JPH0317295B2 - - Google Patents
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- Publication number
- JPH0317295B2 JPH0317295B2 JP60055821A JP5582185A JPH0317295B2 JP H0317295 B2 JPH0317295 B2 JP H0317295B2 JP 60055821 A JP60055821 A JP 60055821A JP 5582185 A JP5582185 A JP 5582185A JP H0317295 B2 JPH0317295 B2 JP H0317295B2
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- JP
- Japan
- Prior art keywords
- chamber
- electrophoresis
- liquid
- migration
- electrode
- 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.)
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- Peptides Or Proteins (AREA)
- Electrostatic Separation (AREA)
Description
【発明の詳細な説明】
〔発明の利用分野〕
本発明は、たんぱく質、核酸、細胞等の荷電物
質を電気泳動によつて分離する無担体電気泳動装
置に関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Application of the Invention] The present invention relates to a carrier-free electrophoresis device for separating charged substances such as proteins, nucleic acids, cells, etc. by electrophoresis.
〔発明の背景〕
電気泳動法によりたんぱく質等の荷電物質を連
続的に分離する電気泳動装置は、技術文献「エレ
クトロフオレシス1982、３、p235〜243」
（Electrophoresis 1982、３、235−243）に開示
されている。ここに開示されている無担体連続電
気泳動装置は、泳動室両端の電極室内の電極に直
流電圧を印加すると共に、泳動室上部より荷電物
質および分離バツフア（緩衝液）を流下させ、荷
電物質を泳動室内で電気泳動させて分離し、泳動
室下部から抜出す如き構成となつている。このよ
うな装置では、泳動室内での液の温度が均一であ
れば問題なく分離可能である。しかし、実際に
は、ジユール熱の発生によつて液の流れは乱さ
れ、安定した分離を行なう上で問題を残してい
る。[Background of the Invention] An electrophoresis device that continuously separates charged substances such as proteins by electrophoresis is described in the technical document "Electrophoresis 1982, 3, p. 235-243".
(Electrophoresis 1982, 3, 235-243). The carrier-free continuous electrophoresis device disclosed herein applies a DC voltage to electrodes in electrode chambers at both ends of the electrophoresis chamber, and at the same time allows a charged substance and a separation buffer (buffer) to flow down from the top of the electrophoresis chamber. The structure is such that the particles are separated by electrophoresis in the electrophoresis chamber, and then extracted from the lower part of the electrophoresis chamber. In such an apparatus, if the temperature of the liquid in the electrophoresis chamber is uniform, separation can be performed without any problem. However, in reality, the flow of the liquid is disturbed by the generation of Joule heat, which poses a problem in performing stable separation.
本発明の目的は、荷電物質の分離を安定して行
なうことのできる無担体連続電気泳動装置を提供
することである。
An object of the present invention is to provide a carrier-free continuous electrophoresis device that can stably separate charged substances.
本発明は、従来における問題点が泳動室内で発
生するジユール熱による対流混合である点に着目
し、ジユール熱により生じる対流を利用し、この
対流が安定して生じるようにこの対流と同一方向
の循環流を外部に設けた送液装置の送液によつて
発生させ、この循環流と電極への電圧印加による
電気泳動との作用によつて荷電物質を分離し、泳
動室の電極室寄りから荷電物質を抜出すようにし
たことを特徴とする。
The present invention focuses on the fact that a conventional problem is convective mixing due to Joule heat generated in the migration chamber.The present invention utilizes the convection generated by Joule heat, and in order to stably generate this convection, A circulating flow is generated by feeding a liquid from an external liquid feeding device, and charged substances are separated by the action of this circulating flow and electrophoresis caused by voltage application to the electrodes, and the charged substances are separated from the electrophoresis chamber near the electrode chamber. It is characterized by extracting charged substances.
