Multidimensional liquid analysis system and a method for analyzing a liquid sample

Abstract

Multidimensional liquid analysis system (10), comprising: an analysis system of first dimension (12) comprising a first separation column for the chromatographic separation of a liquid mobile phase into an efflux of first dimension (20) with an efflux rate of first dimension; a flow divider (24) for separating the outflow of first dimension (20) into a first outlet partial flow (26) and a second outlet partial flow (28) with a first pressure; a second-dimensional analysis system (14) comprising a second separation column (34) for chromatographically separating the second outlet partial stream (28) into a second-dimensional outflow (38) and an injection valve (32) for directing the samples from the second outlet partial stream (28) into the second separation column (34), wherein the injection valve (32) has an inlet opening (1) for receiving the second outlet partial stream (28), an outlet opening (6) and a first and second separate sample loop (36a, 36b), comprising flow channels that can be alternately positioned in fluidic connection with the second outlet partial stream (28) or the second separation column (34); and a flow metering device (30) for receiving the second exhaust partial flow (28) from the outlet opening (6) of the injection valve (32) along an exhaust flow path (29), wherein the flow metering device (30) comprises a valve for selectively closing or opening the exhaust flow path (29) in response to a control signal (72) from a metering control (74) programmed to allow the second exhaust partial flow (28) from the flow divider (24) at a predetermined second partial exhaust flow rate, wherein the second partial exhaust flow rate is determined by a volume of the second exhaust partial flow (28) enabled by the flow metering device (30) along the exhaust flow path (29) within a defined time interval. wherein the flow metering device (30) includes: (i) a stator (112) with a stator surface (116), a first stator inlet passage (114) extending along the outlet flow path (29) through the stator (112) and opening into the stator surface (116) through a first stator opening (118), a first discharge passage (120) extending along the outlet flow path (29) through the stator (112) and through a second stator opening (124) spaced apart from the first stator opening (118), and a second discharge passage (122) extending along a discharge path (33) through the stator (112) and opening into the stator surface (116) through a third stator opening (126); and (ii) a rotor (128) with a rotor surface (130) which is in fluid-tight contact with the stator surface (116) at an interface, wherein the rotor surface (130) includes a boat (132, 134) designed to receive a partial quantity of liquid in fluid-flow communication with the interface, wherein the rotor (128) is rotatable about an axis of rotation (136) with respect to the stator (112) in order to move the boat (132, 134) successively into several stations spaced apart about the axis, wherein a first station (138) aligns the boat (132, 134) in fluid-flow communication with the outlet flow path (29) at the first stator opening (118) and a second station (140) aligns the boat (132, 134) in fluid-flow communication with the outlet flow path (29) at the second aligns the stator opening (124), with the second and third stator openings (124, 126) being positioned at least when the boat (132, 134) is at the second station (140),are connected to each other in terms of fluid dynamics.

Description

AREA OF INVENTION

[0001] The present invention relates to flow systems used in analytical chemistry, and in particular to a division system for dividing a mobile phase flow in a multidimensional liquid chromatography instrument. BACKGROUND OF THE INVENTION

[0002] A mixture of compounds, or analytes, can be separated by pumping it through a separation device such as a chromatographic column using a process known as liquid chromatography, a variant of which is known as high-performance liquid chromatography (HPLC). The separation of the sample is caused by analytes having different affinities for the chromatographic packing material in the column. The separated sample flows continuously from the chromatographic column, with the separated analytes emerging at different times. The individual compounds comprising the analytes can then be passed through various detection devices, such as an ultraviolet light absorption detector, a mass spectrometer, a fluorescence detector, and the like, which contribute to determining the composition of the sample.The analytes can also be dispensed to a receiving station, where each analyte can be stored in separate containers in a manner known as fraction collection. In some cases, a small amount of the column effluent can be directed to the inlet of another sample analysis device, such as a mass spectrometer, for further analysis of each individual analyte. The dispensing of at least a portion of the column effluent to another liquid analysis device is referred to as "second-dimensional" analysis and is commonly used in complex liquid analysis.

