JPH01262468A - Carrier for chromatography - Google Patents
Carrier for chromatographyInfo
- Publication number
- JPH01262468A JPH01262468A JP63089262A JP8926288A JPH01262468A JP H01262468 A JPH01262468 A JP H01262468A JP 63089262 A JP63089262 A JP 63089262A JP 8926288 A JP8926288 A JP 8926288A JP H01262468 A JPH01262468 A JP H01262468A
- Authority
- JP
- Japan
- Prior art keywords
- group
- glycidyl
- crosslinked polymer
- polymer particles
- particles
- 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
- 238000004587 chromatography analysis Methods 0.000 title claims description 18
- 239000002245 particle Substances 0.000 claims abstract description 86
- 229920006037 cross link polymer Polymers 0.000 claims abstract description 50
- 125000003055 glycidyl group Chemical group C(C1CO1)* 0.000 claims abstract description 33
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 28
- 125000003700 epoxy group Chemical group 0.000 claims abstract description 23
- 238000005342 ion exchange Methods 0.000 claims abstract description 17
- 230000007062 hydrolysis Effects 0.000 claims abstract description 8
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 8
- 238000004255 ion exchange chromatography Methods 0.000 claims description 23
- 239000000463 material Substances 0.000 claims description 22
- 238000012856 packing Methods 0.000 claims description 22
- 239000002253 acid Substances 0.000 abstract description 9
- 238000004132 cross linking Methods 0.000 abstract description 8
- -1 glycidyl ester Chemical class 0.000 abstract description 7
- 239000002270 dispersing agent Substances 0.000 abstract description 4
- 229910052500 inorganic mineral Inorganic materials 0.000 abstract description 4
- 239000011707 mineral Substances 0.000 abstract description 4
- 239000003431 cross linking reagent Substances 0.000 abstract description 3
- 229920001290 polyvinyl ester Polymers 0.000 abstract description 3
- 230000004048 modification Effects 0.000 abstract 2
- 238000012986 modification Methods 0.000 abstract 2
- 238000007142 ring opening reaction Methods 0.000 abstract 2
- 239000000725 suspension Substances 0.000 abstract 1
- 230000002194 synthesizing effect Effects 0.000 abstract 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- 239000003480 eluent Substances 0.000 description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 239000003960 organic solvent Substances 0.000 description 7
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 5
- 239000004372 Polyvinyl alcohol Substances 0.000 description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 5
- 238000007796 conventional method Methods 0.000 description 5
- 239000000178 monomer Substances 0.000 description 5
- 229920002451 polyvinyl alcohol Polymers 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 125000001174 sulfone group Chemical group 0.000 description 5
- 239000008096 xylene Substances 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 238000006116 polymerization reaction Methods 0.000 description 4
- 239000000376 reactant Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2,2'-azo-bis-isobutyronitrile Substances N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000003957 anion exchange resin Substances 0.000 description 3
- 239000000945 filler Substances 0.000 description 3
- 239000003999 initiator Substances 0.000 description 3
- 238000006386 neutralization reaction Methods 0.000 description 3
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 150000005846 sugar alcohols Polymers 0.000 description 3
- 238000010557 suspension polymerization reaction Methods 0.000 description 3
- 238000004448 titration Methods 0.000 description 3
- JJRUAPNVLBABCN-UHFFFAOYSA-N 2-(ethenoxymethyl)oxirane Chemical compound C=COCC1CO1 JJRUAPNVLBABCN-UHFFFAOYSA-N 0.000 description 2
- KUDUQBURMYMBIJ-UHFFFAOYSA-N 2-prop-2-enoyloxyethyl prop-2-enoate Chemical compound C=CC(=O)OCCOC(=O)C=C KUDUQBURMYMBIJ-UHFFFAOYSA-N 0.000 description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 description 2
- KZMGYPLQYOPHEL-UHFFFAOYSA-N Boron trifluoride etherate Chemical compound FB(F)F.CCOCC KZMGYPLQYOPHEL-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- 108010026206 Conalbumin Proteins 0.000 description 2
- 102000019265 Cytochrome c1 Human genes 0.000 description 2
- 108010007528 Cytochromes c1 Proteins 0.000 description 2
- 102000036675 Myoglobin Human genes 0.000 description 2
- 108010062374 Myoglobin Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 102000004338 Transferrin Human genes 0.000 description 2
- 108090000901 Transferrin Proteins 0.000 description 2
- 150000008044 alkali metal hydroxides Chemical class 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- WTEOIRVLGSZEPR-UHFFFAOYSA-N boron trifluoride Chemical compound FB(F)F WTEOIRVLGSZEPR-UHFFFAOYSA-N 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 239000003729 cation exchange resin Substances 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- MVPPADPHJFYWMZ-UHFFFAOYSA-N chlorobenzene Chemical compound ClC1=CC=CC=C1 MVPPADPHJFYWMZ-UHFFFAOYSA-N 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Substances CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- VOZRXNHHFUQHIL-UHFFFAOYSA-N glycidyl methacrylate Chemical compound CC(=C)C(=O)OCC1CO1 VOZRXNHHFUQHIL-UHFFFAOYSA-N 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- FAIDIRVMPHBRLT-UHFFFAOYSA-N propane-1,2,3-triol;prop-2-enoic acid Chemical compound OC(=O)C=C.OC(=O)C=C.OCC(O)CO FAIDIRVMPHBRLT-UHFFFAOYSA-N 0.000 description 2
- 125000001453 quaternary ammonium group Chemical group 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- FDRCDNZGSXJAFP-UHFFFAOYSA-M sodium chloroacetate Chemical compound [Na+].[O-]C(=O)CCl FDRCDNZGSXJAFP-UHFFFAOYSA-M 0.000 description 2
- 229910001220 stainless steel Inorganic materials 0.000 description 2
