DE102008038114B4 - A method for the spatial separation and for the separate detection of cations and anions, which are located in an analyte - Google Patents

Abstract

Method for the spatial separation and separate detection of cations and anions contained in an analyte, comprising the steps of a) introducing an electrolyte with the valves (37, 38) open at the ends of the two branches (11, 12) of the separation channel by actuation a first pump (281) via an open valve (28) to a lower reservoir (26) and the lower branch (22) of the injection channel in an intersection region (2) of the injection channel with a separation channel such that the electrolyte, starting from the crossing region ( 2) through the separation channel in the direction of two separation electrodes (31, 32), which are located on two opposite sides of the separation channel, via reservoirs (35, 36) for waste and the opened valves (37, 38) to the reservoirs for waste ( 35, 36) is conveyed, whereby the entire channels are filled with the electrolyte and residues are discharged via the discharges to the reservoirs (35, 36) for waste, b) Conveying the analyte containing cations and anions by means of a second pump (291) through an open valve (27) to an upper reservoir (25) and the upper branch (21) of the injection channel into the crossing region (2) fills the crossing region (2) of the injection channel with the separation channel, and moreover through the lower branch (22) of the injection channel, the lower reservoir (26) and an opened valve (29), c) closing all valves (27, 28, 29 , 37, 38) and applying an electrical voltage to the two separation electrodes (31, 32), whereby an electric field is formed between the two separation electrodes (31, 32), in which the cations in the direction of the first separation electrode (31) and the anions move towards the second separation electrode (32) so as to spatially separate the cations and anions from one another; and (d) detect the cations by means of a first measurement cell (33) running along the separation channel in the direction of first separation electrode (31) is arranged, and detecting the anions by means of a second measuring cell (32) which is arranged along the separation channel in the direction of the second separation electrode (32).

Description

The invention relates to a method for the spatial separation and for the separate detection of cations and anions, which are located in an analyte.

The invention is in the field of ion analysis, in particular capillary electrophoresis (CE). Conventionally, either cations or anions are determined by capillary electrophoresis. For samples that require determination of both types of ions, two consecutive analyzes are usually performed. These are on the one hand time-consuming and cost-intensive and on the other hand they require twice the amount of analyte. In certain areas, however, there is only a limited amount of analyte available, which among other reasons is one of the reasons for performing the CE in chip format. Due to the high cost and the additional sample requirements, however, this method does not solve the problem of the simultaneous determination of both types of ions.

• - Verfahren, die eine simultane Messung durch die Beeinflussung des elektroosmotischen Flusses (EOF),
• - Verfahren, die durch eine chemische Reaktion die Eigenschaften der Analytionen verändern und
• - Verfahren, die durch rein konstruktive Maßnahmen ohne Beeinflussung der Analyten die simultane Messung ermöglichen.

A. Padarauskas gives in Simultaneous determination of inorganic anions and cations by capillary electrophoresis, Reviews in Analytical Chemistry, Vol. 20, pp. 271-301, 2001 , an overview of procedures that allow the analysis of cations and anions in one step. These procedures can be divided into three groups:

• - methods involving simultaneous measurement by the influence of electroosmotic flow (EOF),
• - Processes that alter the properties of the analyte ions by a chemical reaction and
• - Procedures that allow simultaneous measurement by purely constructive measures without influencing the analytes.

• - Verfahren der Modifizierung bzw. Verstärkung des EOF durch oberflächenaktive Substanzen; siehe C. Johns, W. C. Yang, M. Macka, und P. R. Haddad, Simultaneous separation of anions and cations by capillary electrophoresis with high magnitude, reversed electroosmotic flow, Journal of Chromatography A, Band 1050, S. 217-222, 2004 ],
• - Verfahren zur Modifizierung bzw. Verstärkung des EOF durch pH-Änderung des Leitelektrolyts; siehe J. W. Jorgenson und K. D. Lukacs, Zone electrophoresis in open-tubular glass capillaries, Anal. Chem., Band 53, S. 1298-1302, 1981 oder S. A. Shamsi und N. D. Danielson, Individual and Simultaneous Class Separations of Cationic and Anionic Surfactants Using Capillary Electrophoresis with Indirect Photometrie Detection, Analytic. Chemistry, Band 67, S. 4210-4216, 1995 und
• - Verfahren zur Verstärkung der durch den EOF induzierten Fluidbewegung durch eine zusätzliche Druckdifferenz über dem Trennkanal, wie in der DE 43 15 928 C1 offenbart.

