CN101246150A - Tsiklomitsin molecular engram integral column preparation method - Google Patents
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- CN101246150A CN101246150A CNA2008100525413A CN200810052541A CN101246150A CN 101246150 A CN101246150 A CN 101246150A CN A2008100525413 A CNA2008100525413 A CN A2008100525413A CN 200810052541 A CN200810052541 A CN 200810052541A CN 101246150 A CN101246150 A CN 101246150A
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Abstract
The invention provides a preparing method of Tetracycline molecular imprinting column. The invention includes the following steps: dissolving template molecular Tetracycline (TC) with ultrasound in methanol, adding functional monomer and pore-forming agent (cyclohexanol and lauryl alcohol), oscillating for 6 hours, adding crosslinking agent (ethylene glycol dimethacrylate) and initiator (azobisisobutyronitrile), after ultrasonic degassing, adding nitrogen for 20 minutes, deoxidizing, pouring the solution into 10cm stainless column with bottom sealed, sealing the top end, putting the column into 60 DEG C water for water bath, after 12 hours, obtaining the molecular imprinting column by heat initiation reaction. Connecting the column to the high-pressure pump for washing away the pore-forming agent and template molecular, in the end the molecular imprinting column with good separation effect is obtained. Compared with traditional bulk polymerization method, the invention has the characteristics of simple preparation for avoiding process of lapping, good molecular recognition ability and so on. The molecular imprinting column provided by the invention is used as a liquid chromatogram filling, and can realize separation, enrichment and purification of Tetracycline antibiotics.
Description
[technical field]:
The invention belongs to technical field of bioengineering, relate to the preparation of molecular engram integral column, specifically, relate to the preparation that TCs has the molecular engram integral column of specific recognition.
[background technology]:
TCs (tetracyclines, TCs) be a class broad-spectrum antibiotic that produces by streptomycete, as feed addictive, in livestock-raising, be used for preventing, controlling and treat the diseases such as disease and pest of aquatic products animal and plant, this just is easy to cause the medicament residue of animal derived food, so human health has been constituted potential harm.Many countries are to the residual enforcement routine monitoring of TCs, and the maximum residue limit of TCs is 0.1mg/kg in China's regulation animal derived food.
At present the teracycline antibiotic residues detection method mainly contains in the agricultural product: 1. microbial method: poor specificity, the sensitivity of detection is low, detect limit for height (＞3mg/kg), use seldom at present; 2. enzyme linked immunosorbent assay (Enzyme-Linked ImmunosorbentAssay, ELISA): be to carry out main, the most popular method of rapid screening at present, sensitivity higher (0.1-1 μ g/L), but the main dependence on import of detectable, the cost height can be suitable for scene, the fast detecting of some tractable samples (urine sample, blood etc.), but when other compound of containing in the sample with the microbiotic structure similar, false positive results may occur, have a strong impact on the result and judge.3. chromatography: wherein high performance liquid chromatography (High Performance Liquid Chromatography, HPLC) and vapor-phase chromatography (Gas Chromatography GC), is the teracycline antibiotic residues quantitative detection method of generally acknowledging in the world.But there is the sample pretreatment process complexity in these class methods, costs an arm and a leg, analysis speed waits shortcoming slowly, are not suitable for scene, fast detecting analysis.
Because agricultural product mesostroma complicated component, the most conventional detection method often can't accurately detect the trace antibiotic residue from these complex samples, directly cause method for quick such as ELISA problem, HPLC and GC isochrome spectrometries such as false positive height to occur and length consuming time (more than 3 days) occurs.Therefore, how to detect from complex sample that tetracycline is residual to have become important research project in the food safety detection work, wherein the pre-treatment of sample is the committed step during antibiotic residue detects in the food.Solid-Phase Extraction (solid-phase extraction is arranged, SPE) be a technology that is specifically designed to the sample purifying and concentrates that develops rapidly in recent years, being the mode standard of carrying out sample pretreatment in a lot of fields, also is the mainstream technology in the sample pre-treatments during present antibiotic residue detects.The core of SPE is a filler, and the filling kind that can be applicable to SPE at present mainly contains: acticarbon, silica gel silicic acid, magnesium aluminium oxide etc.Adsorbent in these conventional filler and the acting force between the target substance in the Solid-Phase Extraction are nonspecific, have therefore limited further developing of solid phase extraction techniques.
Molecular imprinting is a kind of bionical recognition methods of high selectivity, and advantages such as selectivity simple to operate with it, polymkeric substance is good, stable performance, low price are used for Solid-Phase Extraction, chromatographic column filler.The report of using it for the Solid-Phase Extraction TCs is also arranged before.As Caro, E. etc. have prepared terramycin (OTC) molecularly imprinted polymer and it has successfully been extracted TCs (Caro, the E. of animal tissue as the Solid-Phase Extraction materials with mass polymerization; Marc é, R.M.; Cormack, P.A.G.; Sherrington, D.C.; Borrull, F.Synthesis and application of an oxytetracycline imprinted polymer for the solid-phaseextraction of tetracycline antibiotics.Anal.Chim.Acta 2005,552,81-86).
