CN114749161A - Molecular imprinting microsphere of dioscin, solid-phase extraction device and preparation method - Google Patents
Molecular imprinting microsphere of dioscin, solid-phase extraction device and preparation method Download PDFInfo
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- CN114749161A CN114749161A CN202210482281.3A CN202210482281A CN114749161A CN 114749161 A CN114749161 A CN 114749161A CN 202210482281 A CN202210482281 A CN 202210482281A CN 114749161 A CN114749161 A CN 114749161A
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- dioscin
- molecularly imprinted
- phase extraction
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- diosgenin
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- HDXIQHTUNGFJIC-UHFFFAOYSA-N (25R)-spirost-5-en-3beta-ol 3-O-<O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranoside> Natural products O1C2(OCC(C)CC2)C(C)C(C2(CCC3C4(C)CC5)C)C1CC2C3CC=C4CC5OC1OC(CO)C(O)C(O)C1OC1OC(C)C(O)C(O)C1O HDXIQHTUNGFJIC-UHFFFAOYSA-N 0.000 title claims abstract description 103
- VNONINPVFQTJOC-RXEYMUOJSA-N Collettiside III Natural products O([C@@H]1[C@@H](O)[C@H](O[C@H]2[C@H](O)[C@H](O)[C@@H](O)[C@H](C)O2)[C@H](CO)O[C@@H]1O[C@@H]1CC=2[C@@](C)([C@@H]3[C@H]([C@H]4[C@@](C)([C@H]5[C@H](C)[C@@]6(O[C@H]5C4)OC[C@H](C)CC6)CC3)CC=2)CC1)[C@H]1[C@H](O)[C@H](O)[C@@H](O)[C@H](C)O1 VNONINPVFQTJOC-RXEYMUOJSA-N 0.000 title claims abstract description 103
- VNONINPVFQTJOC-ZGXDEBHDSA-N dioscin Chemical compound O([C@@H]1[C@@H](CO)O[C@H]([C@@H]([C@H]1O)O[C@H]1[C@@H]([C@H](O)[C@@H](O)[C@H](C)O1)O)O[C@@H]1CC2=CC[C@H]3[C@@H]4C[C@H]5[C@@H]([C@]4(CC[C@@H]3[C@@]2(C)CC1)C)[C@@H]([C@]1(OC[C@H](C)CC1)O5)C)[C@@H]1O[C@@H](C)[C@H](O)[C@@H](O)[C@H]1O VNONINPVFQTJOC-ZGXDEBHDSA-N 0.000 title claims abstract description 103
- CJNUQCDDINHHHD-APRUHSSNSA-N dioscin Natural products C[C@@H]1CC[C@@]2(OC1)O[C@H]3C[C@H]4[C@@H]5CC=C6C[C@H](CC[C@@H]6[C@H]5CC[C@]4(C)[C@H]3[C@@H]2C)O[C@@H]7O[C@H](CO)[C@@H](O[C@@H]8O[C@@H](C)[C@H](O)[C@@H](O)[C@H]8O)[C@H](O)[C@H]7O[C@@H]9O[C@@H](C)[C@H](O)[C@@H](O)[C@H]9O CJNUQCDDINHHHD-APRUHSSNSA-N 0.000 title claims abstract description 103
- VNONINPVFQTJOC-UHFFFAOYSA-N polyphyllin III Natural products O1C2(OCC(C)CC2)C(C)C(C2(CCC3C4(C)CC5)C)C1CC2C3CC=C4CC5OC(C(C1O)OC2C(C(O)C(O)C(C)O2)O)OC(CO)C1OC1OC(C)C(O)C(O)C1O VNONINPVFQTJOC-UHFFFAOYSA-N 0.000 title claims abstract description 103
- 239000004005 microsphere Substances 0.000 title claims abstract description 83
- 238000002414 normal-phase solid-phase extraction Methods 0.000 title claims abstract description 60
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 153
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 76
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 49
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 45
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 claims abstract description 32
- DWCSNWXARWMZTG-UHFFFAOYSA-N Trigonegenin A Natural products CC1C(C2(CCC3C4(C)CCC(O)C=C4CCC3C2C2)C)C2OC11CCC(C)CO1 DWCSNWXARWMZTG-UHFFFAOYSA-N 0.000 claims abstract description 31
- WQLVFSAGQJTQCK-VKROHFNGSA-N diosgenin Chemical compound O([C@@H]1[C@@H]([C@]2(CC[C@@H]3[C@@]4(C)CC[C@H](O)CC4=CC[C@H]3[C@@H]2C1)C)[C@@H]1C)[C@]11CC[C@@H](C)CO1 WQLVFSAGQJTQCK-VKROHFNGSA-N 0.000 claims abstract description 31
- WQLVFSAGQJTQCK-UHFFFAOYSA-N diosgenin Natural products CC1C(C2(CCC3C4(C)CCC(O)CC4=CCC3C2C2)C)C2OC11CCC(C)CO1 WQLVFSAGQJTQCK-UHFFFAOYSA-N 0.000 claims abstract description 31
- 238000005406 washing Methods 0.000 claims abstract description 26
- DBCAQXHNJOFNGC-UHFFFAOYSA-N 4-bromo-1,1,1-trifluorobutane Chemical compound FC(F)(F)CCCBr DBCAQXHNJOFNGC-UHFFFAOYSA-N 0.000 claims abstract description 21
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 21
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims abstract description 21
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Substances CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 claims abstract description 21
- 235000012239 silicon dioxide Nutrition 0.000 claims abstract description 19
- 239000002245 particle Substances 0.000 claims abstract description 18
- 238000006116 polymerization reaction Methods 0.000 claims abstract description 17
- 238000003756 stirring Methods 0.000 claims abstract description 17
- 239000000945 filler Substances 0.000 claims abstract description 13
- 229920000642 polymer Polymers 0.000 claims abstract description 12
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims abstract description 11
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 10
- 230000007935 neutral effect Effects 0.000 claims abstract description 8
- 238000001132 ultrasonic dispersion Methods 0.000 claims abstract description 7
- 238000010438 heat treatment Methods 0.000 claims abstract description 6
- 238000001035 drying Methods 0.000 claims abstract description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000001301 oxygen Substances 0.000 claims abstract description 4
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 4
- 239000003480 eluent Substances 0.000 claims abstract description 3
- 238000002156 mixing Methods 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 18
- 239000013504 Triton X-100 Substances 0.000 claims description 10
- 229920004890 Triton X-100 Polymers 0.000 claims description 10
- 239000006185 dispersion Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 45
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 29
- 238000001179 sorption measurement Methods 0.000 description 24
- 229910052681 coesite Inorganic materials 0.000 description 23
- 229910052906 cristobalite Inorganic materials 0.000 description 23
- 229910052682 stishovite Inorganic materials 0.000 description 23
- 229910052905 tridymite Inorganic materials 0.000 description 23
