GB2597469A

GB2597469A - Pressurizing fluid by expanding membrane

Info

Publication number: GB2597469A
Application number: GB2011331.2A
Authority: GB
Inventors: Bäuerle Martin
Original assignee: Agilent Technologies Inc
Current assignee: Agilent Technologies Inc
Priority date: 2020-07-22
Filing date: 2020-07-22
Publication date: 2022-02-02
Also published as: GB202011331D0

Abstract

A pressurising unit 250 comprising first 320 and second 330 chambers separated by a membrane 340 that expands into the first chamber when there is a pressure difference between the chambers. Fluid to be pressurized is introduced into the first chamber and substantially isolated. Pressurising fluid is introduced into the second chamber in order to pressurise the fluid isolated in the first chamber by expansion of the membrane. Pressurising the fluid may mean increasing or reducing the pressure of the fluid or at least reducing pressure pulsations within the fluid. A sample dispatcher for a fluid separator comprises a sampling path having a sampling volume, the pressurizing unit and an injection switching unit. A software program or product may execute a pressurisation method using the unit. The unit may be used as part of a high-pressure liquid chromatography (HPLC) system.

Description

Intellectual Property Office Application No G1320113312 RTM Date:20 January 2021 The following terms are registered trade marks and should be read as such wherever they occur in this document: Agilent Intellectual Property Office is an operating name of the Patent Office www.gov.uk/ipo

DESCRIPTION

PRESSURIZING FLUID BY EXPANDING MEMBRANE BACKGROUND ART

[0001] The present invention relates to pressurizing fluid in particular for a sample dispatching preferably for chromatographic sample separation.

[0002] In high performance liquid chromatography (HPLC), a liquid has to be provided usually at a very controlled flow rate (e. g. in the range of microliters to milliliters per minute) and at high pressure (typically 20-100 M Pa, 200-1000 bar, and beyond up to currently 200 MPa, 2000 bar) at which compressibility of the liquid becomes noticeable. For liquid separation in an HPLC system, a mobile phase comprising a sample fluid (e.g. a chemical or biological mixture) with compounds to be separated is driven through a stationary phase (such as a chromatographic column packing), thus separating different compounds of the sample fluid which may then be identified. The term compound, as used herein, shall cover compounds which might comprise one or more different components.

[0003] The mobile phase, for example a solvent, is pumped under high pressure typically through a chromatographic column containing packing medium (also referred to as packing material or stationary phase). As the sample is carried through the column by the liquid flow, the different compounds, each one having a different affinity to the packing medium, move through the column at different speeds. Those compounds having greater affinity for the stationary phase move more slowly through the column than those having less affinity, and this speed differential results in the compounds being separated from one another as they pass through the column. The stationary phase is subject to a mechanical force generated in particular by a hydraulic pump that pumps the mobile phase usually from an upstream connection of the column to a downstream connection of the column. As a result of flow, depending on the physical properties of the stationary phase and the mobile phase, a relatively high-pressure drop is generated across the column.

[0004] The mobile phase with the separated compounds exits the column and passes through a detector, which registers and/or identifies the molecules, for -1 -example by spectrophotometric absorbance measurements. A two-dimensional plot of the detector measurements against elution time or volume, known as a chromatogram, may be made, and from the chromatogram the compounds may be identified. For each compound, the chromatogram displays a separate curve feature also designated as a "peak". Efficient separation of the compounds by the column is advantageous because it provides for measurements yielding well defined peaks having sharp maxima inflection points and narrow base widths, allowing excellent resolution and reliable identification and quantitation of the mixture constituents. Broad peaks, caused by poor column performance, so called "Internal Band Broadening" or poor system performance, so called "External Band Broadening" are undesirable as they may allow minor components of the mixture to be masked by major components and go unidentified.

[0005] Sample injectors in chromatography systems are provided for injecting the sample fluid into the mobile phase. Such sample injectors typically comprise a needle for aspirating the sample fluid e.g. from a sample vial. For injecting the (aspirated) sample fluid into the mobile phase, the needle can be inserted into a corresponding needle seat fluidically coupled to a chromatographic column for separating compounds of the sample fluid in the mobile phase.

[0006] W02010139359A1, by the same applicant, discloses prepressurization of the sample fluid before injection into the mobile phase in order to reduce or avoid adverse effects resulting from pressure artefacts. Such pressure artefacts may result from the sample fluid having a different pressure than the mobile phase before injecting the sample fluid into the mobile phase. Decreasing the pressure difference between sample fluid and mobile phase can reduce or even avoid such adverse pressure effects.

DISCLOSURE

[0007] It is an object of the invention to provide an improved pressurization of fluid, in particular for prepressurization of a sample fluid for sample dispatching, preferably for chromatographic sample separation. The object is solved by the independent 30 claims. Further embodiments are shown by the dependent claims.

[0008] A preferred embodiment provides a pressurizing unit for pressurizing fluid. -2 -

The pressurizing apparatus comprises a first chamber, a second chamber, and a membrane between the first chamber and the second chamber. The membrane is configured to expand into the first chamber resulting from a pressure different between the first chamber and the second chamber. In other words, the membrane is configured to expand into the first chamber when a pressure in the second chamber is higher than in the first chamber, and vice versa. The pressurizing unit is configured to introduce a fluid to be pressurized into the first chamber and to substantially (at least to an extent allowing pressurizing the fluid isolated within the first chamber) isolate the first chamber, and to introduce a pressurizing fluid into the second chamber in order to pressurize the fluid isolated within the first chamber by expanding the membrane into the first chamber. This allows to pressurize fluid, e.g. in order to remove or at least reduce pressure pulsations in the fluid, increase or reduce pressure of the fluid, or the like.

[0009] The term "pressurize" as used herein can mean increasing pressure as well as decreasing pressure, depending on the respective application and pressure conditions in the first chamber and in the second chamber.

