Sealing a fluid sample using a strong solvent

Abstract

A sample separation device (10) for separating a fluid sample (120), wherein the sample separation device (10) comprises a fluid driver (20) for driving a mobile phase and the fluid sample (120) injected into the mobile phase, a sample separation unit (30) for separating the fluid sample (120) in the mobile phase, and a control unit (70) configured for pinching the fluid sample (120) between two mobile phase portions (122, 124) of the mobile phase, wherein at least one of the mobile phase portions (122, 124) is arranged directly adjacent to the fluid sample (120) and has a higher solvent strength compared to a solvent of the fluid sample (120).

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a sample separation apparatus, a method of separating a fluid sample, and a fluid flow. BACKGROUND

[0002] In liquid chromatography, a fluid sample and an eluent (liquid mobile phase) can be pumped through conduits and a separation unit, e.g. a column in which separation of sample components takes place. The column can comprise a material capable of separating different components of the fluid sample. The separation unit can be connected through conduits to other fluidic components, e.g. a sampler or injector, a detector. Prior to the fluid sample being introduced into a separation path between a fluid drive unit, in particular a high pressure pump, and the separation unit, a predetermined amount of fluid sample shall be injected from a sample source, e.g. a sample container, into a sample loop via an injection needle by a respective movement of a piston within a metering apparatus. Thereafter, an injector valve is switched in order to introduce the injection amount of fluid sample from the sample loop of the metering path into the separation path between the fluid drive unit and the separation unit for subsequent separation. As a result, the fluid sample is injected into a mobile phase, e.g. a solvent or a solvent composition. The exact composition of the mobile phase is important for the precise separation of the fluid sample.

[0003] However, it can be difficult to direct the fluid sample through the conduits without creating artifacts on the sample separation apparatus and precision of the separation. SUMMARY

[0004] It is an object of the present disclosure to be able to direct the fluid sample correctly through the conduits without creating artifacts on the sample separation apparatus and / or precision of the separation. This object is solved by the independent claims. Additional embodiments are shown by the dependent claims.

[0005] According to an exemplary embodiment of the present disclosure, a sample separation apparatus for separating a fluid sample is provided, wherein the sample separation apparatus comprises a fluid drive for driving a mobile phase and a fluid sample injected into the mobile phase, a sample separation unit for separating the fluid sample in the mobile phase, and a control unit configured for pinching the fluid sample between two mobile phase portions of the mobile phase, wherein at least one of the mobile phase portions is arranged directly adjacent to the fluid sample and has a higher solvent strength compared to a solvent of the fluid sample.

[0006] According to another exemplary embodiment, a method of separating a fluid sample is provided, wherein the method comprises: driving a mobile phase and the fluid sample; injecting the fluid sample into the mobile phase; separating the fluid sample in the mobile phase by a sample separation unit; and pinching the fluid sample between two mobile phase portions of the mobile phase, wherein at least one of the mobile phase portions is arranged directly adjacent to the fluid sample and has a higher solvent strength compared to a solvent of the fluid sample.

[0007] According to yet another exemplary embodiment, a fluid flow is provided, comprising a chromatographic sample directly between two mobile phase portions having a higher solvent strength compared to a solvent of the chromatographic sample.

[0008] In the context of the present application, the term "sample separation device" can particularly denote any apparatus capable of separating different fractions of a fluid sample by applying a certain separation technique, in particular liquid chromatography.

[0009] In the context of the present application, the term "fluid sample" can particularly denote any liquid and / or gaseous medium (which optionally further comprises solid particles) to be analyzed. Such a fluid sample can comprise a plurality of molecular or particulate fractions, e.g. small mass molecules or large mass biomolecules (e.g. proteins), which shall be separated. Separating the fluid sample into individual fractions can involve a certain separation criterion (e.g. mass, volume, chemical property, etc.) with which the separation is performed.

[0010] In the context of the present application, the term "mobile phase" can particularly denote any liquid and / or gaseous medium capable of serving as a fluid carrier for the fluid sample during separation. The mobile phase can be a solvent or a solvent composition (e.g. consisting of water and an organic solvent such as ethanol or acetonitrile). In an isocratic separation mode of a liquid chromatography apparatus, the mobile phase can have a constant composition over time. However, in a gradient mode, the composition of the mobile phase can change over time, in particular for desorbing fractions of the fluid sample which have previously been adsorbed onto a stationary phase of the separation unit.

[0011] In the context of the present application, the term "fluid driver" can particularly denote an entity capable of driving a fluid (i.e. a liquid and / or a gas, which optionally comprises solid particles), in particular the fluid sample and / or the mobile phase. For example, the fluid driver can be a pump (e.g. embodied as a piston pump or a peristaltic pump) or other high-pressure source. For example, the fluid driver can be a high-pressure pump, e.g. capable of driving the fluid at a pressure of at least 100 bar, in particular at least 500 bar.

[0012] The term“separation unit” can specifically denote a fluidic member through which a fluid sample is transported and which is configured such that upon guiding the fluid sample through the separation unit, the fluid sample will be separated into different groups of molecules or particles. An example of a separation unit is a liquid chromatography column, which is capable of capturing or retarding and selectively releasing different fractions of a fluid sample.

[0013] In the context of the present application, the term“mobile phase portion is arranged directly adjacent to the fluid sample” can specifically denote that the mobile phase portion and the fluid sample flowing in the fluidic conduit are provided directly next to each other without any other medium (e.g. a gas or other liquid) in between. In particular, the mobile phase portion and the fluid sample can be in direct physical contact with each other, i.e. can abut each other. For example, the mobile phase portion and the fluid sample can form a continuous uninterrupted liquid flow.

[0014] In the context of the present application, the term“solvent strength” can specifically denote the ability of a fluid to elute or desorb a fluid sample that is adsorbed on a stationary phase of a sample separation unit (e.g. a chromatography column). In other words, for a fluid sample that is fixed at a sample separation unit, a fluid with a higher solvent strength can have a stronger elution power compared to a fluid with a lower solvent strength. Thus, the terms“weak” and“strong” solvent can relate to the elution power of the respective solvent in releasing a fluid sample from a sample separation unit. Descriptively, a solvent with a high solvent strength can prevent, inhibit or reduce adsorption of a fluid sample at a stationary phase and can increase the solubility of this fluid sample in a solvent with a higher solvent strength. For example, an increase in solvent strength can be achieved by adding a salt or increasing the salt concentration of a solvent. However, also the base material of a solvent can have an influence on the solvent strength. For example, the solvent strength of water is lower than that of organic solvents such as ethanol, methanol or acetonitrile (ACN).

[0015] According to exemplary embodiments of this disclosure, a plug for a fluid sample to be separated into individual fractions can be arranged directly adjacent to a mobile phase portion having a higher solvent strength than the solvent of the fluid sample itself. This has the advantage that the fluid sample can then be forced through a fluid conduit toward a sample separation unit for sample separation without the risk of fluid sample material adhering to or being adsorbed onto the conduit wall, etc. If this were to occur, the high solvent strength could cause erroneously adsorbed fluid sample to be released again from the conduit wall, etc. Therefore, the mobile phase portion with higher solvent strength can hold the fluid sample together and also prevent the fluid sample from being undesirably carried over from one separation run to another. This advantageous effect is specifically facilitated by the direct fluid connection between the fluid sample and the mobile phase portion with higher solvent strength, as this promotes proper interaction between the fluid sample and the high solvent strength mobile phase. Therefore, a direct sequence of the sample plug and the mobile phase plug with higher solvent strength enables sample separation without the risk of contamination or sample loss. Furthermore, the direct adjacent sequence of the fluid sample and the mobile phase portion with higher solvent strength can effectively suppress the risk of unwanted precipitation of the fluid sample. Advantageously, by adding a mobile phase portion with higher solvent strength directly adjacent to the fluid sample, unwanted sample loss, retention, and precipitation can be effectively prevented, particularly at critical parts of the sample separation device (e.g., switching positions at fluid valves). While a mobile phase portion with higher solvent strength may involve slight self-desorption of the fluid sample at the sample separation unit, this phenomenon has proven acceptable in certain situations and can even be suppressed by simple measures (described below).

