DE2051715A1 - Continuous deflection electrophoresis - in chamber of nonplanar cross section - Google Patents

Abstract

Separating chamber for continuously deflection electrophoresis in a buffer flow, with inlets and outlet for the electro-buffer, the chamber buffer and the substance to be separated, is arranged so that the separating chamber between the electrodes is not flat in cross-section, but has one or more angles or curves in it. Specif the separation chamber is built up of an internal block, external block and electrode block clamped together with seals and electrode membranes between the electrode block and the other two. The arrangement enables the chamber to be varied in its dimensions.

Description

Separation chamber for continuous deflection electrophoresis Die The invention relates to a separation chamber for continuous deflection electropnoresis in a carrier-free buffer stream, with electrodes, with a separating chamber and supply and discharge lines for the electrode buffer, the chamber buffer and the substance to be separated.

For continuous electrophoretic separation in the free buffer stream A number of separation chambers have been developed, almost without exception based on the following Principle: A buffer curtain is placed between two plane-parallel plates Thickness d moved evenly in the direction of fall. In the buffer stream is at one point continuously the substance mixture to be separated injected as Strip of the bandwidth a perpendicular to the plane-parallel plates wanders down. Under the influence of an electric direct current field, the field lines of which are horizontal run, the substance particles are deflected depending on their charge. Your hiking trails form angles with the direction of flow of the buffer stream, the size of which depends on the specific Properties of the particles, the field strength and the buffer flow rate depends. The bandwidth a is determined by the cross section of the chamber and by the ratio Dosing speed of the substance mixture and the buffer flow speed (dosing ratio) given. It increases during the separation through diffusion, turbulence, electro-osmosis and The, rmoconvexion. The condition for the complete separation of two substances is fulfilled if the bandwidths do not overlap at the time of removal and if the effective width b of a single removal device is not one than the distance between the individual bands. at the extraction point. The dimensions the separation chambers of known devices are, for example, L x W x d = 50 x 50 x 0.05 cm or 50 x 10 x 0.05 cm. Devices with separation chambers are also used, the dimensions of which are L - 18 to 45 cm, B = 2.5 to 5.0 cm and d = 0.15 cm.

In the known devices, because of the in the vicinity of the electrodes Inhomogeneities in the electric field are not used over the entire width of the separating chamber so that the device must be made wider than it is at maximum utilization the available separation chamber width would actually be necessary. Further is the problem, pairs of chamber plates of this size, which are also still of all four Pages out through the electrode and buffer feed or suction device under quite high mechanical stress can be set, precisely at the correct distance d to hold and stabilize against fluctuations in buffer pressure, technically only very complex to solve and therefore Cause of increased susceptibility to failure. Also is the large intrinsic volume of the separation chamber when working with small sample lengths disadvantageous. So it is hardly possible to shorten the residence time of the substance in the chamber than to hold for about 30 minutes, which makes working with sensitive substances in particular be hindered.

The present invention is therefore based on the object of a To create separation chamber that can be assembled in a few simple steps and in its main dimensions is variable, which is also mechanically so stable that, for example, changes in the Buffer pressure and uneven pressure of the seals have no effect on the shape the Chamber have. In addition, high field strengths, intensive Cooling and high buffer flow rates can be achieved.

The separation chamber according to the invention is characterized in that it between the electrodes a non-flat, single or multiple angled or has a curved cross-section.

In this embodiment, for example, the effective area the separation chamber are placed in one plane, while the ineffective parts of the separation chamber lie in a different level, so that overall the actual The width of the chamber can be reduced significantly.

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

The inner surface of the separation chamber can thereby be an inner block and their outer surface is formed by an outer block. There are inner block, Outer block and electrode block provided to accommodate cooling devices. Owns the separation chamber, for example, a U-shaped cross-section, so the outer block the end a front block and two side blocks, and the inner block from a cooling block exist.

Outer and inner block are expediently about seals and Electrode membranes pressed onto the electrode block, the electrode-side Chamber ends lie over the slots in the electrode spaces.

To allow precise adjustment of the panel thickness, it is expedient to design the individual blocks so 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 leading through the electrode block into the cooling block H can be provided, which counteracts the forces that reduce the chamber thickness would cause.

The outer and inner blocks can each consist of one or more parts exist, by their mutual displacement separating chambers of variable thickness and Shape can be made.

In order to also change the electrodes as quickly and easily as possible can, the electrodes are advantageously plugged into the electrode spaces executed, the plug connections sealing the electrode channel and adjust the electrodes in the electrode spaces.

