CN106632529B - A kind of shell tetrose monomer separation extracting method based on molecular imprinting technology - Google Patents
A kind of shell tetrose monomer separation extracting method based on molecular imprinting technology Download PDFInfo
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- C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
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Abstract
The shell tetrose monomer separation extracting method based on molecular imprinting technology that the invention discloses a kind of, steps are as follows for the separating and extracting process: cloth-bag type filtering-nanofiltration membrane-shell tetrose molecular cngram resin absorption-shell tetrose molecular cngram resin elutes-removes zinc ion-freeze-drying.Shell tetrose monomer separation extracting method based on molecular imprinting technology of the invention compared with prior art, has the characteristics that separate process steps are simple, production cost is low, shell tetrose monomer purity is high, prepare with scale can be achieved.
Description
Technical field
The present invention relates to ocean oligosaccharide monomer separation purifying research fields, specifically a kind of to be based on molecular engram skill
The shell tetrose monomer separation extracting method of art.
Background technique
Oligosaccharides (oligosaccharides) and glycoconjugate are information substances important in organism, in life process
It is played an extremely important role during cell recognition, signal transduction and regulation.Chitosan oligosaccharide
(Chitooligosaccharides, COS) is by ocean chitin through product that is deacetylated, obtaining after degradation, generally
It is to be formed by 3-10 Glucosamine by β -1,4- glucosides key connection.Chitosan oligosaccharide molecular weight low energy is dissolved in water, and has excellent
In the bioactivity of chitosan, it is widely used in fields such as health care product, biochemical industry material and food.Ocean shell is few at present
There are the following problems for the separation preparation of sugar monomer: (1) purity is low.It is heavy using substep since chitosan oligosaccharide separation purifying technique is complicated
The conventional separation methods such as drop method and membrane separation process, it is difficult to obtain the chitooligose monomer of high-purity.Therefore, presently commercially available ocean shell
Oligosaccharide product is all largely the chitosan oligosaccharide mixture of low polymerization degree, constrains the high-valued development and utilization of chitosan oligosaccharide.(2) it produces
It measures small.Currently used exclusion chromatography, ion-exchange chromatography, polyacrylamide gel electrophoresis, cellulose acetate film electricity
The instruments separation side such as swimming method, high performance liquid chromatography and high performance capillary electrophoresis, quick preparative liquid chromatograph and perfusion chromatography
Method can only prepare a small amount of chitooligose monomer (usually in mg grades of levels), it is difficult to realize the scale separation system of chitooligose monomer
It is standby.(3) price.High-purity ocean oligosaccharide monomer (polymerization degree n=4 ~ 6) is, price grade packaged with mg currently on the market
It is expensive.Such as the shell tetrose of high-purity is to six sugar monomer of shell, 1500 ~ 1600 yuan/10mg of selling price or so.Therefore, high-purity
Different polymerization degree chitooligose monomer prepare with scale, become restrict its application and development technical bottleneck.Find new separation
Methods and techniques, and realize that its prepare with scale is one of the difficulties of chitosan oligosaccharide development and utilization.In conclusion due to shell widow
The otherness of physicochemical properties and chemical structure is very small between sugar monomer (n=4 ~ 6), and conventional separation and extraction technology exists
Its extraction field carries out large-scale promotion use and is restricted, and therefore, chitooligose monomer scale separation and Extraction is difficult, becomes system
The about technical bottleneck of its application and development.New separation method and technology are found, and realizes that its prepare with scale is chitooligose monomer
One of difficulties of development and utilization.
