JPS63157973A - Liquid chromatography - Google Patents
Liquid chromatographyInfo
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- JPS63157973A JPS63157973A JP61305681A JP30568186A JPS63157973A JP S63157973 A JPS63157973 A JP S63157973A JP 61305681 A JP61305681 A JP 61305681A JP 30568186 A JP30568186 A JP 30568186A JP S63157973 A JPS63157973 A JP S63157973A
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- 238000004811 liquid chromatography Methods 0.000 title claims abstract description 9
- 239000002245 particle Substances 0.000 claims abstract description 126
- 239000003463 adsorbent Substances 0.000 claims abstract description 26
- 239000011247 coating layer Substances 0.000 claims abstract description 22
- 229910052586 apatite Inorganic materials 0.000 claims abstract description 21
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims abstract description 21
- 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 abstract description 21
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 16
- 239000006185 dispersion Substances 0.000 claims abstract description 13
- 210000004027 cell Anatomy 0.000 claims abstract description 12
- 210000003463 organelle Anatomy 0.000 claims abstract description 10
- VSIIXMUUUJUKCM-UHFFFAOYSA-D pentacalcium;fluoride;triphosphate Chemical compound [F-].[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 VSIIXMUUUJUKCM-UHFFFAOYSA-D 0.000 claims abstract description 10
- 150000001875 compounds Chemical class 0.000 claims abstract description 9
- 239000011521 glass Substances 0.000 claims abstract description 8
- 239000000463 material Substances 0.000 claims abstract description 6
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 5
- 239000000919 ceramic Substances 0.000 claims abstract description 3
- 229910052587 fluorapatite Inorganic materials 0.000 claims abstract description 3
- 229910052751 metal Inorganic materials 0.000 claims abstract description 3
- 239000002184 metal Substances 0.000 claims abstract description 3
- 239000011347 resin Substances 0.000 claims abstract description 3
- 229920005989 resin Polymers 0.000 claims abstract description 3
- 239000007788 liquid Substances 0.000 claims description 6
- 229940077441 fluorapatite Drugs 0.000 claims description 2
- 238000011049 filling Methods 0.000 abstract description 2
- 239000007921 spray Substances 0.000 abstract 1
- 239000013078 crystal Substances 0.000 description 21
- -1 NA-Fist plasmids Chemical class 0.000 description 17
- 238000013375 chromatographic separation Methods 0.000 description 12
- 239000000843 powder Substances 0.000 description 11
- 239000000725 suspension Substances 0.000 description 10
- 239000011163 secondary particle Substances 0.000 description 7
- 239000007858 starting material Substances 0.000 description 6
- 239000011324 bead Substances 0.000 description 5
- 239000011575 calcium Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 230000007704 transition Effects 0.000 description 5
- 230000018044 dehydration Effects 0.000 description 4
- 238000006297 dehydration reaction Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000003513 alkali Substances 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000013076 target substance Substances 0.000 description 3
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 239000000853 adhesive Substances 0.000 description 2
- 230000001070 adhesive effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000004576 sand Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 101100283604 Caenorhabditis elegans pigk-1 gene Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 102100030497 Cytochrome c Human genes 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
- 102000018832 Cytochromes Human genes 0.000 description 1
- 108010075031 Cytochromes c Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 102000008791 Lysozyme C Human genes 0.000 description 1
- 102100033468 Lysozyme C Human genes 0.000 description 1
- 108050000633 Lysozyme C Proteins 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000004931 aggregating effect Effects 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 description 1
- UKFWSNCTAHXBQN-UHFFFAOYSA-N ammonium iodide Chemical compound [NH4+].[I-] UKFWSNCTAHXBQN-UHFFFAOYSA-N 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- XAAHAAMILDNBPS-UHFFFAOYSA-L calcium hydrogenphosphate dihydrate Chemical compound O.O.[Ca+2].OP([O-])([O-])=O XAAHAAMILDNBPS-UHFFFAOYSA-L 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- PWZFXELTLAQOKC-UHFFFAOYSA-A dialuminum;hexamagnesium;carbonate;hexadecahydroxide;tetrahydrate Chemical compound O.O.O.O.[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Al+3].[Al+3].[O-]C([O-])=O PWZFXELTLAQOKC-UHFFFAOYSA-A 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 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
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Abstract
Description
【発明の詳細な説明】 イ、発明の目的 〔産業上の利用分野〕 本発明は、液体クロマトグラフィーに関する。[Detailed description of the invention] B. Purpose of the invention [Industrial application field] The present invention relates to liquid chromatography.
更に詳しくは細胞やオルガネラなどの粒子分散液（又は
懸濁液）からその粒子分散質を効果的にクロマト分離（
分取）することを可能とした手法に関する。More specifically, it is possible to effectively chromatographically separate particle dispersoids from particle dispersions (or suspensions) of cells, organelles, etc.
Regarding the method that made it possible to perform preparative separation.
