CN102671639A - Bamboo-leaf-flavonoids molecular imprinted solid-phase extraction column, preparation and application thereof - Google Patents
Bamboo-leaf-flavonoids molecular imprinted solid-phase extraction column, preparation and application thereof Download PDFInfo
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- CN102671639A CN102671639A CN2012101644967A CN201210164496A CN102671639A CN 102671639 A CN102671639 A CN 102671639A CN 2012101644967 A CN2012101644967 A CN 2012101644967A CN 201210164496 A CN201210164496 A CN 201210164496A CN 102671639 A CN102671639 A CN 102671639A
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Abstract
The invention relates to a bamboo-leaf-flavonoids molecular imprinted solid-phase extraction column, preparation and application thereof, and belongs to the technical field of medicine separation. Template molecule luteolin, functional monomer 4-ethylpyridine and ethylene glycol dimethacrylate which is a cross-linking agent are polymerized into bamboo-leaf-flavonoids molecular imprinted polymer particles in a suspension manner, the molar ratio of template molecules to the functional monomer to the cross-linking agent is 1:(5-8):80. The prepared bamboo-leaf-flavonoids molecular imprinted polymer particles are filled in a polypropylene or glass solid-phase extraction column hollow tube, so that the bamboo-leaf-flavonoids molecular imprinted solid-phase extraction column is prepared. The bamboo-leaf-flavonoids molecular imprinted solid-phase extraction column is used for selectively gathering and purifying bamboo-leaf-flavonoids extracting solution, has the advantages if simplicity, rapidness, high recycling rate and the like, and is wide in application prospect in the field of separation of natural flavonoids compounds.
Description
Technical field
The invention belongs to the medicine separation technology field, specifically, relate to the preparation method of bamboo-leaves flavones molecularly imprinted solid phase extraction column of high selectivity separation, purifying and the enrichment of Flavonoid substances in the extract solution of bamboo leaves.
Background technology
In medicine separated, the pre-treatment process of sample was most important.It is a kind of sample pre-treatments technology comparatively commonly used that column chromatography is separated.SPE (Solid Phase Extraction; SPE) combine development by liquid-solid extraction and column liquid chromatographic technology; With liquid-liquid extraction method (Liquid-Liquid Extraction; LLE) compare, SPE has rate of recovery height, good separating effect, simple to operate, save time, advantage such as laborsaving and be widely used in the pre-treatment of sample.
Present SPE column packing kind positive, anti-phase and ion exchange column and adsorption resin column.Positive SPE column packing is mainly silica gel, aluminium oxide and silmag etc.Anti-phase SPE column packing is mainly C
18Bonded silica gel, cyanic acid bonded silica gel post (CN) etc.These column packings make the thing of carrying altogether that produces interference removed by drip washing through the absorption object, reach the purpose of purification, enrichment; Or impurity stayed on the post, object to be measured is through the SPE post, and sample is cleaned, but can not enrichment component to be measured, can not separate the retention properties impurity more weak than component to be measured, causes to purify not exclusively.
Molecular engram solid phase extraction (Molecularly imprinted solid-phase extraction; MIPSE) be to utilize MIP to select adsorption mechanism to prepare the SPE filler; Not only can improve the SPE clean-up effect; Realize the separation and concentration of the medicine of low concentration, can realize being repeatedly used of SPE post simultaneously, thereby reduce cost.
Bamboo-leaves flavones is the one type of natural products that from the leaf of bamboo, extracts, and has anti-oxidant, the anti-ageing effect of waiting for a long time, and is emerging functional food additives and natural drug.Bamboo-leaves flavones is main with charcoal glycosides flavones, the main component that orientoside, Lutonaretin, Vitexina and different special chaste tree glycosides are bamboo-leaves flavones.Bamboo-leaves flavones product in the market is mainly semifinished product, and the total content of above-mentioned 4 kinds of flavones generally in 10%, is difficult to satisfy market to high-load bamboo-leaves flavones product demand.
Summary of the invention
The objective of the invention is to shortcoming and defect to the separation of existing laboratory, enrichment bamboo-leaves flavones method; A kind of bamboo-leaves flavones molecularly imprinted solid phase extraction column, preparation and application process are provided; Make the bamboo-leaves flavones in its can be fast simple separation and purification extract solution of bamboo leaves, the specificity that can directly apply to bamboo-leaves flavones is selected to separate, efficiently concentrating.
