JPH0780294A - Composite adsorbent - Google Patents
Composite adsorbentInfo
- Publication number
- JPH0780294A JPH0780294A JP5224717A JP22471793A JPH0780294A JP H0780294 A JPH0780294 A JP H0780294A JP 5224717 A JP5224717 A JP 5224717A JP 22471793 A JP22471793 A JP 22471793A JP H0780294 A JPH0780294 A JP H0780294A
- Authority
- JP
- Japan
- Prior art keywords
- hydrophilic polymer
- porous body
- composite adsorbent
- cross
- chitosan
- 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
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は液体クロマトグラフィー
用担体として有用な複合化吸着剤に関する。詳しくは、
本発明は強度の弱い架橋ゲルを、キトサン多孔質体の細
孔内に埋め込んでなる、大きな吸着能と強度を有し、通
液性に優れ、かつ体積変化の少ない吸着剤に関する。FIELD OF THE INVENTION The present invention relates to a composite adsorbent useful as a carrier for liquid chromatography. For more information,
The present invention relates to an adsorbent obtained by embedding a weakly crosslinked gel in the pores of a chitosan porous body, having a large adsorption capacity and strength, excellent liquid permeability, and a small volume change.
【０００２】[0002]
【従来の技術】タンパク質に代表される生体高分子の吸
着剤としては、主に 合成高分子からなる多孔質型のイオン交換体 親水性高分子の架橋ゲルからなるイオン交換体 が用いられている。2. Description of the Related Art As an adsorbent for a biopolymer represented by a protein, a porous ion exchanger mainly composed of a synthetic polymer and an ion exchanger composed of a crosslinked gel of a hydrophilic polymer are used. .
【０００３】上記の合成高分子からなる多孔質型のイ
オン交換体は、これと接触する溶液のｐＨ、塩濃度によ
る体積変化が小さく、液体クロマトグラフィーの担体と
して用いた場合、通液時の耐圧性が良いという利点を有
している。しかし、このイオン交換体は、タンパク質の
分離に用いた場合、疎水的相互作用に基づく非特異吸着
が生起し、クロマトグラムのピークに非対称化が起きた
り、吸着されたタンパク質が吸着されたままで回収でき
ない事があるといった問題点がある。また、合成高分子
に弱塩基性陰イオン交換基を導入してなるイオン交換体
は、イオン交換基の導入量を多くして交換容量を大きく
しても、タンパク質の吸着量が低いという欠点がある。The above-mentioned porous type ion exchanger made of a synthetic polymer has a small volume change due to pH and salt concentration of a solution in contact therewith, and when used as a carrier for liquid chromatography, it has a pressure resistance during liquid passage. It has the advantage of good performance. However, when this ion exchanger is used for protein separation, non-specific adsorption due to hydrophobic interaction occurs, the peak of the chromatogram becomes asymmetric, and the adsorbed protein is recovered as adsorbed. There is a problem that there are things that can not be done. In addition, an ion exchanger obtained by introducing a weakly basic anion exchange group into a synthetic polymer has a drawback that the amount of adsorbed protein is low even if the amount of ion exchange group introduced is increased to increase the exchange capacity. is there.
【０００４】一方、上記のデキストラン、アガロース
等の多糖に代表される親水性高分子の架橋ゲルからなる
イオン交換体は、タンパク質の非特異吸着が殆ど無いと
いう利点がある。しかし、逆にこのイオン交換体は、水
溶液中で著しく膨潤したり、溶液のイオン強度による体
積変化及び、遊離型と負荷型との体積変化が大きく、か
つ機械的強度も小さいという欠点を有する。特に、この
イオン交換体をクロマトグラフィーの担体として使用す
る場合、通液時の圧力損失が大きく、かつ通液により圧
密化するといった欠点がある。On the other hand, the ion exchanger comprising a crosslinked gel of hydrophilic polymer represented by polysaccharides such as dextran and agarose has an advantage that non-specific adsorption of proteins is hardly present. However, conversely, this ion exchanger has drawbacks that it significantly swells in an aqueous solution, undergoes a large volume change due to the ionic strength of the solution, a large volume change between the free type and the loaded type, and has a low mechanical strength. In particular, when this ion exchanger is used as a carrier for chromatography, there are drawbacks in that the pressure loss during the passage of the liquid is large and the liquid is densified.
【０００５】親水性高分子の架橋ゲルが持つ上記の欠点
を克服する為に、いわば「骨格」となる剛直な物質と組
み合わせる試みがこれまでになされている。例えば、米
国特許第４９６５２８９号では、多孔性高分子の細孔内
にゲルを保持した複合体を、ペプチド合成の分野で用い
ている。硬い高分子物質でゲルを包囲するため容積変化
が無く、カラムに充填して用いてもカラムを通過するフ
ロースルーの圧力が変化しないという効果を上げてい
る。しかし、この複合体の多孔性高分子は７５％以上と
いう大きな細孔容積を有しているので、いわゆる“骨
格”部分が少なく、従って強度が弱いと考えられる。さ
らに、この複合体は粉砕されたもので球状ではないた
め、クロマトグラフィーの担体として使用した場合、満
足な分離が得られない。In order to overcome the above-mentioned drawbacks of the crosslinked gel of hydrophilic polymer, attempts have been made so far to combine it with a rigid substance which is a so-called "skeleton". For example, in US Pat. No. 4,965,289, a complex in which a gel is held in the pores of a porous polymer is used in the field of peptide synthesis. Since the gel is surrounded by a hard polymer substance, there is no change in volume, and the effect of not changing the pressure of the flow-through that passes through the column even when the gel is packed in the column and used. However, since the porous polymer of this composite has a large pore volume of 75% or more, it is considered that the so-called "skeleton" portion is small and therefore the strength is weak. Furthermore, since this complex is ground and not spherical, a satisfactory separation cannot be obtained when used as a carrier for chromatography.