以下、本発明の一実施例を第１，２図により説
明する。第１図は本発明による無担体連続電気泳
動装置の正面断面図、第２図は第１図の−矢
視断面図である。図において、１は泳動装置本
体、２は泳動室、３ａ，３ｂは電極室で、泳動室
２と電極室３ａ，３ｂは半透膜４で仕切られてい
る。泳動室２内は濾過膜５により３室２ａ，２
ｂ，２ｃに区分されており、濾過膜５の上端と泳
動室２内壁との間には液が流通するための開孔部
６がある。一方、電極室３ａ，３ｂ内にはそれぞ
れ直流電圧をかけるための電極７ａ，７ｂが設置
されている。泳動室２ａ，２ｃの下部には液循環
用出口ノズル７ａ，７ｂが泳動室２ｂの下部には
液循環用入口ノズル８ａ，８ｂが取りつけられ、
液循環ポンプ９ａ，９ｂとそれぞれ配管１０で接
続されている。泳動室２ｃのほぼ中央部には荷電
物質を含む被処理液の入口ノズル１１があり、泳
動室２ａ，２ｂのそれぞれの下端に被処理液の出
口ノズル１２ａ，１２ｂがある。一方電極室３
ａ，３ｂのそれぞれの下部及び上部には、電極液
の入口ノズル１３ａ，１３ｂ及び出口ノズル１４
ａ，１４ｂが設けられている。
An embodiment of the present invention will be described below with reference to FIGS. 1 and 2. FIG. 1 is a front sectional view of a carrier-free continuous electrophoresis device according to the present invention, and FIG. 2 is a sectional view taken along the - arrow in FIG. In the figure, 1 is the electrophoresis device main body, 2 is an electrophoresis chamber, and 3a and 3b are electrode chambers.The electrophoresis chamber 2 and the electrode chambers 3a and 3b are separated by a semipermeable membrane 4. The inside of the migration chamber 2 is divided into three chambers 2a and 2 by the filter membrane 5.
b and 2c, and there is an opening 6 between the upper end of the filtration membrane 5 and the inner wall of the migration chamber 2 through which the liquid flows. On the other hand, electrodes 7a and 7b for applying a DC voltage are installed in the electrode chambers 3a and 3b, respectively. Exit nozzles 7a, 7b for liquid circulation are installed at the bottom of the migration chambers 2a, 2c, and inlet nozzles 8a, 8b for liquid circulation are installed at the bottom of the migration chamber 2b.
It is connected to liquid circulation pumps 9a and 9b through piping 10, respectively. An inlet nozzle 11 for a liquid to be processed containing a charged substance is located approximately in the center of the migration chamber 2c, and outlet nozzles 12a, 12b for a liquid to be processed are provided at the lower ends of each of the migration chambers 2a, 2b. One electrode chamber 3
Inlet nozzles 13a, 13b and outlet nozzle 14 for the electrode liquid are provided at the lower and upper parts of the electrodes a and 3b, respectively.
a, 14b are provided.
ここで、電極室３ａ，３ｂと泳動室２を仕切る
半透膜４には、たんぱく質などの被処理荷電物質
を通さない孔径を持つ逆浸透膜などが用いられ
る。又、泳動室２内を区分する濾過膜５は、被処
理荷電物質を通過させる孔径を持つている。この
濾過膜５のかわりに孔径0.1〜１mmの多孔板を用
いることも可能である。 Here, as the semipermeable membrane 4 that partitions the electrode chambers 3a, 3b and the migration chamber 2, a reverse osmosis membrane or the like having a pore size that does not allow the passage of charged substances such as proteins is used. Further, the filtration membrane 5 that divides the inside of the migration chamber 2 has a pore size that allows the charged substance to be processed to pass therethrough. It is also possible to use a perforated plate with a pore diameter of 0.1 to 1 mm instead of the filter membrane 5.
