GB2588635A

GB2588635A - Sample injector with fluidic sample mixing

Info

Publication number: GB2588635A
Application number: GB1915716.3A
Authority: GB
Inventors: Pallmann Sebastian; Steflova Jana; Seifried Martin; Spuling Irina; Metzger Benedikt
Original assignee: Agilent Technologies Inc
Current assignee: Agilent Technologies Inc
Priority date: 2019-10-30
Filing date: 2019-10-30
Publication date: 2021-05-05
Anticipated expiration: 2039-10-30
Also published as: GB2588635B; GB201915716D0

Abstract

A sample injector for a chromatography system is configured for injecting a mixed sample fluid into a mobile phase, and comprises a sampling volume 200 configured for receiving and buffering a mixed sample fluid before being injected into the mobile phase, and a switching unit 250 configured for loading the mixed sample fluid into the sampling volume 200 before being injected into the mobile phase, and for injecting the mixed sample fluid into the mobile phase. The injector further comprises a needle 215 fluidically coupled to a fluid drive 245 for aspirating the sample fluid from a receptacle 260. A control unit 70 is configured for firstly mixing the sample fluid in the receptacle 260 by operating the fluid drive 245 to aspirate a volume of mixing fluid (air) through the needle and to introduce at least a portion of the aspirated volume of mixing fluid into the sample fluid within the receptacle 260. The injector enables automated sample mixing.

Description

DESCRIPTION

SAMPLE INJECTOR WITH FLUIDIC SAMPLE MIXING BACKGROUND ART

[0001] The present invention relates to sample injection in particular for chromatographic sample separation.

[0002] For liquid separation in a chromatography 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 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 -1 -also designated as a "peak".

[0005] In preparative chromatography systems, a liquid as the mobile phase is provided usually at a controlled flow rate (e. g. in the range of 1 mL/min to thousands of mL/min, e.g. in analytical scale preparative LC in the range of 1 -5 mL/min and preparative scale in the range of 4 -200 mL/min) and at pressure in the range of tens to hundreds bar, e.g. 20 -600 bar.

[0006] In high performance liquid chromatography (HPLC), a liquid as the mobile phase 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 MPa, 200- 1000 bar, and beyond up to currently 200 MPa, 2000 bar) at which compressibility of the liquid becomes noticeable.

[0007] In preparative chromatography systems used for chromatography fluidically separating samples at a larger volume, typically in the range of 0.1 mL to tens of mL, there often is a need for analysing a smaller volume of such sample prior to running the separation of the larger volume (e.g. in the sense of an "analytical scouting run").

For such purpose, an analytical chromatography system may be used for chromatographically separating smaller sample volumes, typically in the range of 10 uL -50 ul. Such analytical chromatography system may be an HPLC system.

[0008] The Agilent Dual Loop Sampler G2258A, by the applicant Agilent Technologies, Inc., provides a sampling unit for a combined analytical and preparative chromatography system allowing to inject sample into the analytical as well as the preparative chromatography system.

[0009] In several applications, homogenization of sample fluid may be required before sample injection and/or sample fraction reinjection. When preparing dilution mixtures of sample manually, a homogenized concentration profile of sample in the respective dilution matrix can be achieved by shaking, rocking, sonification or centrifugation employing additional instrumentation such as rocking platforms, ultrasonic baths, centrifuges or shakers. This requires tedious transfer of sample vessels from preparation areas/gadgets to target instrumentations and, therefore, relies on human interaction and maintenance efforts. Both diminishes sample throughput, efficiency and increases costs per sample. -2 -

DISCLOSURE

[0010] It is an object of the invention to provide an improved sample injection, preferably for chromatographic sample separation. The object is solved by the independent claims. Further embodiments are shown by the dependent claims.

