JPH0363561A - Device and method for filter type electrophoresis - Google Patents

Abstract

PURPOSE:To obtain a filter type electrophoresis device which is excellent in separationperformance and capable of continuous treatment at a large amounts by performing separation due to electrophoresis in a porous carrier. CONSTITUTION:A rectangular parallelopiped 3 for a porous carrier is formed by closely laminating about 100 sheets of electrophoresis film made of cellulose acetate having 0.5mum mean pore diameter. A cathode 1 and an anode 3 are provided on both sides thereof via the semipermeable hydrated gel layers 4a, 4b wherein substance obtained by partially formalizing PVA is utilized. Buffer soln. is supplied through the feed parts 5a, 5b and raw liquid is supplied through a feed port 6 of the raw liquid to be separated. The separated components are obtained through the liquid collection ports 7a, 7b, 7c, 7d.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field] The present invention relates to a continuous electrophoresis method and apparatus for separating ampholytes such as proteins, nucleic acids, and amino acids using electrophoresis.

"Prior Art" Conventional methods for separating and purifying ampholytes such as proteins, nucleic acids, and amino acids include electrophoresis, membrane separation, and liquid chromatography. Membrane separation is a method of separating substances based on the size of the membrane's pores, and has the advantage of being able to process large quantities continuously, but is unsuitable for separating proteins with similar molecular weights or amino acids. . The liquid chromatography method is a method in which substances are separated through a carrier-packed column, and although it has excellent separation performance, it is a batch operation and is not suitable for large-scale processing on an industrial scale.On the other hand, the electrophoresis method separates substances by passing them through a carrier-packed column. This is a method of separating and purifying in an electric field using the difference. This electrophoresis includes carrier electrophoresis using carriers such as gel and filter paper;
There is carrier-free electrophoresis, which is performed in a free solution without using a carrier.

``Problem to be solved by the invention'' Carrier electrophoresis has excellent separation performance and is widely used for analytical purposes, but it is difficult to recover the separated substances, so preparative It is hardly used for any purpose. In the past, carrier-free electrophoresis has been the focus of attention and research for applications in which large amounts of continuously separated substances are recovered. Regarding carrier-free electrophoresis, as Yuji Matsuo describes in detail in Soko's ``Latest Electrophoresis Methods J'' (3rd edition, 1986, published by Tokoyo Shoten), Joule heat is always generated because electric current is passed through the separation buffer. The heat generated causes convection in the buffer, which disrupts the flow of the buffer and conducts an electric current, reducing the separation of the buffer and the substance.

In order to improve these drawbacks of carrier-free electrophoresis, it is necessary to control the temperature and flow rate of the separation buffer solution with extreme precision, and for this purpose it is necessary to make the separation chamber as thin and flat as possible. However, the throughput was small, which was a problem when scaling up to an industrial scale. In JP-A No. 61-83952, the size of the separation chamber is increased by installing an injection port for the stock solution to be separated and a recovery port for the separated substance above the convective liquid flow caused by heat generation. However, in this method, the stock solution is injected into the separation chamber midway through the flow of the solution, so sufficient separation time cannot be secured, and only a small flow rate can be processed despite the increased size.

A device was developed in 1983 to allow multiple fractions to be fractionated in parallel to the electric field in the separation chamber, and to supply the stock solution to each fractionation chamber.
This is done in No. 26392. This method is very effective in reducing turbulence in liquid flow. However, if the separation chamber is divided into too many parts or divided too finely, it becomes difficult to keep the liquid flow rate in each division chamber constant, and this method is not a satisfactory solution either.

"Objective of the Invention" The object of the present invention is to eliminate the above-mentioned drawbacks of carrier-free electrophoresis, and to provide a filtration-type electrophoresis device that has excellent separation performance and is capable of continuous large-scale processing. .

"Means for Solving the Problems" The object of the invention was achieved by the following method of performing electrophoretic separation in a porous chamber carrier, and an overview thereof will be described below.