[0010] In one embodiment, the pressurizing unit further comprises a pressurizing switching unit fluidically coupling to the first chamber and to the second chamber, and a control unit configured for controlling the pressurizing switching unit to introduce the fluid to be pressurized into the first chamber and to substantially isolate the first chamber, and to introduce the pressurizing fluid into the second chamber in order to pressurize the fluid isolated within the first chamber by expanding the membrane into the first chamber. This allows to control the process of isolation and pressurization by means and operation of the pressurizing switching unit and the control unit.

[0011] The control unit may be configured for controlling the pressurizing switching unit for introducing the fluid to be pressurized into the first chamber by opening the first chamber on at least one side in order to allow the fluid to be pressurized to flow into the first chamber. Alternatively or in addition, the control unit may be configured for isolating the first chamber by (at least substantially) blocking an (or any if plural entries) entry into and an (or any if plural outlets) outlet from the first chamber.

Alternatively or in addition, the control unit may be configured for isolating the fluid to be pressurized within the first chamber by (at least substantially) blocking an entry -3 -into and an outlet from the first chamber. Alternatively or in addition, the control unit may be configured for introducing the pressurizing fluid into the second chamber by opening the second chamber on at least one side of the second chamber in order to allow the pressurizing fluid to flow into the second chamber. Alternatively or in addition, the control unit may be configured for removing the pressurized fluid from the first chamber. Alternatively or in addition, the control unit may be configured for removing the pressurized fluid from the first chamber by opening the first chamber on at least one side of the first chamber.

[0012] In one embodiment, the pressurizing unit further comprises a first pumping unit configured for moving the fluid to be pressurized.

[0013] In one embodiment, the first chamber has a first input and a first output, wherein preferably the fluid to be pressurized is introduced via the first input and the first chamber is isolated by closing the first input and the first output: [0014] In one embodiment, the second chamber has a second input and a second output, wherein preferably the pressurizing fluid is flown through the second chamber from the second input to the second output, at least during isolation of the first chamber.

[0015] In one embodiment, the pressurizing unit further comprises a second pumping unit. The second pumping unit may be configured for moving the pressurizing fluid. Alternatively or in addition, the second pumping unit may be configured for increasing a pressure of the pressurizing fluid in the second chamber in order to pressurize the fluid isolated within the first chamber by expanding the membrane into the first chamber.

[0016] In one embodiment, the pressurizing unit further comprises a housing comprising the first chamber, the second chamber, and the membrane. The housing may comprise a plurality of layers, wherein the first chamber is provided by a first void within the plurality of layers, the second chamber is provided by a second void within the plurality of layers, and the membrane is provided by at least one layer intermediate between the first void and the second void. The plurality of layers may be comprised by a plurality of metal layers. At least one layer of the plurality of layers may be comprised by a material of the group: metal, preferably Stainless Steel (SST), bio -4 -inert metals such as titan, et cetera; plastic materials, preferably a polymer, PEEK, PTFE, PFA, et cetera, typically depending on pressure regime applied; or other suitable materials. Such materials may also be coated (typically on suitable carrier materials), which may be applied e.g. after layer bonding. At least two layers of the plurality of layers may be bonded to each other, preferably by diffusion bonding, ultrasonic bonding, friction or laser welding, et cetera. Preferably, the plurality of layers may be provided by diffusion bonded metal sheets, e.g. as disclosed in W02017025857A1 by the same applicant.

[0017] One embodiment of the present invention provides a sample dispatcher for a fluid separation apparatus. The fluid separation apparatus comprises a mobile phase drive, configured for driving a mobile phase, and a separating device configured for separating a portion of a fluidic sample when comprised within the mobile phase. The sample dispatcher comprises a sampling path having a sampling volume configured for receiving the fluidic sample, a pressurizing unit according to any of the aforementioned embodiments, wherein the fluid to be pressurized is at least a portion of the fluidic sample, the pressurizing fluid is the mobile phase, and the first chamber is the sampling volume, and an injection switching unit, preferably comprising a valve, fluidically coupling to the sampling path, the mobile phase drive, and the separating device. The injection switching unit comprises a loading state configured for loading the fluidic sample into the sampling volume and for pressurizing the fluidic sample within the sampling volume by means of the pressurizing unit, and an injection state configured for injecting at least a portion of the pressurized fluid sample into the mobile phase.

[0018] The sample dispatcher may further comprise a sampling fluid drive configured for moving the fluidic sample and being fluidically coupled to the injection switching unit.

[0019] The sampling path may comprise a needle and a needle seat, wherein in an open position the needle is configured to be separated from the needle seat in order to receive the fluidic sample, and in a closed position the needle is configured to be fluidically sealingly coupled with the needle seat.

[0020] The sampling volume may comprise at least one of a group of: a sample -5 -loop, a sample volume, a trap volume, a trap column, a fluid reservoir, a capillary, a tube, a microfluidic channel structure.

[0021] The sample dispatcher may further comprise a control unit configured to control operation of the sample dispatcher, preferably at least one of: operation of the sampling fluid drive, switching of the injection switching unit, and switching of the pressurizing switching unit.

[0022] One embodiment according to the present invention provides a fluid separation apparatus comprising a mobile phase drive, configured for driving a mobile phase, and a separating device configured for separating a portion of a fluidic sample when comprised within the mobile phase. The fluid separation apparatus comprises a sample dispatcher, according to any one of the aforementioned embodiments, configured for dispatching at least a portion of the fluidic sample to the fluid separation apparatus.

[0023] One embodiment according to the present invention provides a method for pressurizing fluid The method comprises introducing a fluid to be pressurized into a first chamber, wherein a membrane is coupled between the first chamber and a second chamber, the membrane being configured to expand into the first chamber resulting from a pressure different between the first chamber and the second chamber. The method further comprises substantially isolating the first chamber, and pressurizing the fluid isolated within the first chamber by introducing a pressurizing fluid into the second chamber in order to expand the membrane into the first chamber.

