JPH01174505A - Production of composite separating agent - Google Patents
Production of composite separating agentInfo
- Publication number
- JPH01174505A JPH01174505A JP62333023A JP33302387A JPH01174505A JP H01174505 A JPH01174505 A JP H01174505A JP 62333023 A JP62333023 A JP 62333023A JP 33302387 A JP33302387 A JP 33302387A JP H01174505 A JPH01174505 A JP H01174505A
- Authority
- JP
- Japan
- Prior art keywords
- polyvinyl alcohol
- separating agent
- agent
- solution
- separating
- 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.)
- Pending
Links
- 239000002131 composite material Substances 0.000 title claims abstract description 25
- 238000004519 manufacturing process Methods 0.000 title claims description 18
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 80
- 229920002451 polyvinyl alcohol Polymers 0.000 claims abstract description 57
- 239000004372 Polyvinyl alcohol Substances 0.000 claims abstract description 54
- 238000000926 separation method Methods 0.000 claims abstract description 34
- 239000002245 particle Substances 0.000 claims abstract description 31
- 150000003839 salts Chemical class 0.000 claims abstract description 18
- 239000003960 organic solvent Substances 0.000 claims abstract description 17
- 238000006243 chemical reaction Methods 0.000 claims abstract description 14
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 13
- 229920001477 hydrophilic polymer Polymers 0.000 claims abstract description 4
- 150000008282 halocarbons Chemical class 0.000 claims abstract 2
- 239000007864 aqueous solution Substances 0.000 claims description 17
- 238000004132 cross linking Methods 0.000 claims description 15
- 239000006185 dispersion Substances 0.000 claims description 12
- -1 dialdehyde compound Chemical class 0.000 claims description 4
- 239000003377 acid catalyst Substances 0.000 claims 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 abstract description 20
- 150000004676 glycans Chemical class 0.000 abstract description 18
- 229920001282 polysaccharide Polymers 0.000 abstract description 18
- 239000005017 polysaccharide Substances 0.000 abstract description 18
- 102000004169 proteins and genes Human genes 0.000 abstract description 13
- 108090000623 proteins and genes Proteins 0.000 abstract description 13
- 239000000243 solution Substances 0.000 abstract description 13
- 239000011780 sodium chloride Substances 0.000 abstract description 9
- 230000008961 swelling Effects 0.000 abstract description 5
- LEQAOMBKQFMDFZ-UHFFFAOYSA-N glyoxal Chemical compound O=CC=O LEQAOMBKQFMDFZ-UHFFFAOYSA-N 0.000 abstract description 4
- 238000006116 polymerization reaction Methods 0.000 abstract description 3
- 238000007127 saponification reaction Methods 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 229940015043 glyoxal Drugs 0.000 abstract description 2
- 239000012266 salt solution Substances 0.000 abstract description 2
- 239000008187 granular material Substances 0.000 abstract 4
- 230000003115 biocidal effect Effects 0.000 abstract 1
- 239000002612 dispersion medium Substances 0.000 abstract 1
- 238000000746 purification Methods 0.000 abstract 1
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 49
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 24
- 238000001879 gelation Methods 0.000 description 19
- 229920002307 Dextran Polymers 0.000 description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 238000003756 stirring Methods 0.000 description 10
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 8
- 238000011088 calibration curve Methods 0.000 description 7
- 229920005654 Sephadex Polymers 0.000 description 5
- 239000012507 Sephadex™ Substances 0.000 description 5
- 239000002585 base Substances 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 239000002202 Polyethylene glycol Substances 0.000 description 4
- 239000003513 alkali Substances 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 238000010992 reflux Methods 0.000 description 4
- 229920000936 Agarose Polymers 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000003054 catalyst Substances 0.000 description 3
- 229920006217 cellulose acetate butyrate Polymers 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 229910052938 sodium sulfate Inorganic materials 0.000 description 3
- 235000011152 sodium sulphate Nutrition 0.000 description 3
- 230000002269 spontaneous effect Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- SCYULBFZEHDVBN-UHFFFAOYSA-N 1,1-Dichloroethane Chemical compound CC(Cl)Cl SCYULBFZEHDVBN-UHFFFAOYSA-N 0.000 description 2
- BRLQWZUYTZBJKN-UHFFFAOYSA-N Epichlorohydrin Chemical compound ClCC1CO1 BRLQWZUYTZBJKN-UHFFFAOYSA-N 0.000 description 2
- IMROMDMJAWUWLK-UHFFFAOYSA-N Ethenol Chemical compound OC=C IMROMDMJAWUWLK-UHFFFAOYSA-N 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- 108010062374 Myoglobin Proteins 0.000 description 2
- 102000036675 Myoglobin Human genes 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 150000001338 aliphatic hydrocarbons Chemical class 0.000 description 2
- 239000012888 bovine serum Substances 0.000 description 2
- MVPPADPHJFYWMZ-UHFFFAOYSA-N chlorobenzene Chemical compound ClC1=CC=CC=C1 MVPPADPHJFYWMZ-UHFFFAOYSA-N 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 229910052736 halogen Inorganic materials 0.000 description 2
- 150000002367 halogens Chemical class 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- CUNWUEBNSZSNRX-RKGWDQTMSA-N (2r,3r,4r,5s)-hexane-1,2,3,4,5,6-hexol;(z)-octadec-9-enoic acid Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO.OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO.CCCCCCCC\C=C/CCCCCCCC(O)=O.CCCCCCCC\C=C/CCCCCCCC(O)=O.CCCCCCCC\C=C/CCCCCCCC(O)=O CUNWUEBNSZSNRX-RKGWDQTMSA-N 0.000 description 1
- ZORQXIQZAOLNGE-UHFFFAOYSA-N 1,1-difluorocyclohexane Chemical compound FC1(F)CCCCC1 ZORQXIQZAOLNGE-UHFFFAOYSA-N 0.000 description 1
- OCJBOOLMMGQPQU-UHFFFAOYSA-N 1,4-dichlorobenzene Chemical compound ClC1=CC=C(Cl)C=C1 OCJBOOLMMGQPQU-UHFFFAOYSA-N 0.000 description 1
- UWFRVQVNYNPBEF-UHFFFAOYSA-N 1-(2,4-dimethylphenyl)propan-1-one Chemical compound CCC(=O)C1=CC=C(C)C=C1C UWFRVQVNYNPBEF-UHFFFAOYSA-N 0.000 description 1
- APISJZGSHUOIPO-UHFFFAOYSA-N 1-(2-aminoacetyl)oxybutyl 2-aminoacetate Chemical compound NCC(=O)OC(CCC)OC(=O)CN APISJZGSHUOIPO-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- ZNZYKNKBJPZETN-WELNAUFTSA-N Dialdehyde 11678 Chemical class N1C2=CC=CC=C2C2=C1[C@H](C[C@H](/C(=C/O)C(=O)OC)[C@@H](C=C)C=O)NCC2 ZNZYKNKBJPZETN-WELNAUFTSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 229920000896 Ethulose Polymers 0.000 description 1
