WO2012149956A1

WO2012149956A1 - Flow stabilization by capacitive load

Info

Publication number: WO2012149956A1
Application number: PCT/EP2011/057016
Authority: WO
Inventors: Klaus Witt
Original assignee: Agilent Technologies, Inc.
Priority date: 2011-05-03
Filing date: 2011-05-03
Publication date: 2012-11-08

Abstract

A sample separation device (200) for separating a fluidic sample, wherein the sample separation device (200) comprises a first fluid conduit (202) for conducting a fluid, a second fluid conduit (204) diverging from the first fluid conduit (202), a fluidic capacitance (206) arranged in the second fluid conduit (204) and configured for reducing pulsations of the flow in the first fluid conduit (202), a fluidic switch (208) coupled to the fluidic capacitance (206) and being switchable for at least partially discharging the fluidic capacitance (206), and a separation unit (210) arranged in the first fluid conduit (202) and configured for separating the fluidic sample injected into the fluid.

Description

DESCRIPTION FLOW STABILIZATION BY CAPACITIVE LOAD

BACKGROUND ART

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

[0003] The composition of the mobile phase can be adjusted by composing the mobile phase from different fluidic components with variable contributions. Under undesired circumstances, the flow and sometimes also the composition of the delivered mobile phase may be altered or disturbed, which may deteriorate proper operation of the sample separation device.

[0004] US 4,587,993 discloses a high-pressure viscous damper, in particular for use in liquid chromatographs, in which two chambers are separated by an elastic partition, and the one chamber receives a fluid to be damped and the other chamber holds a damping fluid. In the chamber holding the damping fluid an insert is provided within the fluid with a selected volume and temperature expansion coefficient so that by the combination of the insert and damping fluid and the volume and wall material of the second chamber a volume compensation is achieved.

[0005] US 4,245,963 discloses a pump for precise, smooth delivery of liquid, particularly in liquid chromatography systems, featuring two liquid displacement elements mounted for reciprocating movement in chambers connected in series with two check valves, one displacement element serving to accumulate some of the liquid delivered by the first element and to deliver the accumulated liquid while the first element is refilling. [0006] US 4,234,427 discloses a pulse damper for use in high-pressure liquid pumping applications such as l iqu id chromatography and comprises a length of flattened polytetrafluoroethylene tubing that can be coupled to the high-pressure liquid flow line. The flattened tubing is enclosed within a liquid-tight housing structure completely filled with a compressible liquid, and the end of the tubing are coupled to the high-pressure flow line by fittings mounted inside the housing structure. When a transient pressure variation occurs in the flow line, the cross-section of the tubing changes from a flattened elliptical configuration to a more rounded configuration as the pressure pulse temporarily overcomes the stresses that tend to maintain the tubing in its flattened configuration. The restoring force of the compressible liquid in the housing structure surrounding the tubing prevents expansion of the tubing beyond a desired limit during a pulse, so that the tubing cannot burst when its cross-sectional configuration changes. The overall length of the tubing, related to the volume and compressibility of the compressible liquid, the highest anticipated pulse pressure in the flow line, and the pre-determined maximum change to be allowed in the average cross- sectional area of the tubing, is generally much greater than the length of the housing structure, and hence the tubing is folded or coiled with in the housing structure. Dissipation of the transient energy of a pulse in compressing the liquid in the housing structure effectively damps the pulse. [0007] US 4,132,51 1 discloses a damper for use with a high pressure pumping system, such as a liquid chromatography system, which incorporates a reciprocating pump. The device is a generally enclosed canister including an internally formed flow volume. Inlet and outlet passages through the canister communicate with the flow volume, the inlet passage being connectable to receive the high pressure flow. A compressible body, e.g. of Teflon is positioned in the flow volume. The dimensions of the body are slightly smaller than those of the surrounding volume, whereby the high pressure flow passing between the inlet and outlet flows through the space between the body and the internal canister walls. The compression and decompression of the body in response to the pulsations in the flow dissipate the energy carried by the pulses, thereby damping same.

[0008] However, proper operation of a sample separation device may still be difficult when a condition like e.g. flow, solvent composition and/or pressure changes in a sample separation device.

DISCLOSURE

[0009] It is an object of the invention to provide an efficiently operating sample separation device. The object is solved by the independent cla ims . Fu rther embodiments are shown by the dependent claims.

[0010] According to an exemplary embodiment of the present invention, a sample separation device for separating a fluidic sample is provided, wherein the sample separation device comprises a first fluid conduit for conducting a fluid (such as a solvent or solvent composition into which the fluidic sample is to be injected), a second fluid conduit diverging from the first fluid conduit (for instance, the connection portion between first and second fluid conduit may be T-shaped), a fluidic capacitance arranged in the second fluid conduit and configured for reducing pulsations of the flow in the first fluid conduit, a fluidic switch coupled to the fluidic capacitance and being switchable for at least partially (i.e. partially or completely) discharging the fluidic capacitance, and a separation unit arranged in the first fluid conduit (particularly downstream the position at which the second fluid conduit diverges from the first fluid conduit) and configured for separating the fluidic sample injected into the fluid (wherein the injection of the fluidic sample into the fluid may be performed downstream the position at which the second fluid conduit diverges from the first fluid conduit). [001 1 ] According to an exemplary embodiment of the present invention, a method of separating a fluidic sample is provided, wherein the method comprises conducting a fluid through a first fluid conduit, reducing pulsations of the flow in the first fluid conduit by a fluidic capacitance arranged in a second fluid conduit diverging from the first fluid conduit, switching a fluidic switch coupled to the fluidic capacitance for at least partially discharging the fluidic capacitance, and separating the fluidic sample injected into the fluid in a separation unit arranged in the first fluid conduit.

