CN102266758B - Separation and purification medium with saccharosan molecule serving as derivative and preparation method of separation and purification medium - Google Patents
Separation and purification medium with saccharosan molecule serving as derivative and preparation method of separation and purification medium Download PDFInfo
- Publication number
- CN102266758B CN102266758B CN 201110147824 CN201110147824A CN102266758B CN 102266758 B CN102266758 B CN 102266758B CN 201110147824 CN201110147824 CN 201110147824 CN 201110147824 A CN201110147824 A CN 201110147824A CN 102266758 B CN102266758 B CN 102266758B
- Authority
- CN
- China
- Prior art keywords
- ficoll
- aglucon
- 1mmol
- coupling
- separation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention relates to the field of separation and purification, in particular to a separation and purification medium with a saccharosan molecule serving as a derivative and a preparation method of the separation and purification medium. The preparation method comprises the following steps of: 1) activating derived sites on a substrate to obtain an activated substrate; 2) coupling saccharosan with the activated substrate; 3) activating the hydroxide radical of the saccharosan coupled with the activated substrate; and 4) coupling aglucon with the activated hydroxide radical of the saccharosan so as to obtain the separation and purification medium with the saccharosan molecule serving as the derivative. Compared with a chromatographic medium with other polymers as derivatives, the separation and purification medium with the saccharosan molecule serving as the derivative has the advantages of: (1) effectively increasing the surface area, along with (2) rich derived sites, (3) high mass transfer rate, (4) cluster effect and the like.
Description
Technical field
The present invention relates to the separation and purification field, particularly, the present invention relates to a kind of separation and purification medium take the ficoll molecule as extension and preparation method thereof.
Background technology
Separating and purifying technology is one of step of most critical in the biological product production process, accounts for the 60-90% of totle drilling cost.In the past few decades, due to the development of biotechnology, the productive rate of biological product improves constantly.For the raising of the balance biological product level of production, require the downstream purification technology to need that also corresponding raising is arranged.
In separating and purifying technology, medium is the basic factor that affects separating resulting.The optimal design of medium need to be taken into account Static Adsorption ability, mass transfer rate and flow behavior usually.Yet in multiple situation, fixedly phase behaviour affects these character in opposite mode.For example, sephadex shows higher Static Adsorption ability and mass transfer rate, but flow behavior relatively poor (E.Boschetti, J.Chromatogr.A 658 (1994) 207; M.Leonard, J.Chromatogr.B 699 (1997) 3); On the contrary, the macropore rigid particles shows comparatively faster mass transfer rate and good flow behavior, but common Static Adsorption ability general (A.Jungbauer, J.Chromatogr.A 1065 (2005) 3).
Therefore, have the composite of rigid porous structure by considering Static Adsorption ability, mass transfer rate and flow behavior design, thereby larger operating flexibility is provided.By at rigidity porous matrix surface grafting polymerization thing, then described polymer is carried out functionalizedly can preparing above-mentioned composite.Available polymer-groundmass composite material comprises glucan-agarose composite, as SP-Sepharose-XL (GE Healthcare, Piscataway, NJ), it shows high protein binding ability and adsorption dynamics adsorption kinetics (A.Staby between particle faster, I.H.Jensen, J.Chromatogr.A 908 (2001) 149; Y.Yao, A.M.Lenhoff, J.Chromatogr.A 1126 (2006) 107.).Yet the adsorption capacity that the people's such as Stone (G.Carta, J.Chromatogr.A 1216 (2009) 4465 for M.C.Stone, Y.Tao) result of study shows glucan-agarose Ion Exchange Medium is fast reducing with the rising of salinity.This situation is to cause due to shielding construction that the loose linear structure of glucan on dielectric surface forms under certain salinity, this screen layer can be got rid of large molecule outside the glucan layer under high ionic strength, therefore, such medium can only use (B.D.Bowes in limited salinity scope, H.Koku, K.J.Czymmek, A.M.Lenhoff, J.Chromatogr.A 1216 (2009) 7774).Therefore, above-mentioned situation shows, using linear polymer molecule (as glucan) is inappropriate as the grafting of composite in some cases.Proposed to improve the method for fluid bed medium Dynamic Adsorption amount in US Patent No. 6428707 B1, wherein the extension (extender) that is connected on matrix of the method utilization connects aglucon, thereby has improved mass transfer rate and total adsorbance.Yet for making with extra care of biological product, the resolution ratio of fluidised bed chromatography is inadequate.Therefore, still need to design a kind of polymer-groundmass composite material that can be used for the fixed bed chromatogram.
