Patent Publication Number: US-2021190734-A1

Title: Sample metering and injection for liquid chromatography

Description:
RELATED APPLICATION 
     This application claims the benefit of the earlier filing date of U.S. Provisional Patent Application Ser. No. 62/952,871, filed Dec. 23, 2019 and titled “Sample Metering and Injection for Liquid Chromatography,” the entirety of which is incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     The invention relates generally to liquid chromatography systems. More particularly, the invention relates to fluidic networks for loading of a chromatographic sample and injection of the sample into a liquid chromatography system. 
     BACKGROUND 
     High performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC®) systems typically include a pump for delivering a fluid (the “mobile phase”) at a controlled flow rate and composition, an injector to introduce a sample solution into the liquid chromatography system flow (the “mobile phase”), a chromatographic column that contains a packing material or sorbent (the “stationary phase”), and a detector to detect the presence and amount of sample components in the mobile phase leaving the column. When the mobile phase passes through the stationary phase, each component of the sample typically emerges from the column at a different time because different components in the sample typically have different affinities for the packing material. The presence of a particular component in the mobile phase exiting the column is detected by measuring changes in a physical or chemical property of the eluent. By plotting the detector signal as a function of time, chromatographic “peaks” corresponding to the presence and quantities of the components of the sample can be observed. 
     In some fluidic networks employing a sample needle, a syringe is used to prime the fluidic path that includes the sample needle. The syringe is also used to acquire sample from a sample vial or other sample source. Often, bubbles are present in the fluidic path. Priming is generally performed at atmospheric pressure and the flow of the solvent through the fluidic path during priming may not sufficiently displace the bubbles. 
     In various applications, it is desirable to acquire small volumes of sample for analysis. For example, samples as small as 0.1 uL with a standard deviation of sample volume not to exceed 1%. The ability to acquire such small samples may be limited by the presence of bubbles. Consequently, the volumes of acquired sample can vary significantly despite the same intake volume stroke of the syringe. 
     SUMMARY 
     In one aspect, a fluidic network for acquiring and injecting a chromatographic sample includes a metering pump module, a sample needle, a needle seal and an injection valve. The metering pump module includes a metering pump and a pressure transducer in serial fluidic communication. The metering pump module has a first pump port and a second pump port. The sample needle has a needle tip. The needle seal is configured to receive the needle tip of the sample needle. The injection valve has a plurality of valve ports. A first one of the valve ports is in fluidic communication with the first pump port and a second one of the valve ports is in fluidic communication with the needle seal. The injection valve is operable in at least two valve states. When the injection valve is in the first valve state, the injection valve is configured to fluidically terminate the first and second pump ports. When the injection valve is in the second valve state, the injection valve is configured to fluidically couple a third valve port to the first valve port and to fluidically couple a fourth valve port to the second valve port. 
     The third valve port may be fluidically coupled to a source of a solvent flow and the fourth valve port may be fluidically coupled to a chromatographic column. 
     When the needle tip of the sample needle is received in the needle seal, a continuous fluidic path may be defined from the first valve port through the metering pump module, sample needle and needle seal to the second valve port. When the needle tip of the sample needle is received in the needle seal and the injection valve is in the first valve state, the metering pump may be operable to generate a pressure in the continuous fluidic path that exceeds 1,000 psi and, in some examples, may exceed 10,000 psi. 
     In another aspect, a method for injecting a chromatographic sample into a chromatography system flow includes aspirating a chromatographic sample into a sample needle and forming a fluidic path that passes through the sample needle. The fluidic path is terminated at each end and the chromatographic sample is included in the liquid in the fluidic path. A pressure of the liquid in the fluidic path is increased to a value that is substantially equal to a pressure of the liquid chromatography system and the fluidic path is inserted into the liquid chromatography system flow such that the chromatographic sample flows to a chromatography column in the liquid chromatography system. 
     A difference in the increased pressure of the liquid in the fluidic path and the pressure of the liquid chromatography system at a time of insertion may be less than that 10% of the pressure of the liquid chromatography system. 
     Forming the fluidic path may include coupling a needle tip of the sample needle into a needle seal that is in fluidic communication with one of the ends of the fluidic path. 
     Each end of the fluidic path may be terminated at a respective valve port of an injection valve when the injection valve is in a first valve state. Inserting the fluidic path into the liquid chromatography system flow may include switching the injection valve from the first valve state to a second valve state. 
     The method may further include monitoring the pressure of the liquid in the fluidic path while increasing the pressure to determine that the pressure of the liquid in the fluidic path is substantially equal to the pressure of the liquid chromatography system. 
