WO2006021465A1

WO2006021465A1 - Electrophoretic separation in a moving fluid

Info

Publication number: WO2006021465A1
Application number: PCT/EP2005/051092
Authority: WO
Inventors: Christian A. Heid; Klaus Witt
Original assignee: Agilent Technologies, Inc.
Priority date: 2004-08-25
Filing date: 2005-03-10
Publication date: 2006-03-02
Also published as: DE212005000044U1

Abstract

A device for electrophoretic separation and purification of positively and negatively charged compounds (8, 9) and neutral compounds (10) in a hydraulically motivated analyte solution is provided, using a radial or axial electrical field which is generated inside a flow chamber (2) which has preferably a cylindrical geometry. Hydraulic flow forces and electrical forces act upon the analyte solution resulting in deflection (13) of the charged and neutral compounds (8, 9, 10) of the analyte solution, the deflection being a function of the electrical field and of the hydraulic forces being applied on the analyte solution and of the charge of the compound, respectively its iso-electric point, both depending of the pH-value of the solution, which can be predetermined, therefore causing the separation. A method for separation and purification of positively and negatively charged compounds (8, 9) and neutral compounds (10) is performed by this device.

Description

ELECTROPHORETIC SEPARATION IN A MOVING FLUID

DESCRIPTION

[0001] The present invention relates generally to the field of separation devices for conducting fractionation of organic molecules.

BACKGROUND ART

[0002] Separation and purification systems are used to separate and identify biological samples containing a diversity of up to 35 000 different proteins. Depending on the amount of interesting material needed for further analysis, techniques are requested with minimized separation times, offering a product with a high degree of purity.

[0003] One of the methods of separating complex biological samples is by iso-electric separation for example by iso-electric focusing (IEF). IEF is a technique of electrophoresis whereby compounds can be separated on the basis of iso-electric point (pi) within a pH gradient. Iso-electric focusing systems are usually free flowing buffered systems or immobilized buffered systems. To conduct efficient and successful scientific experiments a minimum volume of the compound of interest is needed. When IEF is conducted in a free flowing buffered system, the separation takes hours to receive the needed amount of substance and frequently additional steps such as removal of carrier ampholytes have to be performed when - as usual - the compound of interest has to be separated from an educt solution containing several compounds having close pi values.

[0004] When IEF is conducted in an immobilized buffered system, the compound of interest is trapped in a gel or membrane being located in complex apparatus and therefore recoverable only under increased efforts.

[0005] Founding upon the basics of this technology is the electrophoretic separation of compounds as referred to in US 2003/0104449 A1 to Faupel, Girault, et al., a device has been developed which provides a separation method of compounds in complex mixtures combined with a method for recovery of compounds of interest in order to conduct subsequent analysis. Anyway the problem of separating volumes of a compound of interest in order to use it in subsequent preparative application within an agreeable limit of time and acceptable efforts is even by means of that device not in sight.

DISCLOSURE OF THE INVENTION

[0006] It is an object of the present invention to provide an improved separation of molecules. This object is solved by the independent claims. Preferred embodiments are shown by the dependent claims.

[0007] Embodiments of the present invention address the aforementioned needs in the art to separate reasonable amounts of a desired compound contained in an analyte solution which is comprised of a diversity of i.e. organic molecules; some of which having a very close pi therefore being difficult to separate. The desired compound shall furthermore be obtained in a way that makes it easy to immediately conduct experiments, which may have an analytical as well as a preparative character. The second object has been described in former patents not been answered in an adequate manner since using the separated compounds for preparative applications requires an excellent purity and therefore quality of the educt.

[0008] In one embodiment, the present invention provides a device consisting mainly of a flow chamber wherein the analyte solution containing the compound of interest is introduced in order to carry out the separation by means of electrophoresis. The central improvement in comparison to the separation systems known in the art is the superimposition of hydraulic forces and electronic forces, both acting upon the analysis solution inside the flow chamber. This is achieved by generating a homogeneous electrical field inside the flow chamber while at the same time the analyte solution is moved, or pumped, respectively, by an upstream pump, from an inlet end to an outlet end across the cell. Those compounds carrying charge are then deflected in direction to the corresponding electrode(s) while passing the flow cell, the grade of deflection being generally a function of size/charge of the compound, the electrical field and the hydraulic forces. The compounds remain at those places to which they had been drawn. The compounds being neutral under the given settings leave the flow cell unaffected and are collected by a collecting means. The polarity of the electrodes generating the electrical field can be reversed, thus causing repulsion of the charged compounds, which thus are set free from the places at which they had been kept. This mechanism offers a brilliant possibility to enrich compounds such as organic molecules with a definite charge or pi, respectively, at definite places of the inner surface of the flow chamber, and, if once enough material has been enriched, reversing the electronic attraction in a controlled manner, therefore setting free only those compounds which are repulsed by the used voltage. Once set free and being resolubilized, the compounds of interest exit the flow chamber, too. Therefore, the device offers an option for separating volumes of a compound of interest in order to use it in subsequent preparative application within an agreeable limit of time.