[0011] In one embodiment, a sample injector for a chromatography system comprises a mobile phase drive and a separation unit. The mobile phase drive is configured for driving a mobile phase through the separation unit. The separation unit is configured for chromatographically separating compounds of a sample fluid in the mobile phase. The sample injector is configured for injecting the sample fluid into the mobile phase and comprises a sampling volume configured for receiving and buffering the sample fluid before being injected into the mobile phase. The sample injector further comprises a switching unit having a first switching configuration configured for loading the sample fluid into the sampling volume before being injected into the mobile phase, and a second switching configuration configured for injecting the sample fluid into the mobile phase. The sample injector further comprises a needle fluidically coupled to a fluid drive, in particular a pump, and configured for aspirating the sample fluid from a receptacle. A control unit is configured for mixing the sample fluid in the receptacle by operating the fluid drive to aspirate a volume of mixing fluid through the needle and to introduce (e.g. inject, blow in, or otherwise supply) at least a portion of the aspirated volume of mixing fluid into the sample fluid within the receptacle. This allows to provide mixing of the sample fluid (also referred to as sample fluid homogenization) within the sample injector so that additional mixing of the sample fluid, e.g. external to the sample injector, can be avoided. The mixing and homogenization may be provided fully automatically without requiring human interaction or additional mixing procedures external to the sample injector, thus improving the workflow and/or reproducibility of sample handling.

[0012] In one embodiment of the injector, the sampling volume is or 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.

[0013] In one embodiment of the injector, the sampling volume is configured for receiving and buffering the sample fluid aspirated by the needle before being injected -3 -into the mobile phase.

[0014] In one embodiment of the injector, in the first switching configuration, the mobile phase drive is fluidically coupled to the separation unit, and the sampling volume is decoupled from a flow of the mobile phase provided by the mobile phase drive. This allows to provide a continuous flow of mobile phase from the mobile phase drive to the separation unit, while at the same time the sampling fluid is decoupled from such flow of the mobile phase and may be loaded with sample fluid e.g. after a previous mixing of such sample fluid within the receptacle.

[0015] In one embodiment of the injector, in the second switching configuration, the sampling volume is fluidically coupled to or into a flow of the mobile phase for injecting the sample fluid into the mobile phase. Injection of the respective sample fluid into the respective mobile phase can be provided by either one or both of the well-known injector schemes, namely "loop injection" and "feed injection". In the loop injection scheme (also referred to as flow-through injection), as described e.g. in US20160334031A1, the respective sample loop is coupled between the respective pump and the respective separation unit, so that the respective mobile phase is flown through the sample loop. In the feed injection scheme, as described e.g. in US2017343520A1, the respective sample loop is fluidically coupled to the flow path between the respective pump and the respective separation unit, so that for sample injection a flow through the sample loop (containing the sample fluid to be injected) is combined with the flow of the mobile phase.

[0016] In one embodiment of the injector preferably implementing the feed injection scheme, in the second switching configuration, the sampling volume is fluidically coupled to a coupling point between the mobile phase drive and the separation unit for combining a flow through the sampling volume with a flow of the mobile phase.

[0017] In one embodiment of the injector preferably implementing loop injection scheme, in the second switching configuration, the sampling volume is fluidically coupled between the mobile phase drive and the separation unit.

[0018] In one embodiment of the injector, the mixing fluid is at least one of: a liquid, preferably a solvent, a gas, preferably ambient air. Ambient air has been found -4 -suitable for mixing the sample fluid in several applications, but other gases, such as noble gases, may be applied accordingly. Also or in addition, liquids may be applied, which may also be used for diluting the sample fluid, thus leading to a combined mixing and diluting of the sample fluid.

[0019] In one embodiment of the injector, the needle comprises an elongated shape with an open end for fluid aspiration, and may be or comprise at least one of a conduit and a nozzle. The open end may be preferably coupled to another open end of a fluid path, preferably a needle seat, in a fluid-tight manner. The needle may have a sharpened end e.g. configured for penetrating through a surface (e.g. a cap or other coverage covering the receptacle), but may also be embodied without such sharpened end, e.g. having a blunt end. Further, while the needle is preferably embodied using a substantially rigid material, such as a metal (e.g. stainless steel), ceramic, et cetera, softer materials may be applied as well e.g. allowing to bend the needle (e.g. in the sense of a soft(er) tubing).

[0020] In one embodiment of the injector, at least one of the mobile phase drive and the fluid drive is or comprises at least one of: a syringe, a syringe pump, a peristaltic pump or roller pump, a venturi valve coupled to a fluid flow generating unit, a pump, and a pumping unit comprising a plurality of pumps, a piston pump, preferably a reciprocating piston pump, a dual pump comprising two piston pumps connected in parallel or serial to each other, a multi-stage step-piston pump, and a modulation pump.