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 21
- 239000002904 solvent Substances 0.000 description 21
- 229920000344 molecularly imprinted polymer Polymers 0.000 description 18
- 239000007788 liquid Substances 0.000 description 15
- 239000002699 waste material Substances 0.000 description 14
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 12
- 230000008569 process Effects 0.000 description 11
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 8
- 239000008367 deionised water Substances 0.000 description 8
- 229910021641 deionized water Inorganic materials 0.000 description 8
- 239000000839 emulsion Substances 0.000 description 8
- 239000006260 foam Substances 0.000 description 8
- 239000012071 phase Substances 0.000 description 8
- 238000000926 separation method Methods 0.000 description 8
- 238000003786 synthesis reaction Methods 0.000 description 8
- 238000004587 chromatography analysis Methods 0.000 description 7
- 235000019441 ethanol Nutrition 0.000 description 7
- 239000008187 granular material Substances 0.000 description 7
- 238000001802 infusion Methods 0.000 description 7
- 239000011259 mixed solution Substances 0.000 description 7
- 230000000379 polymerizing effect Effects 0.000 description 7
- 239000000725 suspension Substances 0.000 description 7
- 229910021529 ammonia Inorganic materials 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000003995 emulsifying agent Substances 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 4
- 239000000178 monomer Substances 0.000 description 4
- -1 Steroid saponin compounds Chemical class 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 229930014626 natural product Natural products 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- 229910004298 SiO 2 Inorganic materials 0.000 description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 239000003431 cross linking reagent Substances 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000010413 mother solution Substances 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 239000002861 polymer material Substances 0.000 description 2
- 229930182490 saponin Natural products 0.000 description 2
- 235000017709 saponins Nutrition 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 125000002133 (4-hydroxy-3-iodo-5-nitrophenyl)acetyl group Chemical group OC1=C(C=C(C=C1I)CC(=O)*)[N+](=O)[O-] 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000002159 adsorption--desorption isotherm Methods 0.000 description 1
- 239000012752 auxiliary agent Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000007479 molecular analysis Methods 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3852—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36 using imprinted phases or molecular recognition; using imprinted phases
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- 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
- B01J20/28016—Particle form
- B01J20/28021—Hollow particles, e.g. hollow spheres, microspheres or cenospheres
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
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- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Polymerisation Methods In General (AREA)
Abstract
The invention provides a dioscin molecularly imprinted microsphere, a solid-phase extraction device and a preparation method, wherein the dioscin molecularly imprinted microsphere comprises the following steps: s1, dissolving diosgenin and methacrylic acid in a toluene solvent, performing ultrasonic dispersion, adding ethylene glycol dimethacrylate and azobisisobutyronitrile, and introducing nitrogen to remove oxygen to obtain a prepolymerization solution; s2, adding silicon dioxide particles and a dispersing aid into water, uniformly dispersing by ultrasonic, and carrying out heat treatment to obtain a silicon dioxide solution; and S3, mixing the prepolymerization solution and the silicon dioxide solution, stirring for dispersion, heating for polymerization reaction, settling the obtained polymer by using methanol after the polymerization reaction is finished, washing the diosgenin by using eluent, washing the diosgenin to be neutral, and drying to obtain the dioscin molecularly imprinted microspheres. The dioscin molecularly imprinted microsphere filler adopted by the solid-phase extraction device has strong specificity to dioscin molecules and is not easy to interfere.
Description
Technical Field
The invention relates to the field of detection of dioscin molecules, in particular to a dioscin molecularly imprinted microsphere, a solid-phase extraction device and a preparation method.
Background
Steroid saponin compounds are important components of effective components of traditional Chinese medicines, however, due to the complexity of natural products, the concentration of the steroid saponin compounds in plants is often extremely low, a large number of symbiotic components exist, and the analysis process needs to use a complex and complicated pretreatment process to reduce matrix interference.
In order to reduce matrix interference, ball-shaped resin filler is mostly adopted for pretreatment in the solid-phase extraction process of natural components. However, the commercialized filler lacks specific recognition on saponins, and can adsorb other structural analogs or compounds with similar properties while adsorbing target substances.
In order to improve the effect of the solid-phase extraction packed column, spherical polymer packing is mostly adopted as the solid-phase extraction packing, but an emulsifier is required to be added in the preparation process of the spherical polymer to improve the appearance, and the residue of the emulsifier can interfere with the analysis result of the saponin compound.