- 239000001856 Ethyl cellulose Substances 0.000 description 1
- ZZSNKZQZMQGXPY-UHFFFAOYSA-N Ethyl cellulose Chemical compound CCOCC1OC(OC)C(OCC)C(OCC)C1OC1C(O)C(O)C(OC)C(CO)O1 ZZSNKZQZMQGXPY-UHFFFAOYSA-N 0.000 description 1
- 239000001859 Ethyl hydroxyethyl cellulose Substances 0.000 description 1
- 102000008857 Ferritin Human genes 0.000 description 1
- 108050000784 Ferritin Proteins 0.000 description 1
- 238000008416 Ferritin Methods 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- 241000755266 Kathetostoma giganteum Species 0.000 description 1
- 239000011837 N,N-methylenebisacrylamide Substances 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 241000978776 Senegalia senegal Species 0.000 description 1
- HVUMOYIDDBPOLL-XWVZOOPGSA-N Sorbitan monostearate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O HVUMOYIDDBPOLL-XWVZOOPGSA-N 0.000 description 1
- 102000009843 Thyroglobulin Human genes 0.000 description 1
- 108010034949 Thyroglobulin Proteins 0.000 description 1
- 239000000205 acacia gum Substances 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 238000006359 acetalization reaction Methods 0.000 description 1
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 1
- 238000010533 azeotropic distillation Methods 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 230000001112 coagulating effect Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005056 compaction Methods 0.000 description 1
- 229940117389 dichlorobenzene Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- GKIPXFAANLTWBM-UHFFFAOYSA-N epibromohydrin Chemical compound BrCC1CO1 GKIPXFAANLTWBM-UHFFFAOYSA-N 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 235000019325 ethyl cellulose Nutrition 0.000 description 1
- 229920001249 ethyl cellulose Polymers 0.000 description 1
- 235000019326 ethyl hydroxyethyl cellulose Nutrition 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 238000005469 granulation Methods 0.000 description 1
- 230000003179 granulation Effects 0.000 description 1
- JEGUKCSWCFPDGT-UHFFFAOYSA-N h2o hydrate Chemical compound O.O JEGUKCSWCFPDGT-UHFFFAOYSA-N 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 239000001593 sorbitan monooleate Substances 0.000 description 1
- 235000011069 sorbitan monooleate Nutrition 0.000 description 1
- 229940035049 sorbitan monooleate Drugs 0.000 description 1
- 239000001587 sorbitan monostearate Substances 0.000 description 1
- 235000011076 sorbitan monostearate Nutrition 0.000 description 1
- 229940035048 sorbitan monostearate Drugs 0.000 description 1
- 229960005078 sorbitan sesquioleate Drugs 0.000 description 1
- 239000012798 spherical particle Substances 0.000 description 1
- KUCOHFSKRZZVRO-UHFFFAOYSA-N terephthalaldehyde Chemical compound O=CC1=CC=C(C=O)C=C1 KUCOHFSKRZZVRO-UHFFFAOYSA-N 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229960002175 thyroglobulin Drugs 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
Landscapes
- Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Processes Of Treating Macromolecular Substances (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、複合化分離剤の製造方法に関するものであり
詳しくは、蛋白質、酵素、杭先物質等の生体関連の比較
的高分子量の物質を分離、精製するに好適な複合化分離
剤の製造方法に関するものである。[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for producing a composite separation agent, and more specifically, it relates to a method for producing a composite separation agent, and more specifically, it relates to a method for producing a composite separation agent, and more specifically, it relates to a method for producing a composite separating agent, and more specifically, it relates to a method for producing a complex separation agent, and specifically, it relates to a method for producing a composite separating agent, and more specifically, it relates to a method for producing a complex separation agent, and more specifically, it relates to a method for producing a composite separation agent. The present invention relates to a method for producing a composite separating agent suitable for separating and purifying.
従来、生体関連物質を分離、精製する分離剤としては、
デキストランやアガロース等の多糖類を素材とする分離
剤が知られている。これらの分離剤は、親水性に富んで
いる為、特に蛋白質との非特異的吸着が少ないという特
徴がある。Traditionally, separation agents for separating and purifying biological substances include:
Separating agents made from polysaccharides such as dextran and agarose are known. Since these separation agents are highly hydrophilic, they are characterized by low nonspecific adsorption, especially with proteins.
又水中における膨潤度が10−440ゴ/f−dryで
あり、且つ巨大網目構造を持ち、優れた分離能を有して
いる。特にゲルテ過クロマトグラフィー用として好適な
分離剤と言うことができる。Furthermore, it has a swelling degree in water of 10-440 g/f-dry, a huge network structure, and excellent separation ability. It can be said that it is a separation agent particularly suitable for gelte permeation chromatography.
しかし、これらの分離剤は、優れた分離能を有する反面
、軟質であるため、機械的強度が弱く、高層高、高流速
処理が必要な工業的スケールでは、圧密化の為に通液が
不可能になる場合が多いという問題を有している。However, although these separation agents have excellent separation ability, they are soft and have weak mechanical strength, and on an industrial scale that requires high-rises and high-flow rates, it is difficult to pass the liquid through due to compaction. The problem is that there are many cases where this is possible.
本発明は、巨大網目構造を有する多糖類系分離剤の優れ
た分離能を維持しつつ、上記の分離剤の問題点である機
械的強度を改善した、新規な複合化分離剤の製造方法を
提供することを目的とする。The present invention provides a method for producing a novel composite separation agent that maintains the excellent separation ability of a polysaccharide separation agent having a large network structure and improves the mechanical strength, which is a problem with the above-mentioned separation agents. The purpose is to provide.
そこで、上記分離剤の機械的強度を改善するため複合化
分離剤に関して検討した結果、架橋度カ≠〜10モルチ
で、マクロポアー−造を有するポリビニルアルコール基
体中に包含することにより、本目的に合致する複合化分
離剤が得られることを見出し本発明を完成した。Therefore, in order to improve the mechanical strength of the above-mentioned separating agent, we investigated a composite separating agent, and found that it met this purpose by incorporating it into a polyvinyl alcohol base having a macropore structure with a degree of cross-linking of ~10 mol. The present invention was completed by discovering that a composite separating agent that can be obtained can be obtained.