[0021] In one embodiment of the injector, the needle is configured for aspirating the sample fluid from the receptacle by immersing the needle into the receptacle and driving the fluid drive.

[0022] The sample fluid can be drawn in by the fluid drive from the receptacle by the needle. The drawn in sample fluid may be transported directly to or into the sampling volume, however, are kind of transport mechanisms may be applied accordingly as well, e.g. pushing the sample fluid or a combined draw and push scheme so that the sample fluid, or parts thereof, is partly drawn and partly pushed.

This may be executed by the fluid drive only or in conjunction with a fluid transport devices or mechanisms. -5 -

[0023] In one embodiment of the injector, the control unit is configured to control the needle to immerse into the sample fluid within the receptacle and to operate the fluid drive to aspirate a portion of the sample fluid from the receptacle into the needle.

[0024] In one embodiment of the injector, the control unit is configured to control the needle to move into a position wherein the needle is fluidically coupled to the mixing fluid, preferably by moving the needle beyond the sample fluid within the receptacle, and to operate the fluid drive to aspirate the volume of the mixing fluid via the needle into a conduit fluidically coupled to the needle.

[0025] In one embodiment of the injector, the control unit is configured to control the needle to immerse into the sample fluid within the receptacle and to operate the fluid drive to introduce at least a portion of the aspirated volume of mixing fluid into the sample fluid within the receptacle.

[0026] In one embodiment of the injector, wherein the mixing fluid is ambient air, the control unit is configured to control the needle to move beyond the sample fluid within the receptacle and to operate the fluid drive to aspirate a volume of ambient air via the needle into a conduit fluidically coupled to the needle, and to control the needle to immerse into the sample fluid within the receptacle and to operate the fluid drive to introduce at least a portion of the aspirated ambient air into the sample fluid within the receptacle in order to mix the sample fluid, preferably by generating air bubbles within the sample fluid.

[0027] In one embodiment, a chromatography system comprises a mobile phase drive, a separation unit, wherein the mobile phase drive is configured for driving a mobile phase through the separation unit, and the separation unit is configured for chromatographically separating compounds of a sample fluid in the mobile phase.

The chromatography system further comprises a sample injector according to any of the aforementioned embodiments configured for mixing the sample fluid and for injecting the sample fluid into the mobile phase.

[0028] In one embodiment, a method is provided for mixing and injecting a sample fluid in a chromatography system wherein a mobile phase is driven through a separation unit for chromatographically separating compounds of the sample fluid in the mobile phase. The method comprises aspirating a volume of mixing fluid, mixing -6 -the sample fluid in a receptacle by introducing at least a portion of the aspirated volume of mixing fluid into the sample fluid within the receptacle, aspirating and buffering at least a portion of the mixed sample fluid, and injecting at least a portion of the aspirated and buffered sample fluid into the mobile phase.

[0029] In one embodiment, the method further comprises switching into a first switching configuration for aspirating and buffering the portion of the mixed sample fluid. Preferably, in the first switching configuration, the buffered portion of the mixed sample fluid is fluidically decoupled from a flow of the mobile phase, preferably while the mobile phase is driven through the separation unit.

[0030] In one embodiment, the method further comprises switching into a second switching configuration for injecting the portion of the aspirated and buffered sample fluid into the mobile phase. Preferably, in the second switching configuration, the buffered portion of the mixed sample fluid is fluidically coupled to or into a flow of the mobile phase for injecting the sample fluid into the mobile phase.

[0031] In one embodiment, a method is provided for handling a sample fluid in a chromatography system wherein a mobile phase is driven through a separation unit for chromatographically separating compounds of the sample fluid in the mobile phase. The method comprises chromatographically separating the sample fluid, collecting at least a portion of the chromatographically separated sample fluid in a receptacle, aspirating a volume of mixing fluid, mixing the collected portion of sample fluid in the receptacle by introducing at least a portion of the aspirated volume of mixing fluid into the collected portion of sample fluid within the receptacle, aspirating and buffering at least a portion of the mixed sample fluid, and injecting at least a portion of the buffered sample fluid into the mobile phase for further chromatographic separation. This allows providing homogenization of sample fluid received from a previous separation process before re-injecting for further separation.