[0016] The following sections will explain the sample separation apparatus, the method for separating fluid samples, and additional embodiments of the fluid flow.

[0017] In this embodiment, the control unit is configured to clamp the fluid sample directly between two mobile phase portions having higher solvent strength. Undesirable sample loss, retention, and precipitation can be prevented particularly effectively when both the leading and trailing edges of the fluid sample plug are directly adjacent to the respective mobile phase portions with higher solvent strength.

[0018] In an embodiment, the control unit is configured to supply an additional mobile phase with a lower solvent strength after (particularly directly after) a mobile phase portion with a higher solvent strength following the fluid sample. Specifically, the additional mobile phase portion may have a lower solvent strength compared to the mobile phase(s) with higher solvent strength, and particularly also compared to the solvent of the fluid sample. This subsequent mobile phase portion with a lower solvent strength can improve the focusing of the fluid sample on the sample separation unit because this low-solvent-strength mobile phase enhances the adsorption of the sample on the stationary phase.

[0019] In an embodiment, the control unit is configured to reduce the solvent strength profile between the solvent of the fluid sample and at least one mobile phase portion having a higher solvent strength before the fluid sample reaches the sample separation unit. The term "strength profile" can refer to the difference between the solvent strength of the fluid sample and one or two mobile phase portions having a higher solvent strength immediately preceding and / or following the fluid sample. Advantageously, the solvent strength of one or more mobile phase portions directly adjacent to the fluid sample plug can be high as the fluid sample flows toward the sample separation unit and can be reduced in the final segment of the flow path before the fluid sample reaches the sample separation unit. This ensures that the fluid sample is properly focused on the stationary phase while ensuring that sample loss, precipitation, and retention are strongly suppressed. For example, the control unit can be configured to selectively modify the dilution factor of the fluid sample over time, specifically for the purpose of reducing the solvent strength profile directly before the chromatography column. By taking this measure, the solvent strength profile can be appropriately adjusted (specifically, smoothed) before the fluid sample reaches the sample separation unit. Specifically, distribution smoothing can be achieved by diluting with an additional mobile phase that has a lower solvent strength compared to at least one of the mobile phase portions with higher solvent strength.

[0020] In an embodiment, the control unit is configured to arrange an additional mobile phase portion having a lower solvent strength than the solvent of the fluid sample in direct proximity to the fluid sample before the fluid sample arrives at the sample separation unit. This can be accomplished in such a way that when the fluid sample arrives at the sample separation unit, the fluid sample is no longer in direct proximity to at least one of the mobile phase portions(s) having a higher solvent strength.

[0021] In an embodiment, the sample separation device includes an injector for injecting a fluid sample into a flow path toward the sample separation unit by combining the fluid sample with one or both of the mobile phase portions at a fluid coupling point or fluid confluence. Specifically, this combination of the fluid sample and the mobile phase can be achieved in terms of feed injection. Combining the fluid at a fluid coupling point (e.g., fluid T-point) is a very suitable mechanism for forming a sequence of fluid sample plugs directly connected to one or both mobile phase portions having higher solvent strength.

[0022] In one embodiment, the sample separation device includes an injector for injecting a fluid sample into a flow path toward a sample separation unit. The injector includes: a sample receiving volume for containing the fluid sample prior to injection; a sample actuator configured to draw the fluid sample into the sample receiving volume; and a fluid valve switchable between multiple switching states to selectively couple or separate the sample receiving volume from the flow path. In the injection switching state of the fluid valve, the fluid actuator, the separation unit, and the sample actuator are fluidly coupled via the fluid valve, such that fluid driven by the sample actuator and flowing from the sample receiving volume to the separation unit, and additional fluid driven by the fluid actuator and flowing from the fluid actuator to the separation unit, are combined at a fluid connection upstream of the separation unit. This embodiment is exemplified by... Figures 4 to 7 As shown in the diagram, this feed injection architecture can be advantageously combined with direct connection of the fluid sample to a mobile phase portion with high solvent strength, as already mentioned.

[0023] In an embodiment, the sample separation device includes: an additional fluid actuator for driving an additional mobile phase and a fluid sample separated by the sample separation unit; and an additional sample separation unit for further separating the fluid sample after separation by the sample separation unit. Specifically, the sample separation device may include a fluid switch (e.g., a fluid valve) coupled to the outlet of the sample separation unit and the additional fluid actuator, and the fluid switch is configured to be switchable for transferring the fluid sample separated by the sample separation unit to the additional sample separation unit. Such a two-dimensional sample separation device is, for example, in… Figure 8 and Figure 9 The advantages of direct connection of the fluid sample to the mobile phase portion with high solvent strength are particularly significant in two-dimensional sample separation devices, as the problems of sample loss, sample precipitation, and retention are particularly challenging in two-dimensional chromatography with long flow paths.

[0024] In an embodiment, the sample separation device includes a buffer volume for temporarily buffering a portion of the fluid sample separated by the sample separation unit, and for subsequently transferring the temporary buffered portion of the fluid sample for further separation by an additional sample separation unit. Direct connection of the fluid sample to a mobile phase portion with higher solvent strength allows for free selection of the order in which the buffering and transfer sample portions are performed, without being limited to the "first-in, first-out" concept.

[0025] In an embodiment, the control unit is configured to transfer temporary buffer portions of the fluid sample for further separation in a freely chosen order. Additionally or alternatively, the control unit may be configured to transfer temporary buffer portions of the fluid sample for further separation in an order different from the order in which the temporary buffer portions are inserted into the buffer volume. For example, in a two-dimensional sample separation apparatus (specifically, a chromatographic sample separation apparatus), portions of the fluid sample separated in the first separation dimension may be temporarily buffered in a sample loop connected to a fluid valve. To prevent sample retention, this sample buffering and subsequent transfer to the second separation dimension is typically performed in a "first-in, first-out" (FIFO) architecture, so the order in which sample portions are supplied to the sample loop and the order in which these sample portions are transferred to the second separation dimension are chosen to be the same in conventional methods. By sandwiching portions of the fluid sample between mobile phase portions with higher solvent strength, retention problems can be effectively suppressed, thus the "FIFO" rule is no longer mandatory. In contrast, the order of buffering and transferring sample portions can be freely chosen according to the requirements of the specific application.

[0026] In this embodiment, the control unit is configured to release the fluid sample adsorbed on the sample separation unit by driving the eluent through only a portion of the conduit through which the fluid sample flows. Therefore, guiding the eluent through all the conduits through which the fluid sample also flows becomes unnecessary, as the presence of one or more mobile phase portions with higher solvent strength directly adjacent to the fluid sample prevents the fluid sample from unintentionally adhering to the conduits. Driving the eluent only along the shortest path simplifies the operation of the sample separation device.

[0027] In an embodiment, at least one mobile phase portion with higher solvent strength includes a mobile phase portion disposed directly behind the fluid sample in the flow direction (this can be achieved through corresponding control by a control unit). Descriptively, given the higher solvent strength, this mobile phase portion with higher solvent strength following the fluid sample can serve as a sample collection plug, collecting any sample adsorbed on the conduit wall. This can suppress retention and prevent sample loss.

[0028] In this embodiment, a mobile phase portion immediately following the fluid sample in the flow direction is driven through the sample separation unit (this can be achieved through corresponding control by the control unit). Because this mobile phase portion, which has a higher solvent strength following the fluid sample, can collect lost sample material, it can be intentionally guided through the stationary phase of the sample separation unit so that the sample material recollected within the mobile phase portion can also be separated.

[0029] In an embodiment, at least one mobile phase portion having high solvent strength includes a mobile phase portion disposed immediately preceding the fluid sample in the flow direction (this can be achieved through corresponding control by a control unit). Descriptively speaking, due to the high solvent strength of the mobile phase portion, such a mobile phase portion having high solvent strength preceding the fluid sample can clean the conduit wall, for example, by desorbing any historical sample from the conduit wall.

[0030] In this embodiment, the mobile phase portion immediately preceding the fluid sample in the flow direction is prevented from flowing through the sample separation unit (this can be achieved through corresponding control by the control unit). Given the cleaning function of the mobile phase portion with higher solvent strength preceding the fluid sample (as described in the previous paragraph), contaminants may accumulate in the preceding high-solvent-strength mobile phase portion. To ensure highly accurate sample separation, the mobile phase portion with higher solvent strength preceding the fluid sample can be cut out of the flow stream, thereby being removed from the fluid sample before reaching the sample separation unit.

