CN109400803B - Melamine molecularly imprinted microspheres, preparation method and application - Google Patents
Melamine molecularly imprinted microspheres, preparation method and application Download PDFInfo
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- CN109400803B CN109400803B CN201811107106.6A CN201811107106A CN109400803B CN 109400803 B CN109400803 B CN 109400803B CN 201811107106 A CN201811107106 A CN 201811107106A CN 109400803 B CN109400803 B CN 109400803B
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- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F222/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical and containing at least one other carboxyl radical in the molecule; Salts, anhydrides, esters, amides, imides, or nitriles thereof
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
- B01J20/268—Polymers created by use of a template, e.g. molecularly imprinted polymers
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
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- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
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- C08F222/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical and containing at least one other carboxyl radical in the molecule; Salts, anhydrides, esters, amides, imides, or nitriles thereof
- C08F222/10—Esters
- C08F222/1006—Esters of polyhydric alcohols or polyhydric phenols
- C08F222/102—Esters of polyhydric alcohols or polyhydric phenols of dialcohols, e.g. ethylene glycol di(meth)acrylate or 1,4-butanediol dimethacrylate
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- C08J2335/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical, and containing at least one other carboxyl radical in the molecule, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Derivatives of such polymers
- C08J2335/02—Characterised by the use of homopolymers or copolymers of esters
Abstract
The invention discloses a melamine molecularly imprinted microsphere and a preparation method and application thereof, wherein the melamine molecularly imprinted microsphere is prepared from the following components in millimole: 0.85-1.15mmol of melamine, 5-7mmol of 2-acrylamide-2-methylpropanesulfonic acid, 5-7mmol of alpha-methacrylic acid, 8-12mmol of hydroxyethyl cellulose, 38-42mmol of ethylene glycol dimethacrylate, 0.5-0.7mmol of azobisisobutyronitrile and 50-100ml of toluene. According to the invention, a proper hydrophilic functional monomer is searched, and through screening, the alpha-methacrylic acid and 2-acrylamide-2-methylpropanesulfonic acid are used as the double hydrophilic functional monomer to synthesize the melamine molecularly imprinted polymer suitable for the water phase environment, so that the target object is more effectively identified, the hole structure is effectively prevented from being damaged due to the hydrogen bond and swelling action of the water phase environment, and finally, the detection sensitivity and selectivity of the melamine can be effectively improved.
Description
Technical Field
The invention relates to a molecular imprinting technology, in particular to a melamine molecular imprinting microsphere, a preparation method and application thereof.
Background
The molecular imprinting technique is a technique for preparing a molecular imprinted polymer having a molecular recognition property for a specific target molecule. The molecularly imprinted polymer has the advantages of a biological recognition system and a chemical recognition system, has the characteristics of severe environment resistance, high selectivity, good stability, high mechanical strength, simple preparation and the like, and can selectively recognize target objects enriched in complex samples. Therefore, the molecularly imprinted polymer is widely applied to sample pretreatment technologies such as solid phase extraction, solid phase microextraction, membrane extraction and the like. The solid phase extraction is a solvent-free sample pretreatment technology integrating sampling, extraction, concentration and sample injection. The molecular imprinting is combined with a solid phase extraction phase technology, and the method has high selectivity of the molecular imprinting and high sensitivity of the solid phase extraction.
The melamine is an important triazine nitrogen-containing heterocyclic organic compound and an important nitrogen-containing heterocyclic organic chemical raw material, but some lawbreakers increase the total nitrogen content of the product by adding the melamine into feed or even milk powder, so that the measured value of the protein content is higher. In 9 months 2008, the infant milk powder of sanlu in china is polluted, so that the infant who eats the polluted milk powder has the kidney stone disease, and the reason is that the milk powder contains melamine, so that the detection of the melamine becomes a focus of attention. The national ministry of health 2011 "the 10 th bulletin regarding the limited amount of melamine in food (2011, 10)" states that the limited amount of melamine in infant formula is 1mg/kg, and the limited amount in other food is 2.5 mg/kg.