Molecularly imprinted polymer (MIP) is a kind of new separation material for being based on molecular imprinting technology (MIT), main to use
In the separation, purification and concentration sample of biology or environmental sample complicated and that pretreatment formality is many and diverse.MIP preparation is convenient, is a kind of
Cheap efficiently separates material, it is easy to accomplish the preparative-scale with gram-grade (or even kilogram grade) becomes to complicated body
It is the emerging research direction of separation and Extraction [1,2].With the continuous development of MIT, recent domestic researcher is successfully
The technology is used for the separating-purifying of marine natural products.For example, the Wang little Ru project of Oceanographic Inst. No.1 of State Bureau of Oceanography
MIT is used for the separation and Extraction of marine microorganism alkaloid activity ingredient by group, achieves good separating effect [3].Japanology
MIT is used for the chromatographic isolation of seashells toxin domoic acid by person Takuya Kubo et al. [4], is extracted from toxic shellfish
Saxitoxin domoic acid.For the separation and measurement new method for exploring sugar, MIT is extended to carbohydrate chemistry research again by domestic and foreign scholars
Field.For example, Zhiliang Cheng [5], Yiqun Yang [6] and Manju [7] etc. are by MIT and electrochemical analysis, fluorescence
The technologies such as analysis combine, the separation and measurement for monosaccharide；The Parmpi [8] etc. in the U.S. is prepared for MIP water-setting using MIT
Glue, separation and identification for monosaccharide such as glucose；Sineriz [9] research team of France, with glucose -6-O- sulfate
For template, the application that MIT is identified in glucose -6-O- sulfate is had studied；Okutucu [10] of Turkey etc. is closed using MIT
At with the galactolipin MIP of non-covalent bond recognition mode, it to be used for glucose, mannose, fructose, maltose, lactose, sucrose and cotton
Sugared Selective recognition below the trisaccharides such as sub- sugar achieves good achievement in terms of the Study of recognition of sugar.MIT is led in carbohydrate chemistry
These successful applications in domain, provide important theoretical foundation and technical support for the separation of ocean oligosaccharide monomer.Take a broad view of state
Inside and outside present Research, template molecule are largely trisaccharide (such as gossypose) monosaccharide below and disaccharide, the above oligosaccharides of trisaccharide
The research of separation and identification is rarely reported, and MIT not yet appears in the newspapers in the Separation Research of ocean oligosaccharide.
Summary of the invention
Technical assignment of the invention be to provide a kind of low, preparation the shell tetrose monomer product purity is high of production cost based on
The shell tetrose monomer separation extracting method of molecular imprinting technology.
Technical assignment of the invention realizes that steps are as follows for the separating and extracting process in the following manner:
1) cloth-bag type filters: Enzymatic Hydrolysis of Chitosan liquid being filtered with cloth-bag type filter, and stands clarification 20min, is removed
Remove the granule foreign and insoluble matter in Enzymatic Hydrolysis of Chitosan liquid；
2) nanofiltration membrane: the filtered liquid of cloth-bag type filter is filtered with nanofiltration membrane G5, obtains average mark
Son amount is less than the chitosan oligosaccharide mixed component permeate of 1000DA；
3) shell tetrose molecular cngram resin adsorbs: chitosan oligosaccharide mixed component permeate is flowed through equipped with shell tetrose molecular engram
The resin column of resin realizes four sugar monomer of shell using the shell tetrose in shell tetrose molecular cngram resin selective absorption permeate
With the separation of other chitosan oligosaccharide components；
4) shell tetrose molecular cngram resin elutes: after shell tetrose molecular cngram resin adsorption saturation, being washed with eluant, eluent
It is de-；
5) it removes zinc ion: being filtered after eluent is handled with active carbon G-60, so that shell tetrose and zinc ion separation, shell
Tetrose is tightly held by activated carbon；Active carbon is collected, then shell tetrose is eluted with ethyl alcohol and the mixed liquor of deionized water, is used in combination
Hydrochloric acid solution adjusts solution ph to 2-4；
6) it is freeze-dried: collecting shell tetrose eluent, after vacuum concentration freeze-drying, obtain shell tetrose monomer product.
The synthetic method of shell tetrose molecular cngram resin is as follows in the step 2):
The weight proportion of each raw material in shell tetrose molecular cngram resin are as follows: 65-95 parts of main monomer；Monomer 5-15 parts of coordination；
5-9 parts of pore-foaming agent；0.5-1.5 parts of dispersing agent；0.05-0.3 parts of initiator；
Coordination monomer and initiator are dissolved in dehydrated alcohol by weight ratio, so that coordination monomer and initiator are in nothing
Mass percent concentration in water-ethanol is 10-20%, and then by mixed liquor and main monomer, pore-foaming agent is uniformly mixed；Strongly stirring
It mixes down, this mixed system is added slowly in the three-necked flask containing dispersing agent, be passed through 30 min of nitrogen deoxygenation；With water bath with thermostatic control plus
Heat regulates and controls mixing speed to 72-78 DEG C, and polymerization reaction 6h then heats to 80-90 DEG C and is further continued for reaction 3h；It is cooled to 20-30 DEG C
Afterwards, polymer microballoon is filtered, washed to obtain molecular cngram resin microballoon；The molecular cngram resin microballoon of preparation is packed into extraction
In device, with dehydrated alcohol extraction processing 6h, pore-foaming agent, initiator and unreacted a small amount of residual monomer are removed, after room temperature airing,
Immersion treatment is stirred with eluent solution, washes away imprinted templates molecule and zinc ion, then is washed with deionized water to pH value to 6-
8, it after then being balanced with the zinc ion solution coordination of 0.01mol/L, and is dried in vacuo at 50-70 DEG C, four glycan molecule of shell print is made
Mark resin microsphere.