The first group includes methods for influencing electroosmotic flow (EOF), in which both cations and anions are moved into the measuring cell at one end of the capillary by amplification of the EOF. These methods can be subdivided into

• - Process of modification or reinforcement of the EOF by surface-active substances; please refer C. Johns, WC Yang, M. Macka, and PR Haddad, Simultaneous separation of anions and cations by capillary electrophoresis with high magnitude, reversed electroosmotic flow, Journal of Chromatography A, Vol. 1050, pp. 217-222, 2004 ]
• - Method for modifying or enhancing the EOF by changing the pH of the supporting electrolyte; please refer JW Jorgenson and KD Lukacs, Zone electrophoresis in open-tubular glass capillaries, Anal. Chem., Vol. 53, pp. 1298-1302, 1981 or SA Shamsi and ND Danielson, Individual and Simultaneous Class Separations of Cationic and Anionic Surfactants Using Capillary Electrophoresis with Indirect Photometry Detection, Analytic. Chemistry, Vol. 67, p. 4210-4216, 1995 and
• Method for enhancing the fluid movement induced by the EOF by means of an additional pressure difference across the separation channel, as in US Pat DE 43 15 928 C1 disclosed.

All methods in this group have in common that a measurement of the cations and anions can take place in a conventional quartz glass or polymer CE capillary. In this case, the EOF, which causes a movement of the entire liquid column in the capillary in the direction of the cathode due to the generally negative surface charge, so modified (amplified) that this movement is faster than the movement of the anions in the direction of the anode. In this way, first the cations and then the anions can be detected in a measuring cell between injection volume and cathode. Alternatively, reversing the EOF can also be used to study cations and anions in a measuring cell located between the injection volume and the anode.

The different methods for modifying the EOF have considerable limitations in the simultaneous analysis. It is necessary to adapt the supporting electrolyte very precisely to the respective separating task and to add either surface-active substances or means for adjusting the pH. In order to achieve a change in the fluid movement induced by the EOF via a pressure difference applied to the capillary, a high outlay on equipment is likewise necessary. Moreover, the process of increasing the EOF has the disadvantage that it is only suitable for anions that do not exceed a certain electrophoretic mobility, since they otherwise migrate in the opposite direction, even with elevated EOF. The EOF modification by pressure has not gained acceptance due to peak broadening and concomitant loss of resolution and is therefore slowing today A. Padarauskas, Dual-opposed injection capillary electrophoresis for the simultaneous separation of anionic and cationic Compounds, Current Analytical Chem., Vol. 1, pp. 149-156, 2005 , barely used. Moreover, all methods of EOF modification for simultaneous measurement have a lower resolution in their measurement due to the increased migration speed of the cations; this leads to a lower separation efficiency for a given capillary length. Thus, this method is only partially suitable for the simultaneous analysis of anions and cations.

The second group concerns capillary electrophoresis in combination with complexing agents. A change in the chemical properties of the analyte ions allows the simultaneous study of cations and anions by usually positively charged metal ions, in particular of transition elements by suitable complexing agent usually in a separation upstream reaction step or alternatively in the separation channel itself are converted into negative complexes. This ensures that in a sample solution initially containing cations and anions only the original anions and the cations converted into anionic complexes are contained, which can be detected by conventional CE analysis; please refer J. Kobayashi, M. Shirao, and H. Nakazawa, Simultaneous determination of anions and cations in mineral water by capillary electrophoresis with a chelating agent, J. Liquid Chromatography & Related Technologies, Vol. 21, pp. 1445-1456, 1998 and T Söga and GA Ross, Simultaneous determination of inorganic anions, organic acids, amino acids and carbohydrates by capillary electrophoresis, Journal of Chromatography A, Vol. 837, pp. 231-239, 1999 ,

The simultaneous analysis of cations and anions by complex chemistry is particularly useful for the separation of anions in the formation of complexes of additional cationic transition elements. A proof of z. B. Ammonium is not possible. The charge change by complexation of alkali and alkaline earth metals is also after A. Padarauskas, Dual-opposed injection capillary electrophoresis for the simultaneous separation of anionic and cationic compounds, Current Analytical Chemistry, Vol. 1, pp. 149-156, 2005 , usually not sufficient for simultaneous separation. In addition, in this method, the anions are not detected directly, but only on the complexes formed.

