JPH0338891B2 - - Google Patents
Info
- Publication number
- JPH0338891B2 JPH0338891B2 JP60127230A JP12723085A JPH0338891B2 JP H0338891 B2 JPH0338891 B2 JP H0338891B2 JP 60127230 A JP60127230 A JP 60127230A JP 12723085 A JP12723085 A JP 12723085A JP H0338891 B2 JPH0338891 B2 JP H0338891B2
- Authority
- JP
- Japan
- Prior art keywords
- carrier
- column
- hydrophobic
- pores
- substances
- 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.)
- Expired - Lifetime
Links
- 239000011148 porous material Substances 0.000 claims description 54
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 45
- 239000002245 particle Substances 0.000 claims description 40
- 230000002209 hydrophobic effect Effects 0.000 claims description 27
- 230000005526 G1 to G0 transition Effects 0.000 claims description 14
- 239000003795 chemical substances by application Substances 0.000 claims description 7
- 150000001875 compounds Chemical class 0.000 claims description 7
- 125000001309 chloro group Chemical group Cl* 0.000 claims description 6
- 125000003545 alkoxy group Chemical group 0.000 claims description 4
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 3
- 125000001931 aliphatic group Chemical group 0.000 claims description 2
- 125000002029 aromatic hydrocarbon group Chemical group 0.000 claims description 2
- 125000004432 carbon atom Chemical group C* 0.000 claims description 2
- 239000000126 substance Substances 0.000 description 76
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 63
- ZFXYFBGIUFBOJW-UHFFFAOYSA-N theophylline Chemical compound O=C1N(C)C(=O)N(C)C2=C1NC=N2 ZFXYFBGIUFBOJW-UHFFFAOYSA-N 0.000 description 56
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 50
- 102000004169 proteins and genes Human genes 0.000 description 43
- 108090000623 proteins and genes Proteins 0.000 description 43
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 36
- AQHHHDLHHXJYJD-UHFFFAOYSA-N propranolol Chemical compound C1=CC=C2C(OCC(O)CNC(C)C)=CC=CC2=C1 AQHHHDLHHXJYJD-UHFFFAOYSA-N 0.000 description 32
- 239000000243 solution Substances 0.000 description 31
- 239000007864 aqueous solution Substances 0.000 description 29
- 229960000278 theophylline Drugs 0.000 description 28
- 238000002835 absorbance Methods 0.000 description 24
- 238000011084 recovery Methods 0.000 description 23
- 238000000034 method Methods 0.000 description 22
- 125000001165 hydrophobic group Chemical group 0.000 description 19
- 210000002966 serum Anatomy 0.000 description 19
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 18
- RYYVLZVUVIJVGH-UHFFFAOYSA-N caffeine Chemical compound CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 description 18
- 239000000463 material Substances 0.000 description 18
- 239000000523 sample Substances 0.000 description 18
- 238000001179 sorption measurement Methods 0.000 description 18
- 229910001220 stainless steel Inorganic materials 0.000 description 17
- 239000010935 stainless steel Substances 0.000 description 17
- 229960003712 propranolol Drugs 0.000 description 16
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 14
- 239000003480 eluent Substances 0.000 description 13
- 238000006460 hydrolysis reaction Methods 0.000 description 13
- 238000012856 packing Methods 0.000 description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 12
- 230000007062 hydrolysis Effects 0.000 description 12
- BPSIOYPQMFLKFR-UHFFFAOYSA-N trimethoxy-[3-(oxiran-2-ylmethoxy)propyl]silane Chemical compound CO[Si](OC)(OC)CCCOCC1CO1 BPSIOYPQMFLKFR-UHFFFAOYSA-N 0.000 description 12
- 239000000203 mixture Substances 0.000 description 11
- 239000003960 organic solvent Substances 0.000 description 11
- 102000015427 Angiotensins Human genes 0.000 description 10
- 108010064733 Angiotensins Proteins 0.000 description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 10
- 229940098773 bovine serum albumin Drugs 0.000 description 10
- 238000001914 filtration Methods 0.000 description 10
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 9
- 230000003993 interaction Effects 0.000 description 9
- 239000008363 phosphate buffer Substances 0.000 description 9
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 9
- WTEOIRVLGSZEPR-UHFFFAOYSA-N boron trifluoride Chemical compound FB(F)F WTEOIRVLGSZEPR-UHFFFAOYSA-N 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- 239000000741 silica gel Substances 0.000 description 8
- 229910002027 silica gel Inorganic materials 0.000 description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 7
- 238000000921 elemental analysis Methods 0.000 description 7
- 238000010828 elution Methods 0.000 description 7
- 238000011049 filling Methods 0.000 description 7
- 239000001257 hydrogen Substances 0.000 description 7
- 229910052739 hydrogen Inorganic materials 0.000 description 7
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 description 7
- UFWIBTONFRDIAS-UHFFFAOYSA-N naphthalene-acid Natural products C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 7
- 125000001624 naphthyl group Chemical group 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 6
- 229910052799 carbon Inorganic materials 0.000 description 6
- 239000000945 filler Substances 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- 238000006386 neutralization reaction Methods 0.000 description 6
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 5
- 102000004506 Blood Proteins Human genes 0.000 description 5
- 108010017384 Blood Proteins Proteins 0.000 description 5
- 230000007717 exclusion Effects 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- FTOAOBMCPZCFFF-UHFFFAOYSA-N 5,5-diethylbarbituric acid Chemical compound CCC1(CC)C(=O)NC(=O)NC1=O FTOAOBMCPZCFFF-UHFFFAOYSA-N 0.000 description 4
- 229910015900 BF3 Inorganic materials 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 4
- CXOFVDLJLONNDW-UHFFFAOYSA-N Phenytoin Chemical compound N1C(=O)NC(=O)C1(C=1C=CC=CC=1)C1=CC=CC=C1 CXOFVDLJLONNDW-UHFFFAOYSA-N 0.000 description 4
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 239000013060 biological fluid Substances 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000002523 gelfiltration Methods 0.000 description 4
- 238000004811 liquid chromatography Methods 0.000 description 4
- 239000002207 metabolite Substances 0.000 description 4
- DDBREPKUVSBGFI-UHFFFAOYSA-N phenobarbital Chemical compound C=1C=CC=CC=1C1(CC)C(=O)NC(=O)NC1=O DDBREPKUVSBGFI-UHFFFAOYSA-N 0.000 description 4
- 229960002036 phenytoin Drugs 0.000 description 4
- 229910000077 silane Inorganic materials 0.000 description 4
- 239000000377 silicon dioxide Substances 0.000 description 4
- PYJJCSYBSYXGQQ-UHFFFAOYSA-N trichloro(octadecyl)silane Chemical compound CCCCCCCCCCCCCCCCCC[Si](Cl)(Cl)Cl PYJJCSYBSYXGQQ-UHFFFAOYSA-N 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- LPHGQDQBBGAPDZ-UHFFFAOYSA-N Isocaffeine Natural products CN1C(=O)N(C)C(=O)C2=C1N(C)C=N2 LPHGQDQBBGAPDZ-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 229960001948 caffeine Drugs 0.000 description 3
- VJEONQKOZGKCAK-UHFFFAOYSA-N caffeine Natural products CN1C(=O)N(C)C(=O)C2=C1C=CN2C VJEONQKOZGKCAK-UHFFFAOYSA-N 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 150000002431 hydrogen Chemical class 0.000 description 3
- 230000002452 interceptive effect Effects 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical class CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 229960002695 phenobarbital Drugs 0.000 description 3
- 239000005053 propyltrichlorosilane Substances 0.000 description 3
- 125000005372 silanol group Chemical group 0.000 description 3
- 238000007613 slurry method Methods 0.000 description 3
- DOEHJNBEOVLHGL-UHFFFAOYSA-N trichloro(propyl)silane Chemical compound CCC[Si](Cl)(Cl)Cl DOEHJNBEOVLHGL-UHFFFAOYSA-N 0.000 description 3
- KBPLFHHGFOOTCA-UHFFFAOYSA-N 1-Octanol Chemical compound CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 description 2
- XNWFRZJHXBZDAG-UHFFFAOYSA-N 2-METHOXYETHANOL Chemical compound COCCO XNWFRZJHXBZDAG-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 2
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 2
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 2
- OFBQJSOFQDEBGM-UHFFFAOYSA-N Pentane Chemical class CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 239000003125 aqueous solvent Substances 0.000 description 2
- 229960002319 barbital Drugs 0.000 description 2
- 229920001222 biopolymer Polymers 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- ZSIAUFGUXNUGDI-UHFFFAOYSA-N hexan-1-ol Chemical compound CCCCCCO ZSIAUFGUXNUGDI-UHFFFAOYSA-N 0.000 description 2
- 125000005647 linker group Chemical group 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 229910017604 nitric acid Inorganic materials 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 239000012044 organic layer Substances 0.000 description 2
- 239000005054 phenyltrichlorosilane Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- -1 propamide chains Chemical group 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 238000004381 surface treatment Methods 0.000 description 2