アパタイト系化合物は一般式Ｍ１゜（２０４） ６ｘ２
の基本組成をもつ六方晶系Ｐ６３／ｍに属する化合物で
ある。上記基本組成を倍にした単斜晶系Ｐ２．／ｂとし
てとらえる場合もある。その具体的な代表化合物として
、Ｍ＝Ｃａ、Ｚ＝ Ｐ、Ｘ＝ＯＨ−？ｌ’あるハイドロ
キシアパタイト（Ｈｙｄｒｏｙｙａｐａｔｉｔｅ、 Ｃ
ａ＋ｏ （ＰＯ４） ６ （ＯＨ）２ 、六方晶系単位
格子＝ａ Ａ ｂ ＝１２０°、ａＡｃ＝ｂＡｃ＝９０
°、 Ｉａｌ＝ｌｂｌ＝８．４２人、 １ｃｌ＝ ６．
８８人）、Ｘ＝Ｆである７ ロー ノｙアパタイト、Ｘ
＝ＣＩであるクロロアパタイト等が挙げられる。Apatite compounds have the general formula M1゜(204) 6x2
It is a compound belonging to the hexagonal system P63/m with the basic composition of Monoclinic system P2 with double the above basic composition. /b. As specific representative compounds, M=Ca, Z=P, X=OH-? l' Hydroxyapatite (C
a+o (PO4) 6 (OH)2, hexagonal unit cell = a A b = 120°, aAc = bAc = 90
°, Ial=lbl=8.42 people, 1cl=6.
88 people), 7 low apatite, where X=F,
=CI, such as chloroapatite.
アパタイト系化合物は総じて優れた吸着性を有する。以
下代表的化合物であるハイドロキシアパタイト（ＨＡｐ
と略記する）を具体例にして説明する。Apatite compounds generally have excellent adsorption properties. Hydroxyapatite (HAp), a representative compound, is shown below.
(abbreviated as ) will be explained using a specific example.
ＨＡｐＩｋ子はカラムクロマトグラフィーの固定相吸着
剤（カラム内充填剤）として優れたクロマト分離能（物
質分離・展開能）を有し、該粒子を固定相吸着剤とする
クロマトグラフィー（ＨＡｐクロマトグラフィー）は、
他の吸着剤例えばイオン交換樹脂・活性アルミナ・炭酸
カルシウムなどを用いる場合には困難であった、微細な
構造差を有する物質量の相互分離、例えば分子量１０４
〜１０９ダルトンの生体関連高分子（免疫グロブリン・
インターフェロン・酵素類等の蛋白質類、ＲＮＡ・ Ｄ
ＮＡ拳プラスミド類の核酸類、ウィルス類など）につい
ても鋭敏・高精度に分取操作をすることが可能であり、
例えばバイオテクノロジー分野での遺伝子組換え、細胞
融合、細胞大量倍養法等により合成された各種の目的有
用物質の高純度分離精製等に不可欠な手段として活用さ
れるに至っている。HApIk particles have excellent chromatographic separation ability (substance separation and development ability) as a stationary phase adsorbent (in-column packing material) for column chromatography, and chromatography using these particles as a stationary phase adsorbent (HAp chromatography) teeth,
Mutual separation of substances with minute structural differences, which was difficult when using other adsorbents such as ion exchange resins, activated alumina, and calcium carbonate, for example, molecular weight 104
~109 Dalton bio-relevant polymers (immunoglobulins,
Proteins such as interferon and enzymes, RNA/D
It is also possible to carry out preparative operations with sensitivity and high precision for nucleic acids such as NA-Fist plasmids, viruses, etc.
For example, in the field of biotechnology, it has come to be utilized as an indispensable means for high-purity separation and purification of various useful substances synthesized by genetic recombination, cell fusion, cell mass cultivation, etc.
ＨＡｐ粒子としては下記のような各種タイプがある。There are various types of HAp particles as shown below.
（１）Ａタイプ
チゼリウス式製造法（Ａ、 Ｔｉ５ｅｌｉｕｓ、 Ｓ、
Ｈｊｅｒｔ図、１３２（＋！３５Ｂ））で得たＨＡｐ
結晶粒子、即ちブルスハイト結晶粒子（Ｂｒｕｓｈｉｔ
ｅ：ＧａＨＰＯ４ｍ ２Ｈ２０）を出発物質として該結
晶を水中に懸濁してアルカリを作用させることにより生
成させたＨＡｐ結晶粒子であり、出発物質たるプルスハ
イト結晶粒子の形状を実質的にそのまま引き継いだ１通
常厚さ１〜３Ｂｍ、平均直径（長軸方向）数１１０７ｚ
程度の板状もしくは鱗片状の実質的に単結晶の粒子であ
る。(1) A type Ti5elius manufacturing method (A, Ti5elius, S,
HAp obtained with Hjert diagram, 132 (+!35B))
Crystal grains, namely brushheit crystal grains (Brushite crystal grains)
e: HAp crystal particles produced from GaHPO4m2H20) as a starting material by suspending the crystals in water and reacting with an alkali, and having a normal thickness of 1, which substantially inherits the shape of the starting material, the purushite crystal particles. Size 1-3Bm, average diameter (long axis direction) number 1107z
They are substantially single-crystal particles that are somewhat plate-like or scale-like.
（２）Ｂタイプ ＨＡｐ微細結晶−次粒子を凝集させた二次粒子。(2) B type HAp microcrystal-secondary particles that are aggregated secondary particles.
