JPH01123145A - Filler for column and preparation thereof - Google Patents
Filler for column and preparation thereofInfo
- Publication number
- JPH01123145A JPH01123145A JP62281823A JP28182387A JPH01123145A JP H01123145 A JPH01123145 A JP H01123145A JP 62281823 A JP62281823 A JP 62281823A JP 28182387 A JP28182387 A JP 28182387A JP H01123145 A JPH01123145 A JP H01123145A
- Authority
- JP
- Japan
- Prior art keywords
- group
- filler
- raw material
- hydrophobicity
- hydrophobic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000000945 filler Substances 0.000 title claims abstract description 53
- 125000001165 hydrophobic group Chemical group 0.000 claims abstract description 23
- 125000003277 amino group Chemical group 0.000 claims abstract description 19
- 239000002994 raw material Substances 0.000 claims abstract description 16
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000000741 silica gel Substances 0.000 claims abstract description 8
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 8
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract 3
- 102000004169 proteins and genes Human genes 0.000 claims description 33
- 108090000623 proteins and genes Proteins 0.000 claims description 33
- 239000000463 material Substances 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 23
- 238000012856 packing Methods 0.000 claims description 23
- 239000011148 porous material Substances 0.000 claims description 18
- 238000004519 manufacturing process Methods 0.000 claims description 13
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 10
- 239000000126 substance Substances 0.000 claims description 5
- 238000005342 ion exchange Methods 0.000 claims description 4
- CTKINSOISVBQLD-UHFFFAOYSA-N Glycidol Chemical compound OCC1CO1 CTKINSOISVBQLD-UHFFFAOYSA-N 0.000 claims description 3
- 108010004147 polymyxin acylase Proteins 0.000 claims description 3
- 230000001476 alcoholic effect Effects 0.000 claims description 2
- 239000011521 glass Substances 0.000 claims description 2
- 239000005373 porous glass Substances 0.000 claims description 2
- 125000004432 carbon atom Chemical group C* 0.000 claims 4
- 125000000217 alkyl group Chemical group 0.000 claims 3
- 125000004103 aminoalkyl group Chemical group 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- LYCAIKOWRPUZTN-UHFFFAOYSA-N ethylene glycol Natural products OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 abstract description 6
- 125000000524 functional group Chemical group 0.000 abstract description 6
- 239000003054 catalyst Substances 0.000 abstract description 2
- 125000003827 glycol group Chemical group 0.000 abstract description 2
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 12
- 210000002966 serum Anatomy 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 239000003814 drug Substances 0.000 description 8
- 230000002209 hydrophobic effect Effects 0.000 description 8
- 229940079593 drug Drugs 0.000 description 7
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- DDBREPKUVSBGFI-UHFFFAOYSA-N phenobarbital Chemical compound C=1C=CC=CC=1C1(CC)C(=O)NC(=O)NC1=O DDBREPKUVSBGFI-UHFFFAOYSA-N 0.000 description 6
- AQHHHDLHHXJYJD-UHFFFAOYSA-N propranolol Chemical compound C1=CC=C2C(OCC(O)CNC(C)C)=CC=CC2=C1 AQHHHDLHHXJYJD-UHFFFAOYSA-N 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 229960002695 phenobarbital Drugs 0.000 description 5
- 239000008363 phosphate buffer Substances 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 102000004506 Blood Proteins Human genes 0.000 description 4
- 108010017384 Blood Proteins Proteins 0.000 description 4
- 239000012472 biological sample Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 102000035195 Peptidases Human genes 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- -1 glyceroylpropyl groups Chemical group 0.000 description 3
- 229960003712 propranolol Drugs 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- FTOAOBMCPZCFFF-UHFFFAOYSA-N 5,5-diethylbarbituric acid Chemical compound CCC1(CC)C(=O)NC(=O)NC1=O FTOAOBMCPZCFFF-UHFFFAOYSA-N 0.000 description 2
- 102000000496 Carboxypeptidases A Human genes 0.000 description 2
- 108010080937 Carboxypeptidases A Proteins 0.000 description 2
- 108090000317 Chymotrypsin Proteins 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- 125000002355 alkine group Chemical group 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 229960002376 chymotrypsin Drugs 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 229910001410 inorganic ion Inorganic materials 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 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 2
- 238000003756 stirring Methods 0.000 description 2
- 125000006755 (C2-C20) alkyl group Chemical group 0.000 description 1
- 102100040996 Cochlin Human genes 0.000 description 1
- JBCLFWXMTIKCCB-VIFPVBQESA-N Gly-Phe Chemical compound NCC(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-VIFPVBQESA-N 0.000 description 1
- FEUPVVCGQLNXNP-IRXDYDNUSA-N Gly-Phe-Phe Chemical compound C([C@H](NC(=O)CN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 FEUPVVCGQLNXNP-IRXDYDNUSA-N 0.000 description 1
- 101000748988 Homo sapiens Cochlin Proteins 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 241000589774 Pseudomonas sp. Species 0.000 description 1
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 229940121363 anti-inflammatory agent Drugs 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 229960002319 barbital Drugs 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000011162 core material Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 230000002542 deteriorative effect Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 150000002009 diols Chemical class 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
- 235000013305 food Nutrition 0.000 description 1
- 108010081551 glycylphenylalanine Proteins 0.000 description 1
- 125000005843 halogen group Chemical group 0.000 description 1
- CBOIHMRHGLHBPB-UHFFFAOYSA-N hydroxymethyl Chemical compound O[CH2] CBOIHMRHGLHBPB-UHFFFAOYSA-N 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000011229 interlayer Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 229910000077 silane Inorganic materials 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- 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
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3285—Coating or impregnation layers comprising different type of functional groups or interactions, e.g. different ligands in various parts of the sorbent, mixed mode, dual zone, bimodal, multimodal, ionic or hydrophobic, cationic or anionic, hydrophilic or hydrophobic
Landscapes
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
Description
【発明の詳細な説明】
［産業上の利用分野］
本発明はボア内表面とボア外表面が本質的に異なる機能
を有するクロマトグラのカラム用充填剤及びその製造方
法に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a packing material for a chromatography column in which the inner surface of the bore and the outer surface of the bore have essentially different functions, and a method for producing the packing material.