     In still another aspect, a fluidic network for acquiring and injecting a chromatographic sample includes a metering pump module, a sample needle, a needle seal, a sample valve and a merge valve. The metering pump module includes a metering pump and a pressure transducer in serial fluidic communication with each other. The metering pump module has a first pump port and a second pump port. The sample needle has a needle tip. The needle seal is configured to receive the needle tip of the sample needle. The sample valve is operable in at least a first valve state and a second valve state, and is in fluidic communication with the metering pump module and the sample needle. The merge valve is operable in at least a first valve state and a second valve state, and is in fluidic communication with the sample valve and the needle seal. When the sample valve is in the second valve state and the merge valve is in the first valve state, the metering pump is operable to acquire a sample through the sample needle. When the sample valve is in the second valve state, the merge valve is in the first valve state and the needle tip is in the needle seal, the metering pump is operable to pressurize the fluidic network to a system pressure. When the sample valve is in the second valve state, the merge valve is in the second valve state and the needle tip is in the needle seal, a system flow passes through the fluidic network such that the sample acquired through the sample needle is merged into the system flow. 
     When the sample valve is in the second valve state, the merge valve is in the second valve state and the needle tip is in the needle seal, the system flow may be combined in the fluidic network with the sample acquired through the sample needle such that the sample is diluted by the system flow. 
     When the sample valve is in the second valve state, the merge valve is in the second valve state and the needle tip is in the needle seal, the system flow may pass through the fluidic network such that the sample acquired through the sample needle is injected into the system flow as a fluidic plug. 
     The fluidic network may include a purge solvent reservoir in fluidic communication with the sample valve. Alternatively, the fluidic network may include a fluidic channel fluidically coupled at one end to the sample valve and fluidically coupled at an opposite end to a waste channel. 
     At least one of the sample valve and the merge valve may be a six-port rotary shear seal valve. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The above and further advantages of this invention may be better understood by referring to the following description in conjunction with the accompanying drawings, in which like reference numerals indicate like elements and features in the various figures. Letters may be appended to reference numbers to distinguish from reference numbers for similar features and to indicate a correspondence to other features in the drawings. For clarity, not every element may be labeled in every figure. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. 
         FIG. 1  is a block diagram of an example of a liquid chromatography system. 
         FIGS. 2A, 2B and 2C  are schematic diagrams showing different configurations of a conventional fluidic network used to prime a liquid chromatography system and to inject a chromatographic sample. 
         FIGS. 3A, 3B, 3C and 3D  are schematic diagrams showing different configurations of a fluidic network that can be used for low-pressure metering and injection of a chromatographic sample. 
         FIGS. 4A, 4B and 4C  are schematic diagrams showing different configurations of a high-pressure fluidic network for acquiring and injecting a chromatographic sample. 
         FIG. 5  is a flowchart representation of an example of a method for injecting a chromatographic sample into a chromatography system flow. 
         FIGS. 6A, 6B, 6C, 6D and 6E  are schematic diagrams showing different configurations of another high-pressure fluidic network for acquiring and injecting a chromatographic sample. 
         FIGS. 7A, 7B, 7C, 7D and 7E  are schematic diagrams showing different configurations of still another high-pressure fluidic network for acquiring and injecting a chromatographic sample. 
     
    
    
     DETAILED DESCRIPTION 
     Reference in the specification to “one example” or “an example” means that a particular feature, structure or characteristic described in connection with the example is included in at least one example of the teaching. References to a particular example within the specification do not necessarily all refer to the same example. 
     In brief overview, a fluidic network for acquiring and injecting a chromatographic sample includes a metering pump module, a sample needle, a needle seal and an injection valve. The metering pump module includes a metering pump and a pressure transducer in serial fluidic communication. One of the valve ports of the injection valve is in fluidic communication with the metering pump module and a second one of the valve ports is in fluidic communication with the needle seal. The injection valve is operable in at least two valve states. When the injection valve is in the first valve state, the injection valve is configured to fluidically terminate ports of the pump module. When the injection valve is in the second valve state, the injection valve is configured to fluidically couple a third valve port to the first valve port and to fluidically couple a fourth valve port to the second valve port. One advantage of the fluidic network is the lack of a substantial change in the system pressure at the time of injection. Moreover, only a single valve is required. The chromatography system pump can be primed at atmospheric pressure through the sample needle and to waste therefore there is no need for a vent valve at the system pump. In addition, the flow rate during priming can be substantially greater than the system flow rate used during separations. Another benefit of the fluidic network is that new solvent that has been degassed is constantly provided from the system pump at full pressure and used to push the acquired sample so that there is no means for bubble formation. 
