US20230135114A1 - Sample Injector With Metering Device Balancing Pressure Differences In An Intermediate Valve State - Google Patents

Abstract

A sample injector for use in a fluid separation system for separating compounds of a fluidic sample in a mobile phase, the sample injector comprising a switchable valve, a sample loop in fluid communication with the valve and configured for receiving the fluidic sample, a metering device in fluid communication with the sample loop and configured for introducing a metered amount of the fluidic sample on the sample loop, and a control unit configured for controlling switching of the valve to transfer the sample loop between a low pressure state and a high pressure state via an intermediate state and for controlling the metering device during the intermediate state to at least partially equilibrate a pressure difference in the sample loop between the low pressure state and the high pressure state.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS
• The present application is a continuation application under 37 C.F.R. § 1.53(b) of commonly owned U.S. patent application Ser. No. 16/993,070, filed on Aug. 13, 2020; which is a continuation of U.S. patent application Ser. No. 15/965,402, filed on Apr. 27, 2018; which is a continuation application under 37 C.F.R. § 1.53(b) of commonly owned U.S. patent application Ser. No. 15/622,913, filed on Jun. 14, 2017; which is a continuation application under 37 C.F.R. § 1.53(b) of commonly owned U.S. patent application Ser. No. 14/877,758, filed on Oct. 7, 2015; which is a continuation application under 37 C.F.R. § 1.53(b) of commonly owned U.S. patent application Ser. No. 13/375,884, filed on Dec. 2, 2011; which is the national stage application of International Application No. PCT/EP2009/056795, filed on Jun. 3, 2009; the contents of each of which are incorporated by reference herein in their entireties.
BACKGROUND ART
• The present invention relates to sample injectors, in particular in a high performance liquid chromatography application.
• In high performance liquid chromatography (HPLC, see for instance http://en.wikipedia.org/wiki/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 MPa, 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 with compounds to be separated is driven through a stationary phase (such as a chromatographic column), thus separating different compounds of the sample fluid.
• Valves are commonly used in HPLC applications, for instance injection valves for introducing a liquid sample into a high pressure flowing stream of liquid, a purge valves for positive displacement pumps, flow path switching valves, etc. Such valves used in HPLC applications are often multi-position rotary valve. Examples of multi-position rotary valve are disclosed in U.S. Pat. No. 4,068,528 A (two-position valves) or US 2003/0098076 A1 (multi-function rotary valves or random-access, dual, three-way, rotary switching valves).
• Shear valves, which can be used in multi-way embodiments, are usually formed by a housing and a body defining a stepped cavity in which the rotor or seal is positioned. The housing contains at least two shear seal valve members positioned to be aligned with ports in the rotor (body) to establish communication between the shear seal means. Shear valves are usually provided as rotary valves (such as the aforementioned rotary valves) or translational valves (often also called sliding valves), such as disclosed in EP 0321774 A2.
• A multi-way switching valve provides a means for selectively routing a fluid input flow to the valve to one of more alternate output flows from the valve. A rotary valve is of the type wherein fluid flow is directed by rotating a valve rotor element to discrete angular positions relative to a stationary valve stator element. A dual rotary valve provides two valves in one valve body, both simultaneously operated by the positioning of the valve rotor. Rotary switching valves are commonly used, for example, in HPLC and other analytical methods to selectively direct a flow stream of one or more fluids along alternate paths to an analytical device or containment vessel.
• The aforementioned US 2003/0098076 A1 shows in itsFIG.1 a conventional type of dual, three-way,switching valve220, which includes a disc-shaped rotor with a set of rotor grooves in the front face of the rotor that contacts, in a fluid-tight manner, the face of a cylindrically shaped stator body at a rotor-stator interface. Inlet passages and outlet passages, longitudinally bored through the stator body to the rotor-stator interface, are selectively fluidly coupled through the rotor grooves corresponding to the rotation of the rotor relative to the stator. Pivoting of the rotor enables the rotor grooves to fluidly couple selected passages of the stator, depending on their placement on the rotor and the angular position of the valve rotor. Model 7030 of Rheodyne, L. P. is an example of this type of switching valve.
• WO 2007/109529 discloses methods and apparatus for placing a sample in a chromatographic system. The device and method feature placing samples held in a sample loop to pressurization prior to placing such sample loop in communication with high pressure conduits.
• WO 2008/005845 discloses a method for processing a fluid applied to systems that include a valve unit that has a sample-loading state and a sample-introducing state. The sample-loading state disposes a sample loop in fluidic communication with a sample conduit. The sample-introducing state disposes the sample loop in fluidic communication with a process conduit. The method involves transferring a sample through both the sample conduit and the valve unit so that a leading end of the sample exits the valve unit. After transitioning the valve unit to the sample-loading state and allowing the sample loop to decompress, at least some of the transferred sample is loaded into the sample loop. A fluid-processing instrument includes a value unit and a control unit that manages operation of the instrument. The control unit is configured, for example, to implement the above-described method.
• WO 2006/083776 discloses a method and apparatus for substantially eliminating destructive transients of pressure or flow rate which can degrade the efficiency and useful lifetime of chromatography columns. The system enables a substantially constant flow of mobile phase liquid to be maintained through the chromatography system by eliminating the flow blockage interval associated with the actuation of sample injection valves. The system further provides a method to reduce the pressure and flow rate transients associated with pressurization of the sample loop contents when the sample loop is introduced to chromatography system delivery pressure.
• WO 2006/023828 discloses systems, devices, and methods to mitigate the pressure disturbance associated with the injection of low-pressure analyte samples into a high-pressure HPLC fluid stream, to enhance chromatographic performance related to retention time and reproducibility. An embodiment coordinates the injection run with active pressure control of a binary solvent delivery system to virtually eliminate the customary pressure drop when the low-pressure loop is brought on line. An additional feature is accomplished by forcing a consistent timing relationship between the injection run, the mechanical position of the delivery pump pistons, and the start and subsequent gradient delivery.
