US 3616455 A
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United States Patent Inventor Philipp Adolf Von Miinchhausen Grolmanstr. 44-45, 1 Berlin, 12, Germany Appl. No. 808,046 Filed Mar. 13, 1969 Patented Oct. 26, 1971 Priority May 10, 1968 Germany P 17 67 432.7
ELECTROPHORETIC SEPARATION APPARATUS 12 Claims, 5 Drawing Figs.
3,346,479 10/1967 Natelson 204/301 3,450,624 6/ 1969 Natelson 204/299 3,519,549 7/1970 Grassmann et a]. 204/299 FOREIGN PATENTS 1,164,984 3/1964 Germany 204/299 Primary ExaminerF. C. Edmundson Attorney-Karl F. Ross ABSTRACT: An electrophoretic separation apparatus for the support-free'continuous separation of molecular fractions in a buffer solution has a thin'flat electrophoresis chamber defined between water-cooled plates through which the mixture of fractions and buffer solution is passed and wherein the solution is subjected to a unidirectional field transverse to the direction of mass flow to separate the fractions. Subsequently the separated fractions are sedimented out of their respective portions of buffer solution in collecting vessels ahead of the outlets for the solution. Downstream of the collecting vessels is mechanical or hydrostatic pump or flow-control means which makes the fluid flow through the apparatus.
PATENTED OCT 2 6 I97! SHEET 10F 4 Inventor Phil/pp Ado/f u Mdnchhausen Km: 9
A fforney PATENTEUnm. 25 I971 sum '2 0F 4 Inventor:
Philipp Ado/f v. Miinchhausen by gza l JM.
Aftorn ey PATENTEDOBI 2s ism 3,616,455
SHEET 3 BF 4 Inventor:
Phil/pp Adolf vMUnchhausen by at g: 3C5
Attorney ELECTROPI-IORETIC SEPARATION APPARATUS My invention relates to an electrophoretic apparatus as used in the continuous separation of molecular fractions in a buffer solution.
BACKGROUND OF THE INVENTION The resolution of mixtures of electrically separable fractions, usually biological proteinaceous substances which may include whole cells, through electrophoretic processes, has become increasingly widespread in recent years. Such processes recognize the principle that, when such a mixture of fractions is transported along a mass-flow path in a conductive medium, a unidirectional electric field transverse to the direction of mass flow produces a drift of the charged particles in the direction of the potential gradient, the effects of electrically induced migration on the different fractions are different and that, therefore, the fractions drift to different extents. Collection of the fractions at a series of points along a line parallel to the electric potential gradient and transverse to the massflow direction gives a clear resolution of the different fractions.
The devices hitherto proposed to carry out this process, aside from those using supports or carriers, generally have a series of collecting vessels arranged downstream of some sort of pump arrangement which is connected to the actual separation or resolving chamber where the fractions are spread apart by the electric field. These pumping arrangements inevitably damage the larger fragments, e.g. cells, which cannot be recovered intact. This presents an enormous disadvantage of the devices when used in many biological separations.
OBJECTS OF THE INVENTION It is therefore the principal object of my invention to overcome this disadvantage.
Another object is the provision of an improved thin-layer electrophoresis method and apparatus which can be used for a large number of separation processes without damage to cells and the like.
SUMMARY OF THE INVENTION The collecting vessel or vessels of the instant invention are provided between the pump and the output of the separation or electrophoresis chamber. This collecting vessel is advantageously a burette in which the fractions can sediment. The input opening and output openings of the burette are small (in cross section) in relation to its overall cross section so that the velocity of the buffer solution therein is sufficiently slow to permit this sedimentation.
The pump can be a noncontact peristaltic pump connected to the flexible tubing leading from the output opening of the burette. Since a plurality of burettes will be used for the necessary resolution in the apparatus, this type of pump is advantageous in that it can be connected to a plurality of tubes or hoses and pump several burettes simultaneously.