[0009] In one embodiment of the invention, the flow cell is shaped cylindrically. The electrical field created herein is a homogeneous radial field, generated by axially located electrodes in the centre of the cylinder while the wall of the cylinder is or comprises a second electrode, the electrodes being of opposite polarity.

[00010] In a second embodiment of the invention, the inner surface of the above flow cell is coated with an appropriate substance for taking up the charged compounds being deflected in direction to the cylinder wall. In comparison to the embodiment depicted before, the controllability of enriching the compounds of interest and the subsequent setting free, followed by collection of the fraction in a collection vessel, is even better and therefore greatly facilitating further analysis and preparative use.

[00011] In a third embodiment of the invention, the flow cell is shaped cylindrically, too, but the electrical field created therein is a homogeneous axial field. In this embodiment the cylinder is arranged in an inclined position.

[00012] An additional embodiment of the invention depicts the flow cell of the third embodiment, wherein the inner surface of the above flow cell is coated, too, in order to provide an optimal uptake of the enriched compounds.

[00013] This technology, relying on the separation of proteins according to their charge, provides a very high separation rate and therefore a high degree of resolution. Additionally, an easy handling of the separated compounds facilitates subsequent steps like preparation and analysis, since the "product" is recovered in free flowing solution.

BRIEF DESCRIPTION OF DRAWINGS

[00014] Other objects and many of the attendant advantages of embodiments of the present invention will be readily appreciated and become better understood by reference to the following more detailed description of preferred embodiments in connection with the accompanied drawings.

[00015] FIG. 1a is a schematic over all front view of a device for electraphoretic separation and purification which can be used to carry out the process according to the present invention, the flow chamber having a cylindrical design, arranged in upright position, designed to create a radial electrical field,

[00016] FIG. 1b is a cross section of the flow chamber of FIG.1a,

[00017] FIG. 2a is a schematic over all front view of the device of FIG. 1 , showing the beginning of a separation cycle, initial charging with analyte solution, electrical current flowing and therefore creating the radial field,

[00018] FIG. 2b is a cross section of the flow chamber of FIG. 2a,

[00019] FIG. 3a is a schematic over all front view of the device of FIG.2a during a separation cycle, initial charging with analyze solution, electrical current flowing,

[00020] FIG. 3b is a cross section of the flow chamber of FIG. 3a,

[00021] FIG. 4a is a schematic over all front view of a device of FIG. 3a, during a separation cycle, charged with analyze solution, electrical current flowing, coated with a gel at the inner surface inside the flow chamber,

[00022] FIG. 4b is a view from above into the flow chamber of in Fig.4a,

[00023] FIG. 5 is a schematic over all front view of a device for electrophoretic separation and purification which can be used to carry out the process according to the present invention during a separation cycle, charged with analyze solution, electrical current flowing, creating an axial electrical field, the flow chamber being in a tilted position

[00024] FIG. 6 is a schematic over all front view of the device of FIG. 5 during a separation cycle, charged with analyze solution, electrical current flowing, coated with a gel (16) at the inner surface inside of the flow chamber.

DETAILED DESCRIPTION OF THE INVENTION

[00025] Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts of the devices described or to process steps of the methods described as such devices and methods may vary. It is also to be understood, that the terminology used herein is for purposes describing particular embodiments only and it is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms of "a", "an", and "the" include plural referents until the context clearly dictates otherwise. Thus, for example, the reference to "a positively charged compound" includes two or more such positively charged elements.

[00026] In this specification and in the claims which follow, reference will be made to the following terms which shall be defined to have the herewith explained meanings:

[00027] A flow chamber is a reactor that is used for processes in chemistry requiring a continuous flow of liquid. This reactor is designed to permit continuous flowing of the liquid along a definite flow path.

