JPS62184348A - Separating and refining method for ionic material - Google Patents
Separating and refining method for ionic materialInfo
- Publication number
- JPS62184348A JPS62184348A JP61025869A JP2586986A JPS62184348A JP S62184348 A JPS62184348 A JP S62184348A JP 61025869 A JP61025869 A JP 61025869A JP 2586986 A JP2586986 A JP 2586986A JP S62184348 A JPS62184348 A JP S62184348A
- Authority
- JP
- Japan
- Prior art keywords
- column
- voltage
- separated
- substance
- ferritin
- 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
- 239000000463 material Substances 0.000 title claims abstract description 13
- 238000000034 method Methods 0.000 title claims description 11
- 238000007670 refining Methods 0.000 title 1
- 238000001962 electrophoresis Methods 0.000 claims abstract description 15
- 238000012856 packing Methods 0.000 claims abstract description 11
- 239000012141 concentrate Substances 0.000 claims abstract description 3
- 239000000126 substance Substances 0.000 claims description 30
- 238000000926 separation method Methods 0.000 claims description 14
- 238000004811 liquid chromatography Methods 0.000 claims description 5
- 238000005259 measurement Methods 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 2
- 102000008857 Ferritin Human genes 0.000 abstract description 10
- 108050000784 Ferritin Proteins 0.000 abstract description 10
- 238000008416 Ferritin Methods 0.000 abstract description 8
- 239000007788 liquid Substances 0.000 abstract description 5
- 238000013508 migration Methods 0.000 abstract description 3
- 230000005012 migration Effects 0.000 abstract description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 238000000691 measurement method Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003889 chemical engineering Methods 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
Description
【発明の詳細な説明】
〔発明の利用分野〕
本発明バイオテクノロジーなど化学工学分野一般におけ
る物質の分離精製法に係り、特に液体クロマトグラフィ
ー法と電気泳動法を複合化させた分離精製法における被
分離物質のオンライン濃度計測法に関する。Detailed Description of the Invention [Field of Application of the Invention] The present invention relates to a method for separating and purifying substances in general fields of chemical engineering such as biotechnology, and in particular to methods for separating and purifying substances that combine liquid chromatography and electrophoresis. Concerning online concentration measurement method of separated substances.
従来、物質を分離、精製する代表的な方法としては液体
クロマト法及び電気泳動法が用いられていた。最近、こ
の二つの方法を組み合せた方法が優れた分離精製能力を
持つことが示された（Ｐ。Conventionally, liquid chromatography and electrophoresis have been used as typical methods for separating and purifying substances. Recently, it has been shown that a method that combines these two methods has excellent separation and purification capabilities (P.
Ｈ、Ｏ’ Ｆａｒｒｅｌｌ：サイエンス（Ｓｃｉｅｎｃ
ｅ）’ ２２７巻１５８６頁、１９８５年）、この方法
の原理は、液体クロマト用のカラムにイオン性の被分離
物質に対する相互作用が異なる２種の充填剤を積層し、
当該被分離物質を含む溶液を上方から流すと同時にカラ
ムの両端に電場を掛け２つ剤の充填剤の接合部に被分離
物質を濃縮分離するというものである。この場合、被分
離物質は１例えば、カラムの上方から下方へ流れるが、
被分離物質が負に帯電している場合、電極を上方に設定
すれば被分離物質を上方に電気泳動により逆行させるこ
とができる。カラムに与える電圧をカラムに流れる溶液
の流速との関係で最適に設定すれば被分離物質を２つの
充填剤の接触面に停帯させることが出来、物質を濃縮し
分離することができる。しかしながら、上記公ていす、
その結果が分離条件にフィードバックされていないので
、電圧と流速の関係は正確に設定できていず、濃縮が最
適条件で行われていないと゛いう問題点があった６
〔発明の目的〕
本発明は上記公知例の問題点にもとづいてなされたもの
で、カラム内に濃縮されてきた被分離物質の濃度を計測
し、計測結果をフィードバックし、電気泳動速度と流速
とを正確にバランスさせる手段を提供することにある。H. O' Farrell: Science
e)' Vol. 227, p. 1586, 1985), the principle of this method is that two types of packing materials with different interactions with ionic substances to be separated are stacked on a column for liquid chromatography.
A solution containing the substance to be separated is caused to flow from above and at the same time an electric field is applied to both ends of the column to concentrate and separate the substance to be separated at the junction of the two packing materials. In this case, the substance to be separated flows from the top to the bottom of the column, for example,
When the substance to be separated is negatively charged, by setting the electrode upward, the substance to be separated can be moved upward by electrophoresis. By optimally setting the voltage applied to the column in relation to the flow rate of the solution flowing through the column, the substance to be separated can be stopped at the contact surface of the two packing materials, and the substance can be concentrated and separated. However, the above public chair,
Since the results are not fed back to the separation conditions, the relationship between voltage and flow rate cannot be set accurately, resulting in the problem that concentration is not performed under optimal conditions.6 [Object of the Invention] The present invention This was developed based on the problems of the above-mentioned known examples, and provides a means to measure the concentration of the substance to be separated that has been concentrated in the column, feed back the measurement results, and accurately balance the electrophoresis speed and flow rate. It's about doing.