The third group concerns so-called Dual Opposite Injection Capillary Electrophoresis (DOI-CE), according to P. Kuban and B. Karlberg, Simultaneous determination of small cations and anions by capillary electrophoresis, Analytical Chemistry, Vol. 70, pp. 360-365, 1998 and A. Padarauskas, V. Olsauskaite and G. Schwedt, Simultaneous separation of inorganic anions and cations by capillary zone electrophoresis, Journal of Chromatography A, Vol. 800, pp. 369-375, 1998 injected at both capillary ends and then detected in the middle of the capillary.

With the DOI-CE, the separation of an analyte is also carried out in a single capillary with only one measuring cell. The analyte is injected at both ends into the separation capillary. By applying an electric field in the separation capillary, cations and anions each traverse the separation channel from opposite directions. The measuring cell is positioned in this method between the two ends of the capillary so that cations and anions are not simultaneously in the measuring cell. For a good measurement it is necessary to cleverly position the measuring cell and to delay the injection at one of the two capillary ends.

However, the DOI-CE requires injection of the analyte at both ends of the separation capillary, causing increased sample consumption. In addition, it must be operated for a high equipment cost. Use like that P. Kuban, PC Hauser and V. Kuban in A flow injection-capillary electrophoresis system with high-voltage contactless conductivity detection for automated dual opposite end injection, Electrophoresis, Volume 25, pp. 35-42, 2004 and P. Kuban, M. Reinhardt, B. Müller, and PC Hauser in On-Site Simultaneous Determination of Anions and Cations in Drainage Water Using a Flow Injection Capillary Electrophoresis System with Contactless Conductivity Detection, J. Environmental Monitoring, Volume 6, p. 169-174, 2004 in the flow injection method required time delay, two flow injection units, wherein one of the two units is a specially designed valve which must be decoupled from the separation capillary such that the injection unit is isolated from the separation voltage of several 10 kV applied to the one separation electrode , In addition, a suitable detector position and the appropriate injection delay must be determined for each separation problem.

In the field of isotachophoresis (ITP), WA Thormann and D. Schumacher proposed in Bidirectional analytical isotachophoresis: Simultaneous determination of anionic and cationic boundaries, Electrophoresis, Vol. 6, pp. 10-18, 1985, a method for bidirectional ITP and electrolyte systems suitable therefor in front.

First measurements with such a method of T. Hirokawa, K. Watanabe, Y. Yokota and Y. Kiso, Bidirectional isotachophoresis: I. Verification of bidirectional isotachophoresis and simultaneous determination of anionic and cationic components, Journal of Chromatography A, Vol. 633, pp. 251-259, 1993 , were performed in conventional capillaries.

An implementation of this method in a microanalytical ITP process on a polymer microchip has been reported by JE Perst, SJ Baldock, PR Fielden, NJ Goddard, and BJT Brown, Bidirectional isotachophoresis on a planar chip with integrated conductivity detection, Analyst, Vol. 127, pp. 1413-1419, 2002 , described. Since the ITP, in contrast to CE, does not work with a continuous buffer system but with two different electrolytes, a conducting electrolyte with high electrophoretic mobility and a low-mobility end electrolyte, the coordination of the electrolytes to the analyte composition to be expected is also here and the filling of the chip with the different solutions more expensive than in a continuous electrolyte system, as used in the CE.

To fill both capillary sides with different electrolytes, Prest et al. make the filling from both ends of the separation capillary in the direction of the crossing region of the separation capillary with the injection channel. This method has the disadvantage that gas bubbles formed at the separation electrodes by electrolysis reactions are introduced into the separation channel and a renewed measurement in this case becomes impossible. In addition, Prest et al. ensure in their procedure that the entire volume of liquid that could be contaminated by sample ions has been exchanged before a new measurement can be started; d. H. Before each measurement, the entire liquid content of the separation channel and the reservoirs or the supply lines of the electrolyte must be exchanged up to the separation electrodes.

