WO2012175111A1

WO2012175111A1 - Two-dimensional fluid separation with first separation unit feeding to high-pressure end of second separation unit

Info

Publication number: WO2012175111A1
Application number: PCT/EP2011/060263
Authority: WO
Inventors: Klaus Witt
Original assignee: Agilent Technologies, Inc.
Priority date: 2011-06-20
Filing date: 2011-06-20
Publication date: 2012-12-27
Also published as: EP2721403A1

Abstract

A sample separation apparatus (200) for separating a fluidic sample, wherein the sample separation apparatus (200) comprises a first fluid drive (202) configured for conducting the fluidic sample to be separated through a first separation unit (204), a second fluid drive (206) configured for conducting the separated fluidic sample through a second separation unit (208) downstream of the first separation unit (204), a flow coupler (210) having two fluid inlet terminals (212, 214) and a fluid outlet terminal (216), the fluid outlet terminal (216) being fluidically connectable to the second separation unit (208), and a fluidic valve (218) having fluidic interfaces (222, 224, 226, 228) with the first fluid drive (202), with the second fluid drive (206) and with the two fluid inlet terminals (212, 214) of the flow coupler (210), wherein the fluidic valve (218) is switchable for performing the separation of the fluidic sample so that the first fluid drive (202), the second fluid drive (206) and the flow coupler (210) are in fluid communication with one another in each switching state of the fluidic valve (218).

Description

DESCRIPTION

TWO-DIMENSIONAL FLUID SEPARATION WITH FIRST SEPARATION UNIT FEEDING TO HIGH-PRESSURE END OF SECOND SEPARATION

UNIT

BACKGROUND ART

[0001 ] The present invention relates to a sample separation system.

[0002] In liquid chromatography, a fluidic sample and an eluent (liquid mobile phase) may be pumped through conduits and a column in which separation of sample components takes place. The column may comprise a material which is capable of separating different components of the fluidic analyte. Such a packing material, so-called beads which may comprise silica gel, may be filled into a column tube which may be connected to other elements (like a control unit, containers including sample and/or buffers) by conduits. The composition of the mobile phase can be adjusted by composing the mobile phase from different fluidic components with variable contributions.

[0003] Two-dimensional separation of a fluidic sample denotes a separation technique in which a first separation procedure in a first separation unit is performed to separate a fluidic sample into a plurality of fractions, and in which a subsequent second separation procedure in a second separation unit is performed to further separate the plurality of fractions into sub-fractions. Two- dimensional liquid chromatography (2D LC) may combine two liquid chromatography separation techniques.

[0004] However, when performing a 2D LC measurement, operation of two pumps needs to be coordinated, for instance by correspondingly switching fluidic valves. This may conventionally result in pressure ripples or dips acting on separation units and other components of the fluid separation system, thereby deteriorating the chromatographic performance. DISCLOSURE

[0005] It is an object of the invention to provide an efficiently operating two- dimensional sample separation apparatus. The object is solved by the independent claims. Further embodiments are shown by the dependent claims. [0006] According to an exemplary embodiment of the present invention, a sample separation apparatus for separating a fluidic sample is provided, wherein the sample separation apparatus comprises a first fluid drive configured for conducting the fluidic sample (particularly mixed with a mobile phase) to be separated through a first separation unit, a second fluid drive (which may be arranged downstream of the first fluid drive) configured for conducting the separated fluidic sample (particularly mixed with a further mobile phase) through a second separation unit downstream of the first separation unit, a flow coupler (which for instance may be a separate fluidic member or simply a bifurcation of a fluid conduit) having two (or more) fluid inlet terminals and a (i.e. one or more) fluid outlet terminal, the fluid outlet terminal being fluidically connectable (or connected) to the second separation unit, and a fluidic valve having fluidic interfaces with the first fluid drive, with the second fluid drive and with the two fluid inlet terminals of the flow coupler, wherein the fluidic valve is switchable (for instance manually or under control of a control unit such as a processor) for performing the separation of the fluidic sample so that the first fluid drive and the second fluid drive (and preferably the flow coupler) are in fluid communication with one another (particularly for enabling pressure equilibration) in each switching state of the fluidic valve.

[0007] According to another exemplary embodiment of the present invention, a method of separating a fluidic sample is provided, wherein the method comprises conducting the fluidic sample to be separated through a first separation unit by a first fluid drive, conducting the separated fluidic sample through a second separation unit downstream of the first separation unit by a second fluid drive, wherein a flow coupler is provided having two fluid inlet terminals and a fluid outlet terminal, the fluid outlet terminal being fluidically connected to the second separation unit, and switching a fluidic valve, the fluidic valve having (direct, i.e. without any members in between, or indirect, i.e. with at least one member in between) fluidic interfaces with the first fluid drive and with the second fluid drive and with the two fluid inlet terminals of the flow coupler, for performing the separation of the fluidic sample so that the first fluid drive, the second fluid drive and the flow coupler are in fluid communication with one another in each switching state of the fluidic valve.

[0008] According to still another exemplary embodiment of the present invention, a software program or product is provided, preferably stored on a data carrier, for controlling or executing the method having the above mentioned features, when run on a data processing system such as a computer.

[0009] Embodiments of the invention can be partly or entirely embodied or supported by one or more suitable software programs, which can be stored on or otherwise provided by any kind of data carrier, and which might be executed in or by any suitable data processing unit. Software programs or routines can be preferably applied in the context of fluid separation control. The fluid separation control scheme according to an embodiment of the invention can be performed or assisted by a computer program, i.e. by software, or by using one or more special electronic optimization circuits, i.e. in hardware, or in hybrid form, i.e. by means of software components and hardware components.

[0010] In the context of this application, the term "fluidic sample" may particularly denote any liquid and/or gaseous medium, optionally including also solid particles, which is to be analyzed. Such a fluidic sample may comprise a plurality of fractions of molecules or particles which shall be separated, for instance biomolecules such as proteins. Since separation of a fluidic sample into fractions involves a certain separation criterion (such as mass, volume, chemical properties, etc.) according to which a separation is carried out, each separated fraction may be further separated by another separation criterion (such as mass, volume, chemical properties, etc.), thereby splitting up or separating a separate fraction into a plurality of sub-fractions.

[001 1 ] In the context of this application, the term "fraction" may particularly denote such a group of molecules or particles of a fluidic sample which have a certain property (such as mass, volume, chemical properties, etc.) in common according to which the separation has been carried out. However, molecules or particles relating to one fraction can still have some degree of heterogeneity, i.e. can be further separated in accordance with another separation criterion.

[0012] In the context of this application, the term "sub-fractions" may particularly denote individual groups of molecules or particles all relating to a certain fraction which still differ from one another regarding a certain property (such as mass, volume, chemical properties, etc.). Hence, applying another separation criterion for the second separation as compared to the separation criterion for the first separation allows these groups to be further separated from one another by applying the other separation criterion, thereby obtaining the further separated sub-fractions.