カラム中に分離された物質の濃度の測定にはオンライン
計測が望ましい。分離された物質をカラム外に取り出し
て分析するオフライン計測では分離操作が不連続となり
好ましくない。本発明ではオンライン計測法として如何
なるイオン性物質にも一般的に適用し得る濃度測定法と
してカラム内に掛けられた泳動分離用の電圧のカラム内
各部位における勾配を測定する方法を用いた。イオン性
物質が高濃度に′ａ縮された部位がカラム内に存在すれ
ば、その部位では電圧勾配が周辺部に比して大きく低下
する。すなわち、カラムの上下方向に微小間隔で電極を
配置し、微小電極間の電圧差を測定すれば第１図に一般
的に示すように被分離物質が存在するカラム部位では電
圧勾配は低下し、分離物質がもつとも高濃度で存在する
部位では勾配は極小値を取る。この極小値の絶対値が極
小になるように流速と泳動電圧の関係を制御すれば分離
は最適条件で行われたことになる。電圧勾配は必ずしも
カラムの全長に渡って測定する必要はなく、分離を望む
物質が分離されてくることが予め予定されている部位の
勾配のみを測定しても良いことは勿論である。要するに
本発明においては、分離されたイオン性物質の濃度をカ
ラム内の電圧勾配を測定することにより知り、その結果
により泳動用電圧を制御して分離を高速にかつ至適な条
件で行う。又、本発明によれば、電圧勾配の極小値が最
小になり、一定時間摘持したとき濃縮が完了したものと
判断することができる。Online measurement is desirable for measuring the concentration of substances separated in the column. Off-line measurement, in which separated substances are taken out of the column and analyzed, is not preferred because the separation operation becomes discontinuous. In the present invention, as an online measurement method, a method of measuring the concentration at each location in the column of a voltage applied to the column for electrophoretic separation was used as a concentration measurement method that can be generally applied to any ionic substance. If there is a region in the column where ionic substances are condensed to a high concentration, the voltage gradient at that region will be significantly lower than in the surrounding region. That is, if electrodes are arranged at minute intervals in the vertical direction of the column and the voltage difference between the minute electrodes is measured, the voltage gradient will decrease in the column part where the substance to be separated exists, as generally shown in Figure 1. The gradient takes a minimum value at a site where the separated substance is present at a high concentration. If the relationship between flow rate and electrophoresis voltage is controlled so that the absolute value of this minimum value becomes minimum, separation will be performed under optimal conditions. The voltage gradient does not necessarily need to be measured over the entire length of the column, and it is of course possible to measure only the gradient at a region where the substance desired to be separated is expected to be separated. In short, in the present invention, the concentration of the separated ionic substance is determined by measuring the voltage gradient within the column, and the electrophoresis voltage is controlled based on the results to perform separation at high speed and under optimal conditions. Further, according to the present invention, it can be determined that the concentration is completed when the minimum value of the voltage gradient becomes the minimum and the concentration is maintained for a certain period of time.
以下、本発明の一実施例を第２図により説明する。中空
のカラム４に、まず、充填剤ビオゲルＡ−５０ｍ （バ
イオラド社製品）１９を充填し、次にこの上に別の充填
剤２０（ビオゲルＰ−１０）をのせ、搬送液１５ （１
０ｍＭのトリス酢酸水溶液、ｐＨ７−４）に被分離試料
である蛋白質フェリチンを含む試料溶液１を加え、ポン
プ２を作動させ、フェリチンをカラムに流入せしめた。An embodiment of the present invention will be described below with reference to FIG. The hollow column 4 was first filled with the packing material Biogel A-50m (Bio-Rad product) 19, and then another packing material 20 (Biogel P-10) was placed on top of this, and the carrier liquid 15 (1
Sample solution 1 containing the protein ferritin, which is a sample to be separated, was added to a 0 mM aqueous solution of Tris acetic acid, pH 7-4), and pump 2 was activated to cause ferritin to flow into the column.
次に。next.
電気泳動用電源１１を作動せしめ正極１４をカラム上方
、負極１３を下方に設定しカラム内に電圧勾配を付与し
た。フェリチンを加え終ったら、搬送液１５のみをカラ
ムに加え、電圧測定器３により２つの充填剤の界面付近
に配置した電ｒ４１２ 。The electrophoresis power supply 11 was activated, the positive electrode 14 was set above the column, and the negative electrode 13 was set below, thereby applying a voltage gradient within the column. After adding ferritin, only the carrier liquid 15 was added to the column, and the voltage measuring device 3 was used to place the voltage r412 near the interface between the two packing materials.
