WO2020104575A1 - Method and device for separating ions of the same charge polarity in an electric field - Google Patents

Abstract

The invention relates to a dimensionally reduced free-flow electrophoresis method and to a device for separating ions of the same charge polarity, such as proteins or other ionic biomolecules in aqueous solution, in an electric field. In a separation zone (5), at least one target substance (1) in carrier material (3) is retained in the effective region of an electric field (6) as a result of a force equilibrium between flow force (4) and electrical force (7), while at least one other component (2) of a substance mixture migrates on with the flow of the carrier material (3). Differences in the properties of the target substance and of the contaminating substances of the mixture are utilized in order to increase the purity of the target substance and remove contaminants with the aid of widely different ionic interactions. Typical target substances are, in particular but not exclusively, proteins, nucleic acids, metal ions, low-molecular-weight organic ions and ionic polymers. This method advantageously allows a significantly simplified architecture of the separation device, the method according to the invention thus having higher throughput and, in comparison with the prior art, greater scalability, parallelizability and robustness when used in the preparative purifying or processing of target substances in industrial processes and process scales.

Description

 Method and device for separating ions of the same charge polarity in an electric field

description

Technical field

The invention relates to a method and a device for separating ions of the same charge polarity in the electric field. It is particularly applicable for the separation or purification of ions that are in a carrier, for example for proteins or other ionic biomolecules in aqueous solution.

The separation of ions is an integral part of a multitude of processes for the purification or processing of raw materials or products from mixtures of substances in the chemical, biotechnological and pharmaceutical industries. Differences in the properties of the target substance and the contaminating substances in the mixture are used to increase the purity of the target substance and to separate impurities with the aid of various ionic interactions. Typical target substances are in particular but not exclusively proteins, nucleic acids, metal ions, low molecular weight organic ions and ionic polymers.

The extent of the differences in the properties of the target substance and the impurities required for the separation influences the efficiency and the costs of the target substance processing, the smaller the differences, the more inefficient and cost-intensive the processing. A typical example of this is the purification of recombinantly produced technical or pharmaceutical proteins from a mixture of many thousands of other proteins and charged biomolecules such as nucleic acids in a cell lysate.

The separation of ions usually takes place either via their interaction with an ionic stationary phase (eg ion exchange chromatography, zeolitic ion exchangers) or via the interaction of the ions with an external electrical field. The latter are assigned to the class of electrophoresis processes. The use of electrophoretic separation techniques is for preparative purification of an ionic target substance is limited to processes that do not adversely affect the structural and functional integrity of the target substance. Maintaining the ionic target substance in its native form or at least in a form that is structurally and functionally not detrimental to the intended use is therefore essential for the selection and application of methods and devices for the separation of ions of the same charge polarity in the electrical field.

State of the art

A wide variety of methods and devices for carrying them out are known from the prior art, which allow the separation of ions in the electric field. While the separation of oppositely charged ions can already be achieved using the simplest methods and devices, the separation of ions of the same charge polarity is technically more demanding. In the following, therefore, only those methods will be dealt with which are suitable for separating ions of the same charge polarity in the electric field and which at the same time do not adversely affect the structural and functional integrity of the target substance.

Methods are known in which the ions are separated in an electric field in a macroscopically immobile carrier, in particular the methods of gel electrophoresis and paper electrophoresis. Depending on their ion mobility, the ions move at different speeds through a carrier, the particles of which are not subject to any directional movement or a movement specified by the process. A disadvantage of these processes is their low throughput, especially for large, weakly charged ions with low ion mobility. An improvement in resolution can be achieved by using gels, but with a further disadvantageous reduction in throughput, since the ions in the gel, depending on their size, have an even lower ion mobility than without a gel. Furthermore, these methods are only of limited suitability for the preparative separation of ions, since they are difficult to parallelize and can hardly be operated continuously.

The methods of capillary electrophoresis are also known, in which the separation of the ions takes place in an electrical field which is located between the ends a capillary filled with carrier and mixture of substances rests. As a result of the electroosmotic flow occurring here, the carrier moves through the capillary. The ions of the mixture of substances move according to their ion mobility and interaction with the moving carrier. However, the flow rate is very slow, so that in combination with the small cross-sectional area of the capillaries used (inner diameter in the order of 100 gm) the throughput of such processes is disadvantageously too low for the preparative separation of ions. In this sense, the vulnerability of capillaries is further disadvantageous against macroscopic impurities, in particular particles, precipitated proteins or other ions, which can lead to blockage of the capillary.

The methods of free-flow electrophoresis are known, in which the ions of the substance mixture are moved by a continuous directional flow of a carrier. The ions of the mixture of substances are deflected and thus separated according to their ion mobility by an electric field oriented orthogonally to this flow. Methods of free-flow electrophoresis are also known in which, for the purpose of improving the resolution, the flow and the electric field are not completely orthogonal to one another, so that an electrical force acts on each ion in the electric field both parallel to the direction of flow and orthogonally to the direction of flow. The separation of the ions in the electrical field thus takes place in a two-dimensional plane parallel to the plane spanned by the flow vector and the field strength vector, the ions always continuously with the carrier current from the inlet opening to the outlet of the free-flow electrophoresis. Move the device.