The present invention realizes through following technical scheme:
The preparation method of bamboo-leaves flavones molecularly imprinted solid phase extraction column may further comprise the steps:
⑴ preparation quality concentration is the aqueous dispersant of 3%-3.5%; Function monomer 4-vinylpridine and template molecule cyanidenon are dissolved in the aqueous dispersant; Crosslinking agent GDMA, initator azodiisobutyronitrile are dissolved in respectively in the organic solvent, mix with above-mentioned aqueous dispersant then; And carry out ultrasonic degas; Stir down at 70-75 ℃, under logical nitrogen protection, carry out polymerisation 10-24h, generate the molecularly imprinted polymer particle;
The mol ratio of described template molecule cyanidenon, 4-vinylpridine, GDMA is 1: (5-6): 80, and the volume ratio of GDMA, organic solvent, dispersant solution is 1: 2-3: 20-30.Azodiisobutyronitrile is this area routine techniques, as long as add a small amount of the initiation.
⑵ after reaction finished, polymer beads was isolated in centrifugal filtration; With isolated polymer beads water cyclic washing, use methyl alcohol, washing with acetone more repeatedly again; Extract through Suo Shi at last and further remove the template molecule cyanidenon in the polymer beads, until can not detecting template molecule, vacuum drying then;
⑶ be filled in the prepared polymer beads of ⑵ in polypropylene or the glass column pipe, and sieve plate is loaded onto at the two ends of pipe respectively, promptly makes solid-phase extraction column.
Described aqueous dispersant is the carboxyethyl cellulose aqueous solution;
Described sieve plate is the polyethylene sieve plate, and its aperture is 20 μ m.
The described ultrasonic degas time is 10-20min; Described speed of agitator is 600-700rpm.
Described water is ultra-pure water.
Said polymer beads vacuum drying, temperature are 60 ℃, and be 6 ~ 8h drying time.
The bamboo-leaves flavones molecularly imprinted solid phase extraction column of the present invention's preparation is applied to separation, purifying and the enrichment of bamboo-leaves flavones in the extract solution of bamboo leaves, and concrete steps are following:
⑴ behind the filling bamboo-leaves flavones molecularly imprinted solid phase extraction column, use the acetonitrile activation earlier, use the drip washing of mass concentration 5-15% methanol aqueous solution again,
⑵ add sample extracting solution or sample solution;
⑶ with the drip washing of 15-15% methanol aqueous solution;
⑷ collect eluent with methanol-eluted fractions 2 times, behind 0.45 μ m membrane filtration, collects.
Above-mentioned particular methanol concentration of aqueous solution 10%; The bamboo-leaves flavones molecularly imprinted polymer particle of the present invention preparation is a kind of macromolecule cross-linked polymer with fixed cavitation size, shape and definite arranging functional group, and the stereochemical structure of bamboo-leaves flavones is had the dynamic memory function.The present invention can be applicable to separation, enrichment and the purifying of bamboo-leaves flavones in the extract solution of bamboo leaves, more common liquid-liquid extraction method, C
18Solid phase extraction more simply, fast, more efficient.
The specific embodiment
Further illustrate content of the present invention below in conjunction with embodiment, but the present invention is not restricted to the scope of following embodiment.
Instance 1. bamboo-leaves flavones molecularly imprinted polymer particle solid-phase extraction columns (template molecule and function monomer mol ratio are 1: 5)
⑴ compound concentration is the carboxyethyl cellulose solution of 3%-3.5%; The zinc acetate of 2.5mmol function monomer 4-vinylpridine (4-vp), 0.5mmol template molecule cyanidenon and 5mmol is dissolved in the methyl alcohol ultrasonication 30min; The xylenes of crosslinking agent GDMA (EGDMA), 0.2g initator (AIBN) and the 7.5mL of 7.5mL is mixed; Ultrasonication 20min; With above-mentioned three kinds of solution mixing ultrasonic degas 10min; Stir 10h at 70 ℃ of constant temperature 650rpm.
⑵ with ultra-pure water washing 5 times, use the methyl alcohol filtering and washing again 5 times then with the molecularly imprinted polymer particle centrifugal filtration of above-mentioned preparation, extracts through Suo Shi at last and further remove template molecule.
⑶ will remove the polymer of template molecule, and vacuum drying (60 ℃) obtains bamboo-leaves flavones molecularly imprinted polymer particle.
⑷ be filled in prepared polymer beads in polypropylene or the glass column pipe, and sieve plate is loaded onto at the two ends of pipe respectively, promptly makes solid-phase extraction column.
Instance 2. bamboo-leaves flavones molecularly imprinted polymer particle solid-phase extraction columns (template molecule and function monomer mol ratio are 1: 8)
⑴ compound concentration is the carboxyethyl cellulose solution of 3%-3.5%; The zinc acetate of 4.0mmol function monomer 4-vinylpridine (4-vp), 0.5mmol template molecule cyanidenon and 5mmol is dissolved in the methyl alcohol ultrasonication 30min; The xylenes of crosslinking agent GDMA (EGDMA), 0.2g initator (AIBN) and the 7.5mL of 7.5mL is mixed; Ultrasonication 20min; With above-mentioned three kinds of solution mixing ultrasonic degas 10min; Stir 10h at 70 ℃ of constant temperature 650rpm.