このタイプの粒子は一般に下記のような要領で製造され
る。即ちＧａ（ＯＨ）２． ＣａＣｌ２ 、 Ｃａ （
ＮＯ３） ２、Ｃａ （ＣＨ３０００） ２などｃ７）
Ｃａ塩水溶液又は懸濁液と、ＮＨ４Ｉ ２ＰＯ４、（Ｎ
Ｈ４） ２ ＨＰＯ４、Ｈ３ＰＯ４などのＰ０４塩水
溶液とを塩基性条件下で混合する。そうすると通常０．
１７Ｌｍ以下の微細なＨＡｐ−次結晶粒子が生成する。This type of particle is generally produced as follows. That is, Ga(OH)2. CaCl2, Ca (
NO3) 2, Ca (CH3000) 2, etc.c7)
Ca salt aqueous solution or suspension and NH4I 2PO4, (N
H4) 2 Mix with an aqueous P04 salt solution such as HPO4 or H3PO4 under basic conditions. Then it is usually 0.
Fine HAp-order crystal particles of 17 Lm or less are produced.
その−次粒子懸濁液をスプレー乾燥処理することにより
ＨＡｐ−次粒子が乾燥凝集した直径１〜１０ＩＬｍ程度
の実質的に球状の二次粒子体が得られる。By spray-drying the secondary particle suspension, substantially spherical secondary particles having a diameter of about 1 to 10 ILm are obtained by drying and aggregating the HAp secondary particles.
（３）Ｃタイプ
本出願人の先の提案（特願昭８１−１８１１８８号）に
係る製造法により得られるＨＡｐの実質的に単結晶の粒
子。即ちプルスハイト結晶粒子を出発物質とし、これを
一旦脱水相転移温度以上〜分解温度以下の範囲で加熱し
てモネタイト結晶粒子（Ｍｏｎｅｔｉｔｅ：ＧａＨＰＯ
４）に脱水相転移させ、それにアルカリを作用させてＨ
Ａｐの実質的に単結晶の粒子を生成させる。これにより
得られるＨＡｐ粒子は出発物質たる比較的大型の板状も
しくは鱗片状のプルスハイト結晶粒子が脱水相転移によ
りモネタイトに相転移したときの内部歪み力で結晶体面
内で数μｍ程度の大きさの細片に自然に砕は状態となっ
た厚さ１〜３ｇｍ、直径数μｍの全体的に大きさ・形の
揃ったもので、前記ＡタイプのＨＡｐ粒子即ち出発物質
たるプルスハイト結晶粒子の大きさ争形をそのまま引き
継いだ形態の板状もしくは鱗片状粒子よりも大きさ・形
のバラツキ幅が小さく、該Ａタイプのものよりもクロマ
トグラフィー用吸着剤として優れる。(3) Type C Substantially single-crystal particles of HAp obtained by the production method according to the applicant's previous proposal (Japanese Patent Application No. 81-181188). That is, prusheite crystal particles are used as a starting material, and this is heated in a range from above the dehydration phase transition temperature to below the decomposition temperature to form monetite crystal particles (Monetite: GaHPO
4) is subjected to dehydration phase transition, and alkali is applied to it to transform H
Substantially single crystal particles of Ap are produced. The HAp particles obtained in this way are formed into particles with a size of several μm within the plane of the crystal due to the internal strain force when the relatively large plate-like or scale-like purusheit crystal particles, which are the starting material, undergo a phase transition to monetite due to dehydration phase transition. They are naturally crushed into small pieces with a thickness of 1 to 3 gm and a diameter of several μm, and have an overall uniform size and shape, and are the same size as the above-mentioned type A HAp particles, that is, the pruscite crystal particles that are the starting material. The variation in size and shape is smaller than that of the plate-like or scale-like particles that inherited the same type A type, and it is superior as an adsorbent for chromatography than the A type.
プルスハイト結晶粒子を脱水相転移処理して予め生産し
たモネタイト結晶粒子、又は既存のモネタイト結晶粒子
、若しくはブルスハイトからモネタイトへ至る中間体物
質粒子であって全体的に大きさ・形の揃った、或は揃う
ように処置したものを出発物質として、それにアルカリ
を作用させて得るようにしてもよい。monetite crystal particles pre-produced by dehydration phase transition treatment of bullshite crystal particles, existing monetite crystal particles, or intermediate material particles from bullshite to monetite that are uniform in size and shape as a whole, or It may be obtained by treating the starting material so that it is uniform and treating it with an alkali.
（４）Ｄタイプ
本出願人の先の提案（特願昭８１−４９８８８号）に係
る焼成結合ＨＡｐ粒子。即ち前記Ｂタイプの凝集二次粒
子を更に約３００℃以上〜分解温度以下の温度で加熱焼
成したもので、−次粒子相互が強固に結合して耐圧力崩
壊性に優れる。(4) Type D sintered bonded HAp particles according to the applicant's previous proposal (Japanese Patent Application No. 81-49888). That is, the agglomerated secondary particles of the B type are further heated and calcined at a temperature of about 300° C. or above and below the decomposition temperature, and the secondary particles are strongly bonded to each other and have excellent pressure collapse resistance.
クロマト分離操作する原料液に含まれる目的物質が分子
オーダで溶存しているものである場合には上記各種の７
パタイト化合物粒子は何れもその溶存する目的物質をク
ロマト分離するための固定相吸着剤として有効に利用で
きる。If the target substance contained in the raw material liquid to be subjected to chromatographic separation is dissolved in molecular order, the above 7.