Integral post (monolithic column) is to have drawn the quick separating power of nonporous packing and film and the high power capacity of HPLC porous aggregate, a kind of new and effective liquid chromatography stuffing that does not increase the advantage of these several respects of column resistance again and grow up, and molecular engram integral column (Molecular Imprinting Monolithic Column) has the advantage of molecular engram and integral post concurrently, have efficient, quick, single-minded characteristics, just in time can satisfy the requirement of complex sample pre-treatment.At present, be not the relevant report that template molecule prepares molecular engram integral column also with the TCs.
[summary of the invention]:
The conventional sense method that the objective of the invention is to solve most of teracycline antibiotic residues often can't accurately detect the trace antibiotic residue from complex sample, directly cause method for quick such as ELISA the false positive height to occur, and the problem of length consuming time (more than 3 days) appears in HPLC and GC isochrome spectrometry, a kind of preparation method of tetracycline molecularly imprinted polymer integral post is provided, this method step is simple, can be avoided loaded down with trivial details process of lapping, the imprinted polymer that the molecular recognition performance is good, directly can be used for off-line or online Solid-Phase Extraction pre-treatment, thereby reach the impurity of removing in the complex sample as novel liquid chromatography stuffing, the purpose that the selective enrichment trace amount tetracycline antibiotics is residual.
The preparation method of tetracycline molecular engram integral column provided by the invention may further comprise the steps:
The first, with the tetracycline ultrasonic dissolution in polar solvent, add then function monomer, pore-foaming agent mix, ultrasonic, the effect of leaving standstill formed compound in 4-6 hour;
The second, upwards add crosslinking chemical, initiating agent in the compound that the step forms, ultrasonic degas, the time is 5-10min; Logical nitrogen deoxygenation, the time is 20min; Mixed liquor is injected stainless steel column seal the after heat initiated polymerization;
Three, will go up synthetic molecular engram integral column of step and receive on the high pressure pump, and clean and remove pore-foaming agent and template molecule, obtain the tetracycline molecular engram integral column;
Wherein,
The described polar solvent of the first step is a methyl alcohol; Function monomer is a methacrylic acid; Pore-foaming agent is cyclohexanol and lauryl alcohol mixed liquor;
Second step, described crosslinking chemical was an ethylene glycol dimethacrylate; Initiating agent is an azoisobutyronitrile; Described hot initiation conditions is to react 12 hours in 60 ℃ of water-baths.
The mass ratio of the material of described template molecule, function monomer, crosslinking chemical is 1: 8: 35,1: 8: 40,1: 8: 45,1: 8: 50 or 1: 8: 60; The mass ratio of cyclohexanol and lauryl alcohol was respectively 11: 0,7: 4,6: 5,5: 6,4: 7,2: 9 or 0: 11.
Methanol usage is the minimum amount of solubilized template molecule, and the total consumption of pore-foaming agent is function monomer, crosslinking chemical cumulative volume 2 times, and initiator amount is 1% of a mixed liquor gross mass
Stainless steel column described in second step, column length is 10cm.
The 3rd step, described cleaning fluid was that methyl alcohol or volume ratio are acetonitrile/acetic acid solution of 94: 6.
Advantage of the present invention and good effect:
The tetracycline molecular engram integral column and the polymer phase ratio that traditional bulk polymerization obtains of the present invention's preparation have following advantage:
1, preparation process is simple, synthesizes in the stainless steel column to need not to grind promptly to can be used as the chromatographic stationary phase;
2, the overall fixed that obtains has macroporous structure can guarantee fast mass transfer rate and lower back pressure mutually;
3, the molecular recognition performance is good.
Consider that from using value tetracycline molecular engram integral column provided by the present invention can be realized separation, enrichment and purifying to TCs as a kind of liquid chromatography stuffing, overcomes the shortcoming of present general chromatographic detection method impurity serious interference.
[description of drawings]:
Fig. 1-5 is the molecular engram integral column SEM figure of different dosage of crosslinking agent.
[embodiment]:
Example 1.
At first with template molecule of tetracycline TC 0.1mmol ultrasonic dissolution in 20mmol methyl alcohol, add pore-foaming agent (cyclohexanol 1.10g) function monomer MAA 0.8mmol, make it to interact 6h to form compound, add crosslinking chemical EDMA 4mmol (TC: MAA: EDMA=1: 8: 40), initiator A IBN 20mg again, solution is through ultrasonic degas 5-10min, logical nitrogen deoxygenation 20min is injected into this mixed liquor in the stainless steel column of 10cm then, and heat causes 60 ℃ of water-bath 12h.The molecular engram integral column that makes received use methyl alcohol, acetonitrile/acetate on the high pressure pump respectively (94: 6, v/v) solution cleaned and removes pore-foaming agent and template molecule obtains polymkeric substance 1 (MIP1).