At present, the melamine residue in food and feed is detected at home and abroad by generally adopting liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry and capillary electrophoresis-mass spectrometry. The milk powder and milk product sample matrix is more complex than the feed, which brings difficulty to separation and analysis. A method for detecting melamine in raw milk and dairy products (GB/T22388-. In a complex milk powder matrix, the method has insufficient purification capacity and low selectivity, so that how to analyze melamine in food quickly, accurately and at low cost becomes an urgent problem to be solved.
Disclosure of Invention
Aiming at the conditions of complex operation and weak selectivity of a national standard method and overcoming the defects of the prior art, the invention aims to provide the melamine molecularly imprinted microsphere. The molecular imprinting solid-phase extraction method is adopted, the advantages of molecular imprinting and solid-phase extraction technologies are combined, and the detection sensitivity and selectivity of melamine can be effectively improved.
The second purpose of the invention is to provide a preparation method of the melamine molecularly imprinted microspheres. The preparation method has mild reaction conditions and strong operability, and is suitable for industrial production.
The invention also aims to provide a preparation method of the melamine molecular imprinting solid phase column by adopting the melamine molecular imprinting microspheres as the filler.
The fourth purpose of the invention is to provide a melamine molecular imprinting solid phase column applied to the detection of melamine residual samples in food or feed extracting solution.
One of the purposes of the invention is realized by adopting the following technical scheme: the melamine molecularly imprinted microsphere is prepared from the following components in millimole: 0.85-1.15mmol of melamine, 5-7mmol of 2-acrylamide-2-methylpropanesulfonic acid, 5-7mmol of alpha-methacrylic acid, 8-12mmol of hydroxyethyl cellulose, 38-42mmol of ethylene glycol dimethacrylate and 0.5-0.7mmol of azobisisobutyronitrile.
Further, the melamine molecularly imprinted microsphere is prepared from the following components in millimole: 1mmol of melamine, 6mmol of 2-acrylamide-2-methylpropanesulfonic acid, 6mmol of alpha-methacrylic acid, 10mmol of hydroxyethyl cellulose, 40mmol of ethylene glycol dimethacrylate and 0.6mmol of azobisisobutyronitrile.
The second purpose of the invention is realized by adopting the following technical scheme: a preparation method of melamine molecularly imprinted microspheres comprises the following steps:
dissolving a dispersing agent: weighing hydroxyethyl cellulose dispersing agent with the formula ratio, adding the hydroxyethyl cellulose dispersing agent into water, stirring to dissolve the dispersing agent, and standing and cooling to obtain hydroxyethyl cellulose solution;
the step of preparing the aqueous phase: adding the 2-acrylamide-2-methylpropanesulfonic acid and melamine with the formula ratio into the hydroxyethyl cellulose solution, and stirring to obtain a water phase part;
preparing an organic phase: adding alpha-methacrylic acid, ethylene glycol dimethacrylate and azodiisobutyronitrile in formula amount into toluene, mixing, and ultrasonically dissolving to obtain an organic phase part;
the reaction steps are as follows: slowly dripping the organic phase part into the water phase part under the condition of mechanical stirring, introducing nitrogen to remove oxygen for 15min, and sealing; stirring and reacting for 6-10h at constant temperature and constant speed under the protection of nitrogen, and filtering to obtain microspheres;
and (3) washing: washing residual reactants of the microspheres by water, methanol and acetonitrile in sequence, repeatedly eluting by hydrochloric acid solution until no melamine is detected by ultraviolet visible spectrum detection, and repeatedly cleaning by ultrapure water to be neutral;
and (3) drying: and drying in vacuum to constant weight to obtain the melamine molecularly imprinted microspheres.
Further, in the step of reaction, the constant temperature is 65-75 ℃, and the constant speed is 350-450 rpm.
Further, in the washing step, the volume concentration of the methanol is 99.5%, the volume concentration of the acetonitrile is 99.9%, and the concentration of the hydrochloric acid is 1 mol/L.
Further, the vacuum drying condition is that the vacuum degree is-0.10 mpa; the drying temperature is 20-30 ℃.