すなわち、本発明は巨大網目構造を有する親水性ポリマ
ーからなる分離剤を膨潤させた状態で、ポリビニルアル
コール及び塩類の水溶液と共に有機溶媒からなる分散浴
中に分散造球させ、前記分離剤および塩水溶液を包含し
たポリビニルアルコール粒子を得、次いで該ポリビニル
アルコ−2粒子を自然ゲル化させたのち架橋剤と反応さ
せることを特徴とする複合化分離剤の製造方法を要旨と
するものである。That is, in the present invention, a swollen separating agent made of a hydrophilic polymer having a large network structure is dispersed and spheroidized in a dispersion bath made of an organic solvent together with an aqueous solution of polyvinyl alcohol and a salt, and the separating agent and the salt aqueous solution are dispersed into balls. The gist of the present invention is to provide a method for producing a composite separation agent, which is characterized in that polyvinyl alcohol particles containing the following are obtained, the polyvinyl alcohol-2 particles are spontaneously gelled, and then reacted with a crosslinking agent.
本発明の複合化分離剤は、粒径が2Q〜7000μで、
マクロポア−を有する架橋ポリビニルアルコール粒子中
に、粒径5−zooμの多糖類系分離剤を≠0−10%
(容積比)包含したものである。The composite separation agent of the present invention has a particle size of 2Q to 7000μ,
≠0-10% polysaccharide separating agent with a particle size of 5-zooμ in cross-linked polyvinyl alcohol particles having macropores
(volume ratio).
複合化分離剤は、多糖類系分離剤を膨潤させた状態でポ
リビニルアルコール水溶液と共に分散浴中に分散、造球
することにより、多糖類系分離剤を包含し、そののち、
ポリビニルアルコール水溶液の自然ゲル化現象を利用し
て造球、不溶化したのち架橋反応を行なうことにより製
造できる。The composite separating agent contains the polysaccharide separating agent by dispersing the swollen polysaccharide separating agent in a dispersion bath with an aqueous polyvinyl alcohol solution and forming balls, and then,
It can be produced by making balls by utilizing the natural gelation phenomenon of an aqueous polyvinyl alcohol solution, making it insolubilized, and then carrying out a crosslinking reaction.
架橋ポリビニルアルコール基体のマクロポアは、包含し
た多糖類系分離剤の巨大網目構造のポアーよりも大きく
蛋白質、酵素等の生体高分子が自由に出入できるような
ものが好ましい。The macropores of the crosslinked polyvinyl alcohol base are preferably larger than the pores of the giant network structure of the included polysaccharide separation agent, allowing biopolymers such as proteins and enzymes to freely enter and exit.
このマクロポアはポリビニルアルコール水溶液の自然ゲ
ル化現象を利用することにより形成することが可能であ
る。These macropores can be formed by utilizing the natural gelation phenomenon of an aqueous polyvinyl alcohol solution.
ここで言う自然ゲル化現象とは、ポリビニルアルコール
水溶液が、架橋剤を用いることなく、不溶化することで
ある。The natural gelation phenomenon referred to here means that an aqueous polyvinyl alcohol solution becomes insolubilized without using a crosslinking agent.
ポリビニルアルコール水溶液は放置すると、経時的に粘
度が上昇し、ついにはゲル化することが古くから知られ
ている。It has long been known that when an aqueous polyvinyl alcohol solution is left to stand, its viscosity increases over time and eventually gels.
この現象は、ポリビニルアルコール分子間の水素結合に
より生起すると考えられており、ポリビニルアルコール
水溶液の濃度が高い程又放置温度が低い程、早くゲル化
がおこる。自然ゲル化現象を利用し製造することにより
マクロポアを有する原因は明らかではないが、おそらく
、自然ゲル化現象に伴ない、ポリビニルアルコールが反
応媒体中から析出し、相分離することにより、不均一構
造をとり、多孔質化されるものと考えられる。This phenomenon is thought to occur due to hydrogen bonds between polyvinyl alcohol molecules, and the higher the concentration of the aqueous polyvinyl alcohol solution or the lower the temperature at which it is left, the faster gelation occurs. The reason why macropores are formed by manufacturing using natural gelation phenomenon is not clear, but it is probably due to polyvinyl alcohol precipitating from the reaction medium and phase separation due to natural gelation phenomenon, resulting in a heterogeneous structure. It is thought that this makes the material more porous.
本発明は、架橋度がよ〜10モルチで、マクロポア構造
を有するポリビニルアルコール基体中に、多糖類系分離
剤を包含させた複合化分離剤の製造方法に関するもので
あり、複合化分離剤の製造方法は、多糖類系分離剤を膨
潤させた状態でポリビニルアルコール及び塩類の水溶液
と共に有機溶媒からなる分散浴中に分散、造球させて、
前記分離剤および塩水溶液を包含したポリビニルアルコ
ール粒子を得、次いて該ポリビニルアルコール粒子を自
然ゲル化させたのち架橋剤と反応させることを特徴とす
るものである。包含する分離剤としては、巨大網目構造
を有する架橋デキストラン、アガロース、架橋アガロー
ス等の多糖類系、アクリルアミドとN、Nメチレンビス
アクリルアミドを共重合させたポリアクリルアミド系等
が用いられる。The present invention relates to a method for producing a composite separating agent in which a polysaccharide-based separating agent is incorporated into a polyvinyl alcohol base having a macropore structure and a degree of crosslinking of from 10 to 10 mol. The method involves dispersing the swollen polysaccharide separating agent in a dispersion bath consisting of an organic solvent together with an aqueous solution of polyvinyl alcohol and salts to form balls.
The method is characterized in that polyvinyl alcohol particles containing the separating agent and a salt aqueous solution are obtained, and then the polyvinyl alcohol particles are spontaneously gelled and then reacted with a crosslinking agent. Examples of separating agents that can be used include crosslinked dextran having a large network structure, agarose, polysaccharide systems such as crosslinked agarose, and polyacrylamide systems obtained by copolymerizing acrylamide with N,N methylenebisacrylamide.
前記の分離剤は、乾燥粉末状であることから、水にて膨
潤させた状態でポリビニルアルコール及び塩類の水溶液
に添加する。Since the separating agent is in the form of a dry powder, it is swollen with water and added to the aqueous solution of polyvinyl alcohol and salts.
又、塩類を添加したポリビニルアルコール水溶液中に添
加し膨潤することもできる。Alternatively, it can be added to an aqueous polyvinyl alcohol solution to which salts have been added to cause swelling.
前記分離剤を水にて膨潤させるためには、各分離剤の膨
潤度で異なるが、室温で3〜72時間、沸騰水浴で/−
7時間必要である。In order to swell the separating agent with water, it is necessary to swell the separating agent with water in a boiling water bath for 3 to 72 hours at room temperature, although the degree of swelling varies depending on the degree of swelling of each separating agent.
It takes 7 hours.
多糖類系分離剤のポリビニルアルコール基体中への包含
は、分離剤を膨潤した状態で塩類を添加したポリビニル
アルコール水溶液と共に、有機溶媒からなる分散浴中に
分散、造球させることにより包含させることができる。The polysaccharide separating agent can be incorporated into the polyvinyl alcohol base by dispersing the separating agent in a swollen state together with an aqueous polyvinyl alcohol solution to which salts have been added in a dispersion bath consisting of an organic solvent to form balls. can.