Ficoll is the large molecule that sucrose passes through the formed height cladodification of polymerization of epoxychloropropane.It is fully non-ionic, comprises abundant hydroxyl, therefore has high hydrophily and very easily water-soluble (can obtain the concentration of 50% (w/v) in water).The height cladodification make ficoll have spherical structure, its surface coverage highdensity hydroxyl.Show crosslinked size and the configuration that makes derivatization can not affect ficoll of neutrality or carboxy methylation ficoll by exclusion chromatography, light scattering and viscosimetric analysis etc.The molecular diameter of ficoll molecule commonly used is between a few nanometer to tens nanometers.
Therefore, propose in the present invention with the ficoll molecule as extension, for the preparation of the novel chromatographic media of fixed bed chromatogram.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of the separation and purification medium take the ficoll molecule as extension.
A further object of the present invention has been to provide a kind of separation and purification medium take the ficoll molecule as extension.
According to the preparation method of the separation and purification medium take the ficoll molecule as extension of the present invention, the method comprises the following steps:
1) but the derivatization site on activated substrate obtains activated substrate;
2) ficoll is coupled on activated substrate;
3) hydroxyl of the ficoll of activation and matrix coupling;
4) with the activation hydroxyl coupling of aglucon and ficoll, obtain the separation and purification medium take the ficoll molecule as extension.
Preparation method according to the separation and purification medium take the ficoll molecule as extension of the present invention, described step 1) mesostroma is to be selected from a kind of in agarose based substrate and magnetic-particle matrix, matrix all needs activation when using, all need the lip-deep hydroxyl of activated media, obtain after matrix activation can with the avtive spot of the hydroxyl coupling of ficoll, for example after matrix adopts Epichlorohydrin activation, obtain the active epoxy base with the hydroxyl coupling of ficoll;
According to the preparation method of the separation and purification medium take the ficoll molecule as extension of the present invention, described step 1) in, matrix is agarose based substrate or magnetic-particle matrix.
According to the preparation method of the separation and purification medium take the ficoll molecule as extension of the present invention, described step 1) in, but the method in the derivatization site on activated substrate is:
Cyanogen bromide method, the pH of priming reaction are 9～12, the mass ratio between activating reagent and matrix 1: 5～3: 1,
Or
The allyl glycidyl ether method adopts the NaOH solution of 10g/100ml～50g/100ml as reaction media in priming reaction, the volume mass between activating reagent and matrix is than 1ml: 1g～10ml: 1g,
Or
The allyl bromide, bromoallylene method adopts the NaOH solution of 10g/100ml～50g/100ml as reaction media in priming reaction, the volume mass between activating reagent and matrix is than 1ml: 1g～1ml: 0.1g
Or
Epoxychloropropane, the NaOH solution of the 1g/100ml～10g/100ml of priming reaction be as reaction media, and the volume mass between activating reagent and matrix is than 1ml: 10g～2ml: 1g,
Or
Bis-epoxy reagent method, the NaOH solution of the 1g/100ml～10g/100ml of priming reaction is as reaction media, and the volume mass between activating reagent and matrix is than 1ml: 10g～2ml: 1g.
Described activating reagent is for being selected from cyanogen bromide, allyl glycidyl ether, allyl bromide, bromoallylene, epoxychloropropane, 1, a kind of in 2-ethylene glycol diglycidylether, BDDE, diethylene glycol diglycidyl ether, pentaerythrite triglycidyl ether and pentaerythrite four glycidol ethers.
The pH 8～13 of sucrose and activated substrate coupling, the mass ratio between ficoll and activated substrate 1: 50～1: 10, wherein, the molecular weight of ficoll molecule is 10kDa～1000KDa
preparation method according to the separation and purification medium take the ficoll molecule as extension of the present invention, in described step 2) in, by the hydroxyl on ficoll and the active reaction sites on activated media, as generation couplings such as epoxy radicals, the pH 8～13 of ficoll and activated substrate coupling, mass ratio between ficoll and activated substrate 1: 50～1: 10, wherein the molecular weight of ficoll molecule is 10kDa～1000KDa, in some embodiments of the present invention, preferably, the molecular weight of described ficoll molecule is at 10kDa～400kDa, more preferably, the molecular weight of described ficoll molecule is at 10kDa～70kDa.