[0013] In the context of this application, the term "downstream" may particularly denote that a fluidic member located downstream compared to another fluidic member will only be brought in interaction with a fluidic sample after interaction with the other fluidic member (hence being arranged upstream). Therefore, the terms "downstream" and "upstream" relate to a flowing direction of the fluidic sample.

[0014] In the context of this application, the term "sample separation apparatus" may particularly denote any apparatus which is capable of separating different fractions of a fluidic sample by applying a certain separation technique. Particularly, two separation units may be provided in such a sample separation apparatus when being configured for a two- dimensional separation. This means that the sample is first separated in accordance with a first separation criterion, and is subsequently separated in accordance with a second, different, separation criterion. [0015] The term "separation unit" may particularly denote a fluidic member through which a fluidic sample is transferred and which is configured so that, upon conducting the fluidic sample through the separation unit, the fluidic sample will be separated into different groups of molecules or particles (called fractions or sub-fractions, respectively). An example for a separation unit is a liquid chromatography column which is capable of trapping and selectively releasing different fractions of the fluidic sample.

[0016] In the context of this application, the term "fluid drive" may particularly denote any kind of pump which is configured for conducting a mobile phase and/or a fluidic sample along a fluidic path. A corresponding liquid supply system may be configured for metering two or more liquids in controlled proportions and for supplying a resultant mixture as a mobile phase. It is possible to provide a plurality of solvent supply lines, each fluidically connected with a respective reservoir containing a respective liquid, a proportioning valve interposed between the solvent supply lines and the inlet of the fluid drive, the proportioning valve configured for modulating solvent composition by sequentially coupling selected ones of the solvent supply lines with the inlet of the fluid drive, wherein the fluid drive is configured for taking in liquids from the selected solvent supply lines and for supplying a mixture of the liquids at its outlet. More particularly, the first fluid drive can be configured to conduct the fluidic sample, usually mixed with a mobile phase (solvent composition), through the first separation unit, whereas the second fluid drive can be configured for conducting the fluidic sample, usually mixed with a further mobile phase (solvent composition), after treatment by the first separation unit through the second separation unit.

[0017] In the context of this application, the term "flow coupler" may particularly denote a fluidic component which is capable of unifying flow components from two fluid inlet terminals into one common fluid outlet terminal. For example, a bifurcated flow path may be provided in which two streams of fluids flow towards a bifurcation point are unified to flow together through the fluid outlet terminal. At a bifurcation point where the fluid inlet terminals and the fluid outlet terminal are fluidically connected, fluid may flow from any source terminal to any destination terminal depending on actual pressure conditions, thereby allowing for some sort of equilibration. The flow coupler may act as a flow combiner for combining flow streams from the two fluid inlet terminals further flowing to the fluid outlet terminal. The flow coupler may provide for a permanent (or for a selective) fluid communication between the respective fluid terminals and connected conduits, thereby allowing for a pressure equilibration between these conduits. In certain embodiments, the flow coupler may also act as a flow splitter. [0018] In the context of this application, the term "fluidic valve" may particularly denote a fluidic component which has fluidic interfaces, wherein upon switching the fluidic valve selective ones of the fluidic interfaces may be selectively coupled to one another so as to allow fluid to flow along a corresponding fluidic path, or may be decoupled from one another, thereby disabling fluid communication.

[0019] In the context of this application, the terms "fluid inlet terminals" and "fluid outlet terminal" may particularly indicate that in a general flowing direction of fluid through the device, the fluid will be conducted via at least one of the fluid inlet terminals towards the flow coupler and from there towards the fluid outlet terminal. However, this terminology does not exclude (at least temporarily) other flow directions, for instance a fluid flow from one of the fluid inlet terminals into the other one via the flow combiner, for instance for pressure equilibration purpose. In a similar way, this terminology does also not exclude that, in a certain operation mode, there may also be temporarily a backflowfrom the fluid outlet terminal to at least one of the fluid inlet terminals.

[0020] According to an exemplary embodiment of the invention, a sample separation apparatus with a fluidic path is provided which advantageously allows to perform two-dimensional fluid separation using two different separation units and at the same time suppressing undesired pressure ripples or pressure drops which may conventionally occur when a necessary fluidic valve is switched. According to an exemplary embodiment of the invention, this basically artifact-free operation scheme is enabled by ensuring that fluid communication between the two fluid drives will be always maintained, regardless which switching state the fluidic valve presently assumes. In other words, in each static switching position (i.e. in each position of valve members taken at the beginning or at the end of a switching process) of the fluidic valve, fluid communication is enabled between the fluid drives via the fluidic valves. By ensuring such a fluid-related short circuit between the fluid drive units, it can be prevented that certain fluid conduits or fluidic members (such as separation units like chromatographic columns) experience a sudden pressure drop or pressure increase when switching the valve between a switching position in which the fluid drive units are fluidically decoupled from one another and another switching position in which the fluid drive units are fluidically coupled with one another. Therefore, by the fluidic coupling scheme of embodiments of the present invention, the chromatography separation performance may be significantly improved and the lifetime of the fluidic members of the sample separation system may be increased.

[0021 ] In the following, further exemplary embodiments of the sample separation system will be explained. However, these embodiments also apply to the method, and the software program or product.

[0022] When the sample separation system is a liquid chromatography system such as a HPLC, the first separation unit may be a liquid chromatography column.

[0023] In an embodiment, the first separation unit is arranged between (particularly downstream of) the first fluid drive and (particularly upstream of) the corresponding fluidic interface of the fluidic valve. Therefore, the first fluid drive may be fluidically coupled to its assigned fluidic interface of the fluidic valve indirectly via the first fluid separation unit. Hence, the first fluid drive may be operative to conduct the fluidic sample through the first separation unit. Before the separation by the first separation unit, the first fluid drive may add a mobile phase (i.e. a solvent composition which may be varied over time by the first fluid drive and an assigned proportioning valve) to the fluidic sample. For example, it is possible that the first fluid drive varies a solvent composition over time so as to carry out a gradient run in the first separation unit. Thereby, the fluidic sample may be separated into multiple fluidic components at an outlet of the first separation unit by applying a principle known to a person skilled in the art of liquid chromatography.

[0024] In an embodiment, the second separation unit for further separating the fluidic sample after treatment (usually separation) by the first separation unit may be arranged downstream of the first separation unit and downstream of the fluidic valve so as to further separate the already separated fractions of the fluidic sample into sub-fractions. For this purpose, it may be advantageous that the second separation unit operates in accordance with another separation technique or even separation criterion as compared to the first separation unit. [0025] In an embodiment, the second separation unit is arranged in the fluid outlet terminal of the flow coupler. Therefore, the fluidic sample separated or treated by the first separating unit as well as a solvent provided by the second fluid drive may be mixed at the bifurcation point of the flow coupler and may together be coupled into the second separation unit. [0026] In an embodiment, the flow coupler is configured as a fluidic T-piece, a fluidic Y-piece, or a fluidic X-piece, In case of a fluidic T piece and a fluidic Y piece, two flow streams are combined at one bifurcation point into a single outlet path. In the case of a fluidic X piece, there may be one further fluid conduit. This further fluid conduit can be a second fluid outlet conduit or a third fluid inlet conduit. Other kinds of flow couplers are possible as well.