The coordination monomer is N- [2- (dimethylamino) ethyl]-N'- [(2- hydroxyl -4- ethenylphenyl)] oxamides
Double-core zinc-shell tetrose, main monomer are ethylene glycol dimethacrylate, and pore-foaming agent is petroleum ether 90-120, and initiator is azo
Bis-isobutyronitrile, dispersing agent are polyvinyl alcohol.
The eluant, eluent is the hydrochloric acid solution of 0.1 mol/L.
Ethyl alcohol and the mass ratio of deionized water are 1:1 in the mixed liquor of the ethyl alcohol and deionized water.
Compared to the prior art a kind of shell tetrose monomer separation extracting method based on molecular imprinting technology of the invention, has
There are following characteristics:
1) shell tetrose molecular cngram resin absorbing process, the highly selective separation and Extraction shell four from Enzymatic Hydrolysis of Chitosan liquid are used
The method of sugar monomer, with other water-soluble chitosan oligosaccharide extractive technique (number of patent application 201310660738；201310019782；
201310270184) it compares, realizes the separation of target isolate shell four sugar monomers and other chitosan oligosaccharide components, prepared shell
Tetrose monomer product purity is high.
2) shell tetrose molecular cngram resin absorbing process, four sugar monomer of separation and Extraction shell from Enzymatic Hydrolysis of Chitosan liquid are used
Method, with other chitooligose monomer isolation technics (number of patent application 201110068951；201210146042) horizontal phase is prepared
Than, it can be achieved that kilogram grade prepare with scale.
3) shell tetrose molecular cngram resin absorbing process, four sugar monomer of separation and Extraction shell from Enzymatic Hydrolysis of Chitosan liquid are used
Method, separation-extraction technology is not only simple, but also preparation yield is big, and shell tetrose monomer product production cost is low, the market competitiveness
By force.
Detailed description of the invention
Attached drawing 1 is that the HPLC of Enzymatic Hydrolysis of Chitosan product analyzes map.
Attached drawing 2 is the HPLC map for making four sugar monomer of shell by oneself.
Attached drawing 3 is the MALDI-TOF-MS map of shell tetrose monomer standard product.
Attached drawing 4 is the MALDI-TOF-MS map for making shell tetrose monomer sample by oneself.
Specific embodiment
Embodiment 1:
Stock: 65kg ethylene glycol dimethacrylate (EGDMA) is taken；5kg N- [2- (dimethylamino) ethyl]-N'-
[(2- hydroxyl -4- ethenylphenyl)] oxamides double-core zinc-shell tetrose；5kg petroleum ether 90-120；0.5kg polyvinyl alcohol
(PVA)；0.05kg azodiisobutyronitrile (AIBN)；
Prepare shell tetrose molecular cngram resin: by N- [2- (dimethylamino) ethyl]-N'- [(2- hydroxyl -4- vinyl benzene
Base)] oxamides double-core zinc-shell tetrose and azodiisobutyronitrile be dissolved in dehydrated alcohol by weight ratio, so that N- [2- (two
Methylamino) ethyl]-N'- [(2- hydroxyl -4- ethenylphenyl)] oxamides double-core zinc-shell tetrose and azodiisobutyronitrile be in nothing
Mass percent concentration in water-ethanol is 10%, then by mixed liquor and ethylene glycol dimethacrylate, petroleum ether 90-120
It is uniformly mixed；Under strong stirring, this mixed system is added slowly in the three-necked flask containing polyvinyl alcohol, is passed through nitrogen deoxygenation
30 min；72 DEG C are heated to water bath with thermostatic control, regulates and controls mixing speed, polymerization reaction 6h then heats to 80 DEG C and is further continued for reacting
3h；After being cooled to 20 DEG C, polymer microballoon is filtered, washed to obtain molecular cngram resin microballoon；By the molecular cngram resin of preparation
Microballoon is fitted into extractor, with dehydrated alcohol extraction processing 6h, removes petroleum ether 90-120, azodiisobutyronitrile and unreacted
A small amount of residual monomer after room temperature airing, stirs immersion treatment with the hydrochloric acid solution of 0.1 mol/L, washes away imprinted templates molecule
And zinc ion, then be washed with deionized water to pH value to 6, it is then coordinated with the zinc ion solution of 0.01mol/L after balancing, and 50
DEG C vacuum drying, be made shell tetrose molecular cngram resin microballoon, it is spare.