Disclosure of Invention
In order to overcome the disadvantages of the prior art, the present invention provides molecularly imprinted microspheres for dioscin, a solid-phase extraction device using the molecularly imprinted microspheres for dioscin, and a preparation method thereof.
The invention is realized by the following technical scheme:
the preparation method of the dioscin molecularly imprinted microspheres comprises the following steps:
s1, dissolving diosgenin and methacrylic acid in a toluene solvent, performing ultrasonic dispersion, adding ethylene glycol dimethacrylate and azobisisobutyronitrile, and introducing nitrogen to remove oxygen to obtain a prepolymerization solution;
s2, adding silicon dioxide particles and a dispersing aid into water, uniformly dispersing by ultrasonic, and carrying out heat treatment to obtain a silicon dioxide solution;
s3, mixing the pre-polymerized solution and the silicon dioxide solution, stirring and dispersing, heating for polymerization reaction, settling the obtained polymer by using methanol after the polymerization reaction is finished, washing the diosgenin by using eluent, washing the obtained product to be neutral, and drying the product to obtain the dioscin molecularly imprinted microspheres.
Preferably, in S1, the ratio of diosgenin, methacrylic acid, ethylene glycol dimethacrylate and azobisisobutyronitrile is 41.4 mg: (158-264) μ L: (1710-2090) μ L: 20 mg.
Preferably, in S2, the dispersing aid is triton X-100.
Preferably, in S2, silica fine particles are used
The preparation method is adopted.
Preferably, in S3, the polymerization reaction temperature is 60-70 ℃ and the time is 12-20 h.
Preferably, the ratio of the silicon dioxide to the diosgenin is (30-70) mg: 41.4 mg.
Preferably, in S3, the stirring speed is 8000-20000 rpm.
The dioscin molecularly imprinted microspheres prepared by the preparation method are adopted.
A molecular imprinting solid-phase extraction device for dioscin comprises a solid-phase extraction column, wherein the filler in the solid-phase extraction column is the dioscin molecular imprinting microsphere.
Compared with the prior art, the invention has the following beneficial effects:
the molecular engram microsphere of dioscin for on-line solid phase extraction prepared by the method is characterized in that template molecular dioscin added in the preparation process of the polymer interacts with functional monomer methacrylic acid, the interaction relation between the template molecular dioscin and the functional monomer is fixed under the action of cross-linking agent ethylene glycol dimethacrylate and initiator azobisisobutyronitrile, and a hole structure matched with the molecular structure of the template is left after the template molecular dioscin is eluted, so that the polymer has specific recognition capability on the dioscin and can selectively recognize and adsorb the dioscin from a plurality of structural compounds. Thereby reducing the interference of other coexisting substances in the natural product. The molecularly imprinted microspheres prepared by the method of the invention adopt silicon dioxide particles as an emulsifier in the preparation process, and do not need to add micromolecular surfactants such as sodium dodecyl sulfate and the like, so that the interference of micromolecular emulsifier residues in the application process of conventional polymer materials on the molecular analysis of dioscin can be effectively avoided.
Further, the ratio of diosgenin, methacrylic acid, ethylene glycol dimethacrylate and azobisisobutyronitrile is defined as 41.4 mg: (158-264) μ L: (1710-2090) μ L: 20 mg. Methacrylic acid and ethylene glycol dimethacrylate are too low, the adsorption capacity of the polymer is low, and too high imprinting factors are reduced.
Furthermore, the adopted dispersing auxiliary agent is beneficial to the stability of the emulsion in the polymerization process.
Furthermore, the polymerization reaction temperature is 60-70 ℃, the time is 12-20 h, the temperature is too low or the time is too short, the polymer cannot be formed, the temperature is too high or the time is too long, and the polymer imprinting factor is reduced.
Furthermore, the stirring speed influences the particle size of the microspheres, and when the stirring speed is lower than 8000rpm, the obtained polymer has large particle size distribution difference and poor form. When the rotating speed is higher than 20000rpm, the particle size of the obtained microspheres is too low, and subsequent treatment is not utilized.
The dioscin molecularly imprinted microspheres obtained by the invention have strong specificity to dioscin molecules and are not easily interfered by other substances.
The on-line solid phase extraction device has simple structure, convenient operation and strong adaptability, is beneficial to improving the solid phase extraction efficiency and reducing the interference of the coexisting materials in natural products, and simultaneously avoids the interference of the emulsifier in the conventional polymer materials on the analysis. The device has the advantages of simplicity, convenience, rapidness, high automation degree, strong specificity, difficulty in interference and reusability.
Drawings
Fig. 1 is an online solid-phase extraction device for molecular imprinting of dioscin.
Fig. 2 is a scanning electron microscope image of the molecularly imprinted microsphere filler containing dioscin in example 1, wherein fig. 2a is a magnification of 100 times, and fig. 2b is a magnification of 1000 times.
FIG. 3 is the N of the molecularly imprinted microsphere of dioscin in example 12Adsorption-desorption isotherms.
Fig. 4 is an adsorption kinetic curve of the molecularly imprinted microsphere of dioscin in example 1.
Fig. 5 is an isothermal adsorption curve of the molecularly imprinted microsphere of dioscin in example 1.
FIG. 6 shows the results of the selectivity of example 1 for the identification of different glycosides.
FIG. 7 is a graph showing the change in the amount of adsorption after repeated use in example 1.
FIG. 8 shows the optimization results of the functional monomers in examples 1 to 3.
FIG. 9 shows the results of optimizing the degree of crosslinking in examples 1 to 3.
FIG. 10 shows the results of the reaction temperature optimization in examples 1 to 4.
FIG. 11 shows the results of the reaction time optimization in examples 1 to 4.
FIG. 12 shows the optimized results of the silicon dioxide dosage in examples 1 and 4.