     The present teaching will now be described in more detail with reference to examples shown in the accompanying drawings. While the present teaching is described in conjunction with various examples, it is not intended that the present teaching be limited to such examples. On the contrary, the present teaching encompasses various alternatives, modifications and equivalents, as will be appreciated by those of skill in the art. Those of ordinary skill having access to the teaching herein will recognize additional implementations, modifications and examples, as well as other fields of use, which are within the scope of the present disclosure. 
       FIG. 1  is a block diagram of a conventional liquid chromatography system  10 . The system  10  includes a system processor  12  (e.g., microprocessor and controller) in communication with a user interface device  14  for receiving input parameters and displaying system information to an operator. The system processor  12  communicates with a solvent manager  16  which provides a single solvent or a combination of solvents as a mobile phase. For example, the solvent manager  16  may be capable of supplying an isocratic mobile phase and a gradient mobile phase. A sample from a sample source  20  is injected into the mobile phase upstream from a chromatographic column  22  at an injection valve  24 . The sample source  20  can be a sample reservoir such as a vial or other container that holds a volume of the sample solution. The chromatographic column  22  is coupled to a detector  26  which provides a signal to the system processor  12  that is responsive to various components detected in the eluent from the column  22 . After passing through the detector  26 , the system flow may exit through a waste port. Alternatively, the system  10  may include a diverter valve (not shown) to receive the system flow from the detector  26 . The diverter valve can be used as part of a fraction collection process in which the diverter valve diverts each separated sample component in the system flow to a corresponding collection vessel. 
     In the fluidic networks described below, each network includes one or more configurable valves to configure fluidic paths for the network. Each valve may be in communication with a valve control module used to switch the valve into one of two or more valve states. In some implementations the control module may be a standalone module that also communicates with a processor (e.g., see system processor  12  in  FIG. 1 ) and in other implementations the control module may be integrated into a processor that is also used to execute other processing and/or control functions. 
       FIG. 2A  is a schematic illustration showing a conventional fluidic network used to prime a liquid chromatography system and to inject a sample into the liquid chromatography system flow. The network includes a metering syringe  42 , a metering valve  44 , a pressure transducer  46 , an injection valve  48 , a sample needle  50  and a needle seal  52 . The needle seal  52  includes a spring-loaded conical port to receive the conical tip of the sample needle  50  and form a fluid tight seal. 
     The metering valve  44  includes a first port  44 - 1  in fluidic communication with the metering syringe  42 , a second port  44 - 2  fluidically coupled to a purge solvent reservoir  54  and a third port  44 - 3  fluidically coupled to the pressure transducer  46 . The metering valve  44  can be configured in a first valve state in which the first and second ports  44 - 1  and  44 - 2  are in fluidic communication. The metering valve  44  can be configured in a second valve state in which the first and third ports  44 - 1  and  44 - 3  are in fluidic communication. 
     In one implementation, the injection valve  48  is a six-port rotary shear seal valve. The double arc lines extending between some of the valve ports in the figure indicate internal fluidic paths between the ports. For example, the double arc lines may represent channels formed in the rotor and/or the stator of the rotary shear seal valve that are used to establish internal fluidic communication between two or more of the valve ports. During purge and sample load operations, the injection valve  48  is configured in a first valve state as shown in the figure. The digit n shown on the valve  48 , where n has a value of 1, 2, 3, 4, 5 or 6, is used to indicate a particular valve port  44 - n.    
     To purge the fluidic paths, the metering valve  44  is configured in the first valve state and the syringe  44  operates to drawn in purge solvent from a solvent reservoir  54 . Subsequently, the metering valve  44  is switched to the second valve state and the purge solvent is pushed from the syringe  42  into the fluidic path through the pressure transducer  46  toward the injection valve  48 . This sequence of drawing purge solvent through the first port  44 - 1  when the metering valve  44  is in the first valve state and pushing the solvent out through the third port  44 - 3  when the metering valve  44  is in the second valve state can be repeated until the volume of purge solvent pushed from the metering valve  42  exceeds the total volume of the fluidic paths that pass from the metering valve  44 , through the injection valve  48  and sample needle  50 , and out to waste. In some implementations, the volume of purge solvent supplied may be substantially larger than the total volume of the fluidic paths. In other implementations, the sample needle  50  may be removed from and positioned remote to the needle seal  52 , for example, positioned to dispense to waste, and the purge process is therefore only applied to the fluidic path from the metering valve  44  through the tip of the sample needle  50 . 