- 239000010935 stainless steel Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 150000008053 sultones Chemical class 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 239000012581 transferrin Substances 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- NEBBLNDVSSWJLL-UHFFFAOYSA-N 2,3-bis(2-methylprop-2-enoyloxy)propyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCC(OC(=O)C(C)=C)COC(=O)C(C)=C NEBBLNDVSSWJLL-UHFFFAOYSA-N 0.000 description 1
- PUGOMSLRUSTQGV-UHFFFAOYSA-N 2,3-di(prop-2-enoyloxy)propyl prop-2-enoate Chemical compound C=CC(=O)OCC(OC(=O)C=C)COC(=O)C=C PUGOMSLRUSTQGV-UHFFFAOYSA-N 0.000 description 1
- STMDPCBYJCIZOD-UHFFFAOYSA-N 2-(2,4-dinitroanilino)-4-methylpentanoic acid Chemical compound CC(C)CC(C(O)=O)NC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O STMDPCBYJCIZOD-UHFFFAOYSA-N 0.000 description 1
- JJBFVQSGPLGDNX-UHFFFAOYSA-N 2-(2-methylprop-2-enoyloxy)propyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OC(C)COC(=O)C(C)=C JJBFVQSGPLGDNX-UHFFFAOYSA-N 0.000 description 1
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 1
- WYGWHHGCAGTUCH-UHFFFAOYSA-N 2-[(2-cyano-4-methylpentan-2-yl)diazenyl]-2,4-dimethylpentanenitrile Chemical compound CC(C)CC(C)(C#N)N=NC(C)(C#N)CC(C)C WYGWHHGCAGTUCH-UHFFFAOYSA-N 0.000 description 1
- VFZKVQVQOMDJEG-UHFFFAOYSA-N 2-prop-2-enoyloxypropyl prop-2-enoate Chemical compound C=CC(=O)OC(C)COC(=O)C=C VFZKVQVQOMDJEG-UHFFFAOYSA-N 0.000 description 1
- DBCAQXHNJOFNGC-UHFFFAOYSA-N 4-bromo-1,1,1-trifluorobutane Chemical compound FC(F)(F)CCCBr DBCAQXHNJOFNGC-UHFFFAOYSA-N 0.000 description 1
- KYARBIJYVGJZLB-UHFFFAOYSA-N 7-amino-4-hydroxy-2-naphthalenesulfonic acid Chemical compound OC1=CC(S(O)(=O)=O)=CC2=CC(N)=CC=C21 KYARBIJYVGJZLB-UHFFFAOYSA-N 0.000 description 1
- 229910015900 BF3 Inorganic materials 0.000 description 1
- 239000004342 Benzoyl peroxide Substances 0.000 description 1
- OMPJBNCRMGITSC-UHFFFAOYSA-N Benzoylperoxide Chemical compound C=1C=CC=CC=1C(=O)OOC(=O)C1=CC=CC=C1 OMPJBNCRMGITSC-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- QMXOFBXZEKTJIK-UHFFFAOYSA-N Glycinol Natural products C1=C(O)C=C2OCC3(O)C4=CC=C(O)C=C4OC3C2=C1 QMXOFBXZEKTJIK-UHFFFAOYSA-N 0.000 description 1
- YIVJZNGAASQVEM-UHFFFAOYSA-N Lauroyl peroxide Chemical compound CCCCCCCCCCCC(=O)OOC(=O)CCCCCCCCCCC YIVJZNGAASQVEM-UHFFFAOYSA-N 0.000 description 1
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 1
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- GQPVFBDWIUVLHG-UHFFFAOYSA-N [2,2-bis(hydroxymethyl)-3-(2-methylprop-2-enoyloxy)propyl] 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCC(CO)(CO)COC(=O)C(C)=C GQPVFBDWIUVLHG-UHFFFAOYSA-N 0.000 description 1
- CQHKDHVZYZUZMJ-UHFFFAOYSA-N [2,2-bis(hydroxymethyl)-3-prop-2-enoyloxypropyl] prop-2-enoate Chemical compound C=CC(=O)OCC(CO)(CO)COC(=O)C=C CQHKDHVZYZUZMJ-UHFFFAOYSA-N 0.000 description 1
- JUDXBRVLWDGRBC-UHFFFAOYSA-N [2-(hydroxymethyl)-3-(2-methylprop-2-enoyloxy)-2-(2-methylprop-2-enoyloxymethyl)propyl] 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCC(CO)(COC(=O)C(C)=C)COC(=O)C(C)=C JUDXBRVLWDGRBC-UHFFFAOYSA-N 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 235000019400 benzoyl peroxide Nutrition 0.000 description 1
- KDPAWGWELVVRCH-UHFFFAOYSA-N bromoacetic acid Chemical compound OC(=O)CBr KDPAWGWELVVRCH-UHFFFAOYSA-N 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 229940023913 cation exchange resins Drugs 0.000 description 1
- FOCAUTSVDIKZOP-UHFFFAOYSA-N chloroacetic acid Chemical compound OC(=O)CCl FOCAUTSVDIKZOP-UHFFFAOYSA-N 0.000 description 1
- 238000010908 decantation Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- GYZLOYUZLJXAJU-UHFFFAOYSA-N diglycidyl ether Chemical compound C1OC1COCC1CO1 GYZLOYUZLJXAJU-UHFFFAOYSA-N 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000002612 dispersion medium Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- MPOKJOWFCMDRKP-UHFFFAOYSA-N gold;hydrate Chemical compound O.[Au] MPOKJOWFCMDRKP-UHFFFAOYSA-N 0.000 description 1
- 229910021389 graphene Inorganic materials 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 239000003505 polymerization initiator Substances 0.000 description 1
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 1
- KPFSGNRRZMYZPH-UHFFFAOYSA-M potassium;2-chloroacetate Chemical compound [K+].[O-]C(=O)CCl KPFSGNRRZMYZPH-UHFFFAOYSA-M 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000007870 radical polymerization initiator Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
Landscapes
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、各種のクロマトグラフィー用充填剤を製造す
るために有用なりロマトグラフィー用担体、および該ク
ロマトグラフィー用担体にイオン交換基を導入したイオ
ン交換クロマトグラフィー用充填剤に関し、特にたんば
く質、オリゴペプチド、核酸およびオリゴヌクレオチド
などの生化学物質の分離1分析に利用することができる
イオン交換クロマトグラフィー用充填剤を製造するだめ
に利用することのできるクロマトグラフィー用担体、お
よび該担体にイオン交換基を導入したイオン交換クロマ
トグラフィー用充填剤に関する。Detailed Description of the Invention [Industrial Field of Application] The present invention is useful for producing various chromatography packing materials, and provides a chromatography support and an ion exchange group introduced into the chromatography support. Regarding packing materials for ion-exchange chromatography, it is particularly useful for producing packing materials for ion-exchange chromatography that can be used for separation 1 analysis of biochemical substances such as proteins, oligopeptides, nucleic acids, and oligonucleotides. The present invention relates to a carrier for chromatography that can be used for chromatography, and a packing material for ion exchange chromatography in which an ion exchange group is introduced into the carrier.
イオン交換クロマトグラフィーにより、たんばく質や核
酸などの生化学物質を分離1分析する方法は、従来より
行われている。しかし、従来の方法は、分離1分析に2
0分以上の長時間を要するという難点を有している。近
年、当業界においては、少量の試料全短時間に分離2分
析することが強く要求されてきた。・短時間で試料の分
離1分析を行うには、小さなカラムに粒子径の極めて小
さな粒子を充填することが必要である。従来、イオン交
換クロマトグラフィー用充填剤には、多孔性のポリマー
グルや多孔性のシリカケ゛ルなどが使用されてきた。BACKGROUND ART A method of separating and analyzing biochemical substances such as proteins and nucleic acids using ion exchange chromatography has been conventionally used. However, the conventional method requires only one separation and two analyses.