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 description 2
- RCHUVCPBWWSUMC-UHFFFAOYSA-N trichloro(octyl)silane Chemical compound CCCCCCCC[Si](Cl)(Cl)Cl RCHUVCPBWWSUMC-UHFFFAOYSA-N 0.000 description 2
- ORVMIVQULIKXCP-UHFFFAOYSA-N trichloro(phenyl)silane Chemical compound Cl[Si](Cl)(Cl)C1=CC=CC=C1 ORVMIVQULIKXCP-UHFFFAOYSA-N 0.000 description 2
- 229920003169 water-soluble polymer Polymers 0.000 description 2
- 238000007779 wet slurry method Methods 0.000 description 2
- CHRJZRDFSQHIFI-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;styrene Chemical compound C=CC1=CC=CC=C1.C=CC1=CC=CC=C1C=C CHRJZRDFSQHIFI-UHFFFAOYSA-N 0.000 description 1
- AWFYPPSBLUWMFQ-UHFFFAOYSA-N 2-[5-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]-1,3,4-oxadiazol-2-yl]-1-(1,4,6,7-tetrahydropyrazolo[4,3-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1=NN=C(O1)CC(=O)N1CC2=C(CC1)NN=C2 AWFYPPSBLUWMFQ-UHFFFAOYSA-N 0.000 description 1
- YWIYEOLSZWMXQG-UHFFFAOYSA-N 4-[dichloro(methyl)silyl]butanoyl chloride Chemical compound C[Si](Cl)(Cl)CCCC(Cl)=O YWIYEOLSZWMXQG-UHFFFAOYSA-N 0.000 description 1
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical group CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- VCUFZILGIRCDQQ-KRWDZBQOSA-N N-[[(5S)-2-oxo-3-(2-oxo-3H-1,3-benzoxazol-6-yl)-1,3-oxazolidin-5-yl]methyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C1O[C@H](CN1C1=CC2=C(NC(O2)=O)C=C1)CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F VCUFZILGIRCDQQ-KRWDZBQOSA-N 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 229910018557 Si O Inorganic materials 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000007824 aliphatic compounds Chemical class 0.000 description 1
- 150000001338 aliphatic hydrocarbons Chemical class 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- DNSISZSEWVHGLH-UHFFFAOYSA-N butanamide Chemical group CCCC(N)=O DNSISZSEWVHGLH-UHFFFAOYSA-N 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- AZUXXYCNTKYZJB-UHFFFAOYSA-N chloro(octadecyl)silane Chemical compound CCCCCCCCCCCCCCCCCC[SiH2]Cl AZUXXYCNTKYZJB-UHFFFAOYSA-N 0.000 description 1
- MNKYQPOFRKPUAE-UHFFFAOYSA-N chloro(triphenyl)silane Chemical compound C=1C=CC=CC=1[Si](C=1C=CC=CC=1)(Cl)C1=CC=CC=C1 MNKYQPOFRKPUAE-UHFFFAOYSA-N 0.000 description 1
- GZGREZWGCWVAEE-UHFFFAOYSA-N chloro-dimethyl-octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[Si](C)(C)Cl GZGREZWGCWVAEE-UHFFFAOYSA-N 0.000 description 1
- DBKNGKYVNBJWHL-UHFFFAOYSA-N chloro-dimethyl-octylsilane Chemical compound CCCCCCCC[Si](C)(C)Cl DBKNGKYVNBJWHL-UHFFFAOYSA-N 0.000 description 1
- HXVPUKPVLPTVCQ-UHFFFAOYSA-N chloro-dimethyl-propylsilane Chemical compound CCC[Si](C)(C)Cl HXVPUKPVLPTVCQ-UHFFFAOYSA-N 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 229920006037 cross link polymer Polymers 0.000 description 1
- 230000003544 deproteinization Effects 0.000 description 1
- OSXYHAQZDCICNX-UHFFFAOYSA-N dichloro(diphenyl)silane Chemical compound C=1C=CC=CC=1[Si](Cl)(Cl)C1=CC=CC=C1 OSXYHAQZDCICNX-UHFFFAOYSA-N 0.000 description 1
- QHBMMABVNRSRHW-UHFFFAOYSA-N dichloro-methyl-octylsilane Chemical compound CCCCCCCC[Si](C)(Cl)Cl QHBMMABVNRSRHW-UHFFFAOYSA-N 0.000 description 1
- GNVPGBIHGALKRR-UHFFFAOYSA-N dichloro-methyl-propylsilane Chemical compound CCC[Si](C)(Cl)Cl GNVPGBIHGALKRR-UHFFFAOYSA-N 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 125000001033 ether group Chemical group 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 150000008282 halocarbons Chemical class 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- TVMXDCGIABBOFY-UHFFFAOYSA-N octane Chemical class CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- LIVNPJMFVYWSIS-UHFFFAOYSA-N silicon monoxide Inorganic materials [Si-]#[O+] LIVNPJMFVYWSIS-UHFFFAOYSA-N 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 150000005846 sugar alcohols Polymers 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 125000003944 tolyl group Chemical group 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Description
発明の技術分野
本発明は、二重構造担体に関し、詳しくは、多
孔質シリカ粒子の外部表面に親水性部位を有する
有機固定相が化学結合され、且つ該粒子の細孔内
部表面に疎水性部位を有する有機固定相が化学結
合された高分子物質及び低分子物質を同時に含有
する水溶液から低分子物質のみを選択的に吸着す
る二重構造担体に関する。
背景技術
高分子物質と低分子物質を同時に含有する溶液
から、それぞれ高分子物質と低分子物質を選択的
に分離する操作は、特に高分子物質を多量に含有
する水溶媒系において重要である。即ち、典型的
な例として、低濃度の低分子物質と蛋白質、核酸
等の多量の生体高分子物質を含有している血清に
代表される生体液、或いは、バクテリア、臓器、
植物等のモネジネート抽出液等の水溶液が挙げら
れる。
水溶媒系から有機物質を吸着する方法として
は、ジビニルベンゼン−スチレン等の疎水性架橋
高分子粒子を用いる方法が米国特許第3531463号
公報に開示されているが、高分子物質に対しても
疎水性相互作用により吸着してしまうため、高分
子物質及び低分子物質を同時に含有する溶液には
適当でない。これはまた、オクタデシル基等の疎
水性基を化学結合した汎用の逆相クロマトグラフ
イ用充填剤の場合でも同様である。
本発明者は、既に、疎水性基を化学結合した多
孔質シリカ粒子を用い、該粒子の外部表面に牛血
清アルブミンを吸着させることにより、外部の疎
水性部位を親水性部位に変化させた充填剤を出願
した（特開昭58−223062）。この牛血清アルブミ
ンを吸着せしめた充填剤は、蛋白質が細孔内部に
浸透できない程の細孔径を有しているため、外部
表面のみ牛血清アルビミンが疎水性相互作用で吸
着し、その結果該粒子の外部表面は蛋白質の親水
性部位により親水性となつたものである。このた
め、前述の担体とは異なり、蛋白質等の水溶性高
分子と充填期間の作用が遮蔽され、一方低分子物
質の細孔内部への浸透を妨げない充填剤である。
しかし、この充填剤は、蛋白質が吸着のみによつ
て粒子の外部表面に付着しているため、長時間の
使用においては蛋白質が脱離してしまい、試料中
の蛋白質を吸着したり、また、蛋白質であるため
に保存安定性に不安があり、さらに吸着蛋白質が
巨大なため、細孔が塞がれ吸着能を低下させる
等、不利な点もある。
発明の目的
本発明は、上記の従来公知の分離方法及び疎水
性物質吸着担体とは異なり、多孔質シリカ粒子の
外部表面が高分子物質と疎水性相互作用しない親
水性部位を有する化学結合された有機固定相で構
成され、且つ細孔内部は低分子物質を疎水性相互
作用によつて吸着しうる疎水性部位を有する化学
結合された有機固定相で構成された二重構造担体
を提供するものである。
発明の構成
高分子物質と低分子物質を共に含有する溶液か
ら低分子物質を分離する方法は、低分子物質のも
つ疎水性、或いはイオン性を利用した担体への吸
着により達成されるが、特に疎水性を利用した吸
着による分離は幅広い試料に適応できることから
も効果的な方法として一般に用いられている。し
かし、高分子物質を含有する溶液では疎水的吸着
により、低分子物質ばかりか高分子物質の吸着も
同時に起こつてしまう。したがつて、このように
高分子物質を含有する溶液から低分子物質を吸着
するために、同一粒子内に高分子物質に対して不
活性な部位と、低分子物質に対しては疎水性相互
作用によつて吸着する疎水性部位を有する粒子で
あれば、高分子が吸着することなく低分子物質の
みを吸着でき、効果的であると考え鋭意研究した
結果同一粒子内に必要とされる部位は、細孔内部
と細孔外部に分けられ、細孔外部には、高分子物
質と相互作用しない不活性部位が、又、細孔内部
は低分子物質を捕獲するための疎水性部位が化学
結合された構造が有用であることを見い出した。
即ち、本発明は、平均細孔径が10〜200Å、平
均粒子径が0.1〜1000μの多孔質シリカ粒子を骨格
とし、該粒子の細孔外部表面が十分な親水性部位
を有する化学結合された有機固定相で構成され、
且つ細孔内部表面が十分な疎水性部位を有する化
学結合された有機固定相で構成された二重構造担
体に係るものである。
以下、本発明を具体的に説明する。
本発明の二重構造担体は多孔質シリカ粒子の外
部表面に親水性部位を有する有機固定相が化学結
合され、且つ該粒子の細孔内部表面に疎水性部位
を有する有機固定相が化学結合された平均細孔径
が10〜200Å、平均粒子径が0.1〜1000μの二重構
造担体である。
本発明において多孔質シリカ粒子の平均細孔径
は10〜200Å、好ましくは30〜150Åの範囲であ
る。細孔径が10Å未満では、低分子物質の細孔内
への拡散を妨げ、十分な吸着能が得られず、ま
た、200Åを超えると細孔内への高分子物質の浸
透を許し、疎水性部位への吸着や、低分子物質の
吸着能低下を生じるため好ましくない。また、粒
子径は、0.1〜1000μ、好ましくは３〜500μの範囲
である。0.1μ未満、また、1000μを超えると実用
性が失なわれ好ましくない。親水性部位を有する
有機固定相とは、その化学構造が、下記の一般
式、
TECHNICAL FIELD OF THE INVENTION The present invention relates to a double-structured carrier, and more specifically, an organic stationary phase having hydrophilic sites is chemically bonded to the outer surface of porous silica particles, and hydrophobic sites are attached to the inner surface of the pores of the particles. The present invention relates to a double-structured carrier that selectively adsorbs only low-molecular substances from an aqueous solution containing chemically bonded high-molecular substances and low-molecular substances at the same time. BACKGROUND ART The operation of selectively separating a high molecular weight substance and a low molecular weight substance from a solution containing both high molecular weight substances and low molecular weight substances, respectively, is particularly important in an aqueous solvent system containing a large amount of high molecular weight substances. That is, typical examples include biological fluids such as serum containing low concentrations of low molecular weight substances and large amounts of biopolymer substances such as proteins and nucleic acids, or bacteria, organs, etc.
Examples include aqueous solutions such as moninenate extracts of plants and the like. As a method for adsorbing organic substances from an aqueous solvent system, a method using hydrophobic crosslinked polymer particles such as divinylbenzene-styrene is disclosed in U.S. Patent No. 3,531,463; It is not suitable for solutions containing both high-molecular and low-molecular substances because they are adsorbed due to chemical interaction. This also applies to general-purpose packing materials for reversed-phase chromatography in which hydrophobic groups such as octadecyl groups are chemically bonded. The present inventor has already developed a method of packing that changes the external hydrophobic sites into hydrophilic sites by adsorbing bovine serum albumin onto the external surface of the particles using porous silica particles chemically bonded with hydrophobic groups. (Japanese Patent Application Laid-Open No. 58-223062). This packing material that adsorbs bovine serum albumin has a pore diameter that is large enough that proteins cannot penetrate into the pores, so bovine serum albumin is adsorbed only on the outer surface through hydrophobic interaction, resulting in the particles The external surface of the protein is made hydrophilic by the hydrophilic sites of the protein. Therefore, unlike the above-mentioned carrier, it is a filler that is shielded from the effects of water-soluble polymers such as proteins and during the filling period, while not interfering with the penetration of low-molecular substances into the pores.