Patite compound particles can be effectively used as a stationary phase adsorbent for chromatographically separating the target substance dissolved therein.
目的物質が分子オーダ以上の比較的大径の単体もしくは
凝集粒子として分散している粒子分散液（又は懸濁液）
、例えば数＃Ｌｍオーダの細胞の分散液や〜０．１ｐｍ
オーグのオルガネラの分散液などである場合にはそれ等
の粒子分散質を高精度・能率的にクロマト分離させるた
めには固定相吸着剤としての粒子はそれ等の分散質粒子
よりもはるかに大径の粒子、例えば５０７１ｍとか１０
０ｇｍ、或はそれ以上の大径粒子であることが必要であ
る（細胞やオルガネラ等はカラムのフィルタを通過し、
固定相吸着剤粒子はフィルタを通過しないことが必要）
。A particle dispersion (or suspension) in which the target substance is dispersed as single or aggregated particles with a relatively large diameter of molecular order or more.
, for example, a dispersion of cells on the order of several #Lm or ~0.1 pm.
In the case of dispersions of organelles, etc., in order to perform chromatographic separation of these particle dispersoids with high precision and efficiency, the particles used as a stationary phase adsorbent must be much larger than the dispersoid particles. Particles of diameter, for example 5071m or 10
It is necessary to have large-diameter particles of 0 gm or more (cells, organelles, etc. pass through the filter of the column,
Stationary phase adsorbent particles must not pass through the filter)
.
ところが上記各種の７パタイト化合物粒子は平均粒径数
ｇｍオーダのものを主体とするものであるから分散液の
粒子分散質を高精度・能率的にクロマト分離させるには
粒径的に小さくて適格性がなく、実際上細胞やオルガネ
ラ等のクロマト分離は不能である。However, since the various heppatite compound particles mentioned above mainly have an average particle size on the order of several gm, they are small in size and suitable for chromatographic separation of particle dispersoids in a dispersion liquid with high precision and efficiency. chromatographic separation of cells, organelles, etc. is practically impossible.
本発明は用いる固定相吸着剤粒子に工夫を加えて、細胞
やオルガネラなどの粒子分散液からその粒子分散質を液
体クロマトグラフィーにより高精度・能率的にクロマト
分離することができるようにしたものである。In the present invention, the stationary phase adsorbent particles used are modified to enable highly accurate and efficient chromatographic separation of particle dispersoids from a particle dispersion of cells, organelles, etc. using liquid chromatography. be.
口、発明の構成
〔問題点を解決するための手段〕
本発明は、細胞やオルガネラなどの粒子分散液からその
粒子分散質を液体クロマトグラフィーにより分離するに
当り、カラムに充填使用する固定相吸着剤粒子として、
担体粒子の表面にアパタイト系化合物を被覆層として担
持させてなり、且つ担体粒子の大きさを選定して全体の
粒径な前記粒子分散質をクロマト分離するに十分な大き
さの粒径に調製した粒子を使用する、ことを特徴とする
液体クロマトグラフィー要旨とする。Summary: Structure of the Invention [Means for Solving Problems] The present invention is directed to a stationary phase adsorption system packed in a column and used in separating particle dispersoids from particle dispersions such as cells and organelles by liquid chromatography. As agent particles,
An apatite-based compound is supported as a coating layer on the surface of carrier particles, and the size of the carrier particles is selected to have a particle size large enough to chromatographically separate the particle dispersoid with the overall particle size. The gist of liquid chromatography is that it uses particles of
上記のアパタイト系化合物被覆形の吸着剤粒子の形状争
大きさは核として使用する担体粒子の形状・大きさに略
対応したものとなる。従って担体粒子として所望の形状
・大きさのものを使用することによりそれに略対応した
球形等の所望の形状で、且つ所望の大きさ、例えば５０
ｇｍとか１００ ｇｍとか、或はそれ以上の所望の大粒
径で、しかも粒径のバラツキ範囲が狭く、形状・大きさ
が実質的に揃った吸着剤粒子としての７パタイト系化合
物粒子を自由に調整して得ることができる。The shape and size of the adsorbent particles coated with the apatite compound described above approximately correspond to the shape and size of the carrier particles used as the core. Therefore, by using carrier particles of a desired shape and size, it is possible to obtain a desired shape such as a spherical shape approximately corresponding to the carrier particle and a desired size, for example, 50 mm.
gm, 100 gm, or more, with a narrow range of variation in particle size, and virtually uniform shape and size. You can get it by adjusting.
従って、原料液が細胞やオルガネラ等の粒子分散液であ
る場合において、その粒子分散質をクロマト分離するた
めに粒径的に十分に適合した所望大粒径の吸着剤粒子を
容易に調製して使用できるから、該粒子分散質を液体ク
ロマトグラフィーにより高精度・能率的にクロマト分離
（分取）することが可能となる。Therefore, when the raw material liquid is a particle dispersion of cells, organelles, etc., it is possible to easily prepare adsorbent particles with a desired large particle size that is sufficiently compatible in terms of particle size in order to chromatographically separate the particle dispersoid. Since it can be used, it becomes possible to chromatographically separate (fractionate) the particle dispersoid with high precision and efficiency by liquid chromatography.