[0024] The method may further comprise introducing the fluid to be pressurized into the first chamber by opening the first chamber on at least one side of the first chamber in order to allow the fluid to be pressurized to flow into the first chamber.

[0025] The method may further comprise isolating the first chamber by blocking any entry into and any outlet from the first chamber.

[0026] The method may further comprise isolating the fluid to be pressurized within the first chamber by blocking any entry into and any outlet from the first chamber.

[0027] The method may further comprise introducing the pressurizing fluid into the second chamber by opening the second chamber on at least one side of the second -6 -chamber in order to allow the pressurizing fluid to flow into the second chamber.

[0028] The method may further comprise removing the pressurized fluid from the first chamber.

[0029] The method may further comprise removing the pressurized fluid from the first chamber by opening the first chamber on at least one side of the first chamber.

[0030] The method may further comprise pressurizing the fluid isolated within the first chamber by introducing the pressurizing fluid into the second chamber, and increasing a pressure of the pressurizing fluid in the second chamber in order to expand the membrane into the first chamber.

[0031] The method may further comprise injecting at least a portion of the pressurized fluid into a mobile phase, and chromatographically separating the injected fluid fluidic sample.

[0032] Injection of the fluidic sample into a mobile phase for chromatographic separation in embodiments according to the present invention may be provided using the so-called flow through injection (as described e.g. in US20160334031A1 by the same applicant) and/or the so-called feed injection (as described e.g. in US2017343520A1 by the same applicant).

[0033] In feed injection configuration of the injection switching unit, the mobile phase drive, the separating device, and the sampling path are coupled together in a first coupling point, and the sampling fluid drive is coupled to the sampling path for combining into the first coupling point a flow from the sampling fluid drive with a flow of the mobile phase from the mobile phase drive, wherein the flow from the sampling fluid drive is through the sampling path and containing at least a portion of the fluidic sample.

[0034] In a flow through configuration of the injection switching unit, the sampling path is coupled between the mobile phase drive and the separating device for introducing the portion of the fluidic sample into the mobile phase.

[0035] In a preferred embodiment comprising feed injection and flow through configuration, both sample introduction types, namely feed injection and flow through -7 -injection, can be applied e.g. with the same injection switching unit, thus allowing a user to select the appropriate sample introduction type for a specific application.

[0036] Embodiments of the present invention might be embodied based on most conventionally available HPLC systems, such as the Agilent 1220, 1260 and 1290 Infinity LC Series (provided by the applicant Agilent Technologies).

[0037] One embodiment of an HPLC system comprises a pumping apparatus having a piston for reciprocation in a pump working chamber to compress liquid in the pump working chamber to a high pressure at which compressibility of the liquid becomes noticeable.

[0038] One embodiment of an HPLC system comprises two pumping apparatuses coupled either in a serial or parallel manner. In the serial manner, as disclosed in EP 309596 Al, an outlet of the first pumping apparatus is coupled to an inlet of the second pumping apparatus, and an outlet of the second pumping apparatus provides an outlet of the pump. In the parallel manner, an inlet of the first pumping apparatus is coupled to an inlet of the second pumping apparatus, and an outlet of the first pumping apparatus is coupled to an outlet of the second pumping apparatus, thus providing an outlet of the pump. In either case, a liquid outlet of the first pumping apparatus is phase shifted, preferably essentially by 180 degrees, with respect to a liquid outlet of the second pumping apparatus, so that only one pumping apparatus is supplying into the system while the other is intaking liquid (e.g. from the supply), thus allowing to provide a continuous flow at the output. However, it is clear that also both pumping apparatuses might be operated in parallel (i.e. concurrently), at least during certain transitional phases e.g. to provide a smooth(er) transition of the pumping cycles between the pumping apparatuses. The phase shifting might be varied in order to compensate pulsation in the flow of liquid as resulting from the compressibility of the liquid. It is also known to use three piston pumps having about 120 degrees phase shift. Also other types of pumps are known and operable in conjunction with the present invention.

[0039] The separating device preferably comprises a chromatographic column providing the stationary phase. The column might be a glass, metal, ceramic or a composite material tube (e.g. with a diameter from 50 pm to 5 mm and a length of 1 -8 -cm to 1 m) or a microfluidic column (as disclosed e.g. in EP 1577012 Al or the Agilent 1200 Series H PLC-Chip/MS System provided by the applicant Agilent Technologies). The individual components are retained by the stationary phase differently and separate from each other while they are propagating at different speeds through the column with the eluent. At the end of the column they elute at least partly separated from each other. During the entire chromatography process the eluent might be also collected in a series of fractions. The stationary phase or adsorbent in column chromatography usually is a solid material. The most common stationary phase for column chromatography is silica gel, followed by alumina. Cellulose powder has often been used in the past. Also possible are ion exchange chromatography, reversed-phase chromatography (RP), affinity chromatography or expanded bed adsorption (EBA). The stationary phases are usually finely ground powders or gels and/or are microporous for an increased surface, which can be especially chemically modified, though in EBA a fluidized bed is used.

[0040] The mobile phase (or eluent) can be either a pure solvent or a mixture of different solvents. It can also contain additives, i.e. be a solution of the said additives in a solvent or a mixture of solvents. It can be chosen e.g. to adjust the retention of the compounds of interest and/or the amount of mobile phase to run the chromatography. The mobile phase can also be chosen so that the different compounds can be separated effectively. The mobile phase might comprise an organic solvent like e.g. methanol or acetonitrile, often diluted with water. For gradient operation water and organic solvent is delivered in separate containers, from which the gradient pump delivers a programmed blend to the system. Other commonly used solvents may be isopropanol, THF, hexane, ethanol and/or any combination thereof or any combination of these with aforementioned solvents.

[0041] The sample fluid might comprise any type of process liquid, natural sample like juice, body fluids like plasma or it may be the result of a reaction like from a fermentation broth.