However, since proteins are attached to the external surface of the particles only by adsorption, the proteins in this packing material tend to detach when used for long periods of time, causing the proteins in the sample to be adsorbed or Because of this, there are concerns about storage stability, and furthermore, since the adsorbed proteins are huge, the pores are clogged and the adsorption capacity is reduced, resulting in other disadvantages. Purpose of the Invention The present invention provides that, unlike the conventionally known separation methods and hydrophobic substance adsorption carriers described above, the external surface of porous silica particles is chemically bonded with a hydrophilic site that does not have hydrophobic interaction with a polymeric substance. Provides a double-structured support composed of an organic stationary phase and a chemically bonded organic stationary phase having hydrophobic sites capable of adsorbing low-molecular substances through hydrophobic interaction inside the pores. It is. Structure of the Invention A method for separating a low-molecular substance from a solution containing both a high-molecular substance and a low-molecular substance is achieved by adsorption onto a carrier using the hydrophobicity or ionicity of the low-molecular substance. Separation by adsorption using hydrophobicity is generally used as an effective method because it can be applied to a wide range of samples. However, in a solution containing a high molecular weight substance, not only the low molecular weight substance but also the high molecular weight substance is adsorbed simultaneously due to hydrophobic adsorption. Therefore, in order to adsorb low-molecular substances from solutions containing high-molecular substances, in the same particle there are sites that are inert to high-molecular substances, and sites that are hydrophobic to low-molecular substances. Particles with hydrophobic sites that adsorb by action can adsorb only low-molecular substances without adsorbing polymers, and we believe that this is effective, and after extensive research, we found that the necessary sites within the same particle The pore is divided into the inside and outside of the pore, and the outside of the pore has an inert site that does not interact with polymeric substances, and the inside of the pore has a hydrophobic site that captures low-molecular substances. We have found that combined structures are useful. That is, the present invention has porous silica particles with an average pore diameter of 10 to 200 Å and an average particle diameter of 0.1 to 1000 μ as a skeleton, and the outer surface of the pores of the particles has a chemically bonded organic silica particle having sufficient hydrophilic sites. Consists of a stationary phase,
Moreover, the present invention relates to a double-structured carrier in which the inner surface of the pores is composed of a chemically bonded organic stationary phase having sufficient hydrophobic sites. The present invention will be explained in detail below. In the dual structure support of the present invention, an organic stationary phase having hydrophilic sites is chemically bonded to the external surface of porous silica particles, and an organic stationary phase having hydrophobic sites is chemically bonded to the internal surface of the pores of the particles. It is a double-structured carrier with an average pore diameter of 10 to 200 Å and an average particle diameter of 0.1 to 1000 μ. In the present invention, the average pore diameter of the porous silica particles is in the range of 10 to 200 Å, preferably 30 to 150 Å. If the pore diameter is less than 10 Å, the diffusion of low-molecular substances into the pores will be hindered, and sufficient adsorption capacity will not be obtained.If the pore diameter exceeds 200 Å, the permeation of high-molecular substances into the pores will be allowed, resulting in hydrophobicity. This is not preferable because it causes adsorption to the site and decreases the adsorption ability of low-molecular-weight substances. Further, the particle size is in the range of 0.1 to 1000μ, preferably 3 to 500μ. If it is less than 0.1 μm or more than 1000 μm, practicality will be lost, which is not preferable. An organic stationary phase having a hydrophilic moiety has a chemical structure having the following general formula:
【式】（但し、ｌ：１〜
６） (1)
−（Ｏ−CH2CH2）n−OCH3（但し、ｍ：１〜６）
(2)[Formula] (However, l: 1 to 6) (1) -(O-CH 2 CH 2 ) n -OCH 3 (However, m: 1 to 6)
(2)
【式】（但し、ｎ：１〜４） (3)
−（Ｏ−CH2CH2）p−OH（但し、ｐ：１〜６）(4)
で表わされる残基を有する化合物である。具体的
には、水酸基を分子内に保有する化合物として、
エチレングリコール、グリセリン、ソルビトール
等の多価アルコール類、アミド基では、アセトア
ミド鎖、プロパミド鎖、ブチルアミド鎖等の低級
脂肪酸アミド類、更にエーテル基を有する化合物
群ではポリエチレンオキシドあるいはそのメチ
ル、エチルなどの低級脂肪族化合物のエーテル類
などの酸素原子あるいは窒素原子などよりなる水
素結合可能な部位を有し、かつ、タンパク質との
反応性が小さい化合物が挙げられるが特に、グリ
シドキシプロピルトリメトキシシラン、エチレン
グリコールモノメチルエーテル、ジエチレングリ
コールなどが好ましい。
疎水性部位を有する有機固定相とは、その化学
構造が下記の一般式
（但し、式中R1は炭素数が１から36までの脂肪
族炭化水素基又は芳香族炭化水素基、R2、R3は
クロロ基又はアルコキシ基あるいはメチル基、
R4はクロロ基又はアルコキシ基である）で表わ
されるシラン処理剤である。具体的にはオクタデ
シルトリクロロシラン、オクチルトリクロロシラ
ン、プロピルトリクロロシラン、フエニルトリク
ロロシラン、アクタデシルメチルジクロロシラ
ン、オクチルメチルジクロロシラン、プロピルメ
チルジクロロシラン、ジフエニルジクロロシラ
ン、オクタデシルジメチルクロロシラン、オクチ
ルジメチルクロロシラン、プロピルジメチルクロ
ロシラン、トリフエニルクロロシランなどの多孔
質シリカ粒子との反応により簡便にその表面上に
有機層を導入できるものを挙げることができる
が、特にR1がオクタデシル基であつて、R2、R3、
及びR4がクロロ基であるオクタデシルクロロシ
ランが好ましい。
次に本発明の二重構造担体の製造方法について
説明する。
多孔質シリカ粒子担体の細孔外部表面と内部表
面にそれぞれ別の官能基を導入する方法として
は、多孔質シリカ粒子の細孔内部を適当な充填物
質で埋め、細孔外部表面に親水性部位を有してい
るシラン処理剤を用い親水性基を導入した後、細
孔内部の充填物質を除去し、引き続き疎水性部位
を有するシラン処理剤を用い疎水性基を導入する
方法。あるいは、多孔質シリカ粒子に疎水性基を
導入した後、細孔内部に適当な充填物質を充填
し、細孔外部表面に導入された疎水性基を切断す
る。充填物質を除去した後、引き続き粒子の外部
表面に親水性基を導入する方法などを挙げること
ができる。以下、簡便かつ効果的である後者の方
法について説明する。
多孔質シリカ粒子に疎水基を導入する方法は多
孔質シリカ粒子と適当な疎水性基を有したシラン
処理剤との反応により得ることができる。即ち、
多孔質シリカ粒子を有機溶媒中に分散し、一般式
(5)で表わされるシラン処理剤を加え、20°〜150
℃、好ましくは40°〜120℃、１分以上反応させる
ことにより疎水性基を導入できる。反応温度が20
℃未満では反応時間が長くかかり、150℃を越え
ると多孔質シリカ粒子の均一な表面被覆反応を妨
げるため好ましくない。また、反応時間が１分未
満では、十分な表面被覆反応が達成できないため
好ましくない。反応の経済性および効率性を考慮
すれば30分〜24時間反応させることが好ましい。
次に疎水性基を有する多孔質シリカ粒子の細孔
内部を適当な充填物質で埋め、細孔外部と遮断し
たのち、適当な化学物質を用いて外部表面のシロ
キサン結合のみを切断する。使用される充填物質
としては、例えば、多孔質シリカ粒子とよく混和
し得る水不溶性或いは水難溶性の有機溶剤が挙げ
られる。具体的には、ペンタン、ヘキサン、オク
タン等の脂肪族炭化水素化合物、ベンゼン、トル
エン、キシレン等の芳香族炭化水素化合物、ヘキ
サノール、オクタノール等の炭素鎖６以上の高級
アルコール、或いはクロロホルム、四塩化炭素等
のハロゲン化炭化水素化合物が挙げられるが、特
にトルエンが好ましい。
多孔質シリカ粒子の細孔内部を充填物質で埋め
る方法としては、上記有機溶媒中に表面に疎水基
を導入した多孔質シリカ粒子を分散し攪拌あるい
は超音波処理により、十分に細孔内に浸透させる
方法。クロマト管等に多孔質シリカ粒子を、乾式
法又は、湿式スラリー法で充填し、静圧下或いは
加圧下に上記有機溶媒を通液し細孔内を上記有機
溶媒で置換する方法などを挙げることができる
が、特に、加圧下で湿式スラリー法で置換する方
法が短時間で目的を達成できるので好ましい。
前述の如き有機溶剤で細孔内を置換された多孔
質シリカ粒子は、その外部表面の疎水性基とシリ
カ基材間の結合部であるシロサキン結合（Si−Ｏ
−Si）を切断するため加水分解試薬によつて処理
される。即ち、酸或いはアルカリ水溶液中、20°
〜100℃、好ましくは30°〜90℃、数秒から48時
間、好ましくは１分間から24時間処理することに
よりシロキサン結合を加水分解する。ここで使用
に供される加水分解試薬は、水酸化カリウム、水
酸化ナトリウム、炭酸ナトリウム等のアルカリ水
溶液、或いは塩酸、硫酸、硝酸等の酸性水溶液が
挙げられる。特に、アルカリ性水溶液はシロキサ
ン結合の切断に有効である。
これらの加水分解試薬は、化学結合基とシリカ
間の結合切断に供されるが、粒子の細孔内は水不
溶性、或いは水難溶性有機溶剤で満たされている
ため細孔内部への浸透できず、その結果、粒子外
部表面でのみシロキサン結合の加水分解が進行す
ることになる。
加水分解に費やされる時間は適切に定められな
ければならず、極端に短すぎることは粒子外部表
面の十分な加水分解を妨げ、一方40時間を超える
とシリカ骨格が浸食され、多孔質シリカ粒子とし
ての性能を低下させるとともに、機械的強度を損
なう結果となり好ましくない。
また、反応温度が20℃未満では不十分な表面の
加水分解により、有機層が残り、100℃を超える
と急激な加水分解がおこりシリカ骨格の浸食され
るため好ましくない。
一定時間、加水分解処理に付された多孔質シリ
カ粒子は、素早く酸、或いはアルカリによつて中
和されなければならない。中和に用いられる試薬
は、シロキサン結合の加水分解試薬がアルカリ水
溶液の場合、塩酸、硫酸、硝酸等の希薄水溶液、
及び酢酸等の有機酸の水溶液を、又、用いる加水
分解試薬が酸性水溶液の場合は、水酸化カリウ
ム、水酸化ナトリウム等の希薄水溶液を通液する
か、加圧下に送液することにより達成することが
できる。中和に用いられる試薬は、例えば、副生
塩が酢酸ナトリウムのような水で洗浄される場合
に溶解しやすい酸、アルカリの対を選択すること
が好ましい。
上記の如くして得られる多孔質シリカ粒子は、
その外部表面に加水分解反応によつて生ぜしめた
シラノール基を有し、且つ該粒子の細孔内部表面
は、元の疎水基を保持した構造となる。新たに粒
子外部表面に生じたシラノール基は、前述した一
般式(1)〜(4)で表わされる残基を有する化合物と不
活性な有機溶媒中、20〜90℃、30分〜24時間処理
することにより水溶性高分子物質に不活性な親水
基が導入される。
上記方法において疎水生物質の吸着能が大きい
逆相担体を用いることにより、上記操作を簡略化
することができる。
逆相担体は、その表面に疎水性の化学結合基が
均一に結合されているため、疎水性物質の吸着能
は大である。多孔質シリカ粒子では、細孔内部表
面に比べ細孔外部表面の疎水基の被覆度は極端に
小さい。かかる事実は、粒子の外部表面に化学結
合された疎水性基の吸着能への寄与が小さいこと
を示唆するものである。即ち、粒子外部表面にの
みに化学結合された疎水性基を選択的に除去し得
られるであろう担体は、その性能における変化は
小さく、元の逆相担体としての性能を保持してい
ると考えられる。
したがつて、逆相担体に適当な表面処理を付
し、粒子の外部表面に化学結合された疎水性基を
切断し得られた逆相担体は、細孔外部表面に新し
く生ぜしめたシラノール基を有しており、この担
体を、ついで前述の一般式(1)〜(4)で表わされる適
当なシラン処理剤で同様に処理することにより、
表面処理を付した担体の外部表面に異なつた官能
基を導入することができる。即ち、逆相担体の表
面処理の方法としては、例えば前述の如き有機溶
剤中に分散後、グラスフイルター上でロ別し、す
ばやくシリカ基材と粒子外部に化学結合された疎
水性基との間の結合を加水分解する試薬を通液す
る方法、あるいは、適当なカラム内に逆相担体を
充填し、有機溶剤で置換後、適当な加圧下で上述
の加水分解試薬を送液する方法を挙げることがで
きるが、特に加圧下での処理方法は短時間で目的
を達成し得る方法として、効果的である。
次いで、前記一般式(1)〜(4)で表わされる残基を
有する化合物で同様に処理する。
このようにして得られた親水性部位を粒子外部
表面に有する二重構造担体は、細孔内部には浸透
できない水溶性高分子物質に対して不活性であ
り、一方低分子物質に対しては、細孔内部の疎水
性基によつて捕獲することによつて、従来の逆相
担体と同程度の吸着性能を発揮する。
以上の如く、本発明は、多孔質シリカ粒子に疎
水性基を導入する工程、多孔質シリカ粒子の細孔
内部を水不溶性、或いは水難溶性有機溶剤で置換
する工程、加水分解工程、及びシラン処理工程に
より、該粒子の外部表面にのみ選択的に親水性基
を導入でき、且つ該粒子の細孔内部表面には、疎
水性基を有した二重構造担体を得ることができ
る。さらに、既存の逆相担体の性能を低下させる
ことなく、同様の二重構造担体を得ることができ
る。
発明の効果
本発明の二重構造担体の利用分野は、例えば高
分子物質と低分子物質を同時に含有する水溶液に
本発明担体を投入・混合することにより、その水
溶液から有害物質、或いは妨害物質としての低分
子物質を吸着、除去し、目的の高分子物質を回収
することができる。例えば、血液中の蛋白質代謝
産物や低中分子の有害物質と蛋白質等の高分子物