     To acquire sample, the sample needle  50  is removed from the needle seal  52  and moved to a source of sample (e.g., a sample vial)  55  as shown in  FIG. 2B . The syringe valve  44  is configured in the second valve state and the metering syringe  42  is operated to draw in liquid through the metering valve  44  to cause sample to be drawn from the sample vial  55  into the sample needle  50 . Generally, the volume of sample aspirated into the sample needle  50  is accurately controlled by the metering syringe  42 ; however, any bubbles present in the solvent in the fluidic path between the metering syringe  42  and the tip of the sample needle  50  prior to aspiration can cause the volume of sample acquired to be different from the draw stroke volume of the metering syringe  42 . Moreover, the repeatability of the volumes of acquired samples may be adversely affected by the presence of the bubbles. 
     To inject the acquired sample into the chromatography system flow, the injection valve  48  is switched to the second valve state as shown in  FIG. 2C  in which port  48 - 1  is coupled to port  48 - 2 , port  48 - 3  is coupled to port  48 - 4 , and port  48 - 5  is coupled to port  48 - 6 . Thus, the system flow arriving at port  48 - 6  of the injection valve  48  exits at port  48 - 5 , flows through the sample needle  50  and needle seal  52 , into port  48 - 2  and out from port  48 - 1  of the injection valve  48  toward the chromatography column. 
     The illustrated fluidic network has advantages. Only one high-pressure valve is required and the network does not add substantially to the delay volume of the liquid chromatography system. In addition, multiple cycles of the metering syringe  42  can be used to acquire larger sample volumes. 
     The purge process occurs under atmospheric pressure and, in some instances, bubbles present in the fluidic paths may not be sufficiently removed. Large volumes of purge solvent may be used to further reduce the air in the fluidic paths; however, this may require a large number of cycles of the metering syringe  42  and syringe valve  44  and can add significant time to the purge process. 
       FIG. 3A  shows another fluidic network that can be used for low-pressure metering of a chromatographic sample. The injection valve  48 , sample needle  50  and needle seal  52  are configured as described above with respect to  FIGS. 2A to 2C ; however, there is no metering syringe and no metering valve. The network instead includes a metering pump  56  and a second valve  58  having six ports and shown configured in a first valve state. The metering pump  56  and pressure transducer  46  are in serial fluidic communication and are collectively referred to as a metering pump module. 
     As used herein, a “metering pump” means any pump capable of delivering a precise volume of liquid over a specified time. By way of one non-limiting example, a metering pump can be a positive displacement pump, such as a single piston pump, that is compatible with chromatographic solvents and which has a piston chamber and two fluid lines each extending from a respective pump port to the piston chamber. The illustrated metering pump  56  has a plunger, a first port  56 - 1  and a second port  56 - 2 . The metering pump  56  does not include check valves and can be configured such that the first port  56 - 1  is an inlet and the second port is an outlet or alternatively configured such that the first port  56 - 1  is an outlet and the second port  56 - 2  is an inlet. Preferably, the metering pump  56  has a displacement volume that is greater than the fluid volumes of any of the fluidic paths. The metering pump  56  may utilize a linear actuator capable of operating under high pressures with precise volume control per actuator steps. Alternatively, the metering pump  56  may utilize a lead-screw or ball-screw actuator for low cost and high reliability. Advantageously, the metering pump  56  can be controlled to acquire and dispense accurate volumes of samples and solvents. The second valve  58  has ports  58 - 3  and  58 - 4  blocked so that no fluid enters or exits those ports. In effect, the blocked ports allow the second valve  58  to perform as a pair of check valves for the metering pump  56 . 
     When purging the fluidic paths, the second valve  58  is configured in a first valve state as shown in the figure in which port  58 - 1  is coupled to port  58 - 2  and port  58 - 4  is coupled to port  58 - 5  while the fluidic path from the pressure transducer  46  is dead-ended at port  58 - 4 . Thus, a fluidic path exists between the purge solvent reservoir  54  and the first port  56 - 1  of the metering pump  56 . The second valve  58  is then switched to the second valve state, as shown in  FIG. 3B , so that solvent dispensed from the metering pump  56  during a discharge stroke purges the fluidic path extending from port  56 - 2  through the pressure sensor  46 , second valve  58 , injection valve  48  and sample needle  50 . If the sample needle is positioned as shown in the needle seal  52 , then the fluidic path from the needle seal  52  through ports  48 - 2  and  48 - 3  of the injection valve  48  can also be purged. Conversely, if the sample needle is not inserted into the needle seal  52  but is positioned to dispense to waste, only the fluidic path leading up to and through the sample needle  50  is purged. 