• US 2007/0251302 discloses a flow path switching valve in which an impact due to the pressure change when a flow path is switched is prevented from being generated. A rotor slot allows an analysis infusion pump to be connected to an analytical column, so as to form a flow path (condensing procedure). The rotor of the flow path switching valve is rotated clockwise for 30 degrees, the rotor slot allows the analysis infusion pump, the analytical column, and a trap column be connected. After the pressure in the trap column is raised to the same pressure level as that of the analytical column, the pressure is stabilized, and the pressure difference between the two columns is counteracted (high-pressure procedure). After the pressure between the two columns has been stabilized sufficiently, the rotor is further rotated for 30 degrees, and the trap column and the analytical column are connected in series, so the sample analysis can be performed (dissolution procedure and detection procedure).
• In modern HPLC with pressures rising up to 100 MPa and beyond, life time of sample injectors becomes critical, in particular for the injection valve, as a high pressure load acts on the components particularly when switching between a high pressure operation mode and a low pressure operation mode, which causes excessive wear.
DISCLOSURE
• It is an object of the invention to provide an improved sample injector, in particular for high pressure HPLC applications. The object is solved by the independent claims. Further embodiments are shown by the dependent claims.
• According to an embodiment of the present invention, a sample injector for use in a fluid separation system for separating compounds of a fluidic sample in a mobile phase is provided, the sample injector comprising a switchable valve (i.e. a valve switchable between multiple positions, wherein each position corresponds to an assigned fluid coupling/decoupling characteristic of conduits connectable to the valve), a sample loop in fluid communication with the valve and configured for receiving the fluidic sample, a metering device in fluid communication with the sample loop and configured for introducing a metered amount (for instance a predefined volume or mass) of the fluidic sample on the sample loop, and a control unit configured for controlling switching of the valve to transfer the sample loop between a low pressure state (at which the sample loop may be at a first pressure value) and a high pressure state (at which the sample loop may be at a second pressure value larger than the first pressure value) via an intermediate state and for controlling the metering device during the intermediate state to at least partially (i.e. partially or entirely) equilibrate a pressure difference in the sample loop between the low pressure state and the high pressure state.
• According to another embodiment of the present invention, a fluid separation system for separating compounds of a fluidic sample in a mobile phase is provided, the fluid separation system comprising a mobile phase drive (such as a pumping system) adapted to drive the mobile phase through the fluid separation system, a separation unit (such as a chromatographic column) adapted for separating compounds of the fluidic sample in the mobile phase, and a sample injector having the above mentioned features for introducing the fluidic sample into the mobile phase.
• According to still another embodiment of the present invention, a method of operating a sample injector in a fluid separation system for separating compounds of a fluidic sample in a mobile phase is provided, wherein the method comprises introducing, by a metering device, a metered amount of the fluidic sample on a sample loop in fluid communication with a switchable valve and the metering device, controlling switching of the valve to transfer the sample loop between a low pressure state and a high pressure state via an intermediate state, and controlling the metering device during the intermediate state to at least partially equilibrate a pressure difference in the sample loop between the low pressure state and the high pressure state.
• According to an exemplary embodiment, a switchable valve, a sample loop and a metering device may be arranged in a configuration in which they are always in fluid communication with one another regardless of a present switching state of the valve. The system may be switchable between two or more pressure modes, particularly between a high pressure mode and a low pressure mode. Exemplary embodiments may allow to suppress or even eliminate undesired pressure drops and consequently undesired cavitation effects (such as bubble implosions in the switchable valve or the sample loop in response to a sudden change of the pressure conditions) by softly equilibrating the pressure difference between the two pressure modes in a dedicated intermediate valve state so that a smooth balancing of pressure differences between the low pressure state and the high pressure state can be achieved. Cavitation effects may deteriorate or even delaminate a coating of the valve. By preventing cavitation effects, the lifetime of the sample injector and particularly of the switchable valve may be significantly increased. Furthermore, a sudden pressure increase or decrease may result in disturbances in a flow profile, and may interrupt a column flow during the switching procedure. Also such undesired effects may be efficiently suppressed by exemplary embodiments.
• In the following, further exemplary embodiments of the sample injector will be explained. However, these embodiments also apply to the fluid separation system and to the method.
• According to an exemplary embodiment, the control unit may be configured for controlling switching of the valve to transfer the sample loop from the high pressure state to the low pressure state via the intermediate state and for controlling the metering device during the intermediate state to perform a decompression (or pressure reduction) in the sample loop before transferring the sample loop to the low pressure state. In such an embodiment, the smooth equilibration starts in a high pressure state in which a high pressure of for instance 100 MPa is present at the sample loop and decompresses the sample loop for reducing the pressure towards or up to a low pressure (for instance an atmospheric pressure) before initiating the switching to the actual low pressure state. This may safely prevent sudden decompression of a fluid which may occur in the sample loop when switching from the high pressure to the low pressure.
• Still referring to the previous embodiment, the control unit may be configured for controlling the metering device for performing the decompression by retracting a metering piston of the metering device. Hence, the metering device which is present in the sample injector predominantly for introducing a sample from a vial or the like into the sample loop by retracting and forwarding a metering piston, may be used as well for performing the decompression prior to the switching. Hence, the metering device can be synergistically used for both purposes of sample introduction and pressure equilibration.