It has the disadvantage that it requires a long time of 0 minutes or more. In recent years, there has been a strong demand in the industry to separate and analyze a small amount of sample in a short period of time. - In order to perform sample separation and analysis in a short time, it is necessary to fill a small column with extremely small particles. Conventionally, porous polymer glues, porous silica cells, and the like have been used as packing materials for ion exchange chromatography.
しかし、多孔性のポリマーグルは、粒子径を小さくする
と、機械的強度が小さいため、高圧、高流速での使用が
できないという問題があった。However, when the particle size of porous polymer glue is reduced, its mechanical strength is low, so there is a problem that it cannot be used at high pressure and high flow rate.
一方、多孔性のシリカゲルは、機械的強度は強いが、弱
アルカリ性の条件下で溶解するという欠点があり、粒子
径を小さくするほど、この傾向が著しくなるという問題
があった。On the other hand, although porous silica gel has high mechanical strength, it has the disadvantage that it dissolves under weakly alkaline conditions, and this tendency becomes more pronounced as the particle size becomes smaller.
本発明の目的は、前記従来のイオン交換クロマトグラフ
ィー用充填剤の欠点を克服して、機械的強度にすぐれて
おり、高圧、高流速で使用に耐え、かつ化学的に安定で
弱アルカリ性の条件下で十分安定であり、体積変化のな
い、イオン交換クロマトグラフィー用充填剤を製造する
ために有用なりロマトグラフィー用担体、および該クロ
マトグラフィー用担体にイオン交換基を導入したイオン
交換クロマトグラフィー用充填剤を提供することである
。The purpose of the present invention is to overcome the drawbacks of the conventional packing material for ion exchange chromatography, to provide a packing material with excellent mechanical strength, withstand use under high pressure and high flow rate, and to be chemically stable and suitable for weakly alkaline conditions. A carrier for ion exchange chromatography that is sufficiently stable under the following conditions and has no volume change, and is useful for producing a packing material for ion exchange chromatography. The aim is to provide agents for
本発明によって、上記目的を達成し得るクロマトグラフ
イル用充填剤を製造するために有用なりロマトグラフィ
ー用担体、および該クロマトグラフィー用担体にイオン
交換基を導入したイオン交換クロマトグラフィー用充填
剤が提供される。The present invention provides a chromatography carrier useful for producing a chromatography filler capable of achieving the above object, and an ion exchange chromatography filler in which an ion exchange group is introduced into the chromatography carrier. be done.
すなわち、本発明は、グリシジル基を有する架橋重合体
粒子からなり、該粒子表面のグリシジル基に基づくエポ
キシ基が加水分解によって水酸基に開環変性され、かつ
該粒子内部のグリシ・ゾル基に基づくエポキシ基が自己
架橋されている、平均粒子径が1〜5μmの範囲内にあ
るクロマトグラフィー用担体に関する。That is, the present invention consists of crosslinked polymer particles having glycidyl groups, in which epoxy groups based on glycidyl groups on the surface of the particles are ring-opened and modified to hydroxyl groups by hydrolysis, and epoxy groups based on glycysol groups inside the particles. The present invention relates to a chromatography carrier having self-crosslinked groups and having an average particle size within the range of 1 to 5 μm.
また、本発明は、グリシ・ゾル基を有する架橋重合体粒
子からなり、該粒子表面のグリシジル基に基づくエポキ
シ基が加水分解によって水酸基に開環変性され、かつ該
粒子内部のグリシジル基に基づくエポキシ基が自己架橋
されている、平均粒子径が1〜5μmの範囲内にちるク
ロマトグラフィー用担体の前記水酸基に、イオン交換基
が導入されていることを特徴とするイオン交換クロマト
グラフィー用充填剤に関する。The present invention also provides a cross-linked polymer particle having a glycidyl group, in which an epoxy group based on a glycidyl group on the surface of the particle is ring-opened and modified to a hydroxyl group by hydrolysis, and an epoxy group based on a glycidyl group inside the particle. A packing material for ion-exchange chromatography, characterized in that an ion-exchange group is introduced into the hydroxyl group of a chromatography carrier whose groups are self-crosslinked and whose average particle diameter falls within the range of 1 to 5 μm. .
以下、本発明のクロマトグラフィー用担体、および該ク
ロマトグラフィー用担体にイオン交換基を導入したイオ
ン交換クロマトグラフィー用充填剤について説明する。Hereinafter, the chromatography carrier of the present invention and the ion exchange chromatography packing material in which an ion exchange group is introduced into the chromatography carrier will be explained.
本発明のクロマトグラフィー用充填剤を製造するために
有用なりロマトグラフイー用担体は、以下に示す工程を
経て合成することができる。The chromatographic carrier useful for producing the chromatographic packing material of the present invention can be synthesized through the steps shown below.
l)グリシジル基金有する架橋重合体粒子の合成。l) Synthesis of crosslinked polymer particles with glycidyl foundations.
2)グリシジル基を有する架橋重合体粒子表面のグリシ
ジル基に基づくエポキシ基の水による加水分解。2) Hydrolysis of epoxy groups based on glycidyl groups on the surface of crosslinked polymer particles having glycidyl groups with water.
3)グリシジル基を有する架橋重合体粒子内部のグリシ
ジル基に基づくエポキシ基の自己架橋。3) Self-crosslinking of epoxy groups based on glycidyl groups inside crosslinked polymer particles having glycidyl groups.
さらに詳しく説明すれば、先づ第一工程のグリシジル基
を有する架橋重合体の製造は(4)不飽和カルデン酸の
グリシジルエステルまたはグリシジルモノビニルエーテ
ルと(B)架橋剤としての多価アルコールのポリビニル
エステル全常法に従って水性懸濁重合させることによシ
製造することができる。To explain in more detail, the first step of producing a crosslinked polymer having a glycidyl group consists of (4) glycidyl ester or glycidyl monovinyl ether of unsaturated caldicic acid and (B) polyvinyl ester of polyhydric alcohol as a crosslinking agent. It can be produced by aqueous suspension polymerization according to any conventional method.
囚成分の不飽和カルデン酸のグリシジルエステルとして
は、グリシノルアクリレート、グリシシルメタクリレー
ト等が用いられる。また、(4)成分のグリシジルモノ
ビニルエーテルとしては、アリルグリシジルエーテル、
メタアリルグリシジルエーテル等が用いられる。Glycinol acrylate, glycidyl methacrylate, etc. are used as the glycidyl ester of unsaturated caldenic acid as the prison component. In addition, as component (4) glycidyl monovinyl ether, allyl glycidyl ether,
Metaallyl glycidyl ether and the like are used.