[0012] According to another exemplary embodiment of the present invention, a sample separation device for separating a fluidic sample is provided, wherein the sample separation device comprises a first fluid conduit for conducting a fluid, a second fluid conduit diverging from the first fluid conduit, a fluidic capacitance arranged in the second fluid conduit, a fluidic switch being switchable so that a buffer volume of the fluid is conducted from the first fluid conduit towards the fluidic capacitance in the second fluid conduit, and a separation unit (such as a chromatographic column) arranged downstream in the first fluid conduit and configured for separating the fluidic sample injected into the fluid.

[0013] According to still another exemplary embodiment, a method of separating a fluidic sample is provided, wherein the method comprises conducting the fluid through a first fluid conduit, switching a fluidic switch so that a buffer volume of the fluid is conducted from the first fluid conduit towards a fluidic capacitance arranged in a second fluid conduit diverging from the first fluid conduit, and separating the fluidic sample injected into the fluid in a separation unit arranged in the first fluid conduit.

[0014] In the context of this application, the term "fluidic capacitance" may particularly denote a fluidic member which is capable of temporarily storing or buffering a fluidic volume, particularly in a way that a biasing force is applied by the fluidic capacitance against the filling of its buffering volume. An electrical analogon to such a fluidic capacitance is a capacitor capable of temporarily buffering or storing an electric charge. The skilled person will understand that a fluidic capacitance is to be distinguished from a mere portion of a capillary or fluid conduit but forms a separate fluidic member. It may be a bigger tube, where the liquid volume in it already acts as a hydraulic capacitance.

[0015] In the context of this application, the term "pulsations of the flow" may particularly denote ripples or other discontinuities in the fluidic flow, i.e. in the temporal dependency of the fluid volume flowing into the first fluid conduit per time interval. Such a pulsation, which may have an undesired impact on the separation performance of the separation unit, may be suppressed or smoothed or averaged out or buffered by the fluidic capacitance.

[0016] In the context of this application, the term "fluidic switch" may particularly denote a fluidic member which selectively allows to open or close an assigned fluidic path (such as the first fluid conduit or the second fluid conduit) upon a switching operation which may be initiated manually by a user or automatically by a control unit. Hence, the fluidic switch may selectively enable or disable a fluid flow through an assigned fluid conduit.

[0017] In the context of this application, the term "discharging" may particularly denote draining a fluid amount, which has previously been loaded onto the fluidic capacitance, away from the fluidic capacitance towards a fluidic destination such as a connected fluid conduit directed to or a container like a waste.

[0018] In the context of this application, the term "diverging" may particularly denote that the corresponding fluid conduits have a cross point so that a fluid reaching the cross point (such as a fluidic T-piece) has more than one option for flowing into different connected fluidic paths. Hence, a fluidic bifurcation may be formed at this position.

[0019] In the context of this application, the term "buffer volume" may particularly denote a predefined fixed volume or a variable volume which can be accommodated by the fluidic capacitance.

[0020] According to an exemplary embodiment, a sample separation device is provided in which a sufficiently pulsation-free characteristic of the fluidic flow in the first fluid conduit along which the separation is performed can be ensured. This can be achieved by arranging a fluidic capacitance in a side path (i.e. the bifurcated second fluid conduit) which can be emptied at least partially by a certain switching operation of an assigned fluidic switch. In other words, if emptying of this side path is required, the switch can be selectively opened or closed so that a historical fluid or fluid composition which has previously been stored in the fluidic capacitance can be conducted away from the fluidic capacitance and from the first fluid path. In this context, the term historic fluid or fluid composition may denote a fluid volume which has beforehand been pumped from the first fluid conduit into the second conduit. By conducting it off and preventing it from at least partially flowing back into the first fluidic conduit, it can be advantageously prevented that this historical fluid deteriorates a separation performance in the first fluidic path. Therefore, a chopping emptying of the fluidic capacitance acting as a fluidic damping element can be performed. With such an emptying feature, it can be safely prevented that the historic fluid composition oscillates between the fluidic capacitance and the first fluid conduit in an undesired way which would transport also historical fluid or fluid composition at unpredictable times into the separation unit. Such an undesired mixing of historical fluids with actual fluids, which is of particularly disadvantage in a chromatographic gradient mode, can be efficiently suppressed by arranging the damper in a side path and by switching it for emptying towards another fluidic part differing from the first fluidic part. Therefore, it is possible that a solvent composition at a crossing point, at which the second fluidic path diverges from the first fluidic path, is emptied or at least partly emptied.

[0021 ] Therefore, a fluidic capacitance is located so as to allow to tackle the event of fluctuations or oscillations of a feed rate or delivery rate of a fluidic drive unit such as a pump. Also in the event of another volume disturbance, it is possible to react with a damped pressure control using the fluidic capacitance for smoothing or averaging or low pass filtering any disturbance on a short time scale. In contrast to approaches in which a fluidic capacitance is not dischargeable away from the first fluid conduit, but would for instance be arranged in a dead end of a fluidic path, undesired oscillations of historic fluid mixtures (compositions) could not be safely prevented. Hence, it is for instance possible that, when a volume requirement for buffering is expected (for instance estimated by a pressure sensor or a flow sensor), a volume from the capacitance may be discharged and may be promptly substituted or del ivered additionally by the fluidic drive unit.