Preparation method according to the separation and purification medium take the ficoll molecule as extension of the present invention, in described step 3) in, although the outer surface of ficoll contains a large amount of hydroxyls, but not all hydroxyl all has activity, can with aglucon generation coupling reaction, therefore, as much as possible and the aglucon generation coupling reaction for the hydroxyl surface that makes ficoll, therefore will the hydroxyl on ficoll be activated, its activation method can be selected from step 1) but described in activated substrate on the method in derivatization site.
According to the preparation method of the separation and purification medium take the ficoll molecule as extension of the present invention,
In described step 4) in, described aglucon is:
1) ionic aglucon,
Described ionic aglucon is carboxymethyl, the pH of the activation hydroxyl coupling of itself and ficoll is 7～12, with coupling, the ratio of the substrate reaction of ficoll being arranged is 1mmol: 2g～5mmol: 1g, this class medium Liquidity limit, carry out Selective Separation by the charge differences between target molecule in mixture and impurity molecule, it is suitable for the separation of the target molecule of positively charged; Or
Described ionic aglucon is the diethylamino ethyl, the pH of the activation hydroxyl coupling of itself and ficoll is 7～12, with coupling, the ratio of the substrate reaction of ficoll being arranged is 1mmol: 2g～5mmol: 1g, this class medium adsorpting anion, carry out Selective Separation by the charge differences between target molecule in mixture and impurity molecule, it is suitable for the separation of electronegative target molecule;
Perhaps,
2) hydrophobic aglucon
Described hydrophobic aglucon is butyl, and the pH of the activation hydroxyl coupling of itself and ficoll is 7～12, and with coupling, the ratio of the substrate reaction of ficoll being arranged is 1mmol: 2g～5mmol: 1g; Or
Described hydrophobic aglucon is octyl group, and the pH of the activation hydroxyl coupling of itself and ficoll is 7～12, and with coupling, the ratio of the substrate reaction of ficoll being arranged is 1mmol: 2g～5mmol: 1g; Or
Described hydrophobic aglucon is phenyl, and the pH of the activation hydroxyl coupling of itself and ficoll is 7～12, and with coupling, the ratio of the substrate reaction of ficoll being arranged is 1mmol: 2g～5mmol: 1g; This class medium absorption hydrophobic molecule, it carries out Selective Separation by the hydrophobic difference between target molecule and impurity molecule, and it is suitable for the separation of hydrophobic molecule.
Perhaps,
3) hydrophilic aglucon
Described hydrophilic aglucon ethoxy, the pH of the activation hydroxyl coupling of itself and ficoll is 7～12, with coupling, the ratio of the substrate reaction of ficoll being arranged is 1mmol: 2g～5mmol: 1g; This class medium absorption hydrophilic molecule, it carries out Selective Separation by the hydrophily difference between target molecule and impurity molecule, and it is suitable for the separation of hydrophilic molecule,
Perhaps,
4) Hydrogen Binding Adsorption aglucon
Described Hydrogen Binding Adsorption aglucon is melamine, and the pH of the activation hydroxyl coupling of itself and ficoll is 7～12, and with coupling, the ratio of the substrate reaction of ficoll being arranged is 1mmol: 2g～5mmol: 1g; This class medium separates by the difference of the interaction of hydrogen bond between target molecule and impurity molecule, and it is suitable for providing the separation of the molecule of hydrogen bond receptor or donor.
A further object of the present invention has been to provide a kind of separation and purification medium take the ficoll molecule as extension, but described separation and purification medium comprise matrix, with matrix on derivatization site coupling ficoll and with the aglucon of the hydroxyl coupling of ficoll, wherein the molecular weight of ficoll molecule is 10kDa～1000KDa.