A similar procedure is in K. Bächmann, I. Haumann and T. Groh, Simultaneous Determination of Inorganic Cations and Anions in Capillary Zone Electrophoresis (CZE) with Indirect Fluorescence Detection, Fresenius Journal of Analytical Chemistry, Vol. 343, pp. 901-902, 1992 , also for a construction of conventional capillaries, for capillary electrophoresis. Here, an analyte is injected by lifting the sample reservoir in the two capillaries shown. Thereafter, the sample reservoir is exchanged for a reservoir with conducting electrolyte and an electric field is applied in the two separation capillaries. The electric field is generated in this structure by a single voltage source. Detection takes place at the end of both capillaries by indirect fluorescence. A disadvantage of this, however, is the complex structure of two capillaries with two measuring cells, each working with an optical fluorescence method.

This setup led in professional circles (see P. Kuban and B. Karlberg, Simultaneous determination of small cations and anions by capillary electrophoresis, Analytical Chemistry, Vol. 70, pp. 360-365, 1998 ) considers that this method is unsuitable for practical use. This impression is further substantiated by the fact that this process is not mentioned in reviews for the simultaneous separation of cations and anions. Even Haumann leaves it in his own review article Haumann, J. Boden, A. Mainka, and U. Jegle, Simultaneous determination of inorganic anions and cations bycapillary electrophoresis with indirect UV detection, Journal of Chromatography A, Vol. 895, pp. 269-277, 2000 , disregarded.

Furthermore, from the US 2002/0112959 A1 a method for spatial separation and for the separate detection of cations and anions, which are located in an analyte known. It comprises introducing an electrolyte through an injection channel into an intersection region of the injection channel with a separation channel such that the electrolyte is transported from the crossing region through the separation channel in the direction of two separation electrodes, an introduction of the analyte containing cations and anions, in the Injection channel, applying an electrical voltage to the two separation electrodes, whereby an electric field is formed between the two separation electrodes, in which the cations in the direction of the first separation electrode and the anions move in the direction of the second separation electrode, so that a spatial separation of Cation and the anions from each other and a detection of cations by means of a first measuring cell, which is arranged along the separation channel in the direction of the first separation electrode, and detecting the anions by means of a second measuring cell, which along the separation channel in the direction of the second Trennelek is arranged.

Also are from the WO 2004/076497 A2 and JEPrest et al .: Bidirectional isostachophoresis on a planar chip with integrated conductivity detection analyst Vol. 127 (2002), p. 1413-1419 further methods for the spatial separation and separate detection of cations and anions, which are located in an analyte , known.

Based on this, it is the object of the present invention to propose a method for the spatial separation and for the separate detection of cations and anions which are present in an analyte, which do not have the mentioned disadvantages and limitations.

In particular, the method should offer the possibility of simultaneously separating and detecting cations and anions with a simple, continuously operating electrolyte system, without the need for EOF modifiers or complexing agents.

The object is achieved by a method according to claim 1. The subclaims each describe advantageous embodiments of the invention.

The method according to the invention for the spatial separation and for the separate detection of cations and anions which form constituents of an analyte comprises the method steps a) to d).

According to method step a), an electrolyte is initially introduced from an injection channel into an intersection region of the injection channel with a separation channel. The introduction of an electrolyte with open valves, at the ends of the two branches of the separation channel by actuation of a first pump via an open valve to a lower reservoir and the lower branch of the injection channel in an intersection region of the injection channel with a separation channel such that the electrolyte, from the crossing region through the separation channel in the direction of two separation electrodes, which are located on two opposite sides of the separation channel, through reservoirs for waste and the open valves to the reservoirs for waste is conveyed, whereby the entire channels filled with the electrolyte and residues on the Discharges are discharged at the reservoirs for waste, the promotion takes place hydrodynamically.

In this case, the crossing region of the injection channel with the separation channel does not have to be in the middle of the separation capillary. The two ends of the capillary can rather be adapted to the respective separation task with respect to their length. The geometry of the connection point between separation channel and injection channel can be designed differently. In addition to a simple cross geometry, further geometries can be used, in particular a simple T geometry or a so-called double T geometry.

In a particular embodiment, the filling of the two sides of the separation channel is not carried out simultaneously in both directions, but is preferably carried out sequentially.