内外両表面が異なる機能を有する充填剤は、従来の単一
機能の充填剤ζこ比べて多様かつ高度な応用の可能性を
有しており、現在その一例として、血清などの生体試料
の直接分析あるいはその前処理としての除タンパク処理
に応用され、画期的な効果が得られている。本発明の方
法で製造される充填剤は、多様な可能性を有するが、簡
明を期するため、以下、除タンパク前処理に主眼をおい
て説明する。Packing materials with different functions on both the inner and outer surfaces have the potential for more diverse and advanced applications than conventional fillers with single functions. It has been applied to protein removal treatment as a pretreatment for analysis, and has achieved revolutionary effects. The filler produced by the method of the present invention has various possibilities, but for the sake of simplicity, the following explanation will focus on protein removal pretreatment.
［従来の技術］
血清などのタンパク質を含む生体試料中の薬物などの分
析に、クロマトグラフィー、特に高速液体クロマトグラ
フィー（以下、ＨＰＬＣという）は、非常に有効な手段
として汎用されている。このＨＰ Ｌ Ｃ分析では、通
常逆相系の充填剤が多く用いられているが、試料中のタ
ンパク質は、この充填剤に非可逆的に吸着し、カラムに
つまりを生じるなど重大な障害となるため、ＨＰＬＣ分
析を行う萌に、前処理によって除タンパク処理すること
が不可欠である。このような前処理は手作業で行うため
に非常に労力を要するものである。[Prior Art] Chromatography, particularly high performance liquid chromatography (hereinafter referred to as HPLC), is widely used as a very effective means for analyzing drugs and the like in biological samples containing proteins such as serum. In this HPLC analysis, reversed-phase packing materials are usually used, but proteins in the sample are irreversibly adsorbed to this packing material, causing serious problems such as clogging the column. Therefore, it is essential to pre-process protein removal before performing HPLC analysis. Such preprocessing requires a lot of effort because it is performed manually.
しかし、最近になって、充填剤の内表面を疎水性とし、
かつ外表面を親水性にするなどして、タンパク質が吸着
され難くした新しいタイプの充填剤が開発され、これを
前処理用カラムとして、カラムスイッチングバルブとと
もに）（ＰＬＯに組み込み、オンラインで除タンパク処
理ができるようにし、前述のような生体試料を直接分析
に供することが可能となっている。例として、特開昭５
８−２２３０６２号公報及び特開昭６０−５６２５６号
公報に記載された発明があげばれる。特に、後者の発明
によれば、オクタデシル基を化学結合させたシラン（Ｏ
ＤＳ）にタンパク質をコーティングした充填剤が用いら
れている。このタンパク質コーティングＯＤＳは、ＯＤ
Ｓを充填したカラムに適当な不活性タンパク質溶液を注
入し、このタンパク質を積極的にカラムに吸着させるこ
とによって調製される。ＯＤＳの孔径に比べてタンパク
質はサイズが大きいため、孔の中には進入できず、した
がって孔の内表面には初めから導入されているオクタデ
シル基がそのまま残り、疎水性が保存される。一方、外
表面には吸着したタンパク質に覆われ、もはやタンパク
質を吸着する能力を持たない。However, recently, the inner surface of the filler has been made hydrophobic,
In addition, a new type of packing material was developed that made the outer surface hydrophilic to make it difficult for proteins to be adsorbed.This was used as a pretreatment column (along with a column switching valve) (incorporated into PLO for online protein removal processing). It has become possible to directly analyze biological samples such as those mentioned above.For example,
Examples include inventions described in Japanese Patent Application Laid-open No. 8-223062 and Japanese Patent Application Laid-open No. 60-56256. In particular, according to the latter invention, silane (O
DS) is used as a filler coated with protein. This protein-coated ODS is OD
It is prepared by injecting an appropriate inert protein solution into a column packed with S and actively adsorbing the protein onto the column. Since the protein is larger than the pore size of ODS, it cannot enter the pore, and therefore the octadecyl group introduced from the beginning remains on the inner surface of the pore, preserving hydrophobicity. On the other hand, the outer surface is covered with adsorbed proteins and no longer has the ability to adsorb proteins.
この充填剤のカラムを用いて除タンパク前処理を行うに
は、カラムに適当な緩衝液を送液した後、分析すべき血
清等の試料を注入する。このとき、低分子で比較的疎水
性の高い薬物等の成分は、孔内表面のオクタデシル基に
吸着されて保持されるが、一方、試料中のタンパク質等
はサイズが大きいため、孔の内部には進入できず、かつ
外表面にも吸着されないため、短時間でこのカラムより
溶出してしまう。その後、この前処理用カラムの出口側
流路を分離用カラムに接続し、移動相（溶出液）をより
強い条件に切り換えると、保持されていた薬物などの成
分がこの前処理カラムから離れて、分離カラムへ移動し
、ここで各成分ごとに分離されることになる。To perform protein removal pretreatment using a column made of this packing material, a suitable buffer solution is fed to the column, and then a sample such as serum to be analyzed is injected. At this time, components such as drugs that have low molecular weight and relatively high hydrophobicity are adsorbed and retained by the octadecyl groups on the surface of the pores, but on the other hand, proteins in the sample are large and therefore cannot be stored inside the pores. Since it cannot enter the column and is not adsorbed on the outer surface, it will be eluted from this column in a short time. Then, when the outlet side flow path of this pretreatment column is connected to the separation column and the mobile phase (eluate) is switched to stronger conditions, the retained components such as drugs are separated from this pretreatment column. , to a separation column, where each component is separated.
このようにして調製されたタンパク質コートＯＤＳは短
期の使用に関してはほぼ満足すべき性質を有している。The protein-coated ODS prepared in this way has almost satisfactory properties for short-term use.