Example 2.
Add pore-foaming agent (lauryl alcohol 1.10g), other can get MIP2 with example 1.
Example 3.
Add pore-foaming agent (cyclohexanol 0.20g and lauryl alcohol 0.90g), other can get MIP3 with example 1.
Example 4.
Add pore-foaming agent (cyclohexanol 0.40g and lauryl alcohol 0.70g), other can get MIP4 with example 1.
Example 5.
Add pore-foaming agent (cyclohexanol 0.50g and lauryl alcohol 0.60g), other can get MIP5 (Fig. 2) with example 1.
Example 6.
Add pore-foaming agent (cyclohexanol 0.60g and lauryl alcohol 0.50g), other can get MIP6 with example 1.
Example 7
Add pore-foaming agent (cyclohexanol 0.70g and lauryl alcohol 0.40g), other can get MIP7 with example 1.
Example 8
Add crosslinking chemical EDMA 3.5mmol (TC: MAA: EDMA=1: 8: 35), other can get MIP8 (Fig. 1) with example 5.
Example 9
Add crosslinking chemical EDMA 4.5mmol (TC: MAA: EDMA=1: 8: 45), other can get MIP9 (Fig. 3) with example 5.
Example 10
Add crosslinking chemical EDMA 5.0mmol (TC: MAA: EDMA=1: 8: 50), other can get MIP10 (Fig. 4) with example 5.
Example 11
Add crosslinking chemical EDMA 6.0mmol (TC: MAA: EDMA=1: 8: 60), other can get MIP11 (Fig. 5) with example 5.
The molecular engram integral column SEM that obtains by different dosage of crosslinking agent schemes as can be seen, and along with the increase of degree of crosslinking, the integral post aperture that makes is more little, and post is pressed also high more simultaneously.
In order to verify the selection performance of contrast synthetic product, done following experiment:
Experiment 12.
With 0.5mL/min acetonitrile/acetate (98: 2, v/v) solution is as the separating effect of moving phase test MIP1-MIP9 to sulfamethoxazole (SMO) and tetracycline (TC); With 0.3mL/min acetonitrile/acetate (96: 4, v/v) solution is as the separating effect of moving phase test MIP10 to sulfamethoxazole (SMO) and tetracycline (TC); (90: 10, v/v) solution was as the separating effect of moving phase test MIP11 to sulfamethoxazole (SMO) and tetracycline (TC) with 0.1mL/min acetonitrile/acetate.
The imprinted polymer of each material consumption and different degree of crosslinking is to the separating effect of sulfamethoxazole (SMO) with tetracycline (TC) in table 1 polyreaction
Capacity factor measure (k ') is by formula k '=(t
R-t
0)/t
0Calculate, wherein t
RAnd t
0Represent the retention time of tetracycline (TC) and the dead time of system respectively; Separation factor (α) is by formula α=k '
TC/ k '
SMDCalculate, wherein k '
TCAnd k '
SMDIt is respectively the capacity factor measure of tetracycline (TC) and sulfamethoxazole (SMO).
Claims (5)
1. the preparation method of tetracycline molecular engram integral column is characterized in that, this method may further comprise the steps:
The first, with the tetracycline ultrasonic dissolution in polar solvent, add then function monomer, pore-foaming agent mix, ultrasonic, the effect of leaving standstill formed compound in 4-6 hour;
The second, upwards add crosslinking chemical, initiating agent in the compound that the step forms, ultrasonic degas, the time is 5-10min; Logical nitrogen deoxygenation, the time is 20min; Mixed liquor is injected stainless steel column seal the after heat initiated polymerization;
Three, will go up synthetic molecular engram integral column of step and receive on the high pressure pump, and clean and remove pore-foaming agent and template molecule, obtain the tetracycline molecular engram integral column;
Wherein,
The described polar solvent of the first step is a methyl alcohol; Function monomer is a methacrylic acid; Pore-foaming agent is cyclohexanol and lauryl alcohol mixed liquor;
Second step, described crosslinking chemical was an ethylene glycol dimethacrylate; Initiating agent is an azoisobutyronitrile; Described hot initiation conditions is to react 12 hours in 60 ℃ of water-baths.
2. method according to claim 1, the mass ratio that it is characterized in that the material of template molecule, function monomer, crosslinking chemical is 1: 8: 35,1: 8: 40,1: 8: 45,1: 8: 50 or 1: 8: 60; The mass ratio of cyclohexanol and lauryl alcohol was respectively 11: 0,7: 4,6: 5,5: 6,4: 7,2: 9 or 0: 11.
