CN103265666A - Malathion molecularly imprinted polymer and synthetic method of malathion restricted access media-molecularly imprinted polymer - Google Patents

Abstract

The invention provides a malathion molecularly imprinted polymer and a synthetic method of a malathion restricted access media-molecularly imprinted polymer. The malathion molecularly imprinted polymer is synthesized by using a molecular imprinting technique and a bulk polymerization method. A prepared molecularly imprinted solid phase extraction column has a recovery rate of 97.8% for malathion. The extraction effect is obviously better than that of commercialized solid phase extraction columns. By using the restricted access media-molecular imprinted technology, the precipitation polymerization and suspension polymerization are optimized to synthesize malathion molecularly imprinted microspheres and restricted access media-molecularly imprinted microspheres. The resulting microspheres are made into a solid phase extraction column. Compared with the commercialized solid phase extraction columns, the solid phase extraction column has the advantages of high recovery rate, low standard deviation, and good reproducibility.

Description

The into synthetic method of medium-molecularly imprinted polymer is limit in Malathion molecularly imprinted polymer and Malathion

Technical field

The invention belongs to remains of pesticide detection technique field, particularly limit the into synthetic method of medium-molecular engram microsphere for detection of Malathion residual Malathion molecularly imprinted polymer and Malathion.

Background technology

Pesticide residue exceed standard as an important factor that influences food safety, and it is healthy directly to influence the human consumer, become the primary index that food quality safety is weighed by various countries.Agricultural chemicals can be divided into four big classes such as organic chlorine agriculture chemicals, carbamate chemicals for agriculture, pyrethroid lipid agricultural chemicals, organophosphorus insecticide.Organophosphorus insecticide mainly contains Malathion, Nemacur, terbufos, phorate, Rogor, Chlorpyrifos 94, acephatemet, Trichlorphon, SD-1750, thiophos, Trichlorphon, Volaton etc.Organophosphorus insecticide is prevention and elimination of disease and pests efficiently, and easy to use, economic security, has become the agricultural chemicals of China's present stage usage quantity maximum.Just because its range of application is quite general, organophosphorus insecticide is one of main source of present pesticide residue.In addition, some organophosphorus pesticide has very strong acute toxicity to people, animal, they enter human body through food chain by sucking, eat and absorbing through skin, constantly after the accumulation, can suppress that thereby the activity of Pseudocholinesterase causes dizziness, dim eyesight in the body, feel sick, toxicity symptoms such as hidrosis, myosis, serious even can cause harm such as carcinogenic, teratogenesis, mutagenesis.For this reason, countries in the world are paid much attention to the pesticide residue problem, and the standard of organophosphorus pesticide content in the food is made in European Union, the U.S., the Japanese strictness of all having made laws.China has also promulgated agricultural industry criteria " in vegetables, the fruit in acephatemet etc. 20 agricultural chemicals maximum residue limit " (NY/T150022008) in 2008.Organophosphorus pesticide is preferentially monitored in 10 kinds of listed chemical pesticides of organic pollutant " Black List " at Chinese environmental and has been accounted for 7 kinds, has become the emphasis of pesticide residue analysis.

Usually pesticide concentration is very low in the sample that detects, at mg kg ^-1-fg kg ^-1In the level scope, often contain a large amount of interfering substances in the sample substrate again, so detection difficulty is more and more higher to the requirement of detection technique.The measuring method of organophosphorus pesticide residual quantity has a variety of, and vapor-phase chromatography is the proven technique of most widely used general and method.Its pre-treating process mainly contains liquid-liquid extraction (LLE), Solid-Phase Extraction (SPE), solid-phase microextraction (SPME), liquid-phase micro-extraction (LPME), liquid-liquid micro-extraction (LLME), gel permeation chromatography (GPC), supercritical fluid extraction (SFE), microwave auxiliary extraction (MAE) and accelerated solvent extraction (ASE) etc.Even it should be noted that with same technical measurement, if the sample-pretreating method difference also will directly influence measurement result.As seen pre-treatment is important link in the sample analysis, also is the effective guarantee that improves accuracy in detection.

Solid phase extraction techniques is a kind of sample pre-treatments technology, is developed the seventies in 20th century.Its principle is to utilize the solid adsorbent of filling that the target compound in the liquid sample is adsorbed, thereby realize separating of chaff interference in target compound and the sample, pass through drip washing and wash-out or heating desorption then, finally reach the purpose to target compound separation and enrichment.Commercial solid-phase adsorbent lacks single-minded selectivity, and is lower to the organophosphorus insecticide Detection and Extraction purification efficiency of trace in the sample that contains complex matrices, influences result's accuracy.The molecular engram solid phase extraction sorbent material is as a kind of novel solid phase extraction filler, can carry out selective adsorption to target analytes and gets rid of the interference of sample substrate, can not only reduce the detectability of target analytes like this, can also improve the accuracy of trace analysis.

The target compound of molecular engram solid phase extraction separation and concentration is generally small molecules, if when containing just like macromole such as protein, carbohydrates in the sample, sample at first will be through except albumen or except the processing of sugar.This is because the biomacromolecule in the sample can sex change when running into the anti-phase solid phase extraction filler of hydrophobicity and then adheres to the surface of sorbent material, cause occurring the sorbent material aperture and stop up, reduce the unfavorable phenomenons such as adsorption efficiency of analyte on sorbent material, imitate, reduce column capacity, shorten column life thereby reduce post, not only do not bring into play molecular engram solid phase extraction the high advantage of small molecules analyte selectivity is badly influenced detected result on the contrary.

Limit is advanced the medium Solid-Phase Extraction, and (Restricted Access Matrix-Solid Phase Extraction, RAM-SPE) sorbent material has the dual-use function at macromolecular volume-exclusion and the extraction of small molecules analyte simultaneously concurrently.Limit is advanced the superficies of medium through the wetting ability modification, and inner aperture also can suitably be controlled.According to volume-exclusion mechanism, macromole in the sample substrate advances less than going in the endoporus on the one hand, but making, the hydrophilic layer of outside surface irreversible sex change and absorption can not taken place by the biomacromolecule of exclusion, macromole can namely not be retained in skin by solvent through dead volume or be bordering on dead volume and removed by wash-out; Target compound small molecules in the sample substrate can enter into limit by the aperture to advance medium inner and be retained by hydrophobic interaction and electrostatic interaction on the other hand.Be to contain in the sample biomacromolecule even limit is advanced the advantage of medium, also can effectively extract the target compound small molecules.

Summary of the invention

The object of the invention is to provide Malathion molecularly imprinted polymer and Malathion to limit the into synthetic method of medium-molecularly imprinted polymer, thereby realizes to the selective enrichment of organophosphates with to the accurate analysis of sample.

For achieving the above object, the present invention adopts following technical scheme:

The synthetic method of Malathion molecularly imprinted polymer, template molecule in molar ratio: function monomer: linking agent=1:2～15:20～60, delivery plate molecule Malathion, function monomer α-Jia Jibingxisuan and linking agent ethylene glycol dimethacrylate join in the test tube, add initiator, solvent again, concussion, logical nitrogen, vibration makes it abundant mixing, sealing is reacted 24h in 70 ℃ water bath with thermostatic control; Through grinding, sieving, the Malathion Molecularly Imprinted Polymer of collection cut size 75-150 μ m, wrap with filter paper, earlier with methyl alcohol/glacial acetic acid solution wash-out template molecule in apparatus,Soxhlet's, using pure methyl alcohol behind the wash-out 50h again instead is eluted to no template molecule and detects, dry, get white Malathion molecularly imprinted polymer.

Accurately measure template molecule Malathion 0.067mL, function monomer α-Jia Jibingxisuan 0.170mL, linking agent ethylene glycol dimethacrylate 1.889mL joins in the test tube, add initiator Diisopropyl azodicarboxylate 24.24mg, solvent chloroform 5mL again, concussion, the logical nitrogen 5min 10min that vibrates again makes it abundant mixing, sealing is reacted 24h in 70 ℃ thermostat water bath.Through grinding, after sieving, the Malathion Molecularly Imprinted Polymer of collection cut size 75-150 μ m, wrap with filter paper, use earlier methyl alcohol: Glacial acetic acid (use pure methyl alcohol again instead and be eluted to no template molecule and detect behind the wash-out 50h by 9:1, V/V) solution wash-out template molecule in apparatus,Soxhlet's, dry, get white Malathion molecularly imprinted polymer.

The synthetic method of Malathion molecular blotting polymer microsphere, template molecule in molar ratio: function monomer: linking agent=1:2～15:20～60, delivery plate molecule Malathion, function monomer α-Jia Jibingxisuan and linking agent ethylene glycol dimethacrylate, template molecule Malathion and function monomer α-Jia Jibingxisuan are added round-bottomed flask, add the part chloroform solvent, prepolymerization 1～3h on magnetic stirring apparatus, add linking agent ethylene glycol dimethacrylate and Diisopropyl azodicarboxylate then, add 3 times of chloroform solvents of dosage first again, constant temperature magnetic agitation reaction 10～40h gets the Malathion molecular blotting polymer microsphere;

Get 1g Malathion molecular blotting polymer microsphere, wrap to put into filter paper and place Soxhlet extractor, add 100～360mL methyl alcohol: the acetic acid volume ratio is the mixing solutions backflow wash-out template molecule of 9:1, the new mixing solutions of middle constantly adding, do not detect to there being template molecule to elutriant, put into round-bottomed flask after drying, add 100～360mL methyl alcohol, stirring and refluxing 3～8h removes residual acetic acid, oven dry obtains molecular blotting polymer microsphere behind the suction filtration, collect the microballoon of 45-75 μ m, be the Malathion molecular blotting polymer microsphere.