しかしながら、使用が長期にわたると最初に吸着したタ
ンパク質が溶離をおこし、流れ出して分離カラムに吸着
し、その劣化の原因となったり、また、カラムの保持時
において、微生物などによる腐敗の原因となったりする
おそれがあり、充分な注意が必要であるなどの問題が残
されている。However, if used for a long period of time, the proteins that were initially adsorbed may elute and flow out and adsorb onto the separation column, causing its deterioration.Also, when the column is held, it may cause putrefaction due to microorganisms, etc. Problems remain, including the need for sufficient caution.
除タンパク質前処理の別の方法に、特開昭６１−６５１
５９号公報に記載されたピンカートンらの方法もある。Another method for protein removal pretreatment is disclosed in JP-A-61-651.
There is also the method of Pinkerton et al. described in Publication No. 59.
この方法は充填剤の製法に特徴があり、充填剤の出発物
質としてグリセロイルプロピル基が導入されたシリカゲ
ル（例えば、メルク（Ｍｅｒｃｋ）社から市販されて、
いるＬｉｃｈｒｏｓｏｒｂ Ｄｉｏｌが使用可能であり
、或はシリカゲルに即知の方法でグリセロイルプロピル
基を導入してもよい）あるいは多孔性ガラスを用いる。This method is characterized by the manufacturing method of the filler, and the starting material for the filler is silica gel into which glyceroylpropyl groups have been introduced (for example, commercially available from Merck).
Lichrosorb Diol can be used, or glyceroylpropyl groups can be introduced into silica gel by known methods) or porous glass can be used.
これにカルボニルジイミダゾールを介してオリゴペプチ
ド、典型的にはグリシルフェニルアラニンあるいはグリ
シルフェニルアラニル−フェニルアラニンを結合させる
。この結果、充填剤の内外表面はフェニルアラニンのフ
ェニル基によって疎水性となる。これにタンパク質分解
酵素であるカルボキシペプチダーゼＡもしくはキモトリ
プシンを作用させると、外表面のペプチド結合は切断さ
れ、最初から導入されているグリセロイルプロピル基が
露出し、親水性が回復し、タンパク質に対して非吸着性
となる。An oligopeptide, typically glycylphenylalanine or glycylphenylalanyl-phenylalanine, is bonded to this via carbonyldiimidazole. As a result, the inner and outer surfaces of the filler become hydrophobic due to the phenyl group of phenylalanine. When carboxypeptidase A or chymotrypsin, which is a proteolytic enzyme, is applied to this protein, the peptide bonds on the outer surface are cleaved, the glyceroylpropyl group introduced from the beginning is exposed, the hydrophilicity is restored, and the protein is Becomes non-adsorbent.
しかしながら、酵素は充填剤の孔径より太きいため、こ
の内部には進入できず、従って内表面のペプチドは分解
されずに残り疎水性が保存され、目的にあった性質を有
する充填剤が得られる。However, since the enzyme is larger than the pore size of the filler, it cannot enter into the interior of the filler, so the peptides on the inner surface remain undegraded and remain hydrophobic, allowing a filler with the desired properties to be obtained. .
しかしながら、ピンカートンの方法で得られる充填剤は
、内表面の疎水性が充分満足できるものではないという
間層点がある。すなわち、この製造方法の主眼は、タン
パク質分解酵素によって外表面の疎水性基を分解除去す
る点にあり、したがって、疎水性はペプチドの形で導入
される必要がある。このため、疎水性基が実質的に、フ
ェニルアラニンに限定されるという問題がある。実際、
このカラムを用いて、例えば抗ケイレン剤として使用さ
れるフェノバルビタールを含む血清試料などを前処理す
ると、フェノバルビクールの疎水性はあまり高くないた
め、血清タンパク質が充分に除去される前に、フェノバ
ルビタールが前処理カラムから流失されるおそれがある
。このような問題を解決するためには、疎水性の強さを
種々選択できる充填剤の製造方法が望まれていた。However, the filler obtained by Pinkerton's method has an interlayer point in which the hydrophobicity of the inner surface is not sufficiently satisfactory. That is, the main focus of this production method is to decompose and remove hydrophobic groups on the outer surface using a proteolytic enzyme, and therefore, hydrophobicity must be introduced in the form of a peptide. Therefore, there is a problem that the hydrophobic group is substantially limited to phenylalanine. actual,
If this column is used to pre-treat a serum sample containing phenobarbital, which is used as an anti-inflammatory agent, the hydrophobicity of phenobarbicul is not very high, so the phenobarbital will be removed before the serum proteins are sufficiently removed. Barbital may be washed away from the pretreatment column. In order to solve these problems, there has been a demand for a method for producing fillers that allows for various hydrophobic strengths to be selected.
さらに、多孔性充填剤の内表面と外表面の性質が異なる
充填剤の他の応用例として、例えば、内表面に疎水性基
、外表面にイオン交換基を導入して、試料中の無機イオ
ンと有機物を同時に分析する方法も可能であるが、上記
の従来技術ではこのような性質をもつ充填剤の製造は不
可能であった。Furthermore, as another application example of a porous filler whose inner and outer surfaces have different properties, for example, a hydrophobic group on the inner surface and an ion exchange group on the outer surface can be introduced to absorb inorganic ions in the sample. Although it is possible to simultaneously analyze fillers and organic substances, it has not been possible to produce fillers with such properties using the above-mentioned conventional techniques.
［発明が解決しようとする問題点］
血清等のタンパク質を含む生体試料中の薬物等の分析に
使用する充填剤としては、孔の内表面が疎水性であり、
外表面が親水性であって、タンパク質を吸着しないとい
う性質が必要である。[Problems to be solved by the invention] As a packing material used for analysis of drugs, etc. in biological samples containing proteins such as serum, the inner surface of the pores is hydrophobic,
The outer surface must be hydrophilic and not adsorb proteins.