【０００６】米国特許第４３３６１６１号では、セライ
ト等の無機多孔質体にセルロースなどのキセロゲルを保
持させ、クロマトグラフィーの担体として使用してい
る。その結果として、カラムでの通液性の良さをあげて
いるが、セライト等の無機物質は、一般にアルカリに不
安定なので、クロマトグラフィーの操作上好ましくない
という欠点を有する。[0006] In US Pat. No. 4,336,161, xerogel such as cellulose is held in an inorganic porous material such as Celite and used as a carrier for chromatography. As a result, although the liquid permeability through the column is improved, inorganic substances such as Celite are generally unstable to alkali, and therefore have a drawback that they are not preferable in the operation of chromatography.
【０００７】米国特許第３９６６４８９号には、いわゆ
るマクロネットワーク構造のコポリマーの細孔を、モノ
マーから合成した共重合体のゲルで埋めたハイブリッド
コポリマーのイオン交換体が記載されている。架橋共重
合体ゲルは、架橋度が低い場合、圧力損失が大きい、体
積が変化する等の問題があるが、ハイブリッドコポリマ
ーにすることで通液特性が改善され、圧力損失が少なく
なること、また、イオン交換容量が向上し、リーク挙動
が改善されることを発明の効果としてあげている。しか
し、このハイブリッドコポリマーのネットワーク構造、
すなわち骨格は、スチレン−ジビニルベンゼン共重合体
からなっている。従ってこのものは、疎水性が高く、タ
ンパク質等の生体高分子の分離に使用した場合、非特異
吸着が起きるという欠点を有する。US Pat. No. 3,966,489 describes a hybrid copolymer ion exchanger in which the pores of a so-called macronetwork-structured copolymer are filled with a gel of a copolymer synthesized from monomers. When the degree of crosslinking is low, the cross-linked copolymer gel has problems such as a large pressure loss and a change in volume.However, by using a hybrid copolymer, liquid passing characteristics are improved and the pressure loss is reduced. The effect of the invention is that the ion exchange capacity is improved and the leak behavior is improved. However, the network structure of this hybrid copolymer,
That is, the skeleton is composed of a styrene-divinylbenzene copolymer. Therefore, this is highly hydrophobic and has a drawback that non-specific adsorption occurs when used for separating biopolymers such as proteins.
【０００８】[0008]
【発明が解決しようとする課題】本発明は、親水性高分
子からなる吸着剤が有するタンパク質等の生体高分子に
対する優れた吸着性能を保持しながら、疎水的相互作用
による非特異吸着がなく、かつ、体積変化が少ない吸着
剤を提供せんとするものである。DISCLOSURE OF THE INVENTION The present invention has excellent non-specific adsorption due to hydrophobic interaction while maintaining excellent adsorption performance for biopolymer such as protein possessed by an adsorbent composed of hydrophilic polymer, In addition, it is intended to provide an adsorbent having a small volume change.
【０００９】[0009]
【課題を解決する為の手段】本発明に係る吸着剤は、機
械的強度の大きいキトサン多孔質体の細孔内に、親水性
高分子からなる架橋ゲルを担持した構造を有する複合化
吸着剤である。本発明に係る複合化吸着剤について更に
詳細に説明するに、このものは球状の粒子であり、その
粒径は通常２〜１０００μｍであり、用途により適宜選
択される。例えばカラムに充填してタンパク質等の生体
高分子の吸着に用いる場合には１０〜５００μｍの粒径
のものが好ましい。また分取クロマト、工業用クロマト
の分野では、一般に１００〜２００μｍの粒径のものが
好んで用いられる。また、このものはイオン交換能を有
しており、そのイオン交換容量は少くとも０．３ｍｅｑ
／ｍｌ、通常は０．５ｍｅｑ／ｍｌ以上である。一般に
イオン交換容量が大きい方がタンパク質等の吸着量が大
きいので、１．０ｍｅｑ／ｍｌ以上のイオン交換容量を
有するものが好んで用いられる。The adsorbent according to the present invention is a composite adsorbent having a structure in which a crosslinked gel made of a hydrophilic polymer is supported in the pores of a chitosan porous body having high mechanical strength. Is. The composite adsorbent according to the present invention will be described in more detail. This is a spherical particle, and the particle size thereof is usually 2 to 1000 μm, and is appropriately selected depending on the application. For example, when packed in a column and used for adsorption of biopolymers such as proteins, those having a particle size of 10 to 500 μm are preferable. In the fields of preparative chromatography and industrial chromatography, particles having a particle size of 100 to 200 μm are generally preferably used. This product also has an ion exchange capacity, and its ion exchange capacity is at least 0.3 meq.
/ Ml, usually 0.5 meq / ml or more. In general, the larger the ion exchange capacity is, the larger the adsorption amount of the protein or the like is. Therefore, the one having the ion exchange capacity of 1.0 meq / ml or more is preferably used.
【００１０】本発明に係る複合化吸着剤は、球状のキト
サン多孔質体に親水性高分子を含浸させたのち架橋剤を
反応させて、親水性高分子を架橋させて架橋ゲルとする
ことにより製造することができる。イオン交換基は、親
水性高分子を含浸する前にキトサン多孔質体に導入して
おいてもよいが、キトサン多孔質体内に架橋ゲルを形成
したのちに導入するのが好ましい。架橋ゲルを形成した
後にイオン交換基を導入すると、架橋ゲル自体にもイオ
ン交換基が導入され、架橋ゲル部分における吸着能が高
まると考えられる。なお、キトサン自体の１級アミノ基
もイオン交換基であるのが、通常はこれに加えて更に外
部からイオン交換基を導入するのが好ましい。The composite adsorbent according to the present invention is obtained by impregnating a spherical chitosan porous body with a hydrophilic polymer and then reacting with a crosslinking agent to crosslink the hydrophilic polymer to form a crosslinked gel. It can be manufactured. The ion exchange group may be introduced into the chitosan porous body before impregnation with the hydrophilic polymer, but it is preferably introduced after forming the crosslinked gel in the chitosan porous body. It is considered that when the ion exchange group is introduced after forming the crosslinked gel, the ion exchange group is also introduced into the crosslinked gel itself, and the adsorptivity in the crosslinked gel portion is enhanced. The primary amino group of chitosan itself is also an ion exchange group, but it is usually preferable to introduce an ion exchange group from the outside in addition to this.