Delivery plate molecule Malathion 0.268mL and function monomer α-Jia Jibingxisuan 0.509mL to 250mL round-bottomed flask, add the 40mL chloroform solvent, put into magneton prepolymerization 5h on magnetic stirring apparatus, add linking agent ethylene glycol dimethacrylate 5.642mL and initiator Diisopropyl azodicarboxylate 0.096g then, add the 120mL chloroform solvent at last, the round-bottomed flask that this mixing solutions is housed is put into constant temperature blender with magnetic force, at 70 ℃ and 150r min ^-1Reaction 10h gets the Malathion molecular blotting polymer microsphere;

Take by weighing the synthetic good molecular blotting polymer microsphere of about 1g, it is wrapped to put into filter paper place Soxhlet extractor, add 150mL methyl alcohol: the acetic acid volume ratio is the mixing solutions wash-out template molecule of 9:1, reflux down at 84 ℃, the new mixing solutions of middle constantly adding, do not detect to there being template molecule, after drying, room temperature puts into the 250mL round-bottomed flask, add 150mL methyl alcohol, remove residual acetic acid at 70 ℃ of following stirring and refluxing 5h, dry behind the suction filtration, collected the microballoon of 45-75 μ m, be the Malathion molecular blotting polymer microsphere.

The into synthetic method of medium-molecular blotting polymer microsphere is limit in the Malathion, template molecule in molar ratio: function monomer 1: function monomer 2: linking agent=1:2～20:2～20:20～90, delivery plate molecule Malathion, function monomer 1 α-Jia Jibingxisuan, function monomer 2 glytidyl methacrylate and linking agent ethylene glycol dimethacrylate, with template molecule Malathion and function monomer 1 α-Jia Jibingxisuan and function monomer 2 glytidyl methacrylate, the linking agent ethylene glycol dimethacrylate adds round-bottomed flask, add the initiator chloroform solvent again, constant temperature magnetic agitation reaction 6～30h gets the Malathion and limits into medium-molecular blotting polymer microsphere;

Get and limit the high chloro acid solution of medium-molecular engram microsphere and volume ratio 1:9 to add in the round-bottomed flask, room temperature magnetic agitation reaction 6～30h, suction filtration, thus obtained microsphere constantly stirs in ethanol, acetone, ether successively, filter then, dry under the room temperature, get the Malathion and limit into medium molecule imprinted polymer microballoon.

Delivery plate molecule Malathion 0.268mL, function monomer 1 α-Jia Jibingxisuan 0.509mL, function monomer 2 glytidyl methacrylate 0.199mL, linking agent ethylene glycol dimethacrylate 5.642mL to 250mL round-bottomed flask, add initiator Diisopropyl azodicarboxylate 0.096g and 150mL chloroform solvent again, in the constant temperature blender with magnetic force at 70 ℃ and 150r min ^-1Reaction 10h gets the Malathion and limits into medium-molecular blotting polymer microsphere;

Take by weighing 0.8g and limit into medium-molecular engram microsphere in the 150mL round-bottomed flask, add the high chloro acid solution of 50mL volume ratio 1:9, under the room temperature with 150r min ^-1Rotating speed magnetic agitation reaction 24h, suction filtration, thus obtained microsphere constantly stir in the ethanol of 200mL, acetone, ether successively, filter again, room temperature is dried, and gets the Malathion and limits into medium-molecular blotting polymer microsphere.

The limit that suspends is advanced the synthetic method of medium-molecular engram microsphere, in the polyvinyl alcohol water solution that fills weightmeasurement ratio 1～3%, add the template molecule Malathion, the function monomer α-Jia Jibingxisuan, magnetic agitation 5h prepolymerization forms prepolymerization liquid, other gets glytidyl methacrylate, Vinylstyrene, benzoyl peroxide forms miscible fluid, miscible fluid is slowly dropped in the prepolymerization liquid, 70～90 ℃ of mechanical stirring reaction 3～15h, get microballoon, template molecule Malathion in molar ratio: function monomer α-Jia Jibingxisuan: glytidyl methacrylate: Vinylstyrene=1:2～20:2～20:20～90;

The microballoon of drying is put into the pure water heated and stirred remove polyvinyl alcohol, the limit that must suspend is advanced medium-Molecularly Imprinted Polymer microballoon;

Getting suspends limits the high chloro acid solution of medium-Molecularly Imprinted Polymer microballoon and volume ratio 1:9 to add in the round-bottomed flask, room temperature magnetic agitation reaction 6～30h, suction filtration, thus obtained microsphere constantly stirs in ethanol, acetone, ether successively, filter then, dry under the room temperature, get the suspension Malathion and limit into medium-molecular blotting polymer microsphere.

Taking by weighing the 1g polyvinyl alcohol is dissolved in the 100mL hot water, put into the 250mL round-bottomed flask after the cooling, add Malathion 0.268mL, α-Jia Jibingxisuan 0.509mL, magnetic agitation 5h, make the abundant prepolymerization of template molecule and function monomer form prepolymerization liquid, glytidyl methacrylate 0.794mL, Vinylstyrene 5.606mL, the miscible formation miscible fluid of benzoyl peroxide 65.77mg, slowly miscible fluid is added drop-wise in the prepolymerization liquid 80 ℃, 200r min with dropping funnel ^-1Mechanical stirring reaction 5h gets microballoon;

After the microballoon drying, put into the pure water heated and stirred, polyvinyl alcohol is dissolved in the hot water, suction filtration then, repetitive operation must suspend and limit medium-Molecularly Imprinted Polymer microballoon to removing polyvinyl alcohol fully;

Take by weighing the 0.8mg limit that suspends and advance medium-Molecularly Imprinted Polymer microballoon in the 150mL round-bottomed flask, add the high chloro acid solution of 50mL volume ratio 1/9, under the room temperature with 150r min ^-1Rotating speed magnetic agitation reaction 24h, suction filtration, thus obtained microsphere constantly stir in the ethanol of 200mL, acetone, ether successively, filter again, room temperature is dried, and gets the suspension Malathion and limits into medium-molecular blotting polymer microsphere.

The present invention:

1, utilizes molecular imprinting, adopt bulk polymerization, synthetic Malathion molecularly imprinted polymer.Selection by function monomer, orthogonal experiment is optimized synthesis condition, with template molecule (Malathion): function monomer (α-Jia Jibingxisuan): linking agent (ethylene glycol dimethacrylate) is 1:8:40,70 ℃ of temperature, initiator amount is synthetic molecularly imprinted polymer under 1.5% the condition of monomer and linking agent total mass, carry out absorption property test, imprinted polymer to the Malathion maximal absorptive capacity be 8.62mg g ^-1, but not imprinted polymer is 3.85mg g to the maximal absorptive capacity of Malathion ^-1Obtain by the selectivity experiment, imprinted polymer is respectively 287.97,275.00 to the adsorptive capacity of ethoprophos, phorate, terbufos, Rogor, Malathion, Nemacur, 257.07,300.21,344.11,261.32 μ g g ^-1, but not the adsorptive capacity of imprinted polymer is respectively 142.37,137.58,130.00,143.43,112.34,123.98 μ g g ^-1, the result shows that this polymkeric substance has stronger adsorptive power and template molecule is had good selectivity.The gained imprinted polymer is made solid-phase extraction column and optimize its Solid-Phase Extraction condition: with methanol-water (4:6; V/V) solution is made the drip washing solvent, makes eluting solvent with pure methyl alcohol.The molecularly imprinted solid phase extraction column of preparation reaches 97.8% to the rate of recovery of Malathion, and the rate of recovery of commodity post C18 and Florisil is respectively 82.8% and 75.6%, illustrates that self-control solid-phase extraction column effect of extracting obviously is better than the commodity post.

2, utilize limit to advance medium-molecular imprinting, optimize the molecular engram microsphere and the reaction conditions of limitting medium-molecular engram microsphere of the synthetic Malathion of precipitation polymerization method and suspension polymerization, wherein add new function monomer glytidyl methacrylate and enter the medium-hydrophilic hydroxyl of molecular engram microsphere surface formation top layer to modify limit.Adsorption test shows that limit is advanced medium-molecular engram microsphere and molecular engram microsphere has same specific recognition to template molecule.Selectivity experiment shows, two kinds of limits are advanced medium-molecular engram microsphere the adsorptive capacity of template molecule Malathion is maximum, reach 448.5 and 424.11 μ g g respectively ^-1, with the time limit advance medium-non-molecular engram microsphere to the adsorptive capacity of six kinds of organophosphoruss all less than limitting into medium-molecular engram microsphere, and the adsorptive capacity gap is little.Thus obtained microsphere is made solid-phase extraction column and optimize its Solid-Phase Extraction condition: make the drip washing solvent with 50% acetone solution (V/V) solution, make eluting solvent with pure methyl alcohol.Show by contrast, self-control precipitation and the limit that suspends are advanced the recovery of extraction of medium-molecular engram microsphere solid-phase extraction column all between 88.9%-98.3%, molecular engram microsphere Solid-Phase Extraction and precipitation limit are advanced between medium-molecular engram microsphere Solid-Phase Extraction to contrast, between the equal 89.2%-97.8% of recovery of extraction, and the latter has simplified in the pre-treatment except albumen, except the step of sugar and obtain good effect of extracting; With the contrast of commercialization solid-phase extraction column, the result shows that self-control precipitates and suspension limits medium-molecular engram microsphere solid-phase extraction column to have rate of recovery height (97.8%, 98.3%), standard deviation low (2.25%, 3.15%), characteristics such as favorable reproducibility.The limit of setting up is advanced medium-molecular engram microsphere Solid-Phase Extraction method be applied to the detection of organophosphorus in the honey sample, its linearity range is 0.01-10 μ g mL ^-1, lowest detection is limited to 0.005 μ g mL ^-1, and measured 0.10 and 0.20 μ g mL ^-1The recovery of standard addition of two levels is respectively 95.7%, 97.6% and 96.1%, 98.2% to the rate of recovery of Malathion, and the relative standard deviation scope that replicate(determination) is 6 times is 2.43%, 2.26% and 3.16%, 3.25%.The method of setting up has not only been simplified pre-treatment step, has been improved sensitivity, also greatly reduces cost, has bright prospects in actual applications.