The third purpose of the invention is realized by adopting the following technical scheme: a preparation method of a melamine molecular imprinting solid phase column adopts the melamine molecular imprinting microspheres as the filler; the method specifically comprises the following steps:
a step of column packing: accurately weighing 0.1g of the melamine molecularly imprinted microspheres, and filling the melamine molecularly imprinted microspheres into a 2.5mL medical syringe column with the bottom end blocked by filter paper by a dry method to obtain a solid-phase extraction column;
and (3) activating: and (3) washing the column with 10mL of methanol for activation, then washing with ultrapure water for three times, and blow-drying to obtain the catalyst.
The fourth purpose of the invention is realized by adopting the following technical scheme: a melamine molecular imprinting solid phase column is applied to the detection of melamine residue samples in food or feed extracting solution, and comprises the following steps:
enrichment and elution steps: adding 1mL of food or feed melamine extract into a melamine molecular imprinting solid phase column, eluting with 2mL of water, 2mL of methanol and 3mL of ammonia-methanol solution in sequence, and collecting eluent;
and (3) volume fixing step: and (4) carrying out blowing concentration on the elution liquid nitrogen until the elution liquid nitrogen is dried, dissolving the elution liquid nitrogen by using methanol, and fixing the volume to 1 mL.
Further, in the enrichment elution step, the volume concentration of the methanol is 99.5%, and the volume ratio of the ammonia water to the methanol in the ammonia water-methanol solution is 1: 9.
Compared with the prior art, the invention has the beneficial effects that:
the melamine is a water-soluble substance, and the molecularly imprinted polymer in the prior art is synthesized in an organic solvent, so that the molecularly imprinted polymer is placed in a large amount of aqueous phase environment, and the problems of hole structure damage and the like are caused due to hydrogen bonds and swelling action, and the imprinting effect of the molecularly imprinted polymer is influenced. According to the invention, a proper hydrophilic functional monomer is searched, and through screening, alpha-methacrylic acid (MAA) and 2-acrylamide-2-methylpropanesulfonic Acid (AMPS) are used as a double hydrophilic functional monomer, melamine is used as a target object, ethylene glycol dimethacrylate is used as a cross-linking agent, azobisisobutyronitrile is used as an initiator, and toluene is used as a pore-forming agent, so that the melamine molecularly imprinted polymer suitable for an aqueous phase environment is synthesized, the target object is more effectively identified, the hole structure is effectively prevented from being damaged due to the hydrogen bond and swelling action of the aqueous phase environment, and the detection sensitivity and selectivity of melamine can be effectively improved finally.
(2) The preparation method has mild reaction conditions and strong operability, and is suitable for industrial production.
Drawings
FIG. 1 shows the results of melamine adsorption tests on imprinted polymers (MIPs) of the present invention and a blank polymer;
FIG. 2 is a standard operating curve for melamine;
FIG. 3 is an HPLC chromatogram of a milk powder extract with 2. mu.g/mL melamine added in the analysis of an actual sample;
FIG. 4 is an HPLC chromatogram of an eluate obtained by adding a 2. mu.g/mL melamine milk powder extract to an actual sample analysis and extracting the extract by using an MIP-SPE column;
FIG. 5 is an HPLC chromatogram of a pure milk extract with 2. mu.g/mL melamine added in the analysis of an actual sample;
FIG. 6 is an HPLC chromatogram of an eluate obtained by adding a pure milk extract containing 2. mu.g/mL of melamine to an actual sample and extracting the mixture with a MIP-SPE column.
Detailed Description
In the present invention, all parts and percentages are by weight, unless otherwise specified, and the equipment and materials used are commercially available or commonly used in the art. The methods in the following examples are conventional in the art unless otherwise specified.
The melamine molecularly imprinted microsphere is prepared from the following components in millimole: 0.85-1.15mmol of melamine (target), 5-7mmol of 2-acrylamide-2-methylpropanesulfonic acid (functional monomer 1), 5-7mmol of alpha-methacrylic acid (functional monomer 2), 8-12mmol of hydroxyethyl cellulose (dispersing agent), 38-42mmol of ethylene glycol dimethacrylate (crosslinking agent), 0.5-0.7mmol of azobisisobutyronitrile (initiator) and 50-100ml of toluene (pore-foaming agent).