核としての担体粒子を耐圧力崩壊性を有するものにする
ことにより大容量カラム内に大量に充填使用しても圧力
崩壊せず、カラム内全体の所定の流体流通性が長期にわ
たって安定に保持される。By making the carrier particles as the core resistant to pressure collapse, the pressure does not collapse even when used in large quantities in a large-capacity column, and the specified fluid flowability throughout the column is stably maintained over a long period of time. Ru.
有効成分たるアパタイト系化合約分は核としての担体粒
子表面の被覆層分だけで足り、担体粒子分を含む全体体
積との量比は少く、有効成分たるアパタイト系化合物の
使用量を大幅に少なくすることができ、安価に量産する
ことができる。従って実験室的にも工業的にも低コスト
にクロマト分離操作を実行することが可能となる。The amount of the apatite compound that is the active ingredient is only the coating layer on the surface of the carrier particle as a core, and the ratio to the total volume including the carrier particle is small, so the amount of the apatite compound that is the active ingredient used is significantly reduced. It can be mass-produced at low cost. Therefore, it becomes possible to carry out chromatographic separation operations at low cost both in the laboratory and industrially.
第１図は球形の担体粒子ａと、該担体粒子の表面を覆っ
て固着しているアパタイト系化合物の被覆層すとからな
る吸着剤粒子の一部切欠き拡大模型図である。FIG. 1 is an enlarged partially cutaway schematic diagram of an adsorbent particle consisting of a spherical carrier particle a and a coating layer of an apatite compound that covers and adheres to the surface of the carrier particle.
担体粒子ａは耐圧力崩壊性を有する５例えばガラス、セ
ラミック、金属、樹脂などを所望の形状会大きさの担体
粒子として加工製造したものである。分級した砂粒−砂
鉄・小石など耐圧力崩壊性を有する天然粒状物も利用し
得る。The carrier particles a are produced by processing a material having pressure collapse resistance, such as glass, ceramic, metal, resin, etc., into carrier particles having a desired shape and size. Natural granules that are resistant to pressure collapse, such as classified sand grains, iron sand, and pebbles, can also be used.
被覆層すたるアパタイト系化合物はハイドロキシアパタ
イト、フロールアパタイト、クロロアパタイト等である
。The apatite compound that forms the coating layer includes hydroxyapatite, fluorapatite, chloroapatite, and the like.
担体粒子＆の表面に対するアパタイト系化合物被覆層す
の形成は種々の方法で行うことができる。The apatite compound coating layer can be formed on the surface of the carrier particles by various methods.
例１
例えば０．１ ＃Ｌｍφオーダのアパタイト系化合物微
粉末の適当濃度の懸濁液を５Ｑｐｍφとか１００ ルｍ
φ等の所望形状−大きさの担体粒子に対してスプレー処
理して付着・乾燥させる。これにより担体粒子ａの表面
にアパタイト系化合物微粉末の凝集固着被覆層すが形成
される。Example 1 For example, a suspension of an appropriate concentration of apatite compound fine powder on the order of 0.1 #Lmφ is 5Qpmφ or 100 lm
Carrier particles of a desired shape and size, such as φ, are sprayed to adhere and dry. As a result, an agglomerated and fixed coating layer of fine apatite compound powder is formed on the surface of the carrier particles a.
例２
例１において被覆層すの担体粒子ａの表面への固着力、
被覆層すを構成するアパタイト系化合物微粉末相互の凝
集固化力を増強するために、担体粒子ａにスプレーする
アパタイト系化合物微粉末の懸濁液中に適当なバインダ
剤を適当濃度で溶存させておくこともできる。Example 2 In Example 1, the adhesion force of the coating layer to the surface of carrier particles a,
In order to enhance the mutual coagulation and solidification power of the apatite compound fine powder constituting the coating layer, an appropriate binder agent is dissolved at an appropriate concentration in the suspension of the apatite compound fine powder to be sprayed onto the carrier particles a. You can also leave it there.
例３
例１・例２においてアパタイト系化合物微粉末の凝集固
着被覆層すを形成した担体粒子ａを更に加熱炉等で適当
な温度でシンター（焼成）処理する。これにより担体粒
子８表面に被覆層すが焼付き的に付着し、又被覆層すを
構成するアパタイト系化合物微粉末相互も焼き固められ
て被覆層の機械的強度が向上する。Example 3 The carrier particles a on which the agglomerated and fixed coating layer of fine apatite compound powder was formed in Examples 1 and 2 are further sintered (fired) in a heating furnace or the like at an appropriate temperature. As a result, the coating layer adheres to the surface of the carrier particles 8 in a baking manner, and the apatite compound fine powder constituting the coating layer is also baked and solidified, thereby improving the mechanical strength of the coating layer.
例４
担体粒子８表面に対して適当濃度の接着剤液をスプレー
・浸漬等で適用して接着剤液で濡らし状態にし、次いで
乾燥状態のアパタイト系化合物微粉末をまぶして被覆層
すとして付着させ、乾燥する。更には例３のようにシン
ター処理する。Example 4 An adhesive liquid of an appropriate concentration is applied to the surface of the carrier particles 8 by spraying or dipping to wet the surface with the adhesive liquid, and then a dry apatite compound fine powder is sprinkled on the surface to form a coating layer. ,dry. Furthermore, sinter processing is performed as in Example 3.