Description of drawings

Fig. 1-1 is the synthetic MIP of different monomers, the absorption spirogram of the Malathion of NIP;

Fig. 1-2 (a), Fig. 1-2 (b) are Malathion molecularly imprinted polymer electron-microscope scanning figure;

Fig. 1-3 is the adsorption isotherm line chart of MIP, NIP;

Fig. 1-4 is the scatchard graphic representation of MIP;

Fig. 1-5 is the adsorptive capacity graphic representation over time of MIP;

Fig. 1-6 is the selective adsorption figure of MIP;

Fig. 1-7 is the rate of recovery figure of the Malathion of different drip washing, eluting solvent gained;

Fig. 2-1 is the synthetic MIMs of different monomers, the absorption spirogram of the Malathion of NIMs;

Fig. 2-2(a) is molecular engram microsphere electron-microscope scanning figure, and into medium-molecular engram microsphere electron-microscope scanning figure (b) is limited;

Fig. 2-3 is the equilibrium adsorption lab diagram;

Fig. 2-4 is microballoon adsorption rate figure;

Fig. 2-5 is that microballoon is to the selective adsorption figure of six kinds of organophosphoruss;

Fig. 2-6 is the rate of recovery figure of the Malathion of different drip washing, eluting solvent gained;

Fig. 2-7(a) for the RAM-MISPE mark-on reclaims color atlas, (b) for the MISPE mark-on reclaims color atlas, (c) for the C18SPE mark-on reclaims color atlas, (d) be that Florisil SPE mark-on reclaims color atlas;

Fig. 2-8 is sem photograph, and wherein (a) is the product of 100r min-1, (b) is 150r min-1 product, (c) is the product of 200r min-1;

Fig. 2-9(a) is the product suspension of linking agent for EDMA, (b) is the product suspension of linking agent for TDMA, (c) is the product suspension of linking agent for DVB;

Fig. 2-10 is microballoon adsorption isothermal line in Malathion solution;

Fig. 2-11 is the microsphere polymer adsorption rate;

Fig. 2-12 is that microsphere polymer is to the selectivity of six kinds of organophosphoruss;

Fig. 2-13(a) for the RAM-MISPE1 mark-on reclaims color atlas, (b) reclaim color atlas for the RAM-MISPE2 mark-on.

Embodiment

The synthetic method of embodiment 1, Malathion molecularly imprinted polymer:

Accurately measure template molecule Malathion 0.067mL(0.25mmol), function monomer MAA(α-Jia Jibingxisuan) 0.170mL(2mmol), linking agent EDMA(ethylene glycol dimethacrylate) 1.889mL(10mmol) join in the test tube (template molecule: function monomer: linking agent=1:8:40), add initiator A IBN(Diisopropyl azodicarboxylate again) 24.24mg, solvent chloroform 5mL, concussion, the logical nitrogen 5min 10min that vibrates again makes it abundant mixing, sealing is reacted 24h in 70 ℃ thermostat water bath.Through grinding, after sieving, the Malathion molecularly imprinted polymer of collection cut size 75-150 μ m.Wrap with filter paper then, (9/1, V/V) solution wash-out template molecule in apparatus,Soxhlet's is used pure methyl alcohol again instead and is carried out wash-out till no template molecule detects behind the wash-out 50h with methyl alcohol/Glacial acetic acid earlier.Last natural airing namely gets white imprinted polymer.

With under the situation that does not add template molecule, synthesizing blank polymkeric substance (NIP) with above-mentioned duplicate method.

Utilize synthetic imprinted polymer and blank polymkeric substance to carry out following experiment:

Polymkeric substance to the balance of template molecule Malathion in conjunction with experiment

Be function monomer with MAA, synthetic Malathion imprinted polymer under the peak optimization reaction condition.Prepare a series of concentration 0-50 μ g mL ^-1The Malathion aqueous solution, accurately take by weighing the MIP of 20mg, add each 4mL of Malathion solution of above-mentioned concentration respectively, vibration is left standstill 20h, with 4000r min ^-1The centrifuging and taking supernatant liquor, N ₂After drying up, with 1mL n-hexane dissolution template molecule Malathion, air inlet phase chromatographic determination content, the variation by Malathion concentration in the solution of combination front and back finally calculates MIP to the adsorptive capacity of different concentration of substrate solution.

Q=(C ₀-C)*V ₀/m

Q(μ g mg ^-1)---polymkeric substance is to the adsorptive capacity of Malathion;

C ₀(μ g L ^-1)---the original concentration of Malathion;

C(μ g L ^-1)---the concentration of Malathion in the mother liquor of absorption back;

V ₀(L)---the volume of each standard horse traction sulphur phosphorus solution of getting;

M(mg)---the quality of the polymkeric substance of getting.

Adsorptive capacity with the Malathion solution of the step measurements NIP different concns consistent with aforesaid method.

2. adsorption rate experiment

Get 12 groups of MIP20mg respectively and be put in the centrifuge tube of 5mL, be numbered 1-12, add 2 μ g mL more respectively ^-1Malathion solution 4mL, vibration, standing adsorption be for 1-8 number every 1h mensuration once; 8-12 number is to measure once every 2h.Calculate adsorptive capacity.Replicate(determination) is averaged for 3 times.

3. the selective adsorption of polymkeric substance experiment

Because there is similar molecular structure ethoprophos, terbufos, phorate, Nemacur, Rogor and Malathion, measure the adsorption selectivity of Malathion molecularly imprinted polymer with their mixing solutions.Accurately take by weighing the 20mg polymkeric substance in centrifuge tube, add mixing solutions 4mL(6 kind organophosphorus concentration of standard solution and be respectively 5 μ g mL ^-1), seal, leave standstill 20h.Use gas Chromatographic Determination, and the concentration of six kinds of organophosphoruss in the calculated equilibrium adsorption liquid, and further calculate microballoon to binding capacity Q (the μ g mg of each substrate ^-1), replicate(determination) is averaged for 3 times.

With measuring NIP to 6 kinds of organophosphorus adsorptive capacitys with above-mentioned duplicate method.

4. Solid-Phase Extraction

Accurately take by weighing 100mg MIP and be put in the beaker, add 15mL methyl alcohol, be put in the solid-phase extraction column with wet method dress post, freshly prepd solid-phase extraction column detects with pure methyl alcohol drip washing no Malathion template molecule to the elutant.The pillar for preparing is preserved stand-by.

To make solid-phase extraction column by oneself and use 10mL methyl alcohol successively, and after the activation of 2mL water, get sample on the 20mL Malathion solution, the control flow velocity is 1d s ^-1, use 2mL methanol-water (4:6 then; V/V) the pure methanol-eluted fractions of 1mL is used in drip washing at last.

Above-mentioned experimental result is as follows:

1. the synthetic optimization of molecularly imprinted polymer

1.1 the selection of function monomer

Function monomer and template molecule binding ability are one of important factors of impact polymer recognition performance.Interaction between monomer and the template molecule is more strong, and the selectivity of the recognition site of formation is more strong, and identification and the binding ability of synthetic polymkeric substance are also more strong.Otherwise the well combination of monomer and template, the meeting of formation are weak optionally binding sites, thereby cause the identity of the polymkeric substance that synthesizes also not high.

Be function monomer with 4-vinylpridine (4-VP), α-Jia Jibingxisuan (MAA), acrylamide (AM), methyl methacrylate (MMA), vinylformic acid (AA) respectively, at template molecule: function monomer: linking agent=1:4:20, temperature is synthetic MIP and NIP under 70 ℃ of conditions, and surveys it to the adsorptive capacity of template.The result as Figure 1-1, the adsorptive capacity of MIP has shown that all greater than NIP the template molecule of MIP has stronger affinity and selectivity as seen from the figure, adsorptive power is better.And be that the adsorptive capacity of the Malathion of MIP of monomer is 7.45 μ g mg with MAA ^-1, the adsorptive capacity of NIP is 3.01 μ g mg ^-1, all than all the other corresponding MIP and NIP height, show that the hydrogen bond action between MAA and the Malathion template molecule is the strongest, so the optimum monomer of selecting is MAA.

1.2 the optimization of synthesis condition

The consumption of the main ratio to template molecule and function monomer ratio, template molecule and linking agent of this experiment, temperature of reaction, initiator is optimized.Adopted the orthogonal experiment of 4 factors, 3 levels, shown in table 1-1, orthogonal design table is shown in table 1-2.

Table 1-1 4 factors 3 levels

Factor A: the ratio of template molecule and function monomer

Factor B: the ratio of template molecule and linking agent

Factor C: temperature

Factor D: the consumption of initiator

The synthetic orthogonal design table of table 1-2 Malathion imprinted polymer

1-2 can find out by table, tests the adsorptive capacity maximum of 7 gained, is 7.98 μ g mg ^-1, namely at template molecule: function monomer: linking agent=1:8:40, temperature is 70 ℃, the initiator quality is that the absorption property of the Malathion molecularly imprinted polymer of gained is best under 1.5% the reaction conditions of monomer and linking agent quality.Template molecule: function monomer: linking agent=1:8:40,70 ℃ of temperature are in selected condition, the consumption of function monomer, linking agent all is maximum, temperature also is the highest, may be because of function monomer more for a long time, optionally recognition site is also more many with having of forming of template molecule useful effect, and the adsorptivity of imprinted polymer and selectivity are also more strong, and the absorption quantitative change is big; And more linking agent makes the imprinted polymer that is synthesized have stronger rigidity, has kept the globality of recognition site, thereby makes the absorption quantitative change big; Though low temperature be conducive to template molecule and function monomer with combination, temperature is brought up to and to a certain degree be can significantly improve polymer properties, so under 70 ℃ temperature of reaction, the absorption property of the polymkeric substance of gained is stronger.So the optimum synthesis condition of molecularly imprinted polymer is template molecule: function monomer: linking agent=1:8:40, temperature is 70 ℃, the initiator quality is 1.5% of monomer and linking agent quality.

Contrast the size of four factor extreme differences, the result is R _A＞R _B＞R _D＞R _CTherefore the synthetic significant factors of impact polymer is factor A, i.e. the ratio of template molecule and function monomer.

2. imprinted polymer electron-microscope scanning figure

The synthetic MIP of mass polymerization carries out electron-microscope scanning behind grinding and wash-out.Observe shape and the surface tissue of MIP by electron-microscope scanning figure.With electron-microscope scanning by * 160 times (a) and * 10000 times (b) observe, its result is as Fig. 1-2 (a), 1-2(b) shown in.Can be found out because grinding causes MIP to be irregularly shaped by the electron-microscope scanning figure that amplifies 100 times.Can find out that by the electron-microscope scanning figure that amplifies 10000 times MIP has loose porous surface tissue, for its absorption provides foundation.