In a further embodiment, a capillary tube is used as the pump or flow-control means. The supply of buffer (i.e. its reservoir) for the chamber input is mounted above this tube and a secondary reservoir can be connected to it to form a liquid head in the tube.
According to this principal feature of my invention, the cells or other fragile, bulky fractions are not injured during electrophoretic separation.
A further feature of my invention is that the separation chamber is provided with means enabling its rotation through 90", from a vertical (upright) to a horizontal (recumbent) position. This was previously considered impossible since it was through that the tilt would bring about so-called streaking" but, surprisingly, no such effect is noticed with the water-cooled construction of my invention.
The method of the present invention, therefore, involves the formation of mass-flow path for a buffer solution and the entraining a mixture of fractions to be separated from an inlet side to an outlet side through a thin flat chamber, while applying across this path a unidirectional potential gradient capable of separating the mixture of fractions into discrete portions which arrive at different locations along the outlet side in the direction of the potential gradient; according to this aspect of the invention, a continuous flow of the solution abstracted at each location at the outlet side is induced through an upright elongated sedimentation vessel adapted to retain at least temporarily a portion of the abstracted solution so that sedimentation of the separated fragments occurs in this vessel. Intermittently, the vessel is drained at its bottom between its inlet, which is connected to the electrophoresis chamber, and an outlet connected at an upper location above the inlet and the drain, to a flow control means of the character described. The flow control means may include the noncontact pump mentioned earlier or a capillary through which the solution is driven by a hydrostatic head.
BRIEF DESCRIPTION OF THE FIGURES The foregoing and other features, objects and advantages of my invention will be described in greater detail hereinafter, reference being made to the accompanying drawing in which:
FIG. 1 is a side view of an electrophoresis apparatus according to my present invention;
FIG. 2 is an end view, partly in section, of the embodiment shown in FIG. 1;
FIG. 3 is a side \Yew, partly in section of a further embodiment of my invention;
FIG. 4 is an expanded sectional detail of the chamber according to my invention; and
FIG. 5 is a detail, partly in section, of FIG. I.
SPECIFIC DESCRIPTION FIG. I shows two watcr-cooled'plates 2 and 3 defining a flat, narrow carrier-free unobstructed separation chamber I between them. The plates and the chamber is mounted rotatably by a hinge 31 on a support 34 so that an arm 21 (centered on the hinge and formed with a slot 21a) allows tilting of the chamber upwardly through (see broken lines in FIG. I) and securing the chamber by a locking nut 2Ib.
Attached to the support frame 34 carrying this hinge 31 is a rack in which a plurality of burettes 17 serving as the collecting means or vessels are held in clips (FIG. 2). These burettes 17 are connected through output or discharge tubes or hoses 15 to output of the chamber 1. A valve 16, comprising two bars 16b and 160, can clamp the output hoses l5 shut by means of screws 16a spanning the bars. The burettes I7 have upper stopcock valves 22 and lower stopcock valves I8; an outlet hose 32 made of rubber, silicone rubber, silicones or synthetic resins lead through a variable stepless peristaltic pump 19 to a flask 33.
As seen in FIG. 5, the pump 19 consists of a housing 60 in which a shaft 64 is joumaled. This shaft 64 mounts two disks 63 (only one shown) between which polyvinylchloridc rollers 61 and 62 are rotatably supported. A motor 66 of stepless variable speed is connected through a belt 65 to this shaft 64. The tubes 32 from the outlet openings of a plurality of bulrettes I7 are led between these rollers 6I and 62 and the housing 60 so that, owing to the cyclical compressing effect of the rollers 61 and 62, liquid is pumped through the hoses 32 without being touched by the moving parts of the pump I9. A pump 19 is provided for each rack of burettes 17, since one pump 19 can accommodate a plurality of tubes 32.