質の分類による患者の血液浄化、或いは医薬品等
の生体高分子物質の製造工程に於ける夾雑低分子
物質の除去等が挙げられる。更に、妨害物質とし
ての高分子物質を除去した後、本発明充填剤に捕
獲された低分子物質を引き続いて、適当な有機溶
媒、例えばメタノール、アセトニトリル等で抽出
し、回収することができる。例えば、極端に低レ
ベルで存在する生体液、或いはホモジネート抽出
液中の医薬品や生体関連代謝物、或いは神経伝達
物質、ホルモン、ホルモン様物質等の回収、或い
は菌体から分泌された抗生物質や生理活性物質等
の回収が挙げられる。
一方、近年は特に液体クロマトグラフイの高感
度化により生体成分の分離、分析に有用な手段と
して広範に利用されるようになつて来た。液体ク
ロマトグラフイは、迅速性、ルーチンワークに適
しているため、生体成分の分析に用いられてきた
が、高分子物質を含有する検体の分析では、徐蛋
白操作等の試料の前処理を必要とし、工程を複雑
にし、更には分析結果の再現性等にも問題を有し
ている。本発明の担体は、本来の逆相担体の性能
を保持しているため、液体クロマトグラフイ用カ
ラムに充填することにより、通常の分析カラムと
して使用できる。即ち、本担体はその細孔外部に
親水性部位を有する有機固定相が化学結合されて
いるため、高分子物質に対して不活性となり、蛋
白質等の高分子物質はカラム内を素通りし、しか
も低分子物質は通常の逆相モードで分離分析する
ことができる。この結果、検体のルーチンワー
ク、迅速な測定を可能にしさらに測定誤差を極小
にでき、特に、生体液中の医薬品やその代謝物等
の分析に有用である。
更に、低レベルで生体液中に存在する低分子物
質の分析のためには、本発明担体を濃縮カラムと
して使用することができる。即ち、濃縮カラムに
充填された本発明担体に、微量の目的低分子物質
を含有する多量の検体試料を通液し、高分子物質
の除去を行ないながら、低分子物質を充填剤の細
孔内部の疎水性部に濃縮し、次いで、カラムスイ
ツチング法により、適当な溶離液組成で濃縮され
た目的の低分子物質を本カラムへと導入し分析す
る方法である。例えば、低レベルで薬効を示す医
薬品のモニタリングや、脳や臓器中の微量に存在
する生理活性ペプチド群等の分析、更には、生体
関連代謝物等の濃縮・分離等、臨床学的に有用な
検体試料の分析に利用できる。
以上のごとく、本発明担体は、高分子物質と低
分子物質を同時に含有する水溶液、中に投入する
か、或いは液体クロマトグラフイ用カラムに充填
し、検体試料を注入するか、更にはカラムに充填
し、濃縮カラムとして用いるかする方法によつ
て、対象の検体溶液から目的の高分子物質、或い
は低分子物質を選択的に分離し、回収することが
でき、必要に応じて目的の分析方法に付すること
ができる。
実施例
実施例 １
平均細孔径が70オングストローム、平均粒子径
が10ミクロンのシリカゲル10ｇをトルエン100ml
中に分散し、オクタデシルトリクロロシラン50ｇ
を加え、90℃に保つたオイル浴中で24時間反応し
た。反応液をろ過し、トルエン100ml、アセトン
100mlを洗浄した。このようにして得られた担体
を70％メタノール水溶液中に分散し残存するクロ
ロ基を加水分解するため、60℃で６時間処理し
た。担体をろ別後、メタノール100mlで洗浄し、
100℃で４時間乾燥した。このようにして得られ
た担体の元素分析値は炭素が13.5％、水素が2.1
％であつた。この逆相担体を、内径7.5mm、長さ
30cmのステンレスカラムに、40％エタノールスラ
リーを調製し、エタノールを充填溶媒としてスラ
リー法により充填した。カラム内をトルエンで置
換したのち、該カラムに0.1N水酸化ナトリウム
水溶液を１ml／minの流速で５分間、通液した。
素早く0.1％酢酸水溶液を１ml／minの流速で10
分間通液した。更に、純水を１ml／minの流速で
10分間通液後、メタノールで平衡化後溶離液とし
てメタノールを用い紫外吸収（波長254nｍ）で
ナフタレンの溶出位置を調べたところ、20.5分
（処理前23.4分）であつた。また、カラムから抜
き出したゲルについてメチルレツドの吸着量を調
べたところ、20.3mg／ｇ（処理前14.9mg／ｇ）で
あつた。このゲル５ｇをトルエン50ml中に分散
し、γ−グリシドキシプロピルトリメトキシシラ
ン５ｇを加え80℃で８時間反応させ、ろ液を分離
後、トルエン50ml、アセトン50ml、更にメタノー
ル50mlで洗浄した後、純水100ml中過塩素酸によ
りPHを2.5に調整後、90℃で４時間処理した。次
いでろ過後、メタノールで洗浄した後、100℃で
４時間乾燥した。このようにして得られた担体
0.5ｇを0.5ｇの牛血清アルブミンと1μｇのテオフ
イリンを含有する水溶液１mlに投入し５分間攪拌
した。ろ別後、ろ液を0.2Nリン酸緩衝液（PH6.8）
で平衡化した高速ゲルろ過クロマトグラフイ用カ
ラム（商品名：TSKgel G2000SW、内径7.5mm、
長さ60cm、東洋曹達工業製）に付した。280nｍ
で検出したクロマトグラムを第１図に示した。Ａ
はテオフイリン添加牛血清アルブミン溶液のクロ
マトグラム、Ｂは本発明担体で処理した溶液のク
ロマトグラムであり、１は牛血清アルブミン、２
はテオフイリンの吸光度ピークである。ろ液中の
蛋白質の280nｍの吸光度を調べたところ本発明
担体を投入する前の99％の回収率であつた。一
方、テオフイリンは担体に完全に吸着された。
実施例 ２
平均細孔径が150オングストローム、平均粒子
径が15ミクロンのシリカゲル10ｇを実施例１に準
じてオクタデシル基を有するシラン処理剤で処理
した。このようにして得られた担体の元素分析値
は、それぞれ炭素が15.9％、水素が2.3％であつ
た。このオクタデシル基を結合した逆相担体を実
施例１に準じてステンレスカラムに充填しトルエ
ンで置換後、0.1N水酸化ナトリウムで５分間処
理した。メタノールを溶離液としてナフタレンの
溶出位置を測定したところ、18.8分（処理前20.9
分）であつた。さらに、カラムから抜き出しメチ
ルレツドの吸着量を調べたところ、16.3mg／ｇ
（処理前10.1mg／ｇ）であつた。この担体を実施
例１と同様のγ−グリシドキシプロピルトリメト
キシシランで処理し、更に、過塩素酸で処理し
た。実施例１と同様にこの担体0.5ｇを0.5ｇの牛
血清アルブミンと1μｇのテオフイリンを含有す
る水溶液１mlに投入し、蛋白質の回収率を調べた
ところ、99％であり、テオフイリンは吸着され観
測されなかつた。
実施例 ３
平均細孔径が60オングストローム、平均粒子径
が500ミクロンのシリカゲル10ｇを実施例１と同
様にオクタデシルトリクロロシランを用いて処理
した。このようにして得られた担体の元素分析値
は、それぞれ炭素が12.2％、水素が2.1％であつ
た。この担体を実施例１に準じて、ステンレスカ
ラムに充填し、0.1N水酸化ナトリウムで５分間
処理した。中和、洗浄後、メタノールを溶離液と
してナフタレンの溶出位置を測定したところ、
19.2分（処理前22.6分）であつた。更にカラムか
ら抜き出し、メチルレツドの吸着量を測定したと
ころ、15.2mg／ｇ（処理前11.1mg／ｇ）であつ
た。この担体５ｇを実施例１と同様にγ−グリシ
ドキシプロピルトリメトキシシランで処理し、さ
らに過塩素酸で処理した。このようにして得られ
た担体0.5ｇを実施例１と同様に、蛋白質を含有
する水溶液１mlに投入し、蛋白質の回収率を調べ
たところ、97％であり、テオフイリンは吸着され
観測されなかつた。
実施例 ４
平均細孔径が100オングストローム、平均粒子
径が10ミクロンのシリカゲル10ｇをトルエン100
ml中に分散し、オクチルトリクロロシラン５ｇを
加え、90℃で24時間反応した。反応液をろ過し、
担体をトルエン100ml、アセトン100ml、次いでメ
タノール100mlで洗浄し、100℃で４時間乾燥し
た。このようにして得られたオクチル基が結合さ
れた逆相担体の元素分析値は、それぞれ炭素が
9.5％、水素が1.4％であつた。この担体を、実施
例１に準じて、ステンレスカラムに充填後、
0.1N水酸化ナトリウムで５分間処理した。中和、
洗浄後、メタノールを溶離液として、ナフタレン
の溶出位置を測定したところ、14.5分（処理前
18.1分）であつた。さらにカラムから抜き出し、
メチルレツドの吸着量を測定したところ、19.6
mg／ｇ（処理前14.2分）であつた。この担体５ｇ
を、実施例１と同様にγ−グリシドキシプロピル
トリメトキシシランで処理し、更に過塩素酸で処
理をした。このようにして得られた担体0.5ｇを
実施例１と同様に、蛋白質を含有する水溶液１ml
に投入し、蛋白質の回収率を調べたところ、98％
であり、テオフイリンは吸着され観測されなかつ
た。
実施例 ５
平均細孔径が100オングストローム、平均粒子
径が10ミクロンのシリカゲル10ｇをトルエン100
ml中に分散し、プロピルトリクロロシラン５ｇを
加え、90℃で24時間反応した。反応液をろ過した
のち、トルエン100ml、アセトン100ml、次いでメ
タノール100mlで洗浄したのち、100℃で４時間乾
燥した。このようにして得られたプロピル基が結
合された逆相担体の元素分析値は、それぞれ炭素
が5.4％、水素が1.2％であつた。この担体を、実
施例１と同様にステンレスカラムに充填し、
0.1N水酸化ナトリウム水溶液で処理をした。中
和、洗浄後、メタノールを溶離液としてナフタレ
ンの溶出位置を測定したところ、10.2分（処理前
13.8分）であつた。さらにカラムから抜き出し、
メチルレツドの吸着量を調べたところ、23.1mg／
ｇ（処理前16.9mg／ｇ）であつた。このようにし
て得られた担体を100℃で４時間乾燥したのち、
実施例１と同様にγ−グリシドキシプロピルトリ
メトキシシランで処理し、更に過塩素酸で処理し
た。この担体0.5ｇを実施例１と同様に蛋白質を
含有する水溶液１mlに投入し蛋白質の回収率を調
べたところ、97.5％であり、テオフイリンは吸着
され観測されなかつた。
実施例 ６
平均細孔径が100オングストローム、平均粒子
径が10ミクロンのシリカゲル10ｇをトルエン100
ml中に分散し、フエニルトリクロロシラン５ｇを
加え、90℃で24時間反応した。反応液をろ過した
のち、トルエン100ml、アセトン100ml、次いでメ
タノール100mlで洗浄したのち、100℃で４時間乾
燥した。このようにして得られたフエニル基を結
合した逆相担体の元素分析値は、それぞれ炭素が
7.8％、水素が1.6％であつた。この担体を、実施
例１と同様にステンレスカラムに充填し、0.1N
水酸化ナトリウム水溶液で５分間処理した。中
和、洗浄後、メタノールを溶離液としてナフタレ
ンの溶出位置を測定したところ、14.8分（処理前
16.5分）であつた。さらにカラムから抜き出し、
メチルレツドの吸着量を調べたところ、17.5mg／
ｇ（処理前10.4mg／ｇ）であつた。このようにし
て得られた担体を100℃で４時間乾燥したのち、
実施例１と同様にγ−グリシドキシプロピルトリ
メトキシシランで処理し、次いで過塩素酸で処理
した。この担体を100℃で４時間乾燥したのち、
この担体0.5ｇを実施例１と同様な蛋白質を含有
する水溶液１mlに投入し、蛋白質の回収率を調べ
たところ、96％であり、一方テオフイリンは吸着
され観測されなかつた。
実施例 ７
実施例１に準じて、平均細孔径が70オングスト
ローム、平均粒子径が10ミクロンのシリカゲル10
ｇにオクタデシル基を導入し、更にステンレスカ
ラムに充填し、加水分解処理をした。この担体５
ｇを、トルエン50ml中に分散し、４−（メチルジ
クロロシリル）ブチリルクロライド2.5ｇを加え
80℃で４時間反応させ、次いでジオキサン50ml
中、アンモニア水５mlを加え、50℃で18時間処理
した。ろ過、洗浄後100℃で４時間乾燥した。こ
のようにして得られた担体0.5ｇを実施例１と同
様に0.5ｇの牛血清アルブミンと1μｇのテオフイ
リンを含有する水溶液１mlに投入し、蛋白質の回
収率を調べたところ、96.5％であり、テオフイリ
ンは吸着され観測されなかつた。
実施例 ８
実施例１で得られたオクタデシル基を導入した
逆相担体を、実施例１に準じてステンレスカラム
に充填し、0.1N水酸化ナトリウム水溶液で処理
した。このようにして得られた担体５ｇを実施例
１と同様にγ−グリシドキシプロピルトリメトキ
シシランで処理したのち、ジオキサン50ml中、三
フツ化ホウ素存在下にソルビトール2.5ｇを60℃
で８時間反応した。この担体0.5ｇを実施例１と
同様に蛋白を含有する水溶液１mlに投入し、蛋白
質の回収率を調べたところ、97％であり、テオフ
イリンは吸着され観測されなかつた。
実施例 ９
実施例１で得られたオクタデシル基を導入した
逆相担体を、実施例１に準じてステンレスカラム
に充填し、0.1N水酸化ナトリウム水溶液で処理
した。このようにして得られた担体を、実施例１
と同様にγ−グリシドキシプロピルトリメトキシ
シランで処理したのち、ジオキサン50ml中、三フ
ツ化ホウ素存在下に、エチレングリコールモノメ
チルエーテル25ｇを加え、60℃で８時間反応し
た。この担体0.5ｇを実施例１と同様に蛋白質を
含有する水溶液１ml中に投入し、蛋白質の回収率
を調べたところ、95％であり、更にテオフイリン
は吸着され観測されなかつた。
実施例 10
実施例１で得られたオクタデシル基を導入した
逆相担体を実施例１に準じてステンレスカラムに
充填し、0.1N水酸化ナトリウム水溶液で処理し
た。このようにして得られた担体を、実施例１と
同様にγ−グリシドキシプロピルトリメトキシシ
ランで処理したのち、ジオキサン50ml中、三フツ
化ホウ素存在下に、ジエチレングリコール2.5ｇ
を加え、60℃で８時間反応した。この担体0.5ｇ
を実施例１と同様に蛋白質を含有する水溶液中に
投入し、蛋白質の回収率を調べたところ、96％で
あり、テオフイリンは吸着され観測されなかつ
た。
実施例 11
平均細孔径が70オングストローム、平均粒子径
が10ミクロンのシリカゲル10ｇをトルエン100ml
中に分散し、オクタデシルトリクロロシラン2.5
ｇとプロピルトリクロロシラン2.5ｇを加え、90
℃で24時間反応した。反応液をろ過したのち、ト
ルエン100ml、アセトン100ml、更にメタノール
100mlで洗浄したのち、70％メタノール水溶液中
で80℃で８時間処理した。このようにして得られ
た担体を100℃で４時間乾燥した。この担体の元
素分析値は、それぞれ炭素が10.3％、水素が1.5
％であつた。この担体を実施例１に準じてステン
レスカラムに充填し、0.1N水酸化ナトリウム水
溶液で処理した。メタノールを用いたナフタレン
の溶出位置は、15.4分（処理前18.1分）であつ
た。更にメチルレツドの吸着量は、17.9mg／ｇ
（処理前11.3mg／ｇ）であつた。このようにして
得られた担体を実施例１と同様にγ−グリシドキ
シプロピルトリメトキシシランで処理し、更に過
塩素酸水溶液中で処理した。このようにして得ら
れた担体0.5ｇを実施例１と同様に、蛋白質を含
有する水溶液中に投入し、蛋白質の回収率を調べ
たところ、97％であり、また、テオフイリンは吸
着され観測されなかつた。
実施例 12
実施例１で得られたオクタデシル基を導入した
逆相担体を、実施例１に準じてアルカリ水溶液で
加水分解し、更にγ−グリシドキシプロピルトリ
メトキシシランで処理した。このようにして得ら
れた担体５ｇをジオキサン中100mlに分散し三フ
ツ化ホウ素存在下に、グリセリン2.5ｇとジエチ
レングリコール2.5ｇを加え、50℃で８時間反応
した。ろ過し純水100mlで洗浄したのち、100℃で
４時間乾燥した。このようにして得られた担体
0.5ｇを実施例１に準じて蛋白質を含有する水溶
液１ml中に分散し蛋白質の回収率を調べたとこ
ろ、97％であり、更にテオフイリンは吸着され観
測されなかつた。
実施例 13
実施例１で得られた担体0.1ｇを血清１ml中に
分散し、10分間攪拌した。担体をろ別後、ろ液を
実施例１と同様に、高速液体クロマトグラフイに
付した。その結果を第２図に示す。Ｃはテオフイ
リン添加標準血清のクロマトグラム、Ｄは本発明
担体で処理したテオフイリン添加標準血清のクロ
マトグラムであり、３は血清蛋白質の吸光度ピー
クであり、２はテオフイリンの吸光度ピークであ
る。ろ液中の蛋白質の280nｍの吸光度から、蛋
白質は99％回収され、一方低分子物質のピークは
観測されず、選択的に吸着された。
実施例 14
実施例２、４及び８で得られた担体0.1ｇを血
清１mlにそれぞれ分散し、実施例９と同様に10分
間攪拌した。担体をろ別後、ろ液を実施例１と同
様に、高速液体クロマトグラフイに付した。
280nｍの吸光度から蛋白質の回収率はそれぞ
れ97、96、及び98％であり、一方、テオフイリン
のピークは観測されず、担体に選択的に吸着され
た。
実施例 15
実施例１で得られた担体を内径4.6mm、長さ25
cmのステンレスカラムにスラリー法により充填し
た。このカラムを高速液体クロマトグラフイに装
着し0.1Nリン酸緩衝液（PH5.0）80体積とメタノ
ール20体積の溶離液でカラムを平衡化し、血清１
ml中にテオフイリン5μｇとカフエイン5μｇを含
有する試料を100μ注入し、紫外吸光度計の
273nｍで検出した。その結果を第３図に示す。
３は血清蛋白質、２はテオフイリン、４はカフエ
インの吸光度ピークである。蛋白質は排除限界位
置（Vo）に溶出し、一方テオフイリン、及びカ
フエインは疎水性相互作用により完全に分離され
た。面積法より求めたテオフイリン、及びカフエ
インの回収率はそれぞれ99％、及び98％であつ
た。
実施例 16
実施例２で得られた担体を実施例15と同様なカ
ラムに充填し、高速液体クロマトグラフイに装着
した。0.05Nリン酸緩衝液（PH4.5）65体積とアセ
トニトリル35体積の溶離液で平衡化し、血清１ml
中にフエニトイン5μｇとフエノバルビタール5μ
ｇを含有する試料を100μ注入し、紫外吸光度
計の220nｍで検出した。その結果を第４図に示
す。それぞれ３は血清蛋白質、５はフエノバルビ
タール、６はフエニトインの吸光度ピークであ
る。蛋白質は排除限界位置に溶出し、フエニトイ
ンとバルビタールは疎水性相互作用により分離さ
れ、それぞれ98.5％、及び98％で回収された。
実施例 17
実施例４で得られた担体を実施例15と同様なカ
ラムに充填し、高速液体クロマトグラフイに装着
した。0.05Nリン酸緩衝液（PH2.5）85体積とアセ
トニトリル15体積の溶離液で平衡化し、血清１ml
中にアンジオテンシン1μｇとアンジオテンシ
ン1μｇを含有する試料100μ注入し、紫外吸
光度計の210nｍで検出した。蛋白質は排除限界
位置に溶出し、一方、アンジオテンシン、及び
アンジオテンシンは疎水性相互作用により完全
に分離し、それぞれ92％、及び90％の回収率であ
つた。
実施例 18
実施例６で得られた担体を実施例15と同様なカ
ラムに充填し、高速液体クロマトグラフイに装着
した。0.1Nリン酸緩衝液（PH5.0）80体積とメタ
ノール20体積の溶離液で平衡化し、血清１ml中に
テオフイリン10μｇ、及びカフエイン10μｇを含
有する試料100μを注入し、紫外吸光度計の
273nｍで検出した。蛋白質は排除限界位置に溶
出し、一方、テオフイリン、及びカフエインは疎
水性相互作用により完全に分離し、それぞれ99
％、及び98％の回収率であつた。
実施例 19
実施例11で得られた担体を実施例15と同様なカ
ラムに充填し、高速液体クロマトグラフイに装着
した。0.1Nリン酸緩衝液（PH5.0）80体積とメタ
ノール20体積の溶離液で平衡化し、血清１ml中に
テオフイリン10μｇ、及びカフエイン10μｇを含
有する試料100μを注入し、紫外吸光度計273n
ｍで検出した。蛋白質は排除限界位置に溶出し、
一方、テオフイリン、及びカフエインは疎水性相
互作用により完全に分離し、それぞれ98％、及び
96％の回収率であつた。
実施例 20
実施例１で得られた担体を内径４mm、長さ５cm
のステンレスカラムにスラリー法により充填し、
試料の前処理用カラムとして、試料前処理装置
（商品名：PT−8000東洋曹達工業製）に装着し
た。分析用カラムにはオクタデシル基が結合され
た逆相充填剤（商品名：TSKgel ODS−120T、