本発明の目的は、内表面と外表面の性質が異なる多孔性
充填剤及びその製造方法を提供することにある。より詳
しくは、本発明は疎水性の内表面及び好ましくは親水性
の外表面を有する充填剤の製造にあたり、内表面の疎水
性及び外表面の親水性をともに化学的に導入された官能
基で形成できるため、内外両表面の性質、特に内表面の
疎水性をもたらす官能基を幅広くデザインすることがで
き、その疎水性を容易に調節することができる製造方法
を提供することにある。An object of the present invention is to provide a porous filler whose inner and outer surfaces have different properties, and a method for producing the same. More specifically, in producing a filler having a hydrophobic inner surface and preferably a hydrophilic outer surface, the present invention provides for both the hydrophobicity of the inner surface and the hydrophilicity of the outer surface to be achieved by chemically introduced functional groups. The object of the present invention is to provide a manufacturing method that allows a wide range of functional groups to be designed to provide properties of both the inner and outer surfaces, particularly hydrophobicity of the inner surface, and to easily adjust the hydrophobicity.
本発明の他の目的は、血清中の成分を分析するための充
填剤として使用されるとき、血清タンパク質を実質的に
吸着せずに、カラムから速やかに流出せしめ、これによ
り、分析の精度を向上させることのできる充填剤及びそ
の製造方法を提供することにある。Another object of the present invention is that when used as a packing material for analyzing components in serum, it allows serum proteins to flow out of the column quickly without substantially adsorbing them, thereby increasing the accuracy of the analysis. An object of the present invention is to provide a filler that can be improved and a method for producing the same.
本発明のさらに他の目的は、クロマトグラフィーのタン
パク前処理用カラムの充填剤として使用した時、タンパ
ク質の流出により分離カラムを劣化させるおそれがなく
、あるいはカラム保持時の腐敗の問題がない充填剤及び
その製造方法を提供することにある。Still another object of the present invention is to provide a packing material that, when used as a packing material for a column for protein pretreatment in chromatography, does not cause the risk of deteriorating the separation column due to protein leakage or the problem of corruption during column retention. An object of the present invention is to provide a method for manufacturing the same.
［問題点を解決するための手段］
本発明に係るカラム用充填剤の製造方法では、実質的に
タンパク質を浸透させない孔径をもつ、多孔性材料から
なる充填剤原料の内表面及び外表面の両面に、アミド結
合を有する疎水性基を結合させ、次いで該アミド結合に
ポリミキシン・アシラーゼを作用させて、前記外表面に
結合した疎水性基についてのみアミド結合を切断してア
ミノ基を生ぜしめ、必要に応じて該アミノ基をさらに別
の基に化学修飾することよりなることを特徴としている
。[Means for Solving the Problems] In the method for producing a column packing material according to the present invention, both the inner and outer surfaces of a packing material material made of a porous material having a pore size that does not substantially allow protein to penetrate. A hydrophobic group having an amide bond is bonded to the amide bond, and then polymyxin acylase is allowed to act on the amide bond to cleave the amide bond only for the hydrophobic group bonded to the outer surface to generate an amino group. It is characterized by further chemically modifying the amino group into another group depending on the conditions.
本発明の方法の好ましい態様の要点を第１図に示す。こ
の図かられかるとおり、本発明の方法は大まかに３つの
ステップからなる。A summary of a preferred embodiment of the method of the invention is shown in FIG. As can be seen from this figure, the method of the present invention roughly consists of three steps.
最初のステップは、実質的にタンパク質を浸透させない
孔径をもつ、多孔性材料からなる充填剤原料を用意し、
この原料にアミド結合を有する疎水性基を導入する。こ
のような原料としては、アミド結合を有する疎水性の基
が導入可能であり、かつ該原料の孔が内部に該疎水性の
基を導入できる大きさであること等が必要とされるが、
これらの条件を満たすものであれば、実質的に任意の充
填剤原料が利用可能である。例えば、原料がシリカゲル
タイプのものであるときは、その表面に官能基として第
１図に示すように、
が存在するから、この末端の水酸基に対してγ−アミノ
プロピル・トリエトキシシランのような試薬を反応させ
て、末端アミノ基を形成し、次にこのアミノ基にＲ，Ｃ
ＯＣｌ２（式中、Ｒ８は疎水性を付与する基、例えば炭
素原子数２ないし２０のアルキル基またはフェニル基そ
の他である）を好ましくはトリエチルアミンのような塩
基性触媒の存在下で反応させて、アミド結合を有する疎
水性基を充填剤の内外両表面に２ステツプで導入するこ
とができる。その際、市販品として入手可能な、Ｓ ｐ
ｈｅｒｉｓｏｒｂ ５ Ｎ Ｈ＊ （フェーズ・セバレ
ーシッン社製） 、Ｌｉｃｈｒｏｓｏｒｂ Ｎ Ｈ＊
（メルク社製）、などのアミノ樹脂を使用すれば、上記
末端アミノ基の形成ステップが省略できるから都合がよ
い。The first step is to prepare a filler material consisting of a porous material with a pore size that is virtually impermeable to proteins.
A hydrophobic group having an amide bond is introduced into this raw material. Such a raw material is required to be capable of introducing a hydrophobic group having an amide bond, and that the pores of the raw material are large enough to allow the introduction of the hydrophobic group into the inside.
Substantially any filler raw material can be used as long as it meets these conditions. For example, when the raw material is a silica gel type, there is a functional group on its surface as shown in Figure 1. The reagents are reacted to form a terminal amino group, which is then labeled with R,C
OCl2 (wherein R8 is a group imparting hydrophobicity, such as a C2-C20 alkyl group or a phenyl group, etc.) is reacted, preferably in the presence of a basic catalyst such as triethylamine, to form an amide. Hydrophobic groups with bonds can be introduced on both the inner and outer surfaces of the filler in two steps. At that time, Sp, which is available as a commercial product,
herisorb 5 NH* (manufactured by Phase Sebareshing Co., Ltd.), Lichrosorb NH*
(manufactured by Merck & Co., Ltd.) is convenient because the step of forming the terminal amino group can be omitted.
あるいはシリカゲルから！ステップで疎水性基を都合よ
く導入することも出来る。Or from silica gel! Hydrophobic groups can also be conveniently introduced in a step.
本発明の方法における、次のステップは、前記充填剤の
外表面のアミド結合を選択的に切断する工程である。本
発明においてこの切断工程をポリミキンアシラーゼで行
うことが、特に重要である。The next step in the method of the present invention is to selectively cleave the amide bonds on the outer surface of the filler. It is particularly important in the present invention that this cleavage step is performed with polymykin acylase.