[0042] The fluid is preferably a liquid but may also be or comprise a gas and/or a supercritical fluid (as e.g. used in supercritical fluid chromatography -SFC -as disclosed e.g. in US 4,982.597 A). -9 -

[0043] The pressure in the mobile phase might range from 2-200 MPa (20 to 2000 bar), in particular 10-150 MPa (100 to 1500 bar), and more particular 50-130 MPa (500 to 1300 bar).

[0044] The HPLC system might further comprise a detector for detecting separated compounds of the sample fluid, a fractionating unit for outputting separated compounds of the sample fluid, or any combination thereof. Further details of HPLC system are disclosed with respect to the aforementioned Agilent HPLC series, provided by the applicant Agilent Technologies.

[0045] Embodiments of the invention can be partly or entirely embodied or supported by one or more suitable software programs or products, 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 or by the control unit, e.g. a data processing system such as a computer, preferably for executing any of the methods described herein.

[0046] In the context of this application, the term "fluidic sample" or "sample fluid" 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.) or finer separated by the first separation criterion, thereby splitting up or separating a separate fraction into a plurality of sub-fractions.

[0047] 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 one or more certain properties of the group of: mass, charge, volume, chemical or physical properties or interaction, 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. As well the term "fraction" may denote a portion of a solvent containing the aforementioned group of molecules.

[0048] 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. The terms "downstream" and "upstream" may also relate to a preferred direction of the fluid flow between the two members being in downstream-upstream relation.

[0049] In the context of this application, the term "sample separation apparatus", "fluid separation apparatus" or similar 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 apparatus 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 at least one or some of the fractions resulting from the first separation are subsequently separated in accordance with a second, different, separation criterion or more finely separated in accordance with the first separation criterion.

[0050] The term "separation unit", "separation device" or similar 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 or retaining and selectively releasing different fractions of the fluidic sample.

[0051] In the context of this application, the term "fluid drive", "mobile phase drive" or similar may particularly denote any kind of pump which is configured for forcing a flow of mobile phase and/or a fluidic sample along a fluidic path. A corresponding liquid supply system may be configured for delivery of a single liquid or of 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 drive the fluidic sample, usually mixed with, or injected into a flow of a mobile phase (solvent composition), through the first-dimension separation apparatus, whereas the second fluid drive can be configured for driving the fluidic sample fractions, usually mixed with a further mobile phase (solvent composition), after treatment (e.g. elution) by the first-dimension separation unit through the second-dimension separation apparatus.

[0052] In the context of this application, the term "flow coupler" or "coupling point" 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. 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. A respective coupling point may be configured as one of the group consisting of a fluidic T-piece, a fluidic Y-piece, a fluidic X-piece, microfluidic junction, a group of at least 3 ports of a rotary valve, connectable together in at least one of configurations of the said rotary valve and a multi-entry port of a rotary valve.

[0053] In the context of this application, the term "valve" or "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.

[0054] In the context of this application, the term "loop" may particularly be understood as a fluid conduit allowing to temporarily store an amount of fluid, which may later be fully or partly retrieved from the loop. Preferably, such loop has an elongation along the flow direction of the fluid and a limited mixing characteristic (e.g. resulting from dispersion), so that a spatial variation in composition in the fluid will be at least substantially maintained along the elongation of the loop. Accordingly, the term "sample loop" may be understood as a loop configured to temporarily store an amount of sample fluid. Further accordingly, a sample loop is preferably configured to at least substantially maintain a spatial variation in the sample fluid (along the flow direction of the sample), as e.g. resulting from a previous chromatographic separation of the sample fluid, during temporarily storing of such sample fluid.

[0055] In the context of this application, the term "couple", "coupled", "coupling", or similar, in particular in context with "fluidic" or "fluidically", may particularly be understood as providing a fluidic connection at least during a desired time interval.

Such fluidic connection may not be permanent but allows a (passive and/or active) transport of fluid between the components fluidically coupled to each other at least during such desired time interval. Accordingly, fluidically coupling may involve active and/or passive components, such as one or more fluid conduits, switching elements (such as valves), et cetera.

[0056] The fluid separation apparatus may be configured to drive the mobile phase through the system by means of a high pressure, particularly of at least 400 bar, more particularly of at least 1000 bar.

BRIEF DESCRIPTION OF DRAWINGS

[0057] 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.

[0058] Fig. 1 illustrates a liquid chromatography system according to an exemplary embodiment.

[0059] Figures 2 and 3 illustrate an embodiment of the mobile phase drive 20 supplied by the solvent supply 25 according to the present invention.

[0060] Figures 4 illustrate an embodiment of the sample dispatcher 40 according to the present invention.

[0061] Referring now in greater detail to the drawings, Fig. 1 depicts a general schematic of a liquid separation system 10. A mobile phase drive 20 (such as a pump) receives a mobile phase from a solvent supply 25, typically via a degasser 27, which degases the mobile phase and thus reduces the amount of dissolved gases in it. The mobile phase drive 20 provides a pressurized mobile phase at an output 21 and drives the (pressurized) mobile phase through a separating device 30 (such as a chromatographic column). A sample dispatcher 40 (also referred to as sample introduction apparatus, sample injector, etc.) is provided between the mobile phase drive 20 and the separating device 30 in order to subject or add (often referred to as sample introduction) portions of one or more sample fluids into the flow of a mobile phase (denoted by reference numeral 200, see also Fig. 2). The separating device 30 is adapted for separating compounds of the sample fluid, e.g. a liquid. 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.

[0062] The separating device 30 may comprise a stationary phase configured for separating compounds of the sample fluid. Alternatively, the separating device 30 may be based on a different separation principle (e.g. field flow fractionation).