ポリビニルアルコールとしては重合度数十〜数十、好ま
しくは200−2000で、ケン化度りQモルチ以上、
好ましくはり!そルチ以上のものが用いられる。ポリビ
ニルアルコールは2〜30%、好ましくは!〜lj%と
なるように水に溶解して自然ゲル化を行なう。しかしポ
リビニルアルコール水溶液だけでは自然ゲル化に長時間
を要することから自然ゲル化を促進するためにポリビニ
ルアルコール水溶液に塩を添加する。塩としては塩化ナ
トリウム、硫酸ナトリウム等のポリビニルアルコールを
沈殿凝析する効果があるものを用いればよい。Polyvinyl alcohol has a polymerization degree of several tens to several tens, preferably 200-2000, a saponification degree of Q mol or more,
Preferably! Something more than that is used. Polyvinyl alcohol is 2-30%, preferably! It is dissolved in water to form a natural gel to a concentration of ~lj%. However, since it takes a long time for natural gelation to occur with only an aqueous polyvinyl alcohol solution, a salt is added to the aqueous polyvinyl alcohol solution in order to promote natural gelation. As the salt, one having the effect of precipitating and coagulating polyvinyl alcohol, such as sodium chloride and sodium sulfate, may be used.
その使用量は塩の種類等により異なるが、通常、3%以
上、ポリビニルアルコールが析出しない範囲内の濃度で
添加される。The amount used varies depending on the type of salt, etc., but it is usually added at a concentration of 3% or more, within a range where polyvinyl alcohol does not precipitate.
特に塩化ナトリウムの添加量に関しては、ポリビニルア
ルコールの重合度、ケン化度、及び濃度により異なるが
、たとえば、重合度2001ケン化度りt%以上のポリ
ビニルアルコール70%水溶液を用いる場合には、/−
/コチ(好ましくは3〜10%)、7%ポリビニルアル
コール水溶液を用いる場合には、/−77%(好ましく
はよ一/3%)の量で添加される。In particular, the amount of sodium chloride added varies depending on the degree of polymerization, degree of saponification, and concentration of polyvinyl alcohol. −
/ flathead (preferably 3 to 10%), and when using a 7% polyvinyl alcohol aqueous solution, it is added in an amount of /-77% (preferably 1/3%).
さらに、塩を添加したポリビニルアルコール水溶液に水
酸化ナトリウム等のアルカリを添加することにより、自
然ゲル化をより促進することもできる。Furthermore, natural gelation can be further promoted by adding an alkali such as sodium hydroxide to the polyvinyl alcohol aqueous solution to which a salt has been added.
自然ゲル化は塩を添加したポリビニルアルコー−ル水溶
液および膨潤させた多糖類系分離剤と共に有機溶媒中に
て分散造球後行なう。Natural gelation is carried out after dispersion in an organic solvent together with a salt-added polyvinyl alcohol aqueous solution and a swollen polysaccharide separating agent.
有機溶剤としては、通常、トルエン、ベンゼン、クロル
ベンゼン、ジクロルベンゼン等の芳香族炭化水素および
そのハロゲン誘導体、n −へブタン、n−ヘキサン、
流動パラフィン、シクロヘキサン、ジクロルメタン、ジ
クロルエタン等の脂肪族炭化水素、脂肪族炭化水素およ
びそのハロゲン誘導体などが用いられる。これらの有機
溶剤は通常は単独で用いられるが、2種以上を混合して
用いても差支えない。The organic solvent usually includes aromatic hydrocarbons such as toluene, benzene, chlorobenzene, dichlorobenzene and their halogen derivatives, n-hebutane, n-hexane,
Aliphatic hydrocarbons such as liquid paraffin, cyclohexane, dichloromethane, dichloroethane, aliphatic hydrocarbons and their halogen derivatives, etc. are used. These organic solvents are usually used alone, but two or more types may be used in combination.
有機溶剤の使用量はポリビニルアルコール水溶液の2(
容量)倍以上、好ましくは2〜6(容量)倍である。ま
た有機溶剤中には分散安定剤トシテ、エチルセルロース
、セルロースアセテートブチレート、エチルヒドロキシ
エチルセルロース等の油溶性セルロース、アラビアゴム
、ソルビタンセスキオレエート、ソルビタンモノオレエ
ート、ソルビタンモノステアレート等の油溶性分散安定
剤を添加するのが好ましい。The amount of organic solvent used is 2 (2) of the polyvinyl alcohol aqueous solution.
(capacity) times or more, preferably 2 to 6 (capacity) times. In addition, dispersion stabilizers such as Toshite, ethyl cellulose, cellulose acetate butyrate, and ethyl hydroxyethyl cellulose, as well as oil-soluble dispersion stabilizers such as gum arabic, sorbitan sesquioleate, sorbitan monooleate, and sorbitan monostearate, are included in the organic solvent. It is preferable to add an agent.
その量は有機溶剤に対して通常0.0r−10%、好ま
しくは0./ −1%である。The amount is usually 0.0r-10%, preferably 0.0% to the organic solvent. / -1%.
自然ゲル化は分散造粒後、0−10℃、好ましくは3〜
30℃で、2〜200時間、好ましくは5−ioo時間
行なう。Natural gelation occurs at 0-10℃ after dispersion granulation, preferably from 3 to 10℃.
It is carried out at 30° C. for 2 to 200 hours, preferably 5-ioo hours.
自然ゲル化後、架橋反応を行なう。After spontaneous gelation, a crosslinking reaction is performed.
架橋剤としては、グリオキザール、グルタルアルデヒド
、テレフタルアルデヒド等のジアルデヒド化合物; /
、2,3.≠−ジェポキシブタン等のジェポキシ化合物
;エチレングリコールジグリシジルエーテル、/、弘−
ブタンジオールジグリシシルエーテル等のグリシジルエ
ーテル化合物;エピクロルヒドリン、エビブロモヒドリ
ン等のエピハロヒドリン化合物などポリビニルアルコー
ル間に2個以上の炭素原子を有する架橋部分を形成し得
るものが用いられる。好適には、アセタール化架橋が形
成されるジアルデヒド化合物が用いられる。架橋剤は、
ポリビニルアルコールの全水酸基に対し通常1モルチ以
上用いる。As a crosslinking agent, a dialdehyde compound such as glyoxal, glutaraldehyde, terephthalaldehyde; /
, 2, 3. ≠-Jepoxy compounds such as jepoxybutane; ethylene glycol diglycidyl ether, /, Hiro-
Glycidyl ether compounds such as butanediol diglycyl ether; epihalohydrin compounds such as epichlorohydrin and shrimp bromohydrin, which can form a crosslinking moiety having two or more carbon atoms between polyvinyl alcohols, are used. Preferably, dialdehyde compounds are used in which acetalization crosslinks are formed. The crosslinking agent is
It is usually used in an amount of 1 mol or more based on the total hydroxyl groups of polyvinyl alcohol.