In the subsequent process step b), the analyte, which simultaneously contains cations and anions, introduced by means of a second pump through an open valve to an upper reservoir and the upper branch of the injection channel in the crossing region of the injection channel with the separation channel, so that he Fills intersection of the injection channel with the separation channel, as well as through the lower branch of the injection channel, the lower reservoir and an open valve. The promotion is preferably carried out by hydrodynamic or electrokinetic injection.

Then, according to method step c), all valves are closed and an electric voltage is applied to the two separation electrodes. As a result, an electric field is formed between the two separation electrodes. In this field, the cations and anions of the analyte move into the opposite parts of the separation channel due to their charge from the injection volume. In the two parts of the separation channel there is a spatial separation of the different ions according to their electrophoretic mobility.

At the two branches of the separation capillary, preferably in the vicinity of their ends, in each case a measuring cell for detecting the ions is attached. Thus, according to method step d), the cations are detected by means of a first measuring cell, which is arranged along the separating channel in the direction of the first separating electrode, and the anions are arranged by means of a second measuring cell, which is arranged along the separating channel in the direction of the second separating electrode.

The detection of cations and anions can be done by different methods. The method of contactless conductivity detection (CCD) is preferred. A measurement by means of further electrochemical and optical methods is also possible.

The contactless conductivity measurement is particularly preferred for the detection of ions, since negative effects such as electrode corrosion, biofouling and electrolysis reactions, which can occur in the direct conductivity measurement, are avoided. Compared to indirect optical methods, the incorporation of optically active substances into the conducting electrolyte can be dispensed with. A marking of the ions to be analyzed with corresponding markers, as in the fluorescence measurement, is eliminated because the conductivity of a property of the ions is measured itself, which is also the basis of the method of electrophoretic separation. In addition, this method is characterized by low costs for the measuring technique compared to optical methods, which gain additional importance when using two measuring cells on a chip.

The main difference between the present bidirectional capillary electrophoresis method and the method of Prest et al. Isotachophoresis lies in the type of filling of the separation capillaries with electrolyte and their rinsing after separation. According to the invention, the electrolyte, for which only one solution for the separation is required here, is conveyed from the crossing region of the separation channel with the injection channel in the direction of the two separation electrodes through the separation channel. This ensures that the resulting electrolysis gases are not introduced into the separation channel. Furthermore, the waste products of the separation are not discharged again counter to the separation direction by the separation channel, but promoted in the separation direction from the separation channel on the separation electrodes out into two separate waste reservoirs. As a result, the amount of electrolyte required for flushing and filling the separation capillary can be reduced. The filling of the two sides of the separation channel takes place simultaneously in both directions or in succession.

The present invention thus offers the possibility of simultaneously separating and detecting cations and anions. In contrast to known processes, the addition of EOF modifiers and complexing agents is dispensed with. Compared to ITP, separation is possible with a simple, continuous, electrolyte system that allows for easy setup of an associated measurement system. Integration on a microfluidic CE chip greatly simplifies the design.

Although the method according to the invention, like the DOI-CE, is based on the different directions of movement of cations and anions in an electric field. In contrast to the DOI-CE, however, the separation from a central, common analyte volume is carried out here. From this volume, cations and anions move in opposite directions according to the principle of electrophoresis in a common separation capillary. At each end of the capillary is a measuring cell for detecting the individual ions, each separated according to their electrophoretic mobilities.

The present method also opens up the possibility of preferably using two independent voltage sources for the separation. The combination with two voltage sources allows for a precise adjustment of the separation voltages for cations on the one hand and anions on the other hand, on the other hand can be achieved so that in the region of the injection cross a potential with a low voltage, in particular in the range below 10 volts. The chip can therefore be easily isolated from the sampling point. Thus, without complex measures for electrical insulation of the analysis, the supply of the analyte and, in the case of sampling directly from a process, the fluidic connection with an external sample reservoir to ground and thus an entry of the separation voltage in an external reservoir or a Prevent process. In addition, the voltage for the separation of the cations and anions can be adjusted separately.

Furthermore, the central sample volume makes it possible to reduce analyte consumption by 50% compared to DOI-CE and, moreover, to save a sampling device, which considerably simplifies the structure. The additional expense of a second measuring cell is low when using the contactless conductivity measurement (CCD) by the cheap electronic components.

