Free-flow electrophoresis method for separating analytes

Abstract

The present invention is related to a free-flow electrophoresis method for separating at least one analyte of interest from a mixture of analytes, wherein the method uses a separation medium comprising two or more individual separation media, wherein the two or more individual separation media differ in their pH value, and wherein each of the two or more individual separation media comprise at least one anion of at least one acid and at least one cation of at least one base, wherein the at least one acid is the same in each of the two or more individual separation media and the at least one base is the same in each of the two or more individual separation media.

Claims

1. A free-flow electrophoresis method for separating at least one analyte of interest from a mixture of analytes, wherein the method comprises flowing a separation medium through a separation chamber in a flow direction; applying an electric field in the separation medium by an anode and a cathode, wherein the anode and the cathode are located at a distance from each other and the separation medium flows between the anode and the cathode, and applying the mixture of analytes to the separation medium, whereupon the at least one analyte of interest is separated from the mixture of analytes, or applying the mixture of analytes to the separation medium, and applying an electric field in the separation medium by an anode and a cathode, wherein the anode and the cathode are located at a distance from each other and the separation medium flows between the anode and the cathode, whereupon the at least one analyte of interest is separated from the mixture of analytes; collecting fractions of the separation medium with at least one fraction comprising the at least one analyte of interest separated from the mixture of analytes; characterized in that the separation medium comprises two or more individual separation media, wherein the two or more individual separation media differ in their pH value, and wherein each of the two or more individual separation media comprise at least one anion of at least one acid and at least one cation of at least one base, wherein the at least one acid is the same in each of the two or more individual separation media and the at least one base is the same in each of the two or more individual separation media; the at least one anion of the at least one acid is the same in each of the two or more individual separation media and the at least one cation of the at least one base is the same in each of the two or more individual separation media; and if the analyte of interest has a pI of >7, the analyte of interest is separated at an optimum pH range pH.sub.opt, wherein pH.sub.opt is determined as follows:
pI0.6<pH.sub.optpI or if the analyte of interest has a pI of <7, the analyte of interest is separated at an optimum pH range pH.sub.opt, wherein pH.sub.opt is determined as follows:
pIpH.sub.opt<pI+0.6.

2. The free-flow electrophoresis method of claim 1, wherein the pH value of the two or more individual separation media is set prior to carrying out the free-flow electrophoresis method.

3. The method of claim 1, wherein the pH value of the two or more separation media increases from the anode to the cathode.

4. The method of claim 1, wherein the separation medium comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more individual separation media.

5. The method of claim 1, wherein either (i) the pKa value of the acid is from about 3 to 8, (ii) the pKa value of the base is from about 4 to 10, or (iii) the pKa value of the acid is from about 3 to 8 and the pKa value of the base is from about 4 to 10.

6. The method of claim 1, wherein the concentration of the anion in at least one or each of the at least two individual separation media is from about 3-100 mM.

7. The method of claim 1, wherein in case of a cationic separation the concentration of the cation in at least one or each of the at least two individual separation media is from 5-50 mM.

8. The method of claim 1, wherein in case of an anionic separation the concentration of the anion in at least one or each of the at least two individual separation media is from 5-50 mM.

9. The method of claim 1, wherein either (i) the concentration of the acid is up to 500 mM at the pH of the individual separation medium at the cathode or at the pH of the border stabilization medium at the cathode, (ii) the concentration of the base is up tp 500 mM at the pH of the individual separation medium at the anode or at the pH of the border stabilization medium at the anode, or (iii) the concentration of the acid is up to 500 mM at the pH of the individual separation medium at the cathode or at the pH of the border stabilization medium at the cathode and the concentration of the base is up to 500 mM at the pH of the individual separation medium at the anode of at the pH of the border stabilization medium at the anode.

10. The method of claim 1, wherein the anion bears a single negative charge at the pH value of the individual separation media and/or wherein the cation bears a single positive charge at the pH value of the individual separation media.

11. The method of claim 1, wherein each of the two or more individual separation media comprises at least one anion of two or more acids.