According to the separation and purification medium take the ficoll molecule as extension of the present invention, wherein, described aglucon is:
1) ionic aglucon,
Described ionic aglucon is carboxymethyl, and the ratio of itself and ficoll is 1mmol: 10g～1mmol: 1g; Or
Described ionic aglucon is the diethylamino ethyl, and the ratio of itself and ficoll is 1mmol: 10g～1mmol: 1g;
Perhaps,
2) hydrophobic aglucon
Described hydrophobic aglucon is butyl, and the ratio of itself and ficoll is 1mmol: 10g～1mmol: 1g; Or
Described hydrophobic aglucon is octyl group, and the ratio of itself and ficoll is 1mmol: 10g～1mmol: 1g; Or
Described hydrophobic aglucon is phenyl, and the ratio of itself and ficoll is 1mmol: 10g～1mmol: 1g; Or
Perhaps,
3) hydrophilic aglucon
Described hydrophilic aglucon is ethoxy, and the ratio of itself and ficoll is 1mmol: 10g～1mmol: 1g;
Perhaps,
4) Hydrogen Binding Adsorption aglucon
Described Hydrogen Binding Adsorption aglucon is melamine, and the ratio of itself and ficoll is 1mmol: 10g～1mmol: 1g;
According to the separation and purification medium take the ficoll molecule as extension of the present invention, the mass ratio of described matrix and ficoll is 100: 1～2: 1.
According to the separation and purification medium take the ficoll molecule as extension of the present invention, described matrix is a kind of in agarose based substrate or magnetic-particle matrix.
In some embodiments of the present invention, can carry out crosslinked to improve stability after being coupled to the ficoll molecule on described matrix.
In another aspect of the present invention, the use of described separation and purification medium is provided, it can be used for by fixed bed the separation of protein, polypeptide, natural little molecule etc.In some embodiments of the present invention, described protein can be from natural origin or from the DNA recombinant expression system.In some embodiments of the present invention, described polypeptide can be from natural origin or by chemical synthesis.
The present invention is by adopting spherical ficoll molecule to replace glucan as the derivative of the coupling aglucon of medium, can effectively avoid the formation of shielding construction under certain salinity, avoided large molecule to be got rid of outside the ficoll layer under high ionic strength, thus the adsorbance of raising medium.Such medium is compared with generic media, and on medium, ligand density significantly improves, and adsorption site improves, and static adsorbance improves; This medium is compared with the medium take glucan as extension, because ficoll is spherical molecule, therefore can overcome glucan formed screen layer under certain salinity of linear molecule shape, thereby improved target molecule and had mass transfer rate and flow behavior between the medium of extension, so its adsorbance further improves.
Compare as the chromatographic media of extension with adopting other polymer, can have following advantage with the ficoll molecule as extension: (1) effectively improves surface area; (2) has abundant available derivatization site; (3) mass transfer rate is fast; (4) cluster effect etc.
Description of drawings
Fig. 1 is the synthetic route of a kind of separation and purification medium in the present invention;
Fig. 2 is the chromatogram that melamine-ficoll in the present invention-agarose medium separates thymopeptide-5.
The specific embodiment
Preparation and the application of the separating medium of the melamine as ligand of embodiment 1 take ficoll 70 as extension
1, the preparation take ficoll 70 (mean molecule quantity is as 70kDa) as the agarose matrix of extension
(1) take Sepharose HP as matrix, adopt the epoxychloropropane method to activate (as shown in Figure 1).
The Sepharose HP medium that 15g is drained is added in the 100ml triangular flask, then adds NaOH solution, 22.5ml water and the 50mg NaBH of 2.5ml epoxychloropropane, 10ml 2M
4Mixture was shaken two hours with 240rpm on constant temperature oscillator under 40 ℃; With the reacted medium of deionized water rinsing to remove residual solvent.
(2) use Na
2S
2O
3Method is measured the content of epoxy radicals in activated media.
The activated media that 1g is drained is added in the 50ml triangular flask, adds the Na of 10ml 1.3M
2S
2O
3Solution, under room temperature, reaction is 2 hours, adds 2-3 to drip phenolphthalein indicator, uses the standard salt acidometric titration, and the density that obtains epoxy radicals on anakmetomeres is 72 μ mol/g.
(3) coupling of ficoll
5g ficoll (mean molecule quantity is 70kDa) is dissolved in the 20ml aqueous solution, and the Sepharose HP matrix of getting the above-mentioned activation of 10g joins in the ficoll solution that configures, and adds NaOH solution and the 20mg NaBH of 10ml 2M
4, concussion reaction 10 hours.