この酵素は、一般の酵素に比べて極めて基質特異性が小
さく、極めて多様なアミド結合を切断する能力を有する
。そして、酵素としては例外的ともいえるこの特異性の
低さが、本発明の方法を可能ならしめたのである。すな
わち、ポリミキンアシラーゼは、その特異性の低さの故
に、疎水性を導入する官能基（第１図のＲ，）を自由に
デザインすることが可能となり、充填剤に要求される疎
水性の強さに応じてＣ１からＣ２゜程度までの直鎖また
は枝別れアルキン基（フェニル基、ハロゲン原子、ニト
ロ基その他の疎水性成分で置換されていてもよい）から
選択が可能である。また％Ｒ１としてフェニル基、置換
フェニル基その他のアリール基等の種々の性質・性能の
ものから自由にデザインすることが可能である。This enzyme has extremely low substrate specificity compared to general enzymes and has the ability to cleave a wide variety of amide bonds. This low specificity, which can be said to be exceptional for an enzyme, made the method of the present invention possible. In other words, due to its low specificity, polymicin acylase allows the functional group that introduces hydrophobicity (R, in Figure 1) to be freely designed, thereby achieving the hydrophobicity required for the filler. It is possible to select from linear or branched alkyne groups (which may be substituted with phenyl groups, halogen atoms, nitro groups, or other hydrophobic moieties) having a C1 to C2 degree degree depending on the strength of the alkyne group. Furthermore, %R1 can be freely designed from among phenyl groups, substituted phenyl groups, and other aryl groups with various properties and performances.
この点は、前記ピンカートンの方法に比べて、本発明の
著しい利点である。何故ならピンカートンの方法では、
用いている酵素がタンパク質分解酵素であるカルボキシ
ペプチダーゼＡもしくはキモトリプシンであり、その高
い特異性のために、導入する疎水性基はアミノ酸によら
なければならないという重大な制約が生じ、結局のとこ
ろフェニルアラニンによるフェニル基という不十分な疎
水性しか実現できず、これが前述の問題を生じているた
めである。This is a significant advantage of the present invention over the Pinkerton method. This is because Pinkerton's method
The enzyme used is carboxypeptidase A or chymotrypsin, which is a proteolytic enzyme, and due to its high specificity, there is a serious constraint that the hydrophobic group introduced must be an amino acid, and in the end, the hydrophobic group introduced must be an amino acid. This is because only the insufficient hydrophobicity of the phenyl group can be achieved, which causes the above-mentioned problems.
本発明の方法における、最後のステップは、上記ステッ
プにより充填剤の外表面に生じたアミノ基（−Ｎ ｉ−
ｔ 、）を、所望によりさらに別の基に変換することよ
りなる（例えば、第１図中の−ＮＨＲｆに変換する：但
しＲ１は所望の性質、好ましくはより良好な親水性を付
与する基である。）外表面の親水性を高め、タンパク質
に対する吸着能力を失わせるために、グリシドールを用
いてアミノ基にグリコール等のアルコール性の基を導入
したり（ｒｔ、＝−ＣＨｆＣＩ（（ＯＨ）ＣＨ２ＯＨ）
、酢酸のような低級脂肪酸によりアシル化することが
できる（ Ｒｔ ＝ ＣＯＣＨｓ ）。このような基
の導入は、従来知られている化学反応を応用して行うこ
とができる。さらに、充填剤の外表面にイオン交換基、
例えば４級アンモニウム基に誘導すれば、本発明の方法
で製造された充填剤は、無機イオンと有機化合物を同時
に分析することが可能となる、そのような応用例として
、食品や医薬品などの分析が挙げられる。The last step in the method of the present invention is to remove the amino groups (-N i-
t, ) to another group if desired (for example, to -NHRf in FIG. 1, where R1 is a group that imparts the desired properties, preferably better hydrophilicity). ) In order to increase the hydrophilicity of the outer surface and lose its ability to adsorb proteins, glycidol is used to introduce an alcoholic group such as glycol into the amino group (rt, =-CHfCI((OH)CH2OH )
, can be acylated with lower fatty acids such as acetic acid (Rt = COCHs). Introduction of such groups can be carried out by applying conventionally known chemical reactions. Furthermore, ion exchange groups on the outer surface of the filler,
For example, when induced into a quaternary ammonium group, the filler produced by the method of the present invention can be used to simultaneously analyze inorganic ions and organic compounds.An example of such an application is the analysis of foods, pharmaceuticals, etc. can be mentioned.
また、本発明に係るカラム用充填剤では、実質的にタン
パク質を浸透させない孔径を有するガラスもしくはシリ
カゲルの多孔性材料の内表面に、を介して結合した疎水
性基−（ＣＨｙ）。−ＮＨＣＯ−Ｒ、（式中、Ｒ８は疎
水性を与える成分であり、ｎは１ないし３の数である）
を有し、モして核材を介して結合した−（ＣＨ＊）−Ｎ
Ｈを基もしくは談話より誘導された基を有してなる、
内表面が疎水性を有し、外表面が前記内表面とは異なる
性質を有することを特徴としている。In addition, in the column packing material according to the present invention, a hydrophobic group -(CHy) is bonded to the inner surface of a porous material such as glass or silica gel having a pore size that does not substantially allow protein to penetrate. -NHCO-R, (wherein R8 is a component that provides hydrophobicity, and n is a number from 1 to 3)
-(CH*)-N bonded via the core material
having a group based on H or a group derived from discourse,
It is characterized in that the inner surface is hydrophobic and the outer surface has different properties from the inner surface.
［実施例］
（実施例１ 充填剤の製造）
本実施例の充填剤は、第２図に示す工程に従って、次の
ように製造した。[Example] (Example 1 Manufacture of filler) The filler of this example was manufactured as follows according to the steps shown in FIG.