     Similar to the fluidic network of  FIGS. 2A to 2C , many cycles of the metering pump  56  and second valve  58  may be required to complete the purge, adding to the time required to prepare the liquid chromatography system for a separation. Moreover, because the purging is performed at atmospheric pressure, bubbles present in the fluidic paths may not be purged, for example, due to surface tension. The fluidic paths where bubbles may remain include the narrow channels extending between the plunger chamber and the ports  56 - 1  and  56 - 2  in the metering pump  56 . 
       FIG. 3C  depicts the fluidic network for a sample load operation. The sample needle  50  is positioned so that at least the needle tip is disposed in a sample vial  55  while the injection valve  48  is in its first valve state and the second valve  58  is in its second valve state. Consequently, an intake stroke of the plunger in the metering pump  56  draws liquid into the second pump port  56 - 2 . This liquid is drawn from the sample needle  50  so that sample from the sample vial  55  is drawn into the sample needle  50 . 
     To inject the acquired sample into the chromatography system flow, the injection valve  48  is switched to the second valve state as shown in  FIG. 3D  so that the system flow arriving at port  48 - 6  of the injection valve  48  exits at port  48 - 5 , flows through the sample needle  50  and needle seal  52 , into port  48 - 2  and out from port  48 - 1  of the injection valve  48  toward the chromatography column. 
       FIG. 4A  is a schematic diagram showing a high-pressure fluidic network for acquiring and injecting a chromatographic sample. Reference is also made to  FIG. 5  which shows a flowchart representation of an example of a method  100  for injecting a chromatographic sample into a chromatography system flow. The fluidic network includes an injection valve  48 , a high-pressure metering pump  60 , a high-pressure pressure transducer  62 , a sample needle  50  and needle seal  52 . Ports  48 - 3  and  48 - 4  of the injection valve  48  are blocked. The high-pressure metering pump  60  is operable to generate fluidic pressures that exceed 7 MPa (1,000 psi) and, in some implementations, operates to generate fluidic pressures that exceed 70 MPa (10,000). The high-pressure pressure transducer  62  is preferably capable of measuring pressures that exceed 7 MPa (1,000 psi) and, in some implementations, measures pressures that exceed 70 MPa (10,000 psi). In other implementations the metering pump  60  and high-pressure pressure transducer  62  generate and measure pressures that exceed 125 MPa (18,000 psi). The high-pressure metering pump  60  and high-pressure pressure transducer  62  are in serial fluidic communication and are collectively referred to as a metering pump module. 
     A chromatographic sample is acquired (step  110 ) by positioning the tip of the sample needle  50  in a sample source such as a sample vial  55 . The sample is acquired under atmospheric pressure by retracting the plunger of the metering pump  60  so that sample is drawn through the needle tip into the sample needle  50  toward the metering pump  60 . In this single valve fluidic network no multiple draws (plunger cycles) can be made and the maximum sample volume that can be acquired is limited by the volume of a pump displacement stroke. Once the desired volume of sample is acquired, the tip of the sample needle  50  is moved into the needle seal  52  as shown in  FIG. 4B  to form (step  120 ) a fluidic path through the sample needle  50  that is terminated (blocked) at each end. The metering pump  60  is then operated to pre-compress (step  130 ) the liquid in the fluidic path defined between the two blocked ports  48 - 3  and  48 - 4  of the injection valve  48 . The pressure of the pre-compressed liquid is monitored using the high-pressure transducer  62 . When the measured pressure of the pre-compressed liquid is substantially equal (e.g., within 10%) to the liquid chromatography system pressure, the injection valve  48  is switched to a second valve state as shown in  FIG. 4C  such that port  48 - 1  is coupled to port  48 - 2  and ports  48 - 5  is coupled to port  48 - 6 . As illustrated, the system flow into port  48 - 6  of the injection valve  48  flows out through port  48 - 5  and through the high-pressure metering pump  60 , pressure transducer  62 , sample needle  50  and needle seal  52  before passing into port  48 - 2  and out through port  48 - 1  of the injection valve  48  to the chromatography column. Thus, the formed fluidic path is inserted (step  140 ) into the liquid chromatography system flow. 
     Advantageously, there is no substantial change in the system pressure at the time of injection and only a single valve is required. Moreover, the chromatography system pump can be primed at atmospheric pressure through the sample needle  50  and to waste thereby eliminating the need for a vent valve at the system pump. This allows the flow rate during priming to be substantially greater than the system flow rate used to perform a separation where the system flow passes through the chromatographic column. In one example, the system pump is part of a quaternary solvent manager in which the system pump receives a low-pressure (e.g., atmospheric) flow containing contributions of different solvents from a gradient proportioning valve. Flow from the system pump can similarly be used to prime the high-pressure metering pump  60  at atmospheric pressure with the flow exiting the tip of the sample needle  50  to waste. Another benefit of the illustrated high-pressure fluidic network is that new solvent that has been degassed is constantly provided from the system pump at full pressure and used to push the acquired sample. Consequently, there is no source for bubble formation. 