[0031] In this embodiment, at least one of the mobile phase portions arranged directly adjacent to the fluid sample has a lower solvent strength compared to the solvent of the fluid sample (this can be achieved through corresponding control by the control unit). Therefore, it may be sufficient for only one side of the fluid sample to be directly connected to the mobile phase portion with a relatively high solvent strength. The other side can be connected to the mobile phase portion with a relatively low solvent strength. This combines the advantages of suppressing sample retention, loss, and precipitation with proper focusing of the fluid sample on the sample separation unit.

[0032] The embodiments disclosed herein can be implemented in conventionally available HPLC systems, such as the Agilent 1200 (or 1290) series rapid separation LC system or the Agilent 1150 HPLC series (both provided by the applicant, Agilent Technologies, Inc., see www.aqilent.com, which is incorporated herein by reference).

[0033] One embodiment of the sample separation device includes a pump having a pump piston that reciprocates within a pump chamber to compress a liquid within the pump chamber to high pressure (at which the compressibility of the liquid becomes apparent). The pump can be configured to know (through operator input, notification from another module of the instrument, etc.) or otherwise acquire the solvent properties.

[0034] The separation unit of the sample separation apparatus preferably includes a chromatography column that provides a stationary phase (see, for example...). http: / / en.wikipedia.org / wiki / Column chromatography The column can be a glass or steel tube (e.g., 50 μm to 5 mm in diameter and 1 cm to 1 m in length) or a microfluidic column (e.g., as disclosed in the EP 1577012 or Agilent 1200 series HPLC-Chip / MS system provided by the applicant, Agilent Technologies). As the components propagate through the column with the eluent at different rates, they are retained by the stationary phase in different ways and are at least partially separated from each other. At the end of the column, they elute one component at a time, or at least not all components simultaneously. The eluent may also be collected in a series of fractions throughout the chromatography process. The stationary phase or adsorbent in column chromatography is typically a solid material. The most commonly used stationary phases in column chromatography are silica gel, surface-modified silica gel, and alumina. Cellulose powder was frequently used in the past. Other types of stationary phases include ion exchange chromatography, reversed-phase chromatography (RP), affinity chromatography, or expanded bed adsorption (EBA). The stationary phase is typically a finely ground powder or gel, and / or micropores, to increase surface area.

[0035] The mobile phase (or eluent) can be a pure solvent or a mixture of different solvents (e.g., water and an organic solvent such as ACN or acetonitrile). For example, the amount of retention and / or mobile phase for the compound of interest can be adjusted to run the chromatography. The mobile phase can also be selected to enable efficient separation of different compounds or fractions from the fluid sample. The mobile phase may include an organic solvent, such as methanol or acetonitrile, typically diluted with water. For gradient operations, water and organics are packaged in separate vials, and a gradient pump delivers a programmed mixture from the vials into the system. Other commonly used solvents include isopropanol, THF, hexane, ethanol, and / or any combination thereof, or any combination of the above solvents.

[0036] Fluid samples can include, but are not limited to, any type of process liquid, natural sample (e.g., juice), body fluid (e.g., plasma), or fluid samples can be, for example, the result of a reaction from fermentation broth.

[0037] The pressure in the mobile phase generated by fluid drive can be 2-200 MPa (20 to 2000 bar), especially 10-150 MPa (150 to 1500 bar), and even more especially 50-120 MPa (500 to 1200 bar).

[0038] Sample separation equipment (e.g., an HPLC system) may also include: a detector for detecting the separated compounds in a fluid sample; a fractionation unit for outputting the separated compounds from the fluid sample; or any combination thereof. Further details of such HPLC systems are disclosed in the Agilent 1200 Series Fast Separation LC System or the Agilent 1150 HPLC Series, both provided by the applicant, Agilent Technologies, Inc., available at www.aqilent.com, which are incorporated herein by reference.

[0039] Embodiments of this disclosure may be implemented or supported, in part or in whole, by one or more suitable software programs, which may be stored on or otherwise provided by any type of data carrier, and which may be executed in or by any suitable data processing unit. The software programs or routines may preferably be applied in or by a control unit. Attached Figure Description

[0040] Other objects and numerous accompanying advantages of the embodiments of this disclosure will be readily appreciated and better understood by referring to the following more detailed description of the embodiments in conjunction with the accompanying drawings. Features that are substantially or functionally identical or similar will be indicated by the same reference numerals.

[0041] Figure 1 A liquid sample separation apparatus according to an embodiment of the present disclosure is shown, specifically for high performance liquid chromatography (HPLC).

[0042] Figure 2 The exemplary embodiments shown herein can be used... Figure 1 The fluid flow obtained by the sample separation equipment.

[0043] Figure 3 A liquid sample separation apparatus for implementing feed injection according to an embodiment of the present disclosure is shown, specifically for high-performance liquid chromatography (HPLC).

[0044] Figures 4 to 7 An injector in different switching states is shown according to an exemplary embodiment of the present disclosure.

[0045] Figure 8A two-dimensional sample separation apparatus according to an exemplary embodiment of the present disclosure is shown.

[0046] Figure 9 A two-dimensional sample separation apparatus with feed injection is shown according to an exemplary embodiment of the present disclosure.

[0047] Figure 10 A sample separation apparatus with ring injection according to an exemplary embodiment of the present disclosure is shown.

[0048] The illustrations in the attached diagram are schematic. Detailed Implementation

[0049] Before describing the accompanying drawings in more detail, some basic considerations of this disclosure will be summarized based on exemplary embodiments that have been developed.

[0050] According to exemplary embodiments of this disclosure, fluid samples can be encased in a strong solvent. Therefore, strong solvents can be used for the transport and encapsulation of fluid samples. In embodiments, this can be advantageously combined with the concepts of feed injection and / or two-dimensional sample separation.

[0051] Conventionally, sample plugs can be sandwiched in a weak solvent to enhance initial sample adsorption relative to the column (this can be described as stacking). In essentially the opposite way, exemplary embodiments of this disclosure sandwich the sample in a strong solvent, specifically using feed injection techniques to introduce the sample onto the separation column.

[0052] In traditional chromatography applications, it is generally considered advantageous to sandwich the fluid sample within a section of the chromatography column containing a weak solvent (also referred to as a plug) in the flow path, especially when the original solvent of the sample is strong. This helps the sample components to be initially adsorbed into the stationary phase in the chromatography column (for trapping) and prevents the breakthrough or peak distortion of weakly retained sample components.

[0053] However, the inventors have discovered that this conventional method also carries the risk of sample precipitation or adhesion of sample components to the inner walls of the liquid chromatography system. These risks are particularly evident in systems where the entire sample path is not eluted by a gradient flow and therefore not exposed to a strong solvent composition, such as feed injection systems, two-dimensional liquid chromatography combined with feed injection, or two-dimensional liquid chromatography combined with a partitioned buffer in a dedicated buffer loop and the subsequent pullback of the partitioned buffer for further injection.

[0054] To effectively mitigate the described risks, exemplary embodiments of this disclosure advantageously sandwich the sample in a strong chromatographic solvent (or more precisely, a strong chromatographic solvent compared to the solvent of the fluid sample itself). More advantageously, any peak distortion and breakthrough hazards can be readily mitigated by subsequently reducing the solvent strength using appropriate solvent modulation during feed injection.

[0055] Specifically, it is possible and advantageous to use a strong chromatographic solvent (relative to the chromatographic system involved in the second separation dimension) as a mask solvent and as a solvent supplied by a modulating or metering injector. This concept can also be advantageously applied to other separation tasks, and is not limited to applications related to two-dimensional liquid chromatography involving feed injection techniques. Specifically, a time-varying dilution factor can be provided during the feed injection of the sample sandwiched in the strong solvent. Additionally or alternatively, the composition of the diluent (main solvent stream) can be provided to better control the solvent composition downstream of the feed injection point.