Most preferably, the melamine molecularly imprinted microsphere is prepared from the following components in millimole: 1mmol of melamine, 6mmol of 2-acrylamide-2-methylpropanesulfonic acid, 6mmol of alpha-methacrylic acid, 10mmol of hydroxyethyl cellulose, 40mmol of ethylene glycol dimethacrylate, 0.6mmol of azobisisobutyronitrile and 75ml of toluene.
The preparation method of the melamine molecularly imprinted microspheres comprises the following steps:
dissolving a dispersing agent: weighing hydroxyethyl cellulose dispersing agent with the formula ratio, adding the hydroxyethyl cellulose dispersing agent into water, stirring to dissolve the dispersing agent, and standing and cooling to obtain hydroxyethyl cellulose solution;
the step of preparing the aqueous phase: adding the 2-acrylamide-2-methylpropanesulfonic acid and melamine with the formula ratio into the hydroxyethyl cellulose solution, and stirring to obtain a water phase part;
preparing an organic phase: adding alpha-methacrylic acid, ethylene glycol dimethacrylate and azodiisobutyronitrile in formula amount into toluene, mixing, and ultrasonically dissolving to obtain an organic phase part;
the reaction steps are as follows: slowly dripping the organic phase part into the water phase part under the condition of mechanical stirring, introducing nitrogen to remove oxygen for 15min, and sealing; stirring and reacting for 6-10h at constant temperature and constant speed under the protection of nitrogen, wherein the constant temperature is 65-75 ℃, and the constant speed is 350-450 rpm; filtering to obtain microspheres;
and (3) washing: washing residual reactants of the microspheres by water, methanol and acetonitrile in sequence, repeatedly eluting by hydrochloric acid solution until no melamine is detected by ultraviolet visible spectrum detection, and repeatedly cleaning by ultrapure water to be neutral; the methanol and the acetonitrile are chromatographic pure solvents, the volume concentration of the methanol is 99.5 percent, the volume concentration of the acetonitrile is 99.9 percent, and the concentration of the hydrochloric acid is 1 mol/L.
And (3) drying: vacuum drying to constant weight, wherein the vacuum drying condition is that the vacuum degree is-0.10 mpa; the drying temperature is 20-30 ℃; thus obtaining the melamine molecularly imprinted microspheres.
The invention also provides a preparation method of the melamine molecular imprinting solid phase column, which adopts the melamine molecular imprinting microspheres as the filler; the method specifically comprises the following steps:
a step of column packing: accurately weighing 0.1g of the melamine molecularly imprinted microspheres, and filling the melamine molecularly imprinted microspheres into a 2.5mL medical syringe column with the bottom end blocked by filter paper by a dry method to obtain a solid-phase extraction column;
and (3) activating: and (3) washing the column with 10mL of methanol for activation, then washing with ultrapure water for three times, and blow-drying to obtain the catalyst.
Finally, the invention also provides a melamine molecular imprinting solid phase column applied to the detection of melamine residual samples in food or feed extracting solution, which comprises the following steps:
adding 1mL of food or feed melamine extract into a melamine molecular imprinting solid phase column, eluting with 2mL of water, 2mL of methanol and 3mL of ammonia-methanol solution in sequence, and collecting eluent; the volume concentration of the methanol is 99.5%, and the volume ratio of the ammonia water to the methanol in the ammonia water-methanol solution is 1: 9.
And (4) blowing and concentrating the elution liquid nitrogen to be dry, dissolving the elution liquid nitrogen by using methanol, fixing the volume to 1mL, using the solution as a sample to be detected, and finally detecting the sample in a high performance liquid chromatography.
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.
Example 1Melamine molecularly imprinted microspheres
The melamine molecularly imprinted microsphere is prepared from the following components in millimole: 0.85mmol of melamine, 5mmol of 2-acrylamide-2-methylpropanesulfonic acid, 5mmol of alpha-methacrylic acid, 8mmol of hydroxyethyl cellulose, 38mmol of ethylene glycol dimethacrylate, 0.5mmol of azobisisobutyronitrile and 50ml of toluene.
