DE2051715B2 - SEPARATION CHAMBER FOR CONTINUOUS DEFLECTION ELECTROPHORESIS - Google Patents

Description

via seals (K) and electrode membranes (L) 35 from all four sides through the elec-

are pressed together on the electrode block (E) , with the electrode-side chamber ends above the slots in the electrode spaces.

6. separation chamber according to claim 1 to 3, characterized in that the outer block and inner block can be pressed separately onto the electrode block.

7. separation chamber according to claim I to 5, characterized in that the separation chamber has a U-shaped Has cross section.

8. Separation chamber according to one of the preceding claims, characterized in that a through the electrode block f £? through clamping bolt (H) that clamps blocks (C) and (E) together.

9. Separating chamber according to one of the preceding claims, characterized in that the outer blocks (A, Bi, Bi) and the cooling block (C) each consist of one or more parts, through the mutual displacement of which separating chambers of variable thickness and shape can be produced.

The invention relates to a separation chamber for continuous deflection electrophoresis in the carrier ; ien electrolyte flow, with electrodes, with a separation chamber and supply and discharge lines for the electrode buffer, the chamber buffer and the substance to be separated.

For continuous electrophoretic separation

free buffer stream, a number of separating chambers have been developed, which almost without exception work on the basis of the Principle: Between two plane-parallel electrode and buffer feed or suction devices be placed under quite high mechanical tension, precisely to be kept at the correct distance and against To stabilize buffer pressure fluctuations, technically only very complex to solve and therefore cause increased Susceptibility to failure. The large intrinsic volume of the separation chamber is also important when working with small sample quantities very disadvantageous. So it is hardly possible to shorten the dwell time of the substance in the chamber than Hold for about 30 minutes, which hinders work with sensitive substances in particular. From US-PS 35 09 035 a separation chamber is also known in which the electrode spaces at an angle are arranged to form a planar separation chamber. The connection from the electrode chambers to the separation chamber takes place via narrow slots that are only used to conduct electricity, but in which no electrolyte flow occurs. A flow in the slots must be avoided in this known separation chamber because the slots only extend over a part of the separation chamber, so that an inlet or If electrolyte leaks from the separation space into or out of a slot, the flow in the separation space is impermissible would be disturbed.

In DT-OS 19 23 613, a separating device is finally described in which a horizontal Separation space is limited at the top and bottom by walls that are uniformly ribbed. In the gutters between the ribs, the number of which is about 100 to 200, are supposed to lead the fractions that have already been separated so that remixing by diffusion is avoided. This known separation chamber can therefore only be used in a horizontal position

and is practically useless for particle separation. It is intended for batch operation and is only suitable for a coarse operation in the case of continuous operation Pre-separation.

therefore _he n the present invention, the object S lieft gugrunde to provide a separation chamber, which is mechanically so stable that, for example, changes in the pressure and buffer have unequal pressing of the seals no influence on the shape of the chamber. In addition, “with a small intrinsic volume, high field strengths, intensive cooling and high buffer flood speeds can be achieved. In addition, the chamber should be able to be assembled in a few simple steps and its main dimensions should be variable.

This object is achieved according to the invention in that the separation chamber between the electrodes has a non-flat, single or multiple angled or arched cross-section

It is useful here to combine the electrodes in a common block so that the Electrodes practically form a unit.

The inner surface of the separation chamber can be through an inner block and its outer surface through the inner surface an outer block can be formed. Thereby are inner block, and if desired also outer block and Electrode block provided for holding cooling devices. Has the separation chamber, for example a U-shaped cross-section, the outer block can consist of a front block and two side blocks, and the Inner block consist of a cooling block.

The outer and inner block are expediently (via interposed seals and electrode membranes pressed onto the electrode block, with the electrode-side chamber ends above the slots of the electrode spaces.

In order to enable the plate thickness to be set precisely, it is advisable to design the individual blocks in such a way that the outer block and inner block can be pressed separately onto the electrode block. In the case of a U-shaped chamber, a clamping bolt H leading through the electrode block into the cooling block can be provided, which clamps the blocks C and E together so that the thickness of the separating chamber remains constant.