The simple chip architecture, each with its own capillary sections and measuring cells for the measurement of cations and anions, enables the flexible use of the chip for different separation tasks without costly retrofitting of the measuring cell and determination of a suitable injection delay. By constructing a measuring cell of microvalves and micropumps, which are detachably connected to the CE chip and serve the fluid delivery both for rinsing, conditioning, filling the chip with electrolyte solution and for injecting the analyte solution, an automated hydrodynamic injection can be realized. Combined with a single process sampling peripheral, Bidirectional CE offers a flexible measurement method for the automated analysis of very small volumes in laboratory and process applications.

• 1 Schematische Darstellung der Spülung, Konditionierung bzw. Befüllung des Trennkanals mit Elektrolytlösung gemäß Verfahrensschritt a)
• 2 Schematische Darstellung der Injektion des Analyts in das Injektionskreuz des CE-Chips gemäß Verfahrensschritt b)
• 3 Schematische Darstellung der elektrokinetischen Injektion des Analyts in das Injektionskreuz des CE-Chips gemäß Verfahrensschritt b)
• 4 Schematische Darstellung der Trennung und des Nachweises der Anionen und Kationen im Analyten gemäß den Verfahrensschritten c) und d)
• 5 Schematische Darstellung der hydrodynamischen Spülung, Konditionierung bzw. Befüllung des Trennkanals mit Elektrolytlösung gemäß Verfahrensschritt a), wobei ein fluidisches System aus Pumpen und Ventilen zur Förderung und Steuerung der Analytlösung zum Einsatz kommt.
• 6 Schematische Darstellung der hydrodynamischen Injektion des Analyts in das Injektionskreuz des CE-Chips gemäß Verfahrensschritt b), wobei ein fluidisches System aus Pumpen und Ventilen zur Förderung und Steuerung der Analytlösung zum Einsatz kommt.
• 7 Schematische Darstellung der Trennung und des Nachweises der Anionen und Kationen des Analyten gemäß den Verfahrensschritten c) und d), wobei ein fluidisches System aus Pumpen und Ventilen eingesetzt wird, um die fluidischen Verbindungen des CE-Chips gegenüber der Umgebung zu schließen.
• 8 Elektropherogramm von gleichzeitig getrennten Anionen und Kationen, wobei die Trennung entsprechend 1 bis 4 in einem zur Umgebung offenen CE-Chip erfolgt.
• 9 Elektropherogramm von gleichzeitig getrennten Anionen und Kationen, wobei die Trennung entsprechend 5 bis 7 in einem zur Umgebung durch ein fluidisches System abgeschlossenen CE-Chip erfolgt.

The invention is explained in more detail below with reference to exemplary embodiments and the figures. The figures show:

• 1 Schematic representation of the rinsing, conditioning or filling of the separation channel with electrolyte solution according to method step a)
• 2 Schematic representation of the injection of the analyte into the injection cross of the CE chip according to method step b)
• 3 Schematic representation of the electrokinetic injection of the analyte into the injection cross of the CE chip according to method step b)
• 4 Schematic representation of the separation and detection of the anions and cations in the analyte according to process steps c) and d)
• 5 Schematic representation of the hydrodynamic purging, conditioning or filling of the separation channel with electrolyte solution according to method step a), wherein a fluidic system of pumps and valves for the promotion and control of the analyte solution is used.
• 6 Schematic representation of the hydrodynamic injection of the analyte in the injection cross of the CE chip according to process step b), wherein a fluidic system of pumps and valves for the promotion and control of the analyte solution is used.
• 7 Schematic representation of the separation and detection of the anions and cations of the analyte according to the process steps c) and d), wherein a fluidic system of pumps and valves is used to close the fluidic connections of the CE chip to the environment.
• 8th Electropherogram of simultaneously separated anions and cations, the separation corresponding 1 to 4 takes place in a CE chip open to the environment.
• 9 Electropherogram of simultaneously separated anions and cations, the separation corresponding 5 to 7 in a sealed to the environment by a fluidic system CE chip.

In 1 is the rinsing, conditioning or the filling of the separation channel with an electrolyte solution according to process step a) shown schematically. For this purpose, an electrolyte from the upper reservoir 25 over the upper branch 21 of the injection channel in the crossing area 2 introduced the injection channel with the separation channel. Following this, the electrolyte is passed through the separation channel in the direction of two reservoirs 35, 36 located at the end of the left branch 11 and at the end of the right branch 12 of the separation channel, carried so long, until the electrolyte fills the entire separation channel and the injection channel.