【００１１】本発明に係る複合化吸着剤の製造法につい
て更に詳細に説明すると、原料として用いる球状のキト
サン多孔質体は、キトサンをジイソシアネートその他の
架橋剤で架橋したものである。架橋度及び多孔度は、架
橋反応のやり方およびその際の架橋剤の使用量により適
宜選択できる。通常は０．０１〜１．０μｍの孔径を有
する細孔の容積が樹脂体積の７０〜９５％を占めるよう
な、極めて多孔質のものを用いるのが好ましい。すなわ
ちキトサン多孔質体は、十分な量の親水性高分子の架橋
ゲルを担持できる細孔容積と、タンパク質等の生体高分
子が内部に拡散して行くことのできる細孔直径を有して
いなければならない。キトサン多孔質体には予じめイオ
ン交換基を導入しておいてもよい。The method for producing the composite adsorbent according to the present invention will be described in more detail. The spherical porous chitosan used as a raw material is obtained by crosslinking chitosan with a diisocyanate or other crosslinking agent. The degree of crosslinking and the porosity can be appropriately selected depending on the method of the crosslinking reaction and the amount of the crosslinking agent used at that time. Usually, it is preferable to use an extremely porous material in which the volume of pores having a pore diameter of 0.01 to 1.0 μm occupies 70 to 95% of the resin volume. That is, the chitosan porous material must have a pore volume capable of supporting a sufficient amount of hydrophilic polymer crosslinked gel and a pore diameter through which biopolymers such as proteins can diffuse inside. I have to. An ion-exchange group may be previously introduced into the chitosan porous body.
【００１２】もう一方の原料である親水性高分子として
は、デキストラン、アガロース等の多糖類や、ポリビニ
ルアルコール等の親水性合成高分子が用いられる。通常
はタンパク質等の生体高分子の非特異吸着が殆ん無い多
糖類、特にデキストランが用いられる。親水性高分子の
分子量は１０4 〜２×１０6 、特に１０5 〜１０6 の範
囲にあるのが好ましい。分子量が小さ過ぎるとゲルの形
成が困難となる。逆に分子量が大き過ぎると、溶液にし
たときの粘度が大きくなり、キトサン多孔質体内への拡
散・浸透が困難となる。As the other hydrophilic polymer which is a raw material, polysaccharides such as dextran and agarose, and hydrophilic synthetic polymers such as polyvinyl alcohol are used. Usually, a polysaccharide having almost no nonspecific adsorption of biopolymer such as protein, especially dextran is used. The molecular weight of the hydrophilic polymer is preferably 10 4 to 2 × 10 6 , particularly 10 5 to 10 6 . If the molecular weight is too small, gel formation becomes difficult. On the other hand, if the molecular weight is too large, the viscosity of the solution becomes large and it becomes difficult to diffuse and permeate into the chitosan porous body.
【００１３】この親水性高分子を溶媒、通常は水に溶解
して水溶液とし、これに前述のキトサン多孔質体を加え
て常温ないし加温下に保持し、水溶液をキトサン多孔質
体に含浸させる。含浸に用いる親水性高分子水溶液の濃
度は通常５〜３０（重量）％である。一般に低濃度の水
溶液を含浸させると高含水率の架橋ゲルが生成し、逆に
高濃度の水溶液を含浸させると低含水率の架橋ゲルが生
成する傾向がある。This hydrophilic polymer is dissolved in a solvent, usually water, to form an aqueous solution, and the above-mentioned chitosan porous body is added thereto and kept at room temperature or under heating to impregnate the chitosan porous body with the aqueous solution. . The concentration of the hydrophilic polymer aqueous solution used for impregnation is usually 5 to 30 (weight)%. Generally, when a low concentration aqueous solution is impregnated, a crosslinked gel having a high water content is produced, and when a high concentration aqueous solution is impregnated, a low water content crosslinked gel tends to be produced.
【００１４】親水性高分子の水溶液を含浸させたキトサ
ン多孔質体は、次いでこれに架橋剤を反応させて多孔質
体内に親水性高分子の架橋ゲルを生成させる。このとき
親水性高分子だけでなくキトサン自体も架橋剤と反応す
ると考えられ、従ってキトサン−親水性高分子間の架橋
などキトサンも架橋に関与すると考えられる。架橋剤と
しては、エピクロルヒドリン等のエピハロヒドリン、グ
ルタルアルデヒド等のジアルデヒド化合物、メチレンビ
ス（フェニルイソシアネート）等のジイソシアネート化
合物の如き、ＯＨ基と反応性を有する官能基を２個以上
有する化合物が用いられる。なお、親水性高分子として
キトサンの如きアミノ基を有する化合物を用いる場合に
は、１，８−ジクロロオクタンのようなジハライドを架
橋剤として用いることもできる。The chitosan porous body impregnated with the aqueous solution of the hydrophilic polymer is then reacted with a crosslinking agent to form a crosslinked gel of the hydrophilic polymer in the porous body. At this time, it is considered that not only the hydrophilic polymer but also chitosan itself reacts with the cross-linking agent, and therefore chitosan is also involved in the cross-linking such as the cross-linking between chitosan and the hydrophilic polymer. As the crosslinking agent, a compound having two or more functional groups reactive with an OH group, such as epihalohydrin such as epichlorohydrin, dialdehyde compound such as glutaraldehyde, and diisocyanate compound such as methylenebis (phenylisocyanate) is used. When a compound having an amino group such as chitosan is used as the hydrophilic polymer, a dihalide such as 1,8-dichlorooctane can be used as a crosslinking agent.