12. The method of claim 1, wherein the free-flow electrophoresis method is interval free-flow electrophoresis method.

13. The method of claim 1, wherein the free-flow electrophoresis method is a continuous free-flow electrophoresis method.

14. The method of claim 1, wherein the analytes are selected from the group comprising cells, cell compartments, nanobeads, nanodiscs, viruses and any compounds.

15. The method of claim 14, wherein the compounds are biological and chemical compounds.

16. The method of claim 15, wherein the compounds are charged biological and chemical compounds.

17. A free-flow electrophoresis method for separating at least one analyte of interest from a mixture of analytes, wherein the method comprises: flowing a separation medium through a separation chamber in a flow direction; applying an electric field in the separation medium by an anode and a cathode, wherein the anode and the cathode are located at a distance from each other and the separation medium flows between the anode and the cathode, and applying the mixture of analytes to the separation medium, whereupon the at least one analyte of interest is separated from the mixture of analytes, or applying the mixture of analytes to the separation medium, and applying an electric field in the separation medium by an anode and a cathode, wherein the anode and the cathode are located at a distance from each other and the separation medium flows between the anode and the cathode, whereupon the at least one analyte of interest is separated from the mixture of analytes; and collecting fractions of the separation medium with at least one fraction comprising the at least one analyte of interest separated from the mixture of analytes; characterized in that the separation medium comprises two or more individual separation media, wherein the two or more individual separation media differ in their pH value, and wherein each of the two or more individual separation media comprise at least one anion of at least one acid and at least one cation of at least one base, wherein the at least one acid is the same in each of the two or more individual separation media and the at least one base is the same in each of the two or more individual separation media, wherein each of the two or more individual separation media comprises at least one cation of two or more bases.

18. A free-flow electrophoresis method for separating at least one analyte of interest from a mixture of analytes, wherein the method comprises flowing a separation medium through a separation chamber in a flow direction; applying an electric field in the separation medium by an anode and a cathode, wherein the anode and the cathode are located at a distance from each other and the separation medium flows between the anode and the cathode, and applying the mixture of analytes to the separation medium, whereupon the at least one analyte of interest is separated from the mixture of analytes, or applying the mixture of analytes to the separation medium, and applying an electric field in the separation medium by an anode and a cathode, wherein the anode and the cathode are located at a distance from each other and the separation medium flows between the anode and the cathode, whereupon the at least one analyte of interest is separated from the mixture of analytes; and collecting fractions of the separation medium with at least one fraction comprising the at least one analyte of interest separated from the mixture of analytes; characterized in that the separation medium comprises two or more individual separation media, wherein the two or more individual separation media differ in their pH value, and wherein each of the two or more individual separation media comprise at least one anion of at least one acid and at least one cation of at least one base, wherein the at least one acid is the same in each of the two or more individual separation media and the at least one base is the same in each of the two or more individual separation media, wherein each of the two or more individual separation media comprises at least one anion of two or more acids and at least one cation of two or more bases, wherein each of the two or more individual separation media comprises at least one anion of two or more acids and at least one cation of two or more bases.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present invention will now be further illustrated by the following drawings and examples, from which further features, embodiments and advantages of the invention may be taken, whereby

(2) FIG. 1 is an illustration of an embodiment of the method of the invention where the analyte of interest is an anion and the method thus provides an anionic separation;

(3) FIG. 2 is an illustration of an embodiment of the method of the invention where the analyte of interest is a cation and the method thus provides a cationic separation;

(4) FIG. 3 is an illustration of an embodiment of the method of the invention where the analyte comprises an analyte of interest which is a cation and an analyte of interest which is an anion, and the method is thus a simultaneous anionic separation and a cationic separation;

(5) FIG. 4 is an illustration of the effect arising from pH step-gradients on separation efficiency and a sharpening of the analyte bands;