       FIG. 6A  shows an alternative high-pressure fluidic network which can also be used to dilute a chromatographic sample during injection. The fluidic network includes a high-pressure metering pump  60 , high-pressure transducer  62 , sample valve  64  and merge valve  66 . As used herein, a “merge valve” means any valve that can be used to inject a discrete fluidic plug of sample into the mobile phase of the liquid chromatography system and to alternatively be used to merge a flow of sample with a concurrent flow of mobile phase in the liquid chromatography system to achieve a dilution of the sample. The sample and merge valves  64  and  66  can be configured in different valve states such that the active fluidic paths are determined by the particular valve state of each valve. 
     The sample valve  64  operates under high system pressure (e.g., pressures that may exceed 18,000 psi (125 MPa)) and is operable in at least two valve states. In some examples, the sample valve  64  is a six-port rotary shear seal valve. Port  64 - 1  is coupled to a flush solvent reservoir  54 , port  64 - 3  is coupled to a high-pressure transducer  62 , port  64 - 4  is coupled to a sample needle  50 , port  64 - 5  is coupled to the merge valve  66  and port  64 - 6  is coupled to a metering pump  60 . A degasser (not shown) may be provided between the flush solvent reservoir  54  and the sample valve  64 . Port  64 - 2  is fluidically terminated, that is, blocked so that no fluid enters or exits the port. The sample valve  64  is shown in a first (offline) valve state in which ports  64 - 1  and  64 - 6  are in fluidic communication, ports  64 - 2  and  64 - 3  are in fluidic communication and ports  64 - 4  and  64 - 5  are in fluidic communication. The sample valve  64  can be reconfigured to a second (online) valve state in which the internal valve coupling paths are effectively rotated either clockwise (or counterclockwise) by 60° with respect to those shown in the figure. Thus, when the sample valve  64  is in the second valve state, port  64 - 1  is in fluidic communication with port  64 - 2 , port  64 - 3  is in fluidic communication with port  64 - 4  and port  64 - 5  is in fluidic communication with port  64 - 6 . 
     The merge valve  66  has six merge valve ports  66 - 1  to  66 - 6 . Ports  66 - 4  and  66 - 6  are terminated so that no fluid can enter or exit these ports. Port  66 - 1  is fluidically coupled to the needle seal  52 , port  66 - 2  is fluidically coupled to the chromatographic column, port  66 - 3  is fluidically coupled to a source of mobile phase (e.g., solvent manager  16  in  FIG. 1 ) and port  66 - 5  is fluidically coupled to port  64 - 5  of the sample valve  64 . The merge valve  66  is configurable in at least three valve states. As illustrated, the merge valve  66  is in a first (bypass) valve state in which ports  66 - 2  and  66 - 3  are in internal fluid communication with each other so that the chromatography system flow entering at port  66 - 3  can exit at port  66 - 2  and flow to the chromatographic column. The merge valve  66  is also configurable in two other valve states, a dilution state and a gradient state, as discussed further below with respect to  FIG. 6D  and  FIG. 6E , respectively. 
     As illustrated, the sample valve  64  is in the first valve state and the merge valve  66  is in the bypass state. Bold lines in the figure and in subsequent figures indicate active fluidic paths. A flow of mobile phase passes through the merge valve  66  to the chromatographic column. The metering pump  60  is operated to draw in liquid under atmospheric pressure. As described above, port  64 - 2  on the sample valve  64  is terminated therefore operation of the metering pump  60  results in purge solvent being aspirated from the purge solvent reservoir  54 . The volume of aspirated purge solvent is accurately controlled and pre-fills the metering pump  60  with a volume of purge solvent that is slightly greater that the volume of sample to be injected into the mobile phase. By way of a non-limiting numerical example, a 24 μL volume of flush solvent may be aspirated for a 20 μL volume sample injection. 
     To load the sample, the sample valve  64  is reconfigured to the second (online) valve state as shown in  FIG. 6B . Thus the metering pump  60  is terminated at the left side due to the terminations at ports  66 - 4  and  66 - 6  of the merge valve  66  and the right side of the metering pump  60  communicates with the sample needle  50 . In this fluidic network, the metering pump  60  is operated to continue to draw in liquid under atmospheric pressure which results in sample being aspirated into the sample needle  50  from a sample vial  55 . The volume of aspirated sample is accurately controlled by the operation of the metering pump  60 . The extra volume of acquired purge solvent relative to the acquired volume of sample is used to ensure in a later step that the acquired sample is fully pushed through the volume of the fluidic path between the acquired solvent in the sample needle  50  and the location of merging with the mobile phase at the merge valve  66 , and to accommodate dispersion in the fluidic path. 