• Additionally or alternatively, the control unit may be configured for controlling switching of the valve to transfer the sample loop from the low pressure state to the high pressure state via the intermediate state and for controlling the metering device during the intermediate state to perform a precompression in the sample loop before transferring the sample loop to the high pressure state. Hence, the pressure equilibration feature can be applied also in a configuration in which a switch from the low pressure mode to the high pressure is initiated so that in the intermediate valve state the pressure may be slowly or continuously increased so that a subsequent switch from the intermediate state to the high pressure state of the valve does not generate an intense pressure pulse since the pressure difference has already been equilibrated smoothly beforehand.
• Still referring to the previous embodiment, the control unit may be configured for controlling the metering device for performing the precompression by pushing forward the metering piston. As mentioned above, the metering device may predominantly act for introducing a sample from a vial into the sample loop but may be, according to the described exemplary embodiment, used as well for effecting a pressure increase in the sample loop during the intermediate state for reducing a mechanical load which conventionally acts on the components of the sample injector upon suddenly switching from the low pressure mode to the high pressure mode.
• In an embodiment, the metering device may be configured as a high pressure metering device. In other words, the metering device may be configured for providing pressure values which are significantly higher than only several bars, thereby providing the structural and functional capability of equilibrating the pressure in the sample loop between high and low pressure modes with typical pressure values which can be present in the sample injector of a liquid chromatography device such as a HPLC. This may require to substitute conventional metering devices (like syringe pumps, capable of operating at a pressure value of several bars only) by a high pressure metering device which may be capable of providing significantly larger pressures such as about 10 MPa, particularly at least about 50 MPa, more particularly at least about 100 MPa or more.
• The metering device may be configured for providing basically the same pressure as a mobile phase drive, particularly a pumping system, adapted to drive a mobile phase through a separation column of the fluid separation system. Such a mobile phase drive may be provided to drive a mobile phase such as a solvent composition comprising, for instance, a mixture of water and an organic solvent such as ACN, for conducting the same through a separation column of a liquid chromatography device. The mentioned metering device may provide a pressure of for instance 100 MPa, whereas sample introduction into the sample loop using a metering device is in many conventional cases performed not significantly above an atmospheric pressure or the like. Hence, the metering device may then be operated in different pressure modes, for instance a low pressure mode for introducing a sample from a vial or the like into the sample loop or in a high pressure state for bringing the sample loop smoothly to a high pressure value as provided by the mobile phase drive before switching a valve from an intermediate to a high pressure state.
• The metering device may also be configured for increasing a pressure in the sample loop, before switching the sample loop from the low pressure state to the high pressure state, to or towards a system pressure of a mobile phase drive, particularly a pumping system, adapted to drive a mobile phase through a separation column of the fluid separation system. Hence, the metering device may dampen the pressure drop between a mode in which the sample loop is in fluid communication with the mobile phase drive and a mode in which the sample loop is out of fluid communication with the mobile phase drive.
• According to an exemplary embodiment, the valve may comprise a first valve member and a second valve member, wherein at least one of the first and the second valve members is adapted to be moved with respect to the other, wherein one of the first and second valve members comprises a plurality of ports and the other comprises at least one groove for fluidly coupling respective ones of the ports in dependency on a relative movement position of the first and the second valve members with respect to each other. In other words, fluid paths may be formed by at least two of the ports and at least one of the grooves which can selectively be brought in or out of fluid communication with these ports. In contrast to conventional approaches in which such a valve only has an initial state and a final state and a switching between the initial state and the final state suddenly increases or decreases a pressure, a valve according to an exemplary embodiment may have a stable intermediate state between the initial and the final state to which intermediate state the valve may be brought for performing equilibration between a high pressure and a low pressure using the metering device in the sample loop.
• Still referring to the previous embodiment, the plurality of ports and the at least one groove may be designed so that in the intermediate state of the sample loop succeeding the low pressure state and preceding the high pressure state of the sample loop, a pumping system adapted to drive the mobile phase through a separation column is still in fluid communication with the separation column, and the sample loop is no longer at an atmospheric pressure and not yet in fluid communication with the separation column. Such an embodiment corresponds to a switch from a bypass mode to a main pass mode (for instance to a switch fromFIG.4 viaFIG.3 toFIG.2). Therefore, in the intermediate state, the sample loop may still be fluidly decoupled from the pumping system but can be already brought to a higher pressure as compared to 1 bar before being switched to the start/inject state.
• Additionally or alternatively, the plurality of ports and the at least one groove may further be designed so that in the intermediate state of the sample loop succeeding the high pressure state and preceding the low pressure state of the sample loop, a pumping system adapted to drive the mobile phase through a separation column is still in fluid communication with the separation column, and the sample loop is no longer at a pressure of the pumping system and not yet at an atmospheric pressure. Such an embodiment corresponds to a switch from a main pass mode to a bypass mode (for instance to a switch fromFIG.2 viaFIG.3 toFIG.4). Therefore, in the intermediate state, the sample loop may be already fluidly decoupled from the pumping system and can be already brought to a lower pressure before being switched to the loading state.