(6) FIG. 5 is a diagram illustrating the result of separating an analyte consisting of a mixture of amphoteric dyes and non-amphoteric dye SPADNS (Trisodium 2-(4-sulfophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonate) using the method of the invention realizing a pH step-gradient, whereby the method was carried out as an interval free-flow electrophoresis method. The X-axis indicates the fractions obtained at the fraction points, the Y-axis on the left side indicates the pH value measured in the individual fractions (depicted as full diamonds), and the Y-axis on the right side indicates optical density measured at 420 nm (depicted as full columns);

(7) FIG. 6 is a diagram illustrating the result of separating an analyte of interest, namely non-amphoteric dye SPADNS (trisodium 2-(4-sulfophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonate), from a mixture consisting of a mixture of amphoteric dyes and said non-amphoteric dye SPADNS using a free-flow electrophoresis method of the prior art realizing a constant pH value across the separation medium, whereby the method was carried out as an interval free-flow electrophoresis method. The X-axis indicates the fractions obtained at the fraction points, the Y-axis on the left side indicates the pH value measured in the individual fractions (depicted as full diamonds), and the Y-axis on the right side indicates optical density measured at 420 nm (depicted as full columns);

(8) FIG. 7 is a diagram illustrating the result of separating an analyte of interest, namely SPADNS (trisodium 2-(4-sulfophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonate), from a mixture consisting of a mixture of amphoteric dyes and said non-amphoteric dye SPADNS using the method of the invention realizing a pH step-gradient, whereby the method was carried out as a continuous electrophoresis method. The X-axis indicates the fractions obtained at the fraction points, the Y-axis on the left side indicates the pH value measured in the individual fractions (depicted as full diamonds), and the Y-axis on the right side indicates optical density measured at 420 nm (depicted as full columns);

(9) FIG. 8 is a diagram illustrating the result of separating an analyte of interest, namely SPADNS (trisodium 2-(4-sulfophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonate), from a mixture consisting of a mixture of amphoteric dyes and said non-amphoteric dye SPADNS using a free-flow electrophoresis method of the prior art realizing a constant pH value across the separation medium, whereby the method was carried out as a continuous free-flow electrophoresis method. The X-axis indicates the fractions obtained at the fraction points, the Y-axis on the left side indicates the pH value measured in the individual fractions (depicted as full diamonds), and the Y-axis on the right side indicates optical density measured at 420 nm (depicted as full columns);

(10) FIG. 9 is a diagram illustrating the result of separation of a monoclonal antibody pre-purified by chromatographic techniques using the method of the invention. The X-axis indicates the fractions obtained at the fraction points, the Y-axis on the left side indicates the pH value measured in the individual fractions (depicted as full diamonds), and the Y-axis on the right side indicates optical densities measured at 420 nm, 515 nm and 595 nm;

(11) FIG. 10 is the result of a silver stain of an antibody analysed by isoelectric focusing on a PAGE gel after separating the antibody from a sample containing the antibody using the method of the invention; and

(12) FIG. 11 is an illustration of a device of the invention for free-flow electrophoresis used in the performing of the method of the invention.

DESCRIPTION OF PREFERRED EMBODIMENTS

(13) FIG. 1 is an illustration of an embodiment of the method of the invention. In the embodiment shown the separation is an anionic separation. In such anionic separation the analyte of interest is an anion. Separation medium S consists of individual separation media 2, 3, 4, 5, 6, 7 and 8 which differ in their pH value. Due to such difference in pH value a step-gradient is formed over separation medium S. Separation medium S is flanked on the side of the cathode by border stabilization medium 9 and on the side of the anode by border stabilization medium 1. Both border stabilization medium 1 and border stabilization medium 9 have a conductivity which is increased compared to conductivity of individual separation media 2, 3, 4, 5, 6, 7 and 8. As the analyte of interest is an anion Analyte A which is a mixture of analytes or a sample, is applied to individual separation medium 7 which is closer to the cathode than to the anode (+) and thus to an individual separation medium having a comparatively higher pH value than most of the other individual separation media. Under the influence of the electric field the analyte of interest contained in Analyte A which is a mixture of analytes or a sample, being moved along the direction of the separation medium will be separated from the remaining analytes. As the analyte of interest is an anion it will move to the anode as indicated in FIG. 1 by the arrow direction of migration of analyte.