[00028] "Educt" is generally the substance being the initial material for a chemical or physical reaction leading to a "product" then.

[00029] "Product" is generally the substance that is obtained, when the educt has been exposed to at least one chemical reaction.

[00030] A liquid such as an analyte solution can be forced to move through a chemical reactor such as a flow chamber by means of a "motive source". In the easiest case, gravity is the source that leads to a movement of a liquid along a flow chamber, which is arranged vertically. Generally pumps are used as "motive sources" in order to achieve an economic flow-through. It has to be understood, that in the present invention there is no restriction for the use of any pumps; some of the embodiments of the present invention require the use of pumps creating a pH-gradient.

[00031] A "collector" is generally a collecting means suitable for recovery of liquids or compounds dissolved in liquids.

[00032] A "recycling device" is generally a device suitable for transferring product back to the chemical process.

[00033] Features that are substantially or functionally equal or similar will be referred to with the same reference signs.

[00034] Referring now to FIG. 1, the device of the present invention for electrophoretic separation and purification of analyte solution and subsequent recovery of the product comprises a motive source 12 for moving the analyte solution, which is a conventional pump 12a in this embodiment, one flow chamber 2 which is of cylindrical geometry, having one inlet 3 at the top of the cylinder and one outlet 4 at the bottom of the cylinder. Furthermore, a power source 7 is comprised in order to generate an electrical field by usage of two electrodes that are cathode and, respectively, anode. The electrical field of this embodiment is a radial field 6a, since the wall of the cylindrical flow chamber 2 is providing the one electrode element 5b and an axial element 5a in congruence with the longitudinal axis of the flow chamber 2, which is located upright, is the second electrode.

[00035] Of course, the flow chamber 2 can be generally designed in another way, as long as a homogeneous, or at lest a predictable electrical field is provided, which allows for good controllability of the separation process. A capillary is another example for a cylindrically shaped flow chamber 2 that fulfills the requirements of the present invention. Also it is only necessary that a homogeneous electrical field is only partly existing within the flow chamber. It has to be understood, that the device of the present invention, and therefore the components of which it is built, is not restricted to a definite size; it can be realized in lab size as well as in miniaturized size in order to be integrated in existing apparatus or analysis-lines. Furthermore, any other type of pump can be used.

[00036] FIG. 1 b shows clearly the cylindrical diameter of the flow chamber 2, one electrode 5a is positioned exactly in the center of the cylinder while the other electrode 5b is integrated in the wall of the flow chamber 2. The other electrode has not necessarily to be integrated into the wall, it can also be close to the wall or to a part of it of to the complete circumference of the cylinder, as long as a radial electrical field is at least partly generated inside the chamber.

[00037] Referring now to FIG. 2a, the device of FIG.1 a is shown, indicating now the first step of the separating process: An analyte solution stream 1 is entering the flow chamber 2 via the inlet 3 and an electrical current is flowing, therefore initiating a separation cycle. A collecting vessel 18 is placed below the outlet 4 of the flow chamber 2 to receive the product.

[00038] FIG. 2b shows a cross section of the flow chamber 2 of FIG. 2a, pointing out clearly by arrows that a radial electrical field 6a is created. In this case, the cathode is provided by the axial element 5a in the center of the cylinder while the anode is provided by the wall. The polarity can be reversed.

[00039] Referring now to FIG. 3a, one can see that the separation process is running, indicated by the continuously flowing analyte stream 1, whose compounds start being separated after the stream has entered the flow chamber 2, which has been shown in FIGS. 1 and 2. Once having entered the flow chamber 2, the negatively charged compounds 5b leave their flow path directing them from the inlet 3 to the outlet 4 and move to the chamber wall, being the anode herein, while the positively charged compounds 8 move to the cathode, which is provided by the axial element 5a; the deflection being caused by the electrical field 7, causing movement according to the mobility. The electrical field acts in parallel to the hydraulic forces that are generated by pumping the analyte solution. The neutral compounds 10 flow along the flow path, therefore leaving the flow chamber 2 when they have reached the outlet 4 and "falling" then into a colleting vessel 18.

[00040] Of course, other means such as absorption strips or other devices suitable for the collection of liquid can be used generally instead of a collecting vessel 18.

[00041] FIG. 3b is another view of the situation indicated in FIG. 3a. It shows the flow chamber 2 in cross section and the settling of charged compounds at anode and cathode while neutral compounds 10 are not deflected.