[0056] Now refer to the attached diagram for more details. Figure 1 A general schematic diagram of a liquid separation system, exemplified as a sample separation apparatus 10 according to exemplary embodiments of the present disclosure, is depicted. A fluid drive 20 (e.g., a piston pump) receives the mobile phase from a solvent source 25 via a degassing unit 27, which degasses and thus reduces the amount of dissolved gas in the mobile phase. The fluid drive 20 drives the mobile phase through a separation unit 30 (e.g., a chromatography column) including a stationary phase. A sampler or injector 40 implementing a fluid valve 95 may be provided between the fluid drive 20 and the separation unit 30 to apply or add (generally referred to as sample introduction) a sample fluid to the mobile phase, such that the fluid sample and the mobile phase can be provided toward a separation path in which actual sample separation occurs. The stationary phase of the separation unit 30 is configured for separating compounds from the sample liquid. A detector 50 is provided for detecting the separated compounds from the sample fluid. A fractionation unit 60 may be provided for discharging the separated compounds from the sample fluid.

[0057] While the mobile phase can consist of only one solvent, it can also be a mixture of multiple solvents. This mixing can be low-pressure mixing and provided upstream of the fluid drive 20, such that the fluid drive 20 has received and pumped the mixed solvent as the mobile phase. Alternatively, the fluid drive 20 may include multiple separate pumping units, each receiving and pumping a different solvent or mixture, such that mixing of the mobile phase (as received by the separation unit 30) occurs at high pressure and downstream of the fluid drive 20 (or as part of the fluid drive 20). The composition of the mobile phase can remain constant over time (i.e., a so-called isocratic mode) or vary over time (i.e., a so-called gradient mode).

[0058] A data processing unit or control unit 70 (which may be a PC or workstation) may be coupled (as indicated by the dashed arrow) to one or more devices in the sample separation apparatus 10 to receive information and / or control operations. For example, the control unit 70 may control the operation of the fluid drive 20 (e.g., setting control parameters) and receive information from it regarding actual operating conditions (e.g., output pressure at the pump outlet, etc.). Optionally, the control unit 70 may also control the operation of the solvent source 25 (e.g., setting the solvent or solvent mixture to be supplied) and / or the operation of the degassing unit 27 (e.g., setting control parameters and / or transmitting control commands), and may receive information from it regarding actual operating conditions (e.g., solvent composition supplied over time, vacuum level, etc.). The control unit 70 may also control the operation of the sampling unit or injector 40 (e.g., controlling sample injection, or synchronizing sample injection with the operating conditions of the fluid drive 20). The separation unit 30 may also be controlled by the control unit 70 (e.g., selecting a specific flow path or column, setting the operating temperature, etc.), and sending information (e.g., operating conditions) back to the control unit 70. Accordingly, detector 50 can be controlled by control unit 70 (e.g., regarding spectral or wavelength settings, setting time constants, starting / stopping data acquisition), and information (e.g., regarding detected sample compounds) can be sent to control unit 70. Control unit 70 can also control the operation of fractionation unit 60 (e.g., combining data received from detector 50) and provide data feedback.

[0059] Figure 1 A liquid supply device 150 is also shown, configured to meter liquids in a controlled proportion and to supply the result as a mobile phase. In the illustrated example, the liquid supply device 150 includes two reservoirs 109 and 111, each including a corresponding solvent component A (water in this example) and B (a buffer, i.e., a salt dissolved in the solvent in this example). Additionally, one or more additional and / or alternative reservoirs may be provided, such as an additional reservoir including an organic solvent, another reservoir including an optional organic modifier, etc. Each of the reservoirs 109 and 111 is fluidly connected via a corresponding liquid supply line 117 and via a degassing unit 27 using a proportioning unit 87 (which may be configured as a proportioning valve). The proportioning unit 87 is configured to connect a selected liquid supply line 117 to a supply line or conduit 119 leading to the fluid drive 20 and to switch between different liquid supply lines 117. The supply line or conduit 119 is connected to the inlet of the fluid drive 20. Therefore, solvent mixing is performed at the low-pressure side of the fluid driver 20 by metering or proportioning a series of fluid components.

[0060] Figure 2This illustrates the flow along the flow direction 103 through the conduit 101 according to an exemplary embodiment of the present disclosure (also shown in...). Figure 1 The fluid flow 130 (shown in the figure) can be generated by... Figure 1 Liquid supply equipment 150 is provided. For example, Figure 2 The catheter 101 shown can correspond to Figure 1 The conduit between the injector 40 and the sample separation unit 30.

[0061] like Figure 2 As shown, the fluid flow 130 provided upstream of the sample separation unit 30 may include a plug for the chromatographic sample 120 located directly between two mobile phase portions 122, 124, which have a higher solvent strength than the solvent of the chromatographic sample 120 itself. Advantageously, the control unit 70 is configured to control the dosing unit 87, the injector 40, and the fluid valve 95 for directly clamping the plug of the fluid sample 120 between the two mobile phase portions 122, 124, which have a higher solvent strength than the solvent of the fluid sample 120. Figure 2 As shown, the control unit 70 can also be configured to supply an additional mobile phase portion 126 with a lower solvent strength directly following the mobile phase portion 122 with a higher solvent strength after the fluid sample 120. Correspondingly, the control unit 70 can also be configured to supply another additional mobile phase portion 127 with a lower solvent strength directly before the mobile phase portion 124 with a higher solvent strength. Advantageously, the additional mobile phase portions 126 and 127 can have lower solvent strengths compared to the mobile phase portions 122 and 124 with higher solvent strengths, and also compared to the solvent of the fluid sample 120.

[0062] For example, the fluid sample 120 itself may be located within a mobile phase, which may be a mixture between water and an organic solvent of a first concentration. Each mobile phase portion 122, 124 may be a mixture between water and the organic solvent of a second concentration, wherein the second concentration may be higher than the first concentration. Additional mobile phase portions 126, 127 may be a mixture between water and the organic solvent of a third concentration, wherein the third concentration may be lower than the first and second concentrations.

[0063] Using according to Figure 2The described fluid grouping sequence prevents sample retention, loss, and precipitation while achieving adequate focusing of the fluid sample 120 on the sample separation unit 30. The high solvent strength mobile phase section 122, located directly behind (or downstream) the fluid sample 120, can desorb and collect any fluid sample inadvertently adsorbed onto the inner wall of the conduit 101. The high solvent strength mobile phase section 124, located directly in front of (or upstream) the fluid sample 120, can desorb and collect any historical contaminants that may be present on the inner wall of the conduit 101. The low solvent strength mobile phase section 126 can help to properly focus the fluid sample 120 onto the stationary phase of the sample separation unit 30.

[0064] To further improve the focusing of the fluid sample 120 at the sample separation unit 30, the liquid supply device 150 (and specifically, the control unit 70) may also be configured to arrange corresponding additional mobile phase portions 123, 125, having a lower solvent strength compared to the solvent of the fluid sample 120, directly adjacent to the fluid sample 120 before the fluid sample 120 reaches the sample separation unit 30. For example... Figure 2 As schematically shown, the additional mobile phase portion 123 can be directly inserted between the mobile phase portion 122 and the fluid sample 120. Correspondingly, the additional mobile phase portion 125 can be directly inserted between the mobile phase portion 124 and the fluid sample 120. (Reference) Figure 1 The opportunity to supply additional mobile phase portions 123, 125 can be achieved, for example, through additional fluid valves and / or additional fluid coupling points (not shown) downstream of injector 40 and upstream of sample separation unit 30. As a result, it can be ensured that when fluid sample 120 arrives at sample separation unit 30, fluid sample 120 is no longer directly adjacent to high solvent intensity mobile phase portions 122, 124. Therefore, fluid sample 120 can be properly focused on the stationary phase of sample separation unit 30.

[0065] The moving phase portion 122, arranged directly behind or after the fluid sample 120 in the flow direction, can be driven through the sample separation unit 30 together with the fluid sample 120, that is, directly after driving the fluid sample 120 through the sample separation unit 30. Because the moving phase portion 122 can collect the residue of sample material lost from the plug of the fluid sample 120, driving the moving phase portion 122 through the sample separation unit 30 can advantageously ensure that the vast majority of sample material is actually separated.