アパタイト系化合物の微粉末はハイドロキシアパタイト
では前述ＢタイプのＣａ塩とＰＯ４塩の化学反応により
生成される微細な一次結晶粒子を利用することができる
。Ａ−Ｄタイプの粒子の微細分を分級して利用すること
もできる。大径粒子を微粉に砕き処理して使用すること
もできる。For the fine powder of the apatite compound, in the case of hydroxyapatite, fine primary crystal particles produced by the chemical reaction of the above-mentioned B type Ca salt and PO4 salt can be used. It is also possible to classify and utilize fine fractions of A-D type particles. Large particles can also be used after being crushed into fine powder.
担体粒子ａの表面に形成するアパタイト系化合物被覆層
すの層厚は薄いものでよく、例えば担体粒子ａの粒径の
１／ｌＯ以下でも実用に十分に供し得る。The thickness of the apatite compound coating layer formed on the surface of the carrier particles a may be thin; for example, a thickness of 1/1O or less of the particle size of the carrier particles a may be sufficient for practical use.
担体粒子ａは球形に限らず、立方体Ｏ直方体・板状・円
柱体Φ円錐体・角錐体・鯖片状令イガ状体など任意の形
状にすることができる。The carrier particles a are not limited to a spherical shape, but can be in any shape such as a cube, a rectangular parallelepiped, a plate, a cylinder, a cone, a pyramid, a mackerel-like shape, and a bur-like shape.
実施例１
Ａ、固定相用吸着剤粒子の調製
担体粒子ａとして平均粒径１１００ＪＬの粒度バラツキ
範囲の小さい球形ガラスピーズを用いた。これにＣａ
（ＯＨ） ２ とＮＨ４Ｈ２ＰＯ４とより生成した、微
細で実質的に格子欠陥のない即ちストイキオメトリツク
（Ｓｔｏｉｃｈｉｏｍｅｔｒｉｃ）なハイドロキシアバ
タイ）（ＨＡｐ）結晶粒子の懸濁水（ＨＡｐ結晶粒子径
０．１ｇｍオーダ、懸濁濃度１．６５％）をスプレー処
理して乾燥することにより個々の球形ガラスピーズの外
表面にＨＡｐ微粉末の薄い（層厚ｌ〜２ＩＬｍ程度）凝
集固着被覆層すを形成した。次いで該被覆層すを形成し
た球形ガラスピーズａを電気炉に入れて６５０°Ｃ・３
０分間加熱処理した。これにより被覆層すが担体粒子ａ
たる球形ガラスピーズの表面に焼付き的に強固に付着し
、又被覆層すを構成するＨＡｐ微粉末相互も強固に結合
化状態となった、大粒径の被覆形吸着剤粒子を簡単に得
ることができた。Example 1 A. Preparation of adsorbent particles for stationary phase As carrier particles a, spherical glass beads with an average particle size of 1100 JL and a small particle size variation range were used. In this Ca
(OH) 2 and NH4H2PO4, a suspension of fine, substantially lattice defect-free, ie, stoichiometric (HAp) crystal particles (HAp crystal particle size on the order of 0.1 gm, A thin (layer thickness of about 1 to 2 IL m) cohesive and fixed coating layer of fine HAp powder was formed on the outer surface of each spherical glass beads by spraying and drying a suspension of 1.65% (suspension concentration: 1.65%). Next, the spherical glass beads a with the coating layer formed thereon were placed in an electric furnace at 650°C.
Heat treatment was performed for 0 minutes. As a result, the coating layer is covered with carrier particles a.
Easily obtain large-sized coated adsorbent particles that firmly adhere to the surface of barrel-shaped spherical glass beads in a seizing manner, and also firmly bond the HAp fine powder constituting the coating layer to each other. I was able to do that.
Ｂ、クロマト分離
上記Ａで調製した吸着剤粒子を固定相（カラム内充填剤
）として使用してウサギ赤血球の等張懸濁液（サンプル
）からの赤血球クロマト分離を下記のような条件で実行
した。第２図はそれにより得たクロマトグラムである。B. Chromatographic separation Using the adsorbent particles prepared in A above as the stationary phase (column packing material), chromatographic separation of red blood cells from an isotonic suspension (sample) of rabbit red blood cells was performed under the following conditions. . FIG. 2 is a chromatogram obtained thereby.
■カラム：内径６■、吸着剤充填有効長３３ｃｍ、ｌＱ
Ｑ＃Ｌｍフィルタ
■サンプル：ウサギの赤血球原液０．３ｍｌ■検定：流
量＝ １．０ｍｌ ／ｗｉｎ、圧力＝ １．５ｋｇ／ｃ
ｍ２■緩衝液： １０−３ＭＫＰ（ＫユＨＰＯ４、ＫＨ
２ＰＯ４の等モル混合液） 、 ０．９％ＮａＣｌ
第２図のクロマトグラムにおいて横軸のフラクション（
ｆｒａｃｔｉｏｎ、分画）４は装置スタートＳ→８．５
ｍｌ後から８．５ｍＪＬ後まで（中心７．５ｎ＋Ｊ１
） 、フラクション１１はスタートＳ→２（１，５ｍｊ
２後から２２．５ｍ文後まで（中心２１．５ｍ文）の部
分を示した。■Column: Inner diameter 6■, adsorbent filling effective length 33cm, lQ
Q#Lm filter Sample: Rabbit red blood cell stock solution 0.3ml Test: flow rate = 1.0ml/win, pressure = 1.5kg/c
m2 ■ Buffer: 10-3MKP (KyuHPO4, KH
Equimolar mixture of 2PO4), 0.9% NaCl In the chromatogram in Figure 2, the fraction on the horizontal axis (
fraction, fraction) 4 is device start S → 8.5
From after ml to after 8.5mJL (center 7.5n+J1
), fraction 11 is start S → 2 (1,5 mj
The part from after 2 to 22.5m sentence (center 21.5m sentence) is shown.