[00042] Referring now to FIG.4a, one can see that the separation process is running, too, as explained above with reference to FIG. 3a, the inside surface of the chamber wall being coated with a gel in order to absorb the negatively charged compounds 9 attracted by the chamber wall, which acts as cathode. Since the axial element 5a acting as anode is not coated, the positively charged compounds 8 may remain in solution, as the neutral compounds 10 do. With time neutral and positively charged compounds 8,10 follow the flow path, leave the flow chamber2 via the outlet 4 and "fall" into the colleting vessel 18.

[00043] Of course, the coating of gel or any other suitable material is not restricted to the inside surface of the chamberwall, which makes sense in the above embodiment, thus offering a large surface area for the uptake of separated compounds, but can just as well be applied on an axial element 5a, or in case of more than one axial elements 5a acting as electrode, on all of them.

[00044] FIG. 4b shows the cross section of the flow chamber 2 of FIG. 4a, showing in detail the settling of charged compounds 8,9 at anode and cathode while neutral compounds 10 are not deflected. It can be seen how negatively charged compounds 9 are trapped in the gel coating the inner surface of the chamber wall 2, which is the anode.

[00045] Referring now to FIG. 5, one can see the device of the present invention for electrophoretic separation and purification of analyte solution in an alternative embodiment: The basic components are a motive source 12 for moving the analyte solution, herein not a conventional pump but a pump providing a pH-gradient, one flow chamber 2 of cylindrical geometry, having one inlet 3 at the top of the cylinder and one outlet 4 at the bottom of the cylinder, and a power source 7 in order to generate an electrical field by use of cathode and anode. But in comparison to the above embodiments, the electrical field depicted in FIG. 5 is an axial field, with the anode integrated in the top and the cathode integrated in the bottom of the cylindrical flow chamber 2, which is arranged in a tilted position. The inlet 3, is a central opening in the anode, the outlet 4 is located at the lowest point of the flow chamber 2.

[00046] FIG. 6 shows the device of FIG. 5, wherein the inner surface of the top and the bottom are coated with gel.

[00047] Referring to FIG. 5 and FIG. 6, one can see that the separation process is running, indicated analogous to FIG. 2. Unlike as FIG. 1 to 4, the negatively charged compounds 9 are deflected in direction to the top, which is the anode, since the electrical forces caused by the electrical field 7 are stronger than the hydraulic forces, while the positive charged compounds 8 move to the bottom, which is the cathode. In this embodiment, the neutral compounds 10 exit the flow chamber 2 via the outlet 4 and "fall" into a colleting vessel 18, the positively charged compounds 8 remain in the flow chamber at the bottom, attracted and "fixed" due to the electrical forces.

[00048] Versions of any of the above embodiments facilitate the enrichment of desired compounds, when at least parts of the inner surface of the flow chamber 2 being an electrode are coated with a gel or an other matrix suitable for the uptake of the preferred compound.

[00049] Furthermore, the electrical field can be designed in a way that only the negatively charged compounds are kept in the flow chamber 2, while the positively charged compounds 8 exit together with the neutral ones. [00050] The device of the present invention can be coupled to downstream separation and analytical devices.

[00051] The analyte solution, which is exposed to the process performed in the device of the present invention, contains a sample that is constituted preferably of biological compounds, more preferably organic compounds, and most preferably, proteins, protein derivatives, protein isoforms, enzymes, antigens, antibodies, peptides or nucleic acids, lipids or carbohydrates. Proteins and their derivatives are the most interesting compounds discussed in the present document; they have a net neutral charge at a defined pH value, which is defined as their iso-electric point. Due to the extraordinary property of becoming iso-electric, the proteins are predestined for the method explained below. A mixture of proteins can be separated into pure fractions by buffering the mixture in order to obtain a definite pH value, wherein only proteins having this very pH value as their pi become iso-electric while the other components of the mixture remain charged. The analyte solution referred to in the present invention contains furthermore buffer solution and reagents or other additives.

