JP5458239B2 - 分離方法 - Google Patents
分離方法 Download PDFInfo
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- JP5458239B2 JP5458239B2 JP2009221301A JP2009221301A JP5458239B2 JP 5458239 B2 JP5458239 B2 JP 5458239B2 JP 2009221301 A JP2009221301 A JP 2009221301A JP 2009221301 A JP2009221301 A JP 2009221301A JP 5458239 B2 JP5458239 B2 JP 5458239B2
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- phospholipid
- calcium
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- eluate
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- 238000000926 separation method Methods 0.000 title claims description 46
- 150000003904 phospholipids Chemical class 0.000 claims description 142
- 239000011575 calcium Substances 0.000 claims description 90
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 76
- 238000001179 sorption measurement Methods 0.000 claims description 54
- 239000002502 liposome Substances 0.000 claims description 53
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 52
- 229910052791 calcium Inorganic materials 0.000 claims description 52
- 238000002835 absorbance Methods 0.000 claims description 47
- 239000000945 filler Substances 0.000 claims description 38
- 239000012488 sample solution Substances 0.000 claims description 38
- 239000007788 liquid Substances 0.000 claims description 31
- -1 calcium phosphate compound Chemical class 0.000 claims description 24
- 239000001506 calcium phosphate Substances 0.000 claims description 22
- 229910000389 calcium phosphate Inorganic materials 0.000 claims description 22
- 235000011010 calcium phosphates Nutrition 0.000 claims description 22
- 239000000523 sample Substances 0.000 claims description 22
- 239000003960 organic solvent Substances 0.000 claims description 19
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims description 17
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims description 17
- 238000005194 fractionation Methods 0.000 claims description 13
- 238000011049 filling Methods 0.000 claims description 11
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 10
- 239000003463 adsorbent Substances 0.000 description 66
- 229960005069 calcium Drugs 0.000 description 46
- 239000000872 buffer Substances 0.000 description 35
- 150000002632 lipids Chemical class 0.000 description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 25
- 238000000034 method Methods 0.000 description 21
- 229960001714 calcium phosphate Drugs 0.000 description 20
- 210000002969 egg yolk Anatomy 0.000 description 20
- 239000002245 particle Substances 0.000 description 20
- 108010000912 Egg Proteins Proteins 0.000 description 19
- 102000002322 Egg Proteins Human genes 0.000 description 19
- 235000013345 egg yolk Nutrition 0.000 description 19
- 239000002904 solvent Substances 0.000 description 17
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 16
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 16
- 239000000787 lecithin Substances 0.000 description 16
- 235000010445 lecithin Nutrition 0.000 description 16
- 229940067606 lecithin Drugs 0.000 description 16
- 229910019142 PO4 Inorganic materials 0.000 description 14
- 239000010452 phosphate Substances 0.000 description 14
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 238000003756 stirring Methods 0.000 description 12
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 239000011324 bead Substances 0.000 description 9
- 239000002158 endotoxin Substances 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 241000511976 Hoya Species 0.000 description 7
- 239000007864 aqueous solution Substances 0.000 description 7
- 230000008859 change Effects 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 239000003480 eluent Substances 0.000 description 7
- 229910001220 stainless steel Inorganic materials 0.000 description 7
- 239000010935 stainless steel Substances 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 210000003617 erythrocyte membrane Anatomy 0.000 description 6
- 239000012535 impurity Substances 0.000 description 6
- 239000000463 material Substances 0.000 description 6
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- 239000000725 suspension Substances 0.000 description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 5
- 239000001110 calcium chloride Substances 0.000 description 5
- 229910001628 calcium chloride Inorganic materials 0.000 description 5
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 235000002597 Solanum melongena Nutrition 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 239000012496 blank sample Substances 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 239000012064 sodium phosphate buffer Substances 0.000 description 4