Ｃ１再クロマト分１１１１ （１）
第３図は上記第２図のクロマトグラムにおけるフラクシ
ョン４部分を下記のような条件で再クロマト分離して得
たクロマトグラムである。C1 rechromatogram 1111 (1) FIG. 3 is a chromatogram obtained by rechromatographically separating fraction 4 in the chromatogram shown in FIG. 2 above under the following conditions.
■カラム：前記Ｂの■と同じ、充填固定相も同じ。■Column: Same as ■ in B above, packed stationary phase is also the same.
■サンプル＝４分画１．８ｍ文
■検定二流量＝ １．ｈｕ ／ｌ１ｉｎ、圧力＝ １．
５ｋｇ／ｃｍ２■緩衝液： １０−２ＭＫＰ 、 ０．
９％ＮａＣＩＤ、再りロマト分＃（２）
第４図は前記第２図のクロマトグラムにおけるフラクシ
ョン１１部分を下記のような条件で再クロマト分離して
得たクロマトグラムである。■Sample = 4 fractions 1.8m ■Certification 2 flow rate = 1. hu/l1in, pressure=1.
5kg/cm2 Buffer: 10-2MKP, 0.
9% NaCID, rechromatogram #(2) FIG. 4 is a chromatogram obtained by rechromatographically separating fraction 11 in the chromatogram shown in FIG. 2 under the following conditions.
■カラム：前記Ｂの■と同じ、充填固定相も同じ。■Column: Same as ■ in B above, packed stationary phase is also the same.
■サンプル：１１分画１．８１文
■検定二流量＝ １．（１４１／ｗｉｎ、圧力＝ １．
５ｋｇ／ｃｍ２■緩衝液：前記Ｂの■と同じ。■Sample: 11 fractions 1.81 sentences ■Certification 2 flow rate = 1. (141/win, pressure = 1.
5kg/cm2 ■ Buffer: Same as B above.
実施例２
Ａ、固定相用吸着剤粒子・の調製
担体粒子ａとして平均粒径５０ＩＬｍの粒バラツキ範囲
の小さい球形ガラスピーズを用い、他は実施例１のＡと
同様の要領で被覆形吸着剤粒子を調製した。Example 2 A. Preparation of adsorbent particles for stationary phase A coated adsorbent was prepared in the same manner as in Example 1 A except that spherical glass beads with an average particle size of 50 ILm and a small particle variation range were used as carrier particles a. Particles were prepared.
Ｂ、クロマト分離
上記Ａで調製した吸着剤粒子を固定相として使用して
ａ、ウシ血清アルブミン（ＢＳＡ、ｂｏｖｉｎｅ ５ｅ
ｒｕ＋ｗ ａｌｂ冒１ｎ）
ｂ、リゾチーム（ｌｙｓｏｚｙｍｅ）
Ｃ，チトクロームｃ （ｃｙｔｏｃｈｒｏ＋＊ｅ ｃ）
以上ａ−ｃの３種混合懸濁液のクロマト分離を下記のよ
うな条件で実行した。第５図はそれにより得たクロマト
グラムである。B. Chromatographic separation using the adsorbent particles prepared in A above as the stationary phase a. Bovine serum albumin (BSA, bovine 5e)
ru+w alb affected 1n) b, lysozyme C, cytochrome c (cytochro+*e c)
Chromatographic separation of the mixed suspension of the three species a to c above was carried out under the following conditions. FIG. 5 is a chromatogram obtained thereby.
■カラム：内径６■璽、吸着剤充填有効長３３ｃｍ、フ
ィルタ５ＩＬｍ
■サンプル：濃度比、ＢＳＡ ：リゾチーム：チトクロ
ームＣの混合液０．５閣文
■検定二流量＝ ０．５ｍＪｌｌ ／ｗｉｎ、圧力＝
１ｋｇ／ａｍ２■緩衝液： ＫＰ、初期濃度を１Ｏ−３
として傾き３．５ＩＩＩＭＩＩ１文の線形グラジェント
を形成させる。■Column: Inner diameter 6 ■Silver, effective length filled with adsorbent 33cm, filter 5ILm ■Sample: Concentration ratio, BSA:Lysozyme:Cytochrome C mixture 0.5mm ■Certification flow rate = 0.5mJll /win, Pressure =
1kg/am2 Buffer: KP, initial concentration 1O-3
A linear gradient with a slope of 3.5IIIMII1 is formed.