Steps are as follows for separation and Extraction shell tetrose monomer process:
1) cloth-bag type filters: the Enzymatic Hydrolysis of Chitosan liquid 100kg of mass percentage content 5% is carried out with cloth-bag type filter
Filtering, and clarification 20min is stood, remove the granule foreign and insoluble matter in Enzymatic Hydrolysis of Chitosan liquid；
2) nanofiltration membrane: the filtered liquid of cloth-bag type filter is filtered with nanofiltration membrane G5, obtains average mark
Son amount is less than the chitosan oligosaccharide mixed component permeate of 1000DA；
3) shell tetrose molecular cngram resin adsorbs: chitosan oligosaccharide mixed component permeate is flowed through equipped with four sugar of 100kg shell
The resin column of sub- trace resin, it is vertical l/h of control sample introduction flow velocity 30, saturating using shell tetrose molecular cngram resin selective absorption
The shell tetrose in liquid is crossed, realizes the separation of four sugar monomer of shell and other chitosan oligosaccharide components；
4) shell tetrose molecular cngram resin elutes: after shell tetrose molecular cngram resin adsorption saturation, with 25kg0.1 mol/L
Hydrochloric acid solution eluted, vertical l/h of control elution flow rate 20 is collected into 25 kilograms of eluent of the tetrose containing shell；
5) it removes zinc ion: being filtered after eluent is handled with 4kg active carbon G-60, so that shell tetrose and zinc ion point
From shell tetrose is tightly held by activated carbon；Active carbon is collected, is then eluted shell tetrose with 10kg ethyl alcohol and the mixed liquor of deionized water
Get off, the mass ratio of ethyl alcohol and deionized water is 1:1, and adjusts solution ph to 2 with hydrochloric acid solution；
6) it is freeze-dried: collecting shell tetrose eluent, after vacuum concentration freeze-drying, obtain 1.2kg shell tetrose monomer product,
Four sugar monomer recovery rate 1.2% of shell, product purity 93.6%.
Embodiment 2:
Stock: 80kg ethylene glycol dimethacrylate (EGDMA) is taken；10kg N- [2- (dimethylamino) ethyl]-N'-
[(2- hydroxyl -4- ethenylphenyl)] oxamides double-core zinc-shell tetrose；7kg petroleum ether 90-120；1kg polyvinyl alcohol (PVA)；
0.15kg azodiisobutyronitrile (AIBN)；
Prepare shell tetrose molecular cngram resin: by N- [2- (dimethylamino) ethyl]-N'- [(2- hydroxyl -4- vinyl benzene
Base)] oxamides double-core zinc-shell tetrose and azodiisobutyronitrile be dissolved in dehydrated alcohol by weight ratio, so that N- [2- (two
Methylamino) ethyl]-N'- [(2- hydroxyl -4- ethenylphenyl)] oxamides double-core zinc-shell tetrose and azodiisobutyronitrile be in nothing
Mass percent concentration in water-ethanol is 15%, then by mixed liquor and ethylene glycol dimethacrylate, petroleum ether 90-120
It is uniformly mixed；Under strong stirring, this mixed system is added slowly in the three-necked flask containing polyvinyl alcohol, is passed through nitrogen deoxygenation
30 min；75 DEG C are heated to water bath with thermostatic control, regulates and controls mixing speed, polymerization reaction 6h then heats to 85 DEG C and is further continued for reacting
3h；After being cooled to 25 DEG C, polymer microballoon is filtered, washed to obtain molecular cngram resin microballoon；By the molecular cngram resin of preparation
Microballoon is fitted into extractor, with dehydrated alcohol extraction processing 6h, removes petroleum ether 90-120, azodiisobutyronitrile and unreacted
A small amount of residual monomer after room temperature airing, stirs immersion treatment with the hydrochloric acid solution of 0.1 mol/L, washes away imprinted templates molecule
And zinc ion, then be washed with deionized water to pH value to 7, it is then coordinated with the zinc ion solution of 0.01mol/L after balancing, and 60
DEG C vacuum drying, be made shell tetrose molecular cngram resin microballoon, it is spare.