⑵ with ultra-pure water washing 5 times, use the methyl alcohol filtering and washing again 5 times then with the molecularly imprinted polymer particle centrifugal filtration of above-mentioned preparation, extracts through Suo Shi at last and further remove template molecule.
⑶ will remove the polymer of template molecule, and vacuum drying (60 ℃) obtains bamboo-leaves flavones molecularly imprinted polymer particle.
⑷ be filled in prepared polymer beads in polypropylene or the glass column pipe, and sieve plate is loaded onto at the two ends of pipe respectively, promptly makes solid-phase extraction column.
The preparation and the performance evaluation of instance 3. bamboo-leaves flavones molecularly imprinted solid phase extraction columns
With 3mL polypropylene solid phase extraction column blank pipe as solid phase extraction column; Place the polyethylene sieve plate of one deck 20 μ m in the bottom of solid-phase extraction column; Accurately take by weighing the molecularly imprinted polymer of 200mg embodiment 1 or 2; Dry method is packed in the pillar, beats gently to add one deck sieve plate above the compacting again, promptly gets the bamboo-leaves flavones molecular imprinted solid phase extraction cartridge.
⑵ with 2mL acetonitrile rinse pillar, and maintenance 2min, removes residual liquid, keeps moistening, does not let eluent drain off before the last appearance;
⑶ last appearance.Get the 10mL extract solution of bamboo leaves, add in the molecularly imprinted solid phase extraction column.Contain cyanidenon, orientoside, Lutonaretin, Vitexina, Quercetin and Saponaretin standard items in the extract.
⑷ drip washing, the drip washing of 5mL10% methyl alcohol, flow velocity < 2mL/>min.
⑸ wash-out adds 10mL methyl alcohol, vacuumizes wash-out, collects eluent, and behind 0.45 μ m membrane filtration, HPLC analyzes.
Effect of the present invention does, this solid-phase extraction column absorption bamboo-leaves flavones effect is better, adding concentration when being 10mg/L, 6 kinds of bamboo-leaves flavones the rate of recovery between 80%-120%, RSD is between 3%-5%.
Instance 4. molecular imprinted solid phase extraction cartridges are applied to separation, the purifying of bamboo-leaves flavones
⑴ take by weighing the commercially available bamboo-leaves flavones powder of 10g sample, and with 100 times of 10% dissolve with ethanol, dilutions, filtration or centrifugal obtains the bamboo-leaves flavones sample solution.
⑵ take by weighing the bamboo-leaves flavones molecularly imprinted polymer of 500mg preparation, according to instance 2 methods filling solid-phase extraction column.
⑶ add the activation of 2mL acetonitrile in the solid-phase extraction column of filling, use the drip washing of 2mL10% methyl alcohol again.
⑷ pipette the bamboo-leaves flavones sample solution of ⑴ preparation in the 50mL step, adds in the solid-phase extraction column of activation; After treating that sample solution drenches down fully,, discard leacheate with the drip washing of 5mL10% methyl alcohol; Use the 5mL methanol-eluted fractions again 2 times.
⑸ decompression is concentrated down in 45 ℃ with the eluent of collecting, and the bamboo-leaves flavones sample that obtains separating detects through HPLC, and general flavone content is 86.20% in the gained bamboo-leaves flavones sample.
Claims (9)
1. the preparation method of a bamboo-leaves flavones molecularly imprinted solid phase extraction column may further comprise the steps:
⑴ preparation quality concentration is the aqueous dispersant of 3%-3.5%; Function monomer 4-vinylpridine and template molecule cyanidenon are dissolved in the aqueous dispersant; Crosslinking agent GDMA, initator azodiisobutyronitrile are dissolved in respectively in the organic solvent, mix with above-mentioned aqueous dispersant then; And carry out ultrasonic degas; Stir down at 70-75 ℃, under logical nitrogen protection, carry out polymerisation 10-24h, generate the molecularly imprinted polymer particle;
The mol ratio of described template molecule cyanidenon, 4-vinylpridine, GDMA is 1: (5-6): 80, and the volume ratio of GDMA, organic solvent, dispersant solution is 1: (2-3): (20-30);
⑵ after reaction finished, polymer beads was isolated in centrifugal filtration; With isolated polymer beads water cyclic washing, use methyl alcohol, washing with acetone more repeatedly again; Extract through Suo Shi at last and further remove the template molecule cyanidenon in the polymer beads, until can not detecting template molecule, vacuum drying then;
⑶ be filled in the prepared polymer beads of ⑵ in polypropylene or the glass column pipe, and sieve plate is loaded onto at the two ends of pipe respectively, promptly makes solid-phase extraction column.
2. according to the method for claim 1, it is characterized in that described aqueous dispersant is the carboxyethyl cellulose aqueous solution.