3. the discussion of absorption property test

3.1 balance is in conjunction with experiment

The adsorption isothermal line of MIP and the Malathion of NIP as Figure 1-3.As seen from the figure, the adsorptive capacity of the template molecule of MIP and NIP Malathion increases along with the rising of Malathion strength of solution, and absorption is during same concentrations, and the adsorptive capacity of the template molecule of MIP Malathion all exceeds much than NIP.This shows, compares with NIP, and MIP has stronger avidity because of the specific adsorption effect to the Malathion, and this is the embodiment of imprinted polymer trace specific adsorption, and the Malathion of NIP is non-specific adsorption.

Test the data that obtain according to the equilibrium adsorption of MIP among Fig. 1-3 and be used for the Scatchard analysis.By Scatchard equation Q/C=(Q _Max-Q) Kd ^-1, wherein C is the equilibrium concentration of Malathion in solution, and Kd is the balance dissociation constant of binding site, and Q is that polymkeric substance adsorbs the adsorptive capacity when reaching balance, Q _MaxBe the maximum of binding site performance binding capacity), obtain the Scatchard curve of MIP with the Q mapping of Q/C, shown in Fig. 1-4.

Can find out that by Fig. 1-4 the Scatchard curve is nonlinear relationship, but linear respectively in high density and lower concentration, relation conefficient is respectively 0.9936,0.9975, and this shows that the Malathion imprinted polymer has the binding site of 2 kinds of non-equivalences to the Malathion.The dissociation constant K of the binding site of low-affinity ₁=7.91 μ g mL ^-1, maximum performance binding constant Q _Max1=8.23 μ gmg ^-1The dissociation constant K of the binding site of high-affinity ₂=6.75 μ g mL ^-1, Q _Max2=8.91 μ g mg ^-1

3.2 adsorption rate test

Measured the time dependent adsorptive capacity of polymkeric substance, i.e. adsorption rate is as Fig. 1-5.As seen from the figure, along with the increase of time, the adsorptive capacity of polymkeric substance increases gradually, and absorption is tending towards state of saturation behind the 7h.Adsorptive capacity changes by rapidly to slowing down gradually, may be because utilize the synthetic MIP of mass polymerization because grinding has caused deep mixed hole, shallow hole is conducive to adsorb fast template molecule, and when the absorption of these holes reach saturated after, the Malathion enters inner dark hole can be slower, so cause adsorption rate to descend gradually, finally reach adsorption equilibrium after saturated fully.

3.3 selective adsorption research

Imprinted polymer and non-imprinted polymer have been measured to the selective adsorption of Malathion, phorate, Nemacur, Rogor, ethoprophos, terbufos.The typical curve equation of six kinds of organophosphoruss sees Table 1-3.

The typical curve equation of table 1-3 Malathion, phorate, Nemacur, Rogor, ethoprophos, terbufos

MIP, NIP to the selective adsorption data of various organophosphorus pesticides shown in Fig. 1-6.As seen from the figure, the adsorptive capacity of the ethoprophos of MIP, phorate, terbufos, Rogor, Malathion, Nemacur is respectively 287.97 μ g g ^-1, 275.00 μ g g ^-1, 257.07 μ g g ^-1, 300.21 μ g g ^-1, 344.11 μ g g ^-1, 261.32 μ g g ^-1, and the adsorptive capacity of NIP is respectively 142.37 μ g g ^-1, 137.58 μ g g ^-1, 130.00 μ g g ^-1, 143.43 μ g g ^-1, 112.34 μ g g ^-1, 123.98 μ g g ^-1, the adsorptive capacity maximum of hence one can see that the template molecule of MIP Malathion is 344.11 μ g g ^-1Because MIP has the specific hole with Malathion molecule stereo structure complementation, the Malathion is played specific adsorption, and slightly little to the adsorptive capacity of its analogue, illustrated that MIP has good selective adsorption performance, and its analog has also been played certain specific recognition effect.And NIP is little to the adsorptive capacity difference of each organophosphorus, because NIP does not have the specific recognition site, its functional group is to distribute arbitrarily, and what risen is non-specific adsorption.

3.4 Solid-Phase Extraction condition optimizing

Solid-Phase Extraction is divided into activation, goes up sample, drip washing and four steps of wash-out, wherein drip washing and elution step are decisive steps, because drip washing can be removed on polymkeric substance by the impurity of non-specific adsorption, allow the specific recognition effect of recognition site and determinand obtain maximization.And wash-out will reach the purpose that just analyte can be washed fully and reduce as much as possible the introducing of impurity with small volume of solution simultaneously.

In the Solid-Phase Extraction step, getting 80mg MIP is loaded in the solid-phase extraction column, go up sample with 20mL200 μ g L-1 Malathion acetone-water solution again with after the methyl alcohol activation, then 1mL methanol, ethanol/water, acetonitrile/water (0:10,1:9,2:8,3:7,4:6,5:5,6:4,7:3,8:2,9:1,10:0; V/V) solution carries out drip washing and wash-out, and the solution that elutes is collected in the centrifuge tube, N ₂Dry up the back and use the 1mL n-hexane dissolution, dissolved the back and crossed anhydrous Na ₂SO ₄The post organic membrane also is collected in the sample introduction bottle, and (GC-FPD) measures and calculate its rate of recovery with gas-chromatography.

Its result is shown in Fig. 1-7, when carrying out drip washing with the volume ratio of methanol-water, alcohol-water, acetonitrile-water less than the solution of 4:6, it does not have elutive power to the Malathion, considers that methyl alcohol polarity is strong better to the elute effect of impurity, so select 2mL methanol-water (4:6; V/V) solution is as the drip washing solvent.And when carrying out drip washing with the pure methyl alcohol of 1mL, up to 95.54%, it is 85.31% that straight alcohol is done the eluent rate of recovery to the Malathion rate of recovery, and the rate of recovery of pure acetonitrile is the poorest to be 75.87%.So the best eluting solvent of selecting is the pure methyl alcohol of 1mL.

4.MISPE contrast with the commodity post

With MISPE and commercialization solid-phase extraction column (C ₁₈And florisil) compares.The results are shown in Table 1-4.The result shows that MISPE obviously is better than with C enrichment, the recovering effect of six kinds of organophosphoruss ₁₈With the florisil solid-phase extraction column, illustrate that MISPE is a kind of pre-treating process of highly selective.

Different column extractors are to six kinds of organophosphorus recovery of standard addition in the table 1-4 honey sample

(n=6, mark-on level: 0.20 μ g mL-1).

The synthetic method of embodiment 2, Malathion molecular engram microsphere (precipitation polymerization method):

(1) delivery plate molecule Malathion (0.268mL, 1.0mmol) and function monomer MAA (0.509mL, 6.0mmol) to the 250mL round-bottomed flask, add the 40mL chloroform solvent, put into magneton prepolymerization 5h on magnetic stirring apparatus, (5.642mL 30.0mmol) with initiator A IBN (0.096g), adds the 120mL chloroform solvent at last to add linking agent EDMA then.The round-bottomed flask that this mixing solutions is housed is put into constant temperature blender with magnetic force, and the temperature of constant temperature blender with magnetic force and rotating speed are set at 70 ℃ and 150r min respectively ^-1React 10h with this understanding.Get molecular engram microsphere.

Synthetic ratio and the method for non-molecular engram microsphere are the same, just do not add the template molecule Malathion.

(2) take by weighing the synthetic good molecular engram microsphere of about 1g, it is wrapped to put into filter paper place Soxhlet extractor, add 150mL methyl alcohol: the acetic acid volume ratio is that the mixing solutions of 9:1 comes the wash-out template molecule, reflux down at 84 ℃, the new mixing solutions of middle constantly adding is not till elutriant can detect template molecule.Put into the 250mL round-bottomed flask after room temperature is dried, add 150mL methyl alcohol, remove residual acetic acid at 70 ℃ of following stirring and refluxing 5h, oven dry obtains molecular blotting polymer microsphere behind the suction filtration, collects the microballoon of 45-75 μ m.

Embodiment 3, limit are advanced the synthetic method (precipitation polymerization method) of medium-Malathion molecular engram microsphere: comprise that limit advances the removal of template molecule in synthetic, the microballoon of medium-molecular engram microsphere and the hydrolysis of microsphere surface oxirane ring.

(1) limit is advanced the synthetic of medium-molecular engram microsphere: at embodiment 2(1) mixing solutions in add a kind of new monomer GMA(glytidyl methacrylate) (0.199mL, 6.0mmol), the ratio that is template molecule, MAA, GMA and EDMA is 1:6:6:30, adds initiator A IBN0.146g and 150mL chloroform solvent in the 250mL round-bottomed flask.Other are with embodiment 2(1).

The step that template molecule is removed is with embodiment 2(2).

Advance medium-non-molecular engram microsphere (not adding template molecule) with same ratio and method preparation limit.

(2) limit is advanced the hydrolysis of medium-molecular engram microsphere surface oxirane ring

Take by weighing 800mg and limit into medium-molecular engram microsphere or limit to advance medium-non-molecular engram microsphere in the 150mL round-bottomed flask, add the 50mL high chloro acid solution (1/9, V/V), flask is placed on the magnetic stirring apparatus, at room temperature with 150r min ^-1Tachyphylaxis 24h.After reaction finished, suction filtration constantly stirred microballoon in the ethanol of 200mL, acetone, ether successively, filters then, at room temperature dries, and it is stand-by that the microballoon for preparing is put into moisture eliminator.

Embodiment 4, the limit that suspends are advanced the synthetic method (suspension polymerization) of medium-molecular engram microsphere:

(1) takes by weighing the 1g polyvinyl alcohol and be dissolved in the 100mL hot water, put into the 250mL round-bottomed flask after the cooling, add Malathion (0.268mL, 1.0mmol), α-Jia Jibingxisuan (0.509mL, 6.0mmol), magnetic agitation 5h makes the abundant prepolymerization of template molecule and function monomer.Add GMA0.794mL(6.0mmol again), DVB5.606mL (40.0mmol), BPO65.77mg dissolve in small beaker, slowly is added drop-wise in the flask mechanical stirring reaction 5h(rotating speed 200r min under 80 ℃ of conditions then with dropping funnel ^-1).