• The plurality of ports and the at least one groove may be designed so that in the high pressure state, the sample loop is in fluid communication with a pumping system adapted to drive a mobile phase drive through a separation column and is in fluid communication with the separation column. Thus, the high pressure state may be characterized by a fluid communication between the mobile phase drive and the sample loop.
• The plurality of ports and the at least one groove may further be designed so that in the low pressure state, the sample loop is not in fluid communication with a pumping system adapted to drive a mobile phase through a separation column and is not in fluid communication with the separation unit. Thus, the low pressure state can be characterized by the absence of the high pressure of the mobile phase drive in the sample loop.
• Moreover, the plurality of ports and the at least one groove may be designed so that a first position of one of the at least one groove is aligned with one of the plurality of ports in the low pressure state, a second position of the one of the at least one groove is aligned with the one of the plurality of ports in the high pressure state, and a third position (differing from the first and second positions) of the one of the at least one groove is aligned with the one of the plurality of ports in the intermediate state. In such an embodiment, a stop position of the one of the plurality of ports may be defined (particularly not only at the first and the second position but also) at a third position of the one of the at least one groove. Therefore, an intermediate valve state may be provided which represents a state selectable by the control unit during which an equilibration between a high pressure and a low pressure may be performed within the sample loop.
• In an embodiment, different ones of the plurality of grooves may have different lengths. Therefore, by length selection and also geometry selection (the grooves may have an arcuate partial circle like appearance but can also have further geometrical features such as a hook or the like), additional design parameters for valve configuration are provided which allow to properly define intermediate state, initial state, end state and optionally further states of the valve.
• Optionally, a pressure sensor may be arranged in the sample loop (particularly between the metering device and the switchable valve) for measuring a pressure in the sample loop. The pressure sensor may provide a measured pressure to the control unit as a feedback signal as a basis for the controlling of at least one of the metering device and the valve.
• The low pressure may be smaller than the high pressure. For example, the low pressure may be an atmospheric pressure (of about 0.1 MPa), whereas the high pressure may be at least 50 MPa, more particularly at least about 100 MPa. With such pressure drops between atmospheric pressure and 50 MPa or even 100 MPa, strong and destructive cavitation effects may occur without the pressure equilibration according to an exemplary embodiment.
• According to one embodiment, the valve may comprise six ports and two grooves. In such a configuration which is shown in the embodiment ofFIG.2 toFIG.4 for example, the intermediate state may be arranged between two other valve states.
• In an alternative embodiment, which is illustrated inFIG.5 toFIG.9, the valve may comprise seven ports, three grooves and may be switchable between six (or more) positions. With such a configuration, the pressure equilibration feature may be further refined.
• The low pressure state of the sample loop may correspond to an operation mode in which the fluidic sample is loaded onto the sample loop and the mobile phase is driven by a mobile phase drive, particularly a pumping system, through a separation column of the fluid separation system. In such an operation mode, a needle can be lifted out of a seat in the sample loop and may be immersed into a vial or the like for loading the sample on the sample loop, which may occur at a relatively low pressure of for instance one atmosphere.
• In contrast to this, the high pressure state of the sample loop may correspond to an operation mode in which the fluidic sample is injected from the sample loop to the separation column and is driven by a mobile phase drive, particularly a pumping system, to be loaded onto the separation column of the fluid separation system. In such an embodiment, the sample which has previously been loaded in the sample loop may then be pumped onto a separation column using the high pressure of the mobile phase drive. Subsequently, the different fractions of the sample which are then retained at fluid separation beads of the separation column may be individually and separately be released from the separation column by a gradient run, i.e. by a variation of a solvent which may be subsequently pumped through the separation column by the mobile phase drive.
• Optionally, the sample injector may comprise a flush conduit configured for flushing at least a part of fluidic conduits of the sample injector. For example for cleaning or rinsing purposes, a flush loop may be provided which allows to clean such fluidic conduits to prevent carryover or the like. Such a flush loop may be properly implemented in the pressure equilibration system according to an exemplary embodiment.
• In an embodiment, the metering device is arranged within the sample loop. In other words, in the described embodiment metering device and sample loop may be always in fluid communication with one another regardless of a switching state of the valve. This architecture may allow for a very simple equilibration of the pressure in the sample loop when transferring the sample loop between a high pressure mode and a low pressure mode.
• According to an exemplary embodiment, an appropriate groove design in a valve may allow to provide an additional intermediate position at which the pump may still be connected with the column and the split loop may be not yet connected with the pump and may be no more connected with a waste (atmospheric pressure). When providing a high pressure capable metering device in the split loop, i.e. a high pressure metering device capable of providing the same pressures as a column pump, it is possible to balance out pressure differences before the switching. For instance, such a high pressure metering device may displace a volume of for example 2 .mu.l to 10 .mu.l with a pressure of about 100 MPa. To achieve such a performance, it is possible to position the high pressure metering device (particularly a piston position thereof) in such a manner that the compression of 2 .mu.l to 10 .mu.l is possible.
• In one embodiment, the shear valve is embodied as a rotary valve, with the first and second shear valve members being rotably moveable with respect to each other. In another embodiment, the shear valve is embodied as a translational valve, such as a slide valve, with the first and second shear valve members being translationally moveable with respect to each other.
• In one embodiment, the shear valve further comprises a housing for housing one of the first and second shear valve members, wherein the housing is pre-stressed (pre-loaded) against the housed shear valve member. This allows reducing breakage or fracture stress, which may occur in the housed shear valve member. The housing is preferably attached to the housed shear valve member by using a shrinking process as known in the art.
