CN117299099A - Boron affinity chromatography medium and preparation method thereof - Google Patents
Boron affinity chromatography medium and preparation method thereof Download PDFInfo
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- CN117299099A CN117299099A CN202311291996.1A CN202311291996A CN117299099A CN 117299099 A CN117299099 A CN 117299099A CN 202311291996 A CN202311291996 A CN 202311291996A CN 117299099 A CN117299099 A CN 117299099A
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- silica gel
- divinylbenzene
- styrene
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- affinity chromatography
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- 239000012501 chromatography medium Substances 0.000 title claims abstract description 67
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 title claims abstract description 57
- 238000001042 affinity chromatography Methods 0.000 title claims abstract description 57
- 229910052796 boron Inorganic materials 0.000 title claims abstract description 57
- 238000002360 preparation method Methods 0.000 title abstract description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 166
- 239000000741 silica gel Substances 0.000 claims abstract description 126
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 126
- CHRJZRDFSQHIFI-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;styrene Chemical compound C=CC1=CC=CC=C1.C=CC1=CC=CC=C1C=C CHRJZRDFSQHIFI-UHFFFAOYSA-N 0.000 claims abstract description 83
- 239000011159 matrix material Substances 0.000 claims abstract description 35
- PWTKGAHFMQUAPN-UHFFFAOYSA-N B(O)(O)O.NC1=NC=CC=C1 Chemical compound B(O)(O)O.NC1=NC=CC=C1 PWTKGAHFMQUAPN-UHFFFAOYSA-N 0.000 claims abstract description 23
- ULVXDHIJOKEBMW-UHFFFAOYSA-N [3-(prop-2-enoylamino)phenyl]boronic acid Chemical compound OB(O)C1=CC=CC(NC(=O)C=C)=C1 ULVXDHIJOKEBMW-UHFFFAOYSA-N 0.000 claims abstract description 22
- 230000004048 modification Effects 0.000 claims abstract description 10
- 238000012986 modification Methods 0.000 claims abstract description 10
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 66
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 48
- 239000003999 initiator Substances 0.000 claims description 27
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 22
- 238000003756 stirring Methods 0.000 claims description 18
- 239000000203 mixture Substances 0.000 claims description 15
- 238000001914 filtration Methods 0.000 claims description 14
- 229910021589 Copper(I) bromide Inorganic materials 0.000 claims description 11
- ZXOFHTCCTUEJQJ-UHFFFAOYSA-N [4-(chloromethyl)phenyl]-trimethoxysilane Chemical compound CO[Si](OC)(OC)C1=CC=C(CCl)C=C1 ZXOFHTCCTUEJQJ-UHFFFAOYSA-N 0.000 claims description 11
- NKNDPYCGAZPOFS-UHFFFAOYSA-M copper(i) bromide Chemical compound Br[Cu] NKNDPYCGAZPOFS-UHFFFAOYSA-M 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 11
- 238000001035 drying Methods 0.000 claims description 7
- 238000010438 heat treatment Methods 0.000 claims description 7
- 238000010992 reflux Methods 0.000 claims description 7
- 239000002253 acid Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- XTNBHGUXJJBSDO-UHFFFAOYSA-N OBO.NC1=CC=CC=N1 Chemical compound OBO.NC1=CC=CC=N1 XTNBHGUXJJBSDO-UHFFFAOYSA-N 0.000 claims description 5
- 238000001179 sorption measurement Methods 0.000 abstract description 30
- 239000011248 coating agent Substances 0.000 abstract description 3
- 238000000576 coating method Methods 0.000 abstract description 3
- 230000000052 comparative effect Effects 0.000 description 18
- 239000000243 solution Substances 0.000 description 16
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 10
- -1 cyclic ester Chemical class 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- ROFVEXUMMXZLPA-UHFFFAOYSA-N Bipyridyl Chemical compound N1=CC=CC=C1C1=CC=CC=N1 ROFVEXUMMXZLPA-UHFFFAOYSA-N 0.000 description 5
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 4
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 4
- 229930091371 Fructose Natural products 0.000 description 4
- 239000005715 Fructose Substances 0.000 description 4
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 4
- 238000013375 chromatographic separation Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 239000003480 eluent Substances 0.000 description 2
- HXITXNWTGFUOAU-UHFFFAOYSA-N phenylboronic acid Chemical compound OB(O)C1=CC=CC=C1 HXITXNWTGFUOAU-UHFFFAOYSA-N 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 229910052710 silicon Inorganic materials 0.000 description 2