[0022] Next, further exemplary embodiments of each of the sample separation devices will be explained. However, these embodiments also apply to each of the methods.

[0023] In an embodiment, the fluidic switch is switchable for at least partially discharging the fluidic capacitance into the second fluid conduit. By taking this measure, it can be prevented that the buffered fluid flows back from the flu id ic capacitance into the first fluid conduit and possibly from there into the separation unit which could disturb a separation experiment as a result of an undesired mixing between actual fluid and historical fluid of a mobile phase. The net bleed flow should be at least the amount of flow which is delivered from the damping device back into the T-piece. [0024] In an embodiment, the sample separation device comprises a control unit configured for switching the fluidic switch for at least partially discharging historic fluid composition from the fluidic capacitance. Such a control unit may be a microprocessor or a central processing unit (CPU) which can supply (for instance electrical) switching signals to the fluidic switch so as to enable desired fluidic communication in fluidic paths or between fluidic paths which are connected by the fluidic switch. Therefore, if the fluidic switch is opened, it is for instance possible that a fluidic connection between the fluidic capacitance and a pressureless waste container is enabled which allows the buffer fluid to be discharged away from the bifurcation point.

[0025] In an embodiment, the fluidic switch is switchable so that a predefined flow of the fluid (i.e. a defined flow value) flows through the first fluid conduit. Thus, the control criteria may be that a fluid flow (fluidic volume per time interval) is at a given value. This value may be constant or may vary over time. Controlling or regulating the flow may also contribute to the smoothing of the flow in addition to the suppression of pulsations resulting from the performance of the fluidic capacitance.

[0026] Particularly, the fluidic switch is switchable so that a flow of the fluid flowing through the first fluid conduit is constant. Hence, the switching logic or control criteria may be as well that the flow is constant, i.e. that ripples are suppressed as much as possible. Such ripples may for instance originate from a reciprocating piston of a fluidic pump when the piston changes its moving direction at reversal points within a reciprocation volume. [0027] In an embodiment, the sample separation device comprises a sensor arranged in the first fluid conduit and being configured for sensing data indicative of an actual flow rate in the first fluid conduit, wherein the fluidic switch is switchable based on the sensed data. Such a sensor may be a pressure sensor sensing an actual pressure value at a certain position in the first fluidic path (for instance directly upstream of the separation unit) or may alternatively be a flow sensor sensing a flow (such as a volume flow of a mass flow, i.e. a flowing volume or a flowing mass per time interval, respectively). The sensor may also be arranged at other positions of the fluidic path. One or more of such sensors may provide information with regard to the present pressure or flow value(s) in the fluidic system and therefore allow to be used as a basis for the switching.

[0028] In an embodiment, the sample separation device is configured for operating the separation unit in a gradient mode. Generally, sample separation devices such as chromatographic columns may be driven in a gradient mode or in an isocratic mode. In an isocratic mode, the composition or mixture of a mobile phase (i.e. the fluid pumped through the first flu id ic conduit) is kept constant over time. Alternatively, the composition may be varied over time, so-called gradient mode. The gradient mode can be advantageous for chromatographic separation, since it allows to separately release different fractions of a fluidic sample which have previously been trapped on a separation column, each fraction leaving the column at a certain composition of the solvent. Thus, in the gradient mode, it is of high importance for a proper separation performance that the fluid mixture is always very precisely adjusted. By arranging the fluidic capacitance in a bifurcated path and being emptied only upon operation of a certain fluidic switch in a flow direction away from the first fluidic conduit, it is possible to safely prevent backflow of historical fluid from side path to main path which could deteriorate a gradient mode in a significant way. Therefore, when operating the separation unit in a gradient mode, the arrangement and operation of the fluidic capacitance according to exemplary embodiments is of particular advantage.

[0029] In an embodiment, the sample separation device comprises a drain, wherein the fluidic switch is switchable so that the fluidic capacitance is at least partially discharged towards the drain. In an embodiment, the drain is a pipe or tube connected to a sink or waste container. Such a drain may be arranged at the end of a fluidic conduit and may be pressureless or may be at a low pressure so that fluid has a tendency to flow towards the drain. In other words, such a drain may be configured so that, in the presence of a pressure in the fluidic conduit, it is possible to conduct or discharge the fluid originating from the fluidic capacitance into the connected drain. [0030] In an embodiment, the sample separation device may comprise a fluid drive unit configured for driving the fluid through the first fluid conduit, wherein the fluid drive unit is configured for delivering additional fluid to at least one of the first fluid conduit and the second fluid conduit to substitute fluid at least partially discharged from the fluidic capacitance. Such a fluid drive unit may be a pump, for instance a high pressure pump (operating at 600 bar or even 1200 bar) and will pump a constant or time varying mixture of solvent, also denoted as the mobile phase, into the first fluidic conduit. In the first fluidic conduit, it is possible that this mobile phase is mixed with a sample supplied via a sample loop from an autosampler, thereby forcing the fluid to flow towards the separation unit. Such a sample injector for injecting the fluidic sample (for instance a biological sample) into the fluid (which may be a solvent or a solvent composition) may inject the fluidic sample into the fluid at a position downstream of the bifurcation between the first and the second fluid conduit but upstream of the separation unit. If fluid is lost by discharging the fluidic capacitance, the fluid drive unit may be controlled so as to provide the additional volume into the system. For example, it is possible to use one and the same control unit for controlling the fluidic switch and for controlling the fluid drive unit in order to synchronize the behavior of the system components.