一方、架橋剤としての(B)成分の多価アルコールのポ
リビニルエステルとしては、エチレングリコールジアク
リレート、エチレングリコールジメタクリレート、プロ
ピレングリコールジアクリレート、プロピレングリコー
ルジメタクリレート、グリセリンジアクリレート、グリ
セリンジアクリレート、グリセリントリアクリレート、
グリセリントリメタクリレート、Kンタエリスリトール
ジアクリレート、ペンタエリスリトールジメタクリレー
ト、ペンタエリスリトールト・リメタアクリレート、ン
ルビトールノメタクリレート、ンルビトールトリメタク
リレート等をあげることができる。On the other hand, as the polyvinyl ester of polyhydric alcohol as the component (B) as a crosslinking agent, ethylene glycol diacrylate, ethylene glycol dimethacrylate, propylene glycol diacrylate, propylene glycol dimethacrylate, glycerin diacrylate, glycerin diacrylate, glycerin triacrylate, etc. acrylate,
Examples include glycerin trimethacrylate, pentaerythritol diacrylate, pentaerythritol dimethacrylate, pentaerythritol trimethacrylate, nrubitol no methacrylate, and nrubitol trimethacrylate.
なお、本発明においては、(4)成分を主成分とし、こ
れと共重合し得る他の単量体全適宜併用することは何ら
差し支えない。In the present invention, component (4) may be used as the main component, and all other monomers that can be copolymerized therewith may be used in combination as appropriate.
(B) 成分の多価アルコールの19リビニルエステル
は、全重合性単量体の1〜30モルチの範囲内、好まし
くは5〜20モルチの範囲内になるように使用される。The 19-rivinyl ester of polyhydric alcohol as component (B) is used in an amount of 1 to 30 moles, preferably 5 to 20 moles, of the total polymerizable monomers.
(B)成分の割合が多くなると架橋重合体粒子表面のグ
リシジル基の量が少くなるので好し、くない。If the proportion of component (B) increases, the amount of glycidyl groups on the surface of the crosslinked polymer particles decreases, which is preferable or not.
1合に際して用いられる開始剤は、通常の懸濁重合に用
いられる一般的なラジカル重合開始剤でよく、例えば2
,2′−アゾビスイソブチロニトリル、2.2′−アゾ
ビス−(2,4−ジメチルバレロニトリル)等のアゾ系
の開始剤や過酸化ベンゾイル、過酸化ラウロイル等の過
酸化物系の開始剤を用いることができる。The initiator used in 1 polymerization may be a general radical polymerization initiator used in normal suspension polymerization, for example 2 polymerization initiators.
, 2'-azobisisobutyronitrile, 2,2'-azobis-(2,4-dimethylvaleronitrile) and other azo-based initiators, and peroxide-based initiators such as benzoyl peroxide and lauroyl peroxide. Agents can be used.
懸濁重合を行うに際しては、水相にポリビニルアルコー
ル、カルボキシメチルセルロース、ゼラチン等の分散安
定剤を加えておくことが好ましく、また囚成分および(
B)成分等の1合性単量体が重合分散媒の水へ舒解する
のを防止するため、水に塩化ナトリウム、硫酸ナトリウ
ム、塩化カルシウム等の塩類を溶解させておいてもよい
。When carrying out suspension polymerization, it is preferable to add a dispersion stabilizer such as polyvinyl alcohol, carboxymethylcellulose, or gelatin to the aqueous phase, and also to add a carrier component and (
In order to prevent monomerizable monomers such as component B from dissolving into the water of the polymerization dispersion medium, salts such as sodium chloride, sodium sulfate, and calcium chloride may be dissolved in the water.
水層の量は、有機層の量とほぼ同容量以上から約10倍
容量までの量が使用される。架橋重合体粒子の粒子径は
、単量体を水相に分散する際の攪拌速度でコントロール
することができ、単量体は0.5〜5μmの油滴に分散
することが好ましい。The amount of the aqueous layer used is from approximately the same volume or more to about 10 times the volume of the organic layer. The particle diameter of the crosslinked polymer particles can be controlled by the stirring speed when dispersing the monomer in the aqueous phase, and the monomer is preferably dispersed into oil droplets of 0.5 to 5 μm.
重合反応は、通常50〜90℃で3〜16時間行なわれ
る。重合終了後架橋重合体粒子は、濾過、水洗し、さら
に有機溶媒で洗浄する。このよう圧して得られたグリシ
ジル基を有する架橋重合体粒子は、実質的に非多孔性で
あり、1〜5μm、好ましくは2〜4μmの粒子径に分
級される。分級は常法により風力分級や有機溶媒中で数
回デカンテーションすることによシ行うことができる。The polymerization reaction is usually carried out at 50 to 90°C for 3 to 16 hours. After completion of polymerization, the crosslinked polymer particles are filtered, washed with water, and further washed with an organic solvent. The crosslinked polymer particles having glycidyl groups obtained by pressing in this manner are substantially non-porous and are classified into particle sizes of 1 to 5 μm, preferably 2 to 4 μm. Classification can be carried out by conventional methods such as air classification or decantation several times in an organic solvent.
第二工程は、第一工程で得られたグリシジル基を有する
架橋重合体粒子に、水に難溶性の有機溶媒を含浸させた
後、この粒子を分散剤および鉱酸を含む水溶液中に懸濁
させ、50〜90℃で1〜5時間加熱することにより、
架橋重合体粒子の表面のグリシジル基に基づくエポキシ
基を加水分解して水酸基に開環変性する。架橋重合体粒
子に含浸させる有機溶媒としては、トルエン、キシレン
、クロルベンゼン、ノンラフインなどが、Sけられる。In the second step, the crosslinked polymer particles having glycidyl groups obtained in the first step are impregnated with an organic solvent that is poorly soluble in water, and then the particles are suspended in an aqueous solution containing a dispersant and a mineral acid. By heating at 50 to 90°C for 1 to 5 hours,
Epoxy groups based on glycidyl groups on the surface of crosslinked polymer particles are hydrolyzed to ring-open and modify into hydroxyl groups. Examples of the organic solvent with which the crosslinked polymer particles are impregnated include toluene, xylene, chlorobenzene, and non-roughin.
また、触媒の鉱酸としては、塩酸、硫酸などがあげられ
る。鉱酸の濃度は、0.05〜0.5規定が好筐しい。Further, examples of the mineral acid of the catalyst include hydrochloric acid and sulfuric acid. The concentration of mineral acid is preferably 0.05 to 0.5 normal.