- 239000010703 silicon Substances 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
- B01J20/103—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate comprising silica
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/3085—Chemical treatments not covered by groups B01J20/3007 - B01J20/3078
Landscapes
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Inorganic Chemistry (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
The invention provides a boron affinity chromatography medium and a preparation method thereof, wherein the boron affinity chromatography medium takes silica gel with poly (styrene-divinylbenzene) coated on the surface as a matrix, 3-acrylamidophenylboronic acid is grafted on the matrix, and then 6-aminopyridine boric acid is adopted for modification, so that the boron affinity chromatography medium is obtained. According to the invention, the surface binding site of the matrix can be increased by coating the poly (styrene-divinylbenzene) on the surface of the silica gel, so that the adsorption capacity of the chromatographic medium is improved; meanwhile, the 6-aminopyridine boric acid is adopted for modification, so that the adsorption sites of the chromatographic medium are increased, and the adsorption capacity can be further improved.
Description
Technical Field
The invention relates to a chromatographic separation technology, in particular to a boron affinity chromatographic medium and a preparation method thereof.
Background
The boron affinity material is a novel affinity material developed based on the principle that phenylboronic acid is structurally changed in different pH environments so as to specifically bind cis-dihydroxyl groups, and has the capability of enriching, extracting and separating various biomolecules (such as saccharides, glycoprotein, nucleotide and the like) containing cis-dihydroxyl structures. Can reversibly react with cis-ortho-dihydroxyl to form five-membered or six-membered cyclic ester in physiological and alkalescent environments. However, most boron affinity materials are prepared by using the bonding reaction of the conventional difunctional compounds, so that the adsorption sites are limited and the adsorption capacity is low.
In addition, the boron affinity material has obviously reduced binding performance with cis-form ortho-dihydroxyl substances under the action of hydrophobic force caused by high-concentration salt ions, and can be only suitable for chromatographic separation of low-concentration salt ion solution, so that the application range is limited.
Disclosure of Invention
The invention aims to provide a boron affinity chromatography medium and a preparation method thereof, which are used for solving the problems of low adsorption capacity and narrow application range of the boron affinity chromatography medium in the prior art.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a boron affinity chromatography medium is prepared by taking silica gel coated with poly (styrene-divinylbenzene) as a matrix, grafting 3-acrylamidophenylboronic acid onto the matrix, and modifying with 6-aminopyridine boronic acid.
Preferably, the silica gel coated with poly (styrene-divinylbenzene) on the surface is obtained by mixing activated silica gel with poly (styrene-divinylbenzene) and curing.
Preferably, the mass ratio of activated silica gel to poly (styrene-divinylbenzene) is 10: (3-5).
Preferably, the activated silica gel is an acid activated silica gel.
Preferably, the activated silica gel is obtained by the following method: adding 5.5-6.0mol/L hydrochloric acid into the silica gel, heating and refluxing for 5-6h, filtering and drying to obtain activated silica gel.
Preferably, the mass ratio of the matrix to the 3-acrylamidophenylboronic acid is 1: (1.8-2.8).
Preferably, the mass ratio of the matrix to the 6-aminopyridine boronic acid is 1: (0.1-0.5).
The invention also provides a preparation method of the boron affinity chromatography medium, which comprises the following steps:
(1) Taking silica gel with the surface covered with poly (styrene-divinylbenzene), adding 3-acrylamidophenylboronic acid into the silica gel, and reacting to obtain grafted silica gel;
(2) And (3) adding 6-aminopyridine boric acid into the grafted silica gel obtained in the step (1), and reacting to obtain the boron affinity chromatography medium.