(2) removal of polyvinyl alcohol

Polyreaction finishes, and after the microballoon drying, polyvinyl alcohol can be attached to microsphere surface sticks together microballoon.Microballoon is put into the pure water heated and stirred, polyvinyl alcohol is dissolved in the hot water, then pumping rate.Repeat this operation until removing polyvinyl alcohol fully.

The limit that suspends is advanced the hydrolysis step of the removal of template molecule in medium-molecular engram microsphere, surperficial oxirane ring with embodiment 3.

Carried out following experiment at embodiment 2,3,4:

1. absorption property test

The measuring method of microballoon absorption property: take by weighing the 20mg microballoon in centrifuge tube, add 4mL Malathion standardized solution, seal, concussion is left standstill 24h under the room temperature; Get supernatant liquor 1mL next day behind the centrifugal 3min of 4000r min-1, use N ₂Dry up, use the 1mL n-hexane dissolution, air feed phase chromatogram (GC-FPD) is measured.

The binding capacity calculation formula:

Q=(C ₁-C)*V ₁/m

Q(μ g mg ^-1)---polymkeric substance is to the adsorptive capacity of Malathion;

C ₁(μ g L ^-1)---the original concentration of Malathion;

C(μ g L ^-1)---the concentration of Malathion in the mother liquor of absorption back;

V ₁(L)---the volume of each standard horse traction sulphur phosphorus solution of getting;

M(mg)---the quality of the polymkeric substance of getting.

1.1 equilibrium adsorption test

Take by weighing ten parts of 20mg microballoons in centrifuge tube and number 1-10, add the 0-50 μ g mL that configures respectively ^-1Malathion standardized solution 4mL, 20h is left standstill in vibration.With 4000r min ^-1The centrifuging and taking supernatant liquor, N ₂After drying up, with 1mL n-hexane dissolution template molecule Malathion, the concentration of air inlet phase chromatographic determination Malathion.Calculate binding capacity Q (the mg g of microballoon according to Malathion change in concentration in the solution ^-1), replicate(determination) is averaged for 3 times.

1.2 the test of adsorption rate

Take by weighing ten parts of 20mg microballoons in centrifuge tube and number 1-10, add the 2 μ g mL that configure respectively ^-1Malathion standardized solution 4mL, vibration; Standing adsorption was surveyed one every one hour, the concentration of Malathion and calculate microballoon to binding capacity Q (the μ g g of Malathion in the calculated equilibrium adsorption liquid ^-1), replicate(determination) is averaged for 3 times.

1.3 selectivity experiment

Accurately take by weighing the 20mg microballoon in centrifuge tube, add mixing solutions 4mL(Malathion, ethoprophos, phorate, terbufos, Rogor and Nemacur concentration of standard solution and be respectively 5 μ g mL ^-1), seal, leave standstill 20h.Use gas chromatographic analysis, measure the concentration of six kinds of organophosphoruss in the equilibrium adsorption liquid and calculate microballoon to binding capacity Q (the μ g g of six kinds of organophosphoruss ^-1), replicate(determination) is averaged for 3 times.

1.4 the preparation of solid-phase extraction column

Take by weighing the 100mg microballoon in beaker, adding the 20mL methyl alcohol wet method diameter of packing into is 8mm, and length is in the solid phase extraction column of 8cm.The column extractor interior lower end is inserted the glass cullet cotton and is sealed, and polymer beads suspended when the upper end was plugged with an amount of glass wool equally with free of liquid feeding.

1.5 Solid-Phase Extraction condition

Earlier use 10mL methyl alcohol respectively, 2mL water activates column extractor, follows sample on the extracting sample solution, and drip speed is about 1mL min under the control solution ^-1, use the 2mL50% acetone solution drip washing removal of impurity then, at last with the pure methanol-eluted fractions target compound of 1mL and collect elutriant.The wash-out liquid nitrogen is blown the nearly back of doing add the 1mL normal hexane, gas chromatographic detection is advanced in concussion.

Used actual sample treatment process is:

Take by weighing 5g honey in centrifuge tube, adding 25mL redistilled water and regulator solution pH value is 6.0.Behind the ultrasonic oscillation 20min, add PSA in sample, concussion again is with 8000r min ^-1Centrifugal 5min.Got final product MISPE, C18SPE behind the sample mark-on of handling well, Florisil SPE carries out the mark-on recovery test.And the sample of crossing RAM-MISPE only need take by weighing 5g in centrifuge tube and add 25mL redistilled water, ultrasonic oscillation 20min.

Above-mentioned experimental result is as follows:

1. precipitation polymerization method prepares molecular engram microsphere and limits medium-molecular engram microsphere

1.1 the preparation of molecular engram microsphere

1.1.1 the selection of function monomer

Function monomer is the committed step thing in the macromolecular polymerization reaction, and its selection depends primarily on the interaction with template molecule.In reaction, function monomer want on the one hand can and template molecule between form and interact, want on the other hand and can and be in correct position with the linking agent polymerization, wherein obtain predetermined orientation and location thereby template molecule is inlayed just.Kind and the number of function monomer are a lot, and this experiment is selected five kinds of monomers, are respectively 4-vinylpridine, α-Jia Jibingxisuan, acrylamide, methyl methacrylate and vinylformic acid.

Take by weighing five parts of Malathion (0.067mL, 0.25mmol) in flask, 4-vinylpridine (the 4-VP that adds 1mmol respectively, 0.2157mL), α-Jia Jibingxisuan (MAA, 0.1696mL), acrylamide (AM, 142.16mg), methyl methacrylate (MMA, 0.2130mL) and vinylformic acid (AA 0.1304mL) as function monomer, adds 40 mL chloroform solvents, put into magneton prepolymerization 5h on magnetic stirring apparatus, (0.9403mL 5mmol) with initiator A IBN (20mg), adds the 120mL chloroform solvent at last to add linking agent EDMA then.Place 65 ℃ of constant temperature blender with magnetic force to react 12h round-bottomed flask, make polymer microballoon, suction filtration sieves, and behind the abundant wash-out of template molecule in the microballoon, measures it respectively to the adsorptive capacity of template molecule Malathion.Meanwhile, use with quadrat method and produce non-imprinted polymer (not adding template molecule).Adsorptivity test-results such as Fig. 2-1, as can be seen from the figure MAA be the trace microballoon of function monomer to the adsorptive capacity maximum of target analytes Malathion, simultaneously also obvious with the contrast of corresponding non-trace microballoon.So this experiment selects for use MAA as function monomer.

1.1.2 single factor experiment

1.1.2.1 the influence of stirring velocity

Stirring velocity is one of important factor that influences the microspherulite diameter size.Stir the shearing force energy dispersed monomer that produces, form small droplets.Stirring velocity is more fast, and then the shearing force of Chan Shenging is more big, and the monomer liquid pearl of formation is more little, and the particle diameter of the microballoon that finally obtains is more little.But too high stirring velocity also can make the monomer liquid pearl movement velocity of dispersion too fast, and the too high trend of its " secondary nucleation " growth that makes of probability that collision mutually takes place between particle strengthens, and the product size distribution is uneven and broaden.So, have only the polymer microballoon of selecting suitable stirring velocity just can prepare desirable particle size.

The massfraction that generates each particle diameter microballoon under the different stirring velocitys of table 2-1 is as follows:

Test-results shows that stirring velocity is 75r min ^-1The time, the microspheres product yield maximum in the 100-200 order particle size range is so the used stirring velocity of subsequent experimental is 75r min ^-1

1.1.2.2 Temperature Influence

Generally speaking, temperature is more low, and the recognition performance that forms polymkeric substance is more good, but temperature is crossed low so that when not reaching the heat decomposition temperature of initiator, polyreaction can not take place.And the temperature of polyreaction is more high, and initiation on the one hand, polymerization and nucleation rate are more fast, and polymer core concentration increases; Thereby medium increases raising polymkeric substance critical chain length to the solubleness of polymkeric substance on the other hand, and the polymer particle number descends.The viscosity reduction of polymeric system, atomic scatterring speed and probability of collision increase mutually, common result is the raising along with system temperature, the median size of polymer beads will increase to some extent.Therefore, temperature also is one of important factor that influences the microspherulite diameter size.

Generate the massfraction of each particle diameter microballoon under the table 2-2 differing temps

Test-results shows that when temperature was 70 ℃, the microspheres product yield maximum in the 100-200 order particle size range was so the used temperature of subsequent experimental is 70 ℃.

1.1.3 orthogonal test

The orthogonal test that designs 3 factors, 3 levels further is optimized the polymerizing condition of the molecular engram microsphere of Malathion.

Table 2-3 3 factors 3 levels

Wherein, factor A is the ratio (mol ratio) of template molecule and function monomer; Factor B is the ratio (mol ratio) of template molecule and linking agent; Factor C is initiator amount.

The orthogonal design table of table 2-4 molecular engram microsphere

By comparing Q _InhaleThe size of value is tested 5 Q that obtain as can be known _InhaleValue is maximum, so the optimum polymerizating condition of polyreaction is A ₂BB ₂C ₃, namely the mol ratio of template molecule and function monomer is 1:6, and the mol ratio of template molecule and linking agent is 1:30, and initiator amount is 2.0%.Contrast each factor extreme difference size as can be known, R _A＞R _C＞R _B, namely the ratio of template molecule and function monomer is the significant factors that influences the microballoon absorption property.

According to the variance analysis data, its result is consistent with the range analysis in the orthogonal test, and the significant factors that influences the microballoon absorption property is still the ratio of template and monomer.