Example 2Melamine molecularly imprinted microspheres
The melamine molecularly imprinted microsphere is prepared from the following components in millimole: 1mmol of melamine, 6mmol of 2-acrylamide-2-methylpropanesulfonic acid, 6mmol of alpha-methacrylic acid, 10mmol of hydroxyethyl cellulose, 40mmol of ethylene glycol dimethacrylate, 0.6mmol of azobisisobutyronitrile and 75ml of toluene.
Example 3Melamine molecularly imprinted microspheres
The melamine molecularly imprinted microsphere is prepared from the following components in millimole: 1.15mmol of melamine, 7mmol of 2-acrylamide-2-methylpropanesulfonic acid, 7mmol of alpha-methacrylic acid, 12mmol of hydroxyethyl cellulose, 42mmol of ethylene glycol dimethacrylate, 0.7mmol of azobisisobutyronitrile and 100ml of toluene.
Examples4Blank polymer, used as a blank control.
The procedure for the preparation of a blank polymer (NIP) was as in example 2 above, except that no melamine was added.
Example 5Preparation method of melamine molecularly imprinted microspheres
The preparation method of the melamine molecularly imprinted microspheres of examples 1 to 4 is as follows:
dissolving a dispersing agent: weighing hydroxyethyl cellulose dispersing agent with the formula ratio, adding the hydroxyethyl cellulose dispersing agent into water, stirring to dissolve the dispersing agent, and standing and cooling to obtain hydroxyethyl cellulose solution;
the step of preparing the aqueous phase: adding the 2-acrylamide-2-methylpropanesulfonic acid and melamine with the formula ratio into the hydroxyethyl cellulose solution, and stirring to obtain a water phase part;
preparing an organic phase: adding alpha-methacrylic acid, ethylene glycol dimethacrylate and azodiisobutyronitrile in formula amount into toluene, mixing, and ultrasonically dissolving to obtain an organic phase part;
the reaction steps are as follows: slowly dripping the organic phase part into the water phase part under the condition of mechanical stirring, introducing nitrogen to remove oxygen for 15min, and sealing; stirring and reacting for 6-10h at constant temperature and constant speed under the protection of nitrogen, wherein the constant temperature is 70 ℃, and the constant speed is 400 rpm; filtering to obtain microspheres;
and (3) washing: washing residual reactants of the microspheres by water, methanol and acetonitrile in sequence, repeatedly eluting by hydrochloric acid solution until no melamine is detected by ultraviolet visible spectrum detection, and repeatedly cleaning by ultrapure water to be neutral; the volume concentration of the methanol is 99.5%, the volume concentration of the acetonitrile is 99.9%, and the concentration of the hydrochloric acid is 1 mol/L.
And (3) drying: vacuum drying to constant weight, wherein the vacuum drying condition is that the vacuum degree is-0.10 mpa; the drying temperature is 20-30 ℃; thus obtaining the melamine molecularly imprinted microspheres.
Example 6Preparation method of melamine molecular imprinting solid phase column
A preparation method of a melamine molecularly imprinted solid phase column adopts melamine molecularly imprinted microspheres of examples 1-4 as a filler; the method specifically comprises the following steps:
a step of column packing: accurately weighing 0.1g of the melamine molecularly imprinted microspheres prepared in example 4, and filling the microspheres into a 2.5mL medical syringe column with the bottom end blocked by filter paper by a dry method to obtain a solid phase extraction column;
and (3) activating: and (3) washing the column with 10mL of methanol for activation, then washing with ultrapure water for three times, and blow-drying to obtain the catalyst.
Example 7Method for detecting melamine residual sample
The melamine molecular imprinting solid phase column is applied to the detection of melamine residual samples in food or feed extracting solution, and comprises the following steps:
adding 1mL of food or feed melamine extract into a melamine molecular imprinting solid phase column, eluting with 2mL of water, 2mL of methanol and 3mL of ammonia-methanol solution in sequence, and collecting eluent; the volume concentration of the methanol is 99.5%, and the volume ratio of the ammonia water to the methanol in the ammonia water-methanol solution is 1: 9.