Preferably, the silica gel coated with poly (styrene-divinylbenzene) on the surface is obtained by the following method: adding 5.5-6.0mol/L hydrochloric acid into silica gel, heating and refluxing for 5-6h, filtering and drying to obtain activated silica gel; adding the activated silica gel into a poly (styrene-divinylbenzene) solution, and stirring at 60-80 ℃ until volatile components volatilize to obtain the silica gel with the poly (styrene-divinylbenzene) coated on the surface.
Preferably, the step (1) specifically includes: dispersing silica gel coated with poly (styrene-divinylbenzene) on the surface into toluene, adding 4-chloromethyl phenyl trimethoxy silane into the toluene, reacting for 8-16h at 100-120 ℃, and filtering to obtain silica gel with a surface fixed initiator; dispersing the silica gel with the surface fixed with the initiator in N, N-dimethylformamide, adding 3-acrylamidophenylboronic acid, 2' -bipyridine and cuprous bromide into the mixture, and stirring the mixture at 90-100 ℃ for reaction for 5-10 hours to obtain grafted silica gel.
Preferably, the amount of the silica gel coated with poly (styrene-divinylbenzene) to toluene is in the range of 0.8 to 1.5g of the silica gel per 10ml of toluene. The mass ratio of the silica gel coated with poly (styrene-divinylbenzene) to the 4-chloromethylphenyl trimethoxysilane is 1: (1.2-1.5). The usage amount of the silica gel of the surface fixed initiator and the N, N-dimethylformamide is 1.2-1.8g of the silica gel of the surface fixed initiator in every 10ml of N, N-dimethylformamide. The mass ratio of the silica gel to the 2,2' -bipyridine to the cuprous bromide of the surface-immobilized initiator is 1:0.5:0.3.
preferably, the step (2) specifically includes: adding 6-aminopyridine boric acid into the grafted silica gel obtained in the step (1), and stirring and reacting for 2-8h at 110-130 ℃ to obtain a boron affinity chromatography medium.
The scheme of the invention at least comprises the following beneficial effects:
(1) The boron affinity chromatography medium takes silica gel with poly (styrene-divinylbenzene) coated on the surface as a matrix, and 3-acrylamidophenylboronic acid is grafted on the matrix, and then 6-aminopyridine boric acid is adopted for modification, so that the boron affinity chromatography medium is obtained. According to the invention, the surface binding site of the matrix can be increased by coating the poly (styrene-divinylbenzene) on the surface of the silica gel, so that the adsorption capacity of the chromatographic medium is improved; meanwhile, for the silica gel matrix, the residual silicon hydroxyl groups are easy to adsorb substances with amino groups, so that the selectivity of the silica gel matrix is poor, and the influence of the silicon hydroxyl groups can be reduced by coating the silica gel with poly (styrene-divinylbenzene), so that the selectivity of the chromatographic medium is improved. In addition, the invention adopts 6-aminopyridine boric acid for modification, which increases the adsorption sites of the chromatographic medium and can further improve the adsorption capacity of the chromatographic medium.
(2) The boron affinity chromatography medium can maintain a better chromatographic separation effect in a high-concentration salt ion solution, and has a wider application range.
Detailed Description
The specific conditions not noted in the examples of the present invention were carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used are not marked for manufacturers, are conventional products which can be obtained through commercial purchase, and the implementation of the technical scheme and the realization of the technical effect of the invention are not influenced by raw materials of different manufacturers and models.
Example 1
In the boron affinity chromatography medium in the embodiment, silica gel with poly (styrene-divinylbenzene) coated on the surface is taken as a matrix, 3-acrylamidophenylboronic acid is grafted on the matrix, and then 6-aminopyridine boric acid is adopted for modification, so that the boron affinity chromatography medium is obtained.