2, the preparation of melamine-ficoll-agarose medium
(1) activation of coupling ficoll on the agarose medium
The Sepharose HP medium of the coupling ficoll that 5g is drained is added in the 100ml triangular flask, then adds 8% NaOH solution, 10ml water and the 20mg NaBH of 1ml epoxychloropropane, 5ml
4Mixture was shaken two hours with 240rpm on constant temperature oscillator under 40 ℃; With the reacted medium of deionized water rinsing to remove residual solvent.And the method in employing step (2) is measured the available activated group density 482 μ mol/g of the agarose medium of the rear coupling ficoll of activation.
(2) coupling of melamine as ligand
The 2g melamine is dissolved in the dimethyl sulphoxide aqueous solution of 100ml 60%, gets the Sepharose HP matrix of the above-mentioned activation of 3g, add the melamine solution of the above-mentioned preparation of 10ml, add 8% NaOH solution and the 10mg NaBH of 5ml
4, concussion reaction 10 hours.Adopting the ligand density of Kjeldahl nitrogen determination coupling is 278 μ mol/g.
3, the separation of thymopeptide-5
The thymopeptide-5 crude product that takes the 10mg chemical synthesis is dissolved in 1ml water with the sample of preparation 10mg/ml.Adopt following condition to separating with above-mentioned sample, adopt chromatographic column (the 10mm i.d. * 30mm) of melamine-ficoll-agarose matrix filling; Flow velocity is 1ml/min; Gradient elution, eluent gradient are from 0-200ml, the acetonitrile solution of 0%-50%; Applied sample amount is 500 μ l (as shown in Figure 4).With the thymopeptide-5 that the inverse bonded phase silica gel chromatographic column of Source15 Analyze ﹠ separate obtains, its purity is 91%, and the rate of recovery is 85%.This laboratory by directly with the prepared medium of melamine coupling Sepharose HP (ligand density is 43 μ mol/g) under optimal conditions to identical thymopeptide-5 sample separation, obtaining result is that purity is 78%, the rate of recovery is 86%.By contrast, obtain higher ligand density by ficoll as the prepared medium of extension, thereby improved the selective of media on target thing separation.
Preparation and the application of embodiment 2, the variety classes separating medium take ficoll as extension
The present inventor has also investigated take ficoll as extension from different aglucons and has carried out coupling, prepares different types of separating medium, and experiment condition is as shown in table 1, and the preparation method that it is concrete is with reference to embodiment 1.
Table 1 take ficoll as extension prepares different types of separating medium
In addition, the product for preparing according to the experiment condition in above-mentioned table 1 is as shown in table 2
The separation and purification medium that table 2 the present invention prepares
According to the different types of separation and purification medium for preparing above, the present invention also estimates the separating effect of different types of material, and its result is as shown in table 3.
The separating effect evaluation of the above-mentioned different types of medium of table 3 to respective substance
| Production code member | The material that separates | The rate of recovery | Purity |
| 1 | Glutathione | 90.8％ | 90.1％ |
| 2 | Glutathione | 89.3％ | 96.4％ |
| 3 | Beta-endorphin | 87.6％ | 86.0％ |
| 4 | Beta-endorphin | 88.2％ | 90.3％ |
| 5 | Oxytocin | 90.0％ | 87.1％ |
| 6 | Oxytocin | 84.2％ | 91.6％ |
| 7 | Oxytocin | 88.6％ | 83.1％ |
| 8 | Thymopeptide-5 | 85.3％ | 87.0％ |
| 9 | Thymopeptide-5 | 88.2％ | 92.1％ |
| 10 | Oxytocin | 82.4％ | 81.6％ |
| 11 | Oxytocin | 88.9％ | 93.4 |
| 12 | Thymopeptide-5 | 86.0％ | 84.0％ |
| 13 | Thymopeptide-5 | 88.3％ | 90.1％ |
Can find out from table 1 and 2, under condition of similarity, the ficoll of 10kDa molecular weight is larger than the coupling amount of the ficoll of 1000kDa molecular weight, and this may be because the ficoll of small-molecular weight can enter in the duct of agarose matrix, and available specific area causes thereby increased.From the results shown in Table 3, the selective and medium ligand density positive correlation of target molecule purifying; And crosslinked ligand density has the amount of the ficoll that directly depends on coupling.Therefore, the present invention has improved the ligand density of medium effectively by the coupling ficoll, thereby has improved separate selective.