[0027] In an embodiment, the fluidic valve comprises a first valve member and a second valve member being movable, particularly being rotatable, relative to one another to thereby adjust different operation modes of the sample separation apparatus. Particularly, when such a fluidic valve is configured as a rotary valve, it may be constituted by a stator and a rotor both having fluid conduits. By rotating the rotor relative to the stator, a desired operation mode may be adjusted. Such a valve may be configured as a shear valve which comprises a first shear valve member as a stator, and a second shear valve member as a rotor. By rotating the second shear valve member, the first and second shear valve member can be moved with respect to each other. The first shear valve member comprises a plurality of ports. Afluid conduit such as a capillary, e.g. a glass or metal capillary, can be coupled to each port respectively. [0028] In an embodiment, the fluidic valve is configured to be switchable to a first operation mode in which the fluidic interface fluidically coupled to the first fluid drive is in fluid communication via the fluidic valve with the fluidic interface fluidically coupled to one of the fluid inlet terminals, and in which the fluidic interface fluidically coupled to the second fluid drive is in fluid communication via the fluidic valve with the fluidic interface fluidically coupled to the other one of the fluid inlet terminals. Thus, in the first operation mode, it is always ensured that the two fluid drives are in fluid communication so that a pressure equilibration continuously remains enabled.

[0029] It is also possible that the fluidic valve is configured to be switchable, starting from the first operation mode, to a second operation mode in which the fluidic interface fluidically coupled to the first fluid drive is in fluid communication via the fluidic valve with the fluidic interface fluidically coupled to the other one of the fluid inlet terminals, and in which the fluidic interface fluidically coupled to the second fluid drive is in fluid communication via the fluidic valve with the fluidic interface fluidically coupled to the one of the fluid inlet terminals. Since also in the second operation mode fluid communication between the two fluid drives remains enabled, pressure drops or ripples are also suppressed in this state. Only during the extremely short time interval for switching the switching valve between the first and the second operation mode

(for instance several milliseconds), the two fluid drives may be fluidically decoupled from one another. However, since this switching time may be as short as 20 ms or even shorter, this will not have a noteworthy impact on the continuous pressure characteristics.

[0030] In an embodiment, the first valve member comprises one or more ports forming the flu id ic interfaces, and the second valve member comprise one or more grooves for fluidically coupling different flu id ic interfaces depending on a switching state of the fluidic valve. Thus, a fluid flow may be enabled between an inlet port, a certain one of the grooves and an outlet port. By rotating the grooves along the arrangement of the ports, different fluid communication and paths can be adjusted, while disabling flow along other paths. [0031 ] In an embodiment, at least one of the first fluid drive and the second fluid drive is a binary fluid pump. The term "binary fluid pump" may particularly relate to a configuration in which the fluid pump pumps a corresponding mobile phase with a composition of two components. For example, when such a solvent composition is used for a chromatography gradient run, the ratio between water as a first solvent and acetonitrile (ACN) as a second solvent may be adjusted so as to trap and plate a release individual fraction on a chromatography column. However, other pumps such as a quaternary pump may be used as well.

[0032] In an embodiment, the fluidic valve is switchable so that pressure conditions in the first separation unit and in the second separation unit remain basically constant upon switching. This may significantly improve the performance of the separation, particularly of the chromatographic separation. The arrangement of the fluidic interfaces of the fluidic valve in relation to the fluid drives and the separation units may allow to achieve these conditions. Without pressure ripples, there will also be no artifacts and no deteriorating impact on the fluid separating material in the separating units.

[0033] In an embodiment, the sample separation apparatus comprises a detector for detecting the separated fluidic sample and being arranged in the fluid outlet terminal downstream of the second separation unit. Thus, a detector for detecting the individual fractions and sub-fractions may be arranged downstream of the second separating unit. Such a detector may operate on the basis of an electromagnetic radiation detection principle. For example, an electromagnetic radiation source may be provided which irradiates the sample passing through a flow cell with primary electromagnetic radiation (such as optical light or ultraviolet light). In response to this irradiation with primary electromagnetic radiation, there will be an interaction of this electromagnetic radiation with the fluidic sample so that resulting secondary electromagnetic radiation may be detected being indicative of the concentration and kind of fluidic fractions.

[0034] In an embodiment, the sample separation apparatus comprises a sample injector for injecting the fluidic sample into a mobile phase and being arranged between the first fluid drive and the first separation unit. In such a sample injector, an injection needle may suck a metered amount of fluidic sample into a connected loop. After driving and inserting such an injection needle in a corresponding seat and upon switching a fluid injection valve, the fluidic sample may be injected into the path between first fluid drive and first separating unit. Upon such a switching operation, a mobile phase transported by the fluid drive and constituted by a solvent composition may be mixed with the fluidic sample.

[0035] In an embodiment, the first fluid drive is operable with a first flow rate (pumped fluid volume per time interval) being smaller than a second flow rate (pumped fluid volume per time interval) according to which the second fluid drive is operable. Due to the two-dimensional separation procedure, the amount of solvent per time interval pumped by the first fluid drive may be significantly smaller than another solvent composition pumped by the second fluid drive. Also a pressure (for instance a pressure value in a range between 50 bar and 400 bar, e.g. 200 bar) applied by the first fluid drive may be smaller than a pressure (for instance a pressure value in a range between 500 bar and 1500 bar, e.g. 800 bar) applied by the second fluid drive. [0036] In an embodiment, the second flow rate is at least five times, particularly is at least ten times, more particularly is at least fifty times, of the first flow rate. For example, a flow rate of the second fluid drive may be in a range between about 1 ml/min and about 10 ml/min, whereas a flow rate of the first fluid drive may be in a range between about 10 ml/min and about 500 μΙ/min.

[0037] In an embodiment, the sample separation apparatus comprises a control device configured for controlling the first separation unit to execute a primary separation sequence within a measurement time interval for separating the fluidic sample into a plurality of fractions, and controlling the second separation unit to execute a plurality of secondary separation sequences within the measurement time interval for further separating at least a part of the separated plurality of fractions into a plurality of sub-fractions. In the context of this application, the term "primary separation sequence" may particularly denote a procedure according to which a fluidic sample is to be separated in the first separation unit. This may include a plurality of steps to be carried out subsequently. The execution of these steps occurs over a so-called measurement time interval. In a preferred embodiment, the primary separation sequence is a gradient run by which the fluidic sample is separated in the first separation unit by changing a ratio of two solvents gradually, thereby selectively trapping and later releasing individual fractions of the fluidic sample on the first separation unit. In the context of this application, the term "plurality of second separation sequences" may particularly denote sequences having a similar or the same characteristic as the first sequence but which are to be executed by the second separation unit. Furthermore, each of the second separation sequences is executed over a time interval being smaller than the measurement time interval relating to the primary separation sequence. In other words, several or many secondary separation sequences may be carried out within a time interval of the primary separation sequence. This means that the fluidic sample is split or separated into the various fractions during execution of the primary separation sequence, whereas the secondary separation sequences chop the separated fractions into further subsections by applying another, at least partially different separation criterion. For instance, a number of secondary separation sequences relating to one primary separation sequence may be in a range between 5 and 1000, particularly between 10 and 100. In the context of this application, the term "measurement interval" may particularly denote a time interval required for executing the primary separation sequence. Such a time interval may be in a range between 1 min and 5 h, particularly between 5 min and 1 h. It may relate to the time required for executing a gradient run on a first separation unit configured as a liquid chromatography column. In accordance with the long-lasting primary separation sequence, the sample can be separated into a plurality of fractions by a first separation criteria (for instance the mass). In the subsequent, at least partially orthogonal secondary separation sequences, each fraction separated during the primary separation sequence can be further separated into a plurality of sub-fractions (particularly in accordance with another separating criterion such as volume of the particles). The result of such a separation can be displayed in a two-dimensional coordinate system, wherein the first separating criterion may be plotted along an abscissa and the second separating criterion may be plotted along an ordinate, or vice versa. In an embodiment, at least one of the primary separation sequence and the plurality of secondary separation sequences relates to a chromatographic gradient run.