Wherein the silica gel coated with poly (styrene-divinylbenzene) on the surface is obtained by mixing activated silica gel with poly (styrene-divinylbenzene) and curing. The activated silica gel is acid activated silica gel. The mass ratio of the activated silica gel to the poly (styrene-divinylbenzene) is 10:3. the mass ratio of the matrix, namely the silica gel with the surface coated with poly (styrene-divinylbenzene), to the 3-acrylamidophenylboronic acid is 1:2.8. the mass ratio of the matrix to the 6-aminopyridine boric acid is 1:0.3.
the preparation method of the boron affinity chromatography medium comprises the following steps:
(1) Dispersing silica gel coated with poly (styrene-divinylbenzene) on the surface into toluene, adding 4-chloromethyl phenyl trimethoxy silane into the toluene, reacting for 8 hours at 120 ℃, and filtering to obtain silica gel with a surface fixed initiator; dispersing the silica gel with the surface fixed with the initiator in N, N-dimethylformamide, adding 3-acrylamidophenylboronic acid, 2' -bipyridine and cuprous bromide into the mixture, stirring the mixture at 90 ℃ for reaction for 10 hours, and filtering the mixture to obtain grafted silica gel.
Wherein the amount of the silica gel coated with poly (styrene-divinylbenzene) and toluene is 0.8g of the silica gel per 10ml of toluene. The mass ratio of the silica gel coated with poly (styrene-divinylbenzene) to the 4-chloromethylphenyl trimethoxysilane is 1:1.2. the amount of the silica gel of the surface-immobilized initiator and the amount of the N, N-dimethylformamide are related to each 10ml of the N, N-dimethylformamide, and the silica gel of the surface-immobilized initiator is 1.2g. The mass ratio of the silica gel to the 2,2' -bipyridine to the cuprous bromide of the surface-immobilized initiator is 1:0.5:0.3.
in this example, the silica gel coated with poly (styrene-divinylbenzene) was obtained by the following method: taking silica gel, adding 5.5mol/L hydrochloric acid into the silica gel, heating and refluxing for 6 hours, filtering and drying to obtain activated silica gel; adding the activated silica gel into a poly (styrene-divinylbenzene) solution, stirring at 60 ℃ until volatile components volatilize, and curing the poly (styrene-divinylbenzene) on the surface of the silica gel to obtain the silica gel with the poly (styrene-divinylbenzene) coated on the surface.
Wherein the volume ratio of the silica gel to the hydrochloric acid is at least 1:5. The poly (styrene-divinylbenzene) solution was an aqueous solution having a concentration of 0.5 mg/mL.
(2) And (2) adding 6-aminopyridine boric acid into the grafted silica gel obtained in the step (1), and stirring and reacting for 2 hours at 130 ℃ to obtain a boron affinity chromatography medium.
Example 2
In the boron affinity chromatography medium in the embodiment, silica gel with poly (styrene-divinylbenzene) coated on the surface is taken as a matrix, 3-acrylamidophenylboronic acid is grafted on the matrix, and then 6-aminopyridine boric acid is adopted for modification, so that the boron affinity chromatography medium is obtained.
Wherein the silica gel coated with poly (styrene-divinylbenzene) on the surface is obtained by mixing activated silica gel with poly (styrene-divinylbenzene) and curing. The activated silica gel is acid activated silica gel. The mass ratio of the activated silica gel to the poly (styrene-divinylbenzene) is 10:4. the mass ratio of the matrix to the 3-acrylamidophenylboronic acid is 1:1.8. the mass ratio of the matrix to the 6-aminopyridine boric acid is 1:0.5.
the preparation method of the boron affinity chromatography medium comprises the following steps:
(1) Dispersing silica gel coated with poly (styrene-divinylbenzene) on the surface into toluene, adding 4-chloromethyl phenyl trimethoxy silane into the toluene, reacting for 12 hours at 100 ℃, and filtering to obtain silica gel with a surface fixed initiator; dispersing the silica gel with the surface fixed with the initiator in N, N-dimethylformamide, adding 3-acrylamidophenylboronic acid, 2' -bipyridine and cuprous bromide into the mixture, and stirring the mixture at 100 ℃ for reaction for 7 hours to obtain grafted silica gel.