Steps are as follows for separation and Extraction shell tetrose monomer process:
1) cloth-bag type filters: the Enzymatic Hydrolysis of Chitosan liquid 100kg of mass percentage content 6% is carried out with cloth-bag type filter
Filtering, and clarification 20min is stood, remove the granule foreign and insoluble matter in Enzymatic Hydrolysis of Chitosan liquid；
2) nanofiltration membrane: the filtered liquid of cloth-bag type filter is filtered with nanofiltration membrane G5, obtains average mark
Son amount is less than the chitosan oligosaccharide mixed component permeate of 1000DA；
3) shell tetrose molecular cngram resin adsorbs: chitosan oligosaccharide mixed component permeate is flowed through equipped with four sugar of 100kg shell
The resin column of sub- trace resin, it is vertical l/h of control sample introduction flow velocity 30, saturating using shell tetrose molecular cngram resin selective absorption
The shell tetrose in liquid is crossed, realizes the separation of four sugar monomer of shell and other chitosan oligosaccharide components；
4) shell tetrose molecular cngram resin elutes: after shell tetrose molecular cngram resin adsorption saturation, with 25kg0.1 mol/L
Hydrochloric acid solution eluted, vertical l/h of control elution flow rate 20 is collected into 25 kilograms of eluent of the tetrose containing shell；
5) it removes zinc ion: being filtered after eluent is handled with 4kg active carbon G-60, so that shell tetrose and zinc ion point
From shell tetrose is tightly held by activated carbon；Active carbon is collected, is then eluted shell tetrose with 10kg ethyl alcohol and the mixed liquor of deionized water
Get off, the mass ratio of ethyl alcohol and deionized water is 1:1, and adjusts solution ph to 3 with hydrochloric acid solution；
6) it is freeze-dried: collecting shell tetrose eluent, after vacuum concentration freeze-drying, obtain 1.3kg shell tetrose monomer product,
Four sugar monomer recovery rate 1.3% of shell, product purity 94.5%.
Embodiment 3:
Stock: 95kg ethylene glycol dimethacrylate (EGDMA) is taken；15kg N- [2- (dimethylamino) ethyl]-N'-
[(2- hydroxyl -4- ethenylphenyl)] oxamides double-core zinc-shell tetrose；9kg petroleum ether 90-120；1.5kg polyvinyl alcohol
(PVA)；0.3kg azodiisobutyronitrile (AIBN)；
Prepare shell tetrose molecular cngram resin: by N- [2- (dimethylamino) ethyl]-N'- [(2- hydroxyl -4- vinyl benzene
Base)] oxamides double-core zinc-shell tetrose and azodiisobutyronitrile be dissolved in dehydrated alcohol by weight ratio, so that N- [2- (two
Methylamino) ethyl]-N'- [(2- hydroxyl -4- ethenylphenyl)] oxamides double-core zinc-shell tetrose and azodiisobutyronitrile be in nothing
Mass percent concentration in water-ethanol is 20%, then by mixed liquor and ethylene glycol dimethacrylate, petroleum ether 90-120
It is uniformly mixed；Under strong stirring, this mixed system is added slowly in the three-necked flask containing polyvinyl alcohol, is passed through nitrogen deoxygenation
30 min；78 DEG C are heated to water bath with thermostatic control, regulates and controls mixing speed, polymerization reaction 6h then heats to 90 DEG C and is further continued for reacting
3h；After being cooled to 30 DEG C, polymer microballoon is filtered, washed to obtain molecular cngram resin microballoon；By the molecular cngram resin of preparation
Microballoon is fitted into extractor, with dehydrated alcohol extraction processing 6h, removes petroleum ether 90-120, azodiisobutyronitrile and unreacted
A small amount of residual monomer after room temperature airing, stirs immersion treatment with the hydrochloric acid solution of 0.1 mol/L, washes away imprinted templates molecule
And zinc ion, then be washed with deionized water to pH value to 8, it is then coordinated with the zinc ion solution of 0.01mol/L after balancing, and 70
DEG C vacuum drying, be made shell tetrose molecular cngram resin microballoon, it is spare.