分散剤としては、ポリビニルアルコール、ポリビニルア
ルコールなどがあげられる。分散剤の濃度は0.5〜1
0重量%が好ましい。Examples of the dispersant include polyvinyl alcohol and polyvinyl alcohol. The concentration of dispersant is 0.5-1
0% by weight is preferred.
第三工程として架橋重合体粒子内部のグリシジル基に基
づくエポキシ基を自己架橋させ、架橋重合体粒子の機械
的強度全増強する。自己架橋は、グリシジル基に基づく
エポキシ基と反応しない有機溶媒中で第二工程で製造し
た表面に水酸基を有する架橋重合体粒子を常法により、
三フフ化ホウ累エーテラート全触媒として、20℃〜8
0℃で1〜5時間加熱することよシ行われる。この際に
用いられる有機溶媒としては、トルエン、キシレン、ジ
オキサン、n−パラフィンなどがあげられる。In the third step, the epoxy groups based on the glycidyl groups inside the crosslinked polymer particles are self-crosslinked to fully enhance the mechanical strength of the crosslinked polymer particles. Self-crosslinking is performed by adding the crosslinked polymer particles having hydroxyl groups on the surface produced in the second step in an organic solvent that does not react with epoxy groups based on glycidyl groups by a conventional method.
As a total catalyst of boron trifluoride accumulated etherate, 20℃~8
This is done by heating at 0°C for 1 to 5 hours. Examples of organic solvents used in this case include toluene, xylene, dioxane, and n-paraffin.
以上のごとくして得られる表面に水酸基ケ有し、かつ内
部のグリシジル基に基づくエポキシ基が自己架橋してい
る架橋重合体粒子は、平均粒子径が1〜5μmの範囲内
であって、水酸基にイオン交換基金容易に導入すること
が可能であり、従ってイオン交換クロマトグラフィー用
充填剤全製造するだめのクロマトグラフィー用担体とし
て極めて有用なものである。The crosslinked polymer particles obtained as described above have hydroxyl groups on the surface and self-crosslinked epoxy groups based on internal glycidyl groups have an average particle diameter within the range of 1 to 5 μm, and have hydroxyl groups. It is possible to easily introduce an ion-exchange fund into the ion-exchange chromatography packing material, and therefore it is extremely useful as a chromatography support for the production of all ion-exchange chromatography packing materials.
架橋重合体粒子の平均粒子径が1μm未満では、カラム
に充填した場合、カラム圧が高くなり、溶離液が流れ難
くなる。一方、架橋重合体粒子の平均粒子径が5μmを
越える場合は、表面積が小さくなり、架橋重合体粒子の
表面に導入されるイオン交換基の量が少なくなる。If the average particle diameter of the crosslinked polymer particles is less than 1 μm, the column pressure will be high when packed in a column, making it difficult for the eluent to flow. On the other hand, when the average particle diameter of the crosslinked polymer particles exceeds 5 μm, the surface area becomes small and the amount of ion exchange groups introduced onto the surface of the crosslinked polymer particles decreases.
次に、上記クロマトグラフィー用担体の水酸基に、イオ
ン交換基を導入する方法について説明する1、
水酸基に導入されるイオン交換基としては、(イ)スル
ホン基、(ロ)カルボキシル基、CJジエチルアミンエ
チル基、に)第四級アンモニウム基などがあげられる。Next, a method for introducing an ion exchange group into the hydroxyl group of the above-mentioned chromatography carrier will be explained. 1. The ion exchange group introduced into the hydroxyl group includes (a) a sulfone group, (b) a carboxyl group, and a CJ diethylamine ethyl group. groups, etc.) quaternary ammonium groups, etc.
イオン交換容量は、50〜500M当量/9が好ましい
。The ion exchange capacity is preferably 50 to 500 M equivalent/9.
スルホン基の導入は、第三工程で製造した表面に水酸基
を有する架橋重合体粒子を有機溶媒中でアルカリ金属の
水酸化物を触媒とし、グロノソンスルトンまたは1,4
−ブタンスルトンと反応させることにより行われる、反
応温度および反応時間は、通常室温〜100℃で2〜2
4時間である。The introduction of sulfone groups is carried out by treating the crosslinked polymer particles having hydroxyl groups on the surface produced in the third step with glonosone sultone or 1,4
-The reaction temperature and reaction time are usually room temperature to 100°C for 2 to 2 hours.
It is 4 hours.
カルボキシル基の導入は、第三工程で製造した表面に水
酸基金有する架橋重合体粒子にモノクロル酢酸やモノブ
ロム酢酸等の7・ログン化酢酸、またはモノクロル酢酸
ナトリウム、モノクロル酢酸カリウム等のハロゲン化酢
酸塩を水酸化ナトリウムまたは水酸化カリウムの存在下
で反応させることによって行われる。Introduction of carboxyl groups is carried out by adding 7-logonated acetic acid such as monochloroacetic acid or monobromoacetic acid, or halogenated acetate such as sodium monochloroacetate or potassium monochloroacetate to the crosslinked polymer particles having a hydroxyl group on the surface produced in the third step. It is carried out by reacting in the presence of sodium hydroxide or potassium hydroxide.
ジエチルアミンエチル基の導入は、第三工程で製造した
表面に水酸基を有する架橋重合体粒子をアルカリ金属の
水酸化物の水溶液中で塩酸β−・ノエチルアミノエチル
クロライドと反応させることにより行うことができる。Introduction of diethylamine ethyl groups can be carried out by reacting the crosslinked polymer particles having hydroxyl groups on the surface produced in the third step with hydrochloric acid β-noethylaminoethyl chloride in an aqueous solution of alkali metal hydroxide. can.
反応温度は、室温〜80℃で、反応時間は1〜8時間が
好ましい。アルカリ金属の水酸化物の水溶液の濃度は、
5〜40重量%が好ましい。The reaction temperature is preferably room temperature to 80°C, and the reaction time is preferably 1 to 8 hours. The concentration of an aqueous solution of alkali metal hydroxide is
5 to 40% by weight is preferred.
第四級アンモニウム基は、ジエチルアミノエチル基を導
入した架橋重合体粒子を有機溶媒中でハロダン化メチル
またはハロゲン化エチルと反応させることにより導入さ
れる。The quaternary ammonium group is introduced by reacting crosslinked polymer particles into which diethylaminoethyl groups have been introduced with methyl halide or ethyl halide in an organic solvent.