Wherein the amount of the silica gel coated with poly (styrene-divinylbenzene) and toluene is 1.5g of the silica gel per 10ml of toluene. The mass ratio of the silica gel coated with poly (styrene-divinylbenzene) to the 4-chloromethylphenyl trimethoxysilane is 1:1.3. the amount of the silica gel of the surface-immobilized initiator and the amount of the N, N-dimethylformamide are related to each 10ml of the N, N-dimethylformamide, and the silica gel of the surface-immobilized initiator is 1.8g. The mass ratio of the silica gel to the 2,2' -bipyridine to the cuprous bromide of the surface-immobilized initiator is 1:0.5:0.3.
in this example, the silica gel coated with poly (styrene-divinylbenzene) was obtained by the following method: adding 6.0mol/L hydrochloric acid into the silica gel, heating and refluxing for 5 hours, filtering and drying to obtain activated silica gel; adding the activated silica gel into a poly (styrene-divinylbenzene) solution, stirring at 70 ℃ until volatile components volatilize, and curing the poly (styrene-divinylbenzene) on the surface of the silica gel to obtain the silica gel with the poly (styrene-divinylbenzene) coated on the surface.
Wherein the volume ratio of the silica gel to the hydrochloric acid is at least 1:5. The poly (styrene-divinylbenzene) solution was an aqueous solution having a concentration of 0.5 mg/mL.
(2) And (3) adding 6-aminopyridine boric acid into the grafted silica gel obtained in the step (1), and stirring and reacting for 5 hours at 120 ℃ to obtain a boron affinity chromatography medium.
Example 3
In the boron affinity chromatography medium in the embodiment, silica gel with poly (styrene-divinylbenzene) coated on the surface is taken as a matrix, 3-acrylamidophenylboronic acid is grafted on the matrix, and then 6-aminopyridine boric acid is adopted for modification, so that the boron affinity chromatography medium is obtained.
Wherein the silica gel coated with poly (styrene-divinylbenzene) on the surface is obtained by mixing activated silica gel with poly (styrene-divinylbenzene) and curing. The activated silica gel is acid activated silica gel. The mass ratio of the activated silica gel to the poly (styrene-divinylbenzene) is 10:5. the mass ratio of the matrix to the 3-acrylamidophenylboronic acid is 1:2. the mass ratio of the matrix to the 6-aminopyridine boric acid is 1:0.1.
the preparation method of the boron affinity chromatography medium comprises the following steps:
(1) Dispersing silica gel coated with poly (styrene-divinylbenzene) on the surface into toluene, adding 4-chloromethyl phenyl trimethoxy silane into the toluene, reacting for 16 hours at 110 ℃, and filtering to obtain silica gel with a surface fixed initiator; dispersing the silica gel with the surface fixed with the initiator in N, N-dimethylformamide, adding 3-acrylamidophenylboronic acid, 2' -bipyridine and cuprous bromide into the mixture, and stirring the mixture at 95 ℃ for reaction for 5 hours to obtain grafted silica gel.
Wherein the amount of the silica gel coated with poly (styrene-divinylbenzene) and toluene is 1.2g of the silica gel per 10ml of toluene. The mass ratio of the silica gel coated with poly (styrene-divinylbenzene) to the 4-chloromethylphenyl trimethoxysilane is 1:1.5. the amount of the silica gel of the surface-immobilized initiator and the amount of the N, N-dimethylformamide are related to each 10ml of the N, N-dimethylformamide, and the silica gel of the surface-immobilized initiator is 1.5g. The dosage relationship of the silica gel, the 2,2' -bipyridine and the cuprous bromide of the surface-immobilized initiator is 1:0.5:0.3.
in this example, the silica gel coated with poly (styrene-divinylbenzene) was obtained by the following method: taking silica gel, adding 5.8mol/L hydrochloric acid into the silica gel, heating and refluxing for 6 hours, filtering and drying to obtain activated silica gel; adding the activated silica gel into a poly (styrene-divinylbenzene) solution, stirring at 80 ℃ until volatile components volatilize, and curing the poly (styrene-divinylbenzene) on the surface of the silica gel to obtain the silica gel with the poly (styrene-divinylbenzene) coated on the surface.