内径4.6mm、長さ25cm、東洋曹達工業製）を用い、
0.05Nリン酸緩衝液（PH2.5）70体積とアセトニト
リル30体積の溶離液を１ml／minの流速で通液
し、平衡化した。前処理用カラムには、0.05Nリ
ン酸緩衝液（PH2.5）を1.5ml／minの流速で通液
した。人血清0.5mlに0.1μｇプロプラノロールを
加えたものを前処理カラムに注入した。５分後、
流路を変更し、分析カラムへ前処理カラムに吸着
した物質を導入し、紫外吸光度計の215nｍで検
出した。面積法によるプロプラノロールの回収率
を調べたところ、98.5％であつた。又、前処理カ
ラムから溶出した溶液をフラクシヨンし、280n
ｍの吸光度による蛋白質の回収率を調べたとこ
ろ、98％であつた。更に、前処理カラムから溶出
されたフラクシヨン100μを、0.2Nリン酸緩衝
液（PH6.8）を溶離液とし、高速液体クロマトグ
ラフイ用充填剤（商品名：TSKgel G2000SW内
径7.5mm、長さ60cm、東洋曹達工業製）に注入し、
紫外吸光度計の215nｍで測定したところ、プロ
プラノロールのピークは観測されなかつた。その
結果を第５図に示す。Ｅはプロプラノロール添加
標準血清のクロマトグラム、Ｆは本発明充填剤で
処理したプロプラノロール添加標準血清のクロマ
トグラムであり、３は血清蛋白質の吸光度ピーク
であり、７はプロプラノロールの吸光度ピークで
ある。
実施例 21
実施例２で得られた担体を実施例20と同様にス
テンレスカラムに充填し、試料の前処理用充填剤
として用いた。人血清0.5ml中に各0.1μｇのアン
ジオテンシンとアンジオテンシンをそれぞれ
加え、実施例20と同様に前処理に付した。試料注
入の５分後、流路を変更し、分析カラムに前処理
カラム吸着物質を導入し、紫外吸光度計の220n
ｍで測定したところ、アンジオテンシン、及び
アンジオテンシンは分離し、面積法による回収
率は、それぞれ98％、及び97％であつた。更に、
前処理カラムからの溶出液をフラクシヨンし、実
施例20と同様にゲルろ過用充填剤で測定したとこ
ろ、アンジオテンシン、及びアンジオテンシン
のピークは観測されなかつた。
実施例 22
実施例４で得られた担体を実施例20と同様にス
テンレスカラムに充填し、試料の前処理用充填剤
として用いた。人血清0.5ml中に各0.1μｇのプロ
プラノロールを加え、実施例20と同様に前処理カ
ラムに付した。前処理５分後、流路を変更し分析
カラムに導入し、紫外吸光度計で検出したとこ
ろ、98％でプロプラノロールが回収された。又、
前処理カラムからの溶出液をフラクシヨンし実施
例20と同様にゲルろ過用カラムで測定したとこ
ろ、プロプラノロールのピークは観測されなかつ
た。
実施例 23
実施例５で得られた担体を実施例20と同様にス
テンレスカラムに充填し、試料の前処理用充填剤
として用いた。人血清0.5ml中に0.1μｇのプロプ
ラノロールを加え、実施例20と同様に前処理カラ
ムに付した。前処理カラムに５分間通液後、流路
を変更し分析カラムに導入し、紫外吸光度計で検
出したところ、97％でプロプラノロールが回収さ
れた。又、前処理カラムからの溶出液をフラクシ
ヨンし、実施例20と同様のゲルろ過用カラムで測
定したところ、プロプラノロールのピークは観測
されなかつた。
実施例 24
実施例７で得られた担体を実施例20と同様にス
テンレスカラムに充填し、試料の前処理用充填剤
として用いた。人血清0.5ml中に0.1μｇのプロプ
ラノロールを加え、実施例20と同様に前処理カラ
ムに付した。前処理カラムに５分間通液後、流路
を変更し分析カラムに導入し、紫外吸光度形で検
出したところ、97.5％でプロプラノロールが回収
された。又、前処理カラムからの溶出液をフラク
シヨンし、実施例20と同様のゲルろ過用カラムで
測定したところ、プロプラノロールのピークは観
測されなかつた。[Formula] (where n: 1 to 4) (3) -(O-CH 2 CH 2 ) p -OH (where p: 1 to 6) (4) A compound having a residue represented by the following formula. Specifically, as a compound that has a hydroxyl group in its molecule,
Polyhydric alcohols such as ethylene glycol, glycerin, and sorbitol; amide groups include lower fatty acid amides such as acetamide chains, propamide chains, and butyramide chains; and compounds with ether groups include polyethylene oxide or its lower chains such as methyl and ethyl. Compounds such as ethers of aliphatic compounds that have a hydrogen-bondable site consisting of an oxygen or nitrogen atom and have low reactivity with proteins are mentioned, but in particular, glycidoxypropyltrimethoxysilane, ethylene, etc. Glycol monomethyl ether, diethylene glycol and the like are preferred. An organic stationary phase with a hydrophobic moiety has a chemical structure with the following general formula: (However, in the formula, R 1 is an aliphatic hydrocarbon group or aromatic hydrocarbon group having 1 to 36 carbon atoms, R 2 and R 3 are a chloro group, an alkoxy group, or a methyl group,
R 4 is a chloro group or an alkoxy group). Specifically, octadecyltrichlorosilane, octyltrichlorosilane, propyltrichlorosilane, phenyltrichlorosilane, actadecylmethyldichlorosilane, octylmethyldichlorosilane, propylmethyldichlorosilane, diphenyldichlorosilane, octadecyldimethylchlorosilane, octyldimethylchlorosilane, Examples include propyldimethylchlorosilane, triphenylchlorosilane , etc., which can easily introduce an organic layer onto the surface of the porous silica particles by reaction with the porous silica particles. 3 ,
and octadecylchlorosilane in which R 4 is a chloro group are preferred. Next, the method for manufacturing the double structure carrier of the present invention will be explained. A method for introducing different functional groups into the outer and inner surfaces of the pores of a porous silica particle carrier is to fill the inside of the pores of the porous silica particles with an appropriate filler material, and then add hydrophilic sites to the outer surface of the pores. A method in which a hydrophilic group is introduced using a silanizing agent having a hydrophobic site, the filling material inside the pores is removed, and a hydrophobic group is subsequently introduced using a silanizing agent having a hydrophobic site. Alternatively, after introducing hydrophobic groups into porous silica particles, the insides of the pores are filled with a suitable filling material, and the hydrophobic groups introduced onto the external surfaces of the pores are cut off. Examples include a method of subsequently introducing hydrophilic groups onto the external surface of the particles after removing the filler material. The latter method, which is simple and effective, will be explained below. Hydrophobic groups can be introduced into porous silica particles by reacting porous silica particles with a silanizing agent having a suitable hydrophobic group. That is,
Porous silica particles are dispersed in an organic solvent and the general formula
Add the silane treatment agent represented by (5), and
Hydrophobic groups can be introduced by reacting at 40°C to 120°C for 1 minute or more. reaction temperature is 20
If it is less than 150°C, the reaction time will be long, and if it exceeds 150°C, the uniform surface coating reaction of the porous silica particles will be hindered, which is not preferable. Further, if the reaction time is less than 1 minute, it is not preferable because a sufficient surface coating reaction cannot be achieved. Considering the economy and efficiency of the reaction, it is preferable to react for 30 minutes to 24 hours. Next, the insides of the pores of the porous silica particles having hydrophobic groups are filled with a suitable filling substance to block the outside of the pores, and then only the siloxane bonds on the outside surface are cut using a suitable chemical substance. Examples of the filler material used include water-insoluble or sparingly water-soluble organic solvents that are well miscible with the porous silica particles. Specifically, aliphatic hydrocarbon compounds such as pentane, hexane, and octane, aromatic hydrocarbon compounds such as benzene, toluene, and xylene, higher alcohols with carbon chains of 6 or more such as hexanol and octanol, or chloroform and carbon tetrachloride. Examples include halogenated hydrocarbon compounds such as, among others, toluene is particularly preferred. In order to fill the inside of the pores of porous silica particles with a filler substance, porous silica particles with hydrophobic groups introduced onto the surface are dispersed in the above organic solvent, and the particles are sufficiently penetrated into the pores by stirring or ultrasonic treatment. How to do it. Examples include a method in which porous silica particles are filled into a chromato tube or the like by a dry method or a wet slurry method, and the organic solvent is passed under static pressure or under pressure to replace the inside of the pores with the organic solvent. However, it is particularly preferable to use a wet slurry method under pressure because the purpose can be achieved in a short time. Porous silica particles whose pores have been substituted with an organic solvent as described above have silosaquine bonds (Si-O
-Si) is treated with a hydrolytic reagent to cleave it. That is, in an acid or alkaline aqueous solution at 20°
The siloxane bonds are hydrolyzed by treatment at ~100°C, preferably 30°-90°C, for a few seconds to 48 hours, preferably 1 minute to 24 hours. Examples of the hydrolysis reagent used here include alkaline aqueous solutions such as potassium hydroxide, sodium hydroxide, and sodium carbonate, and acidic aqueous solutions such as hydrochloric acid, sulfuric acid, and nitric acid. In particular, an alkaline aqueous solution is effective in cleaving siloxane bonds. These hydrolysis reagents are used to break bonds between chemical bonding groups and silica, but cannot penetrate into the pores because the pores of the particles are filled with water-insoluble or poorly water-soluble organic solvents. As a result, hydrolysis of siloxane bonds proceeds only on the external surface of the particles. The time spent on hydrolysis must be properly determined; too short will prevent sufficient hydrolysis of the external surface of the particles, while more than 40 hours will erode the silica skeleton and form porous silica particles. This is undesirable because it results in a decrease in performance and a loss in mechanical strength. Further, if the reaction temperature is less than 20°C, insufficient surface hydrolysis will leave an organic layer, whereas if it exceeds 100°C, rapid hydrolysis will occur and the silica skeleton will be eroded, which is not preferable. Porous silica particles that have been subjected to hydrolysis treatment for a certain period of time must be quickly neutralized with acid or alkali. Reagents used for neutralization include dilute aqueous solutions such as hydrochloric acid, sulfuric acid, nitric acid, etc. when the siloxane bond hydrolysis reagent is an alkaline aqueous solution.