3. according to the method for claim 1, it is characterized in that described sieve plate is the polyethylene sieve plate, its aperture is 20 μ m.
4. according to the method for claim 1, it is characterized in that the described ultrasonic degas time is 10-20min; Described speed of agitator is 600-700rpm.
5. according to the method for claim 1, it is characterized in that used water is ultra-pure water.
6. according to the method for claim 1, it is characterized in that, said polymer beads vacuum drying, temperature is 60 ℃, be 6-8h drying time.
7. according to the bamboo-leaves flavones molecularly imprinted solid phase extraction column of the arbitrary method preparation of claim 1-6.
8. the bamboo-leaves flavones molecularly imprinted solid phase extraction column of claim 7 is applied to separation, purifying and the enrichment of bamboo-leaves flavones in the extract solution of bamboo leaves, and concrete steps are following:
⑴ behind the filling bamboo-leaves flavones molecularly imprinted solid phase extraction column, use the acetonitrile activation earlier, use the drip washing of mass concentration 5-15% methanol aqueous solution again;
⑵ add sample extracting solution or sample solution;
⑶ with the drip washing of 15-15% methanol aqueous solution;
⑷ collect eluent with methanol-eluted fractions 2 times, behind 0.45 μ m membrane filtration, collects.
9. according to the application of claim 8, it is characterized in that methanol aqueous solution concentration 10%.
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Cited By (7)
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| CN103331150A (en) * | 2013-07-12 | 2013-10-02 | 中国科学院地理科学与资源研究所 | Cross-linked starch solid phase extraction column, and preparation method and application of solid phase extraction column |
| CN105233801A (en) * | 2015-10-16 | 2016-01-13 | 江苏大学 | Preparation method of boron affiliated dual recognition molecularly imprinted material |
| CN105477433A (en) * | 2015-12-31 | 2016-04-13 | 浙江圣氏生物科技有限公司 | Method for extracting bamboo leaf flavonoid from bamboo leaves |
| CN105921170A (en) * | 2016-05-03 | 2016-09-07 | 新疆大学 | Technical method for novel nano-scale FeZSM-5 catalyst for flue gas denitration |
| CN106478577A (en) * | 2016-10-09 | 2017-03-08 | 中国热带农业科学院热带作物品种资源研究所 | A kind of blumeatin and its process for separation and purification of analog |
| CN111420643A (en) * | 2020-04-15 | 2020-07-17 | 西安交通大学医学院第二附属医院 | Hydrophilic bifunctional monomer anthocyanin molecular imprinting magnetic nanospheres and preparation method and application thereof |
| CN113877552A (en) * | 2021-11-11 | 2022-01-04 | 江南大学 | Rice hull-based magnetic mesoporous silica adsorption material and solid-phase extraction column prepared from same |
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| CN103331150A (en) * | 2013-07-12 | 2013-10-02 | 中国科学院地理科学与资源研究所 | Cross-linked starch solid phase extraction column, and preparation method and application of solid phase extraction column |
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| CN105233801A (en) * | 2015-10-16 | 2016-01-13 | 江苏大学 | Preparation method of boron affiliated dual recognition molecularly imprinted material |
| CN105477433A (en) * | 2015-12-31 | 2016-04-13 | 浙江圣氏生物科技有限公司 | Method for extracting bamboo leaf flavonoid from bamboo leaves |
| CN105477433B (en) * | 2015-12-31 | 2019-03-19 | 浙江圣氏生物科技有限公司 | A method of extracting bamboo-leaves flavones from the leaf of bamboo |
| CN105921170A (en) * | 2016-05-03 | 2016-09-07 | 新疆大学 | Technical method for novel nano-scale FeZSM-5 catalyst for flue gas denitration |
| CN106478577A (en) * | 2016-10-09 | 2017-03-08 | 中国热带农业科学院热带作物品种资源研究所 | A kind of blumeatin and its process for separation and purification of analog |
| CN106478577B (en) * | 2016-10-09 | 2019-04-19 | 中国热带农业科学院热带作物品种资源研究所 | A kind of process for separation and purification of flavanones and its analogue |
| CN111420643A (en) * | 2020-04-15 | 2020-07-17 | 西安交通大学医学院第二附属医院 | Hydrophilic bifunctional monomer anthocyanin molecular imprinting magnetic nanospheres and preparation method and application thereof |
| CN111420643B (en) * | 2020-04-15 | 2022-09-13 | 西安交通大学医学院第二附属医院 | Hydrophilic bifunctional monomer anthocyanin molecular imprinting magnetic nanospheres and preparation method and application thereof |
| CN113877552A (en) * | 2021-11-11 | 2022-01-04 | 江南大学 | Rice hull-based magnetic mesoporous silica adsorption material and solid-phase extraction column prepared from same |
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Application publication date: 20120919 |