Claims (6)
1. the preparation method of the separation and purification medium take the ficoll molecule as extension, is characterized in that, said method comprising the steps of:
1) but the derivatization site on activated substrate obtains activated substrate;
2) ficoll is coupled on activated substrate;
3) hydroxyl of the ficoll of activation and matrix coupling;
4) with the activation hydroxyl coupling of aglucon and ficoll, obtain the separation and purification medium take the ficoll molecule as extension;
In described step 1), matrix is agarose based substrate or magnetic-particle matrix;
In described step 4), described aglucon is:
1) ionic aglucon,
Described ionic aglucon is carboxymethyl, and the pH of the activation hydroxyl coupling of itself and ficoll is 7～12, and with coupling, the ratio of the substrate reaction of ficoll being arranged is 1mmol:2g～5mmol:1g;
Or
Described ionic aglucon is the diethylamino ethyl, and the pH of the activation hydroxyl coupling of itself and ficoll is 7～12, and with coupling, the ratio of the substrate reaction of ficoll being arranged is 1mmol:2g～5mmol:1g;
Perhaps,
2) hydrophobic aglucon
Described hydrophobic aglucon is butyl, and the pH of the activation hydroxyl coupling of itself and ficoll is 7～12, and with coupling, the ratio of the substrate reaction of ficoll being arranged is 1mmol:2g～5mmol:1g;
Or
Described hydrophobic aglucon is octyl group, and the pH of the activation hydroxyl coupling of itself and ficoll is 7～12, and with coupling, the ratio of the substrate reaction of ficoll being arranged is 1mmol:2g～5mmol:1g;
Or
Described hydrophobic aglucon is phenyl, and the pH of the activation hydroxyl coupling of itself and ficoll is 7～12, and with coupling, the ratio of the substrate reaction of ficoll being arranged is 1mmol:2g～5mmol:1g;
Perhaps,
3) hydrophilic aglucon
Described hydrophilic aglucon ethoxy, the pH of the activation hydroxyl coupling of itself and ficoll is 7～12, with coupling, the ratio of the substrate reaction of ficoll being arranged is 1mmol:2g～5mmol:1g;
Perhaps,
4) Hydrogen Binding Adsorption aglucon
Described Hydrogen Binding Adsorption aglucon is melamine, and the pH of the activation hydroxyl coupling of itself and ficoll is 7～12, and with coupling, the ratio of the substrate reaction of ficoll being arranged is 1mmol:2g～5mmol:1g.
2. the preparation method of the separation and purification medium take the ficoll molecule as extension according to claim 1, is characterized in that, in described step 1), but the method in the derivatization site on activated substrate is:
Cyanogen bromide method, the pH of priming reaction are 9～12, the mass ratio 1:5～3:1 between activating reagent and matrix,
Or
The allyl glycidyl ether method adopts the NaOH solution of 10g/100ml～50g/100ml as reaction media in priming reaction, the volume mass between activating reagent and matrix is than 1ml:1g～10ml:1g,
Or
The allyl bromide, bromoallylene method adopts the NaOH solution of 10g/100ml～50g/100ml as reaction media in priming reaction, the volume mass between activating reagent and matrix is than 1ml:1g～0.1g,
Or
Epoxychloropropane, the NaOH solution of the 1g/100ml～10g/100ml of priming reaction be as reaction media, and the volume mass between activating reagent and matrix is than 1ml:10g～2ml:1g,
Or
Bis-epoxy reagent method, the NaOH solution of the 1g/100ml～10g/100ml of priming reaction is as reaction media, and the volume mass between activating reagent and matrix is than 1ml:10g～2ml:1g.
3. the preparation method of the separation and purification medium take the ficoll molecule as extension according to claim 1, it is characterized in that, in described step 2) in, the pH8 of ficoll and activated substrate coupling～13, mass ratio 1:50～1:10 between ficoll and activated substrate, wherein, the molecular weight of ficoll molecule is 10kDa～1000KDa.