The promotion of the electrolyte takes place by hydrodynamic injection from the upper reservoir 25 into the separation channel through a suitably adjusted pressure gradient between the reservoir 25 and the reservoirs 26 . 35 . 36 , The pressure difference can be adjusted for example by means of a pump or by controlling the gas pressure over the reservoirs.

If the separation channel is filled with electrolyte, then, as in 2 represented, the analyte, which simultaneously contains anions and cations, according to process step b) from the upper reservoir 25 over the upper branch 21 of the injection channel in the crossing area 2 the injection channel with the separation channel and beyond by the lower branch 22 of the injection channel into the reservoir 26 promoted until the intersection area 2 completely filled with analyte.

In an alternative procedure, as in 3 shown by applying a voltage between the electrodes 23, 24 located in the upper reservoir 25 or in the lower reservoir 26 located, according to process step b) that portion of the analyte containing cations (anions), from the upper reservoir 25 over the upper branch 21 of the injection channel in the crossing area 2 introduced the injection channel with the separation channel and the portion of the analyte containing anions (cations), from the lower reservoir 26 over the lower branch 22 of the injection channel in the crossing area 2 introduced the injection channel with the separation channel.

Subsequently, how 4 shows, according to process step c) an electrical voltage an.die two separation electrodes 31 , 32, which forms an electric field between the two separation electrodes 31, 32. In this example, therefore, the cations move toward the separation electrode 31 and the anions toward the separation electrode 32 so that cations and anions are spatially separated.

The detection of the cations according to process step d) takes place, as also in 4 represented by means of a measuring cell 33, along the left branch 11 of the separation channel in the direction of the separation electrode 31 is arranged while detecting the anions by means of the measuring cell 34 takes place on the right branch 12 of the separation channel in the direction of the separation electrode 32 is arranged. As measuring cells 33 . 34 CCD sensors were used. 5 schematically shows the hydrodynamic flushing, conditioning or filling of the separation channel with electrolyte solution according to process step a), wherein a fluidic system consisting of pumps and valves, for the promotion and control of the analyte solution is used. By operating the pump 281, a conditioning solution or an electrolyte solution with open valves is supplied via a feed 37 . 38 at the ends of the two branches 11 . 12 of the separation channel over the opened valve 28 , the reservoir 26 and the lower branch 22 of the injection channel into the branches 11 . 12 promoted the separation channel. By promoting the solution over the reservoirs 35 . 36 and the open valves 37 . 38 The entire CE chip is filled with solution and any residues on the discharges to the reservoirs 35 . 36 discharged.

If the separation channel is sufficiently filled with electrolyte, then, as in 6 shown, the valves 28 . 37 . 38 closed as well as the pump 281 off. By the pump 291, the analyte containing anions and cations, according to step b) via a supply through the open valve 27 , the reservoir 25 and the upper branch 21 of the injection channel in the crossing area 2 the injection and the separation channel and beyond by the lower branch 22 of the injection channel, the reservoir 26 and the open valve 29 promoted. Excess analyte will be via a drain that is connected to the pump 291 equipped, discharged from the system.

Subsequently, as in 7 represented, according to method step c) all valves 27 . 28 . 29 . 37 . 38 closed and an electrical voltage to the two separation electrodes 31 . 32 created, resulting in between the two separation electrodes 31 , 32 forms an electric field. In the present example, the cations therefore move in the direction of the separation electrode 31 and the anions in the direction of the separation electrode 32 so that cations and anions are spatially separated.

The detection of the cations according to process step d) takes place, as also in 4 represented by means of a measuring cell 33, along the left branch 11 of the separation channel in the direction of the separation electrode 31 is arranged while detecting the anions by means of the measuring cell 34 takes place on the right branch 12 of the separation channel in the direction of the separation electrode 32 is arranged. As measuring cells 33 . 34 In each case CCD sensors were used. Various attempts have been made to separate a mixture of the three anions Cl ^- , SO ₄ ^2- , F ^- and the three cations NH ₄ ⁺ , Na ⁺ , Li ^{+ in} both electrokinetic and hydrodynamic injection. Exemplary results, in which the detection of the ions by means of measuring cells based on the principle of non-contact conductivity measurement (CCD) are in the 8th and 9 shown.