[0038] In an embodiment, the first separation unit and the second separation unit are configured so as to execute the respective sample separation in accordance with different separation criteria, particularly in accordance with at least partially but not completely orthogonal separation criteria. In this context, the term "orthogonal" may particularly denote the conventional but not very accurate understanding that two different separation criteria in a two- dimensional liquid chromatography system relate to completely decoupled parameters. This is not the case in practice, since for instance a separation with regard to mass and a separation with regard to volume of particles such as molecules are not completely decoupled. Exemplary embodiments of the invention make benefit of this cognition and propose to adjust the parameters under a consideration of the fact that the separation criteria of the two separation units are not completely independently from one another.

[0039] In an embodiment, the first separation unit and the second separation unit are configured so as to execute the respective sample separation on identical separation media but with different operating conditions. Such operating conditions may be different solvents, different steepness of elution gradients, different column temperatures, and/or different pressures, so that the separation criteria are partially but not completely orthogonal. However, not or not only the separation units may relate to non-completely orthogonal separation, but additionally or alternatively it is possible that the partial orthogonality is achieved by using a similar or even the same separation technique, but by adjusting the apparatus properties so that a partial orthogonality is obtained. For example, it is possible to use twice the same separation column, but to operate it at different temperature and/or with different solvents or solvent compositions.

[0040] In an embodiment, the first separation unit and/or the second separation unit may be configured for performing a separation in accordance with liquid chromatography, supercritical-fluid chromatography, capillary electrochromatography, electrophoresis and gas chromatography. However, alternative separating technologies may be applied as well.

[0041 ] In an embodiment, the sample separation apparatus is configured as a two-dimensional liquid chromatography sample separation apparatus, particularly being a comprehensive two-dimensional liquid chromatography apparatus.

[0042] The first and/or second separation unit may be filled with a separating material. Such a separating material which may also be denoted as a stationary phase may be any material which allows an adjustable degree of interaction with a sample so as to be capable of separating different components of such a sample. The separating material may be a liquid chromatography column filling material or packing material comprising at least one of the group consisting of polystyrene, zeolite, polyvinylalcohol, polytetrafluorethylene, glass, polymeric powder, silicon dioxide, and silica gel, or any of above with chemically modified (coated, capped etc) surface. However, any packing material can be used which has material properties allowing an analyte passing through this material to be separated into different components, for instance due to different kinds of interactions or affinities between the packing material and fractions of the analyte. [0043] At least a part of the first and/or second separation unit may be filled with a fluid separating material, wherein the fluid separating material may comprise beads having a size in the range of essentially 0.1 μιτι to essentially 50 μιτι. Thus, these beads may be small particles which may be filled inside the separation section of the microfluidic device. The beads may have pores having a size in the range of essentially 0.01 μιτι to essentially 0.2 μιτι. The fluidic sample may be passed through the pores, wherein an interaction may occur between the fluidic sample and the surface of the pores.

[0044] The sample separation apparatus may be configured as a fluid separation system for separating components of the sample. When a mobile phase including a fluidic sample passes through the fluidic device, for instance by applying a high pressure, the interaction between a filling of the column and the fluidic sample may allow for separating different components of the sample, as performed in a liquid chromatography device.

[0045] However, the sample separation apparatus may also be configured as a fluid purification system for purifying the fluidic sample. By spatially separating different fractions of the fluidic sample, a multi-component sample may be purified, for instance a protein solution. When a protein solution has been prepared in a biochemical lab, it may still comprise a plurality of components. If, for instance, only a single protein of this multi-component liquid is of interest, the sample may be forced to pass the columns. Due to the different interaction of the different protein fractions with the filling of the column (for instance using a gel electrophoresis device or a liquid chromatography device), the different samples may be distinguished, and one sample or band of material may be selectively isolated as a purified sample. [0046] The sample separation apparatus may be implemented in different technical environments, like a sensor device, a test device, a device for chemical, biological and/or pharmaceutical analysis, a capillary electrophoresis device, a capillary electrochromatography device, a liquid chromatography device, a gas chromatography device, an electronic measurement device, or a mass spectroscopy device. Particularly, the fluidic device may be a High Performance Liquid device (HPLC) device by which different fractions of an analyte may be separated, examined and/or analyzed.

[0047] The sample separation unit element may be a chromatographic column for separating components of the fluidic sample. Therefore, exemplary embodiments may be particularly implemented in the context of a liquid chromatography apparatus.

[0048] The sample separation apparatus may be configured to conduct the mobile phase through the system with a high pressure, particularly of at least 600 bar, more particularly of at least 1200 bar. [0049] The sample separation apparatus may be configured as a microfluidic device. The term "microfluidic device" may particularly denote a fluidic device as described herein which allows to convey fluid through microchannels having a dimension in the order of magnitude of less than 500 μιτι, particularly less than 200 μιτι, more particularly less than 100 μιτι or less than 50 μιτι or less. The sample separation apparatus may also be configured as a nanofluidic device. The term "nanofluidic device" may particularly denote a fluidic device as described herein which allows to convey fluid through nanochannels having even smaller dimensions than the microchannels.

BRIEF DESCRIPTION OF DRAWINGS [0050] Other objects and many of the attendant advantages of embodiments of the present invention will be readily appreciated and become better understood by reference to the following more detailed description of embodiments in connection with the accompanied drawings. Features that are substantially or functionally equal or similar will be referred to by the same reference signs.

[0051 ] Fig. 1 illustrates a liquid chromatography system according to an exemplary embodiment.

[0052] Fig. 2 illustrates a sample separation apparatus according to an exemplary embodiment.

[0053] Fig. 3 illustrates a diagram showing a part of a secondary separation sequence performed with a second dimension separation column and also indicates the time of a switching of the fluidic valve.

[0054] Fig. 4 illustrates a primary separation sequence according to which the first dimension chromatographic column is operated.

[0055] Fig. 5 illustrates another diagram showing multiple secondary separation sequences as performed by a second dimension liquid chromatography column.