• In one embodiment, the fluid path of this shear valve comprises a groove. In one embodiment, one or more of the ports of the shear valve comprise a through hole having an opening fluidly coupling with the fluid path dependent on the moving position. In one embodiment, wherein the first shear valve member comprises a plurality of ports, the second shear valve member comprises the at least one fluid path for fluidly coupling respective ones of the port independency on a relative movement position of the first and second shear valve member with respect to each other. In a further embodiment, the second shear valve member is adapted to be moved with respect to the first shear valve member. Preferably, the second shear valve member is provided as rotor or slider moving on the first shear valve member, which is embodied as static member and not moving. A drive might be provided for moving the shear valve member to be moved. Alternatively or in addition, the shear valve member to be moved might also be moved manually. A valve drive and control unit (e.g. gearbox+motor+encoder+central processing unit, CPU), might be provided for controlling movement of the shear valve member to be moved.
• The shear valve is preferably adapted to conduct a liquid in the at least one fluid path at a high pressure at which compressibility of the liquid becomes noticeable, such as pressure in the range of 20-200 MPa, and particularly 50-120 MPa.
• The shear valve can be a sample injection valve for introducing a liquid sample into a high pressure flowing stream of liquid, a high pressure purge valve for a positive displacement pump, or a flow path switching valve for switching from one flow path to another flow path.
• The shear valve might be embodied in an HPLC sample injector adapted to introduce a sample fluid into a mobile phase. The mobile phase is to be driven by a mobile phase drive through a separation unit for separating compounds of the sample fluid in the mobile phase. A sample loop is provided for receiving the sample fluid. The shear valve is provided for switching the sample loop between the mobile phase drive and the separation unit for introducing the sample fluid into the mobile phase.
• Embodiments of the present invention might be embodied based on most conventionally available HPLC systems, such as the Agilent 1200 Series Rapid Resolution LC system or the Agilent 1100 HPLC series (both provided by the applicant Agilent Technologies—see www.agilent.com—which shall be incorporated herein by reference).
• One embodiment 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.
• One embodiment comprises two pumping apparatuses coupled either in a serial or parallel manner. In the serial manner, as disclosed in EP 309596 A1, 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 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 (for instance 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 for instance 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.
• The separating device preferably comprises a chromatographic column (see for instance http://en.wikipedia.org/wiki/Column chromatography) providing the stationary phase. The column might be a glass or steel tube (for instance with a diameter from 50 .mu.m to 5 mm and a length of 1 cm to 1 m) or a microfluidic column (as disclosed for instance in EP 1577012 or the Agilent 1200 Series HPLC-Chip/MS System provided by the applicant Agilent Technologies, see for instance http://www.chem.agilent.com/Scripts/PDS.asp?|Page=38308). For example, a slurry can be prepared with a powder of the stationary phase and then poured and pressed into the column. 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 one at a time. 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, though in EBA a fluidized bed is used.
• The mobile phase (or eluent) can be either a pure solvent or a mixture of different solvents. It can be chosen for instance to minimize the retention of the compounds of interest and/or the amount of mobile phase to run the chromatography. The mobile phase can also been chosen so that the different compounds can be separated effectively. The mobile phase might comprise an organic solvent like for instance methanol or acetonitrile, often diluted with water. For gradient operation water and organic is delivered in separate bottles, 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.
• 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.
• 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 50-120 MPa (500 to 1200 bar).
• The HPLC system might further comprise a sampling unit for introducing the sample fluid into the mobile phase stream, 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 Agilent 1200 Series Rapid Resolution LC system or the Agilent 1100 HPLC series, both provided by the applicant Agilent Technologies, under www.agilent.com which shall be in cooperated herein by reference.
• Embodiments of the invention can be partly or entirely embodied or supported by one or more suitable software programs, which can be stored on or otherwise provided by any kind of data carrier, and which might be executed in or by any suitable data processing unit. Software programs or routines can be preferably applied in or by the control unit.
BRIEF DESCRIPTION OF DRAWINGS
• 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 drawing(s). Features that are substantially or functionally equal or similar will be referred to by the same reference sign(s).
• FIG.1 shows a liquid separation system, in accordance with embodiments of the present invention, for instance used in high performance liquid chromatography (HPLC).
• FIG.2 toFIG.4 shows an exemplary embodiment of a sample injector according to the present invention in different operation modes.
• FIG.5 toFIG.9 shows another exemplary embodiment of a sample injector according to the present invention in different operation modes.
• Referring now in greater detail to the drawings,FIG.1 depicts a general schematic of aliquid separation system10. Apump20 receives a mobile phase from asolvent supply25, typically via adegasser27, which degases and thus reduces the amount of dissolved gases in the mobile phase. Thepump20—as a mobile phase drive—drives the mobile phase through a separating device30 (such as a chromatographic column) comprising a stationary phase. A sampling unit40 (compare the detailed description ofFIG.2 toFIG.9) can be provided between thepump20 and the separatingdevice30 in order to subject or add (often referred to as sample introduction) a sample fluid into the mobile phase. The stationary phase of the separatingdevice30 is adapted for separating compounds of the sample liquid. Adetector50 is provided for detecting separated compounds of the sample fluid. A fractionatingunit60 can be provided for outputting separated compounds of sample fluid.
• While the mobile phase can be comprised of one solvent only, it may also be mixed from plural solvents. Such mixing might be a low pressure mixing and provided upstream of thepump20, so that thepump20 already receives and pumps the mixed solvents as the mobile phase. Alternatively, thepump20 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 device30) occurs at high pressure and downstream of the pump20 (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.