FIG. 13 shows the results of optimizing the stirring speeds in examples 1, 6 to 7.
Detailed Description
For a further understanding of the invention, reference will now be made to the following examples, which are provided to illustrate further features and advantages of the invention, and are not intended to limit the scope of the invention as set forth in the following claims.
The preparation method of the dioscin molecularly imprinted microspheres comprises the following steps:
1) preparing silicon dioxide particles;
2) dissolving diosgenin and methacrylic acid in a toluene solvent to form an oil phase, performing low-temperature ultrasonic dispersion and pre-assembly, adding ethylene glycol dimethacrylate and azobisisobutyronitrile, and removing oxygen by nitrogen after dissolving to obtain a pre-polymerization solution.
3) Adding silicon dioxide particles and a dispersing aid triton X-100 into water, uniformly dispersing by using ultrasonic waves, and carrying out constant-temperature water bath to obtain a silicon dioxide solution.
4) Adding the prepolymerization solution obtained in the step 2) into the silica solution obtained in the step 3), and vigorously stirring and dispersing. And (4) carrying out constant-temperature water bath reaction, removing foam floating on the surface layer, and then settling the polymer by using methanol. Washing machine
Washing with stripping solution to remove diosgenin, washing with water to neutrality, and drying at 60 deg.C to obtain diosgenin molecularly imprinted microsphere.
The silicon dioxide particles in the step 1) adopt
The preparation method specifically comprises the following steps: adding 15-20 mL of water, 10-12 mL of ammonia water and 40-60 mL of methanol into the flask, and stirring on a magnetic stirrer. In addition, 50-60 mL of methanol and 5-7 mL of TEOS are precisely measured, mixed and then rapidly added into the solution. Stirring for 10-12 hours at 25 ℃. And (4) performing ultra-high speed centrifugal separation. After washing with absolute ethyl alcohol and deionized water, vacuum drying is carried out at 50 ℃.
The proportion of diosgenin, methacrylic acid, ethylene glycol dimethacrylate and azobisisobutyronitrile in the step 2) is 41.4 mg: (158-264) μ L: (1710-2090) μ L: 20 mg.
The adding proportion of the silicon dioxide to the triton X-100 in the step 3) is (30-70) mg: 0 to 20 μ L. The ratio of silicon dioxide to diosgenin is (30-70) mg: 41.4 mg.
In the step 4), the stirring speed is 8000-20000 rpm, the constant-temperature water bath temperature is 60-70 ℃, and the reaction time is 12-20 h.
The construction of the dioscin molecular imprinting on-line solid-phase extraction device comprises the following steps:
dispersing the obtained dioscin molecularly imprinted microspheres in methanol and filling the methanol in an online solid-phase extraction column, wherein the filling amount of the online solid-phase extraction column is 100-400 mg, washing the column with the methanol, connecting a micro injection pump with the solid-phase extraction column through a three-way valve, and connecting the solid-phase extraction column with a liquid chromatogram through a ten-way switching valve to construct the solid-phase extraction online analysis device.
Example 1: preparation method of dioscin molecularly imprinted microspheres
1) A250 mL flask was charged with 17mL of water, 11.5mL of ammonia, and 50mL of methanol and stirred on a magnetic stirrer. Separately, 58mL of methanol and 7mL of TEOS were precisely measured, mixed, and then the above solution was quickly added thereto, followed by stirring at 25 ℃ for 10 hours. And (4) performing centrifugal separation at 10000r/min for 10 min. Washing with absolute ethyl alcohol and deionized water for 3 times to obtain SiO 2The granules were dried under vacuum at 50 ℃ for 24 hours.
2)41.4mg of diosgenin and 176 mu L of methacrylic acid are dissolved in 1800 mu L of toluene solvent, treated for 5 minutes by an ultrasonic instrument, and self-assembled for 12 hours at 25 ℃. Adding 1900 mu L of ethylene glycol dimethacrylate and 20mg of azobisisobutyronitrile, and introducing nitrogen for 15 minutes to obtain a prepolymerization solution;
3) mu.L of triton X-100 was added to 10mL of water followed by 60mg of SiO2Ultrasonic dispersion to form a stable suspension to obtain SiO2The solution was maintained at a temperature of 70 ℃.
4) In SiO2The pre-polymerization solution was added to the solution and homogenized using 16000rpm to obtain a stable Pickering emulsion. Polymerizing for 16 hours at 60 ℃ to obtain the dioscin molecularly imprinted microspheres. Removing foam in the mixed solution, and settling methanol to obtain the microspheres. After washing the microspheres with water and ethanol, the microspheres were eluted using a methanol/acetic acid (90:10) soxhlet extractor until no diosgenin was detected. Washing with water to neutrality, and drying at 60 deg.C to obtain dioscin Molecularly Imprinted Polymer (MIPs).
The synthesis and elution methods of the blank molecularly imprinted polymers (NIPs) are the same, except that the template molecule diosgenin is not added in the synthesis process.
200mg of dioscin molecularly imprinted microspheres are mixed with methanol and transferred to a solid phase extraction column, and the methanol is continuously injected to compact the filler. The dioscin molecular imprinting online solid-phase extraction device is constructed by referring to fig. 1. The device specifically includes dioscin solid-phase extraction unit (specifically can adopt solid-phase extraction post 1), the unit is cut to the valve (specifically can adopt solid-phase extraction post 2), the ration shifts the unit (specifically can adopt quantitative ring 3), sample/solvent provides the unit (be used for providing required sample or solvent, specifically can adopt syringe pump 4), still include chromatogram infusion unit (specifically can adopt chromatographic pump 5), chromatogram mobile phase holds the unit (specifically can adopt solvent bottle 6), chromatography column unit (specifically can adopt C18 chromatographic column 7), detecting element (specifically can adopt ultraviolet detector 8) and waste liquid collection unit (specifically can adopt waste liquid bottle 9).