This can be achieved by passing through an aqueous solution of an organic acid such as acetic acid or, if the hydrolysis reagent used is an acidic aqueous solution, a dilute aqueous solution such as potassium hydroxide or sodium hydroxide, or by pumping the solution under pressure. be able to. As the reagent used for neutralization, it is preferable to select, for example, an acid-alkali pair that easily dissolves the by-product salt when it is washed with water, such as sodium acetate. The porous silica particles obtained as described above are
The particles have silanol groups generated by a hydrolysis reaction on their outer surfaces, and the inner surfaces of the pores of the particles have a structure that retains the original hydrophobic groups. The silanol groups newly generated on the external surface of the particles are treated with a compound having a residue represented by the general formulas (1) to (4) described above in an inert organic solvent at 20 to 90°C for 30 minutes to 24 hours. By doing so, inert hydrophilic groups are introduced into the water-soluble polymeric substance. In the above method, the above operation can be simplified by using a reversed phase carrier having a high adsorption capacity for hydrophobic biological substances. The reversed-phase carrier has hydrophobic chemical bonding groups uniformly bonded to its surface, and therefore has a high ability to adsorb hydrophobic substances. In porous silica particles, the degree of coverage of hydrophobic groups on the outer surface of the pores is extremely small compared to the inner surface of the pores. This fact suggests that the contribution of the hydrophobic groups chemically bonded to the external surface of the particles to the adsorption capacity is small. In other words, the carrier obtained by selectively removing the hydrophobic groups chemically bonded only to the external surface of the particles shows only a small change in its performance and retains the performance as the original reversed-phase carrier. Conceivable. Therefore, the reversed-phase support obtained by subjecting the reversed-phase support to an appropriate surface treatment and cleaving the hydrophobic groups chemically bonded to the external surface of the particles has the ability to absorb the silanol groups newly generated on the external surface of the pores. By subsequently treating this carrier in the same manner with a suitable silane treatment agent represented by the above-mentioned general formulas (1) to (4),
Different functional groups can be introduced onto the external surface of the surface-treated carrier. That is, as a method for surface treatment of a reversed phase support, for example, after dispersing it in an organic solvent as described above, it is filtered on a glass filter, and then the silica base material and the hydrophobic group chemically bonded to the outside of the particles are quickly separated. A method of passing a reagent that hydrolyzes the bond of However, a treatment method under pressure is particularly effective as it can achieve the objective in a short time. Next, it is treated in the same manner with a compound having a residue represented by the above general formulas (1) to (4). The thus obtained double-structured carrier having hydrophilic sites on the external surface of the particles is inactive against water-soluble polymer substances that cannot penetrate into the pores, while being inactive against low-molecular substances. By capturing the particles with hydrophobic groups inside the pores, it exhibits adsorption performance comparable to that of conventional reversed-phase supports. As described above, the present invention includes a step of introducing a hydrophobic group into porous silica particles, a step of replacing the inside of the pores of the porous silica particles with a water-insoluble or poorly water-soluble organic solvent, a hydrolysis step, and a silane treatment. Through this step, it is possible to selectively introduce hydrophilic groups only to the outer surface of the particles, and to obtain a double-structured carrier having hydrophobic groups on the inner surfaces of the pores of the particles. Furthermore, a similar double-structured support can be obtained without reducing the performance of existing reversed-phase supports. Effects of the Invention The field of application of the double-structured carrier of the present invention is, for example, by adding and mixing the carrier of the present invention into an aqueous solution containing both a high-molecular substance and a low-molecular substance, and removing harmful substances or interfering substances from the aqueous solution. It is possible to adsorb and remove low molecular weight substances and recover the desired high molecular weight substances. For example, blood purification of patients by classifying protein metabolites and harmful substances of low and middle molecular weight in the blood and high molecular weight substances such as proteins, or removal of contaminant low molecular weight substances in the manufacturing process of biopolymer substances such as pharmaceuticals. etc. Further, after removing the high molecular weight substances as interfering substances, the low molecular weight substances captured by the filler of the present invention can be subsequently extracted and recovered with a suitable organic solvent such as methanol, acetonitrile, etc. For example, recovery of pharmaceuticals, biologically related metabolites, neurotransmitters, hormones, hormone-like substances, etc. from biological fluids or homogenate extracts that exist at extremely low levels, or recovery of antibiotics and physiological substances secreted from bacterial cells. Examples include recovery of active substances, etc. On the other hand, in recent years, liquid chromatography has come to be widely used as a useful means for separating and analyzing biological components, especially as the sensitivity has increased. Liquid chromatography has been used to analyze biological components because it is quick and suitable for routine work, but when analyzing samples containing polymeric substances, sample pretreatment such as deproteinization is required. This complicates the process, and furthermore, there are problems with the reproducibility of analysis results. Since the carrier of the present invention retains the performance of the original reversed-phase carrier, it can be used as a normal analytical column by being packed in a column for liquid chromatography. In other words, this carrier has an organic stationary phase with hydrophilic sites chemically bonded to the outside of its pores, making it inert to polymeric substances, allowing polymeric substances such as proteins to pass through the column without any problem. Low molecular weight substances can be separated and analyzed in normal reversed phase mode. As a result, routine work and rapid measurement of specimens can be performed, and measurement errors can be minimized, making it particularly useful for analyzing pharmaceuticals and their metabolites in biological fluids. Furthermore, the support of the present invention can be used as a concentration column for the analysis of low molecular weight substances present in biological fluids at low levels. That is, a large amount of a sample sample containing a trace amount of a target low-molecular substance is passed through the carrier of the present invention packed in a concentration column, and while the high-molecular substance is removed, the low-molecular substance is transferred into the pores of the packing material. In this method, the target low-molecular substance concentrated with an appropriate eluent composition is introduced into the main column and analyzed using a column switching method. For example, it can be used to monitor pharmaceuticals that exhibit medicinal efficacy at low levels, to analyze bioactive peptides that exist in trace amounts in the brain and organs, and to concentrate and separate biologically relevant metabolites. Can be used to analyze specimen samples. As described above, the carrier of the present invention can be placed into an aqueous solution containing both a high molecular weight substance and a low molecular weight substance, or packed into a column for liquid chromatography, and then an analyte sample can be injected into the column. By filling the column and using it as a concentration column, it is possible to selectively separate and recover the target high-molecular substance or low-molecular substance from the target sample solution, and if necessary, the target analysis method can be used. can be attached to Examples Example 1 10 g of silica gel with an average pore size of 70 angstroms and an average particle size of 10 microns is added to 100 ml of toluene.
50g of octadecyltrichlorosilane dispersed in
was added and reacted for 24 hours in an oil bath kept at 90°C. Filter the reaction solution and add 100ml of toluene and acetone.
100ml was washed. The carrier thus obtained was dispersed in a 70% methanol aqueous solution and treated at 60° C. for 6 hours to hydrolyze the remaining chloro groups. After filtering the carrier, wash it with 100ml of methanol,
It was dried at 100°C for 4 hours. The elemental analysis values of the support thus obtained were 13.5% carbon and 2.1% hydrogen.
It was %. This reversed phase carrier has an inner diameter of 7.5 mm and a length of
A 40% ethanol slurry was prepared in a 30 cm stainless steel column, and filled by the slurry method using ethanol as the filling solvent. After replacing the inside of the column with toluene, a 0.1N aqueous sodium hydroxide solution was passed through the column at a flow rate of 1 ml/min for 5 minutes.
Quickly add 0.1% acetic acid aqueous solution at a flow rate of 1ml/min for 10 minutes.
The solution was passed for a minute. Furthermore, pure water was added at a flow rate of 1 ml/min.
After passing through the solution for 10 minutes, the solution was equilibrated with methanol, and the elution position of naphthalene was examined by ultraviolet absorption (wavelength 254 nm) using methanol as an eluent, and it was found to be 20.5 minutes (23.4 minutes before treatment). Furthermore, when the amount of methyl red adsorbed on the gel extracted from the column was examined, it was 20.3 mg/g (14.9 mg/g before treatment). Disperse 5 g of this gel in 50 ml of toluene, add 5 g of γ-glycidoxypropyltrimethoxysilane, react at 80°C for 8 hours, separate the filtrate, wash with 50 ml of toluene, 50 ml of acetone, and further 50 ml of methanol. After adjusting the pH to 2.5 with perchloric acid in 100 ml of pure water, the mixture was treated at 90°C for 4 hours. After filtration, the mixture was washed with methanol and dried at 100°C for 4 hours. The carrier thus obtained
0.5 g was added to 1 ml of an aqueous solution containing 0.5 g of bovine serum albumin and 1 μg of theophylline, and the mixture was stirred for 5 minutes. After filtration, add the filtrate to 0.2N phosphate buffer (PH6.8)
High-speed gel filtration chromatography column (product name: TSKgel G2000SW, inner diameter 7.5 mm,
60cm long, attached to Toyo Soda Kogyo). 280nm
Figure 1 shows the chromatogram detected. A
is a chromatogram of a solution of bovine serum albumin added with theophylline, B is a chromatogram of a solution treated with the carrier of the present invention, 1 is bovine serum albumin, 2 is a chromatogram of a solution treated with the carrier of the present invention.
is the absorbance peak of theophylline. When the absorbance at 280 nm of the protein in the filtrate was examined, the recovery rate was 99% before adding the carrier of the present invention. On the other hand, theophylline was completely adsorbed onto the carrier. Example 2 10 g of silica gel having an average pore diameter of 150 angstroms and an average particle diameter of 15 microns was treated with a silane treatment agent having an octadecyl group according to Example 1. The elemental analysis values of the carrier thus obtained were 15.9% carbon and 2.3% hydrogen. This reversed phase support having octadecyl groups bonded thereto was packed into a stainless steel column according to Example 1, replaced with toluene, and then treated with 0.1N sodium hydroxide for 5 minutes. When the elution position of naphthalene was measured using methanol as an eluent, it was found to be 18.8 minutes (20.9 minutes before treatment).
minute). Furthermore, when the adsorption amount of methyl red was extracted from the column, it was found to be 16.3 mg/g.