Table 2-5 orthogonal experiment analysis of variance table

* remarkable

1.2 limit is advanced the preparation of medium-molecular engram microsphere

In synthetic process, limit is advanced medium-molecular engram microsphere and has been added a kind of new monomer GMA on the basis of original monomer M AA.The energy open loop under the hydrolysis of perchloric acid of oxirane ring among the GMA forms hydrophilic hydroxyl and is distributed in microsphere surface, the hydrophilic outside surface of this one deck has guaranteed that biomacromolecule irreversible sex change and absorption can not take place at the outside surface of sorbent material, such sorbent material can produce not keep macromole like this, and macromolecular substance is at dead volume or be bordering under the situation of dead volume and removed by wash-out; The determinand small molecules then keeps by hydrophobic interaction and electrostatic interaction.The structural characteristics of this kind just can guarantee that this new adsorbent can be got rid of interference under the situation of the existence of biomacromolecule, realize the highly selective extraction to the small molecules analyte.GMA should add after template and the abundant prepolymerization of monomer again, can avoid the template of GMA and monomer to interact like this and form the influence of hydrogen bond.

The amount that adds GMA is the key that synthetic limit is advanced medium-molecular engram microsphere.Test is optimized the ratio of GMA and MAA under the optimum synthesis condition of molecular engram microsphere.GMA:MAA is that adsorptive capacity that 1:2,1:1,2:1 make microballoon is respectively 11.6,14.3 and 13.1mg g ^-1Test-results shows, the adsorptive capacity maximum when GMA:MAA is 1:1.

Therefore, limitting the best synthesizing formula of medium-molecular engram microsphere is template molecule: function monomer (MAA): new monomer (GMA): linking agent (EDMA)=1:6:6:30, amount of initiator are 2.0%, and temperature is 70 ℃, and rotating speed is 200r min ^-1, chloroform is pore-creating agent.Under this reaction conditions, the into adsorptive capacity maximum of medium-molecular engram microsphere is limit in the Malathion of gained.

1.3 molecular engram microsphere and limit are advanced medium-molecular engram microsphere electron-microscope scanning figure

By scanning electron microscope molecular engram microsphere and limit being advanced medium-molecular engram microsphere form characterizes.By Fig. 2-2 as can be known, it is good and size is more even to observe the microballoon form after amplifying 100 times clearly, can guarantee that to a certain extent microballoon has good adsorptivity.

1.4 absorption test

1.4.1 absorption property test

Measured molecular engram microsphere, non-molecular engram microsphere, limit medium-molecular engram microsphere and limit to advance the adsorptive capacity of medium-non-molecular engram microsphere in different concns Malathion solution, namely adsorption isothermal line is seen Fig. 2-3.The concentration of Malathion solution from 0 to 50 μ g mL wherein ^-1In the comparison diagram curve as can be known, along with the increase of Malathion strength of solution, the adsorptive capacity that molecular engram microsphere and limit are advanced medium-molecular engram microsphere also significantly increases thereupon, finally reaches adsorption equilibrium.Find out molecular engram microsphere all rightly and limit medium-molecular engram microsphere that the adsorptive capacity of Malathion is respectively 14.41mg g by Fig. 2-3 ^-1With 13.43mg g ^-1, obvious adsorptive capacity greater than each self-corresponding non-trace microballoon.The result shows and advances in molecular engram microsphere and limit that exist in medium-molecular engram microsphere can be to the active binding site of template molecule Malathion specific combination.This explanation, template molecule has stayed the trace hole in the process of microballoon preparation, and the active binding site in these holes has determined high affinity and the specific recognition that microballoon demonstrates when adsorbing template molecule again just.

The trace factor (α) is to judge an index of imprinted polymer specific recognition.α is the ratio (α of imprinted polymer and non-imprinted polymer ₁=Q _MIMs/ Q _NIMs, α ₂=Q _RAM-MIMs/ Q _RAM-NIMs).By calculating α ₁=3.32, α ₂=3.31.Two numbers are so approaching, and this explanation is advanced the specific recognition that the new monomer that adds in medium-molecular engram microsphere building-up process does not reduce microballoon in limit.Therefore, limit medium-molecular engram microsphere and molecular engram microsphere that template molecule is had same avidity and specific recognition.

1.4.2 adsorption rate test

Measure the adsorption rate of microballoon, namely changed the adsorptive capacity of microballoon in time, as Fig. 2-4.As seen from the figure, increase in time, the adsorptive capacity of microballoon also increases gradually, and 0-6h adsorbs and almost is the linear trend increase, and molecular engram microsphere and limit are advanced medium-molecular engram microsphere after 6h does not all have variation to tend to balance to the adsorptive capacity of Malathion substantially.Experimental result explanation, the adsorption rate of microballoon is very fast, can reach adsorption equilibrium very soon.

1.4.3 selective adsorption test

Measure molecular engram microsphere, non-molecular engram microsphere, limit medium-molecular engram microsphere and limit to advance the solid-phase extraction column of medium-non-molecular engram microsphere to the adsorption selectivity of Malathion, ethoprophos, phorate, terbufos, Rogor and Nemacur.

Microballoon the results are shown in Figure the selective adsorption of six kinds of organophosphoruss.By Fig. 2-5 as can be known, molecular engram microsphere and limit are advanced medium-molecular engram microsphere to the adsorptive capacity maximum of template molecule to the template molecule Malathion, reach 468.15 and 448.5 μ g g respectively ^-1, and ethoprophos, phorate, terbufos, Rogor and Nemacur had certain absorption and adsorptive capacity obviously greater than the adsorptive capacity of each self-corresponding non-trace microballoon.This shows in molecular engram microsphere and to have the functional group that contains in the hole that is complementary with the specific three-dimensional arrangement of Malathion molecule and these holes with the Malathion interaction of molecules, so the trace microballoon has specific selectivity to template.And the hole that in non-trace microballoon, not is not complementary with the template molecule architecture, functional group also is to distribute arbitrarily, therefore non-imprinted polymer does not have specific selectivity to template molecule.The trace microballoon also has certain absorption to other five kinds of organophosphorus molecules, this is because the molecular structural formula of organophosphorus has similarity, all contain and to form interactional functional group with function monomer, this explanation trace microballoon can all show the selectivity of special efficacy to this organophosphorus, actual sample is detected have positive effect.

1.5 limit is advanced medium-molecular engram microsphere Solid-Phase Extraction condition optimizing

The Solid-Phase Extraction step comprises activation, goes up sample, drip washing and wash-out.Wherein drip washing and wash-out are the two important steps, and extraction results is had direct influence.

1.5.1 the selection of drip washing solvent and eluting solvent

The purpose of drip washing and elution step is respectively will obtain the minimum and maximum rate of recovery to target analytes.

After the limit for preparing advanced the activation of medium-molecular engram microsphere solid-phase extraction column, get 200 μ g L ^-1Malathion standardized solution 20mL go up sample, adopt methyl alcohol, ethanol, acetonitrile and the aqueous acetone solution of different content as eluent solution and elute soln (organic solvent per-cent is 0%-100%) respectively, collect elutriant and analyze, the results are shown in Figure 2-6.Experimental result shows that 50% aqueous acetone solution can guarantee that drip washing goes out more matrix under the situation that does not wash out target analytes, thereby reaches drip washing effect preferably, so select 50% aqueous acetone solution as the drip washing solvent; 100% pure methanol-eluted fractions effect is best, wash-out target analytes fully almost, and the rate of recovery is 99.84%, so select 100% pure methyl alcohol as eluting solvent.

1.5.2 the selection of drip washing solvent and eluting solvent consumption

The limit for preparing is advanced medium-molecular engram microsphere solid-phase extraction column Activate with 10mL methyl alcohol successively,2mL water After the activation, get 200 μ g L ^-1Malathion standardized solution 20mL go up sample, respectively with 0.5,1.0,1.5,2.0 and 50% acetone solution and the methyl alcohol of 2.5mL carry out drip washing and wash-out.The results are shown in Table 2-6.According to experimental result, selecting 2.0mL is the eluent volume, and 1.0mL is the eluent volume.

The rate of recovery of table 2-6 different volumes drip washing and wash-out target analytes

1.6 the linearity range of method and detection limit

Dispose standardized solution (the 0.01-10 μ g mL of a series of organophosphoruss ^-1), use gas Chromatographic Determination, the results are shown in Table 2-7.

Linearity range and the detectability of table 2-7 method

1.7 the rate of recovery of method and precision

Utilize the limit of setting up to advance medium-molecular engram microsphere Solid-Phase Extraction method honey sample is carried out the mark-on recovery test, mark-on concentration is respectively 0.10 μ g mL ^-1With 0.20 μ g mL ^-1The result is as table 2-8.

The table 2-8 rate of recovery and Precision test result (n=6)

1.8 limit is advanced medium-molecular engram microsphere Solid-Phase Extraction, molecular engram microsphere Solid-Phase Extraction and the contrast of commodity post

With self-control RAM-MISPE, MISPE and commercialization solid-phase extraction column (C ₁₈And Florisil) compares, the results are shown in Figure 2-7.As can be seen, the peak value of each organophosphorus that RAM-MISPE and MISPE mark-on obtain after reclaiming all is higher than C from color atlas ₁₈And Florisil, illustrate that RAM-MISPE and MISPE obviously are better than C to enrichment, the recovering effect of six kinds of organophosphoruss ₁₈With the Florisil solid-phase extraction column.Honey sample needs to have except albumen, except this step of sugar before crossing MISPE, C18 and Florisil solid-phase extraction column, and does not need sample is handled before crossing RAM-MISPE.So RAM-MISPE be a kind of simple, fast, pre-treating process that selectivity is high.

The different column extractors of table 2-9 are to recovery of standard addition (n=6, the mark-on level: 0.20 μ g mL of six kinds of organophosphoruss ^-1)

1.9 actual sample is measured

Honey sample is purchased in local market or market.The limit of setting up is advanced medium-molecular engram microsphere Solid-Phase Extraction method be applied to the actual detected of organophosphorus in the honey sample.The concentration that detects Ethoprophos and Malathion in the honey sample 1 is 15.63 μ gL ^-1With 16.22 μ g L ^-1The concentration of detected Ethoprophos and Terbufos is 12.81 μ g L in the honey sample 3 ^-1With 10.74 μ g L ^-1

Detected organophosphorus concentration (μ g L in the table 2-10 honey sample ^-1)

Nd: do not detect

2. the selection of suspension polymerization condition

2.1 the selection of rotating speed

Rotating speed is that control microspherulite diameter scope is key condition.Stirring velocity is more fast, and then the shearing force of Chan Shenging is more big, and the monomer liquid pearl of formation is more little, and the final polyalcohol microspherulite diameter that forms is more little.In the test rotating speed of reaction is selected, the product that obtains under the different rotating speeds such as Fig. 2-8.The result shows, at 200r min ^-1The microspherulite diameter that makes under the condition is minimum and even.