4. the preparation method of the separation and purification medium take the ficoll molecule as extension according to claim 1, it is characterized in that, in described step 3), cyanogen bromide method, allyl glycidyl ether method, allyl bromide, bromoallylene method, epoxychloropropane, bis-epoxy reagent method are adopted in the activation of the hydroxyl of ficoll.
5. the separation and purification medium take the ficoll molecule as extension of arbitrary described method preparation of according to claim 1 to 4, it is characterized in that, but described separation and purification medium comprise matrix, with matrix on derivatization site coupling ficoll and with the aglucon of the activation hydroxyl coupling of ficoll, wherein the molecular weight of ficoll molecule is 10kDa～1000KDa;
Described aglucon is:
1) ionic aglucon,
Described ionic aglucon is carboxymethyl, and the ratio of itself and ficoll is 1mmol:10g～1mmol:1g; Or
Described ionic aglucon is the diethylamino ethyl, and the ratio of itself and ficoll is 1mmol:10g～1mmol:1g;
Perhaps,
2) hydrophobic aglucon
Described hydrophobic aglucon is butyl, and the ratio of itself and ficoll is 1mmol:10g～1mmol:1g; Or
Described hydrophobic aglucon is octyl group, and the ratio of itself and ficoll is 1mmol:10g～1mmol:1g; Or
Described hydrophobic aglucon is phenyl, and the ratio of itself and ficoll is 1mmol:10g～1mmol:1g;
Perhaps,
3) hydrophilic aglucon
Described hydrophilic aglucon is ethoxy, and the ratio of itself and ficoll is 1mmol:10g～1mmol:1g;
Perhaps,
4) Hydrogen Binding Adsorption aglucon
Described Hydrogen Binding Adsorption aglucon is melamine, and the ratio of itself and ficoll is 1mmol:10g～1mmol:1g;
Described matrix is a kind of in agarose based substrate or magnetic-particle matrix, but the derivatization site of described matrix is hydroxyl.
6. the separation and purification medium take the ficoll molecule as extension according to claim 5, is characterized in that, the mass ratio of described matrix and ficoll is 100:1～2:1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110147824 CN102266758B (en) | 2011-03-11 | 2011-06-02 | Separation and purification medium with saccharosan molecule serving as derivative and preparation method of separation and purification medium |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110057889 | 2011-03-11 | ||
| CN201110057889.3 | 2011-03-11 | ||
| CN 201110147824 CN102266758B (en) | 2011-03-11 | 2011-06-02 | Separation and purification medium with saccharosan molecule serving as derivative and preparation method of separation and purification medium |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102266758A CN102266758A (en) | 2011-12-07 |
| CN102266758B true CN102266758B (en) | 2013-06-05 |
Family
ID=45049327
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110147824 Active CN102266758B (en) | 2011-03-11 | 2011-06-02 | Separation and purification medium with saccharosan molecule serving as derivative and preparation method of separation and purification medium |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102266758B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108176386B (en) * | 2018-01-16 | 2020-11-06 | 武汉轻工大学 | Melamine bonded chromatographic stationary phase, preparation method thereof and metal ion modified melamine bonded chromatographic stationary phase |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008022355A3 (en) * | 2006-08-18 | 2008-10-23 | Perkinelmer Las Inc | Methods and reagents for detecting phosphomonoester groups |
| CN101348520A (en) * | 2007-12-14 | 2009-01-21 | 北京康铭优盛生化技术有限公司 | Method for separating and purifying sea-mussel mucin by using mixing adsorption chromatography |
| CN101456898A (en) * | 2007-12-10 | 2009-06-17 | 中国科学院过程工程研究所 | Method for separating and purifying polypeptide by using hydrogen bond adsorption chromatogram |
| CN101504398A (en) * | 2009-03-19 | 2009-08-12 | 北京康铭优盛生化技术有限公司 | Synthesizing method for agarose hydrophilic adsorption chromatography medium with melamine as ligand |
| CN101955514A (en) * | 2009-07-14 | 2011-01-26 | 杭州华锦药业股份有限公司 | Method for synthesizing agarose gel hydrogen bond adsorbing chromatography medium by using quercetin as genin |
-
2011