【００１５】架橋反応は通常は触媒の存在下に行なわれ
る。触媒は架橋剤の種類に応じて適宜選択するが、例え
ば架橋剤がハロヒドリンの場合には水酸化ナトリウム等
のアルカリが用いられ、ジアルデヒド化合物の場合には
塩酸等の鉱酸が用いられる。架橋反応は、親水性高分子
を含浸させたキトサン多孔質体を反応媒体中に懸濁させ
ておき、これに架橋剤を添加して反応させればよい。架
橋剤の量は目的とする複合化吸着剤の物性に応じて適宜
定めればよい。一般に架橋剤の量を多くすると、生成す
る複合化吸着剤の水中での膨潤度が小さくなる。架橋剤
の量が多過ぎると、架橋が進みすぎて親水性高分子の特
性が損なわれることがあるので注意を要する。The crosslinking reaction is usually carried out in the presence of a catalyst. The catalyst is appropriately selected depending on the kind of the cross-linking agent. For example, when the cross-linking agent is halohydrin, alkali such as sodium hydroxide is used, and when it is a dialdehyde compound, mineral acid such as hydrochloric acid is used. The cross-linking reaction may be carried out by suspending a chitosan porous material impregnated with a hydrophilic polymer in a reaction medium and adding a cross-linking agent to the suspension. The amount of the crosslinking agent may be appropriately determined according to the physical properties of the target composite adsorbent. Generally, when the amount of the cross-linking agent is increased, the degree of swelling of the formed composite adsorbent in water decreases. If the amount of the cross-linking agent is too large, the cross-linking may proceed too much and the properties of the hydrophilic polymer may be impaired, so caution is required.
【００１６】また、架橋反応が触媒によって制御可能な
場合には、架橋剤と親水性高分子の混合溶液をキトサン
多孔質体に含浸させ、次いでこの多孔質体を適当な反応
媒体中に懸濁させ、これに触媒を添加して架橋反応を進
行させることもできる。さらに、温度等の反応条件によ
って架橋反応を制御できる場合には、親水性高分子、架
橋剤および触媒を予じめキトサン多孔質体に含浸させ、
次いでこれを反応触媒に懸濁させ、昇温して架橋反応を
進行させることもできる。これらの架橋反応の反応媒体
としては、多孔質体に含浸させてある親水性高分子等を
抽出せず、且つ架橋反応に不活性なものであれば任意の
溶媒を用いることができる。このような溶媒としては例
えば、トルエン、ジクロロベンゼン、ニトロメタン等が
あげられる。なお架橋反応の反応温度や反応時間など
は、架橋剤により異なるが、通常は５〜９０℃、好まし
くは３０〜９０℃で１〜１０時間反応させればよい。When the crosslinking reaction can be controlled by a catalyst, the chitosan porous material is impregnated with a mixed solution of a crosslinking agent and a hydrophilic polymer, and the porous material is then suspended in a suitable reaction medium. Then, a catalyst may be added to this to promote the crosslinking reaction. Further, when the crosslinking reaction can be controlled by the reaction conditions such as temperature, the hydrophilic polymer, the crosslinking agent and the catalyst are preliminarily impregnated into the chitosan porous body,
Next, this may be suspended in the reaction catalyst and the temperature may be raised to allow the crosslinking reaction to proceed. As a reaction medium for these crosslinking reactions, any solvent can be used as long as it does not extract the hydrophilic polymer impregnated in the porous body and is inert to the crosslinking reaction. Examples of such a solvent include toluene, dichlorobenzene, nitromethane and the like. The reaction temperature and reaction time of the cross-linking reaction vary depending on the cross-linking agent, but the reaction is usually carried out at 5 to 90 ° C, preferably 30 to 90 ° C for 1 to 10 hours.
【００１７】キトサン多孔質体に担持する架橋ゲルの担
持量は任意であるが、担持量が少なすぎると複合化吸着
剤としての性能が十分に発現しない。架橋ゲルの担持量
は、キトサン多孔質体に対し通常は１０（重量）％以上
であり、好ましくは５０（重量）％以上である。担持量
の上限は、キトサン多孔質体の細孔容積と架橋ゲルの含
水率により規制される。担持量の上限は通常キトサン多
孔質体に対し２５０（重量）％以下、特に２００（重
量）％以下である。The amount of the crosslinked gel supported on the chitosan porous body is optional, but if the amount is too small, the performance as a composite adsorbent will not be sufficiently exhibited. The supported amount of the crosslinked gel is usually 10 (wt)% or more, preferably 50 (wt)% or more with respect to the chitosan porous body. The upper limit of the supported amount is regulated by the pore volume of the chitosan porous body and the water content of the crosslinked gel. The upper limit of the supported amount is usually 250 (wt)% or less, especially 200 (wt)% or less with respect to the chitosan porous body.