[0063] While the mobile phase can be comprised of one solvent only, it may also be mixed of plurality of solvents. Such mixing might be a low pressure mixing and provided upstream of the mobile phase drive 20, so that the mobile phase drive 20 already receives and pumps the mixed solvents as the mobile phase. Alternatively, the mobile phase drive 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 mobile phase drive 20 (or as part thereof). The composition (mixture) of the mobile phase may be kept constant over -14-time, the so-called isocratic mode, or varied over time, the so-called gradient mode.

[0064] 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 mobile phase drive 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. monitoring the level or amount of the solvent available) and/or the degasser 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 sample dispatcher 40 (e.g. controlling sample introduction or synchronization of the sample introduction with operating conditions of the mobile phase drive 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. The data processing unit 70 might also process the data received from the system or its part and evaluate it in order to represent it in adequate form prepared for further interpretation.

[0065] Figure 2 illustrates in more detail parts of an embodiment of the liquid separation system 10 in accordance with the schematic representation of Figure 1, namely an embodiment of the mobile phase drive 20 supplied by the solvent supply 25. In the embodiment of Figure 2, the mobile phase drive 20 is provided as a binary pump comprising two pumping units 200A and 200B, which may be embodied as disclosed e.g. in the aforementioned EP309596A1. The pumping unit 200A comprises -in a parallel coupling -a primary pump 210A and a secondary pump 215A. The pumping unit 200B comprises -also in a parallel coupling -a primary pump 210B and a secondary pump 215B.

[0066] The solvent supply 25 is provided by a first solvent 25A and a second solvent 25B, which are preferably different solvents e.g. in order to provide a gradient having a solvent composition varying over time. The first solvent 25A is supplied -via a first Y-connector 220A -to inputs of both primary pump 210A and secondary pump 215A, and the second solvent 25B is supplied -via a second Y-connector 220B -to inputs of both primary pump 210B and secondary pump 215B. Y-connectors 220A and 220B may also be combined into or by a single casing and/or switching unit.

[0067] Outputs of both primary pump 210A and secondary pump 215A are fed to and combined by a first coupler 225A, which may be a T-junction or the like, and outputs of both primary pump 210B and secondary pump 215B are fed to and combined by a second coupler 225B, which may be a T-junction or the like.

[0068] A seal washing for the two pumping units 200A and 200B may be provided in order to remove residues of e.g. crystalized salt which improved lifetime for the seals. For that purpose, a seal was pump 230 can be provided which is configured for pumping a seal wash solvent 235 to each pump of the two pumping units 200A and 200B, e.g. as schematically depicted in Figure 2 by a serial coupling. A seal wash sensor 240 may be provided to indicate if seal wash fluid is still available and thus ensures the proper function of the sealwash.

[0069] An output 245 of the first coupler 225A provides the pressurized first solvent 25A, and an output 248 of the second coupler 225B provides the pressurized second solvent 25B. Both outputs 245 and 248 are provided to a pressurizing unit 250 which shall be shown and explained in greater detail with respect to Figure 3. The pressurizing unit 250 is provided for reducing pressure pulsations in the pressurized solvent streams respectively supplied at the outputs 245 and 248 and for combining these both solvent streams (from the outputs 245 and 248) into a combined output 255.

[0070] The embodiment of Figure 2 may have an optional pressure sensor 260 coupled to the combined output 255, an optional purge valve 265 for releasing the backpressure in hydraulic line 21 and thus may make it easier to prime the pump after e.g. running out of solvent, and an optional mixer 270 for (additionally) mixing the solvent mixture (of solvents 25A and 25B) as provided by the combined output 255. The output 21 of the mobile phase drive 20 (see also Figure 1) in the embodiment of Figure 2 is downstream of the mixer 270 but may also be anywhere downstream to the pressurizing unit 250 dependent on the implementation of one or more of the optional components of the pressure sensor 260, the purge valve 265, and the mixer 270.

[0071] Figure 3 shows in greater detail the pressurizing unit 250 together with additional components coupled thereto, as indicated by dotted box 280. The pressurizing unit 250 of the embodiment of Figure 3 comprises a pressurizing chamber 300 and a coupler 310 (which may be a T-junction or similar). The pressurizing chamber 300 comprises a first chamber 320, a second chamber 330, and a membrane 340. The membrane 340 is provided between the first chamber 320 and the second chamber 330. The membrane 340 is configured to expand into the first chamber 320 when a pressure in the second chamber 330 is higher than a pressure in the first chamber 320, and vice versa. In other words, a pressure difference between the first chamber 320 and the second chamber 330 will cause the membrane 340 to expand into the one of the first chamber 320 and the second chamber 330 where the pressure is lower than in the other one of the first chamber 320 and the second chamber 330.

[0072] Such expansion of the membrane 340 will reduce the volume into the chamber into which the membrane 340 expands. With the coupler 310 coupling the outlets of both the first chamber 320 and the second chamber 330 together, and both pumping units 200A and 200B actively pumping towards the inlet of the first chamber 320 and the second chamber 330, the first chamber 320 and the second chamber 330 are substantially isolated, so that an expansion of the membrane 340 into one chamber (of the first chamber 320 and the second chamber 330) will accordingly increase the pressure in the other chamber (of the first chamber 320 and the second chamber 330) into which the membrane 340 expands, thus pressurizing the fluid in such other chamber.

[0073] In an example, when the pressure in the first chamber 320 is higher than in the second chamber 330, e.g. resulting in a pressure ripple in the pressurized first solvent 25A, the membrane 340 will expand into the second chamber 330, thus increasing the pressure in the second chamber 330 and accordingly pressurize the fluid in the second chamber 330. This allows e.g. to reduce pressure ripples in the pressurized first solvent 25A and second solvent 25B.