The separation chamber according to the invention can be assembled easily and quickly and change their main dimensions easily and quickly with just a few individual parts. Due to its structure, the separation chamber is mechanically so stable that changes the buffer pressure and uneven pressing of the seals have no effect on the Have the shape of the chamber. The volume of the entire device can be reduced by the uneven Construction of the separation chamber to be reduced to the absolutely necessary minimum and there are also high field strengths, intensive cooling and high buffer flow rates possible.

In particular, with a U-shaped cross section of the separation chamber is a very simple construction of the same possible, which allows a great precision of the chamber shape and offers great mechanical stability. This means that chamber buffers need to and drainage not to be synchronized and level vessels of the same cross-section can be used can be used for the buffer feed. Fear that the distraction of the Substance strips would be adversely affected in this chamber shape is therefore unfounded, because normally the flat face of the chamber for the Separations is used. Since also in the planar arranged chambers close to the electrodes Regions due to pH shifts of the buffer present there are avoided anyway measure is the area of the angular and flat chambers that can be used for separations same width e comparable.

Experiments showed that the one around the corner wandering substance strips in their shape and their angle of deflection unaffected stay.

As a major advantage of the chamber construction according to the invention the common arrangement of the two electrodes in one block must also be considered. The one otherwise required for cleaning the chamber, replacing the electrode membranes, etc. This saves work and time to a large extent. Furthermore the construction of a separation chamber is made possible with the help of the relief bolt, the is almost free of tensile and compressive forces. Another advantage is the easy accessibility of the electrodes. The special construction of the electrode block and the non-planar However, the arrangement of the separation chamber also allows chambers of the most varied of shapes to be built. For example, with a given spacing between the electrode channels, the aspect ratio the chamber changed by varying the side blocks and thus the chamber width can be varied. In order to produce the narrowest possible chamber, the cooling block for example by a mechanically sufficient Stable glass plate replaced and the space between the electrode block, sealing strips and glass plate as a channel can be used for the coolant. The plastics technology also allows Using simple means, chambers can also be produced with non-planar boundary surfaces. If casting or spraying processes are used and the cooling block is negative Shape is used, is the constancy of the chamber thickness for any chamber shape guaranteed to a high degree.

The size of the separation chamber according to the invention is not constructive restricted. It can be varied within wide limits.

The distraction of a given substance can be within very wide limits can be varied by the choice of buffer flow rate and field strength. So are more common, for example, in the separation chamber according to the invention Buffer systems Separations possible at field strengths of 80 to 120 V / cm in continuous operation. 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 turned out to be optimal.

Since in the majority of separation problems short residence times in the Chamber and high throughput rates are desirable for the suction device A drive motor can be provided with flow rates from 3 to 20 ml / minute allowed. This corresponds to dwell times of 7 to 1 minute in the chamber. For special separations, for example in ampholyte gradients, low flow velocities are required he wishes. They are powered by a second, easily replaceable drive motor low running speeds achieved. The small chamber volume is for such Separations are also a significant economic advantage.

Based on the example shown in the accompanying drawing swei sen embodiment, the invention is explained in more detail below. Show it: 1 shows a schematic illustration to explain the principle of operation the chamber; Fig. 2 shows a horizontal section through an inventive design Separation chamber; and FIG. 3 shows a vertical section through the separating chamber shown in FIG Fig. 1 shows a separation chamber with the dimensions B and L, in which between two plane-parallel plates a buffer curtain of thickness d evenly in the direction of fall emotional. The substance mixture injected continuously into the buffer stream at one point migrates downwards as a strip of bandwidth a.

Under the influence of an electric direct current field, its field lines run parallel to the edge B, the substance particles are depending on their charge diverted. Their migration paths form with the direction of flow of the buffer stream Angle, etc., the size of which depends on the specific properties of the particles, the field strength and the buffer flow rate depends. The width of the substance mixture is the cross-section of the chamber (B x d) and the ratio of the dosing speed the substance mixture and the buffer flow rate dependent. You enlarged during the separation to the widths a 'and a ". The condition for the complete Separation of two substances is fulfilled if at the time of removal from the Chamber a 'and a "do not overlap and if the effective width b is a single one Extraction device is smaller than the distance a 'to a ". The separability of substance mixtures is in principle only of the constructive size b and the variables a, a ', a "and #, but not the shape and size of the separation chamber addicted.