(14) FIG. 2 is an illustration of a further embodiment of the method of the invention. In the embodiment shown the separation is a cationic separation. In such separation the analyte of interest is a cation. As the analyte of interest is a cation Analyte A which is a mixture of analytes or a sample, is applied to individual separation medium 3 which is closer to the anode (+) than to the cathode () and thus to an individual separation medium having a comparatively lower pH value than most of the other individual separation media. Under the influence of the electrical field the analyte of interest contained in Analyte A being moved along the direction of the separation medium will be separated from the remaining analytes. As the analyte of interest is a cation it will move to the cathode as indicated in FIG. 2 by the arrow direction of migration of analyte. Otherwise, the method is similar to the one described in connection with FIG. 1.

(15) FIG. 3 is an illustration of a still further embodiment of the method of the invention. In the embodiment shown the separation is a simultaneous cationic separation and anionic separation. Separation medium S consists of individual separation media 2, 3, 4, 5, and 6 which differ in their pH value. Due to such difference in pH value a step-gradient is formed over separation medium S. Separation medium S is flanked on the side of the cathode by border stabilization medium 7 and on the side of the anode by border stabilization medium 1. Both border stabilization medium 1 and border stabilization medium 7 have a conductivity which is increased compared to conductivity of individual separation media. In such separation the analyte comprises an analyte of interest which is a cation and an analyte of interest which is an anion. Because of this, Analyte A which is a mixture of analytes or a sample, is applied to individual separation medium 4 which is about equally distant from both the cathode and the anode and thus to an individual separation medium having a pH value which is in the middle of the pH values defined by the individual separation media 2 and 6. Under the influence of the electrical field the analyte of interest contained in Analyte A being moved along the direction of the separation medium will be separated from the remaining analytes. The cationic analyte of interest being a cation will move to the cathode as indicated in FIG. 3 by the arrow direction of migration of cationic analyte, whereas the anionic analyte of interest being an anion will move to the anode as indicated in FIG. 3 by the arrow direction of migration of anionic analyte. Otherwise, the method is similar to the one described in connection with FIGS. 1 and 2.

(16) FIG. 4 is an illustration of the effect arising from a pH step-gradient on separation efficiency. More specifically, FIG. 4 shows the different band widths of any anionic amphoteric analyte of interest at the interface of individual separation media having different pH values. Band width decreases and the concentration of the analyte increases in the maximum of the band due to a significant decrease in electrophoretic migration of the analyte when passing from one individual separation medium to another individual separation medium. This kind of advantageous effect can be observed irrespective of whether the method of the invention is carried out as an interval free flow electrophoresis or as a continuous free flow electrophoresis.

(17) FIG. 11 is an illustration of a device for free-flow electrophoresis used in performing the method of the invention. The individual separation media inside the vessels 2 to 8 are supplied by means of pump 30 and the media feeding lines 12 to 18 to the media inlets 22 till 28.

(18) Additionally, two border stabilization media inside the vessels 1 and 9 are transported by pump 30 via the feeding lines 11 and 19 and via the media inlets 21 and 29 into the separation medium 31, which functions as the separation area, in the neighborhood of the anode 50 and cathode 60. Furthermore, a counterflow medium is supplied by pump 30 from vessel 10 via the feeding line 20 to the manifold inlets 71, 72 and 73 and will enter to the fractionation area 75.

(19) Analytes are supplied from sample container 36 by means of pump 35 to injection port 37. The thus applied analytes are transported along the flow direction of the separation medium formed by individual separation media 8 to fractionation area 75 comprising fractionation plate 76 with fractionation vials 77. At fraction area 75 individual fractions of the separation medium are collected. The Fig. describes the migration path of two different analytes, if the separation process will be operated as a continuous free-flow electrophoresis method. Voltage is applied to the anode 50 and cathode 60 for the entire period of time while the analyte migrates along the separation area. If operated so as to realize an interval free-flow electrophoresis, voltage is applied to the anode and cathode only for a certain period of time during the migration of the analyte along the separation medium. The migration path of the analytes will look like 2 parallel zones of migration.