3. method according to claim 1 and 2 is characterized in that methanol usage is the minimum amount of dissolving template molecule, and the total consumption of pore-foaming agent is function monomer, crosslinking chemical cumulative volume 2 times, and initiator amount is 1% of a mixed liquor gross mass.
4. method according to claim 1 and 2 is characterized in that described cleaning fluid of the 3rd step is that methyl alcohol or volume ratio are acetonitrile/acetic acid solution of 94: 6.
5. method according to claim 1 and 2 is characterized in that the stainless steel column described in second step, and column length is 10cm.
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| CN101816927A (en) * | 2010-04-30 | 2010-09-01 | 南开大学 | Temperature-sensitive protein molecular engram monolithic column and preparation method and application thereof |
| CN101852791A (en) * | 2010-05-21 | 2010-10-06 | 天津出入境检验检疫局动植物与食品检测中心 | Method for preparing norfloxacin molecular imprinted monolithic column in ionic liquid |
| CN101857664A (en) * | 2010-06-21 | 2010-10-13 | 湖北出入境检验检疫局检验检疫技术中心 | Preparation method of molecular imprinting polymer with specific recognition capability to tetracycline family |
| CN101487822B (en) * | 2009-02-25 | 2012-10-24 | 中国科学院过程工程研究所 | L-phenylalanine analysis detection method based on molecular engram integral column |
| CN102116758B (en) * | 2009-12-30 | 2013-04-10 | 宁波大学 | Tetracycline medicament selective electrode and preparation method thereof |
| CN108421542A (en) * | 2018-03-22 | 2018-08-21 | 河南科技学院 | Application of the liquid metal microballoon as pore-foaming agent in preparing integral post |
| CN110078853A (en) * | 2019-05-20 | 2019-08-02 | 齐鲁工业大学 | Pickering emulsion and prepare tetracycline molecule trace charcoal complex microsphere |
| CN112007621A (en) * | 2020-06-23 | 2020-12-01 | 广东工业大学 | Preparation and application methods of tetracycline antibiotic multi-template molecularly imprinted magnetic composite material |
| CN115245690A (en) * | 2021-04-27 | 2022-10-28 | 中国科学院烟台海岸带研究所 | System and method for enriching organic small molecular compounds in water body |
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| CN101487822B (en) * | 2009-02-25 | 2012-10-24 | 中国科学院过程工程研究所 | L-phenylalanine analysis detection method based on molecular engram integral column |
| CN102116758B (en) * | 2009-12-30 | 2013-04-10 | 宁波大学 | Tetracycline medicament selective electrode and preparation method thereof |
| CN101816927B (en) * | 2010-04-30 | 2012-11-14 | 南开大学 | Temperature-sensitive protein molecular engram monolithic column and preparation method and application thereof |
| CN101816927A (en) * | 2010-04-30 | 2010-09-01 | 南开大学 | Temperature-sensitive protein molecular engram monolithic column and preparation method and application thereof |
| CN101852791B (en) * | 2010-05-21 | 2012-08-29 | 天津出入境检验检疫局动植物与食品检测中心 | Method for preparing norfloxacin molecular imprinted monolithic column in ionic liquid |
| CN101852791A (en) * | 2010-05-21 | 2010-10-06 | 天津出入境检验检疫局动植物与食品检测中心 | Method for preparing norfloxacin molecular imprinted monolithic column in ionic liquid |
| CN101857664A (en) * | 2010-06-21 | 2010-10-13 | 湖北出入境检验检疫局检验检疫技术中心 | Preparation method of molecular imprinting polymer with specific recognition capability to tetracycline family |
| CN108421542A (en) * | 2018-03-22 | 2018-08-21 | 河南科技学院 | Application of the liquid metal microballoon as pore-foaming agent in preparing integral post |
| CN108421542B (en) * | 2018-03-22 | 2020-08-07 | 河南科技学院 | Application of liquid metal microspheres as pore-forming agent in preparation of monolithic column |
| CN110078853A (en) * | 2019-05-20 | 2019-08-02 | 齐鲁工业大学 | Pickering emulsion and prepare tetracycline molecule trace charcoal complex microsphere |
| CN110078853B (en) * | 2019-05-20 | 2021-11-05 | 齐鲁工业大学 | Pickering emulsion and preparation of tetracycline molecular imprinting biochar composite microspheres |
| CN112007621A (en) * | 2020-06-23 | 2020-12-01 | 广东工业大学 | Preparation and application methods of tetracycline antibiotic multi-template molecularly imprinted magnetic composite material |
| CN115245690A (en) * | 2021-04-27 | 2022-10-28 | 中国科学院烟台海岸带研究所 | System and method for enriching organic small molecular compounds in water body |
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