【００１８】架橋反応が終了したならば、濾過してキト
サン多孔質体と架橋ゲルの複合体を取出し、メタノール
やエタノール等の親水性有機溶媒や水で洗浄して未反応
の親水性高分子や反応媒体等を除去する。次いで、この
複合体にイオン交換基を導入して複合化吸着剤とする。
イオン交換基の導入は、モノハロゲノ酢酸、モノハロゲ
ノプロピオン酸等のハロゲノカルボン酸や、ジエチルア
ミノエチルクロライド等のハロゲノアルキルアミンを用
いて行なわれる。すなわち上記で得た架橋ゲルを担持し
た複合体を苛性ソーダ等のアルカリ水溶液に浸漬して複
合体中にアルカリを含浸させ、次いでこれに有機溶媒中
で上記の試薬を加えて反応させればよい。有機溶媒とし
てはエタノール、ｎ−プロパノール、イソプロパノー
ル、ｎ−ブタノール、イソブタノール、ｎ−ペンタノー
ル等のアルコール類を用いるのが好ましい。After the crosslinking reaction is completed, the composite of the chitosan porous material and the crosslinked gel is taken out by filtration and washed with a hydrophilic organic solvent such as methanol or ethanol or water to remove unreacted hydrophilic polymer or The reaction medium etc. are removed. Next, an ion-exchange group is introduced into this composite to obtain a composite adsorbent.
The introduction of the ion exchange group is carried out using a halogenocarboxylic acid such as monohalogenoacetic acid or monohalogenopropionic acid, or a halogenoalkylamine such as diethylaminoethyl chloride. That is, the complex obtained by carrying the crosslinked gel obtained above may be dipped in an aqueous alkali solution such as caustic soda to impregnate the complex with an alkali, and then the above-mentioned reagent may be added to and reacted in an organic solvent. As the organic solvent, it is preferable to use alcohols such as ethanol, n-propanol, isopropanol, n-butanol, isobutanol and n-pentanol.
【００１９】反応は４０〜９０℃で０．５〜１２時間行
なえばよい。一般に複合化吸着剤はイオン交換容量が大
きい方がタンパク質等の生体高分子の吸着量が大きい。
従ってイオン交換基の導入は、イオン交換容量が０．３
〜３．０ｍｅｑ／ｍｌ、特に０．５〜２．０ｍｅｑ／ｍ
ｌとなるように行なうのが好ましい。なお、架橋ゲルを
担持する前のキトサン多孔質体にイオン交換基を導入す
る場合にも上記と同様にして行なうことができる。ま
た、別法として親水性高分子にイオン交換基を導入して
からキトサン多孔質体に含浸させ、次いで架橋剤と反応
させて架橋ゲルを形成させることもできる。The reaction may be carried out at 40 to 90 ° C. for 0.5 to 12 hours. Generally, the larger the ion exchange capacity of the composite adsorbent, the larger the adsorption amount of biopolymer such as protein.
Therefore, the introduction of ion-exchange groups has an ion-exchange capacity of 0.3.
~ 3.0 meq / ml, especially 0.5-2.0 meq / m
It is preferable to carry out so as to be 1. In addition, when introducing an ion exchange group into the chitosan porous body before supporting the crosslinked gel, the same procedure as above can be performed. Alternatively, it is also possible to introduce an ion-exchange group into the hydrophilic polymer, impregnate the chitosan porous body, and then react with a crosslinking agent to form a crosslinked gel.
【００２０】このようにして得られる複合化吸着剤は、
タンパク質等の生体高分子に対し大きな吸着能を有して
いる。例えば牛血清アルブミンに対して１５０ｇ／ｌ−
吸着剤以上、特に２００ｇ／ｌ−吸着剤以上の吸着能を
有する複合化吸着剤を容易に得ることができる。また、
本発明に係る複合化吸着剤では、親水性高分子の架橋ゲ
ルがキトサン多孔質体の細孔内に担持されているため、
架橋ゲルが水中で膨潤したり、周囲の溶液のイオン強度
の影響で体積変化しても、複合化吸着剤そのものの体積
は殆んど変化しない。The composite adsorbent thus obtained is
It has a large adsorption capacity for biopolymers such as proteins. For example, 150 g / l-for bovine serum albumin
It is possible to easily obtain a composite adsorbent having an adsorbability of not less than the adsorbent, particularly not less than 200 g / l-adsorbent. Also,
In the composite adsorbent according to the present invention, the crosslinked gel of the hydrophilic polymer is supported in the pores of the chitosan porous body,
Even if the crosslinked gel swells in water or the volume changes due to the influence of the ionic strength of the surrounding solution, the volume of the composite adsorbent itself hardly changes.
【００２１】なお、本発明に係る複合化吸着剤の製造過
程において、イオン交換基の代りにアフィニティーリガ
ンドを導入すると、アフィニティークロマトグラフィー
の担体が得られる。この担体においても、タンパク質の
非特異吸着が無いという親水性高分子架橋ゲルの特徴が
発揮され、タンパク質の吸着−脱着が容易であり、且つ
タンパク質の変性も起こらない。In the production process of the composite adsorbent according to the present invention, if an affinity ligand is introduced instead of the ion exchange group, a carrier for affinity chromatography can be obtained. Also in this carrier, the characteristic of the hydrophilic polymer cross-linked gel is that there is no non-specific adsorption of proteins, the adsorption-desorption of proteins is easy, and denaturation of proteins does not occur.
【００２２】以下に実施例および比較例により本発明を
さらに具体的に説明するが、本発明は以下の実施例に限
定されるものではない。なお、本明細書において粒径は
水中で膨潤した状態での値である。またイオン交換容量
及び牛血清アルブミン吸着量も、水中で膨潤した状態で
の複合化吸着剤に基づく値である。複合化吸着剤の体積
は、水中で膨潤した複合化吸着剤を１０ｍｌのメスシリ
ンダーに流し込み、振動させて密に充填するようにして
自然沈降させ、その体積を測定することにより求められ
る。The present invention will be described in more detail below with reference to Examples and Comparative Examples, but the present invention is not limited to the following Examples. In addition, in this specification, a particle size is a value in the state swollen in water. Also, the ion exchange capacity and the bovine serum albumin adsorption amount are values based on the complexed adsorbent in a state swollen in water. The volume of the complexed adsorbent is obtained by pouring the complexed adsorbent swollen in water into a 10 ml graduated cylinder, vibrating the mixture so that the complex adsorbent is allowed to spontaneously settle, and measuring the volume.