Example 1: Separation of Analytes Consisting of a Mixture of Amphoteric Dyes and Non-Amphoteric Dye SPADNS

(20) Analytes consisting of a mixture of amphoteric dyes and non-amphoteric dye SPADNS were subject to the method of the invention realizing a pH step-gradient, whereby the method was carried out as an interval free-flow electrophoresis method (see FIG. 5). In order to show the advantage of the method of the invention, the same experiment was carried out using the same method of FF-Interval-zone electrophoresis except that no pH gradient was realized (see FIG. 6).

(21) The experimental details of the experiments underlying FIGS. 5 and 6 were as follows.

(22) The separation was conducted in a FFE system on a 0.2 mm gap.

(23) The individual separation media and border stabilization media were as follows:

(24) TABLE-US-00001 Anode border stabilization 150 mM HCl medium: (inlet 1) 300 BISTRIS 250 mM Mannitol Individual separation medium 1: 10 mM BISTRIS (inlet 2&3) adjusted to pH 4.77 with glutamic acid 250 mM Mannitol Individual separation medium 2: 10 mM glutamic acid (inlet 4) adjusted to pH 5.81 with BISTRIS 250 mM Mannitol Individual separation medium 3: 10 mM glutamic acid (inlet 5) adjusted to pH 6.08 with BISTRIS 250 mM Mannitol Individual separation medium 4: 10 mM glutamic acid (inlet 6-8) adjusted to pH 6.87 with BISTRIS 250 mM Mannitol Cathode border stabilization 200 mM triethylamine medium: 100 mM EtOH amine 50 mM TEA 200 mM glutamic acid 250 mM Mannitol Counterflow medium: 250 mM Mannitol

(25) The sample was injected at individual separation medium 5 with 2000 l/h; separation was conducted at 1200V and 53 mA on a 4.5 Minute interval at a media speed of 40 ml/h.

(26) FIG. 6:

(27) The separation was conducted in a FFE system on a 0.2 mm gap.

(28) The individual separation medium and border stabilization media were as follows:

(29) TABLE-US-00002 Anode border stabilization medium: 150 mM HCl (inlet 1) 300 BISTRIS 250 mM Mannitol Individual separation medium 1: 10 mM glutamic acid (inlets 2-5) 250 mM Mannitol adjusted to pH 6.86 with BISTRIS Separation buffer 2: 10 mM glutamic acid (inlets 6-8) 250 mM Mannitol adjusted to pH 6.87 with BISTRIS Cathode border stabilization 200 mM triethylamine medium: 100 mM EtOH amine 50 mM TEA 200 mM glutamic acid 250 mM Mannitol Counterflow medium: 250 mM Mannitol

(30) The sample was injected at individual separation medium 5 with 2000 l/h; separation was conducted at 1200V and 53 mA on a 4.5 Minute interval at a media speed of 40 ml/h.

(31) The results are shown in FIGS. 5 and 6. FIGS. 5 and 6 display the profile of pH-values and the concentration profiles of the separated analytes inside the sample. In case of the process being FF-interval zone electrophoresis, the use of separation media with pH-gradients will give the surplus of quality of separation of the target analytes (pI 4.0, pI 4.75 and pI 5.3), marked inside the graphs.

Example 2: Separation of an Analyte Consisting of a Mixture of Amphoteric Dyes and Non-Amphoteric Dye SPADNS

(32) Analytes consisting of a mixture of amphoteric dyes and non-amphoteric dye SPADNS were subject to the method of the invention realizing a pH step-gradient, whereby the method was carried out as a continuous free-flow electrophoresis method. In order to show the advantage of the method of the invention, the same experiment was carried out using the same method except that no pH gradient was realized, whereby the results are depicted in FIGS. 7 and 8.