架橋反応は自然ゲル化後、上記の架橋剤を添加して行な
う。又、架橋反応は自然ゲル化後の球状の粒子を濾過し
て有機溶剤を分離し、アセトン、メタノール洗浄したの
ち水中にて行なうこともできる。The crosslinking reaction is carried out by adding the above-mentioned crosslinking agent after natural gelation. Further, the crosslinking reaction can also be carried out in water after filtration of the spherical particles after natural gelation to separate the organic solvent and washing with acetone and methanol.
架橋反応は、自然ゲル化後、0℃ないし100℃、好ま
しくは30℃ないしtO℃で1時間ないし20時間、好
ましくは2時間ないしt時間。The crosslinking reaction is carried out at 0° C. to 100° C., preferably 30° C. to t0° C., for 1 hour to 20 hours, preferably 2 hours to t hours, after spontaneous gelation.
行なう。架橋反応の際、架橋剤に応じて、触媒として塩
酸、硫酸等の酸、又は、水酸化す) IJウム、水酸化
カリウム等のアルカリを用いる。Let's do it. During the crosslinking reaction, an acid such as hydrochloric acid or sulfuric acid, or an alkali such as hydroxide, potassium hydroxide, etc. is used as a catalyst depending on the crosslinking agent.
架橋剤として、ジアルデヒド化合物を用いる場合は、触
媒として酸を用いる。酸の使用量は水層中の濃度が/N
以下になる様に添加するのが好ましい。架橋剤として、
二Iノ・ロヒドリン化合物を用いる場合は触媒としてア
ルカリを用いる。アルカリの使用量は水層中の濃度が2
N以上になる様に添加する。 −
上記のごとく架橋反応を水中にて行なう場合も含め、架
橋反応の際、硫酸ナトリウム、塩化す) IJウム等の
塩又は塩水溶液を添加することが好ましい。When using a dialdehyde compound as a crosslinking agent, an acid is used as a catalyst. The amount of acid used is determined by the concentration in the aqueous layer /N
It is preferable to add the following amounts. As a crosslinking agent,
When using a 2I-rohydrin compound, an alkali is used as a catalyst. The amount of alkali used is when the concentration in the water layer is 2.
Add so that the amount is N or higher. - During the crosslinking reaction, including when the crosslinking reaction is carried out in water as described above, it is preferable to add a salt or an aqueous salt solution such as sodium sulfate or IJum chloride.
塩の添加量は種類等により異なるが、通常6チ以上、ポ
リビニルアルコール水溶液が沈殿凝析を起こす濃度以上
で添加される。例えば塩化ナトリウムでは20%以上、
硫酸ナトリウムでは4%以上添加される。The amount of salt added varies depending on the type, etc., but it is usually added at a concentration of 6 salts or more, which is higher than the concentration at which the polyvinyl alcohol aqueous solution causes precipitation and coagulation. For example, for sodium chloride, 20% or more,
For sodium sulfate, 4% or more is added.
このようにして得られた架橋ポリビニルアルコール粒子
は、濾過分離する。次いでアセトン、メタノール等の水
混合性の有機溶剤で洗浄したのち充分水洗する。The crosslinked polyvinyl alcohol particles thus obtained are separated by filtration. Next, it is washed with a water-miscible organic solvent such as acetone or methanol, and then thoroughly washed with water.
有機溶媒を完全に除去するためには、分離した架橋ポリ
ビニルアルコール粒子を水中で加熱し共沸により有機溶
媒を留去することが好ましい。In order to completely remove the organic solvent, it is preferable to heat the separated crosslinked polyvinyl alcohol particles in water and distill off the organic solvent by azeotropic distillation.
以下に実施例により本発明を更に具体的に説明するが、
本発明は以下の実施例に限定されるものではない。The present invention will be explained in more detail with reference to Examples below.
The present invention is not limited to the following examples.
実施例I
攪拌機と還流冷却管を取りつけたOlS tの三ツロフ
ラスコに、ジクロルエタンxjOmlt入れ、セルロー
スアセテート?°チレート(イーストマンコダック社製
、商品名CAB 311−20)0.21を溶解し分散
浴とした。Example I Into an OlSt three-way flask equipped with a stirrer and a reflux condenser, dichloroethane xjOml was added, and cellulose acetate was added. A dispersion bath was prepared by dissolving 0.21 °C of thyrate (manufactured by Eastman Kodak Company, trade name: CAB 311-20).
別に、攪拌機と還流冷却管を取りつけた200−の三ツ
ロフラスコに、塩化ナトリウムよt1水jOtdを入れ
、攪拌しながら溶解後、これに粉末のポリビニルアルコ
ール(日本合成化学工業株式会社製、商標ゴーセノール
NL−01)2、j?を加えて分散させ?r℃で1時間
加熱して溶解したのち、さらにjN水酸化ナトリウムを
6−加えて攪拌した。Separately, put sodium chloride and t1 water jOtd into a 200-meter Mitsuro flask equipped with a stirrer and a reflux condenser, dissolve it while stirring, and add powdered polyvinyl alcohol (manufactured by Nippon Gosei Kagaku Kogyo Co., Ltd., trademark Gohsenol NL) to this. -01)2,j? Add and disperse? After heating at r°C for 1 hour to dissolve, 6-jN sodium hydroxide was further added and stirred.
架橋デキストラン系分離剤(ファルマシア社製、商品名
セファデックスG j O(Medium ) )は、
攪拌機と還流冷却管を取りつけた200dの三ツロフラ
スコ中の塩化カリウム水溶液(塩化ナトリウムよ?、水
j 011Ll )中にst加えて分散させ2℃℃で7
時間加熱して膨潤させたのち、室温まで冷却した上記の
ポリビニルアルコール溶液中に加えて攪拌した。その後
、上記の有機溶媒からなる分散浴に分散した。室温でl
り時間攪拌しながら放置し自然ゲル化を行なった。The cross-linked dextran-based separation agent (manufactured by Pharmacia, trade name Sephadex G j O (Medium)) is
Add ST to a potassium chloride aqueous solution (sodium chloride, water) in a 200 d Mitsulo flask equipped with a stirrer and a reflux condenser, disperse and heat at 2°C for 7 hours.
After heating for a period of time to swell, it was added to the above polyvinyl alcohol solution which had been cooled to room temperature and stirred. Thereafter, it was dispersed in a dispersion bath consisting of the above organic solvent. l at room temperature
The mixture was allowed to stand for several hours with stirring to allow natural gelation.