Wherein the volume ratio of the silica gel to the hydrochloric acid is at least 1:5. The poly (styrene-divinylbenzene) solution was an aqueous solution having a concentration of 0.5 mg/mL.
(2) And (2) adding 6-aminopyridine boric acid into the grafted silica gel obtained in the step (1), and stirring and reacting for 8 hours at 110 ℃ to obtain a boron affinity chromatography medium.
Example 4
In the boron affinity chromatography medium in the embodiment, silica gel with poly (styrene-divinylbenzene) coated on the surface is taken as a matrix, 3-acrylamidophenylboronic acid is grafted on the matrix, and then 6-aminopyridine boric acid is adopted for modification, so that the boron affinity chromatography medium is obtained.
Wherein the silica gel coated with poly (styrene-divinylbenzene) on the surface is obtained by mixing activated silica gel with poly (styrene-divinylbenzene) and curing. The activated silica gel is acid activated silica gel. The mass ratio of the activated silica gel to the poly (styrene-divinylbenzene) is 10:3.8. the mass ratio of the matrix to the 3-acrylamidophenylboronic acid is 1:2.4. the mass ratio of the matrix to the 6-aminopyridine boric acid is 1:0.3.
the preparation method of the boron affinity chromatography medium comprises the following steps:
(1) Dispersing silica gel coated with poly (styrene-divinylbenzene) on the surface into toluene, adding 4-chloromethyl phenyl trimethoxy silane into the toluene, reacting for 16 hours at 110 ℃, and filtering to obtain silica gel with a surface fixed initiator; dispersing the silica gel with the surface fixed with the initiator in N, N-dimethylformamide, adding 3-acrylamidophenylboronic acid, 2' -bipyridine and cuprous bromide into the mixture, and stirring the mixture at 95 ℃ for reaction for 8 hours to obtain grafted silica gel.
Wherein the amount of the silica gel coated with poly (styrene-divinylbenzene) and toluene is 1.2g of the silica gel per 10ml of toluene. The mass ratio of the silica gel coated with poly (styrene-divinylbenzene) to the 4-chloromethylphenyl trimethoxysilane is 1:1.4. the amount of the silica gel of the surface-immobilized initiator and the amount of the N, N-dimethylformamide are related to each 10ml of the N, N-dimethylformamide, and the silica gel of the surface-immobilized initiator is 1.2g. The mass ratio of the silica gel to the 2,2' -bipyridine to the cuprous bromide of the surface-immobilized initiator is 1:0.5:0.3.
in this example, the silica gel coated with poly (styrene-divinylbenzene) was obtained by the following method: taking silica gel with the particle size of 5 mu m, adding 5.8mol/L hydrochloric acid into the silica gel, heating and refluxing the mixture for 6 hours, and filtering and drying the mixture to obtain activated silica gel; adding the activated silica gel into a poly (styrene-divinylbenzene) solution, stirring at 70 ℃ until volatile components volatilize, and curing the poly (styrene-divinylbenzene) on the surface of the silica gel to obtain the silica gel with the poly (styrene-divinylbenzene) coated on the surface.
Wherein the volume ratio of the silica gel to the hydrochloric acid is at least 1:5. The poly (styrene-divinylbenzene) solution was an aqueous solution having a concentration of 0.5 mg/mL.
(2) And (2) adding 6-aminopyridine boric acid into the grafted silica gel obtained in the step (1), and stirring and reacting for 6 hours at 110 ℃ to obtain a boron affinity chromatography medium.