2 and 3 is a preferred embodiment of the invention Separation chamber shown. The separation chamber is on the one hand by a front block A, and two side blocks B limited. A cooling block C is located within these blocks a coolant channel D, so that a total of a U-shaped cross section the separation chamber results. The side blocks B and the cooling block C opposite arranged is an electrode block E, which, opposite the ends of the U-shaped Separation chamber, electrode channels F containing electrodes.

There are compressibles between the electrode block and the separation chamber Sealing strips K (for example made of silicone), electrode membranes L and compensating strips M arranged. The blocks are clamped together by clamping bolts G, the leading through the side blocks B, holes are designed as elongated holes, so that the thickness of the separation chamber is adjustable. One passed through the electrode block E. Clamping bolt H is used to absorb the forces that reduce the chamber thickness would cause. The chamber buffer solution is supplied through chamber buffer feed lines E. or derived. The substance mixture to be separated is introduced through a feed P.

The electrode buffer is supplied or discharged through connections N. the Electrodes themselves are inserted into the electrode channels by means of ground-in plug connections used so that they can be replaced quickly and easily.

The device described above allows isolation of the separation chamber and electrode space as well as high field strengths and long residence 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 Tragerampholten with the solution of the to be separated Mixture of substances charged, one for training a pH gradient A sufficiently high field applies and the fractions separated in the pH gradient from removes from the removal openings of the separation chamber.

According to a preferred embodiment of the invention, one fills in the electrode spaces separated from the separation chamber by suitable membranes Liquids whose acidity (anode compartment) or basicity (cathode compartment) the desired range of pH gradient limited; e.g. pH range 4 - 8, anode compartment 1 percent Phosphoric acid, cathode compartment 1.5 percent. Aqueous ethanolamine solution.

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

The substance mixture to be separated is preferably fed by means of a dosing pump into the separation chamber.

Example One flows through the anode compartment with 1 per cent. Phosphoric acid, the L'athodeuraum with 1.5 per cent. Ethanoamine solution. Separation chamber and electrode spaces are separated from each other by a dialysis membrane.

The high mechanical stability of the chamber allows the ampholie solution through 6 level vessels to the separation chamber.

the usual separation chamber systems are used for this purpose Chamber buffer feeds used. One can adjust the speed of the pH gradient depending on the residence time of the carrier ampholyte in the separation chamber Determine field strengths of 90 - 100 V / em. The 1 per cent flows here.

Ampholine solution from the level vessels as 11 em wider and 0.05 em thick buffer film through the chamber and is after a distance of 36 em taken by a 48-fold peristaltic pump at the lower end of the chamber. One observes that the conductivity, which is a measure of the setting in ampholyte gradients pH gradient i.st, within about 10 min., dependent from the buffer flow rate, drops to 40-60% of the initial value. the Determination of the pH in the 48 functions was within the scope of the methodological accuracy Corresponding pH gradients with residence times of 25, 50 and 100 minutes. They showed the expected course.

The unexpectedly rapid adjustment of the pH gradient in the drinking chamber suggests that the creation of the gradient and an extensive isoelectric Focusing can be achieved in a single pass if the dwell time is sufficiently large in the separation chamber. This possibility was based on the separations of four proteins, serum albumin (bovine) (Behringwerke A.G., Marburg, electrophoretic Purity 100%), gamma globulin (beef) (Behringwerke A.G., Marburg, pure) (electrophoretically uniform (7)), hemiglobin (cattle) (Serva development laboratory, Heidelberg, 2 x crystallized, pure) and myoglobin (horse) (Serve development laboratory, Heidelberg, pure) checked.

Two types of substance delivery into the chamber were used: a) Dissolving the protein in the ampholine solution of one of the six central vessels. at a protein concentration of 1 mg / ml ampholine solution, 100 min. residence time and a chamber volume of 20 ml, 2 mg of protein per hour are enforced.

b) pumping in the protein solution through the metering device. At a Substance concentration of 10 mg / ml Ampholine-Lsfr., A dosage ratio of substance solution / chamber buffer = 1:50, with a residence time of 100 minutes, about 2 mg protein / hour are also used.

separated.