[0031 ] In an embodiment, the fluidic switch is arranged in the second fluid conduit. In such an embodiment, there is a very close spatial relationship between the fluidic switch and the fluidic capacitance so that the fluidic capacitance will be discharged in a very short time after the switching operation, thereby obtaining a high accuracy. Furthermore, when the fluidic switch is arranged in the second fluid conduit, the fluidic switch will not disturb the separation procedure in the first fluid conduit.

[0032] In an embodiment, the fluidic switch is arranged between the fluidic capacitance and the drain. In such an embodiment, it is possible to use the buffering capacity of the fluidic capacitance also during normal use (when the fluidic switch is closed), and by discharging selectively the fluidic capacitance partially or entirely upon switching the switch.

[0033] In an embodiment, the separation unit is arranged downstream (in a fluid flow direction) of a bifurcation position (or T-position) at which the second fluid conduit diverges from the first fluid conduit. Therefore, the fluid supplied to the separation unit for actual separation can already be basically ripple-free.

[0034] In an embodiment, the fluidic switch is configured to be controllable for selectively enabling or disabling fluid flow through the fluidic switch. For instance, a fluidic switch may be a rotary valve which is capable of being rotated so as to provide fluidic communication between different fluidic connections, depending on the switching state of the valve. It is however also possible that the fluidic switch is a simple open/close valve. [0035] In an embodiment, the fluidic capacitance comprises a hollow container with a (particularly flexible) membrane separating (particularly in a fluid-tight manner) an interior of the hollow container (particularly having a rigid wall) in a first volume, having a flu id inlet and a fluid outlet, and in a second volume, the membrane being deformable under fluid pressure flowing into the container via the fluid inlet. In an embodiment, the second volume is filled with a liquid (or with a gas under a certain pressure). Such an embodiment is shown, for instance, in Fig. 6.

[0036] In an alternative embodiment, the fluidic capacitance comprises a hollow container (particularly having a rigid wall) having a fluid inlet and a fluid outlet and a body (particularly a bendable and compressible rubber body such as a cylinder) being freely movable within the hollow container and being compressible under pressure. Such an embodiment is shown, for instance, in Fig. 7.

[0037] In an alternative embodiment, the fluidic capacitance comprises a (particularly flexible) hollow container having a fluid inlet and a fluid outlet and having a wall being deformable under pressure. Such an embodiment is shown, for instance, in Fig. 8.

[0038] In an alternative embodiment, the fluidic capacitance may be partially or completely constituted by an elasticity of the second fluid conduit and the compressibility of the liquid volume therein. Hence, when the second fluid conduit is formed by a resilient material, it may integrally include also the fluidic capacitance. Alternatively, the fluidic capacitance may be a member being provided separately from the second fluid conduit.

[0039] In an embodiment, the first fluid conduit and the second fluid conduit form a bifurcated fluidic network. In such a bifurcated fluidic network, a fluid originating from a source fluid conduit can flow into different destination fluid conduits at the crossing point. A fluidic T-piece may be foreseen at this crossing point.

[0040] In an embodiment, the sample separation device comprises a further fluidic switch and a further fluidic capacitance arranged in a third fluid conduit, wherein the further fluidic switch is switchable for at least partially discharging the further fluidic capacitance. Therefore, it is possible to implement various arrangements of paired fluidic switches and fluidic capacitances to thereby achieve sophisticated fluid flow control possibilities.

[0041 ] The 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, zeol ite, 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. [0042] At least a part of the 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.

[0043] The sample separation device 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.

[0044] However, the sample separation device 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.

[0045] The sample separation device 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 electron ic measu rement 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. [0046] 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.

[0047] The sample separation device may be configured to conduct a liquid mobile phase through the sample separation element and optionally a further sample separation element. As an alternative to a liquid mobile phase, a gaseous mobile phase or a mobile phase including solid particles may be processed using the fluidic device. Also materials being mixtures of different phases (solid, liquid, gaseous) may be processed using exemplary embodiments.

[0048] The sample separation device 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 device 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 magn itude of less than 500 μ ιτι , particularly less than 200 μιτι, more particularly less than 100 μηη or less than 50 μηη or less. The sample separation device 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 to Fig . 5 show various sample separation systems according to exemplary embodiments of the invention. [0053] Fig. 6 to Fig. 8 show examples of fluidic capacitances implementable in any of the above embodiments of the sample separation systems according to exemplary embodiments of the invention.

[0054] The illustration in the drawing is schematically.

[0055] Referring now in greater detail to the drawings, Fig. 1 depicts a general schematic of a liquid separation system 10. A pump 20 receives a mobile phase (also denoted as fluid) from a solvent supply 25, typically via a degasser 27, which degases and thus reduces the amount of dissolved gases in the mobile phase. The pump 20 - as a mobile phase drive - drives the mobile phase through a separating device 30 (such as a chromatographic column) comprising a stationary phase. A sampling unit 40 can be provided between the pump 20 and the 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 separating device 30 is configured for separating compounds of the sample liquid. Adetector 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.