2.2 the selection of linking agent

Get the polyvinyl alcohol water solution of three parts of 100mL1% with in three 250mL round-bottomed flasks, get Malathion 0.268mL (1.0mmol), MAA0.509mL (6.0mmol), GMA0.794mL(6.0mmol respectively) in three small beakers and add DVB5.606mL (40.0mmol), EDMA7.556mL (40.0mmol), three kinds of linking agents of TDMA12.506mL (40.0mmol) and 1%BPO respectively, slowly be added drop-wise in the flask mechanical stirring reaction 5h(rotating speed 200r min under 80 ℃ of conditions then with dropping funnel ^-1).Polymer microballoon is put into the water stirring heating make polyvinyl alcohol be dissolved in hot water, then pumping rate.Repetitive operation is until removing polyvinyl alcohol fully.Sieve, with the abundant wash-out of template molecule in the polymkeric substance, use 10% high chloro acid solution's hydrolysis again, obtain the suspension polymerization product of three kinds of linking agents at last respectively.

2.2.1 the proterties of three kinds of product suspension

Table 2-11 suspension polymerization product characters

2.2.2 the electron-microscope scanning figure of three kinds of product suspension

Study the morphological structure of microballoon by scanning electron microscope.Three kinds of limits of preparation are advanced medium-trace microballoon amplify 200 times of observations with scanning electron microscope.Electronic Speculum figure such as Fig. 2-9:

By Fig. 2-9 (c) as seen, do the trace microsphere surface smoother that linking agent obtains with DVB, sphericity is best.

2.3 the adsorptive capacity of three kinds of polymerisates

To make polymkeric substance, sieve, behind the abundant wash-out of template molecule in the polymkeric substance, measure it respectively to the relative adsorptive capacity of Malathion molecule.

Being three kinds of polymerisates making of linking agent with EDMA, TDMA and DVB is respectively 8.24mg g to the adsorptive capacity of Malathion ^-1, 6.75mg g ^-1, 11.61mg g ^-1, visible DVB is best as the absorption property of the trace microballoon that linking agent obtains, and is that linking agent is further optimized so test will be adopted DVB.

2.3 orthogonal test

For the further polymerizing condition to the molecular engram microsphere of Malathion is optimized, design the orthogonal test of 4 factors, 3 levels.

Table 2-12 4 factors 3 levels

Wherein, factor A is the ratio (mol ratio) of template molecule and function monomer;

Factor B is the ratio (mol ratio) of template molecule and new function monomer;

Factor C is the ratio (mol ratio) of template molecule and linking agent;

Factor D is initiator amount (accounting for the percentage ratio of function monomer, new function monomer and linking agent quality summation).

The synthetic orthogonal design table of table 2-13 Malathion imprinted polymer

By comparing Q _InhaleThe size of value is tested 5 Q that obtain as can be known _InhaleValue is maximum, so the optimum polymerizating condition of polyreaction is A ₂BB ₂C ₃D ₁, namely the mol ratio of template molecule and function monomer is 1:6, and template molecule is 1:6 with the mol ratio of new function monomer, and the mol ratio of template molecule and linking agent is 1:40, and initiator amount is 1.0%.Contrast the extreme difference size of four factors, R _A＞R _C＞R _B＞R _D, namely the ratio of template molecule and function monomer is the significant factors that influences the microballoon absorption property.

Analyze with variance analysis method, the result is consistent with range analysis again, and the significant factors of impact polymer absorption property is still the ratio of template molecule and function monomer.

Table 2-14 orthogonal experiment analysis of variance table

* remarkable

2.4 absorption test

2.4.1 the research of absorption property

Saturated extent of adsorption is important parameters of quality of weighing sorbent material.Limit of determination advances medium-molecular engram microsphere at different concns (0-60 μ g mL ^-1) the adsorptive capacity of Malathion solution, and curve plotting, i.e. adsorption isothermal line.Limit is advanced medium-molecular engram microsphere and is limit medium-non-molecular engram microsphere that the adsorption isothermal line of Malathion molecule is seen Fig. 2-10 respectively.

Compare two curves as can be known, along with the increase of Malathion concentration, the adsorptive capacity of unit mass microballoon also increases thereupon, finally reaches the state of tending to balance.Can see significantly that it is 13.65mgg to the adsorptive capacity of template molecule that rising limit is advanced medium-molecular engram microsphere ^-1, it advances the adsorptive capacity 3.85mg g of medium-non-molecular engram microsphere much larger than limit ^-1Presentation of results advances medium-molecular engram microsphere building-up process in limit, template molecule has stayed the trace hole in polymer microballoon and there is active binding site in the hole, just because of the existence that these active binding sites are arranged, limit is advanced medium-molecular engram microsphere template molecule is just had high affinity and specific recognition.

2.4.2 the research of adsorption rate

Measured and limit medium-molecular engram microsphere to the adsorptive capacity of template molecule in the different time, namely adsorption rate the results are shown in Figure 2-11.As seen from the figure, in the 0-5h scope, the adsorptive capacity of microballoon in time increase and increase, and increase comparatively fast, adsorptive capacity does not almost change after the 5h, reaches balance substantially.This may be that the absorption initial stage is adsorbed as main with shallow cave and microsphere easily enters because the hole of microballoon has and has shallowly deeply, so that adsorptive capacity increases is fast; After the absorption of shallow cave reaches capacity gradually, mainly be the absorption of dark cave, and template molecule enter dark cave and can be subjected to sterically hindered influence, so adsorb the slack-off adsorption equilibrium that finally reaches.

2.5 limit is advanced the performance test of medium-molecularly imprinted solid phase extraction column

2.5.1 limit is advanced medium-molecularly imprinted polymer selectivity test

Having measured limit advances medium-molecular engram microsphere and limits the solid-phase extraction column of medium-non-molecular engram microsphere to the adsorption selectivity of Malathion, ethoprophos, phorate, terbufos, Rogor and Nemacur.

After the solid-phase extraction column activation for preparing, (Malathion, ethoprophos, phorate, terbufos, Rogor and Nemacur concentration of standard solution are respectively 5 μ g mL with mixing solutions ^-1) the last sample of 4mL, drip washing and wash-out then.Collect elutriant and carry out analytical results such as Fig. 2-12.

As seen from the figure, limit is advanced medium-molecular engram microsphere to the adsorptive capacity maximum of template molecule Malathion, reaches 424.11 μ gg ^-1With the time limit advance medium-non-molecular engram microsphere to the adsorptive capacity of six kinds of organophosphoruss all less than limitting into medium-molecular engram microsphere, and the adsorptive capacity gap is little, explanation template molecule in polymerization process has been brought into play the trace effect, there are difference on the tangible space structure in trace microballoon and non-trace microballoon, thereby present the difference of specific adsorption and specific adsorption.So advancing medium-molecular engram microsphere, synthetic limit can be used for effectively identification Malathion and analog thereof.

2.5.2 recovery of standard addition

The limit of utilize setting up is advanced medium-molecular engram microsphere Solid-Phase Extraction method, and honey sample is carried out mark-on concentration is 0.10 μ gmL ^-1, 0.20 μ g mL ^-1The mark-on recovery test.The result is as table 2-15.

The table 2-15 rate of recovery and Precision test result (n=6)

Limit is advanced medium-molecular engram microsphere Solid-Phase Extraction (RAM-MISPE) and the contrast of commodity post 2.6 suspend

Compare with RAM-MISPE and commercialization solid-phase extraction column (C18 and Florisil).The results are shown in Table 2-16.Display random access memory-MISPE1, RAM-MISPE2 are more or less the same to enrichment, the recovering effect of six kinds of organophosphoruss and obviously are better than with C18 and Florisil solid-phase extraction column as a result.And owing to RAM-MISPE does not need to remove complicated pre-treatments such as albumen to containing macromolecular sample, so RAM-MISPE is a kind of simple, the pre-treating process that selectivity is high.

The different column extractors of table 2-16 are to recovery of standard addition (n=6, the mark-on level: 0.20 μ g mL of six kinds of organophosphoruss ^-1).

3.2.7 actual sample analysis

Honey sample is purchased in local market or market.The limit of setting up is advanced medium-molecular engram microsphere Solid-Phase Extraction method be applied to the actual detected of organophosphorus in the honey sample.The concentration that detects Ethoprophos and Malathion in the honey sample 1 is respectively 14.98 μ g L ^-1With 16.01 μ g L ^-1The concentration of detected Ethoprophos and Terbufos is 12.33 μ g L in the honey sample 3 ^-1With 10.25 μ g L ^-1

Detected organophosphorus concentration (μ g L in the table 2-17 honey sample ^-1)

Nd: do not detect

3.3.3 the precipitation limit is advanced medium-molecular engram microsphere solid-phase extraction column (RAM-MISPE1) and is advanced medium-molecular engram microsphere solid-phase extraction column (RAM-MISPE2) contrast with the limit that suspends

RAM-MISPE1 and RAM-MISPE2 are compared.(Fig. 2-13) can find out by color atlas, the peak value basically identical of six kinds of organophosphoruss of extraction of two kinds of self-control solid-phase extraction columns, and namely extraction efficiency is close, and the rate of recovery sees Table 2-18.Presentation of results self-control limit is advanced medium-molecular engram microsphere solid-phase extraction column recovery of extraction height, favorable reproducibility, before crossing post, do not need to carry out complicated pre-treatment to containing the macromole sample, as except operation such as albumen, separation and enrichment organophosphorus target analytes that just can highly selective be a kind of pre-treating processs efficiently.

Two kinds of self-controls of table 2-18 solid-phase extraction column is to recovery of standard addition (n=6, the mark-on level: 0.20 μ gmL of six kinds of organophosphoruss ^-1).