       FIG. 6C  shows the portion of the liquid chromatography system after the sample needle  50  is moved from the sample vial  55  to the needle seal  52  while the valve states of the sample valve  64  and merge valve  66  remain unchanged. The fluidic path from the left side of the metering pump  60  remains terminated at the merge valve  66  and the fluidic path from the right side of the metering pump  60  through the pressure transducer  62 , sample valve  64  and sample needle  50  is also terminated at the merge valve  66  as the valve port  66 - 1  is not coupled to another valve port. The metering pump  60  is then controlled to push liquid out so that both fluidic paths are brought up to the full system pressure. Pressure transducer  62  is used to confirm that system pressure has been reached. 
     Subsequently, the merge valve  66  is reconfigured to a second (dilution) valve state as shown in  FIG. 6D . Continued operation of the metering pump  60  to dispense liquid results in the acquired sample flowing from the sample needle  50  through the needle seal  52  and then though port  66 - 1  of the merge valve  66 . A small volume of solvent in the fluid lines is first merged with the flow of mobile phase received at port  66 - 3  before the sample plug arrives at port  66 - 3  and is merged with the mobile phase. The sample plug exiting the merge valve  66  at port  66 - 2  is merged with concurrently flowing mobile phase also exiting at port  66 - 2 . To maintain a constant system flow to the chromatographic column, the flow rate from the mobile phase source is decreased while the flow rate from the metering pump  60  is increased. As both the flow of mobile phase and the flow of the sample contribute to the system flow throughout the duration of the sample injection and dilution, the sample dilution ratio is determined by the two flow rates. By way of a non-limiting numerical example, if the mobile phase flow rate at port  66 - 3  is nine times the sample flow rate at port  66 - 1 , the sample concentration in the flow exiting at port  66 - 2  is effectively one-tenth the original sample strength (i.e., the dilution ratio is one-part sample to nine parts diluent (mobile phase)). It will be recognized that a wide range of dilution ratios are possible. 
     Some of the solvent in the fluidic path used to “push” the sample plug is allowed to merge with the mobile phase for a time sufficient to ensure that substantially all the sample has been merged with the mobile phase. Subsequently, the flow rate from the mobile phase source is increased while the flow rate from the metering pump  60  is decreased in a complementary manner to maintain a constant system flow rate to the chromatographic column. “Substantially all the sample” means that any sample remaining in the illustrated fluidic paths is of insignificant volume as to not adversely affect chromatographic results. 
     Referring to  FIG. 6E , the sample valve  64  is reconfigured to the first (offline) valve state, thereby disconnecting fluid communication between the metering pump  60  and the sample needle  50 . In addition, the merge valve  66  is reconfigured to a third (gradient) valve state such that the mobile phase received at port  66 - 3  of the merge valve  60  flows out from port  66 - 5 , through the sample valve  64 , sample needle  50  and needle seal  52  before returning at port  66 - 1  and flowing out port  66 - 2  to the column. The two valves  64  and  66  remain in the illustrated fluidic network for the remainder of the gradient chromatographic separation. The fluidic path through the inside of the sample needle  50  may be cleaned after completion of the separation by passing the mobile phase through the sample needle  50 . 
     The fluidic network of  FIGS. 6A to 6E  can be used with some types of samples to dilute a large volume of the sample during the merging with the mobile phase, including sample volumes that are larger than the displacement volume of the metering pump  60 . By way of a non-limiting numerical example, a metering pump having a 100 μL displacement volume can be used to dilute a 1 mL sample volume by consecutively merging smaller volume (e.g., 100 μL or less) sample plugs with the mobile phase. To do this, a smaller volume sample plug is aspirated into the sample needle  50  and subsequently merged into the flow of mobile phase according to the operation of the metering pump  60  and reconfiguration of the sample valve  64  and merge valve  66  described above. This process of merging a smaller volume sample plug with the mobile phase is repeated a number of times so that the total of the volumes of the smaller sample plugs merged into the mobile phase equals to the full sample volume. The effective dilution ratio is determined by the relative flow rates of the mobile phase and the smaller volumes of sample during the merge times. 