A porous chamber carrier rectangular parallelepiped is housed in a container, a positive electrode is arranged parallel to one surface of the rectangular parallelepiped outside that surface, a negative electrode is arranged outside the opposite surface parallel to the surface, and both electrodes A current is passed between them to form an electric field, and a buffer solution supply port and a separation stock solution supply port are provided on one surface of the porous carrier rectangular parallelepiped parallel to the direction of the buffer solution and the current,
On the other side, a filtration type electrophoresis device is used, which is divided into at least two parts perpendicular to the current direction and each has a liquid collection port, and a current is passed between the two electrodes to separate the buffer solution and separation stock solution respectively. The same flux is supplied to the buffer solution supply port and separation stock solution supply port, and a current is passed through the porous carrier rectangular parallelepiped.
The amphoteric electrolyte dissolved in the buffer solution and separation stock solution is separated by electrophoresis, and the permeated liquid is collected at each liquid collection port.

When this method is used, even if Joule heat is generated, it is disturbed by the porous carrier due to the convection of the buffer solution, so separation accuracy is dramatically improved compared to conventional carrier-free electrophoresis.

The invention will be explained in detail below.

The electrodes used here are mainly flat plate electrodes that are parallel to the plane of the porous carrier rectangular parallelepiped that the electrodes face, but they can basically be the same as those used in conventional carrier-free electrophoresis devices. . Therefore, platinum is the most commonly used material, and silver-silver chloride electrodes are also used.

The porous carrier mentioned here is required to have properties that suppress convection due to Joule heat and do not interfere with electrophoresis of electrolytes such as proteins. Therefore, the diameter of the hole is O,1
It is preferable that the diameter is from μm to 5 μm, and it is also necessary that no protein be adsorbed. In addition, the carrier has an electrolyte on its surface in a buffer solution, so a phenomenon called electroosmosis occurs in which the buffer solution moves toward one electrode when an electric field is applied. When electroosmosis occurs strongly, the electrolyte to be separated moves together with the buffer solution and current flows, making it impossible to separate the electrolyte from the buffer solution sufficiently. As a porous carrier that does not adsorb proteins, has little electroosmosis, and has an appropriate pore size, a layer of cellulose acetate porous membranes or a layer of acrylamide gel, starch, and filter paper may be used.

The shape of the porous carrier is preferably a rectangular parallelepiped, but any shape that allows electrophoretic operation, liquid supply, and liquid collection is sufficient.The size of the rectangular parallelepiped varies depending on the properties of the electrolyte to be separated, separation accuracy, and throughput. come. The length between the electrodes (migration distance) is usually 10 cm or less, and 2 to 5 C1 is sufficient. Liquid supply/
The thickness of the carrier in the discharge direction is determined by the electrophoresis time (average residence time of the liquid) required for separation once the supply rates of the separation stock solution and buffer solution are determined. The electrophoresis time varies greatly depending on the properties of the separated electrolyte, so this cannot be said for boat fishing.However, if the thickness of the carrier is too thick, Joule heat will accumulate and the carrier will generate heat, which is undesirable.
The remaining direction (depth) of the rectangular parallelepiped, which is preferably less than or equal to 0 CI, is determined solely by the amount of liquid to be treated.

Buffer selection is the second most important factor for electrophoresis, next to carrier selection. This is detailed in the bottom section above. The most important aspect of buffer selection is to select a buffer having a pH intermediate between the isoelectric points of the two ampholytes to be separated.

Another aspect is ionic strength. If the ionic strength is strong, the separation will be sharp, but the electrophoresis time will be longer.
This will be determined by applying a current, using a buffer solution, and conducting trial experiments in conjunction with setting voltage and current conditions, which will be described later.

The voltage applied between the two electrodes must be found to find the optimal conditions depending on the distance between the electrodes and the ionic strength of the buffer solution! If the pressure is set too high, the current will increase and the migration speed will become faster, but the generation of Joule heat will increase and the carrier will become hot.