The beneficial effect of this embodiment does:
referring to fig. 2, it can be seen from fig. 2 that the molecular imprinting microsphere of dioscin prepared in example 1 has a relatively uniform particle size, and the average particle size is about 44.31 μm. Meanwhile, the surface of the microsphere is rough, and an adsorption site is provided for identification.
Referring to fig. 3, it can be seen from fig. 3 that the molecularly imprinted polymer of dioscin prepared in example 1 exhibits an isotherm type iv, which means that the molecularly imprinted polymer of dioscin is a macroporous material. The closed points at the upper end and the lower end of the hysteresis loop of the dioscin molecularly imprinted polymer are farther apart, which shows that the pore size distribution range of the surface of the dioscin molecularly imprinted polymer is wider and rougher than that of a blank molecularly imprinted polymer.
Referring to fig. 4, it can be seen from fig. 4 that the molecularly imprinted polymer of dioscin prepared in example 1 has a fast mass transfer rate, and can achieve an adsorption equilibrium within about 10 minutes, with an equilibrium adsorption amount of 42.6 mg/g.
Referring to fig. 5, it can be seen from fig. 5 that the saturation adsorption amount of the molecularly imprinted polymer of dioscin increases with the increase of the concentration of the mother solution prepared in example 1, and the saturation adsorption amount of the molecularly imprinted polymer of dioscin is the maximum and reaches 43.1mg/g when the concentration of the mother solution is 200 μ g/mL.
Referring to fig. 6, it can be seen from fig. 6 that the adsorption amount of the dioscin to the molecularly imprinted polymer of the dioscin prepared in example 1 is the largest, the adsorption amount is 43mg/g, and the imprinting factor is 7.86.
Referring to fig. 7, it can be seen from fig. 7 that the adsorption amount of the dioscin to the dioscin molecularly imprinted polymer prepared in example 1 is maintained at 40mg/g or more after the dioscin molecularly imprinted polymer is repeatedly used for 5 times, indicating that the dioscin molecularly imprinted polymer has good durability.
Example 2: construction method of dioscin molecularly imprinted microspheres
1) A250 mL flask was charged with 17mL of water, 11.5mL of ammonia and 50mL of methanol and stirred on a magnetic stirrer. Separately, 58mL of methanol and 7mL of TEOS were precisely measured, mixed, and then the above solution was rapidly added thereto and stirred at 25 ℃ for 10 hours. And (4) performing centrifugal separation for 10min at a speed of 10000 r/min. Washing with absolute ethyl alcohol and deionized water for 3 times to obtain SiO2The granules were dried under vacuum at 50 ℃ for 24 hours.
2)41.4mg of diosgenin and 158 mu L of methacrylic acid are dissolved in 1800 mu L of toluene solvent, treated for 5 minutes by an ultrasonic instrument, and self-assembled for 12 hours at 25 ℃. 1710 μ L of ethylene glycol dimethacrylate and 20mg of azobisisobutyronitrile were added, and nitrogen gas was introduced for 15 minutes to obtain a prepolymerization solution.
3) 60mg of SiO are added to 10mL of water2Ultrasonic dispersion to form a stable suspension, resulting in a silica solution, maintaining the temperature at 70 ℃.
4) The pre-polymerization solution was added to the silica solution and homogenized using 16000rpm to obtain a stable Pickering emulsion. Polymerizing for 20 hours at 70 ℃ to obtain the dioscin molecularly imprinted microspheres. Removing foam in the mixed solution, and settling methanol to obtain the microspheres. After washing the microspheres with water and ethanol, the microspheres were eluted using a methanol/acetic acid (90:10) soxhlet extractor until no diosgenin was detected. Washed to neutrality by water and dried at 60 ℃.
100mg of dioscin molecularly imprinted microspheres and methanol are mixed and transferred to a solid-phase extraction hollow column, and the methanol is continuously injected to compact the filler. The molecular imprinting online solid-phase extraction device of dioscin is constructed by referring to fig. 1. The device specifically includes dioscin solid-phase extraction unit (specifically can adopt solid-phase extraction post 1), the unit is cut to the valve (specifically can adopt solid-phase extraction post 2), the ration shifts the unit (specifically can adopt quantitative ring 3), sample/solvent provides the unit (be used for providing required sample or solvent, specifically can adopt syringe pump 4), still include chromatogram infusion unit (specifically can adopt chromatographic pump 5), chromatogram mobile phase holds the unit (specifically can adopt solvent bottle 6), chromatography column unit (specifically can adopt C18 chromatographic column 7), detecting element (specifically can adopt ultraviolet detector 8) and waste liquid collection unit (specifically can adopt waste liquid bottle 9).
Example 3: preparation method of dioscin molecularly imprinted microspheres
1) A250 mL flask was charged with 17mL of water, 11.5mL of ammonia, and 50mL of methanol and stirred on a magnetic stirrer. Separately, 58mL of methanol and 7mL of TEOS were precisely measured, mixed, and then the above solution was quickly added thereto, followed by stirring at 25 ℃ for 10 hours. And (4) performing centrifugal separation for 10min at a speed of 10000 r/min. Washing with anhydrous ethanol and deionized water for 3 times to obtain SiO2The granules were dried under vacuum at 50 ℃ for 24 hours.