[0066] Related to the moving phase portion 124 arranged in the flow direction 103 directly in front of or before the fluid sample 120, it is advantageous to optionally prevent the moving phase portion 124 from flowing through the sample separation unit 30. As described above, the moving phase portion 124 can clean the inner wall of the conduit 101 before the fluid sample 120 passes through it. Therefore, the moving phase portion 124 may contain a small amount of residual historical sample and / or contaminants. If this is the case, it is advantageous to optionally remove the moving phase portion 124 from the fluid flow 130 before it reaches the sample separation unit 30. This can then ensure that the historical sample and / or contaminants do not degrade the separation accuracy of the fluid sample 120. The removal or shearing of the moving phase portion 124 upstream of the sample separation unit 30 can be accomplished, for example, by additional fluid valves and / or additional fluid coupling points (not shown) downstream of the injector 40 and upstream of the sample separation unit 30.

[0067] Alternative Figure 2 Compared to the solvent (not shown) of the fluid sample 120, one of the mobile phase portions 122 and 124 arranged directly adjacent to the fluid sample 120 may also have a lower solvent strength. Therefore, it is also possible that only one of the two mobile phase portions 122 and 124 has a higher solvent strength compared to the solvent of the fluid sample 120.

[0068] Figure 3 A sample separation apparatus 10 according to another exemplary embodiment of the present disclosure is depicted, which relates to feed injection.

[0069] Figure 3 Implementation examples and Figure 1 The specific difference in the embodiments is that, according to Figure 3 The fluid valve 95 can enter a switching state, wherein a fluid T-shaped element (see reference numeral 108, indicating the fluid coupling point) is formed within the fluid valve 95, thereby increasing the sample receiving volume of the fluid actuator 20, the separation unit 30, and the injector 40 (see reference numeral 108). Figure 3 The vertical arrow (in the diagram) is fluidly coupled in the injection switching state shown. For example, according to... Figure 2 Fluid sample 120 and mobile phase can be provided as fluid flow 130.

[0070] Figures 4 to 7 Exemplary embodiments according to this disclosure are shown (specifically as) Figure 3 (Example of injector 40) Injector 40 (and connected components) in different switching states.

[0071] according to Figures 4 to 7The injector 40 is configured to inject a fluid (here: liquid) sample into a flow path 104 between a high-pressure fluid driver 20 (configured for pumping a mobile phase, i.e., a definable solvent composition) and a separation unit 30 (implemented as a chromatography column). To separate the fluid sample 120 into multiple fractions, the injector 40 includes a sample ring or sample receiving volume 100 for containing a quantity of the fluid sample 120 prior to injection. The sample driver 102 (which may be implemented as a metering pump or a syringe pump) is configured to, when the fluid valve 95 is switched to a corresponding switching state (see...), ... Figure 6 The fluid sample 120 is driven from the sample receiving volume 100 into the flow path 104. To drive the fluid sample 120 toward the separation unit 30, the piston of the sample driver 102 is controlled to move forward. The sample driver 102 is also configured to draw the fluid sample 120 from the sample container (not shown) into the sample receiving volume 100 via the rearward movement of the piston 188. The fluid valve 95 can be switched to multiple switching states under the control of the control unit 70 (see...). Figures 4 to 7 By switching fluid valve 95, the sample containment volume 100 can be selectively coupled to the flow path 104 (see, for example...). Figure 6 Alternatively, the sample containment volume 100 can be separated from the flow path 104 (see example). Figure 4 or Figure 5 The control unit 70 can be configured to adjust the outlet pressure value and / or outlet volumetric flow rate value (or outlet mass flow rate value), according to which the moving phase and fluid sample 120 are driven to the separation unit 30.

[0072] Fluid valve 95 is a rotatable fluid valve 95 having a rotor and a stator that are rotatable relative to each other, such that the different fluid ports 1-6 of the stator are aligned with the corresponding fluid conduits 110 in the rotor. For example... Figures 4 to 7 As indicated by reference numeral 155 in the accompanying drawings, a portion of the fluid conduit 110 may be implemented as a stator groove, while the remaining portion of the fluid conduit 110 (not indicated by reference numeral 155) is implemented according to... Figures 4 to 7 It is implemented as a rotor slot. Fluid valve 95 is an active fluid valve of injector 40 that is switchable under the control of control unit 70.

[0073] The injector 40 includes a needle 112 and a seat 114 configured to receive the needle 112. Although not shown in the figure, the needle 112 can be driven toward a sample container (not shown) to draw a fluid sample 120 stored in the sample container into the sample receiving volume 100 via a sample actuator 102. The needle 112 is also configured to be driven back to the seat 114 before injection (e.g., Figures 4 to 7 (As shown). Reference numeral 166 in the attached figure indicates waste.

[0074] refer toFigure 4 This shows the purge position of the fluid valve 95 of the injector 40. According to... Figure 4 A fluid drive 20 or analytical pump is fluidly connected to a separation unit 30, which is implemented as a liquid chromatography column. In the washout position shown, a ring or sample containment volume 100, a needle 112, a seat 114, and a sample drive 102 (which is implemented as a metering device) are connected to an optional washout pump 180.

[0075] According to Figure 4 During the switching state, a fluid connection is established from the fluid actuator 20 via fluid ports 1 and 6 of the fluid valve 95 and conduits 110 and 155 to the separation unit 30. An additional fluid connection is established from the flushing pump 180 via fluid ports 2 and 3 of the fluid valve 95 and conduits 110 and 155, sample actuator 102, sample container volume 100, needle 112, and seat 114 back to the fluid valve 95 and from the fluid valve 95 to the waste 166.

[0076] Now for reference Figure 5 When the sample driver 102 is switched, the fluid valve 95 is switched to an extraction and depressurization / compression switching state, wherein the predefined overpressure in the sample holding volume 100 is adjustable before switching the fluid valve 95 to inject the fluid sample into the separation unit 30.

[0077] According to Figure 5 In the extraction and depressurization / compression positions of fluid valve 95, fluid actuator 20 or analytical pump is connected to separation unit 30 or liquid chromatography column. Sample containment volume 100 (also denoted as ring), needle 112, seat 114, and sample actuator 102 or metering device are blocked. Therefore, according to Figure 5 During the switching state, the fluid can be depressurized or compressed within the injector 40. Furthermore, according to... Figure 5 During the switching state, fluid sample 120 can be extracted.

[0078] According to Figure 5 During the switching state, a fluid connection is established from the fluid actuator 20 through fluid ports 1 and 6 of the fluid valve 95 and conduits 110 and 155 to the separation unit 30. The flushing pump 180 is disconnected. An additional fluid connection is established from the sample actuator 102 through the sample container volume 100, needle 112, and seat 114 back to the blocked fluid port 5 of the fluid valve 95.

[0079] refer to Figure 6Fluid valve 95 has been switched to the feed injection position. Now, fluid actuator 20 is fluidly connected to the same flow path 104, which is also fluidly connected to sample actuator 102. Sample receiving volume 100, needle 112, seat 114, and sample actuator 102 are fluidly connected to the valve internal fluid T-shaped element or fluid coupling point 108, which is formed by and located at static fluid port 6 (see reference). Figure 6 By defining the fluid coupling point 108 or the bifurcation point of the fluid T-shaped element by the static fluid port 6 and thus as part of the stator of the fluid valve 95, a specific, precise definition and reproducible fluid coupling point 108 can be established in the case of low dead volume or no dead volume. With the movement of the plunger of the sample driver 102 or the metering device, the previously aspirated fluid sample 120 can be injected into the separation unit 30.