Outer and inner block can each consist of one or more parts, through their mutual Displacement separation chambers of variable thickness and shape can be made.

If the electrodes are housed in a common block, they can be used for replacement or to facilitate cleaning, be designed to be pluggable. The plug connections create the seal of the electrode channel and the adjustment of the electrodes in the electrode chambers. The one else to Cleaning the chamber, labor and time required to replace the electrode membranes, etc. This is largely omitted. In addition, with the help of the relief bolt, the Allows construction of a separating chamber that is almost free of tensile and compressive forces: it is also advantageous the easy accessibility of the electrodes.

The separation chamber according to the invention can be assembled easily and quickly and by means of less Change individual parts in their main dimensions quickly and easily. Due to its structure, the Separation chamber mechanically so stable that changes in the buffer pressure and uneven pressing of the seals no influence on the shape of the chamber ha

The special construction of the electrode block and the non-planar arrangement of the separation chamber allow But it is also possible to build chambers of various shapes. For example, with a given The spacing of the electrode channels changes the aspect ratio of the chamber by varying the side blocks and thus the chamber width can be varied. In order to produce the narrowest possible chamber, the Cooling block z. B. replaced by a mechanically sufficiently stable glass plate and the space between the electrode block, Sealing strips and glass plate can be used as a channel for the coolant. The plastics technology It also allows chambers to be produced with uneven boundary surfaces using simple means. When casting or spraying processes are used and the cooling block is used as a negative mold the constancy of the chamber thickness is guaranteed to a high degree for any chamber shape.

The volume of the entire device can be increased by the uneven structure of the separation chamber necessary minimum are reduced and there are also high field strengths, intensive cooling and large Buffer foot speeds possible.

In particular, if the separating chamber has a U-shaped cross section, it has a very simple construction possible, which allows great precision of the chamber shape and offers great mechanical stability. Through this Chamber buffer inflow and outflow do not need to be synchronized and level vessels can be used the same cross-section can be used for the buffer feed. Fear that the distraction of the Substance strips would be adversely affected in this chamber shape has been found to be unfounded proven. Experiments have shown that the strips of substance migrating around the corner remain unaffected in their shape and their angle of deflection.

The size of the separation chamber according to the invention is not structurally restricted. You can in wide Limits can be varied.

The deflection of a given substance can be varied within very wide limits by the choice of the buffer flow rate and field strengths can be varied. For example, in the separation chamber according to the invention using conventional buffer systems, separations at field strengths of 80 to 120 V / cm in continuous operation possible. To avoid turbulence in the flow and as circular as possible, from wall effects To achieve unaffected strips of substance, chamber thicknesses of 0.5 to 1 mm have proven to be optimal.

Because in the majority of separation problems, low dwell times in the chamber and high throughput rates are desirable, a drive motor can be provided for the suction device, the flow rates from 3 to 20 ml / minute allowed. This corresponds to residence times of 7 to 1 minute in the Chamber. For special separations, e.g. B. in ampholyte gradients low flow velocities are desirable. They are driven by a second, easily replaceable drive motor with low running speeds achieved. The small chamber volume is at the same time a considerable one for such separations economic advantage.

Using the exemplary embodiment shown in the drawing, the invention is illustrated in FIG explained in more detail below. It shows

F i g. 1 shows a horizontal section through a separating chamber designed according to the invention, and

F i g. 2 shows a vertical section through the in FIG. 2 separation chamber shown.

In the F i g. 1 and 2 show a preferred embodiment of the separation chamber according to the invention. The separation chamber is delimited on the one hand by a front block A and two side blocks B. Within these blocks there is a cooling block C with a cooling liquid channel D, so that overall a U-shaped cross section of the separation chamber results. An electrode block E is arranged to Seitenblökken B and the cooling block C opposite to the opposite to the ends of the U-shaped includes separation chamber electrode channels F with electrodes between the electrode block and the separation chamber compressible sealing strips K (z. B. silicone), electrode membranes L and compensating strips M arranged. The blocks are clamped together by clamping bolts G , the holes leading through the side blocks B being designed as elongated holes so that the thickness of the separating chamber can be adjusted.A clamping bolt H passed through the electrode block E is used to absorb the forces that cause a reduction in the chamber thickness would. The chamber buffer solution is fed in or discharged through chamber buffer feed lines E The substance mixture to be separated is fed in through a feed line P The electrode buffer is fed in and out through connections N. The electrodes themselves are inserted into the electrode channels by means of ground-in plug connections, so that they are quick and easy can be exchanged.

