromatography A,2010,1217:3612-3618.), covalent bond polymerization method (Yiwei Tang, Guozhen Fang, Shuo Wang, et al.Anal Bioanal Chem , 2011,401(7): 2275-2282.), the MIPs prepared by these methods all need to be ground into particles with a certain particle size as solid-phase extraction materials and loaded into solid-phase extraction cartridges for use on solid-phase extraction instruments; or The target molecules are extracted and enriched in the form of dispersive extraction, but MIPs are not easy to separate after dispersive extraction, which limits the application of RAC-MIPs in sample pretreatment.

In order to reduce the dependence of MIPs on solid-phase extraction equipment when used as solid-phase extraction materials, and facilitate the separation of MIPs after dispersive extraction, magnetic MIPs with magnetic Fe ₃ O ₄ cores were successfully prepared (Yuling Hu, Yuanwen Li, Ruijin Liu, et al. al. Magnetic molecularly imprinted polymer beads prepared by microwave heating for selective enrichment of β-agonists in pork and pig liversamples. Talanta, 2011, 84:462-470.). The method uses RAC as a template molecule, acrylamide (AM) as a functional monomer, trimethylolpropane trimethylacrylate (TRIM) as a crosslinking agent, azobisisobutyronitrile as an initiator, and modified with PEG-6000 Magnetic RAC molecularly imprinted polymer materials were prepared by ion copolymerization of Fe ₃ O ₄ nanoparticles. However, PEG-6000 and Fe ₃ O ₄ are combined by non-covalent bonds, and the interaction force is weak. The polymerization process is easily affected by environmental factors, which will destroy the combination of Fe ₃ O ₄ and PEG-6000, resulting in Fe ₃ O ₄ is exposed; secondly, PEG-6000-modified Fe ₃ O ₄ does not participate in the polymerization reaction during the preparation of MIPs, which may cause Fe ₃ O ₄ leakage during the elution of template molecules or in the actual application process, resulting in the weakening of the paramagnetism of MIPs and affecting Accuracy of experimental test results.

Contents of the invention

purpose of invention

Aiming at the above-mentioned deficiencies in the prior art, the present invention proposes a preparation method for separating and purifying ractopamine magnetic molecularly imprinted polymers, which mainly solves the problem of the magnetic properties of the existing magnetic ractopamine molecularly imprinted polymers in the process of template elution and practical application. The problem of the leakage of ferric oxide.

Technical solutions

The present invention is achieved through the following technical solutions:

A method for preparing a magnetically imprinted polymer for separating and purifying ractopamine, characterized in that the steps are as follows:

(1) Preparation of magnetic ferric oxide (Fe ₃ O ₄ ) nanospheres:

Put FeCl ₂ 4H ₂ O and FeCl ₃ 6H ₂ O in a three-necked flask, add deionized water after ultrasonic deoxygenation, and mechanically stir under nitrogen protection; then heat the reaction system and add ammonia water drop by drop, and the dropwise addition is complete After the constant temperature crystallization; after the crystallization is completed, the temperature is naturally lowered to room temperature, the black product Fe ₃ O ₄ synthesized in the solution is separated by a magnet, the Fe ₃ O ₄ is washed with deionized water, and the unreacted solvent is removed until the solution is neutral , and the product was dried in vacuum for later use;

(2) Preparation of 3-aminopropyltrimethoxysilane (ATPS) modified ferric oxide material (ATPS-Fe ₃ O ₄ ):

Take the Fe ₃ O ₄ nanospheres prepared in step (1) and place them in a three-necked flask, then add ethanol-water solution, adjust the pH to 4 with glacial acetic acid, add ATPS after ultrasonic dispersion at room temperature, and heat under nitrogen protection and water bath. , and mechanically stirred for 10-15 hours; after the reaction, use a magnet to separate the black product ATPS-Fe ₃ O ₄ , and wash the product with deionized water, absolute ethanol, and absolute ether in sequence until there is no oily suspension, and the product is vacuum-dried for later use ;

(3) Preparation of magnetic Fe ₃ O ₄ functional monomer (MAC-ATPS-Fe ₃ O ₄ ):

Put the ATPS-Fe ₃ O ₄ material prepared in step (2) into a three-necked flask, add toluene and K ₂ CO ₃ , ultrasonically vibrate for 20-30 minutes under ice bath conditions, then stir at room temperature and add methacryloyl chloride dropwise (MAC), after 10 to 12 hours, use a magnet to separate the black product magnetic Fe ₃ O ₄ functional monomer MAC-ATPS-Fe ₃ O ₄ in the solution, and wash it with toluene, ethanol, and acetonitrile in sequence, and put the product in a vacuum drying oven to dry for backup use;

(4) Preparation of magnetic RAC molecularly imprinted polymers (Fe ₃ O ₄ @MIPs):

Put the magnetic Fe ₃ O ₄ functional monomer MAC-ATPS-Fe ₃ O ₄ prepared in step (3) and the template molecule ractopamine in a round-bottomed flask, the solvent is acetonitrile, and after stirring at room temperature for 3-5 hours, add the cross-linking agent and initiator, ultrasonically disperse in an ice bath for 5-10 minutes, heat the mixed solution in a water bath at 60-65°C for 24-30 hours, protect it with _N2 during the reaction, and stir it mechanically; use a magnet to separate the solid phase in the solution after the polymerization and the liquid phase, pour off the upper solution to obtain the Fe ₃ O ₄ @MIPs product; then wash the Fe ₃ O ₄ @MIPs product with a methanol-dichloromethane mixed solution, and then put it in a vacuum drying oven at 40-45°C Dry under vacuum overnight; extract ractopamine from the dried product with methanol-glacial acetic acid mixed solvent until no ractopamine is detected, then put it into a vacuum drying oven after extraction, and dry it in vacuum at 40-45°C for 24-30 hours .