[0080] More specifically, in cases such as Figure 6 In the injection switching state of the fluid valve 95 shown, the fluid actuator 20, the separation unit 30, and the sample actuator 102 are fluidly coupled through the fluid valve 95, such that the fluid (e.g., fluid sample 120) driven by the sample actuator 102 and flowing from the sample receiving volume 100 to the separation unit 30, and the additional fluid (e.g., mobile phase portions 122, 124 clamping the fluid sample 120 and flowing from the fluid actuator 20 to the separation unit 30) driven by the fluid actuator 20 are combined at the fluid coupling point 108 upstream of the separation unit 30 into a series of plugs (e.g., with...). Figure 2 (As shown). The combination of fluid flow at fluid connection 108 is in Figure 6 This is indicated by arrows 177 and 199. Therefore, the fluid flows combine at the fluid coupling point 108 into a common fluid flow toward the separation unit 30 (e.g., a series of plugs 122-120-124, such as...). Figure 2 As shown). In accordance with Figure 6 In the injection switching state, the control unit 70 can also be configured to adjust the volume or mass of the moving phase portions 122, 124 driven by the fluid actuator 20 and the fluid sample 120 driven by the sample actuator 102, which are directed toward the separation unit 30 at the fluid coupling point 108. In the injection switching state of the fluid valve 95, the fluid actuator 20, the separation unit 30, and the sample actuator 102 are fluidly coupled at the fluid coupling point 108 defined by the fluid valve 95. More precisely, according to Figure 6 In this switching state, the fluid coupling point 108 is located inside the active fluid valve 95.

[0081] According to Figure 6During the switching state, a fluid connection is established from the fluid actuator 20 via fluid ports 1 and 6 of the fluid valve 95 and conduits 110 and 155 to the separation unit 30. The flushing pump 180 is disconnected. An additional fluid connection is established from the sample actuator 102 via the sample container volume 100, needle 112, and seat 114 back to fluid port 5 of the fluid valve 95, and from fluid port 5 to fluid coupling point 108. At fluid coupling point 108, the fluid flows originating from the fluid actuator 20 and the sample actuator 102 are combined.

[0082] refer to Figure 7 The injection location is shown.

[0083] According to Figure 7 In the switched position of fluid valve 95, fluid sample 120, sandwiched between moving phase portions 122 and 124, is injected and driven towards separation unit 30 by fluid actuator 20, while sample receiving volume 100 is located downstream of fluid actuator 20 and upstream of separation unit 30. Therefore, according to Figure 7 In the additional injection switching state, fluid valve 95 does not (or no longer) form a fluid T-shaped section between fluid actuator 20, separation unit 30, and sample container volume 100. Instead, a continuous fluid connection is established from fluid actuator 20 via fluid valve 95, sample actuator 102, sample container volume 100, needle 112, seat 114, re-fluid valve 95, and separation unit 30. In this additional injection switching state, the fluid driven by fluid actuator 20 flows through sample actuator 102 and sample container volume 100 before flowing to separation unit 30.

[0084] from Figure 6 and Figure 7 The comparison shows that the main difference lies in the switching position of the fluid valve 95. The control unit 70 is configured to control the switching of the fluid valve 95 in order to select one of the following modes:

[0085] - Feed injection mode, in which the fluid sample is injected during the injection switching state (control). Figure 6 );or

[0086] - Flow mode, in which the fluid sample is injected in an additional switching state (control) Figure 7 ).

[0087] Figure 8 A sample separation apparatus 10 according to yet another exemplary embodiment of the present disclosure is depicted, which relates to two-dimensional sample separation.

[0088] Figure 8 Implementation examples and Figure 1 The specific difference in the embodiments is that, according to Figure 8A second separation dimension was added, in which the fluid sample 120, which had already been separated in the first separation dimension, was further separated in the second separation dimension.

[0089] The operation, acting as a first pump in the first fluid drive unit 20, typically receives the mobile phase from the first solvent source 25 via a first degasser 27. The first fluid drive unit 20 drives the mobile phase (e.g., forming mobile phase portions 122, 124 with high solvent strength) through the first separation unit 30. A sampling unit or injector 40 may be disposed between the first fluid drive unit 20 and the first separation unit 30 to add a fluid sample 120 to the mobile phase. The stationary phase of the first separation unit 30 is configured to separate compounds in the sample liquid.

[0090] The second pump, serving as the second fluid drive unit 20', typically receives another mobile phase from the second solvent source 25' via the second degasser 27'. Through fluid valve 94, Figure 8 The first dimension (reference numerals 20, 30, ...) of the two-dimensional liquid chromatography system can be configured to provide fluid segments or portions to the second dimension (reference numerals 20', 30', ...). The fluid sample 120 is separated into multiple fractions through the first dimension, and each fraction (or a portion / fraction thereof) is modulated into a second separation path and further separated into multiple sub-fractions by the second sample separation unit 30' of the second dimension.

[0091] Detector 50 is provided for detecting the separated compounds in fluid sample 120. An optional additional detector 55 is arranged upstream of fluid valve 94 and can be used to operate sample separation device 10 in a heart-cutting operation. However, it can also be used in integrated mode and any other operating mode for monitoring or evaluating first-dimensional chromatographic data. A corresponding fractionation unit 60 can be provided for outputting the separated compounds from the sample fluid. The treated fluid can also be pumped to waste.

[0092] Control unit 70 can control according to Figure 8 The various components of the sample separation device 10 include, specifically, fluid drive units 20 and 20', injector 40, fluid valve 94, separation units 30 and 30', detectors 50 and 55, proportioning unit 87, etc.

[0093] The first fluid drive unit 20 is therefore configured to drive a first fluid (which consists of an injected fluid sample 120 and a mobile phase, which may include mobile phase portions 122, 124, etc.) along a first flow path 85. The second fluid drive unit 20' is configured to drive a second fluid (which is implemented as a mobile phase) along a second flow path 86. A fluid valve 94 is fluidly coupled to both the first flow path 85 and the second flow path 86 and can be switched by the control unit 70 to transfer a portion of the first fluid from the first flow path 85 to the second flow path 86. Figure 8 The fluid processing device 10 shown is configured as a two-dimensional sample separation device, which is configured to separate a first fluid into multiple fractions (which can be detected by detector 55) and to separate at least one of the multiple fractions of the transferred first fluid into multiple sub-fractions (which can be detected by detector 50).

[0094] Figure 9 A two-dimensional sample separation apparatus 10 with feed injection according to an exemplary embodiment of the present disclosure is shown. The first separation dimension is indicated by reference numeral 141, and the second separation dimension is indicated by reference numeral 143.

[0095] Referring now in more detail to the first separation dimension 141, four different solvent reservoirs 105, 107, 109, and 111 (each reservoir containing a corresponding solvent) are provided as the basis for generating the mobile phase. A proportioning unit 87 (which may be implemented as a quaternary valve) is switchable under the control of the control unit 70 to adjust the solvent composition (e.g., to have a low solvent strength) using the components from the reservoirs 105, 107, 109, and 111.

[0096] In addition, a sample reservoir 190 containing fluid sample 120 is provided. Furthermore, a strong solvent reservoir 191 containing a solvent with high solvent strength is provided. In addition to the fluid valve 95 of the injector 40, the first separation dimension 141 includes an additional fluid valve 97 having a plurality of buffer volumes 160. Each buffer volume 160 is implemented as a loop connected between two designated ports of the additional fluid valve 97. The fluid valve 97 is also fluidly coupled to a sample actuator 102 implemented as a metering syringe. A needle 112 is currently immersed in the solvent reservoir 191 for drawing strong solvent into the fluidly connected buffer volume 160 by withdrawing the piston of the sample actuator 102. Alternatively, the needle 112 may also be immersed in the sample reservoir 190 for drawing fluid sample 120 into the fluidly connected buffer volume 160 by withdrawing the piston of the sample actuator 102. Figure 9 As shown, according to Figure 9The buffer volume 160 connected to needle 112 comprises a series of groups or plugs in the form of a fluid sample 120 with lower solvent strength between mobile phase portions 122, 124 with higher solvent strength. In another mode of operation (not shown), needle 112 can be moved into seat 114 to transfer the fluid group sequence 124-120-122 to (optionally but advantageously) detector 53, and from detector 53 to fluid valve 95 (which may also be represented as an injector valve). This movement of needle 112 is schematically indicated by reference numeral 198. For example, detector 53 can measure the composition of the flowing fluid under the control of control unit 70, which can be used to control the timing of the described operation.

[0097] First Figure 145 shows the solvent intensity distribution along the flowing fluid at the location between needle seat 114 and fluid valve 95, indicated by reference numeral 146. The distribution shown in First Figure 145 is particularly striking because the solvent intensity of the mobile phase portions 122, 124 is significantly higher than that of the fluid sample 120. Advantageously, this prevents carryover, loss, and precipitation of the sample along the flow path to fluid valve 95.