• Adata processing unit70, 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 theliquid separation system10 in order to receive information and/or control operation. For example, thedata processing unit70 might control operation of the pump20 (for instance 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). Thedata processing unit70 might also control operation of the solvent supply25 (for instance setting the solvent/s or solvent mixture to be supplied) and/or the degasser27 (for instance 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.). Thedata processing unit70 might further control operation of the sampling unit40 (for instance controlling sample injection or synchronization sample injection with operating conditions of the pump20). The separatingdevice30 might also be controlled by the data processing unit70 (for instance selecting a specific flow path or column, setting operation temperature, etc.), and send—in return—information (for instance operating conditions) to thedata processing unit70. Accordingly, thedetector50 might be controlled by the data processing unit70 (for instance with respect to spectral or wavelength settings, setting time constants, start/stop data acquisition), and send information (for instance about the detected sample compounds) to thedata processing unit70. Thedata processing unit70 might also control operation of the fractionating unit60 (for instance in conjunction with data received from the detector50) and provides data back.
• Reference numeral90 schematically illustrates a switchable valve which is controllable for selectively enabling or disabling specific fluidic paths withinapparatus10.
• In the following, referring toFIG.2, asample injector200 for use in afluid separation system10 as described inFIG.1 for separating components of a fluidic sample in a mobile phase according to an exemplary embodiment of the invention will be explained.
• Thesample injector200 comprises a switchable valve202 (which corresponds to reference numeral90 inFIG.1), asample loop204 in fluid communication with thevalve202 and configured for receiving the fluidic sample from avial230, ametering pump206 in fluid communication with thesample loop204 and configured for introducing a metered amount of the fluidic sample on thesample loop204, and a control unit208 (such as a microprocessor or a central processing unit, CPU) configured for controlling switching of thevalve202 to transfer thesample loop204 between a low pressure state and a high pressure state via an intermediate state, as will be described below in further detail.Control unit208 is further adapted for controlling themetering device206 to at least partially equilibrate, during the intermediate state, a pressure difference in thesample loop204 between the low pressure state and the high pressure state. Thus, the metering device206 (metering pump) is configured to generate a high pressure (in opposite to conventional syringe pumps). Thismetering device206 is arranged within thesplit loop204. Thesplit loop204 can be compressed. The precompression may be performed up to a system pressure of thepump20.
• As can be derived fromFIG.2 toFIG.4, theswitchable valve202 comprises two valve members which are rotatable with respect to one another. By rotating these two valve members along a rotation axis which is perpendicular to the paper plane ofFIG.2, a plurality ofports216 formed in one of the valve members and a plurality of oblongarcuate grooves218 formed in the other one of the valve members can be selectively brought in or out of fluid communication with one another. Since thevarious ports216 are connected to dedicated ones of fluidic channels of the fluidic system as shown inFIG.2, automatically switching thevalve202 under control of thecontrol unit208 may allow to operate thefluidic system10 in different fluid communication configurations. Thevalve202 is configured as a six port high pressure valve in the embodiment ofFIG.2.
• Fluid communication between thehigh pressure pump20 and theseparation column30 can be accomplished by an according switching state of thevalve202. In such a fluidic path, a high pressure of for instance 100 MPa may be present which may be generated by thehigh pressure pump20. In contrast to this, the pressure state in thesample loop204 may be for instance smaller than 0.1 MPa when introducing a sample into thesample loop204. When this sample loaded onsample loop204 is to be loaded oncolumn30, the pressure insample loop204 is also high, for instance 100 MPa.
• For the purpose of loading the sample on thesample loop204, aneedle224 may be driven out of a correspondingly shapedseat226 using adrive228 so that theneedle224 can be immersed intovial230 accommodating a fluidic sample to be loaded onto thesample loop204. Aloop capillary240 is provided in thesample loop204 for at least partially accommodating the introduced sample.
• In a further operation mode, theneedle224 may be immersed in aflush port232.Waste containers234,236 may be provided for receiving a waste fluid which can be pumped through the fluidic channels shown inFIG.2. Furthermore, for flushing thefluidic system200, fluid from a flushsolvent vial238 may be sucked by aperistaltic pump250 and may be pumped through corresponding channels of the fluidic system shown inFIG.2.
• Themetering device206 is configured as a high pressure metering device, i.e. as a metering device which is capable of providing a pressure of up to 100 MPa in thesample loop204 by correspondingly moving areciprocating piston210 of the highpressure metering device206.
• Before describing further details of thesample injector200, some basic recognitions of the present inventors will be summarized based on which exemplary embodiments of the invention have been developed.
• According to an exemplary embodiment, flow perturbances may be reduced and component lifetime of a HPLC autosampler may be increased by a precompression and/or decompression of its loop volume.
• HPLC injection system used for pressures above 60 MPa (for instance 120 MPa) are conventionally faced with various problems. The volume within the split loop (in the embodiment ofFIG.2, the split loop includes particularly highpressure metering device206,loop capillary240,needle224,needle seat226, seat capillary270) may be exposed to very high pressures in a main pass position which is illustrated inFIG.2. Since liquids (mobile phase and sample) under such high pressures are no longer incompressible, this loop volume is being compressed.
• Furthermore, switching theinjector valve202 to a bypass position as shown inFIG.4 conventionally leads to a very fast decompression of the loop volume because it gets connected to atmospheric pressure suddenly. This fast decompression generates a strong acceleration of the liquid which passes with high flow rates through the channels of theinjector valve202. This high flow rate (also called “water jetting”) may cause delamination of a coating on the valve stator due to cavitation and erosion on the polymeric valve rotor seal.