自然ゲル化後、さらにλj%食塩水ioo、zを加え、
3時間攪拌しながら放置したのち、濾過し、アセトン、
メタノール洗浄後、水洗し再び攪拌機付き!00tgl
の三ツロフラスコに移し、塩化ナトリウム4cOt、水
200.1を入れ攪拌しながら、2j%グルタルアルデ
ヒド水溶液l≠yttlを加えたのち!N塩酸4cal
を加え、攪拌しなから6j’Cで2時間加熱して架橋反
応を行なった。室温まで冷却したのち、濾過し、十分水
洗した。平均粒径220μの架橋デキストラン系分離剤
を約76個を包含した平均粒径700μの乳白潤色の球
状架橋ポリビニルアルコール粒子が得られた。After natural gelation, further add λj% saline solution ioo, z,
After stirring for 3 hours, filter and add acetone,
After washing with methanol, wash with water and add a stirrer again! 00tgl
Transfer to a Mitsuro flask, add 4 cOt of sodium chloride and 200.1 g of water, and add 2j% glutaraldehyde aqueous solution l≠yttl while stirring! N hydrochloric acid 4cal
was added and heated at 6J'C for 2 hours without stirring to carry out a crosslinking reaction. After cooling to room temperature, it was filtered and thoroughly washed with water. Milky white spherical crosslinked polyvinyl alcohol particles with an average particle size of 700μ and containing about 76 crosslinked dextran-based separating agents with an average particle size of 220μ were obtained.
実施例コ
使用した架橋デキストラン系分離剤(ファルマシア社製
、商標セファデックスG100(Medium ) )
の種類、添加量を第1表の如く変えた以外は実施例1と
同様の操作を行なった。Cross-linked dextran-based separating agent used in Examples (manufactured by Pharmacia, trademark Sephadex G100 (Medium))
The same operation as in Example 1 was carried out except that the type and amount of addition were changed as shown in Table 1.
平均粒径210μの架橋デキストラン系分離剤を約/r
個を包含した平均粒径700μの乳白潤色の球状架橋ポ
リビニルアルコール粒子が得られた。Cross-linked dextran-based separating agent with an average particle size of 210 μm
Milky-white spherical crosslinked polyvinyl alcohol particles having an average particle diameter of 700 μm and containing 100% polyvinyl alcohol particles were obtained.
実施例3
攪拌機と還流冷却管を取りつけた200dの三ツロフラ
スコに、塩化ナトリウムlOf、水100dを入れ、攪
拌しながら溶解後、これに粉末のポリビニルアルコール
(ゴーセノールNH−/ r ) 3.! ?、架橋デ
キストラン系分離剤(セファデックスGtso(SF)
) 2.39を加えて分散させりt℃で3時間加熱して
ポリビニルアルコールは溶解し、架橋デキストラン系分
離剤は膨潤させたのち、さらにtN水酸化ナトリウム参
d加えて攪拌した。Example 3 In a 200 d three-meter flask equipped with a stirrer and a reflux condenser, put 100 d of sodium chloride and 100 d of water, dissolve with stirring, and add powdered polyvinyl alcohol (Gohsenol NH-/r) to this. ! ? , cross-linked dextran-based separation agent (Sephadex Gtso (SF)
) 2.39 was added and dispersed, and the mixture was heated at t° C. for 3 hours to dissolve the polyvinyl alcohol and swell the crosslinked dextran-based separation agent. Then, tN sodium hydroxide was further added and stirred.
室温まで冷却したのち、セルロースアセテートブチレー
トの添加量を7,22r tに変えた以外は実施例1と
同様な有機溶媒中に分散し室温でlり時間攪拌しながら
放置し、自然ゲル化を行なった。After cooling to room temperature, the mixture was dispersed in the same organic solvent as in Example 1 except that the amount of cellulose acetate butyrate added was changed to 7.22 rt, and left to stand at room temperature with stirring for 1 hour to allow spontaneous gelation. I did it.
自然ゲル化後、さらに20%塩化ナトリウム水溶液10
0−を加え、3時間攪拌しながら放置したのち、濾過し
1.アセトン、メタノール洗浄後、水洗し再び攪拌機付
き300rdの三ツロフラスコに移し、水to、gを入
れ攪拌しながら、冷却下、粒状の水酸化す) IJウム
72fを溶解したのち、エピクロルヒドリン3rdを加
え攪拌しながら60℃で6時間加熱して架橋反応を行な
った。室温まで冷却したのち、濾過し、十分水洗したの
ちもう一度架橋反応を同一条件で行なった。平均粒径6
?μの架橋デキストラン系分離剤を約70個包含した平
均粒径JjOμの薄い黄色の球状架橋ポリビニルアルコ
ール粒子が得られた。After natural gelation, add 20% sodium chloride aqueous solution 10
After adding 0- and stirring for 3 hours, it was filtered and 1. After washing with acetone and methanol, wash with water and transfer again to a 300rd Mitsuro flask equipped with a stirrer, add tog of water and stir while cooling to form granular hydroxide) After dissolving IJum 72f, add epichlorohydrin 3rd and stir. While heating at 60° C. for 6 hours, a crosslinking reaction was carried out. After cooling to room temperature, it was filtered and thoroughly washed with water, and then crosslinking reaction was performed once again under the same conditions. Average particle size 6
? Pale yellow spherical crosslinked polyvinyl alcohol particles with an average particle diameter of JjOμ containing about 70 μ crosslinked dextran-based separating agents were obtained.
実施例≠
使用したポリビニルアルコールの種類、添加量及び!N
水酸化ナトリウムの、添加量を第1表の如く変えた以外
は実施例3と同様の操作を行なった。平均粒径6rμの
架橋デキストラン系分離剤を約70個包含した平均粒径
3!Oμの薄い黄色の球状架橋ポリビニルアルコール粒
子が得られた。Example ≠ Type of polyvinyl alcohol used, amount added, and! N
The same operation as in Example 3 was carried out except that the amount of sodium hydroxide added was changed as shown in Table 1. The average particle size is 3, which contains about 70 crosslinked dextran-based separating agents with an average particle size of 6rμ! Pale yellow spherical crosslinked polyvinyl alcohol particles of Oμ were obtained.
内径L21111φのガラスカラム(ジャケット付)に
粒径を/≠り〜λり7μに整粒した実施例Vで得られた
分離剤を充填した。分離剤の層高は、jQcIILであ
った。カラムを循環水で2j℃に保ち、カラム上部から
、蒸留水を一定流速で流した。充填層が安定し、カラム
の上部にとりつけた圧力計の指針が一定になったところ
で圧力計の目盛りを読みと9、その値から分離剤を充填
していない、いわゆる空力ラムの状態で前述した操作と
全く同様の操作をした場合の圧力計の読みとり値をさし
引いて圧力損失(Δp・単位Af/cil/ −t O
an −bed )を求めた。A glass column (with a jacket) having an inner diameter of L21111φ was filled with the separating agent obtained in Example V, which had been sized to have a particle size of /≠−λ7μ. The layer height of the separating agent was jQcIIL. The column was maintained at 2J°C with circulating water, and distilled water was flowed from the top of the column at a constant flow rate. When the packed bed became stable and the pointer of the pressure gauge attached to the top of the column became constant, the scale of the pressure gauge was read at 9, and from that value, it was assumed that no separation agent was packed in the so-called aerodynamic ram state, as described above. The pressure loss (Δp unit Af/cil/-t O
an-bed) was determined.