- 229930186217 Glycolipid Natural products 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 230000002411 adverse Effects 0.000 description 3
- 230000004075 alteration Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
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- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000010898 silica gel chromatography Methods 0.000 description 3
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 230000000274 adsorptive effect Effects 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- ZCCIPPOKBCJFDN-UHFFFAOYSA-N calcium nitrate Chemical compound [Ca+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ZCCIPPOKBCJFDN-UHFFFAOYSA-N 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000005416 organic matter Substances 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
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- 229920005989 resin Polymers 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- WWUZIQQURGPMPG-UHFFFAOYSA-N (-)-D-erythro-Sphingosine Natural products CCCCCCCCCCCCCC=CC(O)C(N)CO WWUZIQQURGPMPG-UHFFFAOYSA-N 0.000 description 1
- RYCNUMLMNKHWPZ-SNVBAGLBSA-N 1-acetyl-sn-glycero-3-phosphocholine Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCC[N+](C)(C)C RYCNUMLMNKHWPZ-SNVBAGLBSA-N 0.000 description 1
- 241000272814 Anser sp. Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 229910014497 Ca10(PO4)6(OH)2 Inorganic materials 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 108010038061 Chymotrypsinogen Proteins 0.000 description 1
- 102000018832 Cytochromes Human genes 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
- HECLRDQVFMWTQS-UHFFFAOYSA-N Dicyclopentadiene Chemical compound C1C2C3CC=CC3C1C=C2 HECLRDQVFMWTQS-UHFFFAOYSA-N 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 102000036675 Myoglobin Human genes 0.000 description 1
- 108010062374 Myoglobin Proteins 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 239000004721 Polyphenylene oxide Substances 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- CWRILEGKIAOYKP-SSDOTTSWSA-M [(2r)-3-acetyloxy-2-hydroxypropyl] 2-aminoethyl phosphate Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCCN CWRILEGKIAOYKP-SSDOTTSWSA-M 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- MKJXYGKVIBWPFZ-UHFFFAOYSA-L calcium lactate Chemical compound [Ca+2].CC(O)C([O-])=O.CC(O)C([O-])=O MKJXYGKVIBWPFZ-UHFFFAOYSA-L 0.000 description 1
- 239000001527 calcium lactate Substances 0.000 description 1
- 229960002401 calcium lactate Drugs 0.000 description 1
- 235000011086 calcium lactate Nutrition 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229910010293 ceramic material Inorganic materials 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 230000007893 endotoxin activity Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000605 extraction Methods 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
- 238000010304 firing Methods 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 238000010438 heat treatment Methods 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
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- 238000002955 isolation Methods 0.000 description 1
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- 238000004519 manufacturing process Methods 0.000 description 1
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- 239000007769 metal material Substances 0.000 description 1
- 239000004745 nonwoven fabric Substances 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 1
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 1
- 150000003905 phosphatidylinositols Chemical class 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 229920001707 polybutylene terephthalate Polymers 0.000 description 1
- 229920000570 polyether Polymers 0.000 description 1
- 229920000139 polyethylene terephthalate Polymers 0.000 description 1
- 239000005020 polyethylene terephthalate Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
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- 239000011148 porous material Substances 0.000 description 1
- 239000011164 primary particle Substances 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 125000005372 silanol group Chemical group 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
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- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- WWUZIQQURGPMPG-KRWOKUGFSA-N sphingosine Chemical compound CCCCCCCCCCCCC\C=C\[C@@H](O)[C@@H](N)CO WWUZIQQURGPMPG-KRWOKUGFSA-N 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