8th shows results of a measurement after electrokinetic analyte injection according to the schematic representation in FIG 3 as well as the separation accordingly 4 , On the left, the output signal is given in mV; below is the time in s at which the designated peaks were measured. The following measurement conditions were available: Electric field strength in the separation channel: 220V / cm Length of the branch of the separation channel for anions: 52 mm Length of the branch of the separation channel for cations: 83 mm Cross sectional area of the separation channel: 50 × 50 μm ² Buffer: 10 mM boric acid, 10 mM TRIS, acetic acid, pH 4.5 Method for detection: contactless conductivity measurement (CCD)

In 9 are shown results of a measurement, which after hydrodynamic injection by means of a fluidic system as shown in FIG 6 in a closed CE chip like in 7 shown was performed. On the left, the output signal is given in mV; below is the time in s at which the designated peaks were measured. The following measurement conditions were available: Electric field strength in the separation channel: 240V / cm Length of the branch of the separation channel for anions: 52 mm Length of the branch of the separation channel for cations: 83 mm Cross sectional area of the separation channel: 100 × 100 μm ² Buffer: 1 mM boric acid, 0.5 mM lei (II) acetate, acetic acid, pH 4.5 Method for detection: contactless conductivity measurement (CCD)

LIST OF REFERENCE NUMBERS

1

Carrier (CE chip)

11

Separating channel, left branch

12

Separating channel, right branch

2

crossing area

21

Injection channel, upper branch

22

Injection channel, lower branch

23

Electrode in the upper reservoir

24

Electrode in the lower reservoir

25

upper reservoir

26

lower reservoir

27

Valve to the upper reservoir

28

Valve to the lower reservoir for electrolyte

281

first pump for controlling the flow through valve 28

29

Valve to the lower reservoir for analyte

291

second pump for controlling the flow through valve 29

31

first (separation) electrode on the separation channel, left branch

32

second (separation) electrode on the separation channel, right branch

33

first measuring cell at the separation channel, left branch

34

second measuring cell at the separation channel, right branch

35

first reservoir for waste at the separation channel, left branch

36

second reservoir for waste at the separation channel, right branch

37

Valve to the reservoir for waste at the separation channel, left branch

38

Valve to the reservoir for waste at the separation channel, right branch

Claims (4)

A method for the spatial separation and for the separate detection of cations and anions, which are located in an analyte, comprising the steps of a) introducing an electrolyte with the valves (37, 38) open at the ends of the two branches (11, 12) of the separation channel Actuation of a first pump (281) via an open valve (28) to a lower reservoir (26) and the lower branch (22) of the injection channel in an intersection region (2) of the injection channel with a separation channel such that the electrolyte, starting from the crossing region (2) through the separation channel in the direction of two separation electrodes (31, 32) located on two opposite sides of the separation channel, via reservoirs (35, 36) for waste and the opened valves (37, 38) to the waste reservoirs (35, 36), whereby the entire channels are filled with the electrolyte and residues are discharged via the discharges to the reservoirs (35, 36) for waste, b) conveying the analyte containing cations and anions by means of a second pump (291) through an open valve (27) to an upper reservoir (25) and the upper branch (21) of the injection channel in the crossing region (2) so that it fills the crossing region (2) of the injection channel with the separation channel, and above out through the lower branch (22) of the injection channel, the lower reservoir (26) and an open valve (29), c) closing all the valves (27, 28, 29, 37, 38) and applying an electrical voltage to the two separation electrodes (31, 32), whereby an electric field forms between the two separating electrodes (31, 32), in which the cations in the direction of the first separating electrode (31) and the anions move in the direction of the second separating electrode (32) that a spatial separation of the cations and the anions from each other, and d) detecting the cations by means of a first measuring cell (33) which is arranged along the separation channel in the direction of the first separation electrode (31), and detecting the anions by means of a second measuring cell (32 ) disposed along the separation channel in the direction of the second separation electrode (32). Method according to Claim 1 , wherein the electrolyte is first transported through the separation channel in the direction of one of the two separation electrodes and only then in the direction of the other of the two separation electrodes. Method according to Claim 1 or 2 , wherein for each of the two separation electrodes, a separate voltage source is used. Method according to one of Claims 1 to 3 , wherein the cations and the anions are each detected with a measuring cell based on contactless conductivity measurement.

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