The possibility of continuous operation of free-flow electrophoresis processes is advantageous compared to the previous processes. On the other hand, it is disadvantageous that all methods of free-flow electrophoresis depend on a high level of laminarity and homogeneity of the flow profile in order to achieve the required separation performance. Another disadvantage is the high dilution of the separated ions due to the high consumption of carrier fluid in order to maintain a wide separation zone. The high requirements of the free-flow electrophoresis process for laminarity and homogeneity of the flow profile also require strong cooling of the separation zone and a flat structure the separation zone with large cooling surfaces to prevent thermal convection currents. This has a disadvantageous effect on a throughput-increasing parallelization, which would be necessary for scaling current free-flow electrophoresis processes to industrial production scales.

All of the methods and devices known in the prior art for separating ions of the same charge polarity in the electrical field are primarily suitable as analytical technologies, but have serious disadvantages with regard to their use in the preparative purification or processing of target substances in industrial processes and process scales, in particular with regard to throughput, parallelizability as well as technical robustness and susceptibility to errors.

Task

It is therefore an object of the present invention to provide a method by means of which the separation of ions of the same charge polarity in the electrical field can also be used for the preparative purification or processing of target substances in industrial processes and which, in contrast to the prior art, is better scalable, Features parallelizability and robustness against typical interference factors of electrophoretic processes. In particular, a method is to be specified in order to separate a substance mixture of target substance and impurities in the electrical field in such a way that the purity of the target substance in one of the separation products is higher than in the substance mixture before the separation. The object on which the invention is based is achieved by a method according to claim 1 and an apparatus according to claim 9; preferred embodiments result from the subclaims and the description.

Definitions

To ensure the clarity of some of the terms used in the description, these are defined and explained below and in the course of the description. Ions of the same charge polarity in the sense of the invention are all those ions which have the same charge sign. In this respect, all anions are ions of the same charge polarity and all cations are ions of the same charge polarity.

Mixtures of substances in the sense of the invention are mixtures of ionic, ie electrically charged, components. Components in the sense of the invention are themselves either target substance 1 or impurity 2. A substance mixture in the sense of the invention always consists of at least two components. If a substance mixture contains several target substances 1, the target substances 1 mutually count as impurities 2, since a first target substance 1 can be contaminated by a second target substance 1. Substances which are uncharged in the absence of an electric field 6, but which convert into ions under the influence of an electric field 6 through electrical dispersion, polarization and dissociation, are considered to be components of a mixture of substances in the sense of the invention.

Components in the sense of the invention are, in particular, but not exclusively, proteins, nucleic acids, ionic polymers, lipids, cells, organelles, viruses, vesicles, low-molecular ions, elementary ions, metal complexes, charged precipitates, crystals and ores, and ultimately any conceivable form of charged substances, substances , Particles or molecules as well as their accumulations, complexes or other higher organized structures which, if they appear to the outside as a charged unit (eg cells), can themselves represent mixtures internally.

Each component is characterized by physical, chemical and biological properties. These properties span the parameter space according to the invention. Each component represents a point in the parameter space according to its properties. Each property is a dimension of the parameter space. Properties of the components in the sense of the invention are all characteristics of a component which can be described qualitatively or quantitatively, in particular but not exclusively the charge, the size, the shape, the structure, the flexibility, the mass, the mass distribution, the charge distribution, the electron density distribution, the polarizability , the permittivity, the permeability, the absorption and fluorescence characteristics, the hydrophobicity, the dissociability, the structure, size and binding strength of the carrier shell, the ability to bind other molecules or ions and the structure, location, number and binding strength of these binding sites. A large number of the properties of a component change depending on the environmental conditions of the component, so that the position of a component in the parameter space also depends on the properties of the environment of a component. Important environmental properties within the meaning of the invention are, in particular, but not exclusively, the electrical and magnetic field strength, the flow direction and speed, the temperature, the pressure, the pH, the ionic strength, polarity, permittivity, permeability and type of carrier 3, the pH Buffering and additives in the carrier 3, which interact with the components.

The total force 8 in the sense of the invention results as the resulting force from the sum of all forces acting on a component. According to the invention, the total force 8 relevant for the separation of at least two components is in each case the total force 8 acting on a component in the direction of the main flow direction of the carrier 3.

Carriers 3 in the sense of the invention are fluids which consist of one or more dominant molecule types and mostly have a higher concentration of substance than the individual components of the substance mixture. The molecules of the carrier 3 themselves are advantageously no or only weak ions. Often, the carrier 3 also acts as a solvent of the mixture of substances. Important carriers 3 in the sense of the invention are in particular but not exclusively water and organic solvents such as ethanol, hexane, isopropanol, acetone, diethyl ether or else carbon dioxide, nitrogen or argon. Carriers 3 in the sense of the invention can also be solid powders, provided that they have macroscopically fluid-like behavior. An essential feature of the carrier 3 in the sense of the invention is the mediation of the flow force 4. The flow force 4 and its flow force vector 4V always point in the direction of the macroscopic main flow direction of the carrier 3.

The separation zone 5 in the sense of the invention is the effective range of the electric field 6 in the carrier 3 in which components of the substance mixture can still be retained. In the separation zone 5, the components of the substance mixture are separated into a retained fraction, which remains in the separation zone 5 and in an outflowing fraction, which flows through the flowing carrier 3 from the Separation zone 5 is carried out.