かくして得られるイオン交換クロマトグラフィー用充填
剤は、平均粒子径が1〜5μmの範囲内の実質的に非多
孔性の架橋重合体粒子からな)、架橋重合体粒子の表面
にはイオン交換基が導入されておシ、架橋重合体粒子の
内部は密に架橋されている二重構造からなっている。The thus obtained packing material for ion exchange chromatography consists of substantially non-porous crosslinked polymer particles with an average particle size in the range of 1 to 5 μm), and the surface of the crosslinked polymer particles has ion exchange groups. Once introduced, the interior of the crosslinked polymer particles consists of a densely crosslinked double structure.
従来の技術では、担体の表面積を増加させるために多孔
性担体が用いられていた。本発明は、担体の粒子径’z
1〜5μmと小さくすることによって担体の表面積音大
きくし、担体の機械的強度金玉げるため、担体の内部は
架橋密度を高くし、担体の表面のみ架橋密度を低くし、
担体の水酸基を残し、その水酸基にイオン交換基を導入
してイオン又換樹り旨とするものでちる。In the prior art, porous carriers were used to increase the surface area of the carrier. In the present invention, the particle size of the carrier 'z
By reducing the size to 1 to 5 μm, the surface area of the carrier becomes louder and the mechanical strength of the carrier increases, so the crosslinking density is increased inside the carrier and the crosslinking density is lowered only on the surface of the carrier.
The hydroxyl group of the carrier is left behind, and an ion exchange group is introduced into the hydroxyl group to create an ion exchange effect.
本発明によるイオン交換クロマトグラフィー用充填剤は
、平均粒子径が1〜5μmの範囲内であり、従来のイオ
ン交換クロマトグラフィー用充填剤。The packing material for ion exchange chromatography according to the present invention has an average particle diameter within the range of 1 to 5 μm, and is a conventional packing material for ion exchange chromatography.
すなわち多孔性の担体にイオン交換基金導入したものと
は異り、実質上非多孔性で担体内部が高度に架橋されて
いるので、機械的強度も強く、1〜5 lzmという小
さな粒子径の担体も使用できる特徴がちる。In other words, unlike porous carriers in which ion exchange foundations are introduced, the carrier is substantially non-porous and has a highly cross-linked interior, so it has strong mechanical strength and has a small particle size of 1 to 5 lzm. There are also many features that can be used.
ダン、下、実施例?あげて本発明をさらに詳細に説明す
る。Dan, below, example? The present invention will now be described in further detail.
実施例1
(1) グリシジル基を有する架橋重合体粒子の合成
グリシジルメタクリレ−) 324.1工チレングリコ
ールジメタクリレート36gおよびアゾビスイソブチロ
ニトリル14,9’(H混合し、ポリビニルアルコール
96gを溶解した2、41の水中に加えた。この混合物
を高速攪拌し、油滴の粒子径を0.5〜5μmに調節し
た後、60℃に加温し、10時間保った。次に、反応生
成物を室温で冷却し、水で希釈した。生成した架橋重合
体粒子を遠心分離し、また水中に懸濁し遠心分離を行っ
た。この操作を3回繰り返した後、架橋重合体粒子をア
セトン中に懸濁し、グラスフィルターで濾過し、さらに
グラスフィルター上からアセトンで洗浄を行った。この
架橋重合体粒子を自然乾燥し、次いで10℃で8時間真
空乾燥した後、常法により風力分級し、2〜4μmの粒
子径の架橋重合体粒子110Iを得た。Example 1 (1) Synthesis of crosslinked polymer particles having glycidyl groups Glycidyl methacrylate 36 g of 324.1-ethylene glycol dimethacrylate and azobisisobutyronitrile 14,9' (H mixed), 96 g of polyvinyl alcohol The mixture was stirred at high speed to adjust the particle size of the oil droplets to 0.5 to 5 μm, and then heated to 60°C and kept for 10 hours.Next, the reaction The product was cooled to room temperature and diluted with water. The resulting crosslinked polymer particles were centrifuged and then suspended in water and centrifuged. After repeating this operation three times, the crosslinked polymer particles were diluted with acetone. The crosslinked polymer particles were suspended in water, filtered through a glass filter, and washed with acetone from above the glass filter.The crosslinked polymer particles were air-dried, then vacuum-dried at 10°C for 8 hours, and then air-classified using a conventional method. , crosslinked polymer particles 110I having a particle diameter of 2 to 4 μm were obtained.
(2)架橋重合体粒子表面のグリシジル基に基づくエポ
キシ基の水による加水分解
前記(1)で得られた架橋重合体粒子logにキシレン
30gを加え、架橋重合体粒子にキシレン全含浸させた
。次にポリビニルアルコール4gと濃硫酸1.479を
水100−に溶解した水溶液中に、キシレン全含浸させ
た架橋重合体粒子内部え、激しく攪拌した。この懸濁波
音80℃に加温し、1時間保った。反応換金水で希釈し
、遠心分iii′lI金行った。沈殿した架橋重合体粒
子をグラスフィルター上に移し、水で洗浄し、次にアセ
トンで洗浄した。この架橋重合体粒子を室温で自然乾燥
し、さらに40℃で8時間真空乾燥した。得られた架橋
重合体粒子の表面の水酸基量は、0.6 mM/、!9
であった。(2) Hydrolysis of epoxy groups based on glycidyl groups on the surface of crosslinked polymer particles with water. 30 g of xylene was added to the log of crosslinked polymer particles obtained in (1) above to completely impregnate the crosslinked polymer particles with xylene. Next, the interior of the crosslinked polymer particles completely impregnated with xylene was placed in an aqueous solution containing 4 g of polyvinyl alcohol and 1.479 g of concentrated sulfuric acid dissolved in 100 g of water, and the mixture was vigorously stirred. This suspended wave sound was heated to 80°C and maintained for 1 hour. The reaction mixture was diluted with gold water and centrifuged. The precipitated crosslinked polymer particles were transferred onto a glass filter and washed with water and then with acetone. The crosslinked polymer particles were air-dried at room temperature and further vacuum-dried at 40° C. for 8 hours. The amount of hydroxyl groups on the surface of the obtained crosslinked polymer particles was 0.6 mm/! 9
Met.
(3) 表面に水酸基を有する架橋重合体粒子内部の
グリシジル基に基づくエポキシ基の自己架橋前記(2)
で得られた表面に水酸基金性する架橋重合体粒子109
全ソオキサン50ゴ中に懸濁し。(3) Self-crosslinking of epoxy groups based on glycidyl groups inside crosslinked polymer particles having hydroxyl groups on the surface (2) above
Crosslinked polymer particles 109 with hydroxyl group on the surface obtained in
Suspend in 50 g of total sooxane.