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- 229920003002 synthetic resin Polymers 0.000 description 1
- 239000000057 synthetic resin Substances 0.000 description 1
- 239000002691 unilamellar liposome Substances 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
- 239000002759 woven fabric Substances 0.000 description 1
Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J7/00—Phosphatide compositions for foodstuffs, e.g. lecithin
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
- B01D15/424—Elution mode
- B01D15/426—Specific type of solvent
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/04—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of alkali metals, alkaline earth metals or magnesium
- B01J20/048—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of alkali metals, alkaline earth metals or magnesium containing phosphorus, e.g. phosphates, apatites, hydroxyapatites
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/282—Porous sorbents
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B7/00—Separation of mixtures of fats or fatty oils into their constituents, e.g. saturated oils from unsaturated oils
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
- B01J2220/54—Sorbents specially adapted for analytical or investigative chromatography
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- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Inorganic Chemistry (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Fats And Perfumes (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Description
(1) リン脂質を含有する試料液中から、前記リン脂質を選択的に分離する分離方法であって、
少なくとも表面がリン酸カルシウム系化合物で構成された充填剤に、カルシウムを吸着させるカルシウム吸着工程と、
前記充填剤が、充填空間の少なくとも一部に充填されてなる装置内に、前記試料液を供給する供給工程と、
前記装置内に、有機溶媒系の溶出液を供給して、前記装置内から流出する流出液を、所定量ずつ分画することにより、この分画された各画分の流出液中に、前記リン脂質を分画する分画工程を有することを特徴とする分離方法。
まず、本発明の分離方法について説明するのに先立って、本発明で用いられる吸着装置分離装置)の一例について説明する。
まず、試料としての卵黄を用意し、例えば、この卵黄に含まれる総脂質が抽出された試料液を調製する。
次に、充填剤3に、カルシウムを吸着させる。
次に、前記工程[1]で調製した試料液を、流入管24およびフィルタ部材4を介して吸着剤3に供給して、カラム2(吸着装置1)内を通過させて、前記工程[2]においてカルシウムが吸着された吸着剤3に接触させる。
次に、流入管24からカラム2内に、リン脂質を溶出させるための溶出液として、有機溶媒系の溶出液を供給して、カラム2内から流出管25を介して流出する流出液を、所定量ずつ分画(採取)する。
1.リン脂質リポソームの調製
(レシチンリポソーム含有液)
−1A− まず、卵黄由来のレシチン(和光純薬工業社製、Lot No.126-00812)を用意し、攪拌した後に、このもの60μLを、エタノール1.94mLに加え、ローテーター(TAITEC社製、「RT−50」)を用いて、30rpm×15min(室温)の条件で攪拌した。
その結果を、図2および表1に示す。
(リン酸ナトリウム緩衝液による検討)
−1B− まず、カラム(吸着装置)として、ハイドロキシアパタイトビーズ(CHT TypeII、平均粒径40μm、HOYA社製)を4×10mmのステンレス管に充填したものを用意した。
その結果を、図3に示す。
前記工程−3B−および前記工程−4B−を、以下のように変更したこと以外は、前記リン酸ナトリウム緩衝液による検討と同様にして、充填剤に対するリン脂質リポソームの吸着を試みた。
その結果を、図4に示す。
(リン脂質リポソーム吸着カラムのタンパク質分離能の評価)
[リン脂質リポソーム吸着カラムの製造]
−1C− まず、カラム(吸着装置)として、ハイドロキシアパタイトビーズ(CHT TypeII、平均粒径40μm、HOYA社製)を4×10mmのステンレス管に充填したものを用意した。
−1D− まず、カラム(吸着装置)として、ハイドロキシアパタイトビーズ(CHT TypeII、平均粒径40μm、HOYA社製)を4×10mmのステンレス管に充填したものを用意した。
−1E− まず、前記工程−5C−で得られた、リン脂質リポソームが吸着した充填剤を備えるカラムに対して、各種溶媒(アセトニトリル、イソプロパノール、1M水酸化ナトリウム)100CV(カラム容量)を用いて、それぞれ、洗浄した。
(レシチンの検出:実施例1)
−1F− まず、カラム(吸着装置)として、ハイドロキシアパタイトビーズ(CHT TypeII、平均粒径40μm、HOYA社製)を4×10mmのステンレス管に充填したものを用意した。
その結果を、図8(a)に示す。
その結果を、図8(b)に示す。
その結果を、図9に示す。
−1G− まず、卵黄(朝霧高原たまご)5gを攪拌しつつ、メタノール18mLおよびクロロホルム9mLをこの順で添加し、その後、30分間放置した。
その結果を、図10(a)に示す。
その結果を、図10(b)に示す。
その結果を、図10(c)に示す。
−1H− まず、ガチョウ血球(日本バイオテスト)4gを攪拌しつつ、純水100mLを添加することにより、赤血球を溶血させた。
その結果を、図11(a)に示す。
その結果を、図11(b)に示す。
その結果を、図11(c)に示す。
−1I− まず、Control Standard Endotoxin(以下、端に「エンドトキシン」という。;和光純薬工業社製)を用意し、このもの500ngに、純水2.6mLを加え、ローテーター(TAITEC社製、「RT−50」)を用いて、30rpm×15min(室温)の条件で攪拌した後、超音波処理することにより、エンドトキシンを含有する試料液を得た。
その結果を、図12(a)に示す。
その結果を、図12(b)に示す。
その結果を、図12(c)に示す。
2 カラム
20 吸着剤充填空間
21 カラム本体
22、23 キャップ
24 流入管
25 流出管
3 吸着剤
4、5 フィルタ部材
Claims (10)
- リン脂質を含有する試料液中から、前記リン脂質を選択的に分離する分離方法であって、
少なくとも表面がリン酸カルシウム系化合物で構成された充填剤に、カルシウムを吸着させるカルシウム吸着工程と、
前記充填剤が、充填空間の少なくとも一部に充填されてなる装置内に、前記試料液を供給する供給工程と、
前記装置内に、有機溶媒系の溶出液を供給して、前記装置内から流出する流出液を、所定量ずつ分画することにより、この分画された各画分の流出液中に、前記リン脂質を分画する分画工程を有することを特徴とする分離方法。 - 前記カルシウム吸着工程は、カルシウムを含有するカルシウム含有液を前記充填剤に接触させることにより行われる請求項1に記載の分離方法。
- 前記カルシウム含有液は、塩化カルシウム水溶液である請求項2に記載の分離方法。
- 前記試料液は、リン脂質がリポソームを形成しているリン脂質リポソームを含有する請求項1ないし3のいずれかに記載の分離方法。
- 前記分画工程において、前記溶出液は、有機溶媒としてイソプロパノールを含有する請求項1ないし4のいずれかに記載の分離方法。
- 前記分画工程において、前記溶出液は、イソプロパノールの含有量が0〜80%まで上昇するリニアグラジエント溶液である請求項5に記載の分離方法。
- 前記分画工程において、前記溶出液の流速は、0.1〜10mL/分である請求項1ないし4のいずれかに記載の分離方法。
- 前記分画工程において、前記リン脂質の検出は、前記各画分の190〜230nmの吸光度を観察することにより行われる請求項1ないし7のいずれかに記載の分離方法。
- 前記分画工程において、前記リン脂質の検出は、予め測定した前記溶出液の吸光度を、観察される前記各画分の吸光度から差し引いて行われる請求項1ないし8のいずれかに記載の分離方法。
- 前記リン酸カルシウム系化合物は、ハイドロキシアパタイトを主成分とするものである請求項1ないし9のいずれかに記載の分離方法。
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