JPH0625749B2 - Carrier-free continuous electrophoresis method and apparatus - Google Patents

Description

TECHNICAL FIELD The present invention relates to a technique for separating and purifying charged substances such as proteins, nucleic acids, cells, etc. by an electrophoretic method, which has particularly high separation performance and can be used for large-scale processing. It relates to a possible carrier-free electrophoresis method and device.

[Background of the Invention]

As a conventional method for separating and purifying charged substances such as proteins, nucleic acids, and cells (hereinafter, typically referred to as proteins), there are an electrophoresis method, a membrane separation method, a liquid chromatography and the like. The membrane separation method is a method of separating proteins according to the size of the pores of the membrane, and although continuous treatment is possible, it has the disadvantage of poor protein separation ability, and liquid chromatography is a method of separating proteins through a carrier-filled column. Although it has excellent separation ability, it is not suitable for industrial-scale large-scale processing because it is a batch operation. The electrophoresis method is a method of separating and purifying in an electric field by utilizing the difference in the charge amount of proteins. This electrophoresis method includes a carrier electrophoresis method using a carrier such as a gel and a carrier-free electrophoresis method performed in a free flowing wave without using a carrier. The carrier-electrophoresis method is a batch method, and the carrier-free electrophoretic method capable of continuous treatment is suitable for industrialization in which a large amount of treatment is performed.

For carrier-free electrophoresis, see Electrophor
Kur in essays 1982, 3, 235-243
"New aspects i" by t Hannig
n preparative and analyti
cal continuous free-flow
It is discussed in the literature entitled "cell electrophoresis". Devices based on this principle have already been manufactured by Hirshmann, West Germany.

A method for separating and purifying proteins by the carrier-free electrophoresis method will be described. When a mixture of proteins to be continuously separated is injected from the injection port into a separation buffer solution that flows at a constant rate across the electric field in the separation chamber, each protein has a different charge amount. Are different in moving speed in the separation buffer solution, and thus are deflected and separated while flowing in the separation buffer solution depending on the flow rate of the separation buffer solution. As described above, the present method allows continuous separation, and thus is effective for industrial-scale protein separation and purification.

In order to improve the separation performance with this method, it is important to always keep the flow rate of the separation buffer solution in the separation chamber constant, but since an electric current is passed through the separation buffer solution, Joule heat is always generated, This heat causes a convection phenomenon in the separation buffer solution, and the flow of the separation buffer solution is disturbed, so that the protein separation performance deteriorates. In order to solve this problem, in the conventional carrier-free electrophoresis method, the temperature and flow rate of the separation buffer solution are controlled with high accuracy of ± 0.2%, and the device is downsized to support Joule heat. However, the protein separation performance is not so good.

Furthermore, in order to eliminate the effect of this thermal convection, there is a plan to bring the electrophoretic device to outer space and perform electrophoresis under the condition of zero gravity, which shows how serious this convection is.

[Object of the Invention]

An object of the present invention is to provide a carrier-free continuous electrophoresis method and device which have high separation performance and can be processed in large quantities.

[Outline of Invention]

The present invention, in a carrier-free continuous electrophoresis method of separating a charged substance by treating a liquid to be treated containing a plurality of charged substances by a carrier-free electrophoresis method, charging a DC voltage from both outer sides of the migration chamber, While the buffer solution in the migration chamber is circulated so as to generate a pair of convection that rises in the center of the migration chamber and descends at both ends, the liquid to be treated is supplied to approximately the center of the migration chamber to separate charged substances. Then, the treatment liquid is taken out from the lower part of both outer ends of the migration chamber, which is a carrier-free continuous electrophoresis method.

Another feature of the present invention is that in a carrier-free continuous electrophoresis method in which a liquid to be treated containing a plurality of charged substances is treated by a carrier-free electrophoresis method to separate charged substances, both of the migration chambers filled with a buffer solution are used. The electrophoretic device main body having an electrode chamber formed through a semipermeable membrane on the outside is installed vertically, an inlet nozzle for the liquid to be treated is provided in the approximate center of the electrophoretic chamber, and the treatment liquid is provided below both outer ends of the electrophoretic chamber. In the carrierless continuous electric device, each of the outlet nozzles is provided.

Example of Invention

An embodiment of the present invention will be described below with reference to FIGS. 1 and 2. In FIGS. 1 and 2, reference numeral 1 is a vertically installed electrophoretic device main body, 2 is an electrophoretic chamber provided in a central part of the electrophoretic device main body 1, and 3a and 3b are both ends of the electrophoretic device 2 in the electrophoretic device main body 1. The formed electrode chamber is separated from the migration chamber 2 by a semipermeable membrane 4. 5a and 5b are charging electrodes such as platinum wires provided in the electrode chambers 3a and 3b, 6 is an inlet nozzle for a liquid to be treated containing a charged substance, which is provided on the side surface of the electrophoretic chamber 2 at substantially the center thereof, and 7
Reference numerals a and 7b are outlet nozzles of the treatment liquid which have been electrophoretically separated provided at the lower ends of the electrophoretic chamber 2, and 8a, 8b and 9a and 9b are inlet nozzles of the electrode liquid provided at the lower and upper ends of the electrode chambers 3a and 3b, respectively. And the outlet nozzle.