[0098] The second graph 147 shows the solvent intensity distribution along the flowing fluid at the location indicated by reference numeral 148 between the fluid valve 95 and the sample separation unit 30. Because the mobile phase provided by the fluid actuator 20 has a lower solvent intensity compared to mobile phase portions 122, 124, the distribution shown in the second graph 147 is less obvious than in the first graph 145 (the mobile phase portions 122, 124 modified by the mobile phase provided by the fluid actuator 20 are schematically indicated in the second graph 147 by reference numerals 122' and 124'). Advantageously, this dilution ensures proper focusing of the fluid sample 120 at the inlet of the sample separation unit 30. Therefore, the control unit 70 is configured to reduce the solvent intensity distribution between the fluid sample 120 and the mobile phase portions 122, 124 with higher solvent intensity before the fluid sample 120 reaches the sample separation unit 30. The control unit 70 can be configured to selectively modify the dilution factor of the fluid sample 120 sandwiched between the mobile phase portions 122, 124 over time.

[0099] Then, the fluid sample 120 is adsorbed onto the stationary phase of the sample separation unit 30. Subsequently, the separated fraction of the fluid sample 120 can be released from the stationary phase by supplying a corresponding mobile phase via the fluid driver 20, for example, according to a gradient distribution. Figure 9Consequently, the control unit 70 can be configured to release the fluid sample 120 adsorbed on the sample separation unit 30 by driving the eluent through only a portion of the conduit through which the fluid sample 120 previously flowed. This is advantageously artifact-free because the fluid sample 120 is already sandwiched between the high solvent intensity mobile phase portions 122, 124, thus being protected from unintentional adsorption onto the conduit walls, etc. After sample separation in the sample separation unit 30, the result of the first-dimensional separation can be detected by the detector 55.

[0100] In summary, the first separation dimension 141 achieves the function of sandwiching the fluid sample 120 between the mobile phase portions 122, 124 with higher solvent strength to suppress sample retention. Collaboratively, this can be combined with solvent weakening by combining the fluid sequence 124-120-122 with a mobile phase with lower solvent strength from the fluid driver 20 before reaching the sample separation unit 30. By taking the latter approach, proper sample focusing can be achieved at the inlet of the sample separation unit 30.

[0101] Referring now in more detail to the second separation dimension 143, four different solvent reservoirs 105', 107', 109', and 111' (each reservoir containing a corresponding solvent) are provided as the basis for generating an additional mobile phase. An additional proportioning unit 87' (which may be implemented as a quaternary valve) is switchable under the control of the control unit 70 to adjust the solvent composition using the components from the reservoirs 105', 107', 109', and 111'.

[0102] The second separation dimension 143 includes a fluid valve 94 for supplying a portion of the separated fluid sample 120 to an additional sample separation unit 30', and then to a detector 50 for detection. Furthermore, the second separation dimension 143 includes an additional fluid valve 98 for buffering a portion of the fluid sample 120 that has already been separated in the first separation dimension 141.

[0103] Each of the plurality of buffer volumes 161 (implemented herein as a ring) is connected between two designated ports of the fluid valve 98 and is configured to temporarily buffer a portion of the fluid sample 120 separated by the sample separation unit 30. The temporarily buffered portion of the fluid sample 120 can then be transferred for further separation by an additional sample separation unit 30'. Advantageously, the control unit 70 is configured to transfer the temporary buffered portion of the fluid sample 120 for further separation in a freely chosen order, i.e., not necessarily according to a "first-in, first-out" strategy. More specifically, the control unit 70 is configured to transfer the temporary buffered portion of the fluid sample 120 for further separation in an order different from the order in which the temporary buffered portion is inserted into the buffer volumes 161.

[0104] Figure 9 The second separation dimension 143 is also shown to include another fluid actuator 193 that drives a mobile phase with high solvent intensity stored in an additional mobile phase container 194. Furthermore, additional metering syringes 195 and 196 are provided, with the additional metering syringe 196 cooperating with a sample loop 197.

[0105] exist Figure 9 During operation of the sample separation apparatus 10, needle 112 operates according to containers 190, 191 and sample driver 102, such that the illustrated series of fluid groups 124-120-122 are arranged in the designated buffer volume 160. After needle 112 has been moved into seat 114, the piston of sample driver 102 moving forward transfers fluid group 124-120-122 into injector valve 95. In the appropriate switching state of injector valve 95, fluid group 124-120-122 is diluted by the mobile phase supplied by fluid driver 20 to smooth the distinct solvent intensity distribution of graph 145 to the less distinct solvent intensity distribution of graph 147. The combination of the mobile phase and the series of fluid groups 124-120-122 occurs at fluid coupling point 108 in injector valve 95 via feed injection (i.e., by flow combination at fluid coupling point 108). To ensure focused adsorption of the fluid sample 120 on the sample separation unit 30, the solvent intensity of the mobile phase provided by the fluid driver 20 can be lower than the solvent intensity of the mobile phase portions 122, 124 (and optionally also lower than the solvent intensity of the solvent in which the fluid sample 120 is located).

[0106] In the second separation dimension 143, the pre-separated fluid sample 120 can be guided onto the sample loop 197, as indicated by arrow 151. This can be achieved, for example, by withdrawing the piston of the push-pull metering syringe 196. A fluid actuator 193 (which may be represented as a mask flow pump) can provide a corresponding mobile phase plug (from container 194) with a higher solvent strength compared to the fluid sample 120. Such plugs can be added before and after the fluid sample 120. The corresponding mobile phase plugs can be supplied as indicated by arrow 153. More precisely, the mobile phase pre-plug can be supplied by the fluid actuator 193, followed by a portion of the fluid sample 120 (which is supplied by advancing the piston of the metering syringe 196). After said portion of the fluid sample 120, the mobile phase post-plug can be supplied by the fluid actuator 193. The sequence of pre-plug, fluid sample 120, and post-plug can then be transferred through fluid valve 94 into one of the buffer volumes 161 of fluid valve 98 and properly protected against unwanted adsorption and precipitation. Then, after appropriately switching the fluid valve 94, the feed or metering syringe 195 can supply the sequence of the pre-plug, fluid sample 120, and post-plug to the additional sample separation unit 30' for further separation. Prior to this further separation, the sequence of the pre-plug (with higher solvent strength), fluid sample 120 (with lower solvent strength), and post-plug (with higher solvent strength) can be diluted with a mobile phase from the fluid driver 20' having at least a lower solvent strength than the pre-plug and post-plug for focusing purposes.

[0107] Referring again to the first-dimensional separation 141, solvent modulation can be volume-based or flow-based. However, solvent modulation can also be signal-based (e.g., to compensate for dispersion and / or diffusion). For the latter purpose, a detector 53 can be provided (which can be placed before pressurizing the fluid sample 120).

[0108] Referring again to the push-pull metering injector 196, it can operate in half-peak / half-peak operating mode. For flow sample clamps, a first half-volume of the effluent (e.g., peak or center cut) can be drawn in simultaneously with injection into the lead plug by the mask flow pump. The first half-volume can be injected into the second half-volume. The second half-volume of the effluent can be drawn in simultaneously with injection into the tail plug.

[0109] Still referencing Figure 9 The lead plug and tail plug can be discarded into the waste (e.g., upstream of the separation column in the first separation dimension 141, not shown) because they may contain impurities.

[0110] Furthermore, the individual fragments or slices from the first separation dimension 141 can be modulated to have substantially the same solvent content or concentration. Inverse gradient operations can be performed.

[0111] Figure 10 A fluid handling apparatus with a ring injection sample separation device 10 according to an exemplary embodiment of the present disclosure is shown.