2)41.4mg of diosgenin and 264 mu L of methacrylic acid are dissolved in 1800 mu L of toluene solvent, treated for 5 minutes by an ultrasonic instrument and self-assembled for 12 hours at 25 ℃. Adding 2090 mu L of ethylene glycol dimethacrylate and 20mg of azobisisobutyronitrile, and introducing nitrogen for 15 minutes to obtain a prepolymerization solution;
3) 20 μ L of triton X-100 was added to 10mL of water followed by 60mg of SiO2Ultrasonic dispersing to form stable suspension to obtain SiO2The solution was maintained at a temperature of 70 ℃.
4) In SiO2The pre-polymerization solution was added to the solution and homogenized using 16000rpm to obtain a stable Pickering emulsion. Polymerizing for 16 hours at 60 ℃ to obtain the dioscin molecularly imprinted microspheres. Removing foam in the mixed solution, and settling methanol to obtain the microspheres. After washing the microspheres with water and ethanol, the microspheres were eluted using a methanol/acetic acid (90:10) soxhlet extractor until no diosgenin was detected. Washed to be neutral by water and dried at 60 ℃.
400mg of dioscin molecularly imprinted microspheres and methanol are mixed and then transferred to a solid-phase extraction column, and methanol is continuously injected to compact the filler. The molecular imprinting online solid-phase extraction device of dioscin is constructed by referring to fig. 1. The device specifically includes dioscin solid-phase extraction unit (specifically can adopt solid-phase extraction post 1), the unit is cut to the valve (specifically can adopt solid-phase extraction post 2), the ration shifts the unit (specifically can adopt quantitative ring 3), sample/solvent provides the unit (be used for providing required sample or solvent, specifically can adopt syringe pump 4), still include chromatogram infusion unit (specifically can adopt chromatographic pump 5), chromatogram mobile phase holds the unit (specifically can adopt solvent bottle 6), chromatography column unit (specifically can adopt C18 chromatographic column 7), detecting element (specifically can adopt ultraviolet detector 8) and waste liquid collection unit (specifically can adopt waste liquid bottle 9).
Referring to fig. 8, it can be seen from the bar graph of the effect of the content of the functional monomer methacrylic acid on the adsorption of dioscin in the synthesis process of examples 1 to 3 that the adsorption amount of the molecularly imprinted polymer of dioscin gradually increases with the increase of the addition amount of methacrylic acid, the adsorption amount and the imprinting factor are the largest when the amount of methacrylic acid is 176 μ L, and the change of the adsorption amount is not large but the imprinting factor is decreased when the amount of methacrylic acid is continuously increased.
Referring to fig. 9, it can be seen from the histogram of the effect of the content of the crosslinking agent ethylene glycol dimethacrylate on the adsorption of the dioscin in the synthesis process of examples 1 to 3 that the adsorption amount of the dioscin molecularly imprinted polymer gradually increases with the increase of the addition amount of the ethylene glycol dimethacrylate, the adsorption amount and the imprinting factor are the largest when the dosage of the ethylene glycol dimethacrylate is 1900 μ L, and the change of the adsorption amount is not large but the imprinting factor is reduced when the dosage of the ethylene glycol dimethacrylate is continuously increased.
Example 4: preparation method of dioscin molecularly imprinted microspheres
1) A250 mL flask was charged with 17mL of water, 11.5mL of ammonia, and 50mL of methanol and stirred on a magnetic stirrer. Separately, 58mL of methanol and 7mL of TEOS were precisely measured, mixed, and then the above solution was rapidly added thereto and stirred at 25 ℃ for 10 hours. And (4) performing centrifugal separation for 10min at a speed of 10000 r/min. Washing with anhydrous ethanol and deionized water for 3 times to obtain SiO2The granules were dried under vacuum at 50 ℃ for 24 hours.
2)41.4mg of diosgenin and 176 mu L of methacrylic acid are dissolved in 1800 mu L of toluene solvent, treated for 5 minutes by an ultrasonic instrument and self-assembled for 12 hours at 25 ℃. Adding 1900 mu L of ethylene glycol dimethacrylate and 20mg of azobisisobutyronitrile, and introducing nitrogen for 15 minutes to obtain a prepolymerization solution;
3)10mL of water was added 20. mu.L of triton X-100 followed by 60mg of SiO2Ultrasonic dispersing to form stable suspension to obtain SiO2The solution was maintained at a temperature of 70 ℃.
4) In SiO2The pre-polymerization solution was added to the solution and homogenized using 16000rpm to obtain a stable Pickering emulsion. Polymerizing for 12 hours at 60 ℃ to obtain the dioscin molecularly imprinted microspheres. Removing foam in the mixed solution, and settling methanol to obtain the microspheres. After washing the microspheres with water and ethanol, the microspheres were eluted using a methanol/acetic acid (90:10) soxhlet extractor until no dioscin was detected. Washed to be neutral by water and dried at 60 ℃.
200mg of dioscin molecularly imprinted microspheres are mixed with methanol and transferred to a solid phase extraction column, and the methanol is continuously injected to compact the filler. The dioscin molecular imprinting online solid-phase extraction device is constructed by referring to fig. 1. The device specifically includes dioscin solid-phase extraction unit (specifically can adopt solid-phase extraction post 1), the unit is cut to the valve (specifically can adopt solid-phase extraction post 2), the ration shifts the unit (specifically can adopt quantitative ring 3), sample/solvent provides the unit (be used for providing required sample or solvent, specifically can adopt syringe pump 4), still include chromatogram infusion unit (specifically can adopt chromatographic pump 5), chromatogram mobile phase holds the unit (specifically can adopt solvent bottle 6), chromatography column unit (specifically can adopt C18 chromatographic column 7), detecting element (specifically can adopt ultraviolet detector 8) and waste liquid collection unit (specifically can adopt waste liquid bottle 9).