このような泳動装置１の泳動室２に泳動用のバ
ツフアを流入させ、電極室３ａ，３ｂにもバツフ
アを外部より流入循環させる。液循環ポンプ９
ａ，９ｂにより泳動室２ａ，２ｃ内の液を抜出し
て、中央の泳動室２ｂ内へ流入させ、図中の矢印
で示すような対流循環を起こさせる。このような
循環流のある泳動室２にたんぱく質などの荷電物
質を含む被処理液を入口ノズル１１より連続供給
して、両側の電極７ａ，７ｂに直流電圧をかける
と、荷電物質は正負の荷電状態に応じて循環しな
がら泳動移動し、正に荷電した物質は陰極側（７
ｂ側）負に荷電した物質は陽極側（７ａ側）に引
き寄せられ、それぞれ流動室７ａ，７ｃの下部に
濃縮させる。そして荷電状態により分離された物
質は、出口ノズル１２ａ，１２ｂより連続して抜
出される。 A buffer for electrophoresis is caused to flow into the electrophoresis chamber 2 of such an electrophoresis apparatus 1, and buffer is also introduced from the outside and circulated into the electrode chambers 3a and 3b. Liquid circulation pump 9
The liquid in the electrophoresis chambers 2a and 2c is drawn out by a and 9b and flows into the central electrophoresis chamber 2b, causing convection circulation as shown by the arrows in the figure. When a liquid to be processed containing a charged substance such as protein is continuously supplied from the inlet nozzle 11 to the migration chamber 2 having such a circulating flow and a DC voltage is applied to the electrodes 7a and 7b on both sides, the charged substance becomes positively and negatively charged. Electrophoretic movement occurs while circulating depending on the state, and positively charged substances move toward the cathode side (7
(b side) Negatively charged substances are attracted to the anode side (7a side) and are concentrated in the lower portions of the flow chambers 7a and 7c, respectively. The substances separated depending on the charged state are continuously extracted from the outlet nozzles 12a and 12b.
このような電気泳動操作においては、ジユール
熱による対流は問題とならない。また、この対流
と同一方向の循環流をポンプ９ａ，９ｂにより強
制的に発生させるので、循環流は安定して発生
し、循環流の乱れによる分離能力の低下は防止で
きる。ジユール熱により発生する自然対流平均流
速ｕは、第１図に示すような泳動室の幅をＢ、流
れと直角方向の温度勾配をβとすると、次式で求
めることができる。 In such electrophoretic operations, convection due to Joule heat does not pose a problem. Further, since a circulating flow in the same direction as this convection is forcibly generated by the pumps 9a and 9b, the circulating flow is stably generated, and it is possible to prevent a decrease in the separation capacity due to turbulence of the circulating flow. The average flow velocity u of natural convection generated by Joule heat can be determined by the following equation, where B is the width of the migration chamber as shown in FIG. 1, and β is the temperature gradient in the direction perpendicular to the flow.
ｕ＝ρ0αβgB／1536μ ……(1)
ここに、ρ0：液の平均密度、α：体膨張率、
ｇ：重力の加速度、μ：液の粘度である。 u=ρ 0 αβgB/1536μ ...(1) Here, ρ 0 : Average density of liquid, α : Coefficient of body expansion,
g: acceleration of gravity, μ: viscosity of the liquid.
例えば、ほう酸バツフアのように物性が水に近
い液を用い、泳動室の幅Ｂを２cmとし、ジユーム
熱により５℃／cmの温度勾配ができる場合には、
式(1)より自然対流平均流速ｕは0.53cm／Ｓとな
る。そこでこの場合には、泳動装置内の循環流速
が0.6〜1.0cm／Ｓとなるように液循環ポンプ９
ａ，９ｂの吐出流量を調節する。 For example, if a liquid with physical properties similar to water, such as boric acid buffer, is used, and the width B of the migration chamber is 2 cm, and a temperature gradient of 5°C/cm is created due to room heat,
From equation (1), the average flow velocity u of natural convection is 0.53 cm/S. Therefore, in this case, the liquid circulation pump 9 should be adjusted so that the circulation flow rate in the electrophoresis device is 0.6 to 1.0 cm/S.
Adjust the discharge flow rates of a and 9b.
上述した装置でたんぱく質などの分離を行うた
めには、分離すべき物質同志の帯電を逆にする必
要がある。例えば、等電点7.2のミオグロビンと
等電点11のリゾチウムを分離する場合には、泳動
用バツフアのPHを9.0とすることにより、それぞ
れのたんぱく質の帯電を負と正とに分割すること
ができる。この場合の電気泳動操作においては、
導電率0.1〜５ｍＳ／cm程度のほう酸バツフアを
用い、５〜100V／cm程度の電圧勾配をかけるこ
とにより所期の目的が達成できる。 In order to separate proteins and the like using the above-mentioned apparatus, it is necessary to reverse the charges of the substances to be separated. For example, when separating myoglobin, which has an isoelectric point of 7.2, and lysotium, which has an isoelectric point of 11, by setting the pH of the electrophoresis buffer to 9.0, the charge of each protein can be divided into negative and positive. . In the electrophoresis operation in this case,
The desired purpose can be achieved by using a boric acid buffer with a conductivity of about 0.1 to 5 mS/cm and applying a voltage gradient of about 5 to 100 V/cm.