The device described above allows isolation of the separation chamber and electrode space as well high field strengths and long dwell times in the separation chamber. As a result, the device can be operated in such a way that the separation space is continuously with a solution of a suitable carrier ampholyte with the solution of the substance mixture to be separated loads a field that is high enough to form a pH gradient and that in the pH gradient separated fractions from the removal openings .Her separation chamber removes

According to a preferred embodiment of the invention, the electrode spaces separated from the separation chamber by suitable membranes are filled suitable liquids whose acidity (anode compartment) or basicity (cathode compartment) the desired Range of pH gradient limited; e.g. pH range 4 to 8, anode compartment one percent phosphoric acid, cathode compartment, 1 ^ percent aqueous ethanolamine solution.

The substance mixture to be separated is expediently added to the carrier ampholyte solution before it is converted into the separation space enters

The substance mixture to be separated is preferably introduced into the separation space by means of a metering pump.

example

One percent phosphoric acid flows through the anode compartment and 13 percent ethanoamine solution in the cathode compartment. Separation chamber and electrode spaces are separated from one another by a dialysis membrane. The great mechanical stability of the Chamber allows the ampholia solution to be fed into the separation chamber through six level vessels. at The usual chamber buffer feeds are used for this in the commercially available separating chamber systems. You can adjust the speed of the pH gradient as a function of the residence time of the Determine carrier ampholytes in the separation chamber at field strengths of 90 to 100 V / cm. Here the flows One percent ampholine solution from the level vessels as an 11 cm wide and 0.05 cm thick buffer film through the chamber and is after a running route

ίο taken from 36 cm by a 48-fold hose pump at the lower end of the chamber. One observes that the conductivity in ampholyte gradients is a measure of the setting of the pH gradient, within about 10 minutes, depending on the buffer

'5 flow rate, to 40 to 60% of the initial value The determination of the pH in the 48 fractions resulted in the methodological accuracy matching pH gradients with residence times of 25.50 and 100 minutes. They showed the expected Course.

The unexpectedly rapid setting of the pH gradient in the separation chamber suggests that the creation of the gradient and extensive isoelectric focusing can be achieved in a single pass. can be sufficient if the residence time in the separation chamber is sufficiently long. That possibility was based on the separations of four proteins, serum albumin (cattle) (Behringwerke A.G., Marburg, electrophoretic degree of purity 100%), gamma globulin (Cattle) (Behringwerke A.G., Marburg, pure) (electrophoretically uniform [7]), hemiglobin (cattle) (Serva Development laboratory, Heidelberg, twice crystallized, pure) and myoglobin (horse) (Serve development laboratory, Heidelberg, pure) tested

Two types of substance delivery were used in the chamber:

a) Dissolve the protein in the ampholine solution in one of the six level vessels. At a protein concentration of 1 mg / ml Ampholine-Sol, 100 min. Dwell time and a chamber volume of 20 ml, 2 mg of protein are enforced per hour

b) pumping in the protein solution through the metering device. At a substance concentration of 10 mg / ml Ampholine-Lsg, one dosage ratio of substance solution / chamber buffer = 1:50, one Residence time of 100 minutes is also about 2 mg protein / hour. separated

Results

In Fig. 4 is the separation of the gamma globulin im pH gradients from 3 to 10 shown. The dotted curve represents the course of the extinction at 280 nm in the different fractions after a residence time of 50 min. A globulin solution was injected from 5 mg / ml in one percent ampholine solution in a dosage ratio of 1:50. The solid curve shows the Separation after double residence time (100 min.) And double protein concentration. The separation result

in the latter case was independent of the inlet point at which the globulin sample was injected. this shows that the separation achieved at least with a very extensive focusing of the globulin components different IP in their associated pH

The dashed curve shows the pH value determined in the fractions.