(33) The experimental details of the experiments underlying FIGS. 7 and 8 were as follows.

(34) The separation was conducted in a FFE system on a 0.2 mm gap.

(35) The individual separation media and border stabilization media were as follows:

(36) TABLE-US-00003 Anode border stabilization 150 mM HCl medium: (inlet 1) 300 BISTRIS 250 mM Mannitol Individual separation medium 1: 10 mM BISTRIS (inlets 2&3) 250 mM Mannitol adjusted to pH 4.77 with glutamic acid Individual separation medium 2: 10 mM glutamic acid (inlet 4) 250 mM Mannitol adjusted to pH 5.81 with BISTRIS Individual separation medium 3: 10 mM glutamic acid (inlet 5) 250 mM Mannitol adjusted to pH 6.08 with BISTRIS Individual separation medium 4: 10 mM glutamic acid (inlets 6-8) 250 mM Mannitol adjusted to pH 6.87 with BISTRIS Cathode border stabilization 200 mM triethylamine medium: 100 mM EtOH amine 50 mM TEA 200 mM glutamic acid 250 mM Mannitol Counterflow medium: 250 mM Mannitol

(37) The sample was injected at individual separation medium 5 with 1200 l/h; separation was conducted at 1200V and 53 mA continuously at a media speed of 150 ml/h.

(38) FIG. 8:

(39) The separation was conducted in a FFE system on a 0.2 mm gap.

(40) The individual separation media and border stabilization media were as follows:

(41) TABLE-US-00004 Anode border stabilization medium: 150 mM HCl (inlet 1) 300 BISTRIS 250 mM Mannitol Individual separation medium 1: 10 mM glutamic acid (inlets 2-5) adjusted to pH 6.86 with BISTRIS 250 mM Mannitol Individual separation medium 2: 10 mM glutamic acid (inlets 6-8) adjusted to pH 6.87 with BISTRIS 250 mM Mannitol Cathode border stabilization 200 mM triethylamine medium: 100 mM EtOH amine 50 mM TEA 200 mM glutamic acid 250 mM Mannitol Counterflow medium: 250 mM Mannitol

(42) The sample was injected at individual separation medium with 1200 l/h; separation was conducted at 1200V and 52 mA continuously at a media speed of 150 ml/h.

(43) The results are shown in FIGS. 7 and 8. FIGS. 7 and 8 display the profile of pH-values and the concentration profiles of the separated analytes inside the sample. In case of the process being continuous FF-zone-electrophoresis, the use of separation media with pH-gradients will give the surplus of quality of separation of the target analytes, marked inside the figures.

Example 3: Separation of a Murine Monoclonal Antibody from a Cell Culture Medium

(44) A murine monoclonal antibody was separated from a sample containing the antibody pre-purified by chromatographic techniques (IZE, obtained by using the method of the present invention). Fractions obtained from carrying out the method of the inventions were subject to analysis with PAGIEF (analytical isoelectric focusing on polyacrylamid gels).

(45) The technical details of the method of the invention were as follows.

(46) The separation was conducted in a FFE system on a 0.2 mm gap with a flow rate of the various media of 50 ml/h at a 5 minute interval at 1700V and 120 mA.

(47) 20 l of the sample was diluted with 46.1 separation buffer and injected with 2800 l/h at S1

(48) The individual separation media and border stabilization media were as follows:

(49) TABLE-US-00005 Anode border stabilization medium: 148 mM HCl (inlet 1) 150 mM TEA 250 mM Mannitol Individual separation medium 1: 20 mM TEA (inlet 2) 20 mM glutamic acid 250 mM Mannitol Individual separation medium 2: 10 mM TEA (inlet 3) 10 mM glutamic acid 5 mM NaCl 250 mM Mannitol Individual separation medium 3: 10 mM TEA (inlet 4) 10 mM glutamic acid 250 mM Mannitol adjusted with TEA to pH = 7.53 Individual separation medium 4: 10 mM TEA (inlet 5) 10 mM glutamic acid 250 mM Mannitol adjusted with TEA to pH = 7.83 Individual separation medium 5: 10 mM TEA (inlet 6) 10 mM glutamic acid 250 mM Mannitol adjusted with TEA to pH = 8.15 Individual separation medium 6: 50 mM Tris (inlets 7&8) 15 mM glutamic acid 250 mM Mannitol pH = 8.48 Cathode border stabilizationmedium: 200 mM glutamic acid (inlet 9) 50 mM Tris 300 mM Ammediol 250 mM Mannitol pH = 8.64 Counterflow medium: 250 mM Mannitol