In step (1), the molar ratio of FeCl ₂ 4H ₂ O to FeCl ₃ 6H ₂ O is 1:2; the molar volume ratio of FeCl ₂ 4H ₂ O to deionized water is 1mol:8-10L; the heating temperature of the system 60～65℃; the volume molar ratio of ammonia water and FeCl ₂ 4H ₂ O added is 1.8～2L:1mol; constant temperature crystallization temperature is 60～65℃, and the time is 60～80min; the vacuum drying temperature of the product is 40～ 45°C, the drying time is 24-30h.

The mass volume ratio of _Fe3O4 nanometer microspheres and _ATPS in step (2) is 1g:2～2.5mL; In the ethanol-water solution that adds, the volume ratio of ethanol and water is 1:1; The time of ultrasonic dispersion is 10-20min; the temperature of water bath heating is 60-65°C; the temperature of vacuum drying is 40-45°C, and the drying time is 24-30h.

In step (3), the mass volume ratio of ATPS-Fe ₃ O ₄ to methacryloyl chloride is 100mg:1mL; the mass volume ratio of ATPS-Fe ₃ O ₄ to toluene is 1mg:0.5～1.0mL; ATPS-Fe ₃ O The mass ratio of ₄ to K ₂ CO ₃ is 1:1-1.5; the product is vacuum-dried at a temperature of 40-45° C., and the vacuum-dried time is 24-30 hours.

The crosslinking agent described in step (4) is ethylene glycol dimethacrylate.

The initiator described in step (4) is azobisisobutyronitrile.

In step (4), the mass molar ratio of the magnetic Fe ₃ O ₄ functional monomer MAC-ATPS-Fe ₃ O ₄ to the template molecule ractopamine is 1 g:1 mmol.

In step (4), the mass molar ratio of the magnetic Fe ₃ O ₄ functional monomer MAC-ATPS-Fe ₃ O ₄ to the crosslinking agent is 1g:20mmol; in the step (4), the magnetic Fe ₃ O ₄ functional monomer MAC-ATPS - The mass molar ratio of Fe ₃ O ₄ to the initiator is 1g:2-3mmol.

In step (4), the mass volume ratio of the magnetic Fe ₃ O ₄ functional monomer to the solvent acetonitrile is 1 g:150-200 mL.

The volume ratio of methanol and dichloromethane in the methanol-dichloromethane mixed solution in step (4) is 4:1; the volume ratio of methanol and glacial acetic acid in the methanol-glacial acetic acid mixed solvent in step (4) is 9:1.

Advantages and effects

The present invention has following advantage and beneficial effect:

The RAC magnetic molecularly imprinted polymer was prepared sequentially with ATPS and MAC-modified Fe3O4 nanospheres as functional monomers, which avoided the leakage of Fe3O4 nanospheres during the template elution and practical application of the polymer. The prepared RAC molecularly imprinted polymer has a faster adsorption rate to RAC, can reach adsorption equilibrium in a short time (20min), can meet the needs of rapid detection, and the saturation magnetization of the RAC polymer prepared by the present invention reaches 16emu·g ^-1 , has good paramagnetism, which is convenient for the separation of MIPs after dispersive extraction, and is very suitable for on-site sample processing.

Description of drawings

Figure 1 is the infrared spectrum of Fe ₃ O ₄ , ATPS-Fe ₃ O ₄ , MAC-ATPS-Fe ₃ O ₄ and Fe ₃ O ₄ @MIPs. It can be seen from the figure that Fe ₃ O ₄ nanoparticles were successfully prepared, the modification of ferric oxide by ATPS and MAC in turn was successful, and the molecular imprinting process was also successfully carried out.

Fig. 2 is the XRD pattern of Fe ₃ O ₄ (a), ATPS-Fe ₃ O ₄ (b), MAC-ATPS-Fe ₃ O ₄ (c), Fe ₃ O ₄ @MIP (d). The characteristic peaks 2θ=30.33°, 35.73°, 43.40°, 53.96°, 57.37° and 63.13° of Fe ₃ O ₄ in the figure correspond to ₍ 220), (311), ( ₄₀₀ ), ( 422), (511) and (440) crystal planes, which coincide with the standard Fe ₃ O ₄ characteristic peak diffraction peaks; the XRD characteristic peaks of Fe ₃ O ₄ and molecularly imprinted polymers modified by ATPS and MAC are consistent with Fe ₃ O ₄ is similar, indicating that Fe ₃ O ₄ maintains the characteristic crystal form in molecularly imprinted polymers and maintains good superparamagnetism.

Figure 3 is the hysteresis loop of Fe ₃ O ₄ @MIPs. It can be seen from the figure that the saturation magnetism of the magnetic molecularly imprinted polymer is 16.64emu/g, and the magnetic molecularly imprinted polymer has a good separation effect under an external magnetic field.

Figure 4 is a transmission electron microscope image of Fe ₃ O ₄ @MIPs. It can be seen from the figure that the molecularly imprinted polymer has been successfully prepared on the surface of Fe ₃ O ₄ nanoparticles.

Figure 5 shows the adsorption kinetics of Fe ₃ O ₄ @MIPs. It can be seen from the figure that the magnetic molecularly imprinted polymer can reach adsorption equilibrium within 20 minutes.

Fig. 6 is the pseudo-second-order kinetic equation fitting of Fe ₃ O ₄ @MIPs adsorption kinetic data.

Fig. 7 is the adsorption isotherm curves of Fe ₃ O ₄ @MIPs and Fe ₃ O ₄ @NIPs.