流速(LV :線流速、単位m/Hr)を種々変えて圧
力損失(Δp)を測定したところ第1図に示した結果と
なった。測定した流速範囲内では(LVとして3m/H
r以下)、本発明分離剤を用いた場合のΔpとLVの間
には直線関係が成立し、LV=sm/Hr という高流
速で通液しても、担体粒子の変形、破砕は全く認められ
なかった。When the pressure loss (Δp) was measured while varying the flow velocity (LV: linear flow velocity, unit: m/Hr), the results shown in FIG. 1 were obtained. Within the measured flow velocity range (3 m/H as LV)
When the separation agent of the present invention is used, a linear relationship is established between Δp and LV, and even when the liquid is passed at a high flow rate of LV=sm/Hr, no deformation or crushing of the carrier particles is observed. I couldn't.
なお、比較のために、実施例≠で得られた分離剤を用い
る代わりに、包含した多糖類系分離剤セファデックスc
t、z o (M) (ファルマシア社製)を用いる
、以外は上記と同様の操作を行い、ΔpとLVの関係を
求めた。その結果を第1図に示した。For comparison, instead of using the separating agent obtained in Example≠, the included polysaccharide-based separating agent Sephadex c
The same operation as above was performed except that t, zo (M) (manufactured by Pharmacia) was used to determine the relationship between Δp and LV. The results are shown in Figure 1.
多糖類系分離剤を用いた場合は、流速がLV= 0.3
m/Hrをこえたあたりから、Δpが急激に立ちはじ
め、LV = 0.4 m /Hr以上では、通液が困
難となった。When using a polysaccharide-based separation agent, the flow rate is LV = 0.3
When Δp exceeded m/Hr, Δp began to rise rapidly, and at LV = 0.4 m/Hr or more, it became difficult to pass the liquid.
応用例/
実施例/及びλで得られた複合化分離剤10簡φ×jO
OIII!IHのガラスカラムに充填し十分水洗した後
較正曲線を作成するために以下の操作を行ない、ゲルテ
過りロマトグラフィ用担体としての性能評価を行なった
。Application example / Example / Composite separation agent obtained with λ 10 simple φ x jO
OIII! After filling an IH glass column and thoroughly rinsing with water, the following operations were performed to create a calibration curve, and the performance as a support for Gelte chromatography was evaluated.
分子量既知のデキストランの水溶液(濃度jW/V96
)をiooμt、ポリエチレングリコールの水溶液(濃
度JW/V%)を300μL、上記のカラムにチャージ
し、次いで流速O0≠ml/頭で蒸留水を流し、デキス
トラン、ポリエチレングリコールを溶出した。Aqueous solution of dextran with known molecular weight (concentration jW/V96
), 300 μL of an aqueous solution of polyethylene glycol (concentration JW/V%) was charged to the above column, and then distilled water was flowed at a flow rate of O≠ml/head to elute dextran and polyethylene glycol.
流出液中のデキストラン、ポリエチレングリコールを示
差屈折計を用いて検出し、その溶出位置(ピークのトッ
プ位置)を求めた。Dextran and polyethylene glycol in the effluent were detected using a differential refractometer, and their elution positions (peak top positions) were determined.
第2図にその結果を示した。Figure 2 shows the results.
応用例コ
実施例3及びψで得られた複合化分離剤を内径L2M1
1φXj00tmHのガラスカラムに充填し、t j
OmM NaC!含有s o mMリン酸バッファー(
pH7,≠O)で十分平衡化した後、蛋白質の較正曲線
を作成するために以下の操作を行ない、ゲルテ過りロマ
トグラフィー用担体としての性能評価を行なった。Application example The composite separating agent obtained in Example 3 and ψ was
Packed into a glass column of 1φXj00tmH, t j
OmM NaC! Containing SO mM phosphate buffer (
After sufficient equilibration at pH 7, ≠ O), the following operations were performed to create a protein calibration curve, and the performance as a support for gel gel chromatography was evaluated.
下記に示した分子量の異なる蛋白質を0.1%の濃度に
なる様に、/ j OmM NaCL含有10mMリン
酸バッファー(pH7,ψO)に溶解し、/J−0μt
1上記のカラムにチャージした。チャージ後、流速o、
2ytl/rHRで/ j OmM NaCt含有j
OmMリン酸バッファーを流し蛋白質を溶出した。Proteins with different molecular weights shown below were dissolved in 10mM phosphate buffer (pH 7, ψO) containing /j OmM NaCL to a concentration of 0.1%, /J-0μt.
1 was charged to the above column. After charging, flow rate o,
At 2ytl/rHR/j Contains OmM NaCtj
The protein was eluted by flowing OmM phosphate buffer.
蛋白質の検出はUV計を用いて行なった。Protein detection was performed using a UV meter.
(J r Onm )
なお、比較のため、包含した多糖系分離剤セファデック
スGl!0(M)を用いて上記と同様の操作を行ない、
蛋白質の較正曲線を求めた。(J r Onm ) For comparison, the included polysaccharide separation agent Sephadex Gl! Perform the same operation as above using 0(M),
A protein calibration curve was determined.
蛋白質の溶出位置から下式によりKav、を求め、この
値を分子量に対してプロットし、較正曲線として第3図
に示した。Kav was determined from the elution position of the protein using the following formula, and this value was plotted against the molecular weight, and is shown in FIG. 3 as a calibration curve.
(尚、Voは分子量約200万のブルーデキストランを
用いて求めた値)
第2図より本発明の分離剤を用いて得られた較正曲線は
良好な直線性を示し、ゲル口過クロマトグラフィー用担
体として好適であることがわかる。(Note that Vo is the value determined using blue dextran with a molecular weight of approximately 2 million.) From Figure 2, the calibration curve obtained using the separation agent of the present invention shows good linearity, and is suitable for gel filtration chromatography. It can be seen that it is suitable as a carrier.