【００２３】[0023]
〔実施例１〕膨潤状態のキトサン多孔質体（キトパール
ＢＣＷ−２５０１、富士紡績社製品、陰イオン交換容量
０．５８ｍｅｑ／ｍｌ）１００ｍｌを、デキストラン水
溶液（蒸留水１００ｍｌに、分子量５０万のデキストラ
ン１９．２５ｇを溶解）に浸せきし、室温で６６．３時
間撹拌した。次いでデキストラン水溶液（蒸留水１００
ｍｌに分子量５０万のデキストラン１２．５ｇを溶解）
を加え、５０度で２５時間放置した。更にデキストラン
水溶液（蒸留水１００ｍｌに分子量５０万のデキストラ
ン１２．４９ｇを溶解）を加え、５０℃で４７．５時間
放置した。更にデキストラン水溶液（蒸留水１００ｍｌ
に分子量５０万のデキストラン１２．７２ｇを溶解）を
加え、５０℃で４９．５時間放置した。再度デキストラ
ン水溶液（蒸留水１００ｍｌに分子量５０万のデキスト
ラン１２．７７ｇを溶解）を加え、５０℃で２８．３時
間放置した後、デキストラン溶液（３．８５規定水酸化
ナトリウム水溶液１００ｍｌに分子量５０万のデキスト
ラン１２．６６ｇと水素化ほう素ナトリウム３．８４ｇ
を溶解）を加え、５０℃で１４．５時間放置した。遠心
分離機で、多孔質体の外部に付着した過剰のデキストラ
ン溶液を除去した。デキストラン溶液含浸キトサン多孔
質体を、トルエン９６０ｍｌ中にエチルセルロース３
８．９ｇを溶解した溶液中に加え、５０℃で撹拌し、分
散、懸濁した。懸濁液中にエピクロルヒドリン１６．３
ｍｌを加えて、この温度で８時間撹拌し、架橋反応させ
た。トルエン、５０％エタノール水溶液、脱イオン水で
順次洗浄した。複合体を、遠心分離で水を除去した後、
３規定の水酸化ナトリウム水溶液１００ｍｌ中にいれ、
室温で１時間放置した。１２．９ｇのジエチルアミノエ
チルクロリド塩酸塩を１４．７ｍｌの水に溶解し、更に
イソプロピルアルコール１６０ｍｌと混合した溶液を、
水酸化ナトリウム含浸状態の複合体に加えた。温度を５
０℃まで上げ、撹拌しながら９時間反応させた。反応終
了後、濾過、水洗し、目的の複合化吸着剤を得た。複合
化吸着剤の収率は、原料のキトサン多孔質体の重量に対
し２８７％であった。また、この複合化吸着剤のイオン
交換容量は１．１２ｍｅｑ／ｍｌで、そのうち１級アミ
ノ基は、０．７１ｍｅｑ／ｍｌであった。Example 1 100 ml of a swollen chitosan porous body (Chitopearl BCW-2501, product of Fuji Spinning Co., anion exchange capacity 0.58 meq / ml) was added to an aqueous dextran solution (distilled water 100 ml, dextran 19 having a molecular weight of 500,000). 0.25 g was dissolved) and the mixture was stirred at room temperature for 66.3 hours. Next, dextran aqueous solution (distilled water 100
Dissolve 12.5 g of dextran with a molecular weight of 500,000 in ml)
Was added and the mixture was allowed to stand at 50 degrees for 25 hours. Further, a dextran aqueous solution (12.49 g of dextran having a molecular weight of 500,000 was dissolved in 100 ml of distilled water) was added, and the mixture was left at 50 ° C. for 47.5 hours. Furthermore, dextran aqueous solution (distilled water 100 ml
(12.72 g of dextran having a molecular weight of 500,000 was dissolved) and the mixture was allowed to stand at 50 ° C. for 49.5 hours. A dextran aqueous solution (12.77 g of dextran having a molecular weight of 500,000 was dissolved in 100 ml of distilled water) was added again, and the mixture was allowed to stand at 50 ° C. for 28.3 hours, then, a dextran solution (100 ml of a 3.85N sodium hydroxide aqueous solution having a molecular weight of 500,000) was added. Dextran 12.66g and sodium borohydride 3.84g
Was dissolved), and the mixture was left at 50 ° C. for 14.5 hours. An excess dextran solution adhering to the outside of the porous body was removed with a centrifuge. Chitosan porous material impregnated with dextran solution was mixed with ethyl cellulose 3 in 960 ml of toluene.
8.9 g was added to the dissolved solution, and the mixture was stirred at 50 ° C., dispersed and suspended. Epichlorohydrin 16.3 in suspension
ml was added and the mixture was stirred at this temperature for 8 hours to cause a crosslinking reaction. It was washed successively with toluene, a 50% ethanol aqueous solution, and deionized water. After removing water from the complex by centrifugation,
Pour into 100 ml of 3N aqueous sodium hydroxide,
It was left at room temperature for 1 hour. A solution prepared by dissolving 12.9 g of diethylaminoethyl chloride hydrochloride in 14.7 ml of water and further mixing it with 160 ml of isopropyl alcohol was prepared.
It was added to the complex impregnated with sodium hydroxide. Temperature 5
The temperature was raised to 0 ° C., and the reaction was performed for 9 hours with stirring. After completion of the reaction, the mixture was filtered and washed with water to obtain the target composite adsorbent. The yield of the composite adsorbent was 287% based on the weight of the raw material chitosan porous body. The ion exchange capacity of this composite adsorbent was 1.12 meq / ml, of which the primary amino group was 0.71 meq / ml.