In the above-described configuration, when the migration buffer solution is filled in the migration chamber 2 and the buffer solution is also circulated from the outside into the electrode chambers 3a and 3b, a predetermined voltage is applied to the charging electrodes 5a and 5b, Joule heat is generated by the charging current,
As shown in the figure, convection occurs in the buffer solution in the migration chamber 2. Although Joule heat is uniformly generated in the electrophoretic chamber 2, it is difficult to dissipate the heat in the central portion of the electrophoretic chamber 2.
A temperature difference is generated between the buffer solution at the inner center and the buffer solution at both ends, and the buffer solution at the center in the migration chamber 2 that has the highest temperature has an upward flow and the buffer solution at both ends in the migration chamber 2 has a downward flow. Circulate in 2. When a liquid to be treated having a charge such as a protein is supplied from the inlet nozzle 6 into the migration chamber 2 having such a circulating flow of the buffer solution, the migration chamber 2 is charged according to the positive or negative charge of the charged substance. While flowing by convection,
It moves in the direction of the negative or positive electrode chamber 3b or 3a. In this case, when a DC voltage is applied with the charging electrode 5a being positive and the negative electrode 5b being negative, the positively charged charged substance moves to the electrode chamber 3b side and is concentrated in the lower portion of the migration chamber 2 on the electrode chamber 3b side, The treatment liquid is taken out from the outlet nozzle 7b. Similarly, the negatively charged charged substance moves to the side of the electrode chamber 3a, is concentrated in the lower part of the migration chamber 2 on the side of the electrode chamber 3a, and is taken out from the outlet nozzle 7a as a processing liquid. As a result, it is possible to continuously separate the plurality of charged substances according to the charged state, that is, the positive or negative charge.

Further, the charged substance is concentrated while being moved downward at both ends of the migration chamber 2 by convection of the buffer solution circulating from the center of the migration chamber 2 toward both ends, and is taken out from the lower portions of both ends of the migration chamber 2. Even when the concentration of charged substances becomes high,
It can be easily separated and highly concentrated.

In order to separate the charged substances from the liquid to be treated containing the plurality of charged substances by the electrophoretic method, it is necessary to reverse the charge of the charged substances to be separated. For example, the isoelectric point 7
When separating the myoglobin of 2 from the lysozyme of the isoelectric point 11, by setting the pH of the buffer solution for migration to 9.0, the charge of each protein can be divided into positive and negative polarities. .

FIG. 3 shows another embodiment of the present invention.
The same parts as those in FIG.
In FIG. 3, 10a and 10b are temperature controllers for adjusting the temperature in the electrode chambers 3a and 3b, and 11a and 11b are buffer solutions whose temperature is controlled by the temperature controllers 10a and 10b, respectively. The liquid feed pump circulates into the electrode chambers 3a and 3b, and the buffer liquid is extracted from the electrode chambers 3a and 3b and cooled to a predetermined temperature by the temperature controllers 10a and 10b. By forcibly cooling 3b, convection of the buffer liquid and the liquid to be treated in the migration chamber 2 can be promoted, and the liquid temperature in the migration chamber 2 can be maintained at a predetermined temperature to stabilize the circulating flow due to convection. it can.

In this embodiment, the migration chamber 2 necessary for separating charged substances is used.
Internal convection can be obtained by adjusting the temperature of the buffer solution in the electrode chambers 3a and 3b, and the efficiency of separating charged substances can be improved.

FIG. 4 shows still another embodiment of the present invention, in which a partition plate 12 is installed vertically in the lower center of the migration chamber 2,
The rising convection in the migration chamber 2 is stabilized, and a part of the charged substances electrophoretically separated in the lower part of the electrode chambers 3a, 3b in the migration chamber 2 is prevented from flowing to the central part and remixing. This is intended to improve the separation efficiency of charged substances.

FIG. 5 shows still another embodiment of the present invention, in which a partition plate 13 with a jacket is provided vertically in the lower center of the migration chamber 2.
Is installed and a heat medium is circulated through the jacketed partition plate 13 to maintain the liquid temperature in the central portion of the migration chamber 2 at a predetermined temperature, thereby obtaining stable convection. The separation efficiency is further improved. That is, by constantly maintaining the temperature difference between the central portion and both ends of the migration chamber 2 at a constant value, convection with a constant flow velocity can be obtained, and the turbulence of the liquid in the migration chamber 2 can be reduced to improve the separation performance. Can be increased.