Referring to fig. 10, it can be seen from the bar graph of the effect of the reaction temperature on the adsorption of dioscin in the synthesis process of examples 1 to 4 that the adsorption amount of the molecularly imprinted polymer of dioscin is gradually increased with the increase of the reaction temperature, but the imprinting factor is decreased. The optimum is reached at 60 ℃.
Referring to fig. 11, it can be seen from the bar graph of the effect of the reaction temperature on the adsorption of dioscin in the synthesis process of examples 1 to 4 that the adsorption amount of the molecularly imprinted polymer of dioscin is slightly increased and the imprinting factor is slightly decreased as the reaction time is increased. The optimum is reached at 16 h.
Example 5: preparation method of dioscin molecularly imprinted microspheres
1) Into a 250mL flask was added17mL of water, 11.5mL of ammonia water and 50mL of methanol were stirred on a magnetic stirrer. Separately, 58mL of methanol and 7mL of TEOS were precisely measured, mixed, and then the above solution was quickly added thereto, followed by stirring at 25 ℃ for 10 hours. And (4) performing centrifugal separation for 10min at a speed of 10000 r/min. Washing with anhydrous ethanol and deionized water for 3 times to obtain SiO2The granules were dried under vacuum at 50 ℃ for 24 hours.
2)41.4mg of diosgenin and 176 mu L of methacrylic acid are dissolved in 1800 mu L of toluene solvent, treated for 5 minutes by an ultrasonic instrument and self-assembled for 12 hours at 25 ℃. Adding 1900 mu L of ethylene glycol dimethacrylate and 20mg of azobisisobutyronitrile, and introducing nitrogen for 15 minutes to obtain a prepolymerization solution;
3) mu.L of triton X-100 was added to 10mL of water followed by 30mg of SiO2Ultrasonic dispersion to form a stable suspension to obtain SiO2The solution was maintained at 70 ℃.
4) In SiO2The prepolymerization solution was added to the solution and homogenized using 16000rpm to obtain a stable Pickering emulsion. Polymerizing for 16 hours at 60 ℃ to obtain the dioscin molecularly imprinted microspheres. Removing foam in the mixed solution, and settling methanol to obtain the microspheres. After washing the microspheres with water and ethanol, the microspheres were eluted using a methanol/acetic acid (90:10) soxhlet extractor until no diosgenin was detected. Washed to be neutral by water and dried at 60 ℃.
200mg of dioscin molecularly imprinted microspheres are mixed with methanol and transferred to a solid phase extraction column, and the methanol is continuously injected to compact the filler. The dioscin molecular imprinting online solid-phase extraction device is constructed by referring to fig. 1. The device specifically includes dioscin solid-phase extraction unit (specifically can adopt solid-phase extraction post 1), the unit is cut to the valve (specifically can adopt solid-phase extraction post 2), the ration shifts the unit (specifically can adopt quantitative ring 3), sample/solvent provides the unit (be used for providing required sample or solvent, specifically can adopt syringe pump 4), still include chromatogram infusion unit (specifically can adopt chromatographic pump 5), chromatogram mobile phase holds the unit (specifically can adopt solvent bottle 6), chromatography column unit (specifically can adopt C18 chromatographic column 7), detecting element (specifically can adopt ultraviolet detector 8) and waste liquid collection unit (specifically can adopt waste liquid bottle 9).
Referring to fig. 12, the particle size of the microspheres is affected by the amount of silica added in the synthesis process of examples 1 and 4, the particle size of the microspheres is reduced and the uniformity is enhanced as the amount of silica added is increased, and when the amount of silica added is more than 60mg, the particle size and uniformity are maintained to be substantially constant.
Example 6: preparation method of dioscin molecularly imprinted microspheres
1) A250 mL flask was charged with 17mL of water, 11.5mL of ammonia, and 50mL of methanol and stirred on a magnetic stirrer. Separately, 58mL of methanol and 7mL of TEOS were precisely measured, mixed, and then the above solution was quickly added thereto, followed by stirring at 25 ℃ for 10 hours. And (4) performing centrifugal separation at 10000r/min for 10 min. Washing with absolute ethyl alcohol and deionized water for 3 times to obtain SiO2The granules were dried under vacuum at 50 ℃ for 24 hours.
2)41.4mg of diosgenin and 176 mu L of methacrylic acid are dissolved in 1800 mu L of toluene solvent, treated for 5 minutes by an ultrasonic instrument, and self-assembled for 12 hours at 25 ℃. Adding 1900 mu L of ethylene glycol dimethacrylate and 20mg of azobisisobutyronitrile, and introducing nitrogen for 15 minutes to obtain a prepolymerization solution;
3) 20 μ L of triton X-100 was added to 10mL of water followed by 70mg of SiO2Ultrasonic dispersing to form stable suspension to obtain SiO 2The solution was maintained at 70 ℃.
4) In SiO2The solution was added with the pre-polymerization solution and homogenized using 20000rpm to obtain a stable Pickering emulsion. Polymerizing for 16 hours at 60 ℃ to obtain the dioscin molecularly imprinted microspheres. Removing foam in the mixed solution, and settling methanol to obtain the microspheres. After washing the microspheres with water and ethanol, the microspheres were eluted using a methanol/acetic acid (90:10) soxhlet extractor until no diosgenin was detected. Washed to be neutral by water and dried at 60 ℃.
200mg of dioscin molecularly imprinted microspheres are mixed with methanol and transferred to a solid phase extraction column, and the methanol is continuously injected to compact the filler. The dioscin molecular imprinting online solid-phase extraction device is constructed by referring to fig. 1. The device specifically includes dioscin solid-phase extraction unit (specifically can adopt solid-phase extraction post 1), the unit is cut to the valve (specifically can adopt solid-phase extraction post 2), the ration shifts the unit (specifically can adopt quantitative ring 3), sample/solvent provides the unit (be used for providing required sample or solvent, specifically can adopt syringe pump 4), still include chromatogram infusion unit (specifically can adopt chromatographic pump 5), chromatogram mobile phase holds the unit (specifically can adopt solvent bottle 6), chromatography column unit (specifically can adopt C18 chromatographic column 7), detecting element (specifically can adopt ultraviolet detector 8) and waste liquid collection unit (specifically can adopt waste liquid bottle 9).