Bisection in the sense of the invention is the separation of a quantity into two subsets. In the parameter space according to the invention, bisection takes place by dividing the set of all points of the parameter space into two subsets, which are separated by at least one hyperplane. For the purpose of uniformity and comprehensibility, such a hyperplane is referred to in the description of the invention as a bisection line, regardless of the dimensionality of the parameter space. A bisection line in a one-dimensional parameter space corresponds to a point, in a two-dimensional parameter space a line, in a three-dimensional parameter space a surface, etc. Each bisection line in the sense of the invention is a hyperplane that separates the parameter space into at least two subspaces. Bisection lines in the sense of the invention do not necessarily have to be continuous and connected to one another, the same applies to the partial spaces of the parameter space created by bisection. Bisection lines in the sense of the invention always divide the parameter space into two subsets, one subset of which describes the set of all parameter subspaces in which the total force 8 on a component is greater than zero in the direction of the flow direction of the carrier 3 and of which the other subset the amount describes all parameter subspaces in which the total force 8 on a component is less than or equal to zero in the direction of flow of the carrier 3. In this respect, a bisection line divides the amount of all components in a separation zone 5 into the subset of components in a separation zone 5 the predominant flow force 4 is carried out of the separation zone 5 and into the subset of the components which is retained in the separation zone 5. According to the invention, a bisection line results from the expansion of a parameter space by the dimension of the total force 8 in the direction of the flow direction of the carrier 3 as the set of all points for which this total force 8 is zero.

Iteration in the sense of the invention denotes the multiple execution of the method in a separation stage 10 under different environmental conditions.

Cascading in the sense of the invention denotes the sequential arrangement of a plurality of separation stages 10, a separation taking place in each separation stage 10 under different, but in some cases also the same environmental conditions wherein at least one fraction as a separation product of the previous separation stage 10 is subjected to a further separation in the subsequent separation stage 10 under changed or sometimes the same environmental conditions.

Sensors 11 in the sense of the invention are all devices which are suitable for identifying at least one target substance 1 in the carrier 3 and quantifying them relatively (e.g. as purity in%) or absolutely (e.g. as substance concentration).

solution

According to the invention, the object is achieved by a dimensionally reduced free-flow electrophoresis method, which is characterized in that at least one component of the substance mixture according to the invention in the effective range of the electrical field 6 and / or in the region of force owing to a balance of forces between flow force 4 and electrical force 7 Flow of the carrier is retained, whereas at least one other component of the mixture of substances continues to migrate with the flow of the carrier 3. This method advantageously allows a significantly simplified architecture of the separation device, so that the method according to the invention has a higher scalability, parallelizability and robustness compared to the prior art.

The solution to the problem according to the invention is based on the knowledge that the methods and devices known from the prior art are too complex to meet the industrial requirements for scalability, parallelizability and robustness. Following the principle of "divide and conquer", the method according to the invention is a fundamentally simplified free-flow electrophoresis method in which the separation resolution is reduced to one bisection in order to achieve maximum scalability, parallelizability and robustness of the separation method The total separation resolution required can be achieved by an iterative or cascaded implementation of the method, whereby it is advantageous that, according to the invention, classic search and optimization algorithms can be used to develop a complete work-up process for ionic target substances 1 can be used that allow complete automation of process development according to the invention.

According to the invention, the substance mixture of target substance 1 and impurities 2 is in a flowing carrier 3, so that a flow force 4 acts on target substance 1 and impurities 2, which moves them along their flow vector 4V in the absence of opposing forces.

According to the invention, target substance 1 and impurities 2 in carrier 3 reach at least one separation zone 5, which is located in an electrical field 6, so that an electrical force 7 acts on target substance 1 and impurities 2 according to their charge and other properties along the field strength vector 7V. Flow vector 4V and field strength vector 7V are advantageously arranged antiparallel to one another in at least one point of separation zone 5. According to the invention, a total force 8 acts as a sum of at least the flow force 4 and the electrical force 7 on each component in the separation zone 5.

The amounts of the flow force 4 and the electrical force 7 on a component in the separation zone 5 are particularly but not exclusively dependent on the size, structure, flexibility, charge, charge distribution, hydrate or general carrier shell, the number and distribution of bound ions and other molecules , the hydrophobicity and the thermodynamic and electrochemical environmental parameters (temperature, pressure, pH, total ionic strength, polarity and permittivity of the carrier, etc.) of the component.