Steps are as follows for separation and Extraction shell tetrose monomer process:
1) cloth-bag type filters: the Enzymatic Hydrolysis of Chitosan liquid 100kg of mass percentage content 7% is carried out with cloth-bag type filter
Filtering, and clarification 20min is stood, remove the granule foreign and insoluble matter in Enzymatic Hydrolysis of Chitosan liquid；
2) nanofiltration membrane: the filtered liquid of cloth-bag type filter is filtered with nanofiltration membrane G5, obtains average mark
Son amount is less than the chitosan oligosaccharide mixed component permeate of 1000DA；
3) shell tetrose molecular cngram resin adsorbs: chitosan oligosaccharide mixed component permeate is flowed through equipped with four sugar of 100kg shell
The resin column of sub- trace resin, it is vertical l/h of control sample introduction flow velocity 30, saturating using shell tetrose molecular cngram resin selective absorption
The shell tetrose in liquid is crossed, realizes the separation of four sugar monomer of shell and other chitosan oligosaccharide components；
4) shell tetrose molecular cngram resin elutes: after shell tetrose molecular cngram resin adsorption saturation, with 25kg0.1 mol/L
Hydrochloric acid solution eluted, vertical l/h of control elution flow rate 20 is collected into 25 kilograms of eluent of the tetrose containing shell；
5) it removes zinc ion: being filtered after eluent is handled with 4kg active carbon G-60, so that shell tetrose and zinc ion point
From shell tetrose is tightly held by activated carbon；Active carbon is collected, is then eluted shell tetrose with 10kg ethyl alcohol and the mixed liquor of deionized water
Get off, the mass ratio of ethyl alcohol and deionized water is 1:1, and adjusts solution ph to 4 with hydrochloric acid solution；
6) it is freeze-dried: collecting shell tetrose eluent, after vacuum concentration freeze-drying, obtain 1.4kg shell tetrose monomer product,
Four sugar monomer recovery rate 1.4% of shell, product purity 95.1%.
The analysis detection of four sugar monomer of shell is tested
1. laboratory apparatus
The Breeze HPLC of Waters company, 301 type evaporative light dispersion dispersions；The Asahipak of Shodex company
NH2P-50 4E column；
MALDI-TOF-MS detecting instrument are as follows: the Autoflex of Bruker companyTOF MS；
2. chromatographic condition
Shell tetrose and mixed shell oligosaccharide content use high effective liquid chromatography for measuring, and measuring method is as follows:
The setting of HPLC analysis condition: chromatographic column, Asahipak NH2P-50 4E column (mm/L of 4.6mm ID × 250,
Shodex company)；Mobile phase, φ=75/25,1.0 mL/min of flow velocity；Detector, 301 type evaporative light dispersion dispersions,
30 DEG C of column temperature.
3. HPLC analyzes result
The HPLC of 3.1 enzymolysis products analyzes result
Fig. 1 chromatogram the result shows that, using Shodex NH2P-50 4E nh 2 column (Asahipak) this test condition
Under, the chitosan oligosaccharide of different polymerization degree can be separated, can substantially be calculated in enzymolysis product from peak height and integral area substantially
3-6 sugar accounts for the overwhelming majority of product, wherein 4 sugar, 5 saccharic compositions are most.
3.2 prepare the HPLC map of four sugar monomer of shell
The testing result of homemade shell tetrose is shown in Fig. 2.From Figure 2 it can be seen that under this experiment condition, four oligosaccharide monomer appearance of shell
Sharply, peak type is beautiful, and impurity is obvious by several times.Compare testing result, shell tetrose purity is high.
4. preparing the MALDI-TOF-MS map of four sugar monomer of shell
The molecular weight for losing the molecular formula of hydrochloride is shown in chitosan monomer hydrochloride in Mass Spectrometer Method.Figure
Four saccharide band of shell, 4 hydrochlorides in 3, mass spectrum (ESI positive ion mode) be shown [662+H]+(808-36.5*4=
662) the 663 of display, that is, is printed.Separated obtained sample to be tested is shown identical with four saccharide of shell in Fig. 4
Molecular weight.