[0056] Fig . 6 schematically shows a pressure characteristic as obtained by a sample separation apparatus according to an exemplary embodiment as compared to a conventional sample separation apparatus during carrying out a two-dimensional liquid chromatography experiment.

[0057] Fig. 7 illustrates the sample separation apparatus according to Fig. 2 and shows schematically the different operation modes corresponding to the different valve positions described referring to Fig. 2.

[0058] Fig. 8 illustrates a diagram which shows the result of a two- dimensional liquid chromatography apparatus experiment. [0059] The illustration in the drawing is schematically.

[0060] In an embodiment of a two-dimensional separation technique, a first dimension floats on a resulting pressure level of a second dimension, even while running a gradient. This may also allow to achieve a zero-pressure pulse modulation. In the following, some basic cognitions of the present inventor will be mentioned based on which embodiments of the invention have been developed.

[0061 ] In two-dimensional chromatography (2D LC) systems, usually the individual separations are operated independently. This means that there is one LC arrangement, which has a column for first dimension separation, of which the outlet is injected into the high pressure path of the second dimension upstream of its column. This now leads to some complex arrangements. On one hand it should park a certain amount of sample substance eluting from the first dimension column and on the other hand it should bring the respective sample plug to the second dimension column without disturbance.

[0062] Usually it is possible to start the 2D-LC concept with the first dimension. The outcome of it (at low pressure) now is injected (modulated) into the second dimension. It is very common for low-damping, no-mixing configurations that dynamic changes will result in severe disturbances, both in flow and composition. This brings the fact that on top of the systematic changes due to the fast second gradient there is another class of changes which comes from the modulator.

[0063] A gist of an embodiment of the invention is that, instead of coupling both dimensions in an end-to-head fashion, this approach is like stacking one dimension on top of the other. It may sound simple, but the concept is to feed the outlet of the first dimension directly into the high pressure side of the second dimension. In simple terms, if the second dimension needs 800 bar and the first dimension needs 200 bar, then the first dimension pump feeds against 1000 bar. There is a strong advantage due to the fact that now the separated peaks from the first dimension always elute under exactly the same pressure, which is matched to the actual inlet pressure of the second dimension. This now helps that there is now need to pressurize the injected volume, which is switched into the second dimension. So the modulation is a very smooth transition, which is cycling the valve to generate the impulse, which is needed to trigger the chromatographic separation.

[0064] In terms of operation, the second dimension now can be driven in constant pressure mode (VB-LC). This way the first dimension has a constant pressure at its outlet, which helps for stability. Usually the first dimension runs against a static pressure close to zero. In one case, the first dimension is running on a pressure offset. Further, this concept prevents to bleed any substance to the outside. The valve groove design will support a make-before- break mode, or alternatively the constant pressure VB-LC mode will avoid pressure spikes. The valve design can be pretty simple. Just one bistable flip valve may be sufficient.

[0065] Referring now in greater detail to the drawings, Fig. 1 depicts a general schematic of a liquid separation system 10. A first pump 20 receives a mobile phase (also denoted as fluid) from a first solvent supply 25, typically via a first degasser 27, which degases and thus reduces the amount of dissolved gases in the mobile phase. The first pump 20 - as a mobile phase drive - drives the mobile phase through a first separating device 30 (such as a chromatographic column) comprising a stationary phase. A sampling unit 40 can be provided between the first pump 20 and the first separating device 30 in order to subject or add (often referred to as sample introduction) a sample fluid (also denoted as fluidic sample) into the mobile phase. The stationary phase of the first separating device 30 is configured for separating compounds of the sample liquid.

[0066] A second pump 20' receives another mobile phase (also denoted as fluid) from a second solvent supply 25', typically via a second degasser 27', which degases and thus reduces the amount of dissolved gases in the other mobile phase. By a fluidic valve 90, the first dimension (reference numerals 20, 30, ...) of the two-dimensional liquid chromatography system 10 of Fig. 1 may be fluidically coupled to the second dimension (reference numerals 20', 30', ...). The fluidic sample is separated into multiple fractions by the first dimension, and each fraction is further separated into multiple sub-fractions by the second dimension. The way of switching the fluidic valve 90 and a way of arranging the fluidic paths fluidically coupling the two dimensions will be described below referring to Fig. 2.

[0067] A detector 50 is provided for detecting separated compounds of the sample fluid. A fractionating unit 60 can be provided for outputting separated compounds of sample fluid.

[0068] While each of the mobile phases can be comprised of one solvent only, it may also be mixed from plural solvents. Such mixing might be a low pressure mixing and provided upstream of the pumps 20, 20', so that the respective pump 20, 20' already receives and pumps the mixed solvents as the mobile phase. Alternatively, the pump 20, 20' might be comprised of plural individual pumping units, with plural of the pumping units each receiving and pumping a different solvent or mixture, so that the mixing of the mobile phase (as received by the respective separating device 30, 30') occurs at high pressure und downstream of the pump 20, 20' (or as part thereof). The composition (mixture) of the mobile phase may be kept constant over time, the so called isocratic mode, or varied over time, the so called gradient mode.

[0069] A data processing unit 70, which can be a conventional PC or workstation, might be coupled (as indicated by the dotted arrows) to one or more of the devices in the liquid separation system 10 in order to receive information and/or control operation. For example, the data processing unit 70 might control operation of the pump 20, 20' (e.g. setting control parameters) and receive therefrom information regarding the actual working conditions (such as output pressure, flow rate, etc. at an outlet of the pump). The data processing unit 70 might also control operation of the solvent supply 25, 25' (e.g. setting the solvent/s or solvent mixture to be supplied) and/or the degasser 27, 27' (e.g. setting control parameters such as vacuum level) and might receive therefrom information regarding the actual working conditions (such as solvent composition supplied over time, flow rate, vacuum level, etc.). The data processing unit 70 might further control operation of the sampling unit 40 (e.g. controlling sample injection or synchronization sample injection with operating conditions of the pump 20). The respective separating device 30, 30' might also be controlled by the data processing unit 70 (e.g. selecting a specific flow path or column, setting operation temperature, etc.), and send - in return - information (e.g. operating conditions) to the data processing unit 70. Accordingly, the detector 50 might be controlled by the data processing unit 70 (e.g. with respect to spectral or wavelength settings, setting time constants, start/stop data acquisition), and send information (e.g. about the detected sample compounds) to the data processing unit 70. The data processing unit 70 might also control operation of the fractionating unit 60 (e.g. in conjunction with data received from the detector 50) and provides data back.