The method according to the invention creates a state in at least one point of the separation zone 5 by setting suitable conditions with regard to the flow of the carrier 3 and the strength and shape of the electric field 6, in which the total force 8.2 on at least one impurity 2 and the total force 8.1 on the Target substance 1 are so different that one of the two aforementioned components is retained in the separation zone 5, while the other component moves out of the separation zone 5 and flows off. According to the invention, this leads to a bisection-like separation which detects the purity of the Target substance 1 in the outflowing or in the retained fraction of the separation zone 5 is increased in comparison to the other fraction. In an advantageous embodiment of the invention, the electric field 6 is generated by electrodes 9, which are flowed through as a centrosymmetric hollow body (in particular as a hollow cylinder or torus) or hollow body segments of target substance 1, impurities 2 and carrier 3, so that the electric field 6 the ion current in the separation zone 5 focused and thus increased the selectivity of the bisection. In an advantageous embodiment of the invention, the electric field 6 is generated by electrodes 9 which, in particular as a Faraday cage, are completely or almost completely field-free in at least one point on the inside. In an advantageous embodiment of the invention, the method according to the invention is applied iteratively or cascaded. For this purpose, the fraction which contains at least one target substance 1 is subjected to a further separation according to the invention, which, however, takes place under changed process conditions. This enables the separation of impurities 2 with other properties which influence the separation behavior. In particular, this is continued iteratively and / or cascading until the required purity of at least one target substance 1 is reached. According to the invention, the method according to the invention can be carried out iteratively using the same hardware multiple times as multiple separation stages 10A, 10B and by cascading several separate separation stages 10A, 10B. In an advantageous embodiment of the invention, the separation of ions in the electrical field 6 according to the invention takes place in continuous operation. In an advantageous embodiment of the invention, a plurality of separation stages 10 for carrying out the same separation step (same process parameters) are operated in parallel. On the one hand, this allows the throughput of the overall process to be increased; on the other hand, in iterative or cascaded process applications, a continuous flow can be maintained by suitably interconnecting the separation stages 10. In an advantageous embodiment of the invention, both the retained and the outflowing fraction of a separation zone 5 are monitored with regard to the presence and purity of the target substance 1, in particular, but not exclusively, by optical methods (absorption and fluorescence measurements). In an advantageous embodiment of the invention, the retained fraction is removed from the separation zone 5 regularly or continuously. In some embodiments of the invention, this is done in particular by reversing the polarity of the electrodes 9. In other embodiments, this is done by valves, pumps or other fluid mechanical methods and devices in the area the separation zone 5 or at the exit of the separation stage 10. In some embodiments of the invention, several different target substances 1 are separated from one another. In an advantageous embodiment of the invention, the electrodes 9 are not in direct contact with the separation zone 5, but are convectively separated from the carrier 3 in the separation zone 5 by suitable separators 12 (for example membranes, gels, molecular sieves) in such a way that electrolytically formed gas bubbles do not form can pass through the separation zone 5 while the electric field 6 acts through the separators 12 or is only coupled into the separation zone 5 through them. The separators 12 can be both electrically conductive materials or material mixtures and insulators.

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

Exemplary embodiments and figures

Figure 1, a schematic representation of the inventive method. FIG. 2 shows a schematic representation of the method according to the invention for separating ions of the same charge polarity in the electric field 6 as a bisection in a parameter space.

FIG. 3 shows a schematic representation of the method according to the invention for the separation of ions of the same charge polarity in the electrical field 6 as an iterative bisection in a parameter space with a targeted displacement of the bisection line.

FIG. 4 shows a schematic representation of the method according to the invention for separating ions of the same charge polarity in the electrical field 6 as an iterative bisection in a parameter space with a targeted displacement of the components.

Figure 5 is a schematic representation of the method according to the invention and a device according to the invention for performing the method according to the invention with cascading and sensors.

Figure 6 is a schematic representation of a device according to the invention for performing the method according to the invention with separators, electrode buffers and sensor ports.

Figure 1 shows a schematic representation of the method according to the invention for the separation of ions of the same charge polarity in an electrical field 6. According to the invention, at least one target substance 1 and at least one impurity 2 form the components of the substance mixture to be separated and are located in a flowing carrier 3, so that on the Target substance 1 and the at least one impurity 2 have a flow force 4. From the sum of the flow force 4 and all further forces acting on at least the target substance 1 or on the at least one impurity 2, the total force 8 results, which in each case acts on the target substance 1 or the impurity 2. According to the invention, the total force 8 on each component of the substance mixture according to the invention arises in particular, but not exclusively, from the physical, chemical and biological properties of the respective component, from the properties, the composition and the state of its environment, and from the interactions of the component with its environment. The method according to the invention can be seen in FIG. 1. Outside a separation zone 5, the total force 8 on the target substance 1 and the at least one impurity 2 is dominated by the flow force 4 mediated by the carrier 3, since, according to the invention, all other forces acting on the respective component completely or largely cancel each other out. Accordingly, the components of the mixture of substances according to the invention move outside the separation zone 5 with the flow of the carrier 3 (FIGS. 1, A and C).

According to the invention, the separation zone 5 through which the carrier 3 flows is located in an electrical field 6, the direction 4V of the flow (the flow vector corresponding to the direction of the arrow 4) and the direction 7V advantageously corresponding to the field strength (the field strength vector) in at least one point of the separation zone 5 the direction of arrow 6) are arranged antiparallel to each other. An electrical force 7 acts on each component of the mixture of substances according to the invention within the separation zone 5.

As can be seen from FIG. 1B, the method according to the invention comprises the setting of a state of the flow of carrier 3 and of the electric field 6 in the separation zone 5 such that each component, here the target substance 1 and the impurity 2, of the substance mixture according to the invention an electrical force 7 acts which counteracts the flow force 4 as a function of the physical, chemical and biological properties of the respective component, the properties, the composition and the state of its surroundings, and the interactions of the component with its surroundings, acting on each of the components 1, 2 a differently strong electrical force 7.

As can be seen from FIG. 1B, the state set in the separation zone 5 by means of the method according to the invention separates the target substance 1 from the at least one impurity 2 by setting a component-specific total force 8 such that for one of the aforementioned components (in FIG 1 B is the total force 8.1 for the target substance 1, not shown in the separation zone 5 because zero) the total force 8.1 in the The direction of flow of the carrier 3 is zero or acts counter to the direction of flow of the carrier 3, while the total force 8.2 acts on the other component (total force 8.2 on the impurity 2 in FIG. 1) in the direction of the flow direction of the carrier 3. According to the invention, therefore, only the component with a total force 8 in the direction of the flow direction of the carrier 3 can pass the separation zone 5, whereas the other component is retained in it. According to the invention, this bisection increases either the proportion in the retained fraction (FIG. 1B) or the proportion in the outflowing fraction (see also FIG. 5B) and thus the purity of the target substance 1.