FIG. 2 shows in particular the cooling and buffer-supply system of the apparatus. The interior of the plate 2 is divided by bars 6 to form a zigzag path as shown by arrows 5 through which cooled water is pumped in at 4 and out at 4'. The plate 3 is similarly cooled. Two electrode troughs I0 and I0, flanking the sides of the chamber and parallel to the flow direction, as shown in US. Pat. No. 3,305 ,47l of Feb. 2l, l96l, are mounted on opposite ends of the plate 2, with inlets I3 and I3 and outlets l4 and 14'. An adjustable DC high-voltage source 47 is connected across terminals 12 and 12 of the electrodes l and The buffer solution is fed in from a flask 26 through a valve 9. A manifold 8 supplies this wlution to the whole upper edge (inlet side) ofthe chamber 1.
FIG. 4 best shows the construction of the chamber 1. Two plates 2a and 2b forming the plate 2 and separated by a gasket 48 and the bars 6, and two further plates 3a and 3b separated by a gasket 49 and further spacers 6 provide means for cooling the chamber 1. The inlets 4 and outlets 4 are also seen here.
The electrode troughs l0 and 10' are filled with a solution similar to the buffer solution in the chamber 1, but approximately twice as strong, which is renewed or replenished as described below. In the bases of these troughs l0 and 10 are wads of packing 100 as described in the above-cited patent which serve to permit electrical contact with the buffer solution in the chamber 1.
The sheets 3b and 2b are separated by a gasket 50 and form the separation chamber 1 whose inlets 8 and outlets are shown extending through the gasket 50.
FIG. 3 shows how the connections 4 and 4' are joined to a source of coolant, in this case water, in a reservoir 46. This reservoir 46 is equipped with a cooling coil 45 of a refrigeration unit 44. A pump 46a circulates the water through the plates and the cooler. This cooling system tends to keep down the high temperatures generated by my apparatus.
Furthermore FIG. 3 shows a source 40 of double-strength buffer solution connected through a pump 41 to J-arms 43 which drip this solution into two cups 42 (only one shown) associated with respective electrodes 10 and 10'. The outlets 14 (only one visible) drip into a waste flask 39. The necessity of dripping the solution rises from the fact that simply conveying it through tubes would create a shunt across the electrodes 10 and 10' greatly reducing their efflciency.
The sample to be treated is fed into the chamber 1 through a conduit 7 (FIG. 4) from a flask 38.
The methods of feeding the coolant for the plates 2 and 3, the sample into the chamber 1, and the solution for the electrodes 10 and 10' shown in FIG. 3 are the same as used for the embodiment shown in FIGS. 1 and 2.
FIG. 3 shows burettes or capillary tubes 17' which are mounted between supports 28 and 29 on a frame 27 in a plexiglass housing 30 and are connected as described before to outlet tubes 15 of the chamber 1. Three valves l6, l8 and 53 each consisting of a rod 24 serving to crimp a portion of tubing in a holder 52. This rod 24 rides on a bar 37 formed with a bore 38 in which a handle 36 on the rod 24 engages. A spring 23 mounted on a screw 23a on a support 230 on the bar 37 and on a screw 23b on the rod urges the rod 24 toward the holder 52. On depression of the bar 37, the handle 36 disengages the hole 38 and this spring 23 pulls in the rod 24 thereby crimping the portion of tubing. Withdrawal of the handle 36 such that it reengages in the hole 38, resets the valve formed thereby. The pinch-clamp valves 16 and 18 have the same function as the like numbered valves in FIGS. 1 and 2 and are each provided for one tube 15 or burette 17, respectively.
In addition, the valves 53 are provided between a reservoir 25 located above the capillary tubes 17 and connections on the tops of these tubes 17. Outputs of these tubes 17 are shown at 29'.
The difference in height between the reservoirs 25 and 26 forms a hydrostatic head across the tubes 17' which functions to control the pumping action.
In order to use my apparatus the pumps 41 and 46a are started and the high-voltage source 47 is turned on. A sample is fed in 7 and a short time is preferably waited for some spreading of this to take place under the effect of the voltage. Thereupon the valves 9 and 16 are opened and the pumps 19 are started (FIGS. l and 2) or the valves 53 are opened (FIG. 3). Periodically the valves 8 are opened to sample or recover the sediment in the burettes 17 or [7' and the cups are replaced before resampling.