[0112] according to Figure 10 Under the control of the control unit 70, a mobile phase is supplied by the fluid actuator 20. This mobile phase (which may have a higher solvent strength than the solvent of the fluid sample 120) can be split into two partial flows at the fluid split point 200, as indicated by reference numerals 202 and 204. Partial flow 202 is transferred to a conduit in which it passes through a flow restrictor 206 and is then transferred to the fluid coupling point 108. Partial flow 204 is supplied to the sample actuator 102. The sample actuator 102 is fluidly coupled to the sample receiving volume 100 (implemented here as a sample ring), which is connected to the injector valve 95 of the injector 40. The needle 112 can be immersed in the container 190 containing the fluid sample 120, such that the fluid sample 120 can be drawn into the sample receiving volume 100 by withdrawing the plunger of the sample actuator 102. After the needle 112 is driven back to the seat 114, the plunger of the sample actuator 102 can move forward to supply the fluid sample 120 (followed by a mobile phase with high solvent strength) to the fluid coupling point 108. At the fluid coupling point 108, the fluid sample 120 can be directly clamped between the portions of the mobile phase with higher solvent strength. Therefore, Figure 10 An embodiment is shown in which the injector 40 is configured to inject the fluid sample 120 into a flow path toward the sample separation unit 30 by combining the fluid sample 120 with a mobile phase portion of a strong solvent at a fluid coupling point 108.

[0113] It should be noted that the term "comprising" does not exclude other elements or features, and "a" or "an" does not exclude a plurality. Elements described in conjunction with different embodiments may also be combined. It should also be noted that reference numerals in the claims should not be construed as limiting the scope of the claims.

Claims

1. A sample separation apparatus (10) for separating a fluid sample (120) having a second solvent strength, wherein, The sample separation device (10) includes: A fluid actuator (20) for driving a mobile phase and the fluid sample (120) injected into the mobile phase; A sample separation unit (30) is used to separate the fluid sample (120) from the mobile phase; and A control unit (70) is configured to sandwich the fluid sample (120) between two mobile phase portions (122, 124) of the mobile phase, wherein at least one of the mobile phase portions (122, 124) is arranged directly adjacent to the fluid sample (120) and has a first solvent strength, and wherein the at least one of the mobile phase portions (122, 124) having the first solvent strength is interfacially connected to an additional mobile phase portion (126, 127) having a third solvent strength. Wherein, the strength of the first solvent is higher than the strength of the second solvent and the strength of the third solvent.

2. The sample separation device (10) according to claim 1, wherein, The control unit (70) is configured to directly sandwich the fluid sample (120) between two mobile phase portions (122, 124) that have a higher solvent strength than the solvent of the fluid sample (120).

3. The sample separation device (10) according to claim 1 or 2, wherein, The control unit (70) is configured to supply an additional mobile phase (126) with a lower solvent strength after and immediately following the mobile phase (122) with a higher solvent strength after the fluid sample (120).

4. The sample separation device (10) according to claim 3, wherein, The additional mobile phase portion (126) has a solvent strength that is lower than that of the mobile phase portions (122, 124) and also lower than that of the solvent in the fluid sample (120).

5. The sample separation apparatus (10) according to claim 1 or 2, comprising at least one of the following features: in, The control unit (70) is configured to reduce the solvent intensity distribution between the solvent of the fluid sample (120) and at least one of the mobile phase portions (122, 124) having the first solvent intensity before the fluid sample (120) reaches the sample separation unit (30), and to reduce it by diluting it with an additional mobile phase having a lower solvent intensity compared to the at least one of the mobile phase portions (122, 124) having the first solvent intensity; The control unit (70) is configured to arrange at least one second additional mobile phase portion (123, 125) having a lower solvent strength than the solvent of the fluid sample (120) directly adjacent to the fluid sample (120) before the fluid sample (120) arrives at the sample separation unit (30), such that when the fluid sample (120) arrives at the sample separation unit (30), the fluid sample (120) is no longer directly adjacent to the at least one of the mobile phase portions (122, 124) having the first solvent strength.

6. The sample separation apparatus (10) according to claim 1 or 2, comprising at least one of the following features: in, The control unit (70) is configured to selectively modify the dilution factor of the fluid sample (120) over time; The control unit (70) is configured to release the fluid sample (120) adsorbed on the sample separation unit (30) by driving the eluent through only a portion of the conduit through which the fluid sample (120) flows; The sample separation device (10) includes an injector (40) for injecting the fluid sample (120) into a flow path toward the sample separation unit (30) by combining the fluid sample (120) with one or both of the moving phase portions (122, 124) at a fluid coupling point (108).

7. The sample separation apparatus (10) according to claim 1 or 2, comprising an injector (40) for injecting the fluid sample (120) into a flow path toward the sample separation unit (30), wherein, The injector (40) includes: A sample-containing volume (100) is provided for containing the fluid sample (120) prior to injection; A sample driver (102) is configured to draw the fluid sample (120) into the sample holding volume (100); A fluid valve (95) is switchable between multiple switching states to selectively couple or separate the sample receiving volume (100) from the flow path (104), wherein, in the injection switching state of the fluid valve (95), the fluid actuator (20), the separation unit (30) and the sample actuator (102) are fluidly coupled through the fluid valve (95) such that fluid driven by the sample actuator (102) and flowing from the sample receiving volume (100) to the separation unit (30), and additional fluid driven by the fluid actuator (20) and flowing from the fluid actuator (20) to the separation unit (30), are combined at a fluid coupling point (108) upstream of the separation unit (30).

8. The sample separation apparatus (10) according to claim 1 or 2, comprising: An additional fluid actuator (20') is used to drive an additional mobile phase and the fluid sample (120) separated by the sample separation unit (30); An additional sample separation unit (30') is provided for further separating the fluid sample (120) after separation by the sample separation unit (30).

9. The sample separation device (10) according to claim 8, comprising a second fluid valve (94) coupled to the outlet of the sample separation unit (30) and the additional fluid driver (20'), and configured to be switchable for transferring the fluid sample (120) separated by the sample separation unit (30) to the additional sample separation unit (30').

10. The sample separation apparatus (10) according to claim 8, comprising a buffer volume (161) for temporarily buffering a portion of the fluid sample (120) separated by the sample separation unit (30), and for subsequently transferring the temporarily buffered portion of the fluid sample (120) for further separation by the additional sample separation unit (30').

11. The sample separation apparatus (10) according to claim 10, wherein, The control unit (70) is configured to transfer a portion of the temporary buffer of the fluid sample (120) in a freely selectable order for further separation.

12. The sample separation device (10) according to claim 10, wherein, The control unit (70) is configured to transfer portions of the temporary buffer of the fluid sample (120) for further separation in a sequence different from the sequence in which these portions are inserted into the buffer volume (161).

13. The sample separation apparatus (10) according to claim 1 or 2, further comprising at least one of the following features: The sample separation device (10) is configured as a chromatography sample separation device, a liquid chromatography sample separation device, or a supercritical fluid chromatography sample separation device; The sample separation device (10) includes a detector (50) configured to detect the separated fractions of the fluid sample (120); The sample separation device (10) includes a distillation unit (60) configured to collect the separated fractions of the fluid sample (120); The sample separation device (10) includes an injector (40) configured to inject the fluid sample (120) into the mobile phase.

14. A method for separating a fluid sample (120) having a second solvent strength, wherein, The method includes: Drive the mobile phase and the fluid sample (120); Injecting the fluid sample (120) into the mobile phase, wherein injecting the fluid sample (120) into the mobile phase includes: The fluid sample (120) is sandwiched between two mobile phase portions (122, 124) of the mobile phase, wherein at least one of the mobile phase portions (122, 124) is arranged directly adjacent to the fluid sample (120) and has a first solvent strength, and wherein the at least one of the mobile phase portions (122, 124) having the first solvent strength is interfacially connected to an additional mobile phase portion (126, 127) having a third solvent strength. Wherein, the strength of the first solvent is higher than the strength of the second solvent and the strength of the third solvent; and The fluid sample (120) in the mobile phase is separated by the sample separation unit (30).

15. The method according to claim 14, wherein, The at least one mobile phase portion (122, 124) having the first solvent strength includes a mobile phase portion (122) arranged immediately after the fluid sample (120) in the flow direction, wherein the mobile phase portion (122) arranged immediately after the fluid sample (120) in the flow direction is driven through the sample separation unit (30).

16. The method according to any one of claims 14 to 15, wherein, The at least one mobile phase portion (122, 124) having the first solvent strength includes a mobile phase portion (124) immediately preceding the fluid sample (120) in the flow direction, wherein the mobile phase portion (124) immediately preceding the fluid sample (120) in the flow direction is prevented from flowing through the sample separation unit (30).

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