Results In FIG. 4 the separation of the gammn globulin is in the pH gradient shown from 3 - 10. The dotted curve represents the course of the extinction at 280 mii in the various fractions after a residence time of 50 min. Injected a globulin solution of 5 mg / m1 in 1 per cent. Arnpboline solution in the dosage ratio 1:50. The solid curve shows the separation after double residence time (100 min.) And double protein concentration. The separation result was independent in the latter case of where the globulin sample was injected at the inlet site. This shows that the achieved separation at least a very extensive focusing of the globulin components with different IP in their coming pH range. The dashed curve shows the pll 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 illustrated. In shallower pH gradients to pholine pH 6 - 8) myoglobin and hemoglobin solutions (?, 5 mg / ml) with a residence time of 100 min. and one Desier ratio of 1:50 separated. The dotted lines in Fig. 6 represent the at 415 nm, the solid lines represent the absorbances measured at 280 nm. From FIG the excellent reproducibility of the pH gradients can also be seen in the are represented by the dashed line h.

Since the separation and fractionation takes place continuously, you can Samples are taken at any time for control measurements and, for example, the quality of the Separation will be pursued on an ongoing basis. If you work with such a low substance concentration, that no change in the gradient due to the buffer effect of the substance is feared, the IP can be successively separated substances immediately be compared with each other. However, higher substance concentrations cause Disturbances of the linearity of the gradient (see. Fig. 4), so that in this case the The course of the pH gradient must be determined separately in each case.

Since in the continuous process neither a density gradient is built up is still to be fractionated in a special operation after separation must is the amount of work and time required compared to the discontinuous process very low; especially since the separation due to the high applicable Field strengths occurs faster than in the separation column.

The separations have so far only been in 1 per cent. aqueous ampholine solutions executed in the separation chamber (8) developed by us. Because of the low density the Ampholi.ne-Lbsungon only slightly concentrated protein solutions were separated. However, there is the possibility of density compensation, e.g.

through cane sugar, Picoll etc., or through changes in equipment, that we are currently working on.

Claims (13)

Patent claims' 1. Separation chamber for continuous deflection electrophoresis im Carrier-free buffer 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, d a d It is noted that the separation chamber between the electrodes a cross-section that is not flat, but with a single or multiple angled or arched cross-section owns. 2. Trennkanmer according to claim 1, characterized in that the electrodes are combined in a common block (E). 3. Separation chamber according to claim 1, characterized in that the inner surface the separation chamber by an inner block (C) and its outer surface by an outer block (A, B) are formed, the inner block, outer block and electrode block for receiving of cooling devices can be provided. 4. Separation chamber according to claim 1 and 2, characterized in that Outer and inner block over seals (K) and electrode membranes (L) on the electrode block (E) are pressed, with the electrode-side chamber ends over the slots of the Electrode spaces lie. 5. Separation chamber according to one of the preceding claims, characterized in that that the outer block and inner block can be pressed separately onto the electrode block. 6. Separation chamber according to one of the preceding claims, characterized in that that the separation chamber has a U-shaped cross-section that the outer block consists of a front block A and side blocks (B), and that the inner block consists of one There is a cooling block (C). 7. separation chamber according to claim 5, characterized in that a through the electrode block (E) leading clamping bolt (-Y) is provided that the forces counteracts which would cause a reduction in the chamber thickness. 8. Separation chamber according to one of the preceding claims, characterized in that that the outer and inner block each consist of one or more parts their mutual displacement separating chambers of variable thickness and rorm made can be. 9. Separation chamber according to one of the preceding claims, characterized in that that the electrodes can be inserted into the electrode spaces, the plug connections the sealing of the electrode channel and the adjustment of the electrodes in the electrode spaces cause. 10. Process for the continuous electrophoretic separation of Substances with ampholyte properties, e.g. proteins, by means of isoelectric Focusing, characterized in that the separating space of a separating apparatus according to one of claims 1 to 9 for carrier-free electrophoresis continuously with a solution of a suitable carrier ampholyte with the solution of the substance mixture to be separated charged, creates a field high enough to develop a pH gradient, and the fractions separated in the pII gradient from the removal openings of the separation chamber removes. 11. The method according to claim 10, characterized in that in the electrode spaces separated from the separation chamber by suitable membranes Liquids occur whose acidity (anode compartment) or basicity (cathode compartment) limit the desired range of the pH gradient; e.g. pH range 4 - 8, anode compartment 1 percent. Phosphoric acid, cathode compartment 1.5 percent. aqueous ethanolamine solution. 12. The method according to claim 10, characterized in that the substance mixture to be separated into the carrier ampholyte solution before it enters the Separation space enters. 13. The method according to claim 10, characterized in that the introduces the substance mixture to be separated into the separation space by means of a metering pump.