三フフ化ホウ素エーテラート(55%)0.5mi加え
60℃で8時間加温した。次に、反応換金グラスフィル
ターで濾過し、アセトンで洗浄した後、室温で自然乾燥
した。0.5 mi of boron trifluoride etherate (55%) was added and heated at 60°C for 8 hours. Next, it was filtered through a reaction exchange glass filter, washed with acetone, and then air-dried at room temperature.
(4) スルホン基の導入
前記(3)で得られた表面に水酸基を有し、かつ内部の
グリシジル基に基づくエポキシ基が自己架橋した架橋重
合体粒子2g’tlOmZのツメチルスルホキサイド中
に懸濁し、これにプロ・Pンスルトン0.59および1
0%水酸化ナトリウム1.2.9 ′に加え、攪拌しな
がら30℃で6時間保った。次に得られた反応物を水洗
した。(4) Introduction of sulfone group Into 2g'tlOmZ of the crosslinked polymer particles obtained in (3) above, which have hydroxyl groups on the surface and are self-crosslinked by the epoxy groups based on internal glycidyl groups, Suspend this with pro-P sultone 0.59 and 1
It was added to 1.2.9' of 0% sodium hydroxide and kept at 30° C. for 6 hours with stirring. Next, the obtained reaction product was washed with water.
反応物の1部をとり、中和滴定によりスルホン基の定量
を行った結果、0.15 mmol//g(dry g
el )のスルホン基が認められた。A portion of the reactant was taken and the sulfone group was determined by neutralization titration. As a result, 0.15 mmol//g (dry g
el ) sulfone group was observed.
(5) カルボキシル基の導入
前記(3)で得られた表面に水酸基を有し、かつ内部の
グリシジル基に基づくエポキシ基が自己架橋した架橋重
合体粒子2gを水9−中に懸濁し、これにモノクロル酢
酸ナトリウム3.5g、ヨウ化カリウム2,9.50%
水酸化ナトリウム69 ’、1加え。(5) Introduction of carboxyl groups 2 g of the crosslinked polymer particles obtained in (3) above, which have hydroxyl groups on the surface and are self-crosslinked with epoxy groups based on internal glycidyl groups, are suspended in water (9). Sodium monochloroacetate 3.5g, potassium iodide 2.9.50%
Sodium hydroxide 69', 1 added.
攪拌しながら60℃で3時間保った。次に反応物を水洗
した。反応物を1部と9.0.1規定水酸化ナトリウム
水溶rL金用いて中和滴定を行い、カルボキシル基の定
量全行った。その結果0.2 mmol/9(dry
get )のカルボキシル基が認められた。The mixture was kept at 60° C. for 3 hours while stirring. Next, the reaction product was washed with water. Neutralization titration was performed using 1 part of the reactant and 9.0.1N aqueous sodium hydroxide solution rL gold, and the carboxyl group was completely determined. As a result, 0.2 mmol/9 (dry
get ) carboxyl group was observed.
(6) ジエチルアミンエチル基の導入前記(3)で
得られた表面に水酸基?有し、かつ内部のグリシジル基
に基づくエポキシ基が自己架橋した架橋重合体粒子2.
9全20%水酸化ナトリウム12y中に懸濁し、塩酸β
−ソエチルアミノエチルクロライド1gを加え、攪拌し
ながら60℃で3時間保った。次に反応物音水で洗浄し
た。(6) Introduction of diethylamine ethyl group Is there a hydroxyl group on the surface obtained in (3) above? Crosslinked polymer particles having self-crosslinked epoxy groups based on internal glycidyl groups2.
9 Suspended in total 20% sodium hydroxide 12y, β-hydrochloric acid
-1 g of soethylaminoethyl chloride was added and kept at 60°C for 3 hours with stirring. The reactants were then washed with water.
反応物を1部とり、0.1規定塩酸水溶液全用いて中和
滴定を行った結果、0.25 mmol/′!j(dr
ygel )のジエチルアミノエチル基が認められた。One part of the reactant was taken and subjected to neutralization titration using all 0.1N aqueous hydrochloric acid solution, and the result was 0.25 mmol/'! j(dr
ygel) diethylaminoethyl group was observed.
応用例工
実施例1〜(4)で得られた陽イオン交換樹脂をステン
レスカラム(内径4.6 m X長さ35聾)に充填し
、溶離液囚として20mMリン酸緩衝液Cp+47.0
)全用い、溶離液(B)として20 mM !Jン酸緩
衝液+0.5M塩化ナトリウム(饅7.0)を用い、流
速L5−/min で溶離液囚から溶離液(B)に1
0分間のリニアグラツエン)を行い、■ミオグロビン、
■リボヌクレアーゼA、■チトクロームC1■リゾチー
ムの混合物を分析したところ、第1図に示すように4つ
のたんばく質を5分以内に分離することができた。Application examples The cation exchange resins obtained in Examples 1 to (4) were packed into a stainless steel column (inner diameter 4.6 m x length 35 mm), and 20 mM phosphate buffer Cp + 47.0 was added as an eluent.
) all used, 20 mM as eluent (B)! Using J acid buffer + 0.5M sodium chloride (7.0%), transfer the eluent (B) from the eluent to the eluent (B) at a flow rate of L5-/min.
0 minutes of linear graphene) was performed, and ■ myoglobin,
When a mixture of (1) ribonuclease A, (2) cytochrome C1, and (3) lysozyme was analyzed, four proteins could be separated within 5 minutes as shown in Figure 1.
応用例2
実施例1−(6)で得られた陰イオン交換樹脂をステン
レスカラム(内径4.6×長さ35簡)に充填し、溶離
液(4)として20mMピにラノン(pH6,0)を用
い、溶離i (B)として20 mMビにジノン+05
M塩化ナトリウム(d(6,0)を用い、流速1.5m
+>minで溶離液(イ)から溶離液(B)に10分間
のリニアグラジェントを行い、■コナルブミン、■トラ
ンスフェリン、■オボアルブミン、■トリゾンンインヒ
ビターの混合物を分析したところ、第2図ンζ示すよう
に・1つのたんばく質を6分以内に分離することができ
た。Application Example 2 The anion exchange resin obtained in Example 1-(6) was packed into a stainless steel column (inner diameter 4.6 x length 35 columns), and 20mM lanone (pH 6.0) was added as the eluent (4). ) and 20 mM Bi-Zinone+05 as elution i (B).
Using M sodium chloride (d(6,0), flow rate 1.5 m
A 10-minute linear gradient was performed from eluent (A) to eluent (B) at +>min, and a mixture of ■ conalbumin, ■ transferrin, ■ ovalbumin, and ■ trizone inhibitor was analyzed. As shown in Figure 1, one protein could be separated within 6 minutes.