（１）アミド結合を有する疎水性基の導入５ｐｈｅｒｉ
ｓｏｒｂ ５ Ｎ ）Ｉ ｔ １ ｇにベンゼン２０
ＩＩｌｃを加え、撹拌しながら、ｎ−塩化カプリリル０
．３９ｇ及びトリエチルアミン０．０６ｇを徐々に加え
る。その後、室温で５時間撹拌を続ける。４０ｔａＱの
ベンゼン及び４０＠Ｑのメタノールで洗浄し、５ｐｈｅ
ｒｉｓｏｒｂ ５ Ｎ ＨｔのＮ−カプリル誘導体を得
る。(1) Introduction of hydrophobic group with amide bond 5pheri
sorb 5 N ) I t 1 g to benzene 20
Add IIlc and, while stirring, add n-caprylyl chloride 0.
．． Gradually add 39 g and 0.06 g of triethylamine. Stirring is then continued at room temperature for 5 hours. Washed with 40 taQ benzene and 40@Q methanol, 5phe
An N-caprylic derivative of risorb 5 N Ht is obtained.
（２）アミド結合の切断
上記で得られた５ｐｈｅｒｉｓｏｒｂ ５ Ｎ Ｈｚの
Ｎ−カプリル誘導体１．８９に０．２Ｍりん酸緩衝液（
Ｐ１１７゜５）９０ｍｉ２を加える。これに、シュード
モナス（Ｐｓｅｕｄｏｓｏｎａｓ） ｓｐ、 Ｍ −６
−３株菌体よりトリメンＸ−１００で可溶化したポリミ
キシン・アシラーゼの水溶液３ （１ｍ（！（３００単
位、基質：コリスチン）を加え撹拌すると脂肪酸基の導
入された５ｐｈｅｒｉｓｏｒｂは次第に水になじみ均一
化する。これを５０℃の水浴中で軽く振りながら４０時
間反応させる。遠心分離（７０００Ｇ、３０分間）で酵
素液を除去したシリカを０．１Ｍりん酸緩衝液（ＰＨ７
，５）でよく洗浄し、次いで５０％エチレングリコール
含有０．０１Ｍりん酸緩衝液（ＰＨ７，５）、０．０１
Ｍりん酸緩衝液（ＰＩ（７，５）、水、メタノールの順
序に洗浄し、乾燥する。これにより、外表面にアミノ基
を有する本発明の充填剤を得る。(2) Cleavage of amide bond Add 1.89% of the 5pherisorb 5N Hz N-caprylic derivative obtained above to 0.2M phosphate buffer (
P117゜5) Add 90mi2. In addition, Pseudomonas sp, M-6
- When an aqueous solution of polymyxin acylase solubilized with Trimene This is allowed to react for 40 hours while shaking gently in a water bath at 50°C.The silica from which the enzyme solution has been removed by centrifugation (7000G, 30 minutes) is mixed with 0.1M phosphate buffer (PH7).
, 5), and then 0.01M phosphate buffer containing 50% ethylene glycol (PH7.5), 0.01
Wash with M phosphate buffer (PI(7,5), water, and methanol in this order) and dry. As a result, the filler of the present invention having amino groups on the outer surface is obtained.
（３）充填剤外表面のアミノ基の修飾
前記で得られた充填剤の外表面のアミノ基を修飾するた
めの、この充填剤１９に対して、３ｓ１２のグリシドー
ルを加え、室温で２４時間撹拌する。４０ＩＩＩ２の水
及び４０ｍ（ｌのメタノールで洗浄し、親水性の大きい
グリコール基が外表面に導入された、本発明の充填剤を
得る。(3) Modification of amino groups on the outer surface of the filler To modify the amino groups on the outer surface of the filler obtained above, 3s12 of glycidol was added to this filler 19 and stirred at room temperature for 24 hours. do. Washing with 40 ml of water and 40 ml of methanol yields a filler of the invention in which highly hydrophilic glycol groups have been introduced on the outer surface.
（実施例２ 充填剤の使用例）
実施例１で調製した充填剤を、内径４．６■、長す５ｃ
ｌＩのＨＰＬＣ用ステシステンレスカラムした。このカ
ラムに、下記の条件でサンプルＡ及びサンプルＢを注入
し、フエノバルビタール（１）及びプロプラノロール（
２）の分離を調べた。(Example 2 Example of use of filler) The filler prepared in Example 1 was used in a mold with an inner diameter of 4.6 cm and a length of 5 cm.
A stainless steel column for HPLC was used. Sample A and sample B were injected into this column under the following conditions, and phenobarbital (1) and propranolol (
The separation of 2) was investigated.
サンプルＡ：ヒトコントロール血清に５０μ９／ ｖａ
Ｑのフェノバルビタール及び１００
μＷ／ｌａＱのプロプラノロールを標準物質として添加
したもの
サンプルＢ：ヒトコントロール血清
移動相： １００ｍＭ Ｎ、Ｈ＊Ｐ Ｏａ −１０
（ｌａＭＮ、、ＨＰＯ，−ＣＨ＋ＣＮ （４：４：２）
流速： ０．６ｍ１２／分
検出波長： ２４５ｎｍ
感度： ０．０４ａｕｆｓ
カラム温度：室温
注入量： 夏Ｏｕｌ
得られたクロマトグラムを第３図に示す。サンプル八で
は、ヒト血清タンパク質の溶出ピークに続いて、フェノ
バルビタール（同図（Ａ）中のピークり及びプロプラノ
ロール（同図（Ａ）中のピーク２）が良好に分離され、
相次いで溶出された。Sample A: 50μ9/va in human control serum
Q of phenobarbital and 100 μW/laQ of propranolol were added as standards Sample B: Human control serum Mobile phase: 100 mM N, H*P Oa -10
(laMN,,HPO,-CH+CN (4:4:2)
Flow rate: 0.6 m12/min Detection wavelength: 245 nm Sensitivity: 0.04 aufs Column temperature: Room temperature Injection amount: Summer Oul The obtained chromatogram is shown in FIG. In sample 8, following the elution peak of human serum proteins, phenobarbital (peak 2 in the figure (A)) and propranolol (peak 2 in the figure (A)) were well separated;
They were eluted one after another.
サンプルＢでは、ヒト血清タンパク質の鋭い溶出ピーク
のみが観察された（同図（Ｂ））。In sample B, only a sharp elution peak of human serum proteins was observed ((B) of the same figure).