[0074] It is to be understood that due to the coupler 310 coupling the outlets of both the first chamber 320 and the second chamber 330 together, the pressure level in the first chamber 320 and the second chamber 330 is already substantially equal. On the other hand, pulsations (i.e. temporary variations in pressure with an amplitude typically being significantly smaller than the pressure level at the coupler 310) may result e.g. from the reciprocating movements of the pumping units 200A and 200B, compressibility of the solvents (such as the first solvent 25A and the second solvent 25B or the resulting mixture thereof), elasticity of certain components in the flow path, or other sources, as generally known in chromatography. The pressurizing unit 250 allows reducing (and preferably eliminating) pressure ripples (such as pulsations) in the pressurized first solvent 25A and the pressurized second solvent 25B. In other words, while the pressurized first solvent 25A and the pressurized second solvent 25B in the embodiment of Figures 2 and 3 are already substantially at the same pressure level, the pressurizing unit 250 will mainly act on and balance pressure ripples (such as pulsations) of the pressurized first solvent 25A and the pressurized second solvent 25B, which may be considered as a dynamic pressurizing.

[0075] Figures 4 schematically illustrate in more detail parts of another embodiment of the liquid separation system 10 in accordance with the schematic representation of Figure 1, namely an embodiment of the sample dispatcher 40 configured for dispatching sample fluid into the high-pressure path between the mobile phase drive 20 and the separating device 30. Figure 4A shows an arrangement and process step for drawing up a sample fluid. Figure 4B shows an arrangement and process step for pressurizing the sample fluid (as e.g. drawn according to Figure 4A). Figure 4C shows an arrangement and process step for injecting the sample fluid (e.g. drawn according to Figure 4A and/or pressurized according to Figure 4B) into the mobile phase in the high-pressure path between the mobile phase drive 20 and the separating device 30.

[0076] In Figures 4, the sample dispatcher 40 comprises a needle-seat arrangement 400 comprised of a needle 405 allowing to aspirate sample fluid e.g. from an external vessel 410, a needle seat 415 configured for sealingly receiving the needle 405, and an actuator 420 allowing to move and position the needle 405. In Figure 4A, the actuator 420 has positioned the needle 405 into the external vessel 410, e.g. a vial, well plate or similar, in order to aspirate sample fluid from within the vessel 410 into a sample loop 430 (fluidically) coupled to needle 405. In Figures 4B and 4C, the actuator 420 has positioned the needle 405 into the needle seat 415.

[0077] The sample dispatcher 40 further comprises a metering device 440 allowing to aspirate sample fluid into the sample loop 430 as depicted in Figure 4A. The metering device 440 may have one or more additional functionalities, such as pressurizing and/or depressurizing fluid contained in the sample loop 430, ejecting sample fluid contained in the sample loop 430 into the mobile phase (e.g. in the sense of feed injection as disclosed e.g. in the aforementioned US2017343520A1), or others. Further, the metering device 440 may be (preferably integral) part of a sampling path comprising the sample loop 430, as exemplarily shown in the embodiment of Figures 4, or may be separated or switchable off from the sample loop 430 as e.g. shown in W02014199198A1 by the same applicant.

[0078] Further, the sample dispatcher 40 comprises a switching valve 450 in particular allowing to fluidically couple or decouple the sample loop 430 to or from the high-pressure flow path of the mobile phase between the pump 20 and the separating device 30, as also depicted in the different switching configurations of the Figures 4 and which will be further explained later. In the exemplary embodiment of Figures 4, the switching valve 450 is configured as a three-position rotational valve having six separate ports 1-6 and two rotatable grooves 452 and 454 allowing to fluidically couple adjacent ports, as readily known in the art.

[0079] Further, the sample dispatcher 40 comprises a pressurizing unit 460. In the exemplary embodiment of Figures 4, the pressurizing unit 460 is embodied in accordance with the pressurizing chamber 300, namely having the first chamber 320, the second chamber 330, and the membrane 340 between the first chamber 320 and the second chamber 330. Functioning and operation of the pressurizing unit 460 will be described in more detail later.

[0080] In the embodiment of Figures 4, port 1 is coupled to a first end of a sampling path 455 comprised of and coupling the first chamber 320, the metering device 440, the sample loop 430, and the needle-seat arrangement 400. Port 2 is coupled to a port which may be closed (as indicated by X) with a blind plug 456, in order to be able to hold the pressure in chamber 320 once groove 452 is in its alternate position between port 1 and 2. Port 3 is coupled to a waste 457 allowing to dispose volumes of fluid which may not be required any more. Port 4 is coupled to the other end of the sampling path 455. Port 5 is coupled to the separating device 30. Port 6 is coupled to one end of the second chamber 330, while the other end of the second chamber 330 is coupled to the mobile phase drive 20.

[0081] It is clear that the switching valve 450 may also be embodied by other valve types, such as a translational valve, and that other configurations of ports, coupling grooves, and their respective fluidic connections and couplings are also possible, as well known in the art.

[0082] In Figure 4A, the sample dispatcher 40 is in a sample draw mode allowing to draw sample fluid from the vessel 410 into the sample loop 430 by operation of the metering device 440. Groove 452 is coupling between ports 1 and port 2. At the same time, groove 454 couples between ports 5 and port 6, so that the switching valve 450 couples the mobile phase drive 20 via the second chamber 330 to the separating device 30. Accordingly, the second chamber 330 will be substantially on system pressure (i.e. the pressure provided by the mobile phase drive 20 typically in the range of a few hundred bar up to 2000 bar). While the first chamber 320 is substantially at ambient pressure and typically significantly lower than the system pressure, any expansion of the membrane 340 into the first chamber 320 will not increase pressure (at least not noticeable) in the first chamber 320 and the sampling path 455, because the sampling path 455 is open on at least one end (and in the exemplary embodiment of Figure 4A even on both ends).