- 2011-06-02 CN CN 201110147824 patent/CN102266758B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008022355A3 (en) * | 2006-08-18 | 2008-10-23 | Perkinelmer Las Inc | Methods and reagents for detecting phosphomonoester groups |
| CN101456898A (en) * | 2007-12-10 | 2009-06-17 | 中国科学院过程工程研究所 | Method for separating and purifying polypeptide by using hydrogen bond adsorption chromatogram |
| CN101348520A (en) * | 2007-12-14 | 2009-01-21 | 北京康铭优盛生化技术有限公司 | Method for separating and purifying sea-mussel mucin by using mixing adsorption chromatography |
| CN101504398A (en) * | 2009-03-19 | 2009-08-12 | 北京康铭优盛生化技术有限公司 | Synthesizing method for agarose hydrophilic adsorption chromatography medium with melamine as ligand |
| CN101955514A (en) * | 2009-07-14 | 2011-01-26 | 杭州华锦药业股份有限公司 | Method for synthesizing agarose gel hydrogen bond adsorbing chromatography medium by using quercetin as genin |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102266758A (en) | 2011-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Core–shell metal–organic frameworks as the mixed-mode stationary phase for hydrophilic interaction/reversed-phase chromatography | |
| CN101765458B (en) | Separation matrix | |
| Potter et al. | Porous polymer monoliths for extraction: Diverse applications and platforms | |
| CN101252986B (en) | Manufacture of chromatography matrices | |
| CN103028383B (en) | Silica gel chromatography packing and preparation method thereof | |
| CN105597370B (en) | Isolation medium | |
| CN105561960B (en) | Changeable efficient liquid phase chromatographic stuffing of a kind of chiral selectivity and preparation method thereof | |
| CN108114707B (en) | Silica gel matrix hydrophobic amino acid bonded stationary phase and preparation and application thereof | |
| CN110115992B (en) | Aptamer functionalized polymer column for specific recognition of mycotoxin and preparation method thereof | |
| CN103007905A (en) | Tetraazacalix [2] arene [2] triazine bonded silica gel solid phase extraction material, preparation method and application thereof | |
| Hsieh et al. | Single-step approach for fabrication of vancomycin-bonded silica monolith as chiral stationary phase | |
| CN104289209B (en) | A kind of WCX/HIC for Separation of Proteins difunctional mixed model polymeric matrix chromatographic stationary phases and preparation method thereof | |
| CN102266758B (en) | Separation and purification medium with saccharosan molecule serving as derivative and preparation method of separation and purification medium | |
| Hsieh et al. | Single‐step approach to β‐cyclodextrin‐bonded silica as monolithic stationary phases for CEC | |
| CN110102270B (en) | Aptamer affinity monolithic column for specifically recognizing F2 toxin and preparation method thereof | |
| WO2007058599A1 (en) | Manufacture of a chromatography matrix | |
| Xu et al. | Preparation, characterization, and application of a new stationary phase containing different kinds of amine groups | |
| WO2010059550A1 (en) | New chromatographic media based on phenoxy alkyl and alkoxy-or phenoxy-phenyl alkyl ligands | |
| CN108927113B (en) | Nano-hydroxyapatite functionalized solid phase extraction monolithic column | |
| Wang et al. | Monolithic column with double mixed‐modes of hydrophilic interaction/cation‐exchange and reverse‐phase/cation‐exchange stationary phase for pressurized capillary electrochromatography | |
| CN107674112B (en) | Affinity chromatography medium using heparin as ligand | |
| CN114369182A (en) | Preparation method of porous high-molecular polymer microspheres with amphoteric structures | |
| Yu et al. | RECENT DEVELOPMENT AND APPLICATION OF MONOLITHIC COLUMNS. | |
| CN103203225B (en) | Weak cation exchange/hydrophobic bifunctional hybrid-mode chromatographic stationary phase as well as preparation method and application thereof | |
| CN105601940A (en) | Chitosan derivatization calix [4] arene bonded silica gel stationary phase and preparing method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210125 Address after: 618132 No.74, east section of Taizhong Road, Guanghan City, Deyang City, Sichuan Province Patentee after: Sichuan Guangming Biological Products Co.,Ltd. Address before: 100190 No. two, No. 1, North Haidian District, Beijing, Zhongguancun Patentee before: Institute of Process Engineering, Chinese Academy of Sciences |