• On the other hand does the pump20 deliver flow while thevalve202 switches to a main pass mode shown inFIG.2. During this time, the valve channel is getting deconnected from thepump20. Thepump20 is pumping against the closed channel which results in a pressure increase.
• At the same time thecolumn30 gets deconnected from thepump20 and flow is no longer delivered on top of thecolumn30. Concurrently the system after thecolumn30 is open and via detector cell connected to an atmospheric pressure. This may also cause the column pressure to decrease.
• The above-mentioned problems of conventional systems which may be overcome by the embodiments shown inFIG.2 toFIG.9 have different consequences. Firstly, the jet stream generated during decompression causes damage on rotor seal and stator of thevalve202. This may result in a reduced valve lifetime. Switching thevalve202 furthermore causes pressure/flow disturbances (perturbances) like pressure peaks. This may lead to precision problems of flow rates, etc. Theclosed valve202 causes the column pressure to drop. The reconnectedvalve202 on the other hand forwards the flow generated by thepump20 via split loop to thecolumn30. The pressure may be at reduced level. However, at the beginning of this operation, the column pressure may be still higher as the split loop pressure. In that case there is a possibility for a reverse flow to develop. After this, the pressure starts equilibrating and thepump20 delivers a positive flow towards thecolumn30. The pressure peaks and the reverse flow may conventionally reduce the lifetime of acolumn30.
• Exemplary embodiments of the invention, for instance the systems described inFIG.2 toFIG.9 may overcome these conventional problems by taking particularly the measures explained in the following. In order to reduce the observed effects, a modifiedvalve202 and modified operation procedures are provided. The modifiedvalve202 has flow channels which are different in length (compare different lengths of the arcuate sections of thegrooves218 inFIG.2) and the modified operations include stops to provide an intermediate valve state in an inclined position (compareFIG.3).
• By clockwise turning thevalve202 from main pass (or start/inject) position as shown inFIG.2, thecolumn30 is connected to thepump20 via the split loop orsample loop204. At the inclined position (pre/decompression mode as shown inFIG.3),column30 is connected directly to thepump20. In this inclined position, the split loop (i.e.loop capillary240 plusmetering device206 plusneedle224 plus seat capillary270) is now isolated from thepump20 and thecolumn30 but is still under high pressure. In order to reduce that high pressure,piston210 of themetering device206 can be drawn back a controlled amount for instance until the loop pressure equals atmospheric pressure. For instance, this can be done by using a metering device as disclosed for instance in EP 0,327,658 B1, U.S. Pat. No. 4,939,943 which allows high pressure applications.
• With the loop pressure being brought close to atmospheric pressure, thevalve202 can be again turned clockwise to its bypass position which is shown inFIG.4. This bypass position may also be denoted as a load position. Since there is no pressure gradient between the internal loop pressure and the atmospheric pressure, no water jetting can develop. Therefore, both the delamination of the stator coating and the erosion of the polymer rotor may be eliminated or at least suppressed. The result is an increased lifetime of thevalve202 and of theentire sampling unit200.
• Thevalve202 is in the bypass or load position inFIG.4, and the autosampler is ready to take a sample fromvial230. In a first procedure, theneedle224 may be lifted and moved into thesample vial230 or a well position (for instance of a multi-well plate). Now, thepiston210 of themetering device206 may be drawn back to a controlled preset amount (forinstance 2 .mu.l). Next, theneedle224 is seated in itsseat226, and thesplit loop204 is closed thereby.
• Thevalve202 is then turned counterclockwise to the inclined position shown inFIG.3 where thepump20 is still connected to thecolumn30. Thesplit loop204 is closed on both ends. If now thepiston210 of themetering device206 is moved forward in a controlled manner, its displacement generates a positive pressure and precompresses the trapped volume. This pressure, potentially sensed by apressure sensor220, is being increased until it equals the system pressure.
• This is the trigger to turn thevalve202 completely to the main pass position which is illustrated inFIG.2. Because the pressure of the system and thesplit loop204 are equal at beginning of this operation, there will be only a very small pressure drop causing only minimum flow disturbances. Thepump20 delivers the mobile phase through thesplit loop204 and pushes the sample onto thecolumn30 where the chromatographical separation of the sample may start.
• Hence,FIG.2 toFIG.4 show schematically three positions of theinjection valve202 of theautosampler200 withinHPLC system10 during the injection cycle.
• In the main pass position shown inFIG.2, a start or inject position is shown where the rotor seal flow channels connectpump20 with thesplit loop204 and the seat capillary of thesplit loop204 with theseparation column30.
• In the inclined position shown inFIG.3, the split loop volume gets decompressed or precompressed.
• In the bypass position shown inFIG.4, the flow channels of the rotor seal connect thepump20 directly to theseparation column30 and thesplit loop204 to thewaste outlet236.
• Next, referring toFIG.5 toFIG.9, asample injector500 in aliquid chromatography system10 according to another embodiment of the invention will be explained.
• FIG.5 illustrates a load or bypass position,FIG.6 illustrates a precompress position andFIG.7 illustrates an inject position (or main pass position) of thesample injector500.
• A main difference between thesample injector500 and thesample injector200 is the arrangement of thevalve502 which in an embodiment ofFIG.5 toFIG.9 is configured as a multi-position/seven port high pressure valve.
• Furthermore, in the embodiment ofFIG.5 toFIG.9, three different flushsolvent vials238 are provided and three differentflush ports232 are provided. Selection between three flush channels A, B and C can be performed by correspondingly switching a lowpressure selection valve504. Furthermore, a low pressureflush pump506 is provided for performing the flushing performance.
• Themulti-position valve502 is provided for additionally precompressing, pump priming and pressure testing. All drawn sample gets injected. Additionalflush pump506 may be for instance a syringe pump from the company Tecan. Such an additionalflush pump506 may allow flushing of thesample loop204 using the three flush ports A, B, C (for instance two organic flush ports and one water flush port).
• FIG.8 illustrates the system ofFIG.5 toFIG.7 in a prime pump position, andFIG.9 illustrates thesystem500 in a pressure test position.
• It should be noted that the term “comprising” does not exclude other elements or features and the “a” or “an” does not exclude a plurality. Also elements described in association with different embodiments may be combined. It should also be noted that reference signs in the claims shall not be construed as limiting the scope of the claims.