[00052] In order to carry out the method of electrophoretic separation and purification of an analyte solution as defined above and recovering of the product(s), the educt is introduced into a device of the present invention, in particularly into one of the embodiments shown in FIGS. 1 to 6. The educt is pumped via an inlet 3 into a flow chamber 2, whereby a hydraulic flow is caused. Now, the analyte solution is moved through the flow chamber 2 being driven by hydraulic forces while in parallel electrical forces resulting from an electrical field, which is generated by a power source 7, a cathode and an anode, act upon the analyte solution stream 1 inside the flow chamber 2. This leads to deflection 13 of the charged compounds 8,9, the grade of deflection of any compound 8,9,10 depending on its size/charge ratio, the electrical field 7, the viscosity of the liquid and on said hydraulic forces. Then, the anode attracts the negatively charged compounds 9 and the cathode attracts the positively charged compounds 8, while those compounds 8,9,10 being driven predominantly by hydraulic forces are passing the flow chamber 2 without deflection and leave it. The step of attracting can be reversed into repulsion by changing the polarity of anode and cathode, both repulsion and attraction being controlled by the parameters direction and strength of the electrical field 7, acting relative to the strength of the hydraulic flow, viscosity of the liquid and size/charge ratio of the compounds 8,9,10.

[00053] To generate the electrical field, any voltage may be used as far as it's in the range which is tolerable for the device of the present invention, e.g. 10 to 10 000 volts. Heat resulting from high voltage is suggested to be dissipated by proper cooling.

[00054] The most interesting compounds of the analyze solution referred herein are proteins due to their electrical ambivalence: Proteins have an isoelectric point (pi), defined as the pH at which the protein has a net neutral charge. Therefore, proteins can be fractionated using a pH gradient combined with an electrical field. A pH gradient can be created by using a buffer at a definite pH value. Then, a mixture of proteins can be separated into two fractions as described in the steps above: The "mixture" is pumped through a separation device according the present invention, preferably according to one of the embodiments shown in FIGS. 1 to 6. Those proteins whose iso-electric point is obtained at the given pH-value are neutral then, thus they pass the flow chamber 2 without being deflected and get collected in a collection vessel 18. After a time, all negatively charged proteins/compounds become embedded in the gel or matrix while all positively charged proteins/particles may be attached to the cathode and the neutral proteins/particles remain in the liquid phase.

[00055] The compounds that have been trapped in the flow chamber 2 can be set free in a second step by reversing the polarity of the electrodes; they exit the flow chamber 2 via outlet 4 and get collected in a second collection vessel 18. Both fractions, the one that has been collected in the first collection vessel and the one that has been collected in the second collection vessel, can be further fractionated by using the separation method again at a different pH value. By using additional fractionation rounds, each at different pH, it is possible to use this invention to generate many fractions based on the iso¬ electric points. For instance, fractioning of iso-electric points every 0.5 pH (or even narrower) is possible.

[00056] It has to be noted that some proteins or compounds types may have properties that permanently embed them in the gel or matrix; these properties give this invention another means for fractionation/separation.

[00057] The components which are needed to realize the present invention are already existing in technology, e.g. pumps, capillaries, collection vessels, etc., thus, they are well known instruments which can be combined to a controllable, precise device according to this invention. The separation device described inhere is an easy to operate system, which may be integrated as a module in an existing LC-system. Furthermore, this technique can be performed on a small scale relative to FFE; which can lead to a significantly lower cost and reagent consumption. A further advantage is, that this invention could be automated.

Claims

1. A device for electrophoretic separation of positively and negatively charged compounds (8,9) and neutral compounds (10) in a moved analyte solution, said device comprising: - at least one flow chamber (2) comprising at least one inlet (3) and at least one outlet (4) with an analyte solution stream (1) flowing from the at least one inlet (3) to the at least one outlet (4) through the flow chamber (2),

- a power source (7) and electrodes adapted to generate an electrical field, wherein said electrical field is generated inside the flow chamber (2).

2. The device of claim 1 , wherein one of the electrodes is an anode and one of the electrodes is a cathode.

3. The device of claim 1 or 2, comprising a motive source (12) for moving the analyte solution, thereby causing a hydraulic flow with inherent hydraulic flow forces.

4. The device of claim 3 or any one of the above claims, wherein exposing the analyte solution stream (1) to the hydraulic flow forces and to the electrical field results in deflection (13) of the positively charged compounds (8) to the at least one cathode and of the negatively charged compounds (9) to the at least one anode, the deflection (13) of each compound (8,9) being at least a function of the charge of the compound, of the electrical field and of the hydraulic forces being applied on the analyte solution.

5. The device of claim 1 or any one of the above claims, wherein the electrical field is invertible.

6. The device of claim 1 or any one of the above claims, wherein said flow chamber (2) has a cylindrical geometry with the at least one inlet (3) at the top (14) and the at least one outlet (4) at the bottom (15) of the cylinder, wherein said electrical field inside the flow chamber (2) is a radial field (6a) or an axial electric field with respect to the longitudinal axis of the cylinder.