Comparative example 1
In this comparative example, a boron affinity chromatography medium was prepared in the same manner as in example 4, except that: silica gel is directly used as a matrix, that is, the silica gel coated with poly (styrene-divinylbenzene) on the surface is replaced by silica gel.
Comparative example 2
In this comparative example, a boron affinity chromatography medium was prepared in the same manner as in example 4, except that: and (3) modifying the silica gel without adopting 6-aminopyridine boric acid, namely omitting the step (2), and taking the grafted silica gel obtained in the step (1) as a boron affinity chromatography medium.
Comparative example 3
In this comparative example, a boron affinity chromatography medium was prepared in the same manner as in example 4, except that: in the silica gel with the surface coated with poly (styrene-divinylbenzene), the mass ratio of the activated silica gel to the poly (styrene-divinylbenzene) is 10:2.
comparative example 4
In this comparative example, a boron affinity chromatography medium was prepared in the same manner as in example 4, except that: in the silica gel with the surface coated with poly (styrene-divinylbenzene), the mass ratio of the activated silica gel to the poly (styrene-divinylbenzene) is 10:6.
comparative example 5
In this comparative example, a boron affinity chromatography medium was prepared in the same manner as in example 4, except that: the mass ratio of the matrix to the 6-aminopyridine boric acid is 1:0.6.
experimental example of Effect
To verify the technical effect of the boron affinity chromatography media of the invention, the following tests were performed:
the adsorption capacity test was performed by taking the boron affinity chromatography media prepared in examples 1 to 4 and comparative examples 1 to 5: the adsorption capacity of each boron affinity chromatography medium on the target adsorption substance is measured by adopting a substance containing cis-ortho-dihydroxyl, namely catechol, as the target adsorption substance and adopting an ultraviolet high performance liquid chromatography.
The boron affinity chromatography media prepared in examples 1 to 4 and comparative examples 1 to 5 were taken for adsorption selectivity test: NH at a concentration of 50mmol/L, pH of 8.5 4 Cl-NH 3 Preparing a mixed solution of catechol, aniline, hydroquinone and 5-hydroxytryptamine by using a buffer solution; the concentrations of catechol, aniline, hydroquinone and 5-hydroxytryptamine in the mixed solution are all 5 mug/mL. The boron affinity chromatography medium prepared in examples 1-4 and comparative examples 1-5 was used to extract the above mixed solution by adsorption, then the elution was performed, the eluent was analyzed by ultraviolet high performance liquid chromatography, and the purity of catechol in the eluent was calculated.
The boron affinity chromatography media prepared in examples 1-4 and comparative examples 1-5 were used for carrying out adsorption capacity tests with different ion concentrations: the substance containing cis-ortho-dihydroxyl, namely fructose, is used as a target adsorption substance, and the fructose is added into a phosphate buffer salt solution with the concentration of 0.01mol/L to obtain a sample loading liquid with low ion concentration. And adding fructose into the phosphate buffer salt solution with the concentration of 0.1mol/L to obtain the loading liquid with high ion concentration. The concentration of fructose in each loading solution was 5. Mu.g/mL. And (3) measuring the adsorption capacity of each boron affinity chromatography medium in the sample loading liquid to catechol by adopting an ultraviolet high performance liquid chromatography.
Effect experimental results:
the results are as follows:
as can be seen from the comparison of examples 1-4 and comparative example 1, the boron-affinity chromatography medium of the invention adopts poly (styrene-divinylbenzene) coated on the surface as a matrix, which not only significantly improves the adsorption capacity of the chromatography medium, but also has higher adsorption selectivity, and can efficiently adsorb the target adsorption substance in the solution of high-concentration salt ions. As is clear from the comparison of example 4 with comparative examples 3 to 4, in the silica gel coated with poly (styrene-divinylbenzene), the amount of poly (styrene-divinylbenzene) used was too small to sufficiently exert its effect. However, if the amount of poly (styrene-divinylbenzene) used is too large, the adsorption selectivity of the resulting chromatographic medium is only slightly increased, but the adsorption capacity is rather decreased, and especially in a solution of high-concentration salt ions, the decrease in adsorption capacity is remarkable. As is clear from comparison of examples 1 to 4 with comparative example 2, modification with 6-aminopyridine boronic acid has little influence on adsorption selectivity, but can significantly increase adsorption sites of the chromatographic medium, exhibit higher adsorption capacity, and avoid reduction of adsorption capacity to some extent in a solution of high concentration salt ions. As is clear from the comparison of example 4 with comparative example 5, the excessive amount of 6-aminopyridine boronic acid does not have a significant effect on the increase of adsorption capacity, but rather results in a decrease of adsorption capacity in a solution of high concentration of salt ions.