１２’間の電圧差を信号線８．９を通して計測する。次
に、界面付近の電圧差がその周辺部に等間隔で配した電
極間の電圧差より小さく、かつ、この極小電圧差の絶対
値がなるべく小さくなるように制御信号発生器７より制
御信号５を電気泳動用電源１１に加え泳動用電圧を上下
に変化させる。12' is measured through the signal line 8.9. Next, the control signal generator 7 generates a control signal 5 so that the voltage difference near the interface is smaller than the voltage difference between the electrodes arranged at equal intervals around the interface, and the absolute value of this minimal voltage difference is as small as possible. is applied to the electrophoresis power supply 11 to change the electrophoresis voltage up and down.
界面付近の電圧差が最小の極小値を一定時間維持したと
き、フェリチン１８は界面付近に最適条件下で濃縮分離
され終ったものと判断し、制御信号６を制御信号発生器
７よりポンプ１ｏに加え、界面付近に取りつけた濃縮成
分取出口１７よりフェリチンを取り出した。第２図にお
いて、１６は負極用電極槽である。When the voltage difference near the interface maintains the minimum value for a certain period of time, it is determined that ferritin 18 has been concentrated and separated near the interface under optimal conditions, and the control signal 6 is sent from the control signal generator 7 to the pump 1o. In addition, ferritin was taken out from the concentrated component extraction port 17 installed near the interface. In FIG. 2, 16 is an electrode tank for the negative electrode.
以上、本実施例によれば、泳動用電圧をフィードバック
制御することにより、フェリチンを最適の分離条件下で
、分離濃縮する′ことができた。As described above, according to this example, ferritin could be separated and concentrated under optimal separation conditions by feedback-controlling the electrophoresis voltage.
本発明によれば、分離カラム内を流下する搬送液により
移動する被分離物質の速度と、カラム内を上方に逆向す
る被分離物質の泳動速度とを正確にかつ高速につり合せ
ることができるので、液体クロマトグラフィー法と電気
泳動法を組み合せた分離法を最適分離条件下で用いるこ
とが可能になった。又、電圧勾配を測定することにより
分離濃縮の完了を正確に検知できるようになった。According to the present invention, it is possible to accurately and quickly balance the speed of the substance to be separated, which is moved by the carrier liquid flowing down the separation column, and the migration speed of the substance to be separated, which is moving upward in the column. It has now become possible to use a separation method that combines liquid chromatography and electrophoresis under optimal separation conditions. Furthermore, by measuring the voltage gradient, it has become possible to accurately detect the completion of separation and concentration.
第１図は泳動用電圧の勾配のカラム長方向に対する分布
を示す図である。第２図は本発明の基本構成を示す図で
ある。
１・・・試料溶液、４・・・分離用カラム、１１・・・
電気泳動用電源、１２，１２’・・・電圧勾配測定用電
極、３・・・電圧勾配測定器、２・・・試料導入ポンプ
、１０・・・濃縮物質取り出し用ポンプ、１８・・・濃
縮物質、１９．２０・・・カラム充填剤、７・・・制御
信号発生器。FIG. 1 is a diagram showing the distribution of the gradient of electrophoresis voltage in the column length direction. FIG. 2 is a diagram showing the basic configuration of the present invention. 1... Sample solution, 4... Separation column, 11...
Power source for electrophoresis, 12, 12'... Electrode for voltage gradient measurement, 3... Voltage gradient measuring device, 2... Sample introduction pump, 10... Pump for removing concentrated substance, 18... Concentration Substance, 19.20... Column packing material, 7... Control signal generator.
Claims (1)
ィー用カラムにイオン性の被分離物質を含む溶液を流し
、同時に、カラムの両端に電気泳動用の電圧を掛けて被
分離物質を濃縮分離する方法において、被分離物質の濃
縮度を、この物質が存在するカラム内の部位の電圧勾配
を計測することにより計測し、計測結果にもとづき電気
泳動用電圧を制御し最適条件下で濃縮分離を行うと共に
、濃縮過程の完了を検知することを特徴とするイオン性
物質の分離精製法。1. A solution containing an ionic substance to be separated is passed through a liquid chromatography column containing two or more different types of packing materials, and at the same time, an electrophoretic voltage is applied to both ends of the column to concentrate and separate the substance to be separated. In this method, the degree of concentration of the substance to be separated is measured by measuring the voltage gradient at the location in the column where the substance is present, and the electrophoresis voltage is controlled based on the measurement results to perform concentration separation under optimal conditions. A separation and purification method for ionic substances is characterized in that the completion of the concentration process is also detected.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61025869A JPS62184348A (en) | 1986-02-10 | 1986-02-10 | Separating and refining method for ionic material |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61025869A JPS62184348A (en) | 1986-02-10 | 1986-02-10 | Separating and refining method for ionic material |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS62184348A true JPS62184348A (en) | 1987-08-12 |
Family
ID=12177796
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61025869A Pending JPS62184348A (en) | 1986-02-10 | 1986-02-10 | Separating and refining method for ionic material |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS62184348A (en) |
-
1986
- 1986-02-10 JP JP61025869A patent/JPS62184348A/en active Pending
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