Claims (11)

1. A method of injecting a sample volume into a chromatography column of a chromatography system, the chromatography system comprising:

a sample loop in fluid communication with an injection valve, wherein the sample loop comprises a sample conveying device for loading the sample volume in the sample loop, wherein the sample conveying device comprises a pump volume structure and a moveable element, wherein the moveable element is guidable within the pump volume structure; and

a high-pressure fluidic path in fluid communication with the chromatography column and a high-pressure pump, wherein the method comprising:

flowing an eluent into the high-pressure fluidic path at a pump pressure generated by the high-pressure pump;

isolating the sample loop from the high-pressure fluidic path, wherein the isolating of the sample loop comprises placing the injection valve in a PRESSURE COMPENSATION position;

while the sample loop is isolated from the high-pressure fluidic path, sucking the sample volume into the sample loop from a sample vial by moving the moveable element with a stepping motor relative to the pump volume structure;

while the sample volume is loaded into the sample loop, and while the sample loop is isolated from the high-pressure fluidic path, moving the moveable element relative to the pump volume structure, a predetermined first distance, wherein the predetermined first distance is based at least in part upon a compressibility of the eluent in the sample loop and the pump pressure;

while the compressed sample volume is compressed to the high pressure, connecting the sample loop to the high-pressure fluidic path; and

while the compressed sample volume is compressed to the high pressure, conveying the compressed sample volume from the sample loop to the chromatography column.

2. The method ofclaim 1, wherein the sample loop includes a first connecting piece and a second connecting piece, wherein the first connecting piece is connected to a first sample loop port of the injection valve and to the sample conveying device, wherein the second connecting piece is connected to a second sample loop port of the injection valve and to the sample conveying device, wherein the second connecting piece includes an intake segment and a feed segment, wherein the intake segment and the feed segment are configured to be separated.

3. The method ofclaim 1, wherein in the PRESSURE COMPENSATION position, i) first and second sample loop ports of the injection valve are closed so as to facilitate a pressurization of the sample loop, and ii) first and second high-pressure ports of the injection valve are connected so as to operatively connect the high-pressure pump in fluid communication with the high-pressure fluidic path to the chromatography column, the method further comprising:

determining the compressibility of the eluent with the high-pressure pump.

4. The method ofclaim 1 further including:

after the compressed sample volume has been conveyed from the sample loop to the chromatography column, isolating the sample loop from the high-pressure fluidic path; and

while the sample loop is isolated from the high-pressure fluidic path, moving the moveable element relative to the pump volume structure, a predetermined second distance, to thereby decompress the sample loop to a pressure that essentially corresponds to an atmospheric pressure.

5. The method ofclaim 1, wherein the moveable element is connected to the stepping motor which is operable to move the moveable element within the pump volume structure, and the method further comprises:

measuring a force exerted upon the moveable element by the stepping motor.

6. The method ofclaim 1, wherein the compressibility of the eluent and an elasticity of the sample loop are stored for use by the chromatography system.

7. The method ofclaim 1, wherein the pump volume structure comprises a syringe and the moveable element comprises a plunger.

8. The method ofclaim 1, wherein the sample volume comprises the eluent, wherein the predetermined first distance is also based at least in part upon an elasticity of the sample loop.

9. The method ofclaim 1, wherein the stepping motor comprises an integrated sensor measuring a force applied by the stepping motor on the moveable element.

10. The method ofclaim 1 further comprising:

after the compressed sample volume has been conveyed from the sample loop to the chromatography column, isolating the sample loop from the high-pressure fluidic path; and

while the sample loop is isolated from the high-pressure fluidic path, moving the moveable element relative to the pump volume structure, a predetermined second distance, wherein the predetermined second distance is based at least in part upon a compressibility of an eluent in the sample loop, to thereby decompress the sample loop to a pressure that essentially corresponds to an atmospheric pressure.

11. The method ofclaim 10, wherein the predetermined second distance is also based at least in part upon the pump pressure.

Images