このようにして、複数の等電導を持つたたんぱ
く質の中から有効たんぱく質を分離することがで
きる。 In this way, an effective protein can be separated from a plurality of proteins having equal conductivity.
本発明の他の実施例を第３図に示す。この例で
は、液循環用出口ノズル７ａ，７ｂを中央泳動室
２ｂの上端に設け、液循環用入口ノズル８ａ，８
ｂを両側の泳動室２ａ，２ｃの上端に設けて、液
循環ポンプ９ａ，９ｂに配管したものである。 Another embodiment of the invention is shown in FIG. In this example, outlet nozzles 7a and 7b for liquid circulation are provided at the upper end of the central migration chamber 2b, and inlet nozzles 8a and 8 for liquid circulation are provided at the upper end of the central migration chamber 2b.
b are provided at the upper ends of the migration chambers 2a, 2c on both sides, and are piped to the liquid circulation pumps 9a, 9b.
本実施例においては液循環ポンプ９ａ，９ｂへ
の液循環用出口ノズル７ａ，７ｂ及び入口ノズル
８ａ，８ｂを被処理液の出口ノズル１２ａ，１２
ｂから遠くに離して取りつけることができるの
で、液の強制循環による、出口、入口ノズル７
ａ，７ｂ，８ａ，８ｂ付近での流れの乱れの被処
理液の出口部分への影響を無くすことができ分離
性能が高められる。 In this embodiment, the outlet nozzles 7a, 7b and the inlet nozzles 8a, 8b for liquid circulation to the liquid circulation pumps 9a, 9b are replaced with the outlet nozzles 12a, 12 for the liquid to be treated.
Since it can be installed far away from b, the outlet and inlet nozzles 7 can be installed by forced circulation of the liquid.
The influence of flow turbulence near a, 7b, 8a, and 8b on the exit portion of the liquid to be treated can be eliminated, and separation performance can be improved.
さらに第３図に示すように、液循環ポンプ９
ａ，９ｂへの配管１０、途中にジヤケツト方式な
どの熱交換器１５を組み込むことにより、ジユー
ル熱で加熱された液を冷却して泳動室２ａ，２ｃ
に戻すことができる。これによりたんぱく質など
の荷電物質の熱による変質が軽減できると共に、
泳動室２ｂを上昇してくる液との温度差を顕著に
することができスムースな下降流れが得られ、安
定した分離操作ができる。 Furthermore, as shown in FIG.
By installing a heat exchanger 15 such as a jacket type in the pipe 10 to a and 9b, the liquid heated by Joule heat is cooled and transferred to the migration chambers 2a and 2c.
can be returned to. This can reduce the deterioration of charged substances such as proteins due to heat, and
The temperature difference between the liquid and the liquid rising in the migration chamber 2b can be made significant, a smooth downward flow can be obtained, and a stable separation operation can be performed.
第４図に本発明の他の実施例を示す。第３図の
場合とほぼ同様の構成であるが、液循環ポンプを
１台とし、調節弁１６ａ，１６ｂを介して泳動室
２ａ，２ｃに分散して供給するようにしている点
が異なる。このようにすると、循環用の設備の簡
略化ができる。 FIG. 4 shows another embodiment of the invention. The configuration is almost the same as that shown in FIG. 3, except that there is only one liquid circulation pump, and the liquid is distributed and supplied to the migration chambers 2a, 2c via control valves 16a, 16b. In this way, the circulation equipment can be simplified.