[0083] In Figure 4B, the sample dispatcher 40 is now in a pressurizing mode in order to pressurize (i.e. to increase pressure) in the sampling path 455 for pressurizing the sample fluid contained in the sample loop 430. Grooves 452 and 454 have been moved (clockwise with respect to the position in Figure 4A), with groove 452 only coupling to port 1, so that the sampling path 455 is now blocked on both -20 -ends. Groove 454 still couples between port 5 and port 6, as in Figure 4A, so that the switching valve 450 couples the mobile phase drive 20 via the second chamber 330 to the separating device 30. Accordingly, the second chamber 330 will be substantially on system pressure. On switching (from Figure 4A) into the position of Figure 4B, the sampling path 455 and thus the first chamber 320 is initially at about ambient pressure and at least at significantly lower pressure than the system pressure present in the second chamber 330. With the sampling path 455 being blocked on both ends, the first chamber 320 is now substantially isolated, and the membrane 340 will expand into the first chamber 320 due to the pressure difference between the second chamber 330 and the first chamber 320, thus increasing the pressure in the first chamber 320 and in the sampling path 455 until pressure equilibration is reached. Accordingly, pressure of the sample fluid in the sample loop 430 will also increase and may reach a pressure level close to or in the range of the system pressure (resident in the high-pressure flow path between the mobile phase drive 20 and the separating device 30).

[0084] In Figure 40, the sample dispatcher 40 is now in a sample injection mode in order to inject the sample fluid contained in the sample loop 430 into the mobile phase allowing to chromatographically separate the injected sample fluid by the separating device 30. When switching from the pressurizing mode of Figure 4B into the sample injection mode of Figure 40, the sample fluid has been (pre-)pressurized to a pressure level substantially in the range of the mobile phase (system pressure), so that pressure ripples resulting from significant pressure differences (between the sample fluid and the mobile phase) can be reduced or even be avoided. Such pressure ripples may adversely affect quality of the chromatographic separation (e.g. by inducing fake peaks into the resulting chromatogram) and/or damage components in the liquid separation system 10 (in particular the separating device 30), as a known problem in the art.

[0085] In Figure 40, the grooves 452 and 454 have been moved (clockwise with respect to the position in Figure 4B), so that groove 452 couples between port 1 and port 6, groove 454 couples between port 4 and port 5, and the sampling path 455 is now coupled between the mobile phase drive 20 and the separating device 30. Driven by the mobile phase 20, the mobile phase will transport the sample fluid contained in the sample loop 430 towards and through the separating device 30. With the first -21 -chamber 320 and the second chamber 330 being substantial at the same pressure level, namely system pressure, the membrane 340 will assume its middle position and not expand (at least not substantially) into either one of the first chamber 320 and the second chamber 330.

[0086] The embodiments of Figures 3 and 4 can be individual and separate embodiments but may also be combined in a single liquid separation system 10 (e.g. providing pulsations reduction as well as sample fluid pressurization before injection).

[0087] The pressuring chamber 300 and/or the pressurizing unit 460 is preferably provided to minimise dead volume, i.e. any additional volume in a respective fluid path within the liquid separation system 10. Dead volumes, as generally known in chromatography, can lead to broader peaks and thus less resolution. Furthermore dead volume may lead to longer analysis times as the entire volume need to be flushed before changes in the solvent composition become active at the column.

[0088] The pressuring chamber 300 and/or the pressurizing unit 460 may have more than two chambers (as shown in Figures 3 and 4) with one or more membranes between two or more chambers, e.g. an array of chambers with one or more intermediate membranes.

[0089] The pressuring chamber 300 and/or the pressurizing unit 460 may be provided by a plurality of layers. The first chamber 320 may be provided by a first void within the plurality of layers, the second chamber 330 may be provided by a second void within the plurality of layers, and the membrane 340 may be provided by at least one layer intermediate between the first void and the second void. The plurality of layers may preferably be of or contain one or plural materials of metal, ceramic, polymer, or the like. Preferably, the plurality of layers may be provided by diffusion bonded metal sheets, e.g. as disclosed in W0201 7025857A1 by the same applicant.