(50) The result is shown in FIG. 9. FIG. 9 displays the profile of pH-values and the profiles of concentration of the separated analytes inside the test sample. Following the separation of the test sample, the identical conditions of separation were applied to a sample of the monoclonal antibody.

(51) Fractions 47 to 67 of the FFE run obtained, were subject to the analysis with PAGIEF. The results, as shown FIG. 10, display an excellent quality of separation with a single band purity of the separated isoforms of the monoclonal antibody.

Example 4: Separation of Proteins with pI-Value of 7

(52) Using mixtures of acids and bases with similar values of electrophoretic mobilities protocols can be used for the separation of proteins with a broader range of pI-values. More specifically, the variation of the ratio of the concentrations of the bases allows expanding the pH-range into the alkaline region (pI>7). In a similar approach mixtures of acids with similar values of electrophoretic mobility can be used to expand the pH-range for the separation of proteins with pI values (pI<7).

(53) An example for embodiments of the method of the invention making use of at least one anion of each of two acids and at least one cation of each of two bases is presented in the following.

(54) The technical details of a respective embodiment of the method of the invention were as follows.

(55) The separation was conducted in a FFE system on a 0.2 mm gap with a flow rate of the various media of 50 ml/h at a 5 minute interval at 1700V and 120 mA.

(56) 20 l-50 l of the sample were injected at S1 or S5

(57) The individual separation media and border stabilization media were as follows:

(58) TABLE-US-00006 Anode border stabi- 148 mM HCl lization medium: 150 mM TEA (inlet 1) 30 mM TRIS 250 mM Mannitol pH = 7.29, alternatively pH = 6.70 or 6.40 Individual separation 20 mM TEA + 4 mM TRIS medium 1: (inlet 2) 20 mM HIBA + 4 mM IBA 250 mM Mannitol pH = 6.50, alternatively pH = 6.93 Individual separation 10 mM TEA + 2 mM TRIS medium 2: (inlet 3) 10 mM HIBA + 2 mM IBA 5 mM NaCl 250 mM Mannitol pH = 6.56, alternatively pH = 7.23 Individual separation 10 mM TEA + 2 mM TRIS medium 3: (inlet 4) 10 mM HIBA + 2 mM IBA 250 mM Mannitol adjusted with TEA + TRIS (5:1) to pH = 7.5: Individual separation 10 mM TEA + 2 mM TRIS medium 4: (inlet 5) 10 mM HIBA + 2 mM IBA 250 mM Mannitol adjusted with TEA + TRIS (5:1) to pH = 7.83 Individual separation 10 mM TEA + 2 mM TRIS medium 5: (inlet 6) 10 mM HIBA + 2 mM IBA 250 mM Mannitol adjusted with TEA + TRIS (5:1) to pH = 8.15 Individual separation 10 mM TEA + 2 mM TRIS medium 6: (inlets 7&8) 15 mM HIBA + 3 mM IBA 250 mM Mannitol adjusted with TEA + TRIS (5:1) to pH = 8.48 Cathode border stabi- 200 mM HIBA + 40 mM IBA lizationmedium: (inlet 9) 50 mM Tris 300 mM Ammediol 250 mM Mannitol pH = 8.64 Counterflow medium: 250 mM Mannitol HIBA = Hydroxy-Isobutyric-Acid IBA = Iso-butyric-Acid

(59) The features of the present invention disclosed in the specification, the claims and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof.