MICROCHIP FOR FREE FLOW ELECTROPHORESIS

Abstract

The present invention relates to a Micro-Free Flow Electrophoresis chip for analyzing or separating a sample including a pile (1) comprising at least two plates (2A, 2B), a sheet (3) uniformly disposed between the two plates (2A, 2B), clamping means, each sheet (3) comprising at least two inlets (11) for entry and at least one outlet (12) for exit of a first fluid electrode and a second fluid electrode, the first and second fluid electrodes applying an electric field to a separation chamber (31).

Claims

1. A Micro-Free Flow Electrophoresis chip for analyzing or separating a sample including a pile comprising: at least a first plate and a second plate, a sheet uniformly disposed between the first and second plates, a part of said sheet being hollowed out for designing a fluidic circuit, said fluidic circuit comprising at least two inlets, at least one outlet, and a separation zone comprising at least one separation chamber confined by the plates, clamping means, in-between the sheet and the second plate, n stacks composed of a plate and a sheet, n being zero or a positive integer, each sheet comprising at least two inlets for entry and at least one outlet for exit of: a first fluid electrode intended to flow, in each separation chamber, along a first side of the separation chamber, a second fluid electrode intended to flow, in each separation chamber, along a second side of the separation chamber opposite to the first side, a sample intended to flow, in each separation chamber, between the first and second fluid electrodes, said first and second flowing electrodes in each separation chamber being configured to apply an electric field to the separation chamber.

2. The Micro-Free Flow Electrophoresis chip according to claim 1, wherein, for at least one sheet, the separation zone comprises at least two adjacent separation chambers, wherein the fluidic circuit is configured in such a way that the two adjacent separation chambers are delimited by at least one of the first and second fluid electrodes during their flowing in the fluidic circuit.

3. The Micro-Free Flow Electrophoresis chip according to claim 1, wherein means for generating the electric field within the flowing electrodes is located upstream and/or downstream the pile.

4. The Micro-Free Flow Electrophoresis chip according to claim 1, further comprising a carrier intended to flow between the flowing electrodes and the sample in each separation chamber.

5. The Micro-Free Flow Electrophoresis chip according to claim 1, wherein clamping means comprise glue, screws, and/or springs.

6. The Micro-Free Flow Electrophoresis chip according to claim 1, wherein clamping means comprise two clamping plates.

7. The Micro-Free Flow Electrophoresis chip according to claim 6, wherein said clamping plates are made of metal; or polymer, especially resin; or a suitable insulating material.

8. The Micro-Free Flow Electrophoresis chip according to claim 1, further comprising connectors and tubings for bringing in and out the fluidic circuit, the flowing electrodes, the sample, and optionally the carrier.

9. The Micro-Free Flow Electrophoresis chip according to claim 1, further comprising means for cooling the chip, especially means for circulating a coolant fluid in, over, under or at the sidewall of a plate, a sheet or a stack, wherein the means may be a cooling plate integrated in the stack and/or may be cooling means integrated within at least one clamping plate.

10. The Micro-Free Flow Electrophoresis chip according to claim 1, wherein n is 0 or ranges from 1 to 1000.

11. The Micro-Free Flow Electrophoresis chip according to claim 1, wherein the fluidic circuit is free of any membranes.

12. The Micro-Free Flow Electrophoresis chip according to claim 1, wherein the sheet (3) has a micrometric height.

13. The Micro-Free Flow Electrophoresis chip according to claim 1, wherein the sheet is made of one or more polymers or an inorganic material.

14. The Micro-Free Flow Electrophoresis chip according to claim 1, wherein the exit of each separation chamber is configured to receive a pressure ranging from more than atmospheric pressure to 1000 bars.

15. A network of at least two Micro-Free Flow Electrophoresis chips according to claim 1, wherein a single sample is simultaneously provided to at least two micro-free flow electrophoresis chips.

16. The Micro-Free Flow Electrophoresis chip of claim 10, wherein n ranges from 2 to 100.

17. The Micro-Free Flow Electrophoresis chip of claim 12, wherein the micrometric height ranges from 1 to 600 micrometers.

18. The Micro-Free Flow Electrophoresis chip of claim 17, wherein the micrometric height ranges from 10 to 350 micrometers.

19. The Micro-Free Flow Electrophoresis chip of claim 13, wherein the one or more polymers are polyimide, polyethylene, polyethylene terephtalate, polyamide, epoxy or polycarbonate.

20. The Micro-Free Flow Electrophoresis chip of claim 13, wherein the inorganic material is glass or ceramic.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0104] FIG. 1 is an exploded view of an embodiment of the micro-free flow electrophoresis chip of the invention comprising two plates and a sheet showing a fluidic circuit.

[0105] FIG. 2 is an exploded view of an embodiment of the micro-free flow electrophoresis chip of the invention comprising three plates and two sheets showing a fluidic circuit.

[0106] FIG. 3A is an exploded view of an embodiment of the micro-free flow electrophoresis chip of the invention comprising two clamping plates, a gasket, two glass plates, and a sheet with the fluidic circuit.

[0107] FIG. 3B is a section view of the embodiment shown in FIG. 3A.

[0108] FIG. 4A is a view of the embodiment shown in FIG. 3A when the chip is clamped between the two clamping plates and screws as clamping means.

[0109] FIG. 4B is a section view of the embodiment shown in FIG. 4A.

[0110] FIG. 5A is a view of the embodiment shown in FIG. 4A, wherein the upper clamping plate is displayed slightly transparent to see inside.

[0111] FIG. 5B is a top view of the embodiment shown in FIG. 5A.

[0112] FIG. 6 is a top view of a sheet hollowed out to define a fluidic circuit for a micro-FFE chip according to the invention, where the separation zone is configured to comprise multiple adjacent separation chambers, the sheet being shown in a flowing configuration with no carrier.

[0113] FIG. 7 is a view of a sheet identical to that of FIG. 6, the sheet being shown in a flowing configuration with a carrier.

REFERENCES

[0114] 1Pile,

[0115] 2Plate (in one embodiment, this plate is a glass plate),

[0116] 2AUpper plate (in one embodiment, the upper plate is a glass plate),

[0117] 2BBottom plate (in one embodiment, the bottom plate is a glass plate),

[0118] 3Polymer sheet hollowed out and defining a fluidic circuit 10,

[0119] 30Separation zone,

[0120] 31Separation chamber,

[0121] 31AFirst side of the separation chamber 31,

[0122] 31BSecond side of the separation chamber 31,

[0123] 10Fluidic circuit (also called circuit design),

[0124] 11Inlet, also referred to as inner port,

[0125] 12Outlet, also referred to as outer port,

[0126] 13Clamping plate,

[0127] 14Rubber sheet (in one embodiment, the rubber is made of silicone),

[0128] 15Screw,

[0129] 16Holes for connecting tubings,

[0130] 21First fluid electrode,

[0131] 22Second fluid electrode,

[0132] 23Sample,

[0133] 24Carrier.

[0134] FIG. 1 shows a micro-Free-Flow-Electrophoresis chip which is made with two plates, an upper plate 2A and a bottom plate 2B, both made of glass. The plates squeeze a sheet 3 (laser cut 50 m height sheet), so that the sheet 3 is uniformly disposed between upper and bottom plates (2A, 2B). In one embodiment, the sheet 3 is made of Kapton, a polyimide film developed by DuPont. The sheet 3 is bonded to the glass plates 2A, 2B by epoxy glue.

[0135] The sheet 3 is uniformly disposed between the upper plate 2A and the bottom plate 2B, a part of said sheet 3 being hollowed out for designing a fluidic circuit 10. The fluidic circuit 10 comprises inlets 11, outlets 12, and a separation zone 30 comprising a separation chamber 31 confined by the plates 2A, 2B.

[0136] The sheet 3 forms the walls 31A, 31B of the separation chamber 31. The height of the sheet determines the height of the separation chamber 31. The upper glass slide 2A is drilled with a driller (Dremel) to insert connection tubings (not represented). The fluidic circuit 10 shows five inlets 11 (inner ports) and three outlets 12 (outer ports) and a separation chamber 31.

[0137] A first and a second fluid electrodes, made of highly conductive solutions (up to 0.5M KCl, about 30 mS/cm) are introduced and flown in the separation chamber 31, each along one of the walls 31A, 31B.

[0138] The flowing electrodes are pumped with positive or negative pressure from remote containers, relative to the separation chamber, filled with the highly conductive solutions. Flow-scheme can be such as the highly conductive solutions can be recycled.

[0139] Solid electrodes are in direct contact with the highly conductive solutions.

[0140] Solid electrodes are connected to a power supply so that a voltage can be applied and generate an electric field in the separation chamber. One electrode is a cathode and one electrode is an anode. In one embodiment, solid electrodes are external platinum rod-shaped electrodes each embedded within a glass bottle (Schott) filled with the highly conductive solution; one end of the platinum electrode is in direct contact with the solution whether the other end is connected to a power supply. A standard blue cap from a Schott bottle is specifically adapted to fit with the electrode and the tubings. Three holes have been drilled in each cap: one for introducing the electrode, one to adapt the tubing (PTFE) connecting the bottle to the microchip and one connecting the bottle to the pressure controller. The latter applies a pressure onto the liquids in the closed pressured container so that fluids are pumped into the tubing connected to the microchip and ultimately into the separation chamber.

[0141] Adjacent to and in contact with the flowing electrodes, a carrier solution may be injected.

[0142] Containers are connected to the microchip by tubings of any type, e.g. PEEK or PTFE, or any other means suitable to deal with liquid flows. Carrier solution is pumped with positive pressure into the separation chamber 31.

[0143] Eluents can be sorted to waste or collected in various eluent containers according to the end user needs.

[0144] All the liquids are pumped into the microchip with a pressure controller (Fluigent, MFCS) equipped with 350 mbars and 1000 mbars pressure regulators. Flows at inlets are measured by flow-meters (Fluigent) connected to FRCM monitoring unit (Fluigent).

[0145] In one embodiment, downstream to the flowing electrodes outlets 12 are flow restrictors. They are used to adjust and tune counter pressures at the outlets and so the fluidics within the separation chamber 31.

[0146] FIG. 2 shows another embodiment of the invention. In order to increase the productivity, a so-called DUAL chip was built. In this dual version, two separation chambers work in parallel with only one system pumping fluids, one set of electrodes, etc. This dual microchip shows the possibility of numbering-up the device and hence increase the volume of sample treated.

[0147] In this embodiment, the microchip is built as follows from top layer to bottom layer: [0148] a first (upper) glass plate 2A, [0149] a first laser cut hollowed out sheet 3 with the circuit design 10, [0150] a second (intermediary) glass plate 2B, [0151] a second laser cut hollowed out sheet 3 with the circuit design 10, [0152] a third (bottom) glass plate 2C.

[0153] Downstream to the flowing electrodes outlets may be placed flow restrictors. They are used to adjust and tune counter pressures at the outlets and so the fluidics within the chamber.

[0154] FIG. 3A and FIG. 3B show a further embodiment of the invention including cooling means. In this embodiment, the micro-Free-Flow-Electrophoresis chip of the invention is built as follows from top layer to bottom layer: [0155] an upper clamping plate 13, made for example of aluminum and having a first cooling chamber inside and a window configured to watch the fluidic circuit 10, or to watch at least the separation chamber 31, [0156] a gasket 14, ensuring that the cooling chamber of the upper clamping plate 13 and the separation chamber are watertight. The gasket 14 has been hollowed out so as to accommodate the tubing's connecting flexible pipes to the fluidic circuit 10 of the separation chamber 31, [0157] a first glass plate 2A, which has been hollowed out by drilling. This first glass plate 2A forms the top of the separation chamber 31, [0158] a laser cut hollowed out sheet 3 with the circuit design 10, the sheet 3 being for example a polyimide sheet, [0159] a second glass plate 2B, which forms the bottom of the separation chamber 31, [0160] a bottom clamping plate 13, for example made of aluminum, having a second cooling chamber inside and a window configured to see the fluidic circuit 10, or at least the separation chamber 31, and, optionally, a second gasket ensuring that the second cooling chamber is watertight. When the chip includes two cooling chambers, which are preferably equivalent, heat dissipation is the same at each side of the separation chamber.

[0161] Holes 16 were drilled in the clamping plates, the gasket and the glass plate 2A for connecting tubings. The number of holes corresponds to the sum of inlet and outlet ports. Supplementary holes 16 were drilled in the clamping plates for circulating a cooling fluid.

[0162] As seen on FIG. 4A and 4B, clamping is ensured by screws 15 connecting the upper and the bottom aluminum plates 13, thus providing tightness. The gasket (not represented) served as joint and provided an evenly dispatched pressure onto the glass plates (not represented) following pressures applied by screwing the two plates 13. Interfacing is made with M6 and 1/16 connections. The overall sandwich proved to be free of visible leakage. The microchip is easy to be disassembled and reassembled whenever necessary.

[0163] In one embodiment, a modified syringe pump with several syringes was connected downstream the chip. It commanded the fluidics at the outlets and manages in a tidy manner the flows at the outlets. This was simply done by matching the in and out flux from the chamber. The syringe pump was the driver of a steady flow because it forced flows to evenly exit provided each outlet channel is connected to one syringe. Of course, all syringes have the same setting at a time. In other words, it smoothed small differences in fluidic resistance at the outlets. Eventually syringe pump gave a satisfactory result and allows the collection of fractions directly into syringes.

[0164] In one embodiment, the microchip is as described before, except that upper and bottom clamping plates 13 may be holed laterally. Each plate 13 may have two holes drilled to fit connectors (PEEK, GE Healthcare) where flexible pipes are connected. The second difference is that connectors may be made of plastic with ferrules and rings to tighten the PEEK tubings to the upper glass plate 2A of the separation chamber 31. A third difference is that on each plate a small glass plate may be stuck onto the open window necessary to visualize the streams in the chamber. Once assembled there are three closed chambers, from top to bottom: a first chamber with two pipes connected, a second chamber which is the separation chamber and a third chamber with two pipes. The pipes are used to let a coolant circulate, e.g. water. The pipes are connected to a controlled circulating bath filled with the coolant.

[0165] This microchip enables very long run at higher voltage, for instance 1000 V with a good command of Joule effect thus limiting heating. Thermal energy due to the current is greatly limited to ensure stable separation over time. This is also particularly suitable for the separation of proteins, which are usually prone to irreversible denaturation at temperatures higher than physiological ones. The microchip enables to maintain temperature below 25 C. Separation of two forms of a same GFP was successfully performed with this microchip. GFPmut2A206KSTSHis6 shows a main degradation product due to the loss of some amino acids at the C-terminus. The run demonstrated there is a high separation of the degraded GFP from its non-degraded form.

[0166] FIG. 6 shows another embodiment of a sheet 3, made for example of polyimide, which may be used in a micro-FFE chip similar to the one represented in FIG. 3A to FIG. 5B, in replacement for the sheet 3 shown in these figures. In this embodiment, the separation zone 30 of the sheet 3 comprises multiple adjacent separation chambers 31, more specifically eight adjacent separation chambers 31 in the represented example.

[0167] The sheet 3 is hollowed out for designing a fluidic circuit 10 comprising inlets 11, outlets 12, and the separation zone 30 with the eight separation chambers 31 to be confined by the plates 2A, 2B. In this embodiment, for each pair of adjacent separation chambers 31, there is therebetween a shared fluid electrode (cathode or anode) delimiting the two adjacent separation chambers. The electrodes may be switched, thus making it possible to change the elution outlet.

[0168] In the embodiment of the sheet 3 shown in FIG. 6, the external fluid electrodes flow along respective outer walls of the separation zone 30 of the sheet, whereas the shared internal fluid electrodes do not flow along any wall. In this case, at least one side 31A, 31B of each separation chamber 31 is defined by a shared internal electrode.

[0169] In FIG. 6, the sheet 3 is shown in a flowing configuration with no carrier. The fluidic circuit 10 comprises an anode inlet 11(21) for the introduction of the fluid anode 21 in each separation chamber 31, a cathode inlet 11(22) for the introduction of the fluid cathode 22 in each separation chamber 31, and a sample inlet 11(23) for the introduction of the sample 23 in each separation chamber 31. It is understood that the sheet 3 is hollowed out at different depths to create inlet channels 11(21), 11(22) and 11(23) that are superposed without any junction.

[0170] The portion of the sample that has migrated toward the anode 21 flows with the anode through an anode outlet 12(21), whereas the portion of the sample that has migrated toward the cathode 22 flows with the cathode through a cathode outlet 12(22). Here again, it is understood that the sheet 3 is hollowed out at different depths to create outlet channels 12(21), 12(22) that are superposed without any junction.

[0171] According to one embodiment (not shown), it is possible to have one and the same outlet 12, when it is desired to analyze the sample, without any separation.

[0172] FIG. 7 is a view of a sheet identical to that of FIG. 6, where the sheet 3 is shown in a flowing configuration with a carrier 24. In this case, the fluidic circuit 10 comprises an anode inlet 11(21) for the introduction of the fluid anode 21 in each separation chamber 31, a cathode inlet 11(22) for the introduction of the fluid cathode 22 in each separation chamber 31, a sample inlet 11(23) for the introduction of the sample 23 in each separation chamber 31, and a carrier inlet 11(24) for the introduction of the carrier 24 on each side of each separation chamber 31. It is understood that the sheet 3 is hollowed out at different depths to create inlet channels 11(21), 11(22), 11(23), 11(24) that may be superposed without any junction.

[0173] Such a carrier 24 may advantageously be used to increase the degree of purity of a compound of interest. The carrier 24 prevents the molecules of the sample from diffusing into the fluid electrodes. As illustrated in FIG. 7, such a carrier 24 may be injected from a separate inlet which is independent from the inlets for the sample and the fluid electrodes. As a variant, the carrier 24 may be injected together with the sample or the fluid electrodes.

[0174] Advantageously, in the above embodiments, all fluids including the fluid electrodes 21, 22, the sample 23, and optionally the carrier 24, have laminar flow regime. It has been found experimentally that there is no turbulence when the flow is laminar because the flow is in a self-regulation configuration. Then, no physical separation is necessary between adjacent separation chambers 31.

[0175] According to a variant (not shown), the sheet 3 may be hollowed out so as to obtain multiple parallel separation chambers 31 separated by separation walls of the sheet. In this case, each separation chambers 31 may be delimited by two opposite walls defined by the height of the sheet 3 and the fluid electrodes may flow along respective walls.

EXAMPLES

Example 1

[0176] In a first embodiment, a sample of fluorescein sodium salt (Sigma, ref 46860-25G-F) is injected, at 5 L/min, through the inlet 11 while carrier solutions are flown in, alongside the highly conductive solutions, at 100 L/min, to sheath and focus the sample stream.

[0177] The stream remained stable for more than an hour even when the injection flow was varied down to 0.5 L/min or up to 25 L/min.

[0178] In this embodiment, low conductive carrier was HEPES 10 mM, pH 7.5, HPMC 0.2% (w/v), Tween 20 0.1% (w/v).

[0179] In one embodiment, the highly conductive buffer solution was HEPES 10 mM, pH 7.5, HPMC 0.2% (w/v), Tween 20 0.1% (w/v), Methanol 40% (v/v). 0.5 M KCl and Fluorescein sodium salt used as visual marker for monitoring the flowing electrodes fluidics.

[0180] In another embodiment, the highly conductive solution used as flowing electrode is prepared with HEPES 5 mM, pH 7.5, HPMC 0.2% (w/v), Tween 20 0.1% (w/v), Methanol 40% (v/v), 0.5 M KCl, with conductivity 30 mS/cm. Rhodamine B is added to that buffer. Indeed, Rhodamine B, which is neutral at such pH and shall not migrate under voltage, serves for flowing electrodes fluidic stability monitoring over time.

Example 2

[0181] In a second embodiment, the sample is a mixture made with Rhodamine B, Rhodamine 6G and Fluorescein. It was processed into the separation chamber 31 under a 1.5 kV voltage: at pH 7.5, the said chemicals are respectively neutral, monocationic and dianionic. The sample is injected at 15 L/min, focused by low conductive solution, i.e. carrier 5, at both sides injected evenly at 250 L/min with residence time<5 seconds. The molecules deflected according to expectations and a base-line resolution (Rs>1.5) is achieved. Fluidics were stable, no bubbles were matched and the deflection and separation angle between the streams remained constant during the experiment, which lasted for about 5 minutes.

[0182] The low conductive and the highly conductive solutions are as described in Example 1.

Example 3

[0183] In a third embodiment, the sample was a mix of fluorescein coupled to lysine at different molar ratios. The mix was processed into a chip comprising two stacked chambers (n=1) under a 500 V voltage: at pH 7.5, the said chemicals are neutral or negatively charged. The sample is injected at 11 L/min, focused by low conductive solution at both sides injected at 175 L/min. The molecules deflected according to expectations and a base-line resolution (Rs>1.5) is achieved for at least 3 different components. The streams were stable. At the start and for a few dozen seconds there was a gap between the patterns in the two chambers. Over time electric fields in each chamber stabilize and there was no more any gap at visual inspection. The separation was carried for 30 minutes with steady deflections and no bubbles sighted.

[0184] The low conductive and the highly conductive solutions are as described in Example 1.

Example 4

[0185] In a fourth embodiment, to check separation stability in time, the mixture with small chemicals was injected under a 1.5 kV voltage. The highly conductive solution is as previously but with KCl 0.2 M instead of 0.5 M. Low conductive solution are identical to the one used in Example 1. Sample (same as in Example 2) is injected at 10 L/min, and low conductive solution sheathing the sample at 280 L/min measured through flowmeters. Residence time was less than 5 seconds. The 3 molecules of the mixture did separate well with a very stable fluidic over the course of the run. The experiment lasted for more than two hours with an excellent stability with no bubbles sighted.

Example 5

[0186] In a fifth embodiment, the sample is a mixture of Rhodamine 6G and Fluorescein. It was processed into the chamber under a 1.0 kV voltage: at pH 3.6, the said chemicals are respectively monocationic and neutral. The sample is injected at 1.5 L/min, focused by low conductive solution at both sides injected at 20 and 25 L/min. The molecules deflected according to expectations and a base-line resolution (Rs>1.5) is achieved. Low conductive buffer was citrate 10 mM, pH 3.6, HPMC 0.2% (w/v), Tween 20 0.1% (w/v), Ethanol 70% (v/v). High conductive buffer solution was citrate 10 mM, pH 3.6, HPMC 0.2% (w/v), Tween 20 0.1% (w/v), Methanol 40% (v/v), 0.5 M KCl and Rhodamine 110 used as visual marker for monitoring the flowing electrodes fluidics.