［発明の効果］
以上のようにして製造された本発明のカラム用充填剤は
、その外表面に充分な親水性を有することができ、血清
中のタンパク質を殆ど吸着することがないから、ＨＰＬ
Ｃカラムに充填して血清などの生体試料中に含まれてい
る薬物の分析に特に有用である。タンパク質は、速やか
にカラムから流出し、薬物はその性質に応じて充填剤内
表面の疎水性基に吸着され、吸着の強さに従ってカラム
中で分離される。従って、本発明の充填剤を用いて直接
試料を分析することもでき、あるいはこの充填剤を前処
理カラムに詰めて除タンパク処理に使用することもでき
る。[Effects of the Invention] The column packing material of the present invention produced as described above can have sufficient hydrophilicity on its outer surface and hardly adsorbs proteins in serum.
It is particularly useful when packed in a C column to analyze drugs contained in biological samples such as serum. Proteins quickly flow out of the column, and drugs are adsorbed to hydrophobic groups on the inner surface of the packing material depending on their properties, and separated in the column according to the strength of adsorption. Therefore, the packing material of the present invention can be used to directly analyze a sample, or the packing material can be packed into a pretreatment column and used for protein removal treatment.
また、本発明の充填剤は、内表面の官能基が自由に設計
できるので、その疎水性を自由に！１！節することがで
き、前記ピンカートンの充填剤におけ−る問題点を解決
できる。In addition, the filler of the present invention allows the functional groups on the inner surface to be freely designed, so its hydrophobicity can be adjusted freely! 1! The problem with the Pinkerton filler can be solved.
第１図は本発明のカラム用充填剤を製造する好ましい態
様を説明する工程図であり、第２図は実施例１における
充填剤の製造工程図であ、す、第３図（Ａ）、（Ｂ）は
実施例！で製造された充填剤を、ヒト血清中に含まれる
薬物の分析に使用したときのクロマトグラムである。FIG. 1 is a process diagram illustrating a preferred embodiment of manufacturing the column packing material of the present invention, and FIG. 2 is a manufacturing process diagram of the packing material in Example 1. (B) is an example! This is a chromatogram obtained when the packing material manufactured in 1997 was used for analysis of drugs contained in human serum.
Claims (15)
孔性材料からなる充填剤原料の内表面及び外表面の両面
に、アミド結合を有する疎水性基を結合させ、次いで該
アミド結合にポリミキシン・アシラーゼを作用させて、
前記外表面に結合した疎水性基についてのみアミド結合
を切断してアミノ基を生ぜしめ、必要に応じて該アミノ
基をさらに別の基に化学修飾することによりなる、内表
面が疎水性を有し、外表面が内表面とは異なる性質を有
する多孔性のカラム用充填剤の製造方法。(1) A hydrophobic group having an amide bond is bonded to both the inner and outer surfaces of a filler raw material made of a porous material with a pore size that does not substantially allow protein to penetrate, and then polymyxin acylase is attached to the amide bond. By acting,
The inner surface has hydrophobicity by cleaving the amide bond of only the hydrophobic group bonded to the outer surface to generate an amino group, and if necessary, chemically modifying the amino group into another group. and a method for producing a porous column packing material in which the outer surface has different properties from the inner surface.
ラスであり、該原料の表面にアミド結合を有する疎水性
基を結合させるに際して、該原料表面上の▲数式、化学
式、表等があります▼ の水酸基に炭素原子数２〜２０のアミノアルキル基を導
入し、次にこのアミノ基をアシル化剤でアシル化する、
特許請求の範囲第１項に記載の方法。(2) When the filler raw material is porous silica gel or porous glass, and when bonding a hydrophobic group having an amide bond to the surface of the raw material, there are ▲mathematical formulas, chemical formulas, tables, etc.▼ on the surface of the raw material. Introducing an aminoalkyl group having 2 to 20 carbon atoms into the hydroxyl group, and then acylating this amino group with an acylating agent.
A method according to claim 1.
＿１（式中、Ｒ＿１は疎水性を与える成分であり、ｎは
１ないし３の数である）である、特許請求の範囲第２項
に記載の方法。(3) A hydrophobic group with an amide bond is substituted with the OH of the ▲mathematical formula, chemical formula, table, etc.▼ group on the surface of the raw material -(CH_2)_n-NHCO-R
_1 (wherein R_1 is a component imparting hydrophobicity and n is a number from 1 to 3).
、特許請求の範囲第３項に記載の方法。(4) The method according to claim 3, wherein R_1 is an alkyl group having 2 to 20 carbon atoms.
む基である、特許請求の範囲第３項に記載の方法。(5) The method according to claim 3, wherein R_1 is a phenyl group or a group containing a substituted phenyl group.
学修飾して、アルコール性水酸基を導入することにより
外表面が親水性の充填剤を製造する、特許請求の範囲第
１項乃至第５項のいずれか１項に記載の方法。(6) Claims 1 to 5, in which a filler having a hydrophilic outer surface is produced by chemically modifying amino groups formed on the outer surface with glycidol and introducing alcoholic hydroxyl groups. The method according to any one of the above.
外表面が親水性の充填剤を製造する特許請求の範囲第１
項乃至第５項のいずれか１項に記載の方法。(7) Acetylate the amino groups generated on the outer surface,
Claim 1 for producing a filler whose outer surface is hydrophilic
6. The method according to any one of items 5 to 5.
学修飾して、外表面にイオン交換基が導入された充填剤
を製造する、特許請求の範囲第１項乃至第５項のいずれ
か１項に記載の方法。(8) Any of claims 1 to 5, wherein a filler having an ion exchange group introduced to the outer surface is produced by chemically modifying the amino group formed on the outer surface with an ion exchange group. or the method described in paragraph 1.
ｏｒｂ）５ＮＨ＿２である、特許請求の範囲第１項乃至
第８項のいずれか１項に記載の方法。(9) The raw material for the filler is Spherisolb (Spherisolb).
orb)5NH_2.