[0032] In one embodiment of the liquid separation system, e.g. for providing a chromatographic purification workflow, concentrated samples are being separated (e.g. by the separating device) and collected in distinct fractions by the fractionating unit. The purity of those fractions may determine whether or not the workflow has to be repeated. Therefore, the target compound of the separation may be analyzed -7 -again. In other words, the separated compounds of the sample fluid, or parts thereof, as output by the fractionating unit can be fed back as sample fluid to be injected by the sample injector for further separation. This may require mixing and homogenisation of fractions to ensure that the drawn sample from the given fraction provides a representative result. This can be done by aspirating a volume of mixing fluid (such as ambient air) and introducing at least a portion of the aspirated volume of mixing fluid into the sample fluid within the receptacle before further injection. In contrast to manually mixing or by evaporating the target fractions by means of a rotary evaporator and dissolving the resulting residue in an appropriate solvent, interruptions in the workflow can be avoided leading to an increased cost efficiency as well as a reduced risk of sample loss.

[0033] Embodiments of the present invention allow for an automated homogenization of concentration differences in both sample and fraction mixtures which may be the result of layer formation due to density differences. Those density differences may occur in dilution and fraction collection workflows where either sample matrix and dilution solvent or fractions of different mobile phase compositions are merged. In the latter case, a concentration profile along the time axis of the collection can be the result. Therefore, homogenization may be required to obtain reproducible and representative results. Embodiments of the present invention may allow for an automated purification workflow where sample separation and re-analysis of the target can be conducted within one instrumentation that performs the preparative injection, fraction collection, homogenization of fraction and re-analysis of fractions.

[0034] Embodiments of the present invention may allow for an automated homogenization for liquid sample handling in dilution purposes as well as an automated homogenization of collected fraction of a preparative chromatographic run so that consequent re-analysis of those fractions becomes possible. This way, no human interaction is required within either workflows improving drastically sample handling, throughput, target recovery and, overall, costs per sample. It may further improve reproducibility and minimize human based errors. It may also replace the need for extra instrumentation aiming at homogenization of sample or fractions.

[0035] Embodiments of the present invention might be embodied based on most -8 -conventionally available HPLC systems, such as the Agilent 1220, 1260 and 1290 Infinity LC Series (provided by the applicant Agilent Technologies).

[0036] 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.

[0037] 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.

[0038] 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 cm to 1 m) or a microfluidic column (as disclosed e.g. in EP 1577012 Al or the Agilent 1200 Series HPLC-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 -9 -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.

[0039] 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.

[0040] 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.

[0041] 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).

[0042] 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).

[0043] 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.

[0044] 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.

[0045] In the context of this application, the term "fluidic sample" may particularly denote any liquid and/or gaseous medium, optionally including also solid particles, which is to be analyzed. Such a fluidic sample may comprise a plurality of fractions of molecules or particles which shall be separated, for instance biomolecules such as proteins. Since separation of a fluidic sample into fractions involves a certain separation criterion (such as mass, volume, chemical properties, etc.) according to which a separation is carried out, each separated fraction may be further separated by another separation criterion (such as mass, volume, chemical properties, etc.) or finer separated by the first separation criterion, thereby splitting up or separating a separate fraction into a plurality of sub-fractions.

[0046] 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. Further, layering due to density differences may occur within one or more fractions. The term "fraction" may denote a portion of a solvent containing the aforementioned group of molecules.

[0047] In the context of this application, the term "sub-fractions" may particularly denote individual groups of molecules or particles all relating to a certain fraction which still differ from one another regarding one or more certain properties of the group of: mass, charge, volume, chemical or physical properties or interaction, etc. Hence, applying another separation criterion for the second separation as compared to the separation criterion for the first separation allows these groups to be further separated from one another by applying the other separation criterion, thereby obtaining the further separated sub-fractions. As well the term "sub-fraction" may denote a portion of a solvent containing the aforementioned individual 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. -12-

[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 pod 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 "buffer" or "buffering" may particularly be understood as temporarily storing. Accordingly, the term "buffering fluid" is preferably understood as temporarily storing an amount of fluid, which may later be fully or partly retrieved from such unit buffering the fluid.

[0055] 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.