And (4) blowing and concentrating the elution liquid nitrogen to be dry, dissolving the elution liquid nitrogen by using methanol, fixing the volume to 1mL, using the solution as a sample to be detected, and finally detecting the sample in a high performance liquid chromatography.
1. High selective adsorption performance
The imprinted polymer (MIP) with uniform particle size in example 2 and the blank polymer (NIP) in example 4 were subjected to adsorption experiments with different concentrations of template molecule (melamine) solutions, and the results are shown in fig. 1. From the data in the figure, the adsorption amount (Q) of the imprinted polymer to the melamine is larger than that of the blank polymer, which indicates that the imprinted polymer has excellent specific adsorption to the melamine.
2. Analysis of actual samples
2.1 preparation of Standard Curve and detection Limit
Preparing a melamine solution with a certain concentration, preparing a standard curve, and as shown in fig. 2, the result shows that the melamine concentration is in the range of 0-20 mug/mL, the linear relation of the peak area A to the concentration C is good, and the linear regression equation is that A is 5.062+56.02 xC; the correlation coefficient R was 0.9995, and the detection limit of the method (S/N ═ 3) was 0.008 μ g/mL.
2.2 analysis of actual samples
2 mug/mL of melamine is added into the milk powder extracting solution and the pure milk extracting solution, and compared with HPLC chromatograms of the milk powder extracting solution and the pure milk extracting solution after the MIP-SPE column prepared in the example 2 is extracted in the example 7, as shown in figures 3-6, complex matrixes in the milk powder extracting solution are basically removed, and melamine is selectively separated and enriched, which shows that the MIP-SPE column can be used for quickly separating and detecting the melamine in the milk powder extracting solution and the pure milk extracting solution. As shown in Table 1-2, in the milk powder and the pure milk obtained by adding the standard under different levels, the recovery rates of the melamine molecular imprinting solid phase extraction method are 93.6-101.2% and 94-97.6% respectively, and the relative standard deviations are 3.3% and 1.9% respectively.
TABLE 1 recovery and precision of melamine in milk powder (n ═ 3)
Table 2 recovery and precision of melamine in pure milk (n ═ 3)
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Claims (9)
1. The melamine molecularly imprinted microsphere is characterized by being prepared from the following components: 0.85-1.15mmol of melamine, 5-7mmol of 2-acrylamide-2-methylpropanesulfonic acid, 5-7mmol of alpha-methacrylic acid, 8-12mmol of hydroxyethyl cellulose, 38-42mmol of ethylene glycol dimethacrylate, 0.5-0.7mmol of azobisisobutyronitrile and 50-100ml of toluene;
the preparation method of the melamine molecularly imprinted microspheres comprises the following steps:
dissolving a dispersing agent: weighing hydroxyethyl cellulose dispersing agent with the formula ratio, adding the hydroxyethyl cellulose dispersing agent into water, stirring to dissolve the dispersing agent, and standing and cooling to obtain hydroxyethyl cellulose solution;
the step of preparing the aqueous phase: adding the 2-acrylamide-2-methylpropanesulfonic acid and melamine with the formula ratio into the hydroxyethyl cellulose solution, and stirring to obtain a water phase part;
preparing an organic phase: adding alpha-methacrylic acid, ethylene glycol dimethacrylate and azodiisobutyronitrile in formula amount into toluene, mixing, and ultrasonically dissolving to obtain an organic phase part;
the reaction steps are as follows: slowly dripping the organic phase part into the water phase part under the condition of mechanical stirring, introducing nitrogen to remove oxygen for 15min, and sealing; stirring and reacting for 6-10h at constant temperature and constant speed under the protection of nitrogen, and filtering to obtain microspheres;
and (3) washing: washing residual reactants of the microspheres by water, methanol and acetonitrile in sequence, repeatedly eluting by hydrochloric acid solution until no melamine is detected by ultraviolet visible spectrum detection, and repeatedly cleaning by ultrapure water to be neutral;
and (3) drying: and drying in vacuum to constant weight to obtain the melamine molecularly imprinted microspheres.