[0070] In the following, referring to Fig. 2, a two-dimensional liquid chromatography apparatus 200 according to an exemplary embodiment of the invention will be explained. [0071 ] The sample separation apparatus 200 is capable of separating a fluidic sample, which is injected by a sample injector 236 into a mobile phase, first into a plurality of fractions (each representing a group of molecules) by a first dimension chromatographic column 204. Later, each of these fractions may be further separated into a plurality of sub-fractions by a second dimension chromatographic column 208. The reason why each of the fractions can further be split into a plurality of sub-sections by the second dimension chromatographic column 208 is that the second dimension chromatographic column 208 may be configured so as to have another separation criterion as compared to the first dimension chromatographic column 204. This may for instance achieved by different chemicals, different solvent composition, different temperature, different size or shape of the two separation systems. [0072] The two-dimensional liquid chromatography apparatus 200 comprises a first binary pump 202. The first binary pump 202 is configured for conducting the fluidic sample to be separated through the first dimension chromatographic column 204. For this purpose, the first binary pump 202 provides a mixture from a first solvent 250 (such as water) and a second solvent 252 (such as acetonitrile, ACN). The first binary pump 202 mixes these two solvent compositions to form a mobile phase which is pumped towards the sample injector 236. At the sample injector 236 the actual fluidic sample is added to the mobile phase so that the mixture of the fluidic sample and the mobile phase is then pumped towards the first dimension chromatographic column 204. In the sample injector 236, an injection needle can be immersed into a vial accommodating the fluidic sample (not shown). The fluidic sample may then be sucked into the injection needle and a loop fluidically connected thereto. Subsequently, the injection needle may drive into a seat so as to then inject the fluidic sample into the mobile phase at a position of the autosampler or sample injector 236 in Fig. 2. In the first dimension chromatographic column 204, the different fractions of the fluidic sample are trapped at the separating material of a column and are later individually released from the column during a gradient run. Therefore, at the fluid outlet of the first dimension separation column 204, the various fractions of the sample are already separated.

[0073] Furthermore, a second binary pump 206 is provided which has a significantly higher flow rate as compared to the first binary pump 202. For instance, the flow rate of the second binary pump 206 may be 4 ml/min, whereas a flow rate of the first binary pump 202 may be 100 μΙ/min. As the first binary pump 202, also the second binary pump 206 can provide a mixture of a first solvent 254 with a second solvent 256. The solvents 254, 256 may or may not be the same as the solvents 250, 252. The second binary pump 206 is configured for conducting the already separated or treated fluidic sample conducted via a fluidic valve 218 towards the second dimension chromatographic column 208 which is arranged downstream of the first dimension chromatographic column 204. [0074] A flow coupler 210 is arranged downstream of the fluidic valve 218. The flow coupler 210 has two fluid inlet terminals 212, 214 and one fluid outlet terminal 216. As can be taken from Fig. 2, the fluid outlet terminal 216 is fluidically connected to the second dimension chromatographic column 208. [0075] The fluidic valve 218 has, in the present embodiment, four fluidic interfaces 222, 224, 226, 228. However, in other embodiments, the number of fluidic interfaces may be different and the valve configuration may be different. A first fluidic interface 222 is connected to the first binary pump 202 via the first dimension separation unit 204. A second fluidic interface 224 is connected to the first fluid inlet terminal 212. A third fluidic interface 226 is connected to the second fluid inlet terminal 214. A fourth fluidic interface 228 is directly coupled with the second binary pump 206.

[0076] Furthermore, a control unit 238 (such as a processor, for instance a microprocessor or a central processing unit, CPU) is provided which is capable of controlling all the devices and fluidic components shown in Fig. 2. This is illustrated schematically by the dotted lines directed from the control unit 238 towards the corresponding components.

[0077] Inter alia, the control device 238 is also capable of switching the fluidic valve 218. Particularly, the fluidic valve 218 can be switched by the control device 238 so that the first binary pump 202 and the second binary pump 206 remain always in fluid communication with one another, which holds for all switching states of the fluidic valve 218. Fig. 2 illustrates a first switching state 260 and illustrates a second switching state 270. In both operation modes or switching states, certain grooves 232 and corresponding ports 230 of the two valve members (a rotor and a stator) are aligned such that the above condition is always fulfilled: The binary pump 202 and the binary pump 206 remain always in fluid communication with one another, i.e. are hydraulically coupled. This provides the advantageous effect that no or basically no ripples occur as a result of the switching of the fluidic valve 218, as will be described below in more detail referring to Fig. 6. The operation mode 260 is an operation mode in which the second binary pump 206 is operated in the gradient mode. In contrast to this, in the operation mode 270, the second binary pump 206 pumps a sample towards the flow combiner 210 so that only a slight dilution of the sample occurs here. Depending on the switching state 260, 270, a fluidic conduit 290 or 292 in which the larger flow occurs, is changed. In the respective other fluidic conduit 292 or 290, the smaller flow occurs.

[0078] Furthermore, a detector 234 is provided which is capable of detecting the individualized fractions and sub-fractions of the fluidic sample by an electromagnetic radiation based detection principle. The separated fluidic sample flows through a flow cell and is irradiated with electromagnetic radiation from a light source 280. The beam of the light source 280 is guided through a focusing lens 282, then passes through the flow cell and can be detected by a detector 284. For instance, a fluorescence measurement may be performed. The wavelength regime in which a measurement is carried out can for instance be the optical range or in the ultraviolet range.

[0079] After having passed the detector 234, the fluidic sample will be collected in a waste container 286. It should be mentioned that the waste container 286 is the only position in the whole fluidic path at which the fluidic sample is not under a pressure being higher (particularly significantly higher) than ambient pressure. All other conduits are always under pressure which is advantageous for the pressure ripple suppression characteristics of the present invention.

[0080] The control device 238 is capable of executing a certain sequence of procedural steps for performing the actual two-dimensional liquid separation procedure.

[0081 ] By a rapid switching of the modulator valve 218, it always cuts out a short portion of the fluidic sample which is already separated by the first dimension column 204 very slowly. Only this part of the sample is then guided towards the second dimension column 208. An advantage of this is that due to the modulation valve 218, there are no pressure shocks during switching of the valve 218, since the already separated fluidic sample portion of interest is, at the time of the switching, already at the proper pressure value. Apart from the waste 286 at the very end of the fluidic path, there is no way for the fluid in the fluid conduit of Fig. 2 to escape. Therefore, it is a completely closed fluidic system which is pressure-less only at the very end of the fluidic path (i.e. at the position of the waste 286).

[0082] The architecture of Fig. 2 is significantly simpler than conventional approaches, since a single valve 218 is sufficient. Particularly, the fluidic coupling of the modulator valve 218 with the fluidic T-piece 210 allows the pressure ripples to be suppressed. The pressure which is generated in the second dimension is, via the T-piece 210, always at the outlet of the first dimension, so that the second dimension floats on the first dimension.