[0056] In the context of this application, the term "retain", "retaining", or similar, in particular in context with "unit", may particularly be understood as providing a surface (e.g. a coating) and/or a stationary phase configured for interacting with a fluid in the sense of having a desired retention characteristics with one or more components contained in the fluid. Such desired retention characteristics shall be understood as an intentionally applied retention, i.e. a retention beyond an unintentional side-effect. Accordingly, the term "retaining unit" may be understood as a unit in a fluidic path being configured for interacting with a sample fluid for providing a desired retention characteristic for one or more components contained in the sample fluid.

[0057] 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.

[0058] 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

[0059] 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.

[0060] Fig. 1 illustrates a liquid chromatography system according to an exemplary embodiment.

[0061] Figures 2 illustrate preferred embodiments of a sample injector 40 according to the present invention.

[0062] 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 drives the mobile phase through a separating device 30 (such as a chromatographic column). A sample injector 40 (also referred to as sample introduction apparatus, sample dispatcher, 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. 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. In one embodiment, at least parts of the sample injector 40 and the fractionating unit 60 can be combined, e.g. in the sense that some common hardware is used as applied by both of the sample injector 40 and the fractionating unit 60.

[0063] 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).

[0064] 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 and downstream of the mobile phase drive 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.

[0065] 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 injector 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 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.

[0066] Figures 2A-2F illustrate preferred embodiments of a sample injector 40 according to the present invention.

[0067] Figure 2A shows a sampling volume 200, a sampling unit 210 comprised of a needle 215 and a needle seat 218, and an optional retaining unit 220, all arranged in a serial connection and providing a sampling path 230. Further coupled to the sampling path 230 is a sampling fluid drive 240, which in the embodiment of Figures 2 is comprised of a metering device 245 and an optional fluid pump 248. The fluid pump 248 may be coupled to one or more solvent reservoirs 249 as schematically indicated in the drawings.

[0068] A switching unit 250 is coupled to the sampling path 230, the sampling fluid drive 240, the mobile phase drive 20, and the separating device 30, as will be explained later in more detail, in particular showing the various configurations of the switching unit 250.

[0069] The sampling volume 200 is configured for temporarily storing an amount of the received fluidic sample, and can be any of a sample loop, a sample volume, a trap volume, a trap column, a fluid reservoir, a capillary, a tube, a microfluidic channel 30 structure.

[0070] The retaining unit 220 is configured for receiving and retaining from the sampling volume 200 at least a portion of the fluidic sample stored in the sampling volume 200. The retaining unit 220 may be further configured to show different retention characteristics for different components of the fluidic sample.

[0071] Figure 2A shows an open position of the sampling unit 210 wherein the needle 215 is physically separated from the needle seat 218, thus allowing the needle 215 to receive a fluidic sample e.g. from a sample vessel 260 (e.g. a vial) as indicated in the exemplary representation of Figure 2A. It is clear that instead of the sample vessel 260, the fluidic sample may be provided to the needle 215 by any other way as known in the art, e.g. coupling to an online sampling loop is provided e.g. from a chemical or biological reactor. Further, instead of the needle 215 and the needle seat 218 any other mechanism for fluidically coupling to a fluidic sample can be applied accordingly, e.g. a nozzle, a tubing having an open end to be inserted into the vessel 260 or otherwise contracted with fluidic sample.

[0072] In the exemplary embodiment of Figures 2, the switching unit 250 is a rotational valve having a stator 250S and a rotor 250R, as shown in detail in Figure 2B. The stator 250S has six ports (indicated by reference numerals 1-6 and each port allowing to couple e.g. a fluid conduit thereto as shown e.g. in Figure 2A), a first static groove 251, and a second static groove 252. The first static groove 251 extends from port 1, and the second static groove 252 extends from port 2. The rotor 250R comprises a first (dynamic) groove 253, a second (dynamic) groove 254, and a third (dynamic) groove 255. While Figure 28 shows the stator 250S and the rotor 250R separated from each other, the stator 250S and the rotor 250R are facing each other and are centrally aligned to each other (thus allowing a rotational movement relative to each other between the stator 250S and the rotor 250R as depicted in the different configurations of Figures 2). It is clear that other valve configurations may be applied accordingly.