[0056] While the mobile phase 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 pump 20, so that the pump 20 already receives and pumps the mixed solvents as the mobile phase. Alternatively, the pump 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 separating device 30) occurs at high pressure und downstream of the pump 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.

[0057] 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 (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 (e.g. setting the solvent/s or solvent mixture to be supplied) and/or the degasser 27 (e.g. setting control parameters such as vacuum level) and m ight 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 separating device 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.

[0058] From the embodiment of Fig . 1 , it can be seen that the flow path of the mobile phase typically comprises plural individual components, such as pump 20, separating device 30, sampling unit 40, and detector 50, which are coupled together and which might also be comprised of individual sub-components. Also, fluid conduits, e.g. capillaries, for conducting the fl u id are provided as ind icated by the sol id connections in Fig. 1 .

[0059] Additionally, components 202, 204, 206, 208, 214, 216 and 220 are shown in Fig. 1 which relate to a switchable fluid discharging system for suppressing pulsations in the fluid flowing towards the separation column 30 and wh ich may result, for instance, from reciprocation of a piston (not shown) within the pump 20. This can be achieved by selectively discharging a fluidic capacitance 206 arranged in a side path of the main fluidic conduit of Fig. 1 . The various components 202, 204, 206, 208, 214, 216 and 220 and the way they contribute to the system function will be explained below in more detail referring to Fig. 2 to Fig. 5.

[0060] However, beforehand, some more general considerations in the context of embodiments of the invention will be explained. Embodiments of the invention may provide for a flow stabilization by a capacitive load, particularly a chopper generated bleed flow to control a capacitive load. [0061 ] In gradient Liquid Chromatography (LC) systems often there is a requirement to have both accurate and stable dispensing of flow and still having lowest possible delay volume. This results in the fact that capacitive loads should not be plumbed along the flow stream.

[0062] Modern UHPLC-systems are faced with even increased requirements. In the interest to increase peak capacity (total number of peaks per time interval) several parameters are optimized, including smaller size of packing material, smaller columns, and faster linear speed of solutes during separation faster compositional gradients longer separation beds. While on one hand it increases the need for higher pressures, on the other hand it requires a combination of more precise resolution and fast dynamics of the pump drive, which delivers the desired flow. [0063] For a long time it has been common to add damping, which simply is a capacitive element, say membrane, which is coupled in-between the flow supply and the hydraulic load. However, such a capacitive element adds to the delay volume in a system, which in case of a nanoflow arrangement easily can be of fatal size. [0064] If a capacitive element (capacitance C) is under pressure load (pressure P), then the actual volume V in it is at least of the size V = C ^* P. While actually a decent capacitance is needed for dampening of a given volumetric disturbance at an operating pressure, to keep the total volume low a low capacitance would be necessary. One could consider a T-piece and locating the damping capacitance in a side arm, but now the liquid content inside the damper would be of disturbing nature because it is unknown which composition at what time would bleed back into the flow stream.

[0065] In view of these considerations, embodiments of the present invention have been developed: While the dead volume effect is avoided because the capacitance is connected in a side arm to the flow stream, some decent bleed flow is directed away from the junction. If this bleed flow at all times is larger than the resulting capacitive discharge on a down ramp of the pressure, then a bleeding of false composition back into the flow stream is avoided. In order to still deliver the correct flow to the operational load (say separation column) the flow supply will have to deliver the correct amount in access. Such bleeding could be a constant value, but then it is required to cover a pretty broad range. On one hand the operating pressure may have a large span, for instance 5 MPa to 200 MPa, and on the other hand a gradient will overlay a change in viscosity. A fixed bleed-restrictor would lead to huge dynamics in bleed flow, which in turn under extreme conditions requires a flow supply to dispense an immense amount on top of the operating flow. In order to limit the total flow overhead, but still keep the bleed flow above the capacitive discharge, it is of advantage to have a dynamic adjustment implemented . This way under static conditions, when the operating pressure is flat or at least rising, then the bleed flow can be switched off. Only in the advent of falling pressure levels it then is required to open the bleed channel to the extent which results from the capacitance multiplied by the pressure slope. Such controllable device with sufficient dynamic performance can be implemented by a high-speed switchable valving device, which is operated for instance by a piezo-driven actuator. High speed operation is preferred to use the natural capacitance of our damping device to filter the activation frequency. Running this piezo-driven actuator at frequencies of larger than 100Hz or even above 25kHz will ensure that no pressure cycling or remaining ripple is visible on the pressure plot at the load. While this whole concept is viable already in feed-forward system like flow dispensing arrangements, there is a specific advantage when using a flow sensor driven control . Such flow sensor may have a much higher resolution than the flow supply is able to deliver. In this arrangement by its pure passive action the capacitance will dampen the course, step-wise motion of the flow supply. This will be appreciated when considering an already fine motion of a piston pump dispensing by a stepwise motion in increments of 10Onl . In a nanoflow regime, where the load may be operated with 600nl/min, it results in steps being 10 sec apart. In a hi-speed analytical system this may be already seen as a pulsed flow. But with sufficient capacitance in the side arm, the damping will be efficient to filter the motion ripple, still not adding to the delay volume. Now the flow to the load can be controlled to the resolution of the flow sensor, which can be as good as 10nl or even better.