【００２４】次に、この複合化吸着剤を用いて、タンパ
ク質の吸着実験を行った。複合化吸着剤を０．１Ｎ−水
酸化ナトリウム水溶液に浸漬した。次いで、充分な量の
脱塩水で洗浄したのち、７ｍＭ−リン酸塩酸緩衝液（ｐ
Ｈ６．９）で緩衝化した。これをカラムに充填し、同一
緩衝液で調製した牛血清アルブミン溶液を通液して、そ
の破過曲線を求めた。この結果、牛血清アルブミンの飽
和吸着量は、３４２ｇ／ｌであった。Next, a protein adsorption experiment was conducted using this composite adsorbent. The composite adsorbent was immersed in a 0.1N-sodium hydroxide aqueous solution. Then, after washing with a sufficient amount of demineralized water, 7 mM-phosphate buffer (p
Buffered with H6.9). This was packed in a column and a bovine serum albumin solution prepared with the same buffer was passed through to determine the breakthrough curve. As a result, the saturated adsorption amount of bovine serum albumin was 342 g / l.
【００２５】また、固相物質移動容量係数（ｋP ）を、
破過曲線の実測値と、Ｃｈｅｍｉｃａｌ Ｅｎｇｉｎｅ
ｅｒｉｎｇ Ｓｃｉｅｎｃｅ ３９号 １４８９頁（１
９８４年）に記載のＡｎａｌｙｔｉｃａｌ Ｓｏｌｕｔ
ｉｏｎ ｏｆ ｔｈｅ Ｂｒｅａｋｔｈｒｏｕｇｈ ｆ
ｏｒ Ｒｅｃｔａｎｇｕｌａｒ ＩｓｏｔｈｅｒｍＳｙ
ｓｔｅｍｓに基づく均一モデルによる理論線とのカーブ
フィッティングにより決定し、次式を用いて固相拡散係
数（Ｄs ）を求めた。The solid phase mass transfer capacity coefficient (k P ) is
Measured value of breakthrough curve and Chemical Engine
ering Science No. 39, page 1489 (1
984) Analytical Solut described in
ion of the Breakthrough f
or Rectangular IsothermSy
The solid-phase diffusion coefficient (D s ) was determined by curve fitting with a theoretical line using a uniform model based on the stems and using the following equation.
【００２６】[0026]
【数１】ｋP ＝１５Ｄs ／ｒ0 2 （ｒ0 ：複合化吸着剤
の半径（ｃｍ）） その結果、固相拡散係数は２．４３×１０-9ｃｍ2 ／ｓ
ｅｃであった。## EQU1 ## k P = 15D s / r 0 2 (r 0 : radius of composite adsorbent (cm)) As a result, the solid-phase diffusion coefficient is 2.43 × 10 -9 cm 2 / s.
It was ec.
【００２７】〔比較例１〕デキストランの架橋ゲルから
なる弱塩基性陰イオン交換体（ファルマシア社製品、Ｄ
ＥＡＥ−Ｓｅｐｈａｄｅｘ Ａ−５０）について、実施
例１と同様の要領で、交換容量、タンパク質の飽和吸着
量および固相拡散係数を求めた。その結果、交換容量
０．０５ｍｅｑ／ｍｌ、牛血清アルブミンの飽和吸着量
２６１ｇ／ｌ、固相拡散係数１．８６×１０-9ｃｍ2 ／
ｓｅｃであった。[Comparative Example 1] A weakly basic anion exchanger comprising a crosslinked gel of dextran (D, Pharmacia, D
For EAE-Sephadex A-50), the exchange capacity, the saturated adsorption amount of protein and the solid phase diffusion coefficient were determined in the same manner as in Example 1. As a result, the exchange capacity was 0.05 meq / ml, the saturated adsorption amount of bovine serum albumin was 261 g / l, and the solid phase diffusion coefficient was 1.86 × 10 −9 cm 2 /
It was sec.
【００２８】従って、実施例１の複合化吸着剤は、本比
較例のデキストランの架橋ゲルからなる弱塩基性陰イオ
ン交換体と比べて、固相拡散係数および牛血清アルブミ
ンの平衡吸着量がいずれも大きく、吸着剤として優れた
性能を有する事がわかる。また、本比較例のデキストラ
ン架橋ゲルからなるイオン交換体は、機械的強度が弱
く、カラムに充填し、線速度１０ｃｍ／ｈで通液した場
合、徐々に圧縮され、カラム内圧が増加する。これに対
して、実施例１の複合化吸着剤は、線速度１００ｃｍ／
ｈで通液が可能であり、強度的にも優れている事がわか
る。Therefore, the composite adsorbent of Example 1 has a higher solid phase diffusion coefficient and an equilibrium adsorption amount of bovine serum albumin than the weakly basic anion exchanger composed of the cross-linked dextran gel of this Comparative Example. It is also found that it has excellent performance as an adsorbent. Further, the ion exchanger made of the dextran crosslinked gel of this comparative example has weak mechanical strength, and when it is packed in a column and passed through at a linear velocity of 10 cm / h, it is gradually compressed and the column internal pressure increases. In contrast, the composite adsorbent of Example 1 has a linear velocity of 100 cm /
It can be seen that it is possible to pass the liquid with h and it is also excellent in strength.
【００２９】〔比較例２〕複合化吸着剤の母体として用
いた、キトサン多孔質体（富士紡績社製 キトパールＢ
ＣＷ−２５０１）について実施例１と同様の要領で、牛
血清アルブミンの飽和吸着量および固相拡散係数を求め
た。その結果、飽和吸着量は１３８ｇ／ｌ、固相拡散係
数は０．２３９×１０-9ｃｍ2 ／ｓｅｃであった。従っ
て、実施例１の複合化吸着剤は、その母体のキトサン多
孔質体と比べても、固相拡散係数が１０．２倍で、かつ
牛血清アルブミンの平衡吸着量が約２．５倍と高く、優
れた性能を有する事がわかる。Comparative Example 2 Chitosan porous material (chitopearl B manufactured by Fuji Spinning Co., Ltd.) used as a matrix of the composite adsorbent.