Figure 8 shows the selectivity test of Fe ₃ O ₄ @MIPs and Fe ₃ O ₄ @NIPs for the adsorption of ractopamine and other three structural analogues isoproterenol, phenoxypropofolamine and terbutaline. The results showed that the synthesized Fe ₃ O ₄ @MIPs had a good ability to select and recognize the target molecule RAC.

detailed description

The present invention is described in detail below with reference to accompanying drawing:

The present invention is a preparation method for separating, purifying and enriching ractopamine magnetic molecularly imprinted polymers, in which Fe is covalently modified with 3-aminopropyltrimethoxysilane (ATPS) and methacryloyl chloride (MAC) sequentially. After ₃ O ₄ is used as a functional monomer, RAC is used as a template molecule, azobisisobutyronitrile (AIBN) is used as an initiator, and ethylene glycol dimethacrylate (EDMA) is used as a crosslinking agent to prepare RAC for magnetic nanomolecular imprinting polymerization things. In the present invention, during the preparation process of Fe ₃ O ₄ @MIPs, since the nano-Fe ₃ O ₄ microspheres participated in the polymerization reaction, the problem of leakage of Fe ₃ O ₄ molecules in the process of eluting template molecules and practical application of MIPs was overcome, increasing The number of times magnetic MIPs are used.

The preparation method of the magnetically imprinted polymer for separating and purifying ractopamine of the present invention, the steps are as follows:

(1) Preparation of magnetic ferric oxide (Fe ₃ O ₄ ) nanospheres:

Put FeCl ₂ 4H ₂ O and FeCl ₃ 6H ₂ O in a three-necked flask, add deionized water after ultrasonic degassing, and mechanically stir under the protection of nitrogen; then heat the reaction system and add ammonia water drop by drop, the dropwise addition is complete After the constant temperature crystallization; after the crystallization is completed, the temperature is naturally lowered to room temperature, the black product Fe ₃ O ₄ synthesized in the solution is separated by a magnet, the Fe ₃ O ₄ is washed with deionized water, and the unreacted solvent is removed until the solution is neutral , and the product was dried in vacuum for later use;

(2) Preparation of 3-aminopropyltrimethoxysilane (ATPS) modified ferric oxide material (ATPS-Fe ₃ O ₄ ):

Take the Fe ₃ O ₄ nanospheres prepared in step (1) and place them in a three-necked flask, then add ethanol-water solution (1:1, V:V), adjust the pH to 4 with glacial acetic acid, and add ATPS, under the protection of nitrogen and heating in a water bath, react with mechanical stirring for 10-15 hours; after the reaction, use a magnet to separate the black product ATPS-Fe ₃ O ₄ , and wash the product with deionized water, absolute ethanol, and anhydrous ether until it is free Oily suspension, the product is vacuum-dried for later use;

(3) Preparation of magnetic Fe ₃ O ₄ functional monomer (MAC-ATPS-Fe ₃ O ₄ ):

Put the ATPS-Fe ₃ O ₄ material prepared in step (2) into a three-necked flask, add toluene and K ₂ CO ₃ , ultrasonically vibrate for 20-30 minutes under ice bath conditions, then stir at room temperature and add methacryloyl chloride dropwise (MAC), after 10 to 12 hours, use a magnet to separate the black product magnetic Fe ₃ O ₄ functional monomer MAC-ATPS-Fe ₃ O ₄ in the solution, and wash it with toluene, ethanol, and acetonitrile in sequence, and put the product in a vacuum drying oven to dry for backup use;

(4) Preparation of magnetic RAC molecularly imprinted polymers (Fe ₃ O ₄ @MIPs):

Put the magnetic Fe ₃ O ₄ functional monomer MAC-ATPS-Fe ₃ O ₄ prepared in step (3) and the template molecule ractopamine in a round-bottomed flask, the solvent is acetonitrile, and after stirring at room temperature for 3-5 hours, add the cross-linking agent and initiator, ultrasonically disperse in an ice bath for 5-10 minutes, heat the mixed solution in a water bath at 60-65°C for 24-30 hours, protect it with _N2 during the reaction, and stir it mechanically; use a magnet to separate the solid phase in the solution after the polymerization and the liquid phase, pour off the upper solution to obtain the Fe ₃ O ₄ @MIPs product; then wash the Fe ₃ O ₄ @MIPs product with a methanol-dichloromethane (4:1, V:V) mixed solution, and then put it into In a vacuum drying oven, vacuum-dry overnight at 40-45°C; extract ractopamine from the dried product with a mixed solvent of methanol-glacial acetic acid (9:1, V:V) until no ractopamine is detected. Then put it into a vacuum drying oven, and dry it under vacuum at 40-45°C for 24-30 hours.

In the above step (1), the molar ratio of FeCl ₂ 4H ₂ O to FeCl ₃ 6H ₂ O is 1:2; the molar volume ratio of FeCl ₂ 4H ₂ O to deionized water is 1mol: 8-10L; the system is heated The temperature is 60-65°C; the volume molar ratio of ammonia water to FeCl ₂ ·4H ₂ O is 1.8-2L:1mol; the constant temperature crystallization temperature is 60-65°C, and the time is 60-80min; the vacuum drying temperature of the product is 40 ～45℃, the drying time is 24～30h.

The mass volume ratio of _Fe3O4 nanospheres and _ATPS in the above step (2) is 1g:2～2.5mL; in the added ethanol-water solution, the volume ratio of ethanol to water is 1:1; the time of ultrasonic dispersion 10-20 minutes; the temperature of water bath heating is 60-65°C; the temperature of vacuum drying is 40-45°C, and the drying time is 24-30h.

In the above step (3), the mass volume ratio of ATPS-Fe ₃ O ₄ and methacryloyl chloride is 100mg:1mL; the mass volume ratio of ATPS-Fe ₃ O ₄ and toluene is 1mg:0.5～1.0mL, ATPS-Fe The mass ratio of ₃ O ₄ to K ₂ CO ₃ is 1:1~1.5; the product is dried in vacuum at a temperature of 40~45°C, and the time of vacuum drying is 24~30h.

The crosslinking agent described in the above step (4) is ethylene glycol dimethacrylate.

The initiator described in the above step (4) is azobisisobutyronitrile.

In the above step (4), the mass molar ratio of the magnetic Fe ₃ O ₄ functional monomer MAC-ATPS-Fe ₃ O ₄ to the template molecule ractopamine is 1 g:1 mmol.

In the above step (4), the mass molar ratio of the magnetic Fe ₃ O ₄ functional monomer MAC-ATPS-Fe ₃ O ₄ to the crosslinking agent is 1 g:20 mmol.

In the above step (4), the mass molar ratio of the magnetic Fe ₃ O ₄ functional monomer MAC-ATPS-Fe ₃ O ₄ to the initiator is 1 g:2.0-3.0 mmol.

In the above step (4), the mass volume ratio of the magnetic Fe ₃ O ₄ functional monomer to the solvent acetonitrile is 1 g:150-200 mL.

The volume ratio of methanol and dichloromethane in the methanol-dichloromethane mixed solution in the above-mentioned step (4) is 4:1; the volume ratio of methanol and glacial acetic acid in the methanol-glacial acetic acid mixed solvent in the step (4) is 9:1 .

The present invention will be further described below in conjunction with specific embodiments.

Example 1:

A method for preparing magnetically imprinted polymers for separating and purifying ractopamine, the steps of which are as follows:

(1) Preparation of magnetic ferric oxide nanospheres:

Take 0.03mol FeCl ₂ ·4H ₂ O and 0.06mol FeCl ₃ ·6H ₂ O into a three-necked flask, then add 300mL of deionized water after ultrasonic degassing, and mechanically stir under the protection of nitrogen. Heat the reaction system to 65°C and add 60mL ammonia water dropwise. After the dropwise addition, crystallize at a constant temperature at 65°C for 60 minutes; after the crystallization is complete, let the temperature drop to room temperature naturally, and use a magnet to separate the black product Fe ₃ O ₄ synthesized in the solution. , wash Fe ₃ O ₄ with deionized water, remove unreacted solvent until the solution is neutral, and vacuum-dry the product at 45°C for 24 hours before use.

(2) Preparation of 3-aminopropyltrimethoxysilane (ATPS) modified ferric oxide material (ATPS-Fe ₃ O ₄ ):

Take 1.5 g of Fe ₃ O ₄ nanospheres prepared in step (1) and place them in a three-necked flask, then add 200 to 250 mL of ethanol-water solution (1:1, V:V), adjust the pH to 4 with glacial acetic acid, and Under ultrasonic dispersion for 10 minutes, add 3.268mL ATPS (3-aminopropyltrimethoxysilane), under the protection of nitrogen, heat in a water bath at 60°C, and mechanically stir for 10 hours; after the reaction, use a magnet to separate the black product ATPS-Fe ₃ O ₄ , And successively wash with deionized water, absolute ethanol, and absolute ether until there is no oily suspension, and vacuum-dry at 45°C for 24 hours before use.

(3) Preparation of magnetic Fe ₃ O ₄ functional monomer (MAC-ATPS-Fe ₃ O ₄ ):

Take 400mg of the ATPS-Fe ₃ O ₄ material prepared in step (2) and place it in a three-necked flask, add 200mL of toluene, 0.4gK ₂ CO ₃ , ultrasonically oscillate for 30min in an ice bath, stir at room temperature and add 4mL of methacryloyl chloride dropwise (MAC), after 12 hours, use a magnet to separate the black product magnetic Fe ₃ O ₄ functional monomer MAC-ATPS-Fe ₃ O ₄ , and wash it with toluene, ethanol, and acetonitrile in sequence, and put the product in a vacuum drying oven at 45°C for vacuum drying Reserve after 24 hours.

(4) Preparation of magnetic RAC molecularly imprinted polymers (Fe ₃ O ₄ @MIPs):

Take 100 mg of magnetic Fe ₃ O ₄ functional monomer MAC-ATPS-Fe ₃ O ₄ prepared in step (3) and 0.1 mmol (0.0337 g) template molecule ractopamine RAC to a round bottom flask, the solvent is 20 mL of acetonitrile, and stir at room temperature After 3h, take 2mmol (0.380mL) of cross-linking agent EGDMA and 0.2mmol of initiator AIBN into the round bottom flask, and ultrasonically disperse in an ice bath for 5min; heat the mixed solution in a water bath at 65°C for 24h, and protect it with _N2 during the reaction. and mechanical stirring; after the polymerization, use a magnet to separate the solid phase and liquid phase in the solution, pour off the upper solution to obtain the black product Fe ₃ O ₄ @MIPs; then use methanol-CH ₂ Cl ₂ (4:1, V:V) Wash the Fe ₃ O ₄ @MIPs product with the mixed solution, then put it into a vacuum drying oven, and dry it under vacuum overnight at 45°C; the dried product is extracted by Soxhlet method, that is, methanol-glacial acetic acid (9:1, V:V) Extract ractopamine with a mixed solvent until no ractopamine RAC is detected, then put it into a vacuum drying oven after extraction, and vacuum dry at 45° C. for 24 hours.

Example 2:

A method for preparing magnetically imprinted polymers for separating and purifying ractopamine, the steps of which are as follows:

(1) Preparation of magnetic ferric oxide nanospheres:

Take 0.03mol FeCl ₂ 4H ₂ O and 0.06mol FeCl ₃ 6H ₂ O into a three-necked flask, then add 240mL of deionized water after ultrasonic degassing, stir mechanically under the protection of nitrogen, heat the reaction system to 60°C and gradually Add 54mL of ammonia water dropwise, and crystallize at a constant temperature of 60°C for 80min after the dropwise addition; after the crystallization is completed, let the temperature drop to room temperature naturally, use a magnet to separate the black product Fe ₃ O ₄ synthesized in the solution, and wash Fe ₃ O with deionized water _4. Remove the unreacted solvent until the solution is neutral, and dry the product under vacuum at 40°C for 30 hours before use.

(2) Preparation of 3-aminopropyltrimethoxysilane (ATPS) modified ferric oxide material (ATPS-Fe ₃ O ₄ ):

Take 1 g of Fe ₃ O ₄ nanospheres prepared in step (1) and place them in a three-necked flask, then add 200 to 250 mL of ethanol-water solution (1:1, V:V), adjust the pH to 4 with glacial acetic acid, and Ultrasonic disperse for 20min, add 2mL ATPS (3-aminopropyltrimethoxysilane), under the protection of nitrogen, heat in a water bath at 65°C, and stir mechanically for 15h; after the reaction, use a magnet to separate the black product ATPS-Fe ₃ O ₄ , and sequentially Wash with deionized water, absolute ethanol, and absolute ether until there is no oily suspension, and vacuum-dry at 40°C for 30 hours before use.

(3) Preparation of magnetic Fe ₃ O ₄ functional monomer (MAC-ATPS-Fe ₃ O ₄ ):

Take 500 mg of the ATPS-Fe ₃ O ₄ material prepared in step (2) and put it in a three-necked flask, add 300 mL of toluene, 600 mg of K ₂ CO ₃ , ultrasonically oscillate for 20 min in an ice bath, stir at room temperature and add 5 mL of methacryloyl chloride ( MAC), after 10h, use a magnet to separate the black product magnetic Fe ₃ O ₄ functional monomer MAC-ATPS-Fe ₃ O ₄ in the solution, and wash it with toluene, ethanol, and acetonitrile in sequence, and put the product in a vacuum drying oven for 30 hours at 40°C Backup.

(4) Preparation of magnetic RAC molecularly imprinted polymers (Fe ₃ O ₄ @MIPs):

Take 100mg of the magnetic _Fe3O4 functional monomer MAC- _ATPS - _Fe3O4 prepared in step ( ₃ ) and 0.1mmol (0.0337g) template molecule ractopamine RAC to a round bottom flask, the solvent is 15mL acetonitrile, and stir at room temperature After 5h, take 2mmol (0.380mL) of cross-linking agent EGDMA and 0.25mmol of initiator AIBN into the round-bottomed flask, and ultrasonically disperse in an ice bath for 10min; heat the mixed solution in a water bath at 60°C for 30h, and protect it with _N2 during the reaction. and mechanical stirring; after the polymerization, use a magnet to separate the solid phase and liquid phase in the solution, pour off the upper solution to obtain the black product Fe ₃ O ₄ @MIPs; then use methanol-CH ₂ Cl ₂ (4:1, V:V) Wash the Fe ₃ O ₄ @MIPs product with the mixed solution, then put it in a vacuum drying oven, and dry it under vacuum overnight at 40°C; the dried product is extracted by Soxhlet method, that is, methanol-glacial acetic acid (9:1, V:V) Extract ractopamine with a mixed solvent until no ractopamine RAC is detected, then put it into a vacuum drying oven after extraction, and vacuum dry at 40° C. for 30 hours.

Example 3:

A method for preparing magnetically imprinted polymers for separating and purifying ractopamine, the steps of which are as follows:

(1) Preparation of magnetic ferric oxide nanospheres:

Take 0.03mol FeCl ₂ 4H ₂ O and 0.06mol FeCl ₃ 6H ₂ O into a three-necked flask, then add 270mL of deionized water after ultrasonic degassing, stir mechanically under the protection of nitrogen, heat the reaction system to 62°C and gradually Add 57mL of ammonia water dropwise, and crystallize at a constant temperature of 63°C for 70min after the dropwise addition; after the crystallization is completed, let the temperature drop to room temperature naturally, use a magnet to separate the black product Fe ₃ O ₄ synthesized in the solution, and wash Fe ₃ O with deionized water _4. Remove the unreacted solvent until the solution is neutral, and dry the product under vacuum at 42°C for 28 hours before use.

(2) Preparation of 3-aminopropyltrimethoxysilane (ATPS) modified ferric oxide material (ATPS-Fe ₃ O ₄ ):

Take 1 g of Fe ₃ O ₄ nanospheres prepared in step (1) and place them in a three-necked flask, then add 200 to 250 mL of ethanol-water solution (1:1, V:V), adjust the pH to 4 with glacial acetic acid, and Ultrasonic disperse for 15 minutes, add 2.5mL ATPS (3-aminopropyltrimethoxysilane), under the protection of nitrogen, heat in a water bath at 62°C, and mechanically stir for 13 hours; after the reaction, use a magnet to separate the black product ATPS-Fe ₃ O ₄ , and Wash with deionized water, absolute ethanol, and anhydrous ether in sequence until there is no oily suspension, and dry in vacuum at 42°C for 28 hours before use.

(3) Preparation of magnetic Fe ₃ O ₄ functional monomer (MAC-ATPS-Fe ₃ O ₄ ):

Take 500 mg of the ATPS-Fe ₃ O ₄ material prepared in step (2) and place it in a three-necked flask, add 500 mL of toluene, 750 mg K ₂ CO ₃ , ultrasonically shake for 25 min in an ice bath, stir at room temperature and add 5 mL of methacryloyl chloride ( MAC), after 11h, use a magnet to separate the black product magnetic Fe ₃ O ₄ functional monomer MAC-ATPS-Fe ₃ O ₄ in the solution, and wash it with toluene, ethanol, and acetonitrile in sequence, and put the product in a vacuum drying oven for 28 hours at 42°C. Backup.

(4) Preparation of magnetic RAC molecularly imprinted polymers (Fe ₃ O ₄ @MIPs):

Take 100mg of the magnetic _Fe3O4 functional monomer MAC- _ATPS - _Fe3O4 prepared in step ( ₃ ) and 0.1mmol (0.0337g) template molecule ractopamine RAC into a round bottom flask, the solvent is 18mL acetonitrile, and stir at room temperature After 4h, take 2mmol (0.380mL) of cross-linking agent EGDMA and 0.3mmol of initiator AIBN and add them into a round bottom flask, and disperse them ultrasonically in an ice bath for 8min; heat the mixture in a water bath at 63°C for 28h, and protect it with _N2 during the reaction. and mechanical stirring; after the polymerization, use a magnet to separate the solid phase and liquid phase in the solution, pour off the upper solution to obtain the black product Fe ₃ O ₄ @MIPs; then use methanol-CH ₂ Cl ₂ (4:1, V:V) Wash the Fe ₃ O ₄ @MIPs product with the mixed solution, then put it in a vacuum drying oven, and dry it under vacuum overnight at 42°C; the dried product is extracted by Soxhlet method, that is, methanol-glacial acetic acid (9:1, V:V) Extract ractopamine with a mixed solvent until no ractopamine RAC is detected, then put it into a vacuum drying oven after extraction, and vacuum dry at 42° C. for 28 hours.

Example 4: Preparation of magnetic non-imprinted polymers (Fe ₃ O ₄ @MIPs): the implementation conditions are the same as in Example 1, but no template molecule RAC is added.

The synthesized Fe ₃ O ₄ , ATPS-Fe ₃ O ₄ , MAC-ATPS-Fe ₃ O ₄ , and Fe ₃ O ₄ @MIPs were detected by Fourier transform infrared spectrometer, and the detection results are shown in Fig. 1 . The infrared spectra of a, b, c, and d in Figure 1 are Fe ₃ O ₄ , ATPS-Fe ₃ O ₄ , MAC-ATPS-Fe ₃ O ₄ , Fe ₃ O ₄ @MIPs, respectively. In the infrared spectrum of Fe ₃ O ₄ , the 582cm ^-1 peak is the characteristic absorption peak of Fe-O bond in Fe ₃ O ₄ ; in the ATPS-Fe ₃ O ₄ infrared spectrum, 588cm ^-1 is the Fe-O-Si bond The characteristic absorption peaks of 3442cm ^-1 and 1635cm ^-1 are the characteristic absorption peaks of NH; in the MAC-ATPS-Fe ₃ O ₄ infrared spectrum, 1396cm ^-1 is the absorption peak of the secondary amide bond, and 1548cm ^-1 is the absorption peak of NH The in-plane bending vibration absorption peak; Fe ₃ O ₄ @MIPs spectrum shows that the magnetic molecularly imprinted polymer was successfully prepared.

Experimental example 1:

Add 7 parts of 20mg Fe ₃ O ₄ @MIPs to the acetonitrile solution containing 10 mL of ractopamine with a concentration of 100 mg/L respectively, and then shake the 7 parts of the mixture on a horizontal shaker at 300 rpm at room temperature for 5, 10, 20, 40, 60, 80, 120 minutes. After the shaking time was over, the supernatant was separated and collected by a magnet, and the concentration of unadsorbed ractopamine molecules was measured by high-performance liquid chromatography, and the adsorption capacity was calculated according to the results. The results are shown in Figure 5. It can be seen that the prepared magnetic ractopamine Fe ₃ O ₄ @MIPs has a faster adsorption rate for the target substance, and the adsorption equilibrium can be reached within 20 minutes at the experimental concentration.

According to the pseudo-second-order kinetic equation Fit the adsorption kinetic curve in Figure 5, where q _t represents the adsorption amount of ractopamine magnetic Fe ₃ O ₄ @MIPs at different times, q _e is the theoretical adsorption amount of the second-order kinetic equation, and k is the first-order Kinetic reaction rate constant, t is time. The fitting results are shown in Figure 6. Figure 6 is the pseudo-second-order kinetic equation fitting equation for Fe ₃ O ₄ @MIPs adsorption kinetics data. The Fe ₃ O ₄ @ The correlation coefficient (R ² ) of the quasi-second-order kinetics fitting curve equation of MIPs is 0.9974, and the adsorption capacity during the equilibrium calculated by the formula is 11.1mg/g, which is close to the adsorption capacity during the equilibrium obtained from the experiment, indicating that the prepared by example 4 The adsorption of ractopamine on Fe ₃ O ₄ @MIPs fits the pseudo-second-order kinetic model.