(10.1 mg/g before treatment). This carrier was treated with γ-glycidoxypropyltrimethoxysilane as in Example 1, and further treated with perchloric acid. As in Example 1, 0.5 g of this carrier was added to 1 ml of an aqueous solution containing 0.5 g of bovine serum albumin and 1 μg of theophylline, and the protein recovery rate was 99%, indicating that theophylline was adsorbed and observed. Nakatsuta. Example 3 10 g of silica gel having an average pore diameter of 60 angstroms and an average particle diameter of 500 microns was treated with octadecyltrichlorosilane in the same manner as in Example 1. The elemental analysis values of the carrier thus obtained were 12.2% carbon and 2.1% hydrogen. This carrier was packed into a stainless steel column according to Example 1, and treated with 0.1N sodium hydroxide for 5 minutes. After neutralization and washing, the elution position of naphthalene was measured using methanol as an eluent.
It was 19.2 minutes (22.6 minutes before treatment). Furthermore, when it was extracted from the column and the adsorption amount of methyl red was measured, it was 15.2 mg/g (11.1 mg/g before treatment). 5 g of this carrier was treated with γ-glycidoxypropyltrimethoxysilane in the same manner as in Example 1, and further treated with perchloric acid. In the same manner as in Example 1, 0.5 g of the carrier thus obtained was added to 1 ml of an aqueous solution containing protein, and the protein recovery rate was 97%, indicating that theophylline was adsorbed and was not observed. . Example 4 10 g of silica gel with an average pore size of 100 angstroms and an average particle size of 10 microns was mixed with 10 g of toluene.
ml, added 5 g of octyltrichlorosilane, and reacted at 90°C for 24 hours. Filter the reaction solution,
The carrier was washed with 100 ml of toluene, 100 ml of acetone, and then with 100 ml of methanol, and dried at 100° C. for 4 hours. The elemental analysis values of the reversed-phase support to which octyl groups were bonded were as follows:
9.5%, and hydrogen 1.4%. After filling this carrier into a stainless steel column according to Example 1,
Treated with 0.1N sodium hydroxide for 5 minutes. neutralization,
After washing, we measured the elution position of naphthalene using methanol as an eluent and found that it was 14.5 minutes (before treatment).
18.1 minutes). Furthermore, extract it from the column,
When the adsorption amount of methyl red was measured, it was 19.6
mg/g (14.2 minutes before treatment). 5g of this carrier
was treated with γ-glycidoxypropyltrimethoxysilane in the same manner as in Example 1, and further treated with perchloric acid. In the same manner as in Example 1, 0.5 g of the carrier thus obtained was added to 1 ml of an aqueous solution containing protein.
When we investigated the protein recovery rate, it was 98%.
Theophylline was adsorbed and was not observed. Example 5 10 g of silica gel with an average pore size of 100 angstroms and an average particle size of 10 microns was mixed with 10 g of toluene.
ml, 5 g of propyltrichlorosilane was added, and the mixture was reacted at 90°C for 24 hours. After filtering the reaction solution, it was washed with 100 ml of toluene, 100 ml of acetone, then 100 ml of methanol, and then dried at 100°C for 4 hours. The elemental analysis values of the thus obtained reverse phase carrier to which propyl groups were bonded were 5.4% carbon and 1.2% hydrogen. This carrier was packed into a stainless steel column in the same manner as in Example 1,
It was treated with a 0.1N aqueous sodium hydroxide solution. After neutralization and washing, the elution position of naphthalene was measured using methanol as an eluent, and the result was 10.2 minutes (before treatment).
13.8 minutes). Furthermore, extract it from the column,
When we investigated the adsorption amount of Methyl Red, it was found to be 23.1mg/
g (16.9 mg/g before treatment). After drying the carrier thus obtained at 100°C for 4 hours,
It was treated with γ-glycidoxypropyltrimethoxysilane in the same manner as in Example 1, and further treated with perchloric acid. When 0.5 g of this carrier was added to 1 ml of a protein-containing aqueous solution in the same manner as in Example 1 and the protein recovery rate was examined, it was 97.5%, and theophylline was adsorbed and was not observed. Example 6 10 g of silica gel with an average pore size of 100 angstroms and an average particle size of 10 microns was mixed with 10 g of toluene.
ml, added 5 g of phenyltrichlorosilane, and reacted at 90°C for 24 hours. After filtering the reaction solution, it was washed with 100 ml of toluene, 100 ml of acetone, then 100 ml of methanol, and then dried at 100°C for 4 hours. The elemental analysis values of the thus obtained reversed-phase carrier bonded with phenyl groups show that carbon
7.8%, and hydrogen 1.6%. This carrier was packed into a stainless steel column in the same manner as in Example 1, and 0.1N
It was treated with an aqueous sodium hydroxide solution for 5 minutes. After neutralization and washing, the elution position of naphthalene was measured using methanol as an eluent, and the result was 14.8 minutes (before treatment).
16.5 minutes). Furthermore, extract it from the column,
When we investigated the adsorption amount of Methyl Red, it was found to be 17.5mg/
g (10.4 mg/g before treatment). After drying the carrier thus obtained at 100°C for 4 hours,
It was treated with γ-glycidoxypropyltrimethoxysilane in the same manner as in Example 1, and then with perchloric acid. After drying this carrier at 100°C for 4 hours,
When 0.5 g of this carrier was added to 1 ml of an aqueous solution containing the same protein as in Example 1 and the protein recovery rate was examined, it was 96%, while theophylline was adsorbed and was not observed. Example 7 According to Example 1, silica gel 10 with an average pore size of 70 angstroms and an average particle size of 10 microns was prepared.
An octadecyl group was introduced into g, which was then packed into a stainless steel column and subjected to hydrolysis treatment. This carrier 5
Disperse g in 50 ml of toluene and add 2.5 g of 4-(methyldichlorosilyl)butyryl chloride.
React at 80℃ for 4 hours, then add 50ml of dioxane.
In the middle, 5 ml of ammonia water was added, and the mixture was treated at 50°C for 18 hours. After filtering and washing, it was dried at 100°C for 4 hours. 0.5 g of the carrier obtained in this way was poured into 1 ml of an aqueous solution containing 0.5 g of bovine serum albumin and 1 μg of theophylline in the same manner as in Example 1, and the protein recovery rate was determined to be 96.5%. Theophylline was adsorbed and was not observed. Example 8 The reverse phase carrier into which octadecyl groups were introduced obtained in Example 1 was packed into a stainless steel column according to Example 1, and treated with a 0.1N aqueous sodium hydroxide solution. After treating 5 g of the carrier thus obtained with γ-glycidoxypropyltrimethoxysilane in the same manner as in Example 1, 2.5 g of sorbitol was added in 50 ml of dioxane at 60°C in the presence of boron trifluoride.
It reacted for 8 hours. 0.5 g of this carrier was added to 1 ml of a protein-containing aqueous solution in the same manner as in Example 1, and the protein recovery rate was determined to be 97%, indicating that theophylline was adsorbed and was not observed. Example 9 The reverse phase carrier into which octadecyl groups were introduced obtained in Example 1 was packed into a stainless steel column according to Example 1, and treated with a 0.1N aqueous sodium hydroxide solution. The carrier thus obtained was prepared in Example 1.
After treating with γ-glycidoxypropyltrimethoxysilane in the same manner as above, 25 g of ethylene glycol monomethyl ether was added in 50 ml of dioxane in the presence of boron trifluoride, and the mixture was reacted at 60° C. for 8 hours. 0.5 g of this carrier was poured into 1 ml of a protein-containing aqueous solution in the same manner as in Example 1, and the protein recovery rate was determined to be 95%. Furthermore, theophylline was adsorbed and was not observed. Example 10 The reverse phase carrier into which octadecyl groups were introduced obtained in Example 1 was packed into a stainless steel column according to Example 1, and treated with a 0.1N aqueous sodium hydroxide solution. The carrier thus obtained was treated with γ-glycidoxypropyltrimethoxysilane in the same manner as in Example 1, and then 2.5 g of diethylene glycol was added in 50 ml of dioxane in the presence of boron trifluoride.
was added and reacted at 60°C for 8 hours. 0.5g of this carrier
was poured into an aqueous solution containing protein in the same manner as in Example 1, and the protein recovery rate was determined to be 96%, and theophylline was adsorbed and was not observed. Example 11 10 g of silica gel with an average pore size of 70 angstroms and an average particle size of 10 microns was added to 100 ml of toluene.
Dispersed in octadecyltrichlorosilane 2.5
g and 2.5 g of propyltrichlorosilane, 90
The reaction was carried out at ℃ for 24 hours. After filtering the reaction solution, add 100ml of toluene, 100ml of acetone, and then methanol.
After washing with 100 ml, it was treated in a 70% methanol aqueous solution at 80°C for 8 hours. The carrier thus obtained was dried at 100°C for 4 hours. The elemental analysis values of this carrier are 10.3% carbon and 1.5% hydrogen, respectively.
It was %. This carrier was packed into a stainless steel column according to Example 1, and treated with a 0.1N aqueous sodium hydroxide solution. The elution position of naphthalene using methanol was 15.4 minutes (18.1 minutes before treatment). Furthermore, the adsorption amount of methyl red is 17.9mg/g.
(11.3 mg/g before treatment). The carrier thus obtained was treated with γ-glycidoxypropyltrimethoxysilane in the same manner as in Example 1, and further treated in an aqueous perchloric acid solution. As in Example 1, 0.5 g of the carrier thus obtained was poured into an aqueous solution containing protein, and the protein recovery rate was 97%, and theophylline was observed to be adsorbed. Nakatsuta. Example 12 The reverse phase carrier into which octadecyl groups were introduced obtained in Example 1 was hydrolyzed with an aqueous alkaline solution in the same manner as in Example 1, and further treated with γ-glycidoxypropyltrimethoxysilane. 5 g of the carrier thus obtained was dispersed in 100 ml of dioxane, 2.5 g of glycerin and 2.5 g of diethylene glycol were added in the presence of boron trifluoride, and the mixture was reacted at 50° C. for 8 hours. After filtering and washing with 100 ml of pure water, it was dried at 100°C for 4 hours. The carrier thus obtained
When 0.5 g was dispersed in 1 ml of an aqueous solution containing protein according to Example 1 and the recovery rate of protein was examined, it was 97%, and furthermore, theophylline was adsorbed and was not observed. Example 13 0.1 g of the carrier obtained in Example 1 was dispersed in 1 ml of serum and stirred for 10 minutes. After filtering off the carrier, the filtrate was subjected to high performance liquid chromatography in the same manner as in Example 1. The results are shown in FIG. C is a chromatogram of theophylline-added standard serum, D is a chromatogram of theophylline-added standard serum treated with the carrier of the present invention, 3 is the absorbance peak of serum protein, and 2 is the absorbance peak of theophylline. From the absorbance of the protein in the filtrate at 280 nm, 99% of the protein was recovered, while no peak of low molecular weight substances was observed, indicating that it was selectively adsorbed. Example 14 0.1 g of the carriers obtained in Examples 2, 4, and 8 were each dispersed in 1 ml of serum, and stirred for 10 minutes in the same manner as in Example 9. After filtering off the carrier, the filtrate was subjected to high performance liquid chromatography in the same manner as in Example 1. The recovery rates of the protein based on the absorbance at 280 nm were 97%, 96%, and 98%, respectively, while no theophylline peak was observed, indicating that it was selectively adsorbed onto the carrier. Example 15 The carrier obtained in Example 1 was prepared with an inner diameter of 4.6 mm and a length of 25 mm.
A cm stainless steel column was packed using the slurry method. This column was attached to a high-performance liquid chromatograph, and the column was equilibrated with an eluent of 80 volumes of 0.1N phosphate buffer (PH5.0) and 20 volumes of methanol.
Inject 100 μg of a sample containing 5 μg of theophylline and 5 μg of caffeine per ml, and measure using an ultraviolet absorbance meter.
Detected at 273nm. The results are shown in FIG.
3 is the absorbance peak of serum protein, 2 is theophylline, and 4 is the absorbance peak of caffein. The protein eluted at the exclusion limit (Vo), while theophylline and caffeine were completely separated by hydrophobic interactions. The recovery rates of theophylline and caffein determined by the area method were 99% and 98%, respectively. Example 16 The carrier obtained in Example 2 was packed into a column similar to that in Example 15, and mounted on a high performance liquid chromatograph. Equilibrate with 65 volumes of 0.05N phosphate buffer (PH4.5) and 35 volumes of acetonitrile, and remove 1 ml of serum.