Example 6

[0187] In a sixth embodiment, the sample is a mixture containing B-Phycoerythrin and a GFPmut2 with a 6 Histidine tag called GFPmut2His6. The mixture was processed into the chamber under a 750 V voltage. The sample is injected at 3 L/min, focused by low conductive solution at both sides injected at 50-55 L/min with residence time close to 20 seconds. Both the proteins are acidic with expected pI values of 4.5 and 5.5 respectively for B-Phycoerythrin and GFPmut2His6. Both proteins are expected to go to the anode. The molecules deflected according to expectations and a base-line resolution (Rs>1.5) is achieved. It is to be noted the upper stream is related to B-Phycoerythrin, whether GFP which is less acidic is also less deflected. A third stream is visible in between the two main streams: it is possibly a minor form of B-Phycoerythrin. This result is consistent with deflection patterns observed when these proteins are migrated separately. Low conductive buffer was MES 10 mM, pH 6, HPMC 0.2% (w/v), Tween 20 0.1% (w/v). High conductive buffer solution was same with Methanol 40% (v/v), 0.5 M KCl and Fluorescein sodium salt used as visual marker for monitoring the flowing electrodes fluidics.

Example 7

[0188] In a seventh embodiment, an interesting achieved separation was carried out with two GFPs that cannot be separated otherwise to a baseline separation on a reference chromatography column (Q Sepharose Fast Flow, data not shown). Furthermore, the two GFPs, operated at a pH close to their pI (pH 5), tend to stick to the said reference column and can only be eluted with a highly concentrated sodium hydroxyde solution. Low conductive buffer was Acetate 10 mM, pH 5, HPMC 0.2% (w/v), Tween 20 0.1% (w/v). High conductive buffer solution was Acetate 10 mM, pH 5, HPMC 0.2% (w/v), Tween 20 0.1% (w/v), Methanol 40% (v/v), 0.5 M KCl and Fluorescein sodium salt used as visual marker for monitoring the flowing electrodes fluidics. A binary mixture made of GFPmut2 and GFPmut2His6 was processed into the chamber under a 1000V voltage: at pH 5, the GFPs are close to their pI and are neutral or slightly positively charged. The theoretical pI difference between the two GFPs is 0.37. The sample is injected at 3.5 L/min, focused by low conductive solution at both sides injected at 93 L/min. The molecules deflected according to expectations and a separation is achieved.

Example 8

[0189] In an eighth embodiment, the sample is a mixture of Rhodamine 6G, Rhodamine 110 and Fluorescein, all at 0.33 mM concentration. There is no low conductive buffer as carrier, only highly conductive flowing electrodes and sample. The latter was formulated into Hepes 10 mM, pH7.5, HPMC 0.2% (w/v), Tween 20 0.1% (w/v), Ethanol 50% (v/v). It was injected at 37 L/min through two inlets, focused by high conductive solution from the flowing electrodes at both sides adjacent to wall chambers and at the middle of the separation chamber. Flowing electrodes are high conductive buffer solution that was Hepes 10 mM, pH 7.5, HPMC 0.2% (w/v), Tween 20 0.1% (w/v), Methanol 40% (v/v), 0.5 M KCl.

[0190] The sample is used as visual marker for monitoring the fluidics when the voltage is off, since the flowing electrodes have no added visual marker.

[0191] The sample is sheathed by flowing electrodes and there is a double sample injection thus setting two adjacent separation chambers within one unique physical separation zone, as described above. The sample was processed into the device up to a 1.0 kV voltage.

[0192] At pH 7.5, said chemicals are respectively monocationic, neutral and dianionic. The molecules deflected according to expectations and a base-line resolution (Rs>1.5) is completed between Rhodamine 6G and Fluorescein. Providing Rhodamine 110 is nearly neutral there was no deflection.

[0193] Both Rhodamine 6G (Rh6G) and Fluorescein (FL) got separated in the two separation chambers.