Experimental example 2:

Take 10mL of ractopamine solution whose initial concentration is 20, ₄₀ , 60, 80, 100, 120, 140, 160mg/L respectively in a 50mL volumetric flask, then add 20mg of _Fe3O4 @MIPs and Fe ₃ O ₄ @NIPs polymer was adsorbed on a horizontal shaker at 300rmp for 60 minutes at room temperature. After the time was over, the supernatant was separated and collected with a magnet, and the concentration of unadsorbed ractopamine molecules was measured by high-performance liquid chromatography, and the adsorption capacity was calculated according to the results. The results are shown in Figure 7. Figure 7 shows the isotherm adsorption curves of Fe ₃ O ₄ @MIPs and Fe ₃ O ₄ @NIPs. It can be seen from Figure 7 that the adsorption capacity of Fe ₃ O ₄ @MIPs and Fe ₃ O ₄ @NIPs increases with The initial concentration increases. When the concentration is 120mg/L, the maximum adsorption capacity of Fe ₃ O ₄ @MIPs is 11.38mg/g, and the maximum adsorption capacity of Fe ₃ O ₄ @NIPs is 5.27mg/g. The adsorption capacity of Fe ₃ O ₄ @MIPs for ractopamine was significantly higher than that of Fe ₃ O ₄ @NIPs for ractopamine, indicating that the synthesized Fe ₃ O ₄ @MIPs had specific adsorption for ractopamine.

Experimental example 3:

In order to investigate the specific adsorption of molecularly imprinted polymers to the template molecule RAC, three structural analogues of RAC, terbutaline (TER), phenoxypropofolamine (ISOX), and meta-hydroxyisoproterenol (ISOP), were selected as RAC competitor. The specific operation is as follows:

Accurately weigh 20 mg of Fe ₃ O ₄ @MIPs and Fe ₃ O ₄ @NIPs into a 50 mL volumetric flask, add 10 mL of 100 mg L ^-1 solution of RAC, TER, ISOX, and ISOP, and absorb with a horizontal oscillator at 300 rpm for 60 minutes. After the shaking time was over, the supernatant was separated and collected by a magnet, and the concentration of unadsorbed ractopamine molecules was measured by high-performance liquid chromatography, and the adsorption capacity was calculated according to the results. Then calculate the distribution coefficient (K _d ), selection coefficient (k) and relative selection coefficient (k') according to the following formula.

${\mathrm{<span\; class="notranslate">\; k</span>}}_{\mathrm{<span\; class="notranslate">\; d</span>}}<span\; class="notranslate">\; =</span><span\; class="notranslate">\; \{</span>\frac{{\mathrm{<span\; class="notranslate">\; C</span>}}_{\mathrm{<span\; class="notranslate">\; i</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; C</span>}}_{\mathrm{<span\; class="notranslate">\; f</span>}}}{{\mathrm{<span\; class="notranslate">\; C</span>}}_{\mathrm{<span\; class="notranslate">\; f</span>}}}<span\; class="notranslate">\; \}</span><span\; class="notranslate">\; \&times;</span><span\; class="notranslate">\; \{</span>\mathrm{<span\; class="notranslate">\; V</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; ml</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; \}</span><span\; class="notranslate">\; /</span><span\; class="notranslate">\; \{</span>\mathrm{<span\; class="notranslate">\; m</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; g</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; \}</span><span\; class="notranslate">\; ,</span>\frac{{\mathrm{<span\; class="notranslate">\; k</span>}}_{\mathrm{<span\; class="notranslate">\; d</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; RAC</span>}<span\; class="notranslate">\; )</span>}}{{\mathrm{<span\; class="notranslate">\; k</span>}}_{\mathrm{<span\; class="notranslate">\; d</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; x</span>}<span\; class="notranslate">\; )</span>}}<span\; class="notranslate">\; ,</span>{\mathrm{<span\; class="notranslate">\; k</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; k</span>}}_{<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; Fe</span>}}_{\mathrm{<span\; class="notranslate">\; 3</span>}}{\mathrm{<span\; class="notranslate">\; o</span>}}_{\mathrm{<span\; class="notranslate">\; 4</span>}}<span\; class="notranslate">\; @</span>\mathrm{<span\; class="notranslate">\; MIPs</span>}<span\; class="notranslate">\; )</span>}}{{\mathrm{<span\; class="notranslate">\; k</span>}}_{<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; Fe</span>}}_{\mathrm{<span\; class="notranslate">\; 3</span>}}{\mathrm{<span\; class="notranslate">\; o</span>}}_{\mathrm{<span\; class="notranslate">\; 4</span>}}<span\; class="notranslate">\; @</span>\mathrm{<span\; class="notranslate">\; NIPs</span>}<span\; class="notranslate">\; )</span>}}$

Among them: C _i is the concentration of RAC in the initial solution; C _f is the concentration of RAC in the solution after adsorption; V is the volume of RAC standard solution, M is the amount of polymer; X is the structural analog of competitive adsorption, and RAC is ractopamine. The experimental results are shown in Table 1. It can be seen from Table 1 that the adsorption selectivity of magnetic Fe ₃ O ₄ @MIPs to ractopamine is 1.93 times that of ISOP, 0.517 times that of TERB, and 31 times that of ISOP. The recognition performance is remarkable.

Table 1 Selective adsorption results of magnetic Fe ₃ O ₄ @MIPs and Fe ₃ O ₄ @NIPs

.

Claims (10)

1. a separation, the magnetic blotting method for producing polymer of purification Ractopamine, it is characterised in that: step is as follows:

(1) magnetic ferroferric oxide (Fe is prepared₃O₄) Nano microsphere:

By FeCl₂·4H₂O and FeCl₃·6H₂O is placed in there-necked flask, adds the deionized water after ultrasonic deoxygenation, and nitrogen is protected Lower mechanical agitation；Then it is added dropwise over ammonia after reaction system being heated, thermostatic crystallization after dropping；Temperature is made after crystallization Degree nature is down to room temperature, utilizes the black product Fe of synthesis in Magnet separation solution₃O₄, use deionized water rinsing Fe₃O₄, remove Unreacted solvent is until solution neutral, and product vacuum is the most standby；

(2) ferriferrous oxide material (ATPS-Fe that 3-aminopropyl trimethoxysilane (ATPS) is modified is prepared₃O₄):