[0073] In Figures 2, the mobile phase drive 20 is coupled to port 1, a first end 246 of the metering device 245 is coupled to port 2 while a second end 247 of the metering device 245 is coupled to the sampling volume 200. The fluid pump 248 is coupled to port 3. Port 4 is coupled to waste or any other fluidic unit as indicated by reference numeral 270. Port 5 is coupled to a first end 221 of the retaining unit 220 while the second end 222 of the retaining unit 220 is coupled to the needle seat 218. Port 6 is coupled to the separating device 30.

[0074] In Figure 2A, the switching unit 250 is in a first switching configuration for loading the sample fluid into the sampling volume 200 before being injected into the mobile phase. The first groove 253 is coupling between ports 1 and 6, thus coupling the mobile phase drive 20 to the separating device 30, so that the mobile phase drive 20 drives the mobile phase through the switching unit 250 and the separating device 30. The first groove 253 is overlapping with the first static groove 251. The second groove 254 and the second static groove 252 are overlapping with each other but not reaching to any other port, so that port 2 is blocked, and thus also the first end 246 of the metering device 245 is blocked.

[0075] Before aspirating any sample fluid from the vessel 260, mixing of the sample fluid in the vessel 260 can be provided, e.g. in order to mix segmented fractions or other inhomogeneous characteristics of the sample fluid. With the needle 250 being separated from the needle seat 218 and in the open position as shown in Figure 2A and in detail in Figure 2C, the control unit 70 can operate the metering device 245 to provide a backwards movement (as indicated by the arrow in Figure 2A), which will aspirate a volume of ambient air through the needle 215 into the sampling unit 210, e.g. into the sampling volume 200 and/or any coupled conduit. As shown in detail drawing of Figure 2D, the control unit 70 then operates the needle 215 to immerse into the sample fluid within the vessel 260, preferably so that the needle 215 reaches through an upper surface 262 of sample fluid. By providing a forward movement (opposite to the arrow in Figure 2A) of the metering device 245, at least a portion of the aspirated air can be introduced into the sample fluid within the vessel 260, which will lead to a mixing of the sample fluid, in particular as resulting from the air bubbles 264 moving within the vessel 260. Instead of ambient air, any other suitable mixing fluid can be aspirated and introduced into the sample fluid, such as other gases (e.g. a noble gas) or liquids (which may further lead to a dilution of the sample fluid).

[0076] With the needle 215 still being immersed into the sample fluid within the vessel 260 (e.g. as depicted in Figure 2D), the metering device 245 can be operated to provide a backwards movement (as indicated by the arrow in Figure 2A) allowing the needle 215 to aspirate or retrieve sample fluid from the vessel 260 (e.g. when the needle 215 is immersed into the vessel 260). By further backwards movement of the needle 215, the received sample fluid will also be drawn into the sampling volume 200 and can be temporarily stored therein.

[0077] It is clear that the processes of mixing of the sample fluid and aspirating of the sample fluid into the sampling volume 200 can also be separated and in a different sequence or order than explained above. However, it goes without saying that in such case the needle 215, if not already immersed into the sample fluid, needs to be moved from the open position (as depicted in Figure 2C) to immerse into the sample fluid (as depicted in Figure 2D).

[0078] Figure 2E shows a switching configuration of the switching unit 250 allowing a so-called feed injection of buffered sample fluid into the mobile phase. Rotor 250R has been rotated and is positioned so that the first groove 253 together with the first static groove 251 are coupling ports 1, 6, and 5 fluidically together and representing a first coupling point 259. When the metering device 245 is applying a forward movement (as indicated by the arrow in Figure 2E), fluid content contained in the sampling volume 200 can be pushed (fed) into the mobile phase. In other words, a flow within the sampling path 230 is combined in the first coupling point 259 with a flow of the mobile phase from the mobile phase drive 20, and the combined flow is provided towards the separating device 30.