るガラスもしくはシリカゲルの多孔性材料の内表面に、 ▲数式、化学式、表等があります▼ を介して結合した疎水性基−（ＣＨ＿２）＿ｎ−ＮＨＣ
Ｏ−Ｒ＿１（式中、Ｒ＿１は疎水性を与える成分であり
、ｎは１ないし３の数である）を有し、そして該材料の
外表面には▲数式、化学式、表等があります▼ を介して結合した−（ＣＨ＿２）＿ｎ−ＮＨ＿２基もし
くは該基より誘導された基を有してなる、内表面が疎水
性を有し、外表面が前記内表面とは異なる性質を有する
ことを特徴とする多孔性のカラム用充填剤。(10) On the inner surface of a porous material such as glass or silica gel, which has a pore size that does not substantially allow protein to penetrate, there is a hydrophobic group bonded via -(CH_2)_n-NHC.
O-R_1 (in the formula, R_1 is a component that provides hydrophobicity, and n is a number from 1 to 3), and the outer surface of the material has ▲mathematical formula, chemical formula, table, etc.▼ -(CH_2)_n-NH_2 group or a group derived from the group bonded via -(CH_2)_n-NH_2 group, characterized in that the inner surface has hydrophobicity and the outer surface has different properties from the inner surface. Packing material for porous columns.
ある、特許請求の範囲第１０項に記載の充填剤。(11) The filler according to claim 10, wherein R_1 is an alkyl group having 2 to 20 carbon atoms.
含む基である、特許請求の範囲第１０項に記載の充填剤
。(12) The filler according to claim 10, wherein R_1 is a group containing a phenyl group or a substituted phenyl group.
た外表面の基が、 ▲数式、化学式、表等があります▼（炭素原子数１また
は２ のアルキル基）である、特許請求の範囲第１０項に記載
の充填剤。(13)-(CH_2)_n-NH_2 The group on the outer surface derived from the group is ▲a mathematical formula, a chemical formula, a table, etc.▼(alkyl group having 1 or 2 carbon atoms) Filler according to item 10.
た外表面の基が、 −（ＣＨ＿２）＿ｎ−ＮＨ−ＣＨ（ＯＨ）ＣＨ＿２ＯＨ
である、特許請求の範囲第１０項に記載の充填剤。(14) The group on the outer surface derived from the -(CH_2)_n-NH_2 group is -(CH_2)_n-NH-CH(OH)CH_2OH
The filler according to claim 10, which is
る、特許請求の範囲第１０項乃至第１４項のいずれか１
項に記載の充填剤。(15) Any one of claims 10 to 14, wherein the raw material of the filler is Spherisolve 5NH_2.
Fillers as described in Section.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62281823A JPH01123145A (en) | 1987-11-07 | 1987-11-07 | Filler for column and preparation thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62281823A JPH01123145A (en) | 1987-11-07 | 1987-11-07 | Filler for column and preparation thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01123145A true JPH01123145A (en) | 1989-05-16 |
Family
ID=17644493
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62281823A Pending JPH01123145A (en) | 1987-11-07 | 1987-11-07 | Filler for column and preparation thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01123145A (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5135649A (en) * | 1989-10-02 | 1992-08-04 | Shiseido Company Ltd. | Column packing material with both hydrophobic and hydrophilic groups and process for production thereof |
| WO1994008686A1 (en) * | 1992-10-21 | 1994-04-28 | Cornell Research Foundation, Inc. | Multimodal chromatographic separation media and process for usingsame |
| US5672422A (en) * | 1991-02-28 | 1997-09-30 | Shiseido Company Ltd. | Packing material for column and process for production thereof |
| US5738783A (en) * | 1994-05-09 | 1998-04-14 | Shiseido Company, Ltd. | Liquid chromatograph having a micro- and semi-micro column |
| US6900157B2 (en) * | 2001-09-10 | 2005-05-31 | Tosoh Corporation | Process for production of partially hydrophilized porous adsorbents |
| 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 |
| US7838306B2 (en) | 2000-09-14 | 2010-11-23 | Fresenius Medical Care Deutschland Gmbh | Adsorbent having differently modified surface areas, method for the production thereof and use of the same |
| JP2015527596A (en) * | 2012-09-11 | 2015-09-17 | ダイオネックス コーポレイション | Glycidol functionalized anion exchange stationary phase |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6165159A (en) * | 1984-08-31 | 1986-04-03 | パーデュー・リサーチ・ファウンデーション | Inner-surface reversed-phase filling material for liquid chromatograph-column and manufacture thereof |
-
1987
- 1987-11-07 JP JP62281823A patent/JPH01123145A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6165159A (en) * | 1984-08-31 | 1986-04-03 | パーデュー・リサーチ・ファウンデーション | Inner-surface reversed-phase filling material for liquid chromatograph-column and manufacture thereof |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5135649A (en) * | 1989-10-02 | 1992-08-04 | Shiseido Company Ltd. | Column packing material with both hydrophobic and hydrophilic groups and process for production thereof |
| US5672422A (en) * | 1991-02-28 | 1997-09-30 | Shiseido Company Ltd. | Packing material for column and process for production thereof |
| WO1994008686A1 (en) * | 1992-10-21 | 1994-04-28 | Cornell Research Foundation, Inc. | Multimodal chromatographic separation media and process for usingsame |
| US5738783A (en) * | 1994-05-09 | 1998-04-14 | Shiseido Company, Ltd. | Liquid chromatograph having a micro- and semi-micro column |
| US6063283A (en) * | 1994-05-09 | 2000-05-16 | Shiseido Company, Ltd. | Method for analyzing a sample by using a liquid chromatograph |
| 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 |
| US7838306B2 (en) | 2000-09-14 | 2010-11-23 | Fresenius Medical Care Deutschland Gmbh | Adsorbent having differently modified surface areas, method for the production thereof and use of the same |
| US6900157B2 (en) * | 2001-09-10 | 2005-05-31 | Tosoh Corporation | Process for production of partially hydrophilized porous adsorbents |
| JP2015527596A (en) * | 2012-09-11 | 2015-09-17 | ダイオネックス コーポレイション | Glycidol functionalized anion exchange stationary phase |
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