FIG. 2 shows a schematic representation of the method according to the invention for separating ions of the same charge polarity in the electrical field 6 as bisection in a parameter space using three-dimensional diagrams of the total force 8 acting on the individual components of the substance mixture according to the invention as a function of the flow force 4 and electrical force 7 (FIG. 2 A) and depending on the charge and size (Figure 2 B) of the components. The diagrams are shown as schematic illustrations without units and are simplified in order to represent the method according to the invention in particular with regard to its bisection characteristic.

In the simplest case of the method according to the invention, the total force 8 results exclusively as the sum of the flow force 4 and the electrical force 7. This relationship is shown in FIG. 2A, the electrical force 7 which counteracts the flow force 4 according to the invention being plotted with a negative sign. For each combination of flow force 4 and electrical force 7, a total force 8 results. This plane is crossed (dotted) by a bisection line, along which the total force 8 is zero. This corresponds to the limit case shown in FIG. 1B for the target substance 1, in which the flow force 4 and the electrical force 7 cancel each other out, so that no movement can take place out of the separation zone 5. Components of a substance mixture, the total force 8 of which lies above this bisection line, migrate through the separation zone 5, in particular in the flow direction, and are thus separated from the retained fraction in the separation zone 5 (FIGS. 1B and C for impurity 2). Components of a mixture of substances, the total force 8 of which lies below this bisection line, migrate backwards in the separation zone 5 due to the predominant electrical force 7, in particular against the flow direction, until the components reach a point at which the flow force 4 and electrical force 7 cancel each other out, so that they linger at this point. In this respect, the bisection line forms the boundary of the separation zone 5, in which, according to the invention, components are held which, with the total force 8 acting on them, lie below or on the bisection line.

The flow force 4 and electrical force 7 acting on a component of the mixture of substances according to the invention define the separation behavior of the component in the method according to the invention and are each themselves dependent on the physical, chemical and biological properties of the respective component, on the properties, the composition and the state of the component environment and the interactions of the component with its environment. By way of example, FIG. 2B shows the total force 8 on a component of the mixture of substances according to the invention at a specific flow rate of the carrier 3 and a specific field strength of the electrical field 6 as a function of the charge and the size (as a spherical volume) of the component. As the charge increases, the electrical force 7 directed against the flow force 4 on a component also increases with the same size. The flow cross-section of a component and thus the flow force 4 acting on the component also increase with increasing size. In this respect, the method according to the invention allows bisections to be carried out in the parameter space of the properties of the components of the substance mixture according to the invention and thus separates the components on the basis of their properties.

According to the invention, the parameter space in which the bisections of the method according to the invention are carried out includes all properties of the components of the substance mixture according to the invention which influence the total force 8 on a component in the separation zone 5, but in particular the flow force 4 and the electrical force 7. These properties include in particular but not exclusively the charge, the size, the shape, the structure, the flexibility, the mass, the mass distribution, the charge distribution, the electron density distribution, the Polarizability, hydrophobicity, dissociation, the structure, size and binding strength of the carrier shell, the ability to bind other molecules or ions as well as the structure, location, number and binding strength of these binding sites. For a better representation of the method according to the invention, the high-dimensional parameter space in FIGS. 2 to 4 is, however, limited to two exemplary properties of the components.

The method according to the invention comprises the targeted setting of the environmental parameters, in order in particular, but not exclusively, to directly influence the flow force 4 or the electrical force 7 or to set and vary certain properties of the components of the substance mixture. The direct influence of the flow force 4 and the electrical force 7 serves to shift the bisection line in the parameter space of the component properties. The targeted setting of component properties serves to shift the component in the parameter space.

FIG. 3 shows a schematic representation of the method according to the invention for the separation of ions of the same charge polarity in the electrical field 6 as an iterative bisection in a parameter space with a targeted displacement of the bisection line. The simplified parameter space from FIG. 2B is shown with the charge and size of the component, and in FIGS. 3A and B the total force dependent on these parameters 8. For better visualization of the bisection lines, the representations are not three-dimensional, but two-dimensional from a view along the total force. Axis. The total force 8 is shown analogously to FIG. 2, but the viewer's gaze is along the axis of the total force 8, which results in the two-dimensional representation. The exemplary values and the direction of the total force 8 can be found on the grayscale scale and their orientation on the right edge of the picture. The target substance 1 and a first impurity 2A and a second impurity 2B are arranged in the parameter space. The target substance 1, the first impurity 2A and the second impurity 2B differ from one another in terms of their charge and size. Under the conditions set in FIG. 3A with regard to the flow velocity of the carrier 3 and the electric field strength of the electric field 6, the bisection line shown in dotted lines results. The total force 8 on the first impurity 2A is greater than zero, so the first Contamination 2A lies above the dotted bisection line and is flushed out of the separation zone 5, whereas the target substance 1 and a second contamination 2B are retained therein due to the total forces 8 acting on them.