[0079] Figure 2F shows a different switching configuration (than Figure 2E) of the switching unit 250 allowing a so-called flow through injection of buffered sample fluid into the mobile phase. The first groove 253 is coupling between ports 1 and 2, and the third groove 255 is coupling between ports 5 and 6, so that the sampling path 230 (together with the metering device 245) is switched between the mobile phase drive and the separating device 30. Accordingly, any sample fluid within the sampling path 230 and in particular within the sampling volume 200 will be transported by the mobile phase provided from the mobile phase drive 20 towards and through the separating device 30. In an alternate embodiment, e.g. a so-called fixed loop injection, not shown here, the metering device 245 can be separated from the sampling path 230, so that only the sampling path 230 will be switched between the mobile phase drive 20 and the separating device 30. -20-

[0080] In one embodiment of the liquid separation system 10, e.g. for providing a chromatographic purification workflow, concentrated samples are being separated (by the separating device 30) and collected in distinct fractions by the fractionating unit 60. The purity of those fractions may determine whether or not the workflow has to be repeated. Therefore, the target compound of the separation may be analyzed again. In other words, the separated compounds of the sample fluid, or pads thereof, as output by the fractionating unit 60 may be fed back as sample fluid to be injected by the sample injector 40 for further separation. This may require mixing of fractions to ensure that the drawn sample from the given fraction gives a representative result.

This can be done as illustrated above with respect to Figures 2, namely by aspirating a volume of mixing fluid (such as ambient air) and introducing at least a portion of the aspirated volume of mixing fluid into the sample fluid within the vessel 260 before further injection. In contrast to manually mixing or by evaporating the target fractions by means of a rotary evaporator and dissolving the resulting residue in an appropriate solvent, interruptions in the workflow can be avoided leading to an increased cost efficiency as well as a reduced risk of sample loss. -21 -