ハ、発明の効果
以上のように本発明に依れば、液体クロマトグラフィー
により細胞やオルガネラなどの粒子分散液からその粒子
分散質を高精度・能率的にクロマト分離（分取）するこ
とが可能となるもので、所期の目的がよく達成される。C. Effects of the Invention As described above, according to the present invention, it is possible to chromatographically separate (preparate) particle dispersoids from a particle dispersion of cells, organelles, etc. with high precision and efficiency using liquid chromatography. As a result, the intended purpose is well achieved.
【図面の簡単な説明】
第１図は本発明で用いる吸着剤粒子の構造模型図、第２
図乃至第５図は夫々実施例で実行したクロマト分離操作
で得られたクロマトグラムである。
ａは担体、ｂはその表面のアパタイト系化合物被覆層。
ｕＪｕ’ＩＬ７）ＤａコＵＤｑＪＯ５ｑＤ○[Brief explanation of the drawings] Figure 1 is a structural model diagram of adsorbent particles used in the present invention, Figure 2 is a structural model diagram of adsorbent particles used in the present invention,
Figures 5 to 5 are chromatograms obtained in the chromatographic separation operations carried out in Examples. a is a carrier, and b is an apatite compound coating layer on the surface thereof. uJu'IL7) DakoUDqJO5qD○
Claims (3)
分散質を液体クロマトグラフィーにより分離するに当り
、 カラムに充填使用する固定相吸着剤粒子として、担体粒
子の表面にアパタイト系化合物を被覆層として担持させ
てなり、且つ担体粒子の大きさを選定して全体の粒径を
前記粒子分散質をクロマト分離するに十分な大きさの粒
径に調製した粒子を使用する、 ことを特徴とする液体クロマトグラフィー。(1) When separating particle dispersoids from particle dispersions such as cells and organelles by liquid chromatography, an apatite-based compound is coated on the surface of carrier particles as a coating layer as a stationary phase adsorbent particle to be used in a column. A liquid characterized by using particles that are supported on the particles and whose overall particle size is adjusted to be large enough to chromatographically separate the particle dispersoid by selecting the size of the carrier particles. Chromatography.
、又は樹脂である、特許請求の範囲第（１）項に記載の
吸着剤。(2) The adsorbent according to claim (1), wherein the material of the carrier particles is glass, ceramic, metal, or resin.
フロールアパタイト、又はクロロアパタイトである、特
許請求の範囲第（１）項に記載の吸着剤。(3) Apatite compounds include hydroxyapatite,
The adsorbent according to claim (1), which is fluorapatite or chloroapatite.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61305681A JPS63157973A (en) | 1986-12-22 | 1986-12-22 | Liquid chromatography |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61305681A JPS63157973A (en) | 1986-12-22 | 1986-12-22 | Liquid chromatography |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS63157973A true JPS63157973A (en) | 1988-06-30 |
Family
ID=17948073
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61305681A Pending JPS63157973A (en) | 1986-12-22 | 1986-12-22 | Liquid chromatography |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63157973A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63182563A (en) * | 1987-01-24 | 1988-07-27 | Sangi:Kk | Packing material for chromatograph |
| JPS6413453A (en) * | 1987-07-08 | 1989-01-18 | Asahi Optical Co Ltd | Packing agent for liquid chromatography |
| JPH02147857A (en) * | 1988-11-29 | 1990-06-06 | Kobe Steel Ltd | Spherical carrier for chromatography separation and production thereof |
| GB2282548A (en) * | 1993-10-05 | 1995-04-12 | Asahi Optical Co Ltd | Granular polymer composite for immobilisation of antigen or antibody |
| JP2007290988A (en) * | 2006-04-24 | 2007-11-08 | Kao Corp | Bacterial plaque formation inhibitor |
| JP2008297284A (en) * | 2007-06-04 | 2008-12-11 | Kao Corp | Dental plaque-formation inhibitor |
-
1986
- 1986-12-22 JP JP61305681A patent/JPS63157973A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63182563A (en) * | 1987-01-24 | 1988-07-27 | Sangi:Kk | Packing material for chromatograph |
| JPS6413453A (en) * | 1987-07-08 | 1989-01-18 | Asahi Optical Co Ltd | Packing agent for liquid chromatography |
| JPH02147857A (en) * | 1988-11-29 | 1990-06-06 | Kobe Steel Ltd | Spherical carrier for chromatography separation and production thereof |
| GB2282548A (en) * | 1993-10-05 | 1995-04-12 | Asahi Optical Co Ltd | Granular polymer composite for immobilisation of antigen or antibody |
| US5540995A (en) * | 1993-10-05 | 1996-07-30 | Asahi Kogaku Kogyo Kabushiki Kaisha | Granular polymer composite and production process thereof as well as diagnostic agent |
| GB2282548B (en) * | 1993-10-05 | 1998-04-29 | Asahi Optical Co Ltd | Granular polymer composite and production process thereof |
| US5897953A (en) * | 1993-10-05 | 1999-04-27 | Asahi Kogaku Kogyo Kabushiki Kaisha | Granular polymer composite and production process thereof as well as diagnostic agent |
| JP2007290988A (en) * | 2006-04-24 | 2007-11-08 | Kao Corp | Bacterial plaque formation inhibitor |
| JP2008297284A (en) * | 2007-06-04 | 2008-12-11 | Kao Corp | Dental plaque-formation inhibitor |
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