These electrophoresis conditions can be set relatively easily by conducting an electrophoresis experiment using a thin film made of the same material as the carrier, applying a current, and using a buffer solution.

Finally, we will discuss the necessity of preventing heat generation due to Joule heat. ii When an electric current is passed through the solute, Joule heat is inevitably generated. In carrier-free electrophoresis, heat generated by convection of the liquid escapes to the outside.In zero-filtration electrophoresis, if heat is not actively released, heat will accumulate and the carrier and electrolyte will become hot. Dissolved proteins undergo thermal denaturation.

An effective method for cooling is to provide a space between the electrode and the hydrogel layer and pass a buffer solution there as well. A method of cooling the entire device from outside the device is also often used. Another good method is to set the separation conditions by applying a current, reducing the ionic strength of the buffer solution and the buffer solution as much as possible, and performing fast migration with a small current.

Furthermore, increasing the supply flux of the buffer solution or separation stock solution as much as possible is also effective in reducing the temperature rise.

A more detailed explanation will be given below according to examples.

Embodiment FIG. 1 shows the entire filtration type electrophoresis apparatus of the present invention. FIG. 2 shows a cross section taken along the line AA' in FIG. 3 is a porous carrier rectangular parallelepiped, and in this example, the average pore diameter is 0.5 μm.
It uses approximately 100 layers of cellulose acetate electrophoresis membranes.

1 and 2 are a cathode and an anode, respectively. 4a and 4b are semipermeable hydrogel layers made of partially formalized polyvinyl alcohol.

5a, 5b are buffer solution supply ports, 6 is separation stock solution supply port, 7a
, 7b respectively indicate liquid collection ports. The distance between the electrodes is 25III11. The thickness of the carrier (filtration distance) is 14 am,
The width of the carrier is 100++ m.

Veronal buffer was used as the buffer solution, and human serum diluted 10 times with veronal buffer was used as the separation stock solution. Supply the buffer solution at a flow rate of 18 ml/sin using a pump,
The separation stock solution is supplied at a flow rate of 21 d/win, and 1 d/win is applied between the electrodes.
Apply 5V DC voltage.

Aldagon was obtained from the liquid collector 7a, and T globulin was obtained from the liquid collector 7b.

[Brief explanation of drawings]

FIG. 1 shows an overall view of the filtration type electrophoresis apparatus of the present invention, and FIG. 2 shows a cross section taken along line AA in FIG. The numbers in the figure represent the following. 1: negative electrode 2; positive electrode 3: porous carrier 4a, 4b: hydrogel layer 5a,
5b: Buffer solution supply port 6: Separation stock solution supply port 7a, 7b, 7c, 7d: Liquid collection port 8: Filtration type electrophoresis device 11: w! A edge body 12: frame body 13a
, 13b: II solution chamber 14: Separation stock solution chamber

Claims (2)

[Claims] (1) A porous carrier rectangular parallelepiped is housed in a container, a positive electrode is arranged parallel to one surface of the rectangular parallelepiped outside that surface, and a negative electrode is arranged outside the opposite surface parallel to the surface. , a current is passed between both electrodes to form an electric field, and a buffer solution supply port and a separation stock solution supply port are provided on one side of the porous carrier rectangular parallelepiped parallel to the direction of this current, and the opposite side is provided with a buffer solution supply port and a separation stock solution supply port. A filtration type electrophoresis device, characterized in that it is divided into at least two parts perpendicular to the current direction, each of which is provided with a liquid collection port. (2) A current is passed between both electrodes of the filtration type electrophoresis device according to claim (1), and the buffer solution and the separation stock solution are supplied at the same flux to the buffer solution supply port and the separation stock solution supply port, respectively, and the porous A filtration type electrophoresis method in which amphoteric electrolytes dissolved in a separation stock solution are electrophoresed and separated in a rectangular carrier, and the liquid that passes through each liquid collection port is collected.