Example 7: preparation method of dioscin molecularly imprinted microspheres
1) A250 mL flask was charged with 17mL of water, 11.5mL of ammonia, and 50mL of methanol and stirred on a magnetic stirrer. Separately, 58mL of methanol and 7mL of TEOS were precisely measured, mixed, and then the above solution was rapidly added thereto and stirred at 25 ℃ for 10 hours. And (4) performing centrifugal separation for 10min at a speed of 10000 r/min. Washing with anhydrous ethanol and deionized water for 3 times to obtain SiO2The granules were dried under vacuum at 50 ℃ for 24 hours.
2)41.4mg of diosgenin and 176 mu L of methacrylic acid are dissolved in 1800 mu L of toluene solvent, treated for 5 minutes by an ultrasonic instrument and self-assembled for 12 hours at 25 ℃. Adding 1900 mu L of ethylene glycol dimethacrylate and 20mg of azobisisobutyronitrile, and introducing nitrogen for 15 minutes to obtain a prepolymerization solution;
3) mu.L of triton X-100 was added to 10mL of water followed by 60mg of SiO2Ultrasonic dispersing to form stable suspension to obtain SiO2The solution was maintained at a temperature of 70 ℃.
4) In SiO2The pre-polymerization solution was added to the solution and homogenized using 8000rpm to obtain a stable Pickering emulsion. Polymerizing for 16 hours at 60 ℃ to obtain the dioscin molecularly imprinted microspheres. Removing foam in the mixed solution, and settling methanol to obtain the microspheres. After washing the microspheres with water and ethanol, the microspheres were eluted using a methanol/acetic acid (90:10) soxhlet extractor until no diosgenin was detected. Washed to be neutral by water and dried at 60 ℃.
200mg of dioscin molecularly imprinted microspheres and methanol are mixed and transferred to a solid-phase extraction column, and the methanol is continuously injected to compact the filler. The molecular imprinting online solid-phase extraction device of dioscin is constructed by referring to fig. 1. The device specifically includes dioscin solid-phase extraction unit (specifically can adopt solid-phase extraction post 1), the unit is cut to the valve (specifically can adopt solid-phase extraction post 2), the ration shifts the unit (specifically can adopt quantitative ring 3), sample/solvent provides the unit (be used for providing required sample or solvent, specifically can adopt syringe pump 4), still include chromatogram infusion unit (specifically can adopt chromatographic pump 5), chromatogram mobile phase holds the unit (specifically can adopt solvent bottle 6), chromatography column unit (specifically can adopt C18 chromatographic column 7), detecting element (specifically can adopt ultraviolet detector 8) and waste liquid collection unit (specifically can adopt waste liquid bottle 9).
Referring to fig. 13, the particle size of the microspheres is affected by the stirring rotation speed in the synthesis processes of examples 1, 6 to 7, the particle size of the microspheres is reduced and the uniformity is enhanced with the increase of the rotation speed, and when the rotation speed reaches 16000 revolutions, the particle size and the uniformity are basically maintained constant.
Claims (9)
1. The preparation method of the dioscin molecularly imprinted microspheres is characterized by comprising the following steps:
S1, dissolving diosgenin and methacrylic acid in a toluene solvent, performing ultrasonic dispersion, adding ethylene glycol dimethacrylate and azobisisobutyronitrile, and introducing nitrogen to remove oxygen to obtain a prepolymerization solution;
s2, adding silicon dioxide particles and a dispersing aid into water, uniformly dispersing by ultrasonic, and carrying out heat treatment to obtain a silicon dioxide solution;
s3, mixing the pre-polymerized solution and the silicon dioxide solution, stirring and dispersing, heating for polymerization reaction, settling the obtained polymer by using methanol after the polymerization reaction is finished, washing the diosgenin by using eluent, washing the obtained product to be neutral, and drying the product to obtain the dioscin molecularly imprinted microspheres.
2. The method for preparing dioscin molecularly imprinted microspheres according to claim 1, wherein in S1, the ratio of diosgenin, methacrylic acid, ethylene glycol dimethacrylate and azobisisobutyronitrile is 41.4 mg: (158-264) μ L: (1710-2090) μ L: 20 mg.
3. The method for preparing molecularly imprinted microspheres of dioscin according to claim 1, wherein in S2, the dispersing aid is triton X-100.
4. The method for preparing molecularly imprinted microspheres of dioscin according to claim 1, wherein in S2, silica particles are used 
The preparation method is adopted.
5. The preparation method of the dioscin molecularly imprinted microspheres according to claim 1, wherein in S3, the polymerization reaction temperature is 60-70 ℃ and the time is 12-20 hours.
6. A preparation method of dioscin molecularly imprinted microspheres according to claim 1, wherein the ratio of silicon dioxide to diosgenin is (30-70) mg: 41.4 mg.
7. The method for preparing dioscin molecularly imprinted microspheres according to claim 1, wherein in S3, the stirring speed is 8000-20000 rpm.
8. The molecularly imprinted microsphere of dioscin obtained by the preparation method of any one of claims 1 to 7.
9. A dioscin molecularly imprinted solid phase extraction device, which comprises a solid phase extraction column, wherein the filler in the solid phase extraction column is the dioscin molecularly imprinted microsphere of claim 8.
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