The technical personnel in the technical field can readily realize the present invention with the above specific embodiments,.But it answers
Work as understanding, the present invention is not limited to above-mentioned several specific embodiments.On the basis of the disclosed embodiments, the technology
The technical staff in field can arbitrarily combine different technical features, to realize different technical solutions.
Claims (2)
1. a kind of shell tetrose monomer separation extracting method based on molecular imprinting technology, which is characterized in that the separating and extracting process
Steps are as follows:
1) cloth-bag type filters: Enzymatic Hydrolysis of Chitosan liquid being filtered with cloth-bag type filter, and stands clarification 20min, removes decladding
Granule foreign and insoluble matter in glycan enzymolysis liquid；
2) nanofiltration membrane: the filtered liquid of cloth-bag type filter is filtered with nanofiltration membrane G5, obtains average molecular weight
Chitosan oligosaccharide mixed component permeate less than 1000DA；
3) shell tetrose molecular cngram resin adsorbs: chitosan oligosaccharide mixed component permeate is flowed through equipped with shell tetrose molecular cngram resin
Resin column realize four sugar monomer of shell and its using the shell tetrose in shell tetrose molecular cngram resin selective absorption permeate
The separation of its chitosan oligosaccharide component；
4) shell tetrose molecular cngram resin elutes: after shell tetrose molecular cngram resin adsorption saturation, being eluted with eluant, eluent；
5) it removes zinc ion: being filtered after eluent is handled with active carbon G-60, so that shell tetrose and zinc ion separation, shell tetrose
It is tightly held by activated carbon；Active carbon is collected, is then eluted shell tetrose with ethyl alcohol and the mixed liquor of deionized water, and use hydrochloric acid
Solution adjusts solution ph to 2-4；
6) it is freeze-dried: collecting shell tetrose eluent, after vacuum concentration freeze-drying, obtain shell tetrose monomer product；
The synthetic method of the shell tetrose molecular cngram resin is as follows:
The weight proportion of each raw material in shell tetrose molecular cngram resin are as follows: 65-95 parts of main monomer；Monomer 5-15 parts of coordination；Pore
5-9 parts of agent；0.5-1.5 parts of dispersing agent；0.05-0.3 parts of initiator；
Coordination monomer and initiator are dissolved in dehydrated alcohol by weight ratio, so that coordination monomer and initiator are in anhydrous second
Mass percent concentration in alcohol is 10-20%, and then by mixed liquor and main monomer, pore-foaming agent is uniformly mixed；In strong stirring
Under, this mixed system is added slowly in the three-necked flask containing dispersing agent, 30 min of nitrogen deoxygenation is passed through；It is heated with water bath with thermostatic control
To 72-78 DEG C, regulate and control mixing speed, polymerization reaction 6h then heats to 80-90 DEG C and is further continued for reaction 3h；It is cooled to 20-30 DEG C
Afterwards, polymer microballoon is filtered, washed to obtain molecular cngram resin microballoon；The molecular cngram resin microballoon of preparation is packed into extraction
In device, with dehydrated alcohol extraction processing 6h, pore-foaming agent, initiator and unreacted a small amount of residual monomer are removed, after room temperature airing,
Immersion treatment is stirred with eluent solution, washes away imprinted templates molecule and zinc ion, then is washed with deionized water to pH value to 6-
8, it after then being balanced with the zinc ion solution coordination of 0.01mol/L, and is dried in vacuo at 50-70 DEG C, four glycan molecule of shell print is made
Mark resin microsphere；
The coordination monomer is N- [2- (dimethylamino) ethyl]-N'- [(2- hydroxyl -4- ethenylphenyl)] oxamides double-core
Zinc-shell tetrose, main monomer are ethylene glycol dimethacrylate, and pore-foaming agent is petroleum ether 90-120, and initiator is that azo two is different
Butyronitrile, dispersing agent are polyvinyl alcohol；The eluant, eluent is the hydrochloric acid solution of 0.1 mol/L.
2. a kind of shell tetrose monomer separation extracting method based on molecular imprinting technology according to claim 1, feature
It is, ethyl alcohol and the mass ratio of deionized water are 1:1 in the mixed liquor of the ethyl alcohol and deionized water.
Priority Applications (1)
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| CN111175360B (en) * | 2020-02-24 | 2022-03-04 | 扬州工业职业技术学院 | Preparation method of Sudan red-I molecularly imprinted sensor based on secondary polymerization |
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