[0066] Now referring in more detail to Fig. 2, Fig . 2 shows a sample separation device 200 for separating a fluidic sample. The sample separation device 200 is a chromatographic separation system in which a fluidic sample injected into a fluid can be separated into different fractions, as described in more detail above referring to Fig. 1 .

[0067] Coming back to Fig. 2, the sample separation device 200 comprises a first fluid conduit 202 (which may also be denoted as a main conduit) for conducting the fluid. The first fluid conduit 202 may be a capillary. Furthermore, a second fluid conduit 204 (which may also be denoted as a side conduit), which may be a capillary as well, is arranged to diverge from the first fluid conduit 202. Moreover, a fluidic capacitance 206 (which can be configured as shown in Fig. 6 to Fig. 8) is arranged in the second fluid conduit 202 and is configured for reducing or suppressing pulsations or ripples of the flow in the first fluid conduit 202. Such pulsations may occur due to artifacts resulting from the operation of the pump 218 and/or due to other disturbance. [0068] Moreover, a fluidic switch 208 is coupled to the fluidic capacitance and is switchable, under control of a control unit 21 2, for at least partially discharging the fluidic capacitance 206. Thus, the fluidic capacitance 206, which may still store buffer fluid from previous operation of the sample separation device 200, may be at least partially emptied upon closing the switch 208 (i.e. by enabling fluid communication between the fluidic capacitance 206 and drain or waste 216). Furthermore, a fluidic load such as a separation unit 210 (a chromatographic separation column) is arranged in the first fluid conduit 202 and is configured for separating the fluid based on the chromatographic principle as known by a person skilled in the art.

[0069] Upon switching the fluidic switch 208, i.e. upon closing it under control of the control unit 212, the fluidic path between the fluidic capacitance 206 and waste 216 is enabled so that, due to the pressure conditions within the first fluid conduit 202 and the second flu id condu it 204 (the pump 21 8 pumps the fluid with a pumping rate of typically several hundred bar or more) the fluid will be pressed from the flu id ic capacitance 206 towards the drain 21 6. Thus, any undesired oscillations of such historical fluid in historical composition, which has been previously pumped into the fluidic capacitance 206, back to the first fluid conduit 202 may be prevented by not allowing this historical fluid to flow back from the fluidic capacitance 206 into the fluid conduit 202, but by draining it into the waste 216.

[0070] As can be taken from Fig. 2, the control unit 212 receives pressure data from a pressure sensor 214 sensing an actual pressure value in the first fluid conduit 202 downstream of a T-piece or a bifurcation point 220. However, the pressure sensor 214 may also be arranged at any other appropriate position in the flow path. Based on this pressure value, the control unit 212 may decide that it is time to switch the switch 208, thereby emptying the fluidic capacitance 206 partially or entirely depending on the time interval the switch 208 remains closed. Furthermore, the volume of the drained fluid has to be supplied additionally into the first fluid conduit 202 so that a known and basically constant fluid flow along the first fluid conduit 202 is ensured, thereby keeping any disturbance of the separation procedure as small as possible. For the purpose of synchronization of drain and supply procedures, the control unit 212 may also control operation of the pump 218. The pump 218, in such an embodiment, may pump fluid from a fluid container 222 into the first fluid conduit 202 with a temporarily increased flow rate, until the emptied fluid has been delivered additionally. [0071 ] In the embodiment of Fig. 2, the pump 218 pumps fluid such as a solvent or a solvent composition through the system . At a certain position, as indicated schematically with reference numeral 40 in Fig . 2, an actual fluidic sample to be separated may be injected into the fluid for subsequent separation in separation unit 210. Hence, preferably but not necessarily, the sample injection position 275 is located downstream bifurcation point 220. Furthermore, the sample injection position 275 is located upstream separation unit 210.

[0072] Another embodiment shown in Fig. 3 illustrates a sample separation device 200' in which the pressure sensor 214 of Fig. 2 is substituted by a flow sensor 300. Fig. 3 hence shows the concept of capacitive flow stabilization with the flow sensor 300 in the downstream path.

[0073] It will be readily appreciated to combine this concept not only with isocratic pumping arrangements, but to use this concept by achieving low delay volume in fast gradient operation. Such gradients can be achieved either by a pre-pump proportioning or by an even lower volume arrangement where the concept is used twice, controlling flow in individual channels.

[0074] Fig. 4 shows a sample separation device 200" according to still another exemplary embodiment of the invention in which a concept variation for low-pressure gradient formation is shown. As compared to Fig. 2, more than one fluid container 222, i.e. in this embodiment three fluid containers 222, 400, is provided so as to also allow a mixture of various solvent components by a m ixing un it 402. Therefore, the embodiment of Fig. 4 is particularly suitable for a gradient mode operation of a chromatographic experiment. Particularly, when the chromatography experiment is performed in a gradient mode, the described system may safely prevent undesired backflow of historic fluid from the second fluid conduit 204 back into the first fluid conduit 202, because such a fluid is emptied from the fluidic capacitance 206 via the switch 208 towards the drain 216.