図中屋 蛋白質 分子量(×1O3)等電点/:チログ
ロプリン 667 ≠、j2:フェリ
チン 弘170 4′〜j3:
牛血清r−グロブリン l!タ 1,2〜7
.3V:牛血清フルブミ/ 64.J
4(j 〜4(、rj=卵白アルブミン
≠j 4C046:ミオグロビン
i7.t t、t〜t、λ″7
〔効果〕
本発明方法により得られる複合化分離剤はクロマトグラ
フィー、特に水系ゲルパーミエイシ田ンクロマトグラフ
ィー用の充填剤として有用である。Figure Nakaya Protein Molecular Weight (×1O3) Isoelectric Point/: Thyroglobulin 667 ≠, j2: Ferritin Hiro170 4'~j3:
Bovine serum r-globulin l! Ta 1, 2-7
.. 3V: Bovine serum Fulbumi/64. J
4(j ~4(, rj = ovalbumin
≠j 4C046: Myoglobin
i7. t t, t to t, λ″7 [Effect] The composite separating agent obtained by the method of the present invention is useful as a packing material for chromatography, particularly for aqueous gel permeate chromatography.
本発明方法より得られる複合化分離地は、粒径が20〜
1000μで、マクロボアーを有する架橋ポリビニルア
ルコール粒子中に、粒径j〜200μの多糖類系分離剤
を参〇−10%(容積比)包含されたものである、又本
複合化分離剤は、親水性に富んでいる為、特に蛋白質と
の非特異的吸着が少ないという特徴を有する。従って、
例えば、r−’グロブリン、アルブミン、ミオグロビン
等の各種蛋白質の分離剤として有用である。The composite separated material obtained by the method of the present invention has a particle size of 20 to
The polysaccharide separation agent with a particle size of j to 200μ is contained in crosslinked polyvinyl alcohol particles with a particle size of 1000μ and macropores at a ratio of 0 to 10% (volume ratio). Because of its high properties, it is characterized by low nonspecific adsorption, especially with proteins. Therefore,
For example, it is useful as a separating agent for various proteins such as r-' globulin, albumin, and myoglobin.
加えて、本発明の斯る複合化分離剤はポリビニルアルコ
ール基体の架橋構造により優れた機械的強度を有する。In addition, the composite separating agent of the present invention has excellent mechanical strength due to the crosslinked structure of the polyvinyl alcohol base.
第1図は、本発明の実施例≠で得られた複合化分離剤及
び比較例として多糖類系分離剤を用いて圧力損失と流速
との関係を測定した結果を示すグラフである。図中、横
軸は通液流速(−/hr、 )、縦軸は圧力損失(kf
/cr/l −j Ocm bed )を表わす。
第2図は、本発明の実施例1及びコで得られ複合化分離
剤についてのデキストジン、ポリエチレングリコールの
較正曲線、第3図は実施例3及び≠で得られた複合化分
離剤及び比較例として多糖類系分離剤を用いた場合の蛋
白質の較正曲線である。第2図及び第3図において横軸
は下記式で表わされる保持容量、Kav、値、縦軸は分
子量を表わす。
(尚、Voは分子量約−200万のブルーデキストラン
を用いて求めた値)
出 願 人 三菱化成工業株式会社
代 理 人 弁理士 要否用 −(ほか1名)
第1図
ム、V、 (m/Ar)
第2図
護持e−(
亮3図
にavFIG. 1 is a graph showing the results of measuring the relationship between pressure drop and flow rate using the composite separation agent obtained in Example≠ of the present invention and a polysaccharide separation agent as a comparative example. In the figure, the horizontal axis is the liquid flow rate (-/hr, ), and the vertical axis is the pressure loss (kf
/cr/l −j Ocm bed ). Figure 2 shows the calibration curve of dextodin and polyethylene glycol for the composite separating agent obtained in Examples 1 and 2 of the present invention, and Figure 3 shows the composite separating agent obtained in Example 3 and ≠ and a comparative example. This is a protein calibration curve when a polysaccharide-based separation agent is used as the protein. In FIGS. 2 and 3, the horizontal axis represents the retention capacity, Kav, value expressed by the following formula, and the vertical axis represents the molecular weight. (Vo is the value determined using blue dextran with a molecular weight of approximately -2 million.) Applicant: Mitsubishi Chemical Industries, Ltd. Agent: Patent attorney - (1 other person) Figure 1: M, V, ( m/Ar) Fig. 2 Mamochi e-(Ryo Fig. 3 av
Claims (4)
離剤を膨潤させた状態で、ポリビニルアルコール及び塩
類の水溶液と共に有機溶媒からなる分散浴中に分散造球
させ、前記分離剤および塩水溶液を包含したポリビニル
アルコール粒子を得、次いで該ポリビニルアルコール粒
子を自然ゲル化させたのち架橋剤と反応させることを特
徴とする複合化分離剤の製造方法。(1) A swollen separating agent made of a hydrophilic polymer having a giant network structure is dispersed and spheroidized in a dispersion bath made of an organic solvent together with an aqueous solution of polyvinyl alcohol and a salt to contain the separating agent and the salt aqueous solution. 1. A method for producing a composite separation agent, which comprises obtaining polyvinyl alcohol particles, which are then naturally gelled, and then reacted with a crosslinking agent.
する特許請求の範囲第1項記載の製造方法。(2) The manufacturing method according to claim 1, wherein the crosslinking agent is a dialdehyde compound.
徴とする特許請求の範囲第1項又は第2項記載の製造方
法。(3) The manufacturing method according to claim 1 or 2, wherein the crosslinking reaction is carried out in the presence of an acid catalyst.
する特許請求の範囲第1項、第2項又は第3項記載の製
造方法。(4) The manufacturing method according to claim 1, 2, or 3, wherein the dispersion bath is a halogenated hydrocarbon.
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP62333023A JPH01174505A (en) | 1987-12-29 | 1987-12-29 | Production of composite separating agent |
CA000586983A CA1329800C (en) | 1987-12-29 | 1988-12-23 | Composite separating agent |
EP88121847A EP0324177A1 (en) | 1987-12-29 | 1988-12-29 | Composite separating agent |
US07/651,117 US5114577A (en) | 1987-12-29 | 1991-02-05 | Composite separating agent |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP62333023A JPH01174505A (en) | 1987-12-29 | 1987-12-29 | Production of composite separating agent |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH01174505A true JPH01174505A (en) | 1989-07-11 |
Family
ID=18261414
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP62333023A Pending JPH01174505A (en) | 1987-12-29 | 1987-12-29 | Production of composite separating agent |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH01174505A (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7827693B2 (en) | 2005-03-16 | 2010-11-09 | Toyota Jidosha Kabushiki Kaisha | Method of manufacturing powertrain member |
JP2018013402A (en) * | 2016-07-20 | 2018-01-25 | 学校法人金井学園 | Radiation-sensitive gel indicator, preparation method, using method, and processing method thereof |
-
1987
- 1987-12-29 JP JP62333023A patent/JPH01174505A/en active Pending
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7827693B2 (en) | 2005-03-16 | 2010-11-09 | Toyota Jidosha Kabushiki Kaisha | Method of manufacturing powertrain member |
JP2018013402A (en) * | 2016-07-20 | 2018-01-25 | 学校法人金井学園 | Radiation-sensitive gel indicator, preparation method, using method, and processing method thereof |
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