[0003] An exemplary application for two-dimensional liquid analysis is in the purification of a synthesized compound during the development of a new drug. The synthesis products often contain the desired synthesized compound (with a known molecular weight), reactants, and byproducts, all of which are analytes in the synthesis sample. In this example, a "first-dimensional" analysis performs a separation on an analytical or preparative scale, such as by an HPLC column, with a dedicated detection device, such as a high-flow refractive index detector or an ultraviolet light detector, monitoring the column outflow. A "second-dimensional" analysis may preferably employ a second, separate flow path to capture a portion of the column outflow and direct the flow to a secondary analytical device, such as a mass spectrometer.Such instruments, combined in a “two-dimensional” arrangement, are increasingly used to expand the understanding of the purity of compounds on a liquid scale.

[0004] For a second-dimensional analytical device, such as a mass spectrometer, optimal operation requires a controlled, low mass flow rate of the eluent from the first-dimensional HPLC column containing the analyte. Such mass or flow rates should be easily adjustable and tightly controllable despite variations in the first-dimensional system's flow rate. The flow rate should be reproducibly controlled, facilitating the second-dimensional identification of the purity of an elution tip of the desired synthesized compound, thus simplifying the collection of a pure analyte in individual fractions.An experienced analyst can select a suitable carrier fluid to transfer the analyte into the second-dimensional detector. This second-dimensional carrier fluid may differ from the mobile phase used to perform the preparative first-dimensional separation of the synthesized compound. Certain mobile-phase fluids used for chromatographic separations may contain dissolved buffer salts, which can lead to contamination of other second-dimensional analytical instruments, such as a mass spectrometer. Furthermore, certain organic components of the mobile phase can inhibit the optimal ionization of the analytes required in a mass spectrometer. Appropriate selection of the carrier solvent minimizes the effect of the first-dimensional analyte in the mobile phase on the mass spectrometer.Additionally, the analyte mass transfer rate to the mass spectrometer should be low and should generally represent a small fraction of the total analyte flow rate in the first dimension. A high mass transfer rate to a mass spectrometer can lead to a decaying or persistent signal that distorts the results, and a high mass transfer rate can alter the dielectric properties of the system, causing instantaneous signal loss.

[0005] In some cases, analysts have attempted to work with combined HPLC and mass spectrometry instrumentation by reducing the HPLC mobile-phase flow rate to a less than optimal value, so that the outflow rate from the HPLC separation matches the liquid flow capacity of the mass spectrometer. Such a reduction in the flow rate through the HPLC column tends to decrease the available chromatographic resolution. To avoid this reduction in HPLC resolution, flow dividers have been used in a full-flow scheme to direct part of the flow from the HPLC column or detector outlet to the mass spectrometer inlet and the remainder to another detector or for disposal. Typical commercial flow dividers use resistance tubing elements to split the liquid flow into two or more separate streams.Exemplary flow dividers are described in US patent US 6,289,914 B1 and European patent application EP 0 495 255 A1. Maintaining a flow divider by resistance is difficult when trying to achieve uniform levels. Factors such as variable viscosity of the mobile phase, temperature, and any variations in the flow path during analysis can cause changes in the divider ratio between the respective flow paths. Such variability becomes particularly important when performing multidimensional liquid chromatography.

[0006] An example of a chromatographic application where mobile phase splitting is desirable is two-dimensional liquid chromatography (or LC*LC), where the first-dimensional HPLC column efflux is transferred to a second-dimensional HPLC column, with no portion of the first-dimensional separation being transferred to the second-dimensional column for subsequent second-dimensional separation. It is clear to the average professional in the field of HPLC analysis that various techniques exist for injecting a sample into a chromatographic column. In many cases, a sample volume is formed in a multi-way valve and then injected into the chromatographic column by a fluid force generated by a pump. Samples can be introduced into a flowing mobile phase stream.

[0007] Theoretically, it would be desirable to inject the entire volume of the first-dimensional separation into the column for the second-dimensional separation, although such a strategy can be impractical because the flow rate from the first separation column may be far too high for direct injection into the second. Therefore, analysis of the first-dimensional separation has typically been achieved by collecting the entire flow volume from the first separation column via fraction collection and then re-injecting a representative sample of each fraction into the second-dimensional separation column.