FIG. 6 shows still another embodiment of the present invention. A partition plate 12 is installed vertically in the lower center of the migration chamber 2 and bubble generating nozzles 14a and 14b are provided on both sides of the partition plate 12. By providing the gas outlet nozzle 15 at the upper end of the migration chamber 2 and continuously generating bubbles 16 in the migration chamber 2 from the bubble generation nozzles 14a and 14b, the central portion of the migration chamber 2 is raised. This is to enhance the separation performance by promoting the circulation flow in the migration chamber 2.

In the present embodiment, the description was given of the promotion of convection in the migration chamber 2 by the action of the bubble pump, but as a means for forcibly promoting the convection in the migration chamber 2, a mechanical liquid transfer device such as a submersible pump may be used. .

〔The invention's effect〕

As described above, according to the present invention, a DC voltage is charged from both sides of the migration chamber to form a circulating flow in the buffer solution inside the migration chamber that rises at the center of the migration chamber and falls at both ends, and the liquid to be treated is stored in the migration chamber. Is supplied to separate the charged substances by electrophoresis and the treatment liquid is taken out from the lower part of both ends of the migration chamber, so convection of the buffer liquid due to Joule heat generated by passing an electric current into the buffer liquid is used. You can
In addition, since the charged substance can be stably and continuously treated, the separation performance of the charged substance can be improved and a large amount of treatment can be performed. Further, by taking out the treatment liquid from the lower portions of both ends of the migration chamber, it is possible to prevent convective diffusion of the charged substance and highly concentrate it.

[Brief description of drawings]

FIG. 1 is a front view of an electrophoretic device showing an embodiment of the present invention, FIG. 2 is a sectional view taken along line II-II of FIG. 1, and FIGS.
Each of the drawings is a front view of an electrophoretic device showing another embodiment of the present invention. 1 ... Electrophoresis device body, 2 ... Migration chamber, 3a, 3b ... Electrode chamber, 4 ... Semipermeable membrane, 5a, 5b ... Charging electrode, 6,
8a, 8b ... Inlet nozzle, 7a, 7b, 9a, 9b ...
... Exit nozzle, 10a, 10b ... Temperature controller, 11
a, 11b ... Liquid-sending pump, 12 ... Partition plate, 13 ...
Partition plates with jackets, 14a, 14b ... Bubble generating nozzles, 15 ... Gas outlet nozzles, 16 ... Bubbles

Front Page Continuation (72) Inventor Shoji Yoshinaga 502 Jinritsucho, Tsuchiura-shi, Ibaraki Machinery Research Laboratory, Hiritsu Seisakusho Co., Ltd. In the factory (56) Reference JP 59-52743 (JP, A) JP 58-147639 (JP, A) JP 58-14048 (JP, A)

Claims (7)

[Claims] 1. In a carrier-free continuous electrophoresis method in which a liquid to be treated containing a plurality of charged substances is treated by a carrier-free electrophoresis method to separate charged substances, a DC voltage is charged into the migration chamber from both sides thereof. While the buffer solution in the migration chamber is circulated so as to generate a pair of convection that rises in the center of the migration chamber and falls in both sides, the liquid to be treated is supplied to the substantially center of the migration chamber to remove charged substances. A carrier-free continuous electrophoresis method, characterized in that the treatment liquid is separated and the treated liquid is taken out from the lower portions of both outer ends of the migration chamber. 2. The carrier-free continuous electrophoresis method according to claim 1, wherein both outer side portions of the migration chamber are cooled. 3. A carrier-free continuous electrophoresis apparatus in which a liquid to be treated containing a plurality of charged substances is treated by a carrier-free electrophoresis method to separate the charged substances. The electrophoretic apparatus main body in which the electrode chamber is formed through a permeable membrane is installed vertically, an inlet nozzle for the liquid to be treated is provided substantially in the center of the electrophoretic chamber, and an outlet nozzle for the treating liquid is provided at the lower portions of both outer ends of the electrophoretic chamber. A carrier-free continuous electrophoresis device, characterized in that each is provided with: 4. The carrier-free continuous electrophoresis apparatus according to claim 3, wherein a temperature controller for circulating the cooled buffer solution and a liquid delivery pump are provided in each of the electrode chambers. 5. The carrier-free continuous electrophoresis apparatus according to claim 3, wherein a partition plate extending in the vertical direction is installed at the center of the lower part of the electrophoresis chamber. 6. The carrier-free continuous electrophoresis apparatus according to claim 5, wherein nozzles for generating bubbles are installed on both lower sides of the partition plate of the migration chamber. 7. The carrier-free continuous electrophoresis apparatus according to claim 3, further comprising a partition plate with a jacket extending vertically in the lower center of the electrophoretic chamber and allowing a heat medium to flow therethrough.