Laboratory apparatus and condition:

Agilent 7890 gas-chromatographies, detector are FPD (GC-FPD); HP-5 chromatographic column (30m * 0.25mm, 1.4 μ m, Agilent Technologies, the U.S.); Electronic scanning Electronic Speculum (JSM-6490LV, Japan) HHS-11-4 thermostat water bath (Hangzhou remittance that Instr Ltd.).

GC conditions: injector temperature: 250 ℃; Nitrogen flow rate: 3.0mLmin ^-1The chromatographic column heating schedule: initial temperature is 150 ℃, with 10 ℃ of min ^-1Rise to 240 ℃, keep 11min, total run time is 20min; Adopt no shunt mode; FPD detector temperature: 250 ℃; Sample size: 1 μ L.

Claims (8)

1. the synthetic method of Malathion molecularly imprinted polymer, it is characterized in that, template molecule in molar ratio: function monomer: linking agent=1:2～15:20～60, delivery plate molecule Malathion, function monomer α-Jia Jibingxisuan and linking agent ethylene glycol dimethacrylate join in the test tube, add initiator, solvent again, concussion, logical nitrogen, vibration makes it abundant mixing, and sealing is reacted 24h in 70 ℃ water bath with thermostatic control; Through grinding, sieving, the Malathion Molecularly Imprinted Polymer of collection cut size 75-150 μ m, wrap with filter paper, earlier with methyl alcohol/glacial acetic acid solution wash-out template molecule in apparatus,Soxhlet's, using pure methyl alcohol behind the wash-out 50h again instead is eluted to no template molecule and detects, dry, get white Malathion molecularly imprinted polymer.

2. the synthetic method of Malathion as claimed in claim 1 molecularly imprinted polymer, it is characterized in that, accurately measure template molecule Malathion 0.067mL, function monomer α-Jia Jibingxisuan 0.170mL, linking agent ethylene glycol dimethacrylate 1.889mL joins in the test tube, add initiator Diisopropyl azodicarboxylate 24.24mg, solvent chloroform 5mL again, concussion, the logical nitrogen 5min 10min that vibrates again makes it abundant mixing, and sealing is reacted 24h in 70 ℃ thermostat water bath.Through grinding, after sieving, the Malathion Molecularly Imprinted Polymer of collection cut size 75-150 μ m, wrap with filter paper, earlier use methyl alcohol: glacial acetic acid solution is the wash-out template molecule in apparatus,Soxhlet's, use pure methyl alcohol behind the wash-out 50h again instead and be eluted to no template molecule and detect, dry, get white Malathion molecularly imprinted polymer.

3. the synthetic method of Malathion molecular blotting polymer microsphere, it is characterized in that, template molecule in molar ratio: function monomer: linking agent=1:2～15:20～60, delivery plate molecule Malathion, function monomer α-Jia Jibingxisuan and linking agent ethylene glycol dimethacrylate, template molecule Malathion and function monomer α-Jia Jibingxisuan are added round-bottomed flask, add the part chloroform solvent, prepolymerization 1～3h on magnetic stirring apparatus, add linking agent ethylene glycol dimethacrylate and Diisopropyl azodicarboxylate then, add 3 times of chloroform solvents of dosage first again, constant temperature magnetic agitation reaction 10～40h gets the Malathion molecular blotting polymer microsphere;

Get 1g Malathion molecular blotting polymer microsphere, wrap to put into filter paper and place Soxhlet extractor, add 100～360mL methyl alcohol: the acetic acid volume ratio is the mixing solutions backflow wash-out template molecule of 9:1, the new mixing solutions of middle constantly adding, do not detect to there being template molecule to elutriant, put into round-bottomed flask after drying, add 100～360mL methyl alcohol, stirring and refluxing 3～8h removes residual acetic acid, oven dry obtains molecular blotting polymer microsphere behind the suction filtration, collect the microballoon of 45-75 μ m, be the Malathion molecular blotting polymer microsphere.

4. the synthetic method of Malathion as claimed in claim 3 molecular blotting polymer microsphere, it is characterized in that, delivery plate molecule Malathion 0.268mL and function monomer α-Jia Jibingxisuan 0.509mL to 250mL round-bottomed flask, add the 40mL chloroform solvent, put into magneton prepolymerization 5h on magnetic stirring apparatus, add linking agent ethylene glycol dimethacrylate 5.642mL and initiator Diisopropyl azodicarboxylate 0.096g then, add the 120mL chloroform solvent at last, the round-bottomed flask that this mixing solutions is housed is put into constant temperature blender with magnetic force, at 70 ℃ and 150r min ^-1Reaction 10h gets the Malathion molecular blotting polymer microsphere;

Take by weighing the synthetic good molecular blotting polymer microsphere of about 1g, it is wrapped to put into filter paper place Soxhlet extractor, add 150mL methyl alcohol: the acetic acid volume ratio is the mixing solutions wash-out template molecule of 9:1, reflux down at 84 ℃, the new mixing solutions of middle constantly adding, do not detect to there being template molecule, after drying, room temperature puts into the 250mL round-bottomed flask, add 150mL methyl alcohol, remove residual acetic acid at 70 ℃ of following stirring and refluxing 5h, dry behind the suction filtration, collected the microballoon of 45-75 μ m, be the Malathion molecular blotting polymer microsphere.

5. the into synthetic method of medium-molecular blotting polymer microsphere is limit in the Malathion, it is characterized in that, template molecule in molar ratio: function monomer 1: function monomer 2: linking agent=1:2～20:2～20:20～90, delivery plate molecule Malathion, function monomer 1 α-Jia Jibingxisuan, function monomer 2 glytidyl methacrylate and linking agent ethylene glycol dimethacrylate, with template molecule Malathion and function monomer 1 α-Jia Jibingxisuan and function monomer 2 glytidyl methacrylate, the linking agent ethylene glycol dimethacrylate adds round-bottomed flask, add the initiator chloroform solvent again, constant temperature magnetic agitation reaction 6～30h gets the Malathion and limits into medium-molecular blotting polymer microsphere;

Get and limit the high chloro acid solution of medium-molecular engram microsphere and volume ratio 1:9 to add in the round-bottomed flask, room temperature magnetic agitation reaction 6～30h, suction filtration, thus obtained microsphere constantly stirs in ethanol, acetone, ether successively, filter then, dry under the room temperature, get the Malathion and limit into medium molecule imprinted polymer microballoon.

6. limit the into synthetic method of medium-molecular blotting polymer microsphere as Malathion as described in the claim 5, it is characterized in that, delivery plate molecule Malathion 0.268mL, function monomer 1 α-Jia Jibingxisuan 0.509mL, function monomer 2 glytidyl methacrylate 0.199mL, linking agent ethylene glycol dimethacrylate 5.642mL to 250mL round-bottomed flask, add initiator Diisopropyl azodicarboxylate 0.096g and 150mL chloroform solvent again, in the constant temperature blender with magnetic force at 70 ℃ and 150r min ^-1Reaction 10h gets the Malathion and limits into medium-molecular blotting polymer microsphere;

Take by weighing 0.8g and limit into medium-molecular engram microsphere in the 150mL round-bottomed flask, add the high chloro acid solution of 50mL volume ratio 1:9, under the room temperature with 150r min ^-1Rotating speed magnetic agitation reaction 24h, suction filtration, thus obtained microsphere constantly stir in the ethanol of 200mL, acetone, ether successively, filter again, room temperature is dried, and gets the Malathion and limits into medium-molecular blotting polymer microsphere.

7. the limit that suspends is advanced the synthetic method of medium-molecular engram microsphere, it is characterized in that, in the polyvinyl alcohol water solution that fills weightmeasurement ratio 1～3%, add the template molecule Malathion, the function monomer α-Jia Jibingxisuan, magnetic agitation 5h prepolymerization forms prepolymerization liquid, other gets glytidyl methacrylate, Vinylstyrene, benzoyl peroxide forms miscible fluid, miscible fluid is slowly dropped in the prepolymerization liquid, 70～90 ℃ of mechanical stirring reaction 3～15h, get microballoon, template molecule Malathion in molar ratio: function monomer α-Jia Jibingxisuan: glytidyl methacrylate: Vinylstyrene=1:2～20:2～20:20～90;

The microballoon of drying is put into the pure water heated and stirred remove polyvinyl alcohol, the limit that must suspend is advanced medium-Molecularly Imprinted Polymer microballoon;

Getting suspends limits the high chloro acid solution of medium-Molecularly Imprinted Polymer microballoon and volume ratio 1:9 to add in the round-bottomed flask, room temperature magnetic agitation reaction 6～30h, suction filtration, thus obtained microsphere constantly stirs in ethanol, acetone, ether successively, filter then, dry under the room temperature, get the suspension Malathion and limit into medium-molecular blotting polymer microsphere.

8. limit the into synthetic method of medium-molecular engram microsphere as suspension as described in the claim 7, it is characterized in that,

Taking by weighing the 1g polyvinyl alcohol is dissolved in the 100mL hot water, put into the 250mL round-bottomed flask after the cooling, add Malathion 0.268mL, α-Jia Jibingxisuan 0.509mL, magnetic agitation 5h, make the abundant prepolymerization of template molecule and function monomer form prepolymerization liquid, glytidyl methacrylate 0.794mL, Vinylstyrene 5.606mL, the miscible formation miscible fluid of benzoyl peroxide 65.77mg, slowly miscible fluid is added drop-wise in the prepolymerization liquid 80 ℃, 200r min with dropping funnel ^-1Mechanical stirring reaction 5h gets microballoon;

After the microballoon drying, put into the pure water heated and stirred, polyvinyl alcohol is dissolved in the hot water, suction filtration then, repetitive operation must suspend and limit medium-Molecularly Imprinted Polymer microballoon to removing polyvinyl alcohol fully;

Take by weighing the 0.8mg limit that suspends and advance medium-Molecularly Imprinted Polymer microballoon in the 150mL round-bottomed flask, add the high chloro acid solution of 50mL volume ratio 1/9, under the room temperature with 150r min ^-1Rotating speed magnetic agitation reaction 24h, suction filtration, thus obtained microsphere constantly stir in the ethanol of 200mL, acetone, ether successively, filter again, room temperature is dried, and gets the suspension Malathion and limits into medium-molecular blotting polymer microsphere.

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