The method according to the invention comprises the targeted adjustment of the component environment in order to influence the total force 8 on each individual component and thus its separation behavior. In FIG. 3B this is done by reducing the electric field strength of the electric field 6 compared to the conditions in FIG. 3A, so that the electrical force 7 acting on each component is reduced and the bisection line (dashed line) is shifted towards the higher charge while the flow of the carrier 3 remains constant. Under the ambient conditions shown in FIG. 3B, only the second impurity 2B is retained in the separation zone 5, whereas, in addition to the first impurity 2A, the target substance 1 now also lies above the dashed bisection line and is consequently flushed out of the separation zone 5 in the direction of flow.

The method according to the invention advantageously allows the targeted separation of the target substance 1 from the impurities 2 of the substance mixture according to the invention by cascading or iteration of several bisections. This is shown in FIG. 3 C on the basis of the bisection lines which are characteristic of the ambient conditions from FIGS. 3 A and B. In the first step of the method, all of the first impurities 2A above the dotted bisection line are separated and the target substance 1 and the second impurities 2B below the dotted bisection line remain in the separation zone 5. In the second step of the method, only all second impurities 2B remain below the dashed bisection line in separation zone 5, whereas target substance 1 and any other impurities present above the dashed bisection line, which were not already rinsed out in the first step, are rinsed out of separation zone 5. Consequently, the method according to the invention separates all components between the two bisection lines from the rest of the mixture of substances by double cascading or iteration. According to the invention, the separation resolution of the method can be increased by higher-level cascading or iteration. FIG. 4 shows a schematic representation of the method according to the invention for the separation of ions of the same charge polarity in the electrical field 6 as an iterative bisection in a parameter space with a targeted displacement of the components. The simplified parameter space from FIG. 2B is shown with the charge and size of components 1, 2A, 2B and the total force 8 dependent on these parameters, the total force here also only implicitly due to the distance of components 1, 2A, 2B from the bisection line results. For better visualization of the bisection lines, the representations are not three-dimensional, but two-dimensional from a view along the total force axis. The target substance 1 and the impurities 2A and 2B, which differ in their charge and size, are arranged in the parameter space. The conditions set in FIGS. 4 A and B with regard to the flow velocity of the carrier 3 and the electric field strength of the electric field 6 and the resulting dotted bisection line are identical to one another and are themselves the same as those in FIG. 3A. In FIG. 4A, the total force 8 on the first impurity 2A is greater than zero, so that the first impurity 2A lies above the dotted bisection line and is flushed out of the separation zone 5. In contrast, the target substance 1 and the second impurity 2B lie below the bisection line due to the total forces 8 acting on them and are consequently retained in the separation zone.

The method according to the invention comprises the targeted setting of the component environment in order to influence the total force 8 on each individual component and thus its separation behavior. In addition to the displacement of the bisection line in the parameter space shown as an example in FIG. 3, the total force 8 on each component can also be set according to the invention by changing the position of the components in the parameter space via changes in the ambient conditions. FIGS. 4A and B show the same parameter space under the same conditions with regard to the flow of the carrier 3 and the electric field 6, but the pH of the carrier 3 in FIG. 4B is changed in comparison to FIG. 4A, so that the components are dependent Their hydronium ion dissociatability in FIG. 4B has a different charge than in FIG. 4A. This leads to a shift of the components with respect to the bisection line, the position of the bisection line now remaining constant (in contrast to the method according to FIG. 3). The shift according to the invention of the position of the components of the mixture of substances as a result of changes in the ambient conditions, illustrated here using the example of the pH in the carrier 3, advantageously allows the inventive method to influence the separation behavior of the components under the same conditions with regard to the flow of the carrier 3 and the strength and shape of the electric field 6, since in FIG. 4B, compared to FIG. 4A, target substance 1 is now located above the bisection line and is thus rinsed out of the separation zone 5.

The properties of the components of the substance mixture and thus the position of the components in the parameter space are dependent on a large number of environmental properties, the type and intensity of the dependency again being specific for each component. Important environmental properties within the meaning of the invention are in particular, but not exclusively, the electrical and magnetic field strength, the temperature, the pressure, the pH, the flow direction and flow velocity, ionic strength, polarity, permittivity, permeability and type of carrier 3 and pH buffering and additives in Carrier 3, which interact with the components.

Analogously to the descriptions for FIG. 3, the cascading or iteration of the method according to the invention with different environmental conditions, which lead to the displacement of at least one component of the substance mixture in the parameter space, also allows an increase in the separation resolution by multiple bisection.

It is clear from FIG. 2, FIG. 3 and FIG. 4 that the method according to the invention can advantageously be implemented as a cascaded or iterative interval nesting method. The purification of the target substance 1 thus resembles the numerical search for a value in a search space, this being equivalent to the parameter space of the method according to the invention. In an advantageous embodiment of the invention, typical numerical search algorithms for the development, regulation and quality control of separation processes can therefore be used, which use methods according to the invention. This allows in particular the advantageous automation of the entire development and process management of an industrial separation process. Figure 5 shows a schematic representation of the method according to the invention based on the explanations for Figure 3 and a device according to the invention for performing the method according to the invention with cascading and sensors. Two separation stages 10A, 10B are shown, each comprising at least one anode 9A and one cathode 9K, between which an electrical voltage is present. The voltage causes at least one electric field 6 in the carrier 3, so that an electric force 7 acts on ions in the effective range of the electric field 6. Mediated by the flowing carrier 3, a flow force 4 acts on the ions. In some configurations of the invention, the electrodes 9 are designed as electrodes 9 (in particular as a ring, torus or hollow cylinder) enclosing the carrier 3. The resulting course of the field lines of the electric field 6 in the area between the electrodes 9 can advantageously be used to focus the ionic components in the center of the flowing carrier 3. In an advantageous embodiment of the invention, the electrodes 9 and the electric field 6 acting between them are aligned with the flow direction of the carrier 3 such that the flow vector 4V and field strength vector 7V are arranged antiparallel to one another in at least one point.