It will be appreciated by those skilled in the art that the present invention can be carried out in other embodiments without departing from the spirit or essential characteristics thereof. Accordingly, the above disclosed embodiments are illustrative in all respects, and not exclusive. All changes that come within the scope of the invention or equivalents thereto are intended to be embraced therein.
Claims (10)
1. A boron affinity chromatography medium is characterized in that silica gel coated with poly (styrene-divinylbenzene) is used as a matrix, 3-acrylamidophenylboronic acid is grafted onto the matrix, and then 6-aminopyridine boric acid is used for modification, so that the boron affinity chromatography medium is obtained.
2. The boron-affinity chromatography medium of claim 1, wherein the silica gel coated with poly (styrene-divinylbenzene) is obtained by mixing activated silica gel with poly (styrene-divinylbenzene) and curing.
3. The boron-affinity chromatography medium of claim 2, wherein the mass ratio of activated silica gel to poly (styrene-divinylbenzene) is 10: (3-5).
4. A boron-affinity chromatography medium according to claim 3, wherein the activated silica gel is an acid-activated silica gel.
5. The boron affinity chromatography medium of claim 1, wherein the mass ratio of the matrix to 3-acrylamidophenylboronic acid is 1: (1.8-2.8).
6. The boron affinity chromatography medium of claim 1, wherein the mass ratio of the matrix to the 6-aminopyridine boronic acid is 1: (0.1-0.5).
7. A method for preparing a boron affinity chromatography medium, which is characterized by comprising the following steps:
(1) Taking silica gel with the surface covered with poly (styrene-divinylbenzene), adding 3-acrylamidophenylboronic acid into the silica gel, and reacting to obtain grafted silica gel;
(2) And (3) adding 6-aminopyridine boric acid into the grafted silica gel obtained in the step (1), and reacting to obtain the boron affinity chromatography medium.
8. The method for preparing a boron affinity chromatography medium according to claim 7, wherein the silica gel coated with poly (styrene-divinylbenzene) is obtained by the following method: adding 5.5-6.0mol/L hydrochloric acid into silica gel, heating and refluxing for 5-6h, filtering and drying to obtain activated silica gel; adding the activated silica gel into a poly (styrene-divinylbenzene) solution, and stirring at 60-80 ℃ until volatile components volatilize to obtain the silica gel with the poly (styrene-divinylbenzene) coated on the surface.
9. The method for preparing a boron affinity chromatography medium according to claim 7, wherein the step (1) specifically comprises: dispersing silica gel coated with poly (styrene-divinylbenzene) on the surface into toluene, adding 4-chloromethyl phenyl trimethoxy silane into the toluene, reacting for 8-16h at 100-120 ℃, and filtering to obtain silica gel with a surface fixed initiator; dispersing the silica gel with the surface fixed with the initiator in N, N-dimethylformamide, adding 3-acrylamidophenylboronic acid, 2' -bipyridine and cuprous bromide into the mixture, and stirring the mixture at 90-100 ℃ for reaction for 5-10 hours to obtain grafted silica gel.
10. The method for preparing a boron affinity chromatography medium according to claim 7, wherein the step (2) specifically comprises: adding 6-aminopyridine boric acid into the grafted silica gel obtained in the step (1), and stirring and reacting for 2-8h at 110-130 ℃ to obtain a boron affinity chromatography medium.
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