The separation of the "electrophoretically uniform" serum albumin under the conditions of globulin

separation with 100 min. residence time is shown in FIG. 5 shown. In shallower pH gradients (Ampholine pH 6 to 8) were myoglobin and hemoglobin solutions (2.5 mg / ml) with a residence time of 100 min. and a dosage ratio of 1:50 separately. The dotted lines in Fig.6 represent those at 415nm, the The solid line represents the absorbance measured at 280 nm. From FIG. 6 is also the excellent reproducibility the pH gradients represented by the dashed lines.

Since the separation and fractionation take place continuously, samples can be used for control measurements at any time removed and so z. B. the quality of the separation can be continuously monitored. Will be lower at so Substance concentration worked that no change in the gradient due to the buffer effect of the Substance is to be feared, so the IP of successively separated substances can be directly related to one another be compared. However, higher substance concentrations cause disturbances in the linearity of the Gradients (see FIG. 4), so that in this case the course of the pH gradient is determined separately in each case must become.

Since in the continuous process there is no need to build up a density gradient, even after separation must be fractionated in a special operation, the labor and time required in comparison very low compared to the discontinuous process; especially

ίο the separation due to the high applicable field strengths occurs faster than in the separation column.

The separations have so far only been carried out in one percent aqueous ampholine solutions in the von the separation chamber (8) developed by us. Because of the low density of the ampholine solutions were only slightly concentrated protein solutions separated. However, there is the possibility of density compensation z. B. by cane sugar, Ficoll, etc., or by changes in equipment.

For this purpose 2 sheets of drawings

Claims (5)

Patent claims: 1. Separation chamber for the continuous deflection electrophoresis of a carrier-free Eiektrölytstroms, with electrodes arranged parallel to the electrolyte flow, supply and discharge lines for the electrode buffer, the chamber buffer and the substance to be separated, characterized in that the walls of the separation chamber (TK) are made by mutually displaceable outer blocks (a, Bi, Bi) and an inner, are formed with one wall of these blocks (A. Bi. ^ opposing cooling block (C) which are sealed from each other, wherein the Kühlbl & cks (C) opposite inner sides due to corresponding shaping of the outer side of the Blocks (A. Bx, Bi) and a single or multiple angled or arched separating chamber (TK) with a constant cross section along the flow direction. 2. Separation chamber according to claim 1, characterized in that the electrodes are combined in a common block (Ej / ) adjoining the blocks (Bi, Bi, C) on one side. 3. Separation chamber according to claims 1 and 2, characterized in that the electrodes in the Electrode spaces are insertable and adjustable. 4. Separation chamber according to claim 1 or 2, characterized in that cooling devices are additionally provided in one or more of the outer blocks (A, Bi, Bi) and the electrode blocks (E). 5. Separation chamber according to claim 1 to 3, characterized in that the outer blocks and inner block plates, a buffer curtain with the thickness of the chamber is moved evenly in the direction of fall. The substance mixture to be separated is continuously injected into the buffer stream at one point, which wanders down vertically as a strip between the parallel plates Buffer flow angle, the size of which depends on the specific properties of the particles, the field strength and the buffer flow velocity. The bandwidth extends through the cross-section of the chamber and through the ratio of the metering rate of the substance mixture and the buffer flow rate (metering ratio given. It increases during the separation by diffusion, turbulence, electroosmosis and thermal convection. The condition for the complete separation of two substances is fulfilled if the strips do not overlap at the time of removal and if the effective width b of an individual removal device is smaller than the distance between the individual strips at the removal point. The dimensions of the separation chambers of known devices are, for example, L χ B χ d = 50 χ 50 χ C , 05 cm or 50 χ 10 χ 0.5 cm. In the known devices, because of the inhomogeneities occurring in the vicinity of the electrodes of the electric field are not used the entire width of the separation chamber, so that the device must be made wider than this with maximum utilization of the available separation chamber width would actually be necessary. Furthermore, the problem is chamber plate pairs of this size, the