Claims

CLAIMS1. A sample injector (40) for a chromatography system comprising a mobile phase drive (20) and a separation unit (30), wherein the mobile phase drive (20) is configured for driving a mobile phase through the separation unit (30), and the separation unit (30) is configured for chromatographically separating compounds of a sample fluid in the mobile phase, the sample injector (40) being configured for injecting the sample fluid into the mobile phase and comprising: a sampling volume (200) configured for receiving and buffering the sample fluid before being injected into the mobile phase; a switching unit (250) comprising a first switching configuration (Figure 2A) configured for loading the sample fluid into the sampling volume (200) before being injected into the mobile phase, and a second switching configuration (Figure 2C; Figure 2D) configured for injecting the sample fluid into the mobile phase; a needle (215) fluidically coupled to a fluid drive (245), in particular a pump, and configured for aspirating the sample fluid from a receptacle (260); and a control unit (70) configured for mixing the sample fluid in the receptacle (260) by operating the fluid drive (245) to aspirate a volume of mixing fluid through the needle (215) and to introduce at least a portion of the aspirated volume of mixing fluid into the sample fluid within the receptacle (260).
2. The injector (40) of claim 1, comprising at least one of: the sampling volume (200) is or 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; the sampling volume (200) is configured for receiving and buffering the sample fluid aspirated by the needle (215) before being injected into the mobile phase.
3. The injector (40) of claim 1 or any of the above claims, wherein -22 -in the first switching configuration (Figure 2A), the mobile phase drive (20) is fluidically coupled to the separation unit (30), and the sampling volume (200) is decoupled from a flow of the mobile phase provided by the mobile phase drive (20)
4. The injector (40) of claim 1 or any of the above claims, wherein in the second switching configuration (Figure 2C; Figure 2D), the sampling volume (200) is fluidically coupled to or into a flow of the mobile phase for injecting the sample fluid into the mobile phase.
5. The injector (40) of the preceding claim, comprising at least one of: in the second switching configuration (Figure 2C -"feed injection"), the sampling volume (200) is fluidically coupled to a coupling point between the mobile phase drive (20) and the separation unit (30) for combining a flow through the sampling volume (200) with a flow of the mobile phase; in the second switching configuration (Figure 2D -"loop injection"), the sampling volume (200) is fluidically coupled between the mobile phase drive (20) and the separation unit (30).
The injector (40) of claim 1 or any of the above claims, comprising at least one of: the mixing fluid is at least one of: a liquid, preferably a solvent, a gas, preferably ambient air; the needle (215) comprises an elongated shape with an open end for fluid aspiration, and may be or comprise at least one of a conduit (100) and a nozzle (110), wherein preferably the open end can be coupled to another open end of a fluid path, preferably a needle seat, in a fluid-tight manner; at least one of the mobile phase drive (20) and the fluid drive (245) is or comprises at least one of: a syringe, a syringe pump, a peristaltic pump or roller pump, a venturi valve coupled to a fluid flow generating unit, a pump, and a pumping unit comprising a plurality of pumps, a piston pump, preferably a reciprocating piston pump, a dual pump comprising two piston pumps connected -23-in parallel or serial to each other, a multi-stage step-piston pump, and a modulation pump.
7. The injector (40) of claim 1 or any of the above claims, comprising at least one of: the needle (215) is configured for aspirating the sample fluid from the receptacle (260) by immersing the needle into the receptacle (260) and driving the fluid drive (245); the control unit (70) is configured to control the needle (215) to immerse into the sample fluid within the receptacle (260) and to operate the fluid drive (245) to aspirate a portion of the sample fluid from the receptacle (260) into the needle (215); the control unit (70) is configured to control the needle (215) to move into a position wherein the needle (215) is fluidically coupled to the mixing fluid, preferably by moving the needle (215) beyond the sample fluid within the receptacle (260), and to operate the fluid drive (245) to aspirate the volume of the mixing fluid via the needle (215) into a conduit (100) fluidically coupled to the needle (215); the control unit (70) is configured to control the needle (215) to immerse into the sample fluid within the receptacle (260) and to operate the fluid drive (245) to introduce at least a portion of the aspirated volume of mixing fluid into the sample fluid within the receptacle (260).
8. The injector (40) of claim 1 or any of the above claims: wherein the mixing fluid is ambient air, and the control unit (70) is configured to control the needle (215) to move beyond the sample fluid within the receptacle (260) and to operate the fluid drive (245) to aspirate a volume of ambient air via the needle (215) into a conduit (100) fluidically coupled to the needle (215), and to control the needle (215) to immerse into the sample fluid within the receptacle (260) and to operate the fluid drive (245) to introduce at least a portion of the aspirated ambient air into the sample fluid within the receptacle (260) in order to mix the sample fluid by generating air bubbles within the sample fluid.-24 - 9.
A chromatography system (10) comprising: a mobile phase drive (20), a separation unit (30), wherein the mobile phase drive (20) is configured for driving a mobile phase through the separation unit (30), and the separation unit (30) is configured for chromatographically separating compounds of a sample fluid in the mobile phase, a sample injector (40) according to any of the above claims being configured for mixing the sample fluid and for injecting the sample fluid into the mobile phase.
A method mixing and injecting a sample fluid in a chromatography system (10) wherein a mobile phase is driven through a separation unit (30) for chromatographically separating compounds of the sample fluid in the mobile phase, the method comprising: aspirating a volume of mixing fluid, mixing the sample fluid in a receptacle (260) by introducing at least a portion of the aspirated volume of mixing fluid into the sample fluid within the receptacle, aspirating and buffering at least a portion of the mixed sample fluid, and injecting at least a portion of the aspirated and buffered sample fluid into the mobile phase.
11. The method of the above method claim, further comprising: switching into a first switching configuration for aspirating and buffering the portion of the mixed sample fluid.
12. The method of the preceding claim, wherein in the first switching configuration, the buffered portion of the mixed sample fluid is fluidically decoupled from a flow of the mobile phase, preferably while the mobile phase is driven through the separation unit (30).
13. The method of any of the above method claims, further comprising: -25-switching into a second switching configuration for injecting the portion of the aspirated and buffered sample fluid into the mobile phase.
14. The method of the preceding claim, wherein in the second switching configuration, the buffered portion of the mixed sample fluid is fluidically coupled to or into a flow of the mobile phase for injecting the sample fluid into the mobile phase.A method of handling a sample fluid in a chromatography system (10) wherein a mobile phase is driven through a separation unit (30) for chromatographically separating compounds of the sample fluid in the mobile phase, the method comprising: chromatographically separating the sample fluid, collecting at least a portion of the chromatographically separated sample fluid in a receptacle (260), aspirating a volume of mixing fluid, mixing the collected portion of sample fluid in the receptacle (260) by introducing at least a portion of the aspirated volume of mixing fluid into the collected portion of sample fluid within the receptacle, aspirating and buffering at least a portion of the mixed sample fluid, and injecting at least a portion of the buffered sample fluid into the mobile phase for further chromatographic separation.16 A software program or product, preferably stored on a data carrier, for controlling or executing the method of any of the above method claims, when run on a data processing system such as a computer.-26 -