FIG. 5 shows the course of the method for the two-stage separation described in FIG. 3. The first separation stage 10A with a first separation zone 5A (FIG. 5A) is set with regard to its environmental parameters in such a way that the electrical force 7 on target substance 1 and the second impurity 2B is strong enough to retain both of them in the separation zone 5. The electrical force and the flow force on target substance 1 cancel each other out, so that the target substance remains approximately in the rest position. This is represented by a vertical bar RIA as a neutral, first direction. The electrical force and the flow force at the second impurity 2B add up to a total force which acts on the second impurity upstream in the second direction R2A. The second impurity 2B is thus retained. In contrast, the first impurity 2A is flushed out of the separation zone 5. The second separation stage 10B with a second separation zone 5b (FIG. 5B) operates at a lower field strength (for example due to a low voltage between the electrodes 9), which is represented in the second separation stage 10B by a smaller number of field lines. The electrical force and the flow force on the target substance 1 add up to a total force which acts on the target substance 1 downstream in the first direction R1B. As a result, the electrical force 7 on the target substance 1 is not sufficient to retain it in the separation zone 5 of the second separation stage 10B, so that the latter is conveyed out. On the other hand, the electrical force and the flow force cancel each other out at the second impurity 2B, so that the second impurity 2B remains approximately at rest. This is represented by a vertical bar R2B as a neutral, second direction. As a result, the second impurity 2B remains in the second separation zone 5B of the second separation stage 10B.

According to the invention, different environmental conditions are set either iteratively within a separation stage 10 or cascaded by sequential arrangement of a plurality of separation stages 10A, 10B. In an advantageous embodiment of the invention, the plurality of separation stages 10A, 10B are connected to one another in such a way that a continuous flow of the carrier 3 with substance mixture or with purified target substance 1 is obtained at the entrance to the device and at the exit of the device. In an advantageous embodiment of the invention, the architecture of each individual separation stage 10, which is simple in comparison to devices from the prior art, permits good scalability of the method by parallelizing a plurality of separation sections. In an advantageous embodiment of the invention, the method according to the invention comprises determining the concentration of the target substance 1 at various points in the device, in particular, but not exclusively, within the separation zone 5 and at the entrance or exit of the separation stage 10. In particular, non-invasive, especially optical, capacitive are advantageous and inductive measuring methods. For this purpose, in some embodiments, the device according to the invention has one or more sensors 11 per separation stage 10. Alternatively, some embodiments of the invention include sampling ports in the area of the separation zone 5 and at the input or output of the separation stage 10.

In an advantageous embodiment of the invention, the signal of at least one sensor 11 or values derived from this signal are used as input values for the optimization and control algorithms for process control, these algorithms being carried out and processed on a computer as part of the device according to the invention.

In some configurations of the invention, at least one target substance 1 is marked with a tracer in order to be able to detect it by a sensor 11 according to the invention. In the context of the invention, tracers are in particular, but not exclusively, dyes, fluorescent dyes and magnetic or plasmonic nanoparticles.

In some embodiments of the invention, the composition or the pH of the carrier 3 is changed before, in or behind at least one separation zone 5. In such embodiments, the device has corresponding ports, valves, pumps or other fluid mechanical assemblies which are suitable for changing the composition or the pH of the carrier 3 before, in or behind at least one separation zone 5.

According to the invention, the simple architecture of the device, in particular in the embodiment in the form of an arrangement of a plurality of hollow body electrodes 9 with a focusing electric field 6, is significantly less susceptible to contamination or poor flow homogeneity compared to the prior art.

According to the invention, the simple architecture of the device, in particular in the embodiment as an arrangement of a plurality of hollow body electrodes 9 with a focussing electric field 6, is significantly easier to scale than in the prior art by means of parallelization of separation stages 10.

It is also advantageous compared to the prior art that the method according to the invention does not lead to a dilution of the components in the mixture of substances. In addition is the method according to the invention is able to advantageously retain components in the separation zone 5 and thus to concentrate them.

FIG. 6 shows a schematic representation of a device according to the invention for carrying out the method according to the invention with separators 12, electrode buffers 13 and sensor ports 14. A separation stages 10 is shown, each consisting of at least two anodes 9A and two cathodes 9K, between which an electrical voltage is applied. The electrodes 9 are located in an electrode buffer 13, which is separated convectively by separators 12 from the central separation channel comprising the separation zone 5 with target substance 1, impurities 2 and carrier 3. According to the invention, the electrical field 6 propagates between the electrodes 9 through the separators 12 and can thus exert an electrical force 7 on ions in the effective range of the electrical field 6 in the separation channel.

The separators 12 separate the electrode buffer 13 from the flowing carrier 3, so that electrolytic effects such as gas development or pH drift and the electrodes 9 have no influence on the separation zone 5. In some embodiments of the invention, the electrode buffers 13 are also continuously exchanged via the side ports shown , so that efficient cooling and pH buffering in the electrode area are ensured.