Take Fe prepared by step (1)₃O₄Nano microsphere is placed in there-necked flask, is subsequently adding ethanol-water solution, adjusts with glacial acetic acid Joint pH to 4, adds ATPS, under nitrogen protection, heating in water bath, mechanic whirl-nett reaction 10～15h after ultrasonic disperse under room temperature；Instead With Magnet separation black product ATPS-Fe after should terminating₃O₄, and wash product with deionized water, dehydrated alcohol, absolute ether successively To without oily float, product vacuum is the most standby；

(3) magnetic Fe is prepared₃O₄Function monomer (MAC-ATPS-Fe₃O₄):

ATPS-Fe prepared by step (2)₃O₄Material is placed in there-necked flask, adds toluene and K₂CO₃, super under condition of ice bath Sound oscillation 20～30min, is then stirred at room temperature and is added dropwise over methacrylic chloride (MAC), separates molten with Magnet after 10～12h Black product magnetic Fe in liquid₃O₄Function monomer MAC-ATPS-Fe₃O₄, and rinse successively with toluene, ethanol, acetonitrile, product Put into vacuum drying oven the most standby；

(4) preparation (Fe of magnetic RAC molecularly imprinted polymer₃O₄@MIPs):

Magnetic Fe prepared by step (3)₃O₄Function monomer MAC-ATPS-Fe₃O₄It is placed in round bottom with template molecule Ractopamine In flask, dispersion solvent is acetonitrile, stirs after 3～5h under room temperature, adds cross-linking agent and initiator, ice-bath ultrasonic dispersion 5～ 10min, by mixed liquor heating in water bath polymerization 24～30h, N during reaction at 60～65 DEG C₂Protection, and mechanical agitation；Polymerization knot Utilize the solid phase in Magnet separation solution and liquid phase after bundle, go upper solution, obtain Fe₃O₄@MIPs product；Again with methanol-two Chloromethanes mixed solution rinses Fe₃O₄@MIPs product, then puts it in vacuum drying oven, is vacuum dried at 40～45 DEG C Night；Dried product with methylalcohol-glacial acetic acid mixed solvent is extracted Ractopamine, until without Ractopamine detection being Only, place into after extracting in vacuum drying oven, at 40～45 DEG C, be vacuum dried 24～30h；

Fe in step (2)₃O₄Nano microsphere is 1g:2～2.5mL with the mass volume ratio of ATPS；

ATPS-Fe in step (3)₃O₄It is 100mg:1mL with the mass volume ratio of methacrylic chloride.

Separation the most according to claim 1, the magnetic blotting method for producing polymer of purification Ractopamine, its feature exists In: FeCl in step (1)₂·4H₂O and FeCl₃·6H₂The mol ratio of O is 1:2；FeCl₂·4H₂O and mole body of deionized water Long-pending ratio is 1mol:8～10L；System heating-up temperature is 60～65 DEG C；The ammonia added and FeCl₂·4H₂The Molar ratio of O is 1.8～2L:1mol；Thermostatic crystallization temperature is 60～65 DEG C, and the time is 60～80min；Product vacuum baking temperature is 40～45 DEG C, the time being dried is 24～30h.

Separation the most according to claim 1, the magnetic blotting method for producing polymer of purification Ractopamine, its feature exists In: in the ethanol-water solution that step (2) adds, ethanol is 1:1 with the volume ratio of water；The time of ultrasonic disperse be 10～ 20min；The temperature of heating in water bath is 60～65 DEG C；Vacuum drying temperature is 40～45 DEG C, and the time being dried is 24～30h.

Separation the most according to claim 1, the magnetic blotting method for producing polymer of purification Ractopamine, its feature exists In: ATPS-Fe in step (3)₃O₄It is 1mg:0.5～1.0mL with the mass volume ratio of toluene；ATPS-Fe₃O₄With K₂CO₃Matter Amount ratio is 1:1～1.5；The temperature that product vacuum is dried is 40～45 DEG C, the 24～30h of vacuum drying time.

Separation the most according to claim 1, the magnetic blotting method for producing polymer of purification Ractopamine, its feature exists In: the cross-linking agent described in step (4) is ethylene glycol dimethacrylate.

Separation the most according to claim 1, the magnetic blotting method for producing polymer of purification Ractopamine, its feature exists In: the initiator described in step (4) is azodiisobutyronitrile.

Separation the most according to claim 1, the magnetic blotting method for producing polymer of purification Ractopamine, its feature exists In: magnetic Fe in step (4)₃O₄Function monomer MAC-ATPS-Fe₃O₄With the quality mol ratio of template molecule Ractopamine it is 1g:1mmol。

Separation the most according to claim 1, the magnetic blotting method for producing polymer of purification Ractopamine, its feature exists In: magnetic Fe in step (4)₃O₄Function monomer MAC-ATPS-Fe₃O₄It is 1g:20mmol with the quality mol ratio of cross-linking agent；Step Suddenly magnetic Fe in (4)₃O₄Function monomer MAC-ATPS-Fe₃O₄It is 1g:2～3mmol with the quality mol ratio of initiator.

Separation the most according to claim 1, the magnetic blotting method for producing polymer of purification Ractopamine, its feature exists In: magnetic Fe in step (4)₃O₄Function monomer is 1g:150～200mL with the mass volume ratio of solvent acetonitrile.

Separation the most according to claim 1, the magnetic blotting method for producing polymer of purification Ractopamine, its feature It is: in step (4), methanol dichloromethane mixed solution system for tax payment alcohol is 4:1 with the volume ratio of dichloromethane；First in step (4) In alcohol-glacial acetic acid mixed solvent, methanol is 9:1 with the volume ratio of glacial acetic acid.