Inside is 5μg of phenytoin and 5μg of phenobarbital.
A 100μ sample containing g was injected and detected at 220 nm using an ultraviolet absorbance meter. The results are shown in FIG. 3 is the absorbance peak of serum protein, 5 is the absorbance peak of phenobarbital, and 6 is the absorbance peak of phenytoin. The protein was eluted at the exclusion limit position, and phenytoin and barbital were separated by hydrophobic interactions and recovered at 98.5% and 98%, respectively. Example 17 The carrier obtained in Example 4 was packed into a column similar to that in Example 15, and mounted on a high performance liquid chromatograph. Equilibrate the eluent with 85 volumes of 0.05N phosphate buffer (PH2.5) and 15 volumes of acetonitrile, and remove 1 ml of serum.
100 μg of angiotensin and 100 μg of a sample containing 1 μg of angiotensin were injected into the tube, and the samples were detected using an ultraviolet absorbance meter at 210 nm. The protein was eluted at the exclusion limit position, while angiotensin and angiotensin were completely separated by hydrophobic interactions, with recoveries of 92% and 90%, respectively. Example 18 The carrier obtained in Example 6 was packed into a column similar to that in Example 15, and mounted on a high performance liquid chromatograph. Equilibrate with an eluent of 80 volumes of 0.1N phosphate buffer (PH5.0) and 20 volumes of methanol, inject 100 μg of a sample containing 10 μg of theophylline and 10 μg of caffein in 1 ml of serum, and analyze the sample using an ultraviolet absorbance meter.
Detected at 273nm. The protein elutes at the exclusion limit position, while theophylline and caffeine are completely separated due to hydrophobic interactions, each at 99
%, and the recovery rate was 98%. Example 19 The carrier obtained in Example 11 was packed into a column similar to that in Example 15, and mounted on a high performance liquid chromatograph. Equilibrated with an eluent of 80 volumes of 0.1N phosphate buffer (PH5.0) and 20 volumes of methanol, injected 100 μg of a sample containing 10 μg of theophylline and 10 μg of caffein in 1 ml of serum, and measured with an ultraviolet absorbance meter at 273 nm.
Detected at m. The protein elutes at the exclusion limit position,
On the other hand, theophylline and caffein were completely separated due to hydrophobic interactions, with 98% and 98%, respectively.
The recovery rate was 96%. Example 20 The carrier obtained in Example 1 was prepared with an inner diameter of 4 mm and a length of 5 cm.
Packed into a stainless steel column using the slurry method,
It was attached to a sample pretreatment device (trade name: PT-8000, manufactured by Toyo Soda Kogyo) as a sample pretreatment column. The analytical column uses a reversed phase packing material with octadecyl groups (product name: TSKgel ODS-120T,
(inner diameter 4.6 mm, length 25 cm, manufactured by Toyo Soda Kogyo),
An eluent containing 70 volumes of 0.05N phosphate buffer (PH2.5) and 30 volumes of acetonitrile was passed through the tube at a flow rate of 1 ml/min for equilibration. A 0.05N phosphate buffer (PH2.5) was passed through the pretreatment column at a flow rate of 1.5 ml/min. A mixture of 0.5 ml of human serum and 0.1 μg propranolol was injected into the pretreatment column. 5 minutes later,
The flow path was changed, and the substance adsorbed on the pretreatment column was introduced into the analytical column, and detected using an ultraviolet absorbance meter at 215 nm. The recovery rate of propranolol was examined using the area method and was found to be 98.5%. In addition, the solution eluted from the pretreatment column was fractionated and 280n
The protein recovery rate based on the absorbance of m was 98%. Furthermore, 100μ of the fraction eluted from the pretreatment column was added to a high-performance liquid chromatography packing material (trade name: TSKgel G2000SW, inner diameter 7.5 mm, length 60 cm) using 0.2N phosphate buffer (PH6.8) as the eluent. , manufactured by Toyo Soda Kogyo),
When measured using an ultraviolet absorbance spectrometer at 215 nm, no propranolol peak was observed. The results are shown in FIG. E is a chromatogram of propranolol-added standard serum, F is a chromatogram of propranolol-added standard serum treated with the packing material of the present invention, 3 is the absorbance peak of serum protein, and 7 is the absorbance peak of propranolol. Example 21 The carrier obtained in Example 2 was packed into a stainless steel column in the same manner as in Example 20, and used as a packing material for sample pretreatment. Angiotensin and angiotensin (0.1 μg each) were added to 0.5 ml of human serum and subjected to pretreatment in the same manner as in Example 20. Five minutes after sample injection, the flow path was changed, the pretreatment column adsorbent was introduced into the analytical column, and the 220n
When measured at m, angiotensin and angiotensin were separated, and the recovery rates by area method were 98% and 97%, respectively. Furthermore,
When the eluate from the pretreatment column was fractionated and measured using a gel filtration packing material in the same manner as in Example 20, angiotensin and angiotensin peaks were not observed. Example 22 The carrier obtained in Example 4 was packed into a stainless steel column in the same manner as in Example 20, and used as a packing material for sample pretreatment. Each 0.1 μg of propranolol was added to 0.5 ml of human serum and applied to a pretreatment column in the same manner as in Example 20. After 5 minutes of pretreatment, the flow path was changed and the column was introduced into an analytical column. Propranolol was detected with an ultraviolet absorbance meter, and 98% of propranolol was recovered. or,
When the eluate from the pretreatment column was fractionated and measured using a gel filtration column in the same manner as in Example 20, no propranolol peak was observed. Example 23 The carrier obtained in Example 5 was packed into a stainless steel column in the same manner as in Example 20, and used as a packing material for sample pretreatment. 0.1 μg of propranolol was added to 0.5 ml of human serum, and the mixture was applied to a pretreatment column in the same manner as in Example 20. After passing the solution through the pretreatment column for 5 minutes, the flow path was changed and the solution was introduced into an analytical column. Propranolol was detected at a rate of 97% using an ultraviolet absorbance spectrometer. Furthermore, when the eluate from the pretreatment column was fractionated and measured using the same gel filtration column as in Example 20, no propranolol peak was observed. Example 24 The carrier obtained in Example 7 was packed into a stainless steel column in the same manner as in Example 20, and used as a packing material for sample pretreatment. 0.1 μg of propranolol was added to 0.5 ml of human serum, and the mixture was applied to a pretreatment column in the same manner as in Example 20. After passing the solution through the pretreatment column for 5 minutes, the flow path was changed and the solution was introduced into an analytical column. When detected using ultraviolet absorbance, 97.5% of propranolol was recovered. Furthermore, when the eluate from the pretreatment column was fractionated and measured using the same gel filtration column as in Example 20, no propranolol peak was observed.
第１図は、実施例１におけるテオフイリン添加
牛血清アルブミン溶液のクロマトグラム、第２図
は実施例13におけるテオフイリン添加標準血清の
クロマトグラム、第３図は実施例15におけるテオ
フイリン、及びカフエイン添加の標準血清のクロ
マトグラム、第４図は、実施例16におけるバルビ
タール、及びフエノバルビタール添加の標準血清
のクロマトグラム、第５図は実施例20におけるプ
ロプラノロール添加標準血清のクロマトグラムで
ある。
１……牛血清アルブミン、２……テオフイリ
ン、３……血清蛋白質、４……カフエイン、５…
…フエノバルビタール、６……フエニトイン、７
……プロプラノロール。
Figure 1 is a chromatogram of the theophylline-added bovine serum albumin solution in Example 1, Figure 2 is the chromatogram of the theophylline-added standard serum in Example 13, and Figure 3 is the theophylline- and caffein-added standard in Example 15. 4 is a chromatogram of the standard serum added with barbital and phenobarbital in Example 16, and FIG. 5 is a chromatogram of the standard serum added with propranolol in Example 20. 1...Bovine serum albumin, 2...Theophylline, 3...Serum protein, 4...Caffein, 5...
…Fenobarbital, 6…Phenytoin, 7
...Propranolol.
Claims (1)
細孔内部表面が疎水性部位を有する有機固定相で
化学結合された平均細孔径が10〜200Å、平均粒
子径が0.1〜1000μの多孔質シリカ粒子であること
を特徴とする二重構造担体。 ２ 親水性部位を有する有機固定相が、一般式 【式】（但し、ｌ：１〜 ６） (1) −（Ｏ−CH2CH2）n−OCH3（但し、ｍ：１〜６）
(2) 【式】（但し、ｎ：１〜４） (3) −（Ｏ−CH2CH2）p−OH（但し、ｐ：１〜６）(4) で表わされる残基の少なくとも一種よりなる化合
物である特許請求の範囲第１項記載の二重構造担
体。 ３ 疎水性部位を有する有機固定相が、一般式 （但し、R1：炭素数が１から36までの脂肪族炭
化水素基又は芳香族炭化水素基、R2、R3：クロ
ロ基又はアルコキシ基あるいはメチル基、R4：
クロロ基又はアルコキシ基である）で表わされる
シラン処理剤である特許請求の範囲第１項記載の
二重構造担体。[Claims] 1. An organic stationary phase whose external surface has a hydrophilic site;
1. A double-structured carrier comprising porous silica particles having an average pore diameter of 10 to 200 Å and an average particle diameter of 0.1 to 1000 μ, the inner surface of the pores being chemically bonded with an organic stationary phase having hydrophobic sites. 2 The organic stationary phase having a hydrophilic moiety has the general formula [formula] (where l: 1 to 6) (1) -(O-CH 2 CH 2 ) n -OCH 3 (however, m: 1 to 6)
(2) [Formula] (however, n: 1 to 4) (3) -(O-CH 2 CH 2 ) p -OH (however, p: 1 to 6) At least one type of residue represented by (4) The double structure carrier according to claim 1, which is a compound consisting of. 3 The organic stationary phase having a hydrophobic moiety has the general formula (However, R 1 : aliphatic hydrocarbon group or aromatic hydrocarbon group having 1 to 36 carbon atoms, R 2 , R 3 : chloro group, alkoxy group, or methyl group, R 4 :
The double structure carrier according to claim 1, which is a silanizing agent represented by a chloro group or an alkoxy group.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60127230A JPS61287444A (en) | 1985-06-13 | 1985-06-13 | Double structural carrier |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60127230A JPS61287444A (en) | 1985-06-13 | 1985-06-13 | Double structural carrier |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS61287444A JPS61287444A (en) | 1986-12-17 |
| JPH0338891B2 true JPH0338891B2 (en) | 1991-06-12 |
Family
ID=14954945
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP60127230A Granted JPS61287444A (en) | 1985-06-13 | 1985-06-13 | Double structural carrier |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS61287444A (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63163273A (en) * | 1986-12-26 | 1988-07-06 | Shimadzu Corp | Packing material for liquid chromatography |
| US4778600A (en) * | 1987-06-17 | 1988-10-18 | Dow Corning Corporation | Liquid chromatography dual zone packing materials |
| US5599625A (en) * | 1992-06-17 | 1997-02-04 | Research Corporation Technologies, Inc. | Products having multiple-substituted polysiloxane monolayer |
| JP2000111535A (en) * | 1998-09-30 | 2000-04-21 | Neos Co Ltd | Filler for liquid chromatography |
| JP3995935B2 (en) * | 2000-02-10 | 2007-10-24 | エーザイ・アール・アンド・ディー・マネジメント株式会社 | Chromatographic packing material |
| DE10045434B4 (en) * | 2000-09-14 | 2005-07-14 | Fresenius Hemocare Gmbh | Adsorbent with differently modified surface areas, process for its preparation and use thereof |
| JP2007000687A (en) * | 2005-06-21 | 2007-01-11 | Shimadzu Corp | Method and apparatus for analyzing peptide in biological sample |
| JP4775714B2 (en) * | 2006-11-27 | 2011-09-21 | 株式会社島津製作所 | Protein identification method and protein identification apparatus |
| JP5154973B2 (en) * | 2008-02-20 | 2013-02-27 | 富士シリシア化学株式会社 | Silica gel, chromatographic apparatus, separation method, and method for producing silica gel |
| EP2268393A4 (en) | 2008-04-22 | 2013-08-14 | Ge Healthcare Bio Sciences Ab | Chromatography medium |
| JP4729649B1 (en) * | 2010-12-10 | 2011-07-20 | 株式会社バイオレドックス研究所 | Hydrogen gas-containing calcium carbonate and method for producing the same |
| JP2020516901A (en) * | 2017-04-14 | 2020-06-11 | 株式会社島津製作所 | Detection method and detection device |
| JP7150393B2 (en) * | 2018-06-06 | 2022-10-11 | 国立大学法人九州大学 | Method for treating porous body, adsorbent produced by method for treating porous body, and porous body |
-
1985
- 1985-06-13 JP JP60127230A patent/JPS61287444A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS61287444A (en) | 1986-12-17 |
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