The separators 12 are designed in such a way that the electrical field 6 between the electrodes 9 is coupled through them into the central separation channel and at the same time no target substance 1 can get into the electrode buffer 13. In particular, however, this is not only possible through the use of suitable membranes (e.g. PTFE or cellulose membranes), gels (e.g. polyacrylamide or silica gels), molecular sieves (e.g. zeolites) or other porous devices and arrangements whose pore size is so small that no target substance 1 can get through it.

The device shown in FIG. 6 also has sensor ports 14 on both sides of the separation zone 5, so that monitoring of the separation process is possible using suitable sensors 11. FIG. 6 shows an arrangement of the electrodes 9 according to the invention, which is not designed as a hollow body via ring electrodes, but in which the electrical field 6 generated by the electrodes 9 couples laterally via the separators 12 into the separation channel through which the carrier 3 flows, in order to carry out the method according to the invention . The arrangement of the electrodes 9 shown and the contact areas between separators 12, electrode buffers 13 and the carrier 3 results in the formation of an electrical field 6 according to the invention in the separation zone 5 through which the carrier 3 flows, with the flow vector advantageously in at least one point of the separation zone 5 4V and field strength vector 7V are arranged antiparallel to each other. An electrical force 7 thus acts on each component of the substance mixture according to the invention within the separation zone 5.

Reference symbol list

For the respective interpretation of the reference symbols, the description, claims and figures are to be observed. For better clarity of the figures, components of each figure that are to be named the same - sometimes only once - are provided with a reference symbol. The designation of the other components results from their graphic appearance as well as from the description and the claims.

Claims

Expectations 1. A method for separating ions, in particular of the same charge polarity, in an electrical field 6, the ions comprising at least two components, namely at least one target substance 1 and at least one impurity 2, which are located in a flowing carrier 3, so that the target substance 1 and the impurity 2 have a flow force 4, with this at least one target substance 1 and this at least one impurity 2 entering a separation zone 5 which is located in an electrical field 6, so that in the separation zone 5 the target substance 1 and the impurity 2 an electrical force 7 acts, the flow force 4 acting on the target substance 1 and the impurity 2 and electrical force 6 each forming a total force 8, 8.1, 8.2 in the separation zone 5, in particular in the separation zone 5 the force acting on the target substance 1 Total force 8.1 acts on the target substance 1 in a first direction and the total force 8.2 formed on the impurity 2 acts on the impurity 2 in a second direction,  characterized,  that the first direction and the second direction are at least partially different from one another,  and or that the total force 8.2 on the impurity 2 and the total force 8.1 on the target substance 1 are so different that at least one of the components, namely the impurity 2 or the target substance 1, is retained in the separation zone 5, while the other component, namely the Target substance or the impurity 2, moved out of the separation zone 5. 2. The method according to any one of the preceding claims,  characterized,  that a flow direction of the flowing carrier 3 and a field strength direction of the electric field 6 are arranged antiparallel to one another in at least one point of the separation zone 5. 3. The method according to any one of the preceding claims,  characterized,  that the purity of at least one target substance 1 increases either in the outflowing or in the retained fraction of the separation zone 5 compared to the other fraction;  and or  that in the separation zone 5 the amount of ions 1, 2 is divided into at least two fractions, a concentration of the target substance 1 in a first fraction being increased compared to a concentration of the target substance 1 in a second fraction and / or a concentration of the impurity 2 in a second fraction is higher than a concentration of impurity 2 in a first fraction. 4. The method according to any one of the preceding claims,  characterized,  that several separation steps are cascaded,  and or that at least the target substance 1 and at least one second impurity 2B enter a first separation stage 10A with a first separation zone 5A and then into a second separation stage 10B with a second separation zone 5B, the first viewed in each case relative to a flow direction of the flowing carrier 3 Direction RIA in the first separation zone 5A is different from the first direction R1B of the second separation zone 5B and / or wherein the second direction R2A in the first separation zone 5A is different from the second direction R2B of the second separation zone 5B. 5. The method according to any one of the preceding claims,  characterized,  that the total force 8 on each individual component and thus its separation behavior is influenced by specifically setting the ambient conditions in at least one separation zone 5. 6. The method according to any one of the preceding claims,  characterized,  that a strength of the electric field 6 and / or a flow velocity of the carrier 3 is set separately for at least one separation zone 5. 7. The method according to any one of the preceding claims,  characterized,  that a strength of the electric field 6 is set separately for at least one separation zone 5. 8. The method according to any one of the preceding claims,  characterized,  that the regulation and optimization of the method is carried out using interval nesting algorithms. 9. The method according to any one of the preceding claims,  characterized,  that a concentration of at least one target substance 1 in at least one separation zone 5 or at the exit of at least one separation stage 10 is detected by at least one sensor 11. 10. The device, in particular set up, for carrying out the method according to one of the preceding claims,  comprising at least two electrodes 9 of opposite polarity,  characterized, that these electrodes 9 are arranged such that they are in a flowing carrier 3 with at least one target substance 1 and at least one impurity 2 generate an electric field 6 which, via an electric force 7, retains the at least one target substance 1 or the at least one impurity 2 in the effective area of the electric field 6 of these electrodes 9. 11. The device according to the preceding claim,  characterized,  that the electrodes 9 as a hollow body are flowed through by the carrier 3 with at least one target substance 1 and at least one impurity 2.