[0075] Moreover, Fig. 5 shows a sample separation device 200"' according to still another exemplary embodiment of the invention with a concept variation for high- pressure gradient formation. Fig. 5 shows that more sophisticated fluidic networks can be formed based on basic gists of embodiments of the invention. In the embodiment of the Fig. 5, two parallel fluidic paths are provided, wherein an additional fluidic path upstream of the separation column 210 comprises a further fluidic switch 502 and a further fluidic capacitance 504 arranged in a further fluid conduit 500. The further fluidic switch 502 is switchable for at least partially discharging the further fluidic capacitance 504, in a way as explained above in more detail . Remaining additional components of Fig. 5 are denoted with the same reference numerals as in Fig. 3, but additionally with apostrophes.

[0076] At a T-piece or combination point 506, the two fluidic paths 202, 202' are combined at a downstream position, but still upstream of the separation unit 210.

[0077] Fig. 6 shows a first example for a flu id ic capacitance wh ich can be implemented in any of the embodiments shown above referring to Fig. 1 to Fig. 5. The fluidic capacitance 206 shown in Fig. 6 comprises a hollow container 600 with a rigid wall and a flexible membrane 602 mounted circumferentially to the wall for separating an interior of the hollow container 600 in a first volume, having a fluid inlet 604 and a fluid outlet 606, and in a second volume being free of a fluid inlet and a fluid outlet. The flexible membrane 602 is mounted in a mechanically biased way and is deformable under fluid pressure flowing into the container 600 via the fluid inlet 604. As can be taken from Fig. 6, the second volume can be partially filled with a liquid 608 to provide a counter pressure. When fluid flows along the direction of the arrows of Fig. 6, the fluid can be temporarily stored or buffered in the first fluid volume by deflecting the membrane and can later leave the fluidic capacitance 206 via the fluid outlet 606, when the liquid 608 expands reacting to reduced pressure. One or more optional valves 610, 612 can be foreseen as well to further improve controllability but may also be omitted (such valves are not shown in Fig. 7 and in Fig. 8 but may be foreseen in these embodiments as well).

[0078] Fig. 7 shows an alternative fluidic capacitance 206' which comprises a hollow container 700 with a rigid wall and having a fluid inlet 702 and a fluid outlet 704. A rubber cylinder 706 is almost freely movable within the hollow container 700 and is compressible or at least deformable under application of pressure. [0079] Fig. 8 shows another embodiment of a fluidic capacitance 206" which comprises a flexible hollow container 800. In contrast to Fig. 6 and Fig. 7, the hollow container 800 has a wall being sufficiently elastic and deformable under pressure. Furthermore, a fluid inlet 802 and a fluid outlet 804 are provided so that fluid can be inserted into the fluid capacitance 206" via the fluid inlet 802, thereby deforming the wall and being accommodated within the fluid conduit 206". Later, the fluid can be discharged via the fluid outlet 804.

[0080] It should be noted that a certain fluidic capacitance is a natural behavior or physical property in liquids when under pressure. The fluidic conduit in itself exhibits an elasticity, although small, and the liquid volume in it for instance exhibits a compressibility of the scale 20 - 200 E-4. Depending on the actual construction and size of fluid conduits and required capacitance to achieve sufficient damping, the skilled person will understand that no additional element like in Fig. 6,. Fig. 7, Fig. 8 is needed provided that the fluid conduits are configured so that a sufficiently large fluidic capacitance is contributed by them. So a fluidic capacitance (206) may be an integral component, given by the specific arrangement of the second fluid conduit (204). In other words, although a sufficiently large fluidic capacitance is always needed, it does not necessarily be a separate fluidic member.

[0081 ] 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 device (200) for separating a fluidic sample, the sample separation device (200) comprising a first fluid conduit (202) for conducting a fluid; a second fluid conduit (204) diverging from the first fluid conduit (202); a fluidic capacitance (206) arranged in the second fluid conduit (204) and configured for reducing pulsations of the flow in the first fluid conduit (202); a fluidic switch (208) coupled to the fluidic capacitance (206) and being switchable for at least partially discharging the fluidic capacitance (206); and a separation unit (210) arranged in the first fluid conduit (202) and configured for separating the fluidic sample injected into the fluid.

The sample separation device (200) according to the preceding claim, wherein the fluidic switch (208) is switchable for at least partially discharging the fluidic capacitance (206) out of the second fluid conduit (204).

The sample separation device (200) according to claim 1 or any one of the above claims, comprising a control unit (212) configured for switching the fluidic switch (208) for at least partially discharging the fluidic capacitance (206).

The sample separation device (200) according to claim 1 or any one of the above claims, wherein the fluidic switch (208) is switchable so that a predefined flow of the fluid flows through the first fluid conduit (202).

The sample separation device (200) according to claim 1 or any one of the above claims, wherein the fluidic switch (208) is switchable so that a flow of the fluid flowing through the first fluid conduit (202) is constant.

The sample separation device (200) according to claim 1 or any one of the above claims, comprising a sensor (214) arranged in the first fluid conduit (202) and being configured for sensing data indicative of an actual flow rate in the first fluid conduit (202), wherein the fluidic switch (208) is switchable based on the sensed data.

7. The sample separation device (200) according to claim 1 or any one of the above claims, configured for operating the separation unit (210) in a gradient mode.

8. The sample separation device (200) according to claim 1 or any one of the above claims, comprising a drain (216), wherein the fluidic switch (208) is switchable so that the fluidic capacitance (206) is at least partially discharged towards the drain (216).