本発明によれば、泳動室内で発生するジユール
熱によつて起こる自然対流と同一方向に強制的な
循環流れを起こさせながら、連続的に安定した泳
動操作ができるので、従来装置で起つていたジユ
ール熱による混合を防止でき、安定した分離操作
を実現できる。
According to the present invention, it is possible to perform continuous and stable electrophoresis operation while generating a forced circulation flow in the same direction as the natural convection caused by the Joule heat generated in the electrophoresis chamber. It is possible to prevent mixing due to the heat generated by the slurry and achieve stable separation operations.
第１図は本発明の一実施例に係る無担体連続電
気泳動装置の正面断面図、第２図は第１図の−
矢視断面図を示す平面図、第３図および第４図
は本発明の他の実施例を示す図である。
１……泳動装置本体、２ａ，２ｂ，２ｃ……泳
動室、３ａ，３ｂ……電極室、９ａ，９ｂ……泳
循環ポンプ、１１……被処理液入口ノズル、１２
ａ，１２ｂ……被処理液出口ノズル。
FIG. 1 is a front sectional view of a carrier-free continuous electrophoresis device according to an embodiment of the present invention, and FIG. 2 is a -
A plan view showing a sectional view taken in the direction of arrows, FIGS. 3 and 4 are views showing other embodiments of the present invention. DESCRIPTION OF SYMBOLS 1...Migration device main body, 2a, 2b, 2c...Migration chamber, 3a, 3b...Electrode chamber, 9a, 9b...Migration circulation pump, 11...Processed liquid inlet nozzle, 12
a, 12b... Processed liquid outlet nozzle.
Claims (1)
設け、該電極室内に夫々電極を配し、該半透膜間
を泳動室とし、該泳動室の幅方向中央部に分離さ
れるべき荷電物質を含む被処理液を供給する入口
ノズルを設け、該泳動室の前記電極室寄りに荷電
物質の抜出ノズルを設け、さらに前記泳動室内の
液にジユール熱による対流と同方向の循環流を生
じさせるために前記泳動室内に供給された液を抜
出しこれを再び前記泳動室内に送液する送液装置
を設けたことを特徴とする無担体電気泳動装置。1 Electrode chambers partitioned by semipermeable membranes are provided on both sides of the main container, electrodes are placed in each electrode chamber, and the space between the semipermeable membranes is used as a migration chamber, which should be separated in the center of the width direction of the migration chamber. An inlet nozzle for supplying a liquid to be processed containing a charged substance is provided, a nozzle for extracting the charged substance is provided near the electrode chamber of the electrophoresis chamber, and a circulating flow in the same direction as convection due to Joule heat is provided in the liquid in the electrophoresis chamber. 1. A carrier-free electrophoresis apparatus comprising a liquid feeding device for extracting a liquid supplied into the electrophoresis chamber and feeding the liquid again into the electrophoresis chamber in order to cause the electrophoresis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60055821A JPS61215953A (en) | 1985-03-22 | 1985-03-22 | Carrier-free continuous electrophoretic device |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60055821A JPS61215953A (en) | 1985-03-22 | 1985-03-22 | Carrier-free continuous electrophoretic device |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS61215953A JPS61215953A (en) | 1986-09-25 |
| JPH0317295B2 true JPH0317295B2 (en) | 1991-03-07 |
Family
ID=13009612
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP60055821A Granted JPS61215953A (en) | 1985-03-22 | 1985-03-22 | Carrier-free continuous electrophoretic device |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS61215953A (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10047088C2 (en) | 2000-09-21 | 2002-10-17 | Gerhard Weber | Medium for analytical and preparative electrophoresis |
| DE10063097B4 (en) | 2000-12-18 | 2007-04-19 | Becton, Dickinson And Co. | electrophoresis |
| DE10063096C1 (en) | 2000-12-18 | 2002-09-12 | Gerhard Weber | Electrophoresis device, electrophoresis method using an electrophoresis device and using the electrophoresis device |
| DE102005020134A1 (en) | 2005-04-29 | 2006-11-02 | Becton, Dickinson And Co. | Electrophoresis method, e.g. for analysis and preparation separation of ions, peptides, involves providing boundaries defining separation sub-spaces by adjacent flow of anodic and cathodic stabilization media through respective inlets |
-
1985
- 1985-03-22 JP JP60055821A patent/JPS61215953A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS61215953A (en) | 1986-09-25 |
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