応用例3
応用例2で使用したカラムを溶離液囚として25 mM
リン酸緩衝液+0.2 M塩化ナトリウム(、I(6,
0)−i用い、溶離液(B)として25mMリン酸”−
’< J液+IM塩化ナトリウム(PH6,0)を用い
、流逮15ゴ/・’minで溶離液・囚から溶離液(匂
に30分間のリニアグラジェントを行い、オリゴアゾニ
ール酸の分離を行った。第3図に示すようにオリゴアゾ
ニール酸のオリゴマーを分離することができた。Application example 3 The column used in application example 2 was used as an eluent at 25 mM.
Phosphate buffer + 0.2 M sodium chloride (,I(6,
0)-i, 25mM phosphoric acid as eluent (B)
Using J solution + IM sodium chloride (PH6,0), perform a 30-minute linear gradient from the eluent to the eluent at a flow rate of 15 g/min to separate oligoazonylic acid. As shown in Figure 3, the oligomers of oligoazonylic acid could be separated.
本発明のクロマトグラフィー用担体は1機械的強度にす
ぐれており、かつ化学的に安定で弱アルカリ性の条件下
で十分安定であって、体積変化もないのでイオン交換ク
ロマトグラフィー用充填剤を製造するための担体として
極めて有用なものである。The chromatography carrier of the present invention has excellent mechanical strength, is chemically stable, is sufficiently stable under weakly alkaline conditions, and has no volume change, and thus can be used to produce a packing material for ion exchange chromatography. It is extremely useful as a carrier for
また、本発明により得られるイオン交換クロマトグラフ
ィー用充填剤は、従来のイオン交換クロマトグラフィー
用充填剤に比較して以下のような利点全有している。Moreover, the packing material for ion exchange chromatography obtained according to the present invention has all the following advantages as compared to the conventional packing material for ion exchange chromatography.
1)機械的強度が太きく、1〜5μmの小さな粒子径を
有しているため、高速液体クロマトグラフィー用充填剤
に使用した場合、高性能な分離ができる。1) Since it has high mechanical strength and a small particle size of 1 to 5 μm, it can perform high-performance separation when used as a packing material for high performance liquid chromatography.
2)架橋重合体粒子内部の架橋度が高いため、膨潤度が
小さく、高い塩濃度で使用できる。2) Since the degree of crosslinking inside the crosslinked polymer particles is high, the degree of swelling is low and it can be used at high salt concentrations.
3)実質的に非多孔性のため、イオン交換クロマトグラ
フィー用充填剤の表面のみで分離が行われ、試料ピーク
の拡散による広がシを少くできる。3) Since it is substantially non-porous, separation can be performed only on the surface of the packing material for ion exchange chromatography, reducing the spread of sample peaks due to diffusion.
4)広い一範囲で安定であシ1例えばシリカゲルを担体
とする充填剤では適用できないアルカリ条件下でも安定
に使用できる。4) Stable over a wide range of conditions 1. For example, it can be stably used under alkaline conditions, which is not applicable to fillers using silica gel as a carrier.
【図面の簡単な説明】
第1図は実施例1−(4)で得られた陽イ万ン交換樹脂
を用いて、たんばく質混合物のイオン交換クロマトグラ
フィーを行ったとき得られたクロマトグラムである。
図中■はミオグロビン、■はりボヌクレアーゼーA、■
はチトクロームC1■はりゾチームの各ピークを示す。
第2図は、実施例1−(6)で得られた陰イオン交換樹
脂を用いて、たんばく質混合物のイオン交換クロマトグ
ラフィーを行ったとき得られたクロマトグラムである。
図中■はコナルブミン、■はトランスフェリン、■はオ
ヂアルブミン、・■はンイビーントリグシンインヒビタ
ーの各ピークを示す。
第3図は実施例1−(6)で得られた陰イオン交換樹脂
を用いて、オリゴアゾニール酸のオリゴマーを分離した
クロマトグラムを示す。[Brief explanation of the drawings] Figure 1 is a chromatogram obtained when a protein mixture was subjected to ion exchange chromatography using the cation exchange resin obtained in Example 1-(4). It is. In the figure, ■ is myoglobin, ■ is bonuclease A, and ■ is
indicates each peak of cytochrome C1 and harizozyme. FIG. 2 is a chromatogram obtained when a protein mixture was subjected to ion exchange chromatography using the anion exchange resin obtained in Example 1-(6). In the figure, ■ indicates the peaks of conalbumin, ■ indicates the peaks of transferrin, ■ indicates the peaks of odialbumin, and ■ indicates the peaks of the bean trigsin inhibitor. FIG. 3 shows a chromatogram in which oligomers of oligoazonylic acid were separated using the anion exchange resin obtained in Example 1-(6).
Claims (2)
該粒子表面のグリシジル基に基づくエポキシ基が加水分
解によって水酸基に開環変性され、かつ該粒子内部のグ
リシジル基に基づくエポキシ基が自己架橋されている、
平均粒子径が1〜5μmの範囲内にあるクロマトグラフ
ィー用担体。(1) Consisting of crosslinked polymer particles having glycidyl groups,
An epoxy group based on a glycidyl group on the surface of the particle is ring-opened and modified to a hydroxyl group by hydrolysis, and an epoxy group based on a glycidyl group inside the particle is self-crosslinked.
A carrier for chromatography having an average particle diameter within the range of 1 to 5 μm.
該粒子表面のグリシジル基に基づくエポキシ基が加水分
解によって水酸基に開環変性され、かつ該粒子内部のグ
リシジル基に基づくエポキシ基が自己架橋されている、
平均粒子径が1〜5μmの範囲内にあるクロマトグラフ
ィー用担体の前記水酸基に、イオン交換基が導入されて
いることを特徴とするイオン交換クロマトグラフィー用
充填剤。(2) consisting of crosslinked polymer particles having glycidyl groups,
An epoxy group based on a glycidyl group on the surface of the particle is ring-opened and modified to a hydroxyl group by hydrolysis, and an epoxy group based on a glycidyl group inside the particle is self-crosslinked.
A packing material for ion exchange chromatography, characterized in that an ion exchange group is introduced into the hydroxyl group of the chromatography carrier having an average particle diameter within the range of 1 to 5 μm.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP63089262A JPH01262468A (en) | 1988-04-13 | 1988-04-13 | Carrier for chromatography |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP63089262A JPH01262468A (en) | 1988-04-13 | 1988-04-13 | Carrier for chromatography |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH01262468A true JPH01262468A (en) | 1989-10-19 |
Family
ID=13965843
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP63089262A Pending JPH01262468A (en) | 1988-04-13 | 1988-04-13 | Carrier for chromatography |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH01262468A (en) |
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JP2001099820A (en) * | 1999-09-30 | 2001-04-13 | Sekisui Chem Co Ltd | Method for manufacturing filler for liquid chromatography |
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