[0083] Reference is now made to Fig. 3 which shows a diagram 300 having an abscissa 302 along which a measurement time is plotted and having an ordinate 304 along which a solvent composition of two components A,B which can be mixed by the second fluid drive 206 are plotted. A and B may be the components which refer to reference numerals 254 and 256, respectively, in Fig. 2. As can be taken from Fig. 3, a chromatographic gradient run is applied for performing a separation on the second dimension separation column 208. The switching of the fluidic valve 218 between the operation modes 260 and 270 occurs at a point of time which is denoted in Fig. 3 with reference numeral 306. Hence, at the beginning of a gradient run of Fig. 3 or so-called secondary separation sequence, the switching of the fluidic valve 218 is initiated. [0084] Coming back to the operation principle of the control device 238, Fig. 4 shows a diagram 400 having an abscissa 402 along which a time is plotted and having an ordinate 404 along which a solvent composition as mixed by the first binary pump 202 is plotted. The control device 238 is now configured for controlling the first dimension separation column 204 to execute the primary separation sequence 406 as shown in Fig. 4 within a measurement time interval which is denoted with reference numeral 408 in Fig. 4. In the shown embodiment, the measurement time interval is 30 minutes. Within this time interval, the gradient run in accordance with the primary separation sequence 406 is carried out. [0085] Fig. 5 shows a diagram 500 indicating a plurality of secondary separation sequences 502, each of which being similar to Fig. 3 on another scale. Diagram 500 corresponds to diagram 400, whereas the time axis 342 is shown on another scale. As can be taken from Fig. 5, the control device 238 controls the second dimension separation column 208 to execute all of the plurality of secondary separation sequences 502 within the measurement time interval 408. Each of the secondary separation sequences 502 has a duration of about 20 seconds, compare Fig. 3. Thus, many secondary separation sequences 502 are carried out within one primary separation sequence 406. Thus, each of the fractions already separated by the first dimension chromatographic column 204 can be further separated into a plurality of sub- fractions by the secondary separation column 208.

[0086] Fig. 6 now shows a diagram 600 having an abscissa 602 which corresponds to the time axis, as in Fig. 3 to Fig. 5. However, along an ordinate 604, the pressure value is plotted as experienced in the flow path of a 2D LC apparatus. Fig. 6 shows a first curve 606 having a plurality of ripples. These ripples correspond to a conventional device having fluidic valves which are switched, wherein the fluid at the outlet of the first dimension chromatographic column is pressure-less during the switch, rendering the pressure characteristic unstable. In contrast to this, a curve 608 with no ripples or with strongly suppressed ripples 606 can be obtained with the two-dimensional liquid chromatography apparatus 200 shown in Fig. 2. The reason why this is achieved is the fact that the binary fluid pumps 202, 206 are always in fluid communication regardless of the switching state 260, 270 of the fluidic valve 218.

[0087] Fig. 7 again illustrates part of the sample separation apparatus 200 and shows schematically the first operation mode 260 and the second operation mode 270 corresponding to the different positions of the modulator valve 218.

[0088] In general terms, the fluidic valve 218 is, in the shown embodiment, not so much an ON/OFF valve (although it can be formed with a set of simple ON/OFF valves). Seen from its four ports 222, 224, 226, 228 it is more like a cross-over switch.

[0089] While in switching state or operation mode 260 (here represented by the dotted lines) the fluidic valve 218 connects the inlets straight to the outlets, during switching state or operation mode 270 (here the solid lines) the inlets are cross-wise connected to the outlets. In any of these switching states, the T- junction 210 connects the outlet from the first dimension column 204 and the outlet from the second dimension pump 206. The difference is basically, where and at what flow rate the eluted volume from the first dimension column 204 ends up to be traveling (or stored), while the other branch is driven heavily at high flow rates to forward the second dimension gradient onto the second dimension column 208. On the back-swing of the second dimension gradient this other branch is then filled (flushed) with starting composition of the gradient, which then triggers the flipping of the fluidic valve 218. After switching now the first dimension result elutes into this other branch, while the previously eluted volume from the initial branch is driven by the second dimension pump 206 towards the second dimension column 208 for final separation . It is true that at the same time this second dimension sample plug is diluted by the starting composition at the given actual first dimension flow rate. Purposely this dilutes not only the second dimension sample, but also the matrix that it is dissolved in . By modulating or tuning first dimension and second dimension flows to an advantageous relation, this may improve stacking of sample on the head of the second dimension column 208, further improving resolution and thus peak capacity of the separation system 200.

[0090] Fig. 8 now shows a two-dimensional chromatogram 700 as can be obtained when executing the primary separation sequence of Fig . 4 and the secondary separation sequences of Fig. 5. A first retention time 702 in accordance with the first dimension chromatographic separation (see column 204) is plotted along an abscissa 702, whereas a second retention time in accordance with the second dimension chromatographic separation (see column 208) is plotted along an ordinate 704. As can be taken from Fig. 8, a plurality of peaks 706 can be detected. Due to the non-complete orthogonality of the separation procedures in the column 204 on the one hand and in the column 208 on the other hand, each fraction may be further separated into sub- fractions. [0091 ] It should be noted that the term "comprising" does not exclude other elements or features and the "a" or "an" does not exclude a plurality. Also elements described in association with different embodiments may be combined. It should also be noted that reference signs in the claims shall not be construed as limiting the scope of the claims.

Claims

A sample separation apparatus (200) for separating a fluidic sample, the sample separation apparatus (200) comprising a first fluid drive (202) configured for conducting the fluidic sample to be separated through a first separation unit (204); a second fluid drive (206) configured for at least partially conducting the fluidic sample, after treatment by the first separation unit (204), through a second separation unit (208) downstream of the first separation unit (204); a flow coupler (210) having two fluid inlet terminals (212, 214) and a fluid outlet terminal (216) in fluid communication with one another, the fluid outlet terminal (216) being fluidically connectable to the second separation unit (208); a fluidic valve (218) having fluidic interfaces (222, 224, 226, 228) fluidically coupled to the first fluid drive (202), the second fluid drive (206) and the two fluid inlet terminals (212, 214) of the flow coupler (210); wherein the fluidic valve (218) is switchable for performing the separation of the fluidic sample so that the first fluid drive (202) and the second fluid drive (206) are in fluid communication with one another in each switching state of the fluidic valve (218).

The sample separation apparatus (200) according to claim 1 , comprising the first separation unit (204) for separating the fluidic sample.

The sample separation apparatus (200) according to claim 2, wherein the first separation unit (204) is arranged between the first fluid drive (202) and the fluidic interface (222) of the fluidic valve (218) which the fluidic valve (218) fluidically couples with the first fluid drive (202).

4. The sample separation apparatus (200) according to claim 1 or any one of the above claims, comprising the second separation unit (208) for further separating the fluidic sample after treatment by the first separation unit (204).

5. The sample separation apparatus (200) according to claim 4, wherein the second separation unit (208) is directly fluidically coupled to the fluid outlet terminal (216) of the flow coupler (210).

6. The sample separation apparatus (200) according to claim 1 or any one of the above claims, wherein the flow coupler (210) is configured as one of the group consisting of a fluidic T-piece, a fluidic Y-piece, and a fluidic X-piece,

7. The sample separation apparatus (200) according to claim 1 or any one of the above claims, wherein the fluidic valve (21 8) comprises a first valve member and a second valve member being movable, particularly being rotatable, relative to one another to thereby adjust a respective switching state of the fluidic valve (218) and thereby a corresponding one of different operation modes of the sample separation apparatus (200).

8. The sample separation apparatus (200) according to claim 7, wherein the fluidic valve (218) is configured to be switchable to a first operation mode (260) in which the fluidic interface (222) fluidically coupled to the first fluid drive (202) is in fluid communication via the fluidic valve (218) with the fluidic interface (224) fluidically coupled to one of the fluid inlet terminals (212), and in which the fluidic interface (228) fluidically coupled to the second fluid drive (206) is in fluid communication via the fluidic valve (218) with the fluidic interface (226) fluidically coupled to the other one of the fluid inlet terminals (214).