2. The melamine molecularly imprinted microsphere of claim 1, prepared from: 1mmol of melamine, 6mmol of 2-acrylamide-2-methylpropanesulfonic acid, 6mmol of alpha-methacrylic acid, 10mmol of hydroxyethyl cellulose, 40mmol of ethylene glycol dimethacrylate, 0.6mmol of azobisisobutyronitrile and 75ml of toluene.
3. A method for preparing the melamine molecularly imprinted microsphere as claimed in claim 1, characterized by comprising the following steps:
dissolving a dispersing agent: weighing hydroxyethyl cellulose dispersing agent with the formula ratio, adding the hydroxyethyl cellulose dispersing agent into water, stirring to dissolve the dispersing agent, and standing and cooling to obtain hydroxyethyl cellulose solution;
the step of preparing the aqueous phase: adding the 2-acrylamide-2-methylpropanesulfonic acid and melamine with the formula ratio into the hydroxyethyl cellulose solution, and stirring to obtain a water phase part;
preparing an organic phase: adding alpha-methacrylic acid, ethylene glycol dimethacrylate and azodiisobutyronitrile in formula amount into toluene, mixing, and ultrasonically dissolving to obtain an organic phase part;
the reaction steps are as follows: slowly dripping the organic phase part into the water phase part under the condition of mechanical stirring, introducing nitrogen to remove oxygen for 15min, and sealing; stirring and reacting for 6-10h at constant temperature and constant speed under the protection of nitrogen, and filtering to obtain microspheres;
and (3) washing: washing residual reactants of the microspheres by water, methanol and acetonitrile in sequence, repeatedly eluting by hydrochloric acid solution until no melamine is detected by ultraviolet visible spectrum detection, and repeatedly cleaning by ultrapure water to be neutral;
and (3) drying: and drying in vacuum to constant weight to obtain the melamine molecularly imprinted microspheres.
4. The method for preparing melamine molecularly imprinted microspheres as claimed in claim 3, wherein the constant temperature is 65-75 ℃ and the constant speed is 350-450rpm in the reaction step.
5. The method for preparing melamine molecularly imprinted microspheres according to claim 3, wherein in the washing step, the methanol is 99.5% by volume, the acetonitrile is 99.9% by volume, and the hydrochloric acid is 1 mol/L.
6. The method for preparing melamine molecularly imprinted microspheres according to claim 3, wherein in the drying step, the vacuum drying condition is vacuum degree of-0.10 mpa; the drying temperature is 20-30 ℃.
7. A preparation method of a melamine molecular imprinting solid phase column is characterized in that melamine molecular imprinting microspheres according to claims 1-2 or melamine molecular imprinting microspheres prepared by the preparation method according to any one of claims 3-6 are used as a filler; the method specifically comprises the following steps:
a step of column packing: accurately weighing 0.1g of the melamine molecularly imprinted microspheres, and filling the melamine molecularly imprinted microspheres into a 2.5mL medical syringe column with the bottom end blocked by filter paper by a dry method to obtain a solid-phase extraction column;
and (3) activating: and (3) washing the column with 10mL of methanol for activation, then washing with ultrapure water for three times, and blow-drying to obtain the catalyst.
8. The melamine molecularly imprinted solid phase column is applied to detection of melamine residual samples in food or feed extracting solution, and is characterized in that the melamine molecularly imprinted solid phase column prepared by the preparation method of the melamine molecularly imprinted solid phase column in claim 7 comprises the following steps:
enrichment and elution steps: adding 1mL of food or feed melamine extract into a melamine molecular imprinting solid phase column, eluting with 2mL of water, 2mL of methanol and 3mL of ammonia-methanol solution in sequence, and collecting eluent;
and (3) volume fixing step: and (4) carrying out blowing concentration on the elution liquid nitrogen until the elution liquid nitrogen is dried, dissolving the elution liquid nitrogen by using methanol, and fixing the volume to 1 mL.
9. The application of the melamine molecularly imprinted solid phase column of claim 8 to the detection of melamine residue samples in food or feed extraction liquid, wherein in the step of enrichment elution, the volume concentration of the methanol is 99.5%, and the volume ratio of ammonia to methanol in the ammonia-methanol solution is 1: 9.
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