For CW-2501), the saturated adsorption amount of bovine serum albumin and the solid phase diffusion coefficient were determined in the same manner as in Example 1. As a result, the saturated adsorption amount was 138 g / l and the solid phase diffusion coefficient was 0.239 × 10 -9 cm 2 / sec. Therefore, the composite adsorbent of Example 1 has a solid phase diffusion coefficient of 10.2 times and an equilibrium adsorption amount of bovine serum albumin of about 2.5 times as compared with the matrix chitosan porous body. It can be seen that it is expensive and has excellent performance.
【００３０】[0030]
【発明の効果】本発明に係る複合化吸着剤は、体積変化
が少なく、カラムに充填して使用する際にも大きな線速
度で通液することができる。また、タンパク質等の生体
高分子に対し大きな吸着能を有している。EFFECTS OF THE INVENTION The composite adsorbent according to the present invention has a small volume change, and can be passed through at a large linear velocity even when it is packed in a column and used. In addition, it has a large adsorptivity for biopolymers such as proteins.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 工藤 慶子 神奈川県横浜市緑区鴨志田町1000番地 三 菱化成株式会社総合研究所内 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Keiko Kudo 1000, Kamoshida-cho, Midori-ku, Yokohama-shi, Kanagawa Sanryo Kasei Co., Ltd.
Claims (4)
分子からなる架橋ゲルが担持されていることを特徴とす
るイオン交換能を有する複合化吸着剤。1. A composite adsorbent having an ion-exchange capacity, characterized in that a crosslinked gel made of a hydrophilic polymer is carried in the pores of a chitosan porous body.
上であることを特徴とする請求項１記載の複合化吸着
剤。2. The composite adsorbent according to claim 1, which has an ion exchange capacity of 0.5 meq / ml or more.
ｇ／ｌ以上であることを特徴とする請求項１又は２記載
の複合化吸着剤。3. The saturated adsorption amount of bovine serum albumin is 200.
The composite adsorbent according to claim 1 or 2, wherein the adsorbent is g / l or more.
溶液を含浸させる工程、該親水性高分子に架橋剤を反応
させてキトサン多孔質体−親水性高分子架橋ゲルの複合
体を形成する工程、及びこの複合体にイオン交換基を導
入する工程の各工程を経ることを特徴とする請求項１な
いし３のいずれかに記載の複合化吸着剤の製造法。4. A step of impregnating a spherical chitosan porous body with a hydrophilic polymer solution, and reacting the hydrophilic polymer with a crosslinking agent to form a chitosan porous body-hydrophilic polymer crosslinked gel complex. 4. The method for producing a composite adsorbent according to claim 1, wherein each step of the step of introducing an ion exchange group into the complex and the step of introducing an ion exchange group into the complex are performed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5224717A JPH0780294A (en) | 1993-09-09 | 1993-09-09 | Composite adsorbent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5224717A JPH0780294A (en) | 1993-09-09 | 1993-09-09 | Composite adsorbent |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0780294A true JPH0780294A (en) | 1995-03-28 |
Family
ID=16818149
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP5224717A Pending JPH0780294A (en) | 1993-09-09 | 1993-09-09 | Composite adsorbent |
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| Country | Link |
|---|---|
| JP (1) | JPH0780294A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000012618A1 (en) * | 1998-08-28 | 2000-03-09 | Amersham Pharmacia Biotech Ab | Composite material and its use |
| KR100346577B1 (en) * | 2000-06-20 | 2002-07-26 | 주식회사 티씨 싸이언스 | Carrier specific to liver cell and the carrier-DNA conujugate |
| CN106984282A (en) * | 2017-05-04 | 2017-07-28 | 凤台县双利生物科技有限公司 | A kind of preparation method of the magnetic green substance adsorber of sulfydryl lignocellulosic montmorillonite Composite |
| CN114042439A (en) * | 2021-12-15 | 2022-02-15 | 中国人民解放军96901部队23分队 | Uranium-containing wastewater adsorbent and preparation method thereof |
| CN115261151A (en) * | 2022-08-01 | 2022-11-01 | 江西省安智光电科技有限公司 | Cleaning protective agent and application thereof in preparation of touch screen |
-
1993
- 1993-09-09 JP JP5224717A patent/JPH0780294A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000012618A1 (en) * | 1998-08-28 | 2000-03-09 | Amersham Pharmacia Biotech Ab | Composite material and its use |
| JP2002523759A (en) * | 1998-08-28 | 2002-07-30 | アメルシャム・バイオサイエンシーズ・アクチボラグ | Hybrid substances and their use |
| KR100346577B1 (en) * | 2000-06-20 | 2002-07-26 | 주식회사 티씨 싸이언스 | Carrier specific to liver cell and the carrier-DNA conujugate |
| CN106984282A (en) * | 2017-05-04 | 2017-07-28 | 凤台县双利生物科技有限公司 | A kind of preparation method of the magnetic green substance adsorber of sulfydryl lignocellulosic montmorillonite Composite |
| CN114042439A (en) * | 2021-12-15 | 2022-02-15 | 中国人民解放军96901部队23分队 | Uranium-containing wastewater adsorbent and preparation method thereof |
| CN114042439B (en) * | 2021-12-15 | 2023-06-30 | 中国人民解放军96901部队23分队 | Uranium-containing wastewater adsorbent and preparation method thereof |
| CN115261151A (en) * | 2022-08-01 | 2022-11-01 | 江西省安智光电科技有限公司 | Cleaning protective agent and application thereof in preparation of touch screen |
| CN115261151B (en) * | 2022-08-01 | 2023-12-29 | 江西省安智光电科技有限公司 | Cleaning and protecting agent and application thereof in preparing touch screen |
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