9. The sample separation apparatus (200) according to claim 8, wherein the fluidic valve (218) is configured to be switchable to a second operation mode (270) in which the fluidic interface (222) fluidically coupled to the first fluid drive (202) is in fluid communication via the fluidic valve (21 8) with the fluidic interface (226) fluidically coupled to the other one of the fluid inlet terminals (214), and in which the fluidic interface (228) fluidically coupled to the second fluid drive (206) is in fluid communication via the fluidic valve (218) with the fluidic interface (224) fluidically coupled to the one of the fluid inlet terminals (212).

10. The sample separation apparatus (200) according to claim 7 or any one of the above claims, wherein the first valve member comprises ports (230) forming the fluidic interfaces (222, 224, 226, 228), and the second valve member comprises grooves (232) for fluidically coupling different ports

(230) depending on a switching state of the fluidic valve (218).

1 1 . The sample separation apparatus (200) according to claim 1 or any one of the above claims, wherein at least one of the first fluid drive (202) and the second fluid drive (206) is a binary fluid pump.

12. The sample separation apparatus (200) according to claim 1 or any one of the above claims, wherein the fluidic valve (218) is switchable so that pressure conditions in the first separation unit (204) and in the second separation unit (208) remain constant before and after switching.

13. The sample separation apparatus (200) according to claim 1 or any one of the above claims, comprising a detector (234) for detecting the separated fluidic sample and being arranged downstream of the second separation unit (208).

14. The sample separation apparatus (200) according to claim 1 or any one of the above claims, comprising a sample injector (236) for injecting the fluidic sample into a mobile phase and being arranged between the first fluid drive (202) and the first separation unit (204).

15. The sample separation apparatus (200) according to claim 1 or any one of the above claims, wherein the first fluid drive (202) is operable with a first flow rate being smaller than a second flow rate according to which the second fluid drive (206) is operable.

16. The sample separation apparatus (200) according to claim 15, wherein the second flow rate is at least five times, particularly is at least ten times, more particularly is at least fifty times, of the first flow rate.

17. The sample separation apparatus (200) according to claim 1 or any one of the above claims, wherein the fluidic valve (218) is switchable for performing the separation of the fluidic sample so that the first fluid drive (202) and the second fluid drive (206) are in fluid communication with one another via the flow coupler (210) in each switching state of the fluidic valve (218).

18. The sample separation apparatus (200) according to claim 1 or any one of the above claims, comprising a control device (238) configured for: controlling the first separation unit (204) to execute a primary

separation sequence (406) within a measurement time interval (408) for separating the fluidic sample into a plurality of fractions; controlling the second separation unit (208) to execute a plurality of secondary separation sequences (502) within the measurement time interval (408) for further separating at least a part of the separated plurality of fractions into a plurality of sub-fractions.

19. The sample separation apparatus (200) according to claim 18, wherein at least one of the primary separation sequence (406) and the plurality of secondary separation sequences (502) relates to a chromatographic gradient run.

20. The sample separation apparatus (200) according to claim 1 or any one of the above claims, wherein the first separation unit (204) and the second separation unit (208) are configured so as to execute the respective sample separation in accordance with different separation criteria, particularly in accordance with at least partially but not completely orthogonal separation criteria.

The sample separation apparatus (200) according to any one of claims to 19, wherein the first separation unit (204) and the second separation unit (208) are configured so as to execute the respective sample separation on identical separation media but with different operating conditions, particularly at least one of the group consisting of different solvents, different steepness of elution gradients, different separation unit temperatures, and different pressures, so that the separation criteria are partially but not completely orthogonal.

The sample separation apparatus (200) according to claim 1 or any one of the above claims, comprising at least one of the following features: at least one of the first separation unit (204) and the second separation unit (208) is configured for performing a separation in accordance with one of the group consisting of liquid chromatography, supercritical-fluid chromatography, capillary electrochromatography, electrophoresis and gas chromatography; the sample separation apparatus (200) is configured as a two- dimensional liquid chromatography sample separation apparatus (200), particularly being a comprehensive two-dimensional liquid

chromatography apparatus; the sample separation apparatus (200) is configured to analyze at least one physical, chemical and/or biological parameter of at least one compound of the fluidic sample; the sample separation apparatus (200) comprises at least one of the group consisting of a chromatography device, a liquid chromatography device, an HPLC device, a gas chromatography device, a capillary electrochromatography device, an electrophoresis device, a capillary electrophoresis device, a gel electrophoresis device, and a mass spectroscopy device; the sample separation apparatus (200) is configured to conduct the fluidic sample with a high pressure; the sample separation apparatus (200) is configured to conduct the fluidic sample with a pressure of at least 100 bar, particularly of at least 500 bar, more particularly of at least 1000 bar; the sample separation apparatus (200) is configured to conduct a liquid fluid; the sample separation apparatus (200) is configured as a microfluidic device; the sample separation apparatus (200) is configured as a nanofluidic device; at least one of the group consisting of the first separation unit (204) and the second separation unit (208) is configured for retaining a part of components of the fluidic sample and for allowing other components of the fluidic sample to pass; at least one of the group consisting of the first separation unit (204) and the second separation unit (208) comprises a separation column (30); at least one of the group consisting of the first separation unit (204) and the second separation unit (208) comprises a chromatographic column (30); at least a part of at least one of the group consisting of the first separation unit (204) and the second separation unit (208) is filled with a separating material; at least a part of at least one of the group consisting of the first separation unit (204) and the second separation unit (208) is filled with a separating material, wherein the separating material comprises beads having a size in the range of 1 μιτι to 50 μιτι; at least a part of at least one of the group consisting of the first separation unit (204) and the second separation unit (208) is filled with a separating material, wherein the separating material comprises beads having pores having a size in the range of 0.01 μιτι to 0.2 μιτι.

A method of separating a fluidic sample, the method comprising conducting the fluidic sample to be separated through a first separation unit (204) by a first fluid drive (202); conducting, after treatment by the first separation unit (204) and at least partially by a second fluid drive (206), the fluidic sample through a second separation unit (208) downstream of the first separation unit (204); wherein a flow coupler (210) is provided having two fluid inlet terminals (212, 214) and a fluid outlet terminal (216) in fluid communication with one another, the fluid outlet terminal (216) being fluidically connected to the second separation unit (208); switching a fluidic valve (218), the fluidic valve (218) having fluidic interfaces (222, 224, 226, 228) fluidically coupled to the first fluid drive (202) and to the second fluid drive (206) and to the two fluid inlet terminals (212, 214) of the flow coupler (210), for performing the separation of the fluidic sample so that the first fluid drive (202) and the second fluid drive (206) are in fluid communication with one another in each switching state of the fluidic valve (218).

24. A software program or product, preferably stored on a data carrier, for executing a method according to claim 23, when run on a data processing system (238) such as a computer.