     One advantage of the fluidic network is that the metering pump  60  can be quickly primed using the system flow. For example, the system flow may be a binary or quaternary solvent flow. Another advantage is the ability to perform multiple draw cycles for acquiring sample. In addition, the fluidic path that includes the metering pump  60  and high-pressure transducer  62  is only part of the system path during the injection sequence and therefore this fluidic path does not add to the gradient delay. Still another advantage is that the fluidic network allows for sample dilution without sacrificing the ability to inject sample without dilution. 
       FIG. 7A  shows another high-pressure fluidic network for acquiring and injecting a chromatographic sample. The fluidic network does not support online dilution; however, the ability to acquire a large volume of sample using multiple intake strokes of the metering pump  60  is maintained. The fluidic network is configured similarly to the fluidic network depicted in  FIGS. 6A to 6E  and can be used to perform the method  100  of  FIG. 5 ; however, instead of using the merge valve  66 , a merge valve  70  having a different configuration of internal fluidic coupling paths is used. More specifically, all the internal coupling paths along the rotor surface between valve ports are of equal length. The merge valve  70  can be identical in structure to the sample valve  64  but this is not a requirement. 
     Ports  70 - 5  and  70 - 6  are terminated so that no fluid can enter or exit these ports. Port  70 - 1  is fluidically coupled to the needle seal  52 , port  70 - 2  is fluidically coupled to the chromatographic column, port  70 - 3  is fluidically coupled to the source of mobile phase through a pump and port  70 - 4  is fluidically coupled to port  64 - 5  of the sample valve  64 . The merge valve  70  can be configured in one of two possible valve states. When the merge valve  70  is in the first valve state, as illustrated, port  70 - 1  is coupled to port  70 - 6 , port  70 - 2  is coupled to port  70 - 3  and port  70 - 4  is coupled to port  70 - 5 . Conversely, when the merge valve  70  is in the second valve state, as shown in  FIG. 7E , port  70 - 1  is coupled to port  70 - 2 , port  70 - 3  is coupled to port  70 - 4  and port  70 - 5  is coupled to port  70 - 6 . 
     Referring to  FIG. 7A , the sample valve  64  is shown in its second valve state. The merge valve  70  is in a bypass state in which ports  70 - 2  and  70 - 3  are coupled together so that the chromatography system flow entering at port  70 - 3  can exit at port  70 - 2  and flow to the chromatographic column. 
     To load the sample, the metering pump  60  executes at least a portion of an intake stroke to draw in liquid under atmospheric pressure, resulting in sample being aspirated into the sample needle  50  from the sample vial  55 . The volume of acquired sample is accurately controlled by the operation of the metering pump  60 . If the volume of sample to be acquired is greater than the stroke volume of the metering pump  60 , the sample valve  64  switches to the first valve state after the completion of the initial intake stroke, as shown in  FIG. 7B . The metering pump  60  then executes a reset stroke before the sample valve  64  is switched back to the second valve state, as shown in  FIG. 7C . Additional cycles of operation of the metering pump  60  according to  FIGS. 7B and 7C  can occur to acquire a greater total volume of sample. 
     After the desired volume of sample is acquired, the sample needle  50  is moved from the sample vial  55  to the needle seal  52 , as shown in  FIG. 7D , while the valve states of the sample valve  64  and merge valve  70  remain unchanged. The fluidic path from the left side of the metering pump  60  remains terminated at the merge valve  70  and the fluidic path from the right side of the metering pump  60  through the pressure transducer  62 , sample valve  64  and sample needle  50  similarly remains terminated at the merge valve  70 . The metering pump  60  then executes a portion of a discharge stroke so that the pressure in the two fluidic paths increases to approximately the full system pressure, as confirmed by the pressure transducer  62 . 
     Subsequently, the merge valve  70  is switched to the second valve state as shown in  FIG. 7E . The mobile phase from the system pump received at port  70 - 3  then passes through the fluidic network before returning to port  70 - 1  and back out through port  70 - 2  to the chromatographic column. In this manner, a discrete fluidic plug comprising the volume of acquired sample in the fluidic network is injected into the flow of the mobile phase. 
     In an alternative embodiment, the fluidic network omits the purge solvent reservoir  54  and the fluidic channel (e.g., tubing) coupled at one end to port  64 - 1  of the sample valve  64  may extend to its opposite end to the system waste channel. This embodiment allows for a user to perform a backlash compensation for the metering pump  60 . 
     In the alternative embodiment, the fluidic channel is occupied by the system solvent (e.g., mobile phase). For example, the fluidic network can be configured through valve switching so that the metering pump  60  can acquire the solvent on an intake stroke and subsequently execute a discharge stroke to push the acquired solvent through the fluidic channel to waste to thereby fill the full volume of the fluidic channel. 
     While the invention has been shown and described with reference to specific embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as recited in the accompanying claims.