9. The sample separation device (200) according to the preceding claim, wherein the drain (216) is a waste container.

10. The sample separation device (200) according to claim 1 or any one of the above claims, comprising a fluid drive unit (218) configured for driving the fluid through the first fluid conduit (202), wherein the fluid drive unit (218) is configured for delivering additional fluid to at least one of the first fluid conduit (202) and the second fluid conduit (204) to substitute fluid at least partially discharged from the fluidic capacitance (206).

1 1 . The sample separation device (200) according to claim 1 or any one of the above claims, wherein the fluidic switch (208) is arranged in the second fluid conduit (204).

12. The sample separation device (200) according to the preceding claim, wherein the fluidic switch (208) is arranged between the fluidic capacitance (206) and the drain.

13. The sample separation device (200) according to claim 1 or any one of the above claims, wherein the separation unit (210) is arranged downstream of a bifurcation point (220) at which the second fluid conduit (204) diverges from the first fluid conduit (202).

14. The sample separation device (200) according to claim 1 or any one of the above claims, wherein the fluidic switch (208) is configured to be controllable for selectively enabling or disabling fluid flow through the fluidic switch (208).

15. The sample separation device (200) according to claim 1 or any one of the above claims, wherein the fluidic capacitance (206) comprises a hollow container (600) with a membrane (602) separating an interior of the hollow container (600) in a first volume, having a fluid inlet (604) and a fluid outlet (606), and in a second volume, the membrane (602) being deformable under fluid pressure flowing into the container (600) via the fluid inlet (604).

16. The sample separation device (200) according to the preceding claim, wherein the second volume is filled with a liquid.

17. The sample separation device (200) according to claim 1 or any one of the above claims, wherein the fluidic capacitance (206) comprises a hollow container (700) having a fluid inlet (702) and a fluid outlet (704) and a body (706) being at least partly freely movable within the hollow container (700) and being compressible under pressure.

18. The sample separation device (200) according to claim 1 or any one of the above claims, wherein the fluidic capacitance (206) comprises a hollow container (800) having a fluid inlet (802) and a fluid outlet (804) and having a wall being deformable under pressure.

19. The sample separation device (200) according to claim 1 or any one of the above claims, wherein the fluidic capacitance (206) is at least partly constituted by a natural elasticity of the second fluid conduit (204) and the compressibility of a liquid volume therein.

20. The sample separation device (200) according to claim 1 or any one of the above claims, wherein the first fluid conduit (202) and the second fluid conduit (204) form a bifurcated fluidic network.

21 . The sample separation device (200) according to claim 1 or any one of the above claims, comprising a further fluidic switch (502) and a further fluidic capacitance (504) arranged in a third fluid conduit (500); wherein the further fluidic switch (502) is switchable for at least partia discharging the further fluidic capacitance (504).

The sample separation device (200) according to claim 1 or any one of the above claims, comprising at least one of: the sample separation device (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 device (200) comprises at least one of the group consisting of a sensor device, a test device for testing a device under test or a substance, a device for chemical, biological and/or pharmaceutical analysis, a capillary electrophoresis device, a capillary electrochromatography device, a liquid chromatography device, an HPLC device, a gas chromatography device, a gel electrophoresis device, and a mass spectroscopy device; the sample separation device (200) is configured to conduct the fluid with a high pressure; the sample separation device (200) is configured to conduct the fluid with a pressure of at least 100 bar, particularly of at least 500 bar, more particularly of at least 1000 bar; the sample separation device (200) is configured to conduct a liquid fluid; the sample separation device (200) is configured as a microfluidic device; the sample separation device (200) is configured as a nanofluidic device; the separation unit (210) is configured for retaining a part of components of the fluidic sample and for allowing other components of the fluidic sample to pass the sample separation unit (210); the separation unit (210) comprises a separation column; the separation unit (210) comprises a chromatographic column; at least a part of the separation unit (210) is filled with a separating material; at least a part of the separation unit (210) 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 the separation unit (210) 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.

23. A sample separation device (200) for separating a fluidic sample, the sample separation device (200) comprising a first fluid conduit (202) for conducting a fluid; a second fluid conduit (204) diverging from the first fluid conduit (202); a fluidic capacitance (206) arranged in the second fluid conduit (204); a fluidic switch (208) being switchable so that a buffer volume of the flu id is conducted from the first fluid conduit (202) towards the fluidic capacitance (206) in the second fluid conduit (204); and a separation unit (210) arranged in the first fluid conduit (202) and configured for separating the fluidic sample injected into the fluid.

24. A method of separating a fluidic sample, the method comprising conducting a fluid through a first fluid conduit (202); reducing pulsations of the flow in the first fluid conduit (202) by a fluidic capacitance (206) arranged in a second fluid conduit (204) diverging from the first fluid conduit (202); switching a fluidic switch (208) coupled to the fluidic capacitance (206) for at least partially discharging the fluidic capacitance (206); separating the fluidic sample injected into the fluid in a separation unit (210) arranged in the first fluid conduit (202).

25. A method of separating a fluidic sample, the method comprising conducting a fluid through a first fluid conduit (202); switching a fluidic switch (208) so that a buffer volume of the fluid is conducted from the first fluid conduit (202) towards a fluidic capacitance (206) arranged in a second fluid conduit (204) diverging from the first fluid conduit (202); separating the fluidic sample injected into the fluid in a separation unit (210) arranged in the first fluid conduit (202).