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Claims

CLAIMS1. A pressurizing unit (250; 460) for pressurizing fluid, the pressurizing unit (250; 460) comprising: a first chamber (320), a second chamber (330), and a membrane (340) between the first chamber (320) and the second chamber (330), wherein the membrane (340) is configured to expand into the first chamber (320) resulting from a pressure different between the first chamber (320) and the second chamber (330), wherein the pressurizing unit (250: 460) is configured to introduce a fluid to be pressurized into the first chamber (320) and to substantially isolate the first chamber (320), and to introduce a pressurizing fluid into the second chamber (330) in order to pressurize the fluid isolated within the first chamber (320) by expanding the membrane (340) into the first chamber (320).
2. The pressurizing unit (250; 460) according to the preceding claim, further comprising: a pressurizing switching unit (310, 200; 450) fluidically coupling to the first chamber (320) and to the second chamber (330), and a control unit (70) configured for controlling the pressurizing switching unit (310, 200; 450) to introduce the fluid to be pressurized into the first chamber (320) and to substantially isolate the first chamber (320), and to introduce the pressurizing fluid into the second chamber (330) in order to pressurize the fluid isolated within the first chamber (320) by expanding the membrane (340) into the first chamber (320).
3. The pressurizing unit (250; 460) according to the preceding claim, wherein the control unit (70) is configured for controlling the pressurizing switching unit (310, 200; 450) for at least one of: introducing the fluid to be pressurized into the first chamber (320) by opening -23 -the first chamber (320) on at least one side in order to allow the fluid to be pressurized to flow into the first chamber (320); isolating the first chamber (320) by blocking an entry into and an outlet from the first chamber (320); isolating the fluid to be pressurized within the first chamber (320) by blocking an entry into and an outlet from the first chamber (320); introducing the pressurizing fluid into the second chamber (330) by opening the second chamber (330) on at least one side of the second chamber (330) in order to allow the pressurizing fluid to flow into the second chamber (330); removing the pressurized fluid from the first chamber (320); removing the pressurized fluid from the first chamber (320) by opening the first chamber (320) on at least one side of the first chamber (320).
4. The pressurizing unit (250; 460) according to any one of the above claims, further comprising at least one of: a first pumping unit (200; 440) configured for moving the fluid to be pressurized; the first chamber (320) has a first input and a first output, wherein preferably the fluid to be pressurized is introduced via the first input and the first chamber (320) is isolated by closing the first input and the first output; the second chamber (330) has a second input and a second output, wherein preferably the pressurizing fluid is flown through the second chamber (330) from the second input to the second output, at least during isolation of the first chamber (320).
5. The pressurizing unit (250; 460) according to any one of the above claims, further comprising a second pumping unit (200; 20) configured for at least one of: moving the pressurizing fluid; increasing a pressure of the pressurizing fluid in the second chamber (330) in -24 -order to pressurize the fluid isolated within the first chamber (320) by expanding the membrane (340) into the first chamber (320).
6. The pressurizing unit (250; 460) according to any one of the above claims, further comprising: a housing comprising the first chamber (320), the second chamber (330), and the membrane (340), wherein preferably the housing comprises a plurality of layers, the first chamber (320) is provided by a first void within the plurality of layers, the second chamber (330) is provided by a second void within the plurality of layers, and the membrane (340) is provided by at least one layer intermediate between the first void and the second void.
7. The pressurizing unit (250; 460) according to the preceding claim, further comprising at least one of: the plurality of layers comprises a plurality of metal layers; at least one layer of the plurality of layers is comprised or coated by a material of the group: metal, preferably Stainless Steel (SST) or bio inert metals such as titan, plastic materials, preferably a polymer, PEEK, PTFE, or PFA; wherein at least two layers of the plurality of layers are bonded to each other, preferably by diffusion bonding, ultrasonic bonding, friction or laser welding.
8. A sample dispatcher (40) for a fluid separation apparatus (10), wherein the fluid separation apparatus (10) comprises a mobile phase drive (20; 200), configured for driving a mobile phase, and a separating device (30) configured for separating a portion of a fluidic sample when comprised within the mobile phase; the sample dispatcher (40) comprising: a sampling path (455) having a sampling volume (430) configured for receiving the fluidic sample, a pressurizing unit (250; 460) according to any one of the above claims, wherein the fluid to be pressurized is at least a portion of the fluidic sample, -25 -the pressurizing fluid is the mobile phase, and the first chamber (320) is the sampling volume (430), and an injection switching unit (450), preferably comprising a valve, fluidically coupling to the sampling path (455), the mobile phase drive (20; 200), and the separating device (30); wherein the injection switching unit (450) comprises a loading state configured for loading the fluidic sample into the sampling volume (430) and for pressurizing the fluidic sample within the sampling volume (430) by means of the pressurizing unit (250; 460), and an injection state configured for injecting at least a portion of the pressurized fluid sample into the mobile phase.
9. The sample dispatcher (40) according to the preceding claim, further comprising at least one of: a sampling fluid drive (440) configured for moving the fluidic sample and being fluidically coupled to the injection switching unit (450); the sampling path (455) comprises a needle (405) and a needle seat (415), wherein in an open position the needle (405) is configured to be separated from the needle seat (415) in order to receive the fluidic sample, and in a closed position the needle (405) is configured to be fluidically sealingly coupled with the needle seat (415); the sampling volume (430) comprises at least one of a group of: a sample loop, a sample volume, a trap volume, a trap column, a fluid reservoir, a capillary, a tube, a microfluidic channel structure.
10. The sample dispatcher (40) according to any one of the above claims, further comprising: a control unit (70) configured to control operation of the sample dispatcher (40), preferably at least one of: operation of the sampling fluid drive (440), switching of the injection switching unit (450), and switching of the pressurizing switching unit (310, 200; 450).
11. A fluid separation apparatus (10) comprising a mobile phase drive (20; 200), -26 -configured for driving a mobile phase, and a separating device (30) configured for separating a portion of a fluidic sample when comprised within the mobile phase; the fluid separation apparatus (10) further comprising: a sample dispatcher (40), according to any one of the above claims, configured for dispatching at least a portion of the fluidic sample to the fluid separation apparatus (10).
12. A method for pressurizing fluid, the method comprising: introducing a fluid to be pressurized into a first chamber (320), wherein a membrane (340) is coupled between the first chamber (320) and a second chamber (330), the membrane (340) being configured to expand into the first chamber (320) resulting from a pressure different between the first chamber (320) and the second chamber (330), substantially isolating the first chamber (320), and pressurizing the fluid isolated within the first chamber (320) by introducing a pressurizing fluid into the second chamber (330) in order to expand the membrane (340) into the first chamber (320).
13. The method of the preceding claim further comprising at least one of: introducing the fluid to be pressurized into the first chamber (320) by opening the first chamber (320) on at least one side of the first chamber (320) in order to allow the fluid to be pressurized to flow into the first chamber (320); isolating the first chamber (320) by blocking any entry into and any outlet from the first chamber (320); isolating the fluid to be pressurized within the first chamber (320) by blocking any entry into and any outlet from the first chamber (320); introducing the pressurizing fluid into the second chamber (330) by opening the second chamber (330) on at least one side of the second chamber (330) in order to allow the pressurizing fluid to flow into the second chamber (330); -27 -removing the pressurized fluid from the first chamber (320); removing the pressurized fluid from the first chamber (320) by opening the first chamber (320) on at least one side of the first chamber (320); pressurizing the fluid isolated within the first chamber (320) by introducing the pressurizing fluid into the second chamber (330), and increasing a pressure of the pressurizing fluid in the second chamber (330) in order to expand the membrane (340) into the first chamber (320).
14. The method according to any one of the above claims, further comprising: injecting at least a portion of the pressurized fluid into a mobile phase, and chromatographically separating the injected fluid fluidic sample.
15. A software program or product, preferably stored on a data carrier, for executing a method according to any one of above claims, when run on a data processing system such as a computer.-28 -