7. The device of claim 1 or any one of the above claims, wherein the flow chamber (2) is a capillary.

8. The device of claim 1 or any one of the above claims, wherein the flow chamber (2) is arranged vertically or inclined with respect to the horizontal.

9. The device of claim 1 or any one of the above claims, wherein the electrodes are provided by an axial element (5a) and by an element (5b) extending parallel to the axial element (5a), the elements (5a, 5b) acting as counter electrodes.

10. The device of claim 1 or any one of the above claims, wherein the axial element (5a) is a conductive wire and the element (5b) is the wall of the flow chamber (2).

11. The device of claim 1 or any one of the above claims, wherein the oppositely polarized electrodes are located one at the top (14) and one at the bottom (15) of the cylinder.

12. The device of claim 1 or any one of the above claims, wherein the electrical field is substantially homogeneous.

13. The device of claim 1 or any one of the above claims, wherein an inner surface of the flow chamber (2) is at least partially coated with a gel (16) or another matrix suitable for the retain of separated compounds.

14. The device of claim 1 or any one of the above claims, comprising a collector (18) for collecting compounds of the analytical solution exiting the at least one outlet (4).

15. The device of claim 1 or any one of the above claims, comprising a recycling device (11) to bring collected compounds back into the electrophoretic separation.

16. The device of claim 3 or any one of the above claims, wherein the motive source (12) is a conventional pump (12a).

17. The device of claim 3 or any one of the above claims, wherein the motive source (12) is a pH-gradient creating pump (12b).

18. The device of claim 14 or any one of the above claims, wherein the collector (18) is at least one of a collection vessel, an absorption strip, or another device suitable for the collection of compounds exiting the flow chamber (2).

19. An analyte solution containing at least one of the components: (a) sample comprised of charged compounds (8,9) and neutral compounds (10),

(b) buffer solution, and

(c) reagents or other additives, wherein the charged or neutral compounds (8,9,10) of which the sample is constituted are preferably biological compounds, more preferably organic compounds, and most preferably, proteins, protein derivatives, protein isoforms, enzymes, antigens, antibodies, peptides or nucleic acids, lipids or carbohydrates.

20. The analyte solution of claim 19, wherein constituents of the sample, in particular proteins, have a net neutral charge at a defined pH value defined as iso-electric point.

21. A method for electrophoretic separation of positively and negatively charged compounds (8,9) and neutral compounds (10) in an analyte solution, comprising:

- introducing the analyte solution, in particular the analyte solution of claim 19 or 20, into a device for electrophoretic separation, in particular into a device for electrophoretic separation of claim 1 or any one of the above claims,

- moving the analyte solution stream (1) through the at least one flow chamber (2), - and subjecting the analyte solution stream (1 ) to the electrical field being generated within the flow chamber (2).

22. The method of claim 21 or any one of the above claims, comprising attracting of the positively charged compounds (8) of the analyte solution by the cathode and the negatively charged compounds (9) of the analyte solution by the anode, wherein the strength of attracting is at least a function of the electrical field, viscosity of solution, mobility of compounds and of hydraulic forces.

23. The method of claim 21 or any one of the above claims, comprising flowing of the neutral compounds (10) are through the flow chamber (2) and leaving it via the at least one outlet (4).

24. The method of claim 22 or any one of the above claims, wherein changing of polarity of the electrodes leads to reversing of attraction, thus causing repulsion of the charged compounds (8,9) from the electrodes.

25. The method of claim 21 or any one of the above claims, wherein at least one of the charged compounds (8,9) being deflected is trapped in a gel (16) or another matrix suitable for the retain of separated compounds.

26. The method of claim 24 or any one of the above claims, wherein the repulsion resulting from polarity reversing of the electrodes sets free the at least one charged compound (8,9) being trapped.

27. The method of claim 24 or any one of the above claims, wherein the repulsion is controlled by at least one of the parameters voltage and charge of the charged compounds (8,9,10).

28. The method of claim 21 or any one of the above claims, wherein the charged compounds (8,9,10) exiting the at least one outlet (4) are collected in a collector (18).

29. The method of claim 21 or any one of the above claims, wherein the charged compounds (8,9,10) that have been collected are transferred back into the process.

30. The method of claim 21 or any one of the above claims, wherein the analyte solution is moved by a pH-gradient creating pump (12b).