The improvement described and illustrated is believed to admit of many modifications within the ability of persons skilled in the art, all such modifications being considered within the spirit and scope of the invention except as limited by the appended claims.
What is claimed is:
I. An apparatus for the continuous electrophoretic separation of molecular fractions in a buffer solution, said apparatus comprising:
an electrophoresis chamber having an inlet permitting ad mission of said solution into said chamber and an outlet permitting withdrawal of said buffer therefrom, thereby forming a continuous fluid flow through said chamber;
electrode means in contact with said buffer for creating a unidirectional electric field across said flow;
pump means operatively connected with said outlet for drawing said solution therethrough out of said chamber; and
collecting means between said pump means and said outlet for sedimenting at least one of said fractions.
2. The apparatus defined in claim 1, further comprising conduits connecting said collecting means to said pump means and to said chamber, said collecting means including a collecting vessel having a cross-sectional area substantially greater than the cross-sectional diameter of said conduits to permit sedimentation in said vessel.
3. The apparatus defined in claim 1, further comprising a pair of spaced parallel plates forming said chamber between them and cooling means for preventing overheating of said plates, said outlet comprising a plurality of outlet openings spaced along a wall of said chamber, said inlet being formed along an opposite wall of said chamber.
4. The apparatus defined in claim 3, further comprising means for tilting said plates jointly through substantially from a substantially vertical position to a substantially horizontal position.
5. The apparatus defined in claim 1 wherein said collecting means comprises a substantially vertical burette connected at a lower end thereof to said outlet, and means connecting said burette to said pump means at an upper end of said burette, said burette being provided at said lower end with means for draining off said one of said sedimented fractions.
6. The apparatus defined in claim 1 wherein said pump means comprises a peristaltic pump, said apparatus further comprising a flexible conduit attached to said collecting means, said pump coacting with said flexible conduit.
7 The apparatus defined in claim 1 wherein said collecting means comprises a capillary tube, said pump means comprising means for maintaining a head in said tube.
8. The apparatus defined in claim 1, further comprising valve means between said outlet and said collecting means for selectively prohibiting fluid flow therebetween.
9. An apparatus for the continuous electrophoretic separation of molecular fractions in a buffer solution, said apparatus comprising:
a pair of spaced-apart liquid-cooled plates mounted on said support and defining between them a narrow flat separation chamber having an inlet side and an outlet side opposite one another whereby a mass flow of the bufl'er solution and mixture of fractions traverse said chamber from said inlet side to said outlet side;
means for applying a generally unidirectional field across the mass flow through said chamber to separate said f ractions such that they arrive at different locations along said outlet side;
means at said outlet side for abstracting said fractions from said chamber at said locations; and
tilting means between said support and said plates enabling tilting of said chamber about a substantially horizontal axis through an angle of about 90, and for retaining said cham her in said tilted position.
10. The apparatus defined in claim 9 wherein said plates and said chamber are generally rectangular and said sides are horizontal, said tilting means including a hinge connected to said plates at said outlet side of said chamber enabling swinging of said chamber between a recumbent position and an upright position ll. The apparatus defined in claim 10 wherein said means for abstracting said fractions from said chamber at said outlet side includes:
a respective flexible tube leading from said chamber and communicating therewith at each of said locations;
a respective upright elongated sedimentation vessel connected with each of said tubes and communicating therewith at a location above the bottom of the respective vessel;
means at the bottom of each of said vessels for draining same;
means at an upper portion of each of said vessels above the location at which the respective tube communicates therewith for inducing a flow of said solution through the corresponding tube and vessel whereby said fractions are sedimented at the bottom of said vessels; and
means for selectively closing said tubes.
12. The apparatus defined in claim 11 wherein said vessels are burettes having valves at their bottoms, and said means for selectively closing said tubes are pinch clamps.
12. The apparatus defined in claim 11 wherein said vessels are burettes having valves at their bottoms, and said means for selectively closing said tubes are pinch clamps. 237] 6790103 F i i