[0008] In addition to a flow rate mismatch, the development chromatogram in the first dimension may contain increasing relative concentrations of an organic solvent. This increasing relative concentration of an organic solvent can result from a specific liquid chromatographic strategy in which an organic solvent is injected into the separation column after an aqueous mobile phase. If the relative concentration of an organic solvent increases in the first-dimensional separation, injecting a fixed volume from the first dimension into the second-dimensional chromatograph will further increase the relative organic solvent concentration during the second-dimensional separation. Under the same conditions, injecting large volumes of an organic solvent into the second-dimensional chromatograph is detrimental to the second-dimensional separation.Because variation in the organic solvent concentration over time occurs in the first-dimensional separation, the flow rate exiting a standard resistance flow divider positioned downstream of the first column becomes unpredictable. Analysts therefore find it difficult to determine the actual flow rate of a sample available for injection into the second-dimensional separation column. Knowledge of the sample flow rate is critical for controlling the organic solvent concentration in the second-dimensional column and ensuring that no section of the first-dimensional chromatography remains unsampled in the second-dimensional separation. Typical resistance flow dividers are unable to provide analysts with the necessary information to consistently control second-dimensional analysis.Due to the limitations of standard resistance flow dividers, LC*LC has not found widespread application in engineering.

[0009] A flow-splitting method using a negative displacement pumping scheme is described in US patent application US 2012 / 0240666 A1. The method described in US 2012 / 0240666 A1 uses, for example, a syringe pump that extracts a partial flow from a flow divider positioned upstream of a second-dimensional injection valve. The volumetric flow rate of such a partial flow is determined by the negative displacement pump used to extract the partial flow from the first-dimensional outlet at the flow divider. However, due to the compliance of the syringe pump under pressure, the extraction volume can vary significantly depending on the hydraulic stiffness of the syringe pump, the applied pressure, and the volume of the pressurized fluid in the negative displacement pump. A more consistent flow-splitting scheme is therefore of interest to analysts and is an object of the present invention.Further state of the art is known from US 2005 / 0118075 A1 and WO 2014 / 015049 A2. BRIEF SUMMARY OF THE INVENTION

[0010] The present invention enables the precise control of partial flows in a multidimensional liquid analysis system to meet inlet flow rate requirements for second-dimensional analysis, while ensuring adequate analysis of a representative sample of the total first-dimensional outflow. Controlled flow division can be achieved by directly applying flow metering to one outlet stream from the flow divider, thereby determining the flow output from the other outlet stream of the flow divider. Flow control can be implemented by a valve that introduces a discontinuity in one outlet flow path, bridging the discontinuity at desired time intervals.No additional unsupported volume is inserted between the division point and a detection means attached to the uncontrolled leg of the flow divider, such as a mass spectrometer.

[0011] The multidimensional liquid analysis system according to the invention comprises a first-dimensional analysis system, which includes a first separation column for the chromatographic separation of a liquid mobile phase into a first-dimensional outflow with a first-dimensional outflow rate. The system further comprises a flow divider for separating the first-dimensional outflow into a first outlet stream and a second outlet stream with a first pressure. A second-dimensional analysis system comprises a second separation column for the chromatographic separation of the second outlet stream into a second-dimensional outflow and an injection valve for directing samples from the second outlet stream into the second separation column.The injection valve includes an inlet port for receiving the second outlet partial flow, an outlet port, and separate first and second sample loops that create flow channels which can be alternately positioned in fluidic connection with the second outlet partial flow or the second separation column. The multidimensional liquid analysis system further includes a flow metering device for receiving the second outlet partial flow from the outlet port of the injection valve along an outlet flow path.The flow metering device includes a valve for selectively closing or opening the outlet flow path in response to a control signal from a metering controller programmed to allow the second outlet partial flow from the flow divider at a predetermined second partial outlet flow rate, wherein the second partial outlet flow rate is determined by a volume of the second outlet partial flow enabled by the flow metering device along the outlet flow path within a specified time period.

[0012] The inventive method for analyzing a liquid sample comprises pumping the liquid sample with a first pump to a first-dimensional analysis system comprising a first chromatographic column for identifying a chemical component of the liquid sample, wherein the first-dimensional analysis system provides a first-dimensional liquid outflow at a first-dimensional outflow rate. The method further comprises separating the first-dimensional liquid outflow into a first outlet partial stream and a second outlet partial stream with a first pressure of at least one kilopascal, each of the first and second outlet partial streams being driven by the first pump via the flow divider. The second outlet partial stream is metered along an outlet flow path by selectively closing or opening the outlet flow path with a flow metering device to achieve a second partial outlet flow rate.The second outlet partial stream is directed through a second-dimensional analysis system, which includes a second chromatographic column for identifying the chemical component of the liquid sample. This second-dimensional analysis system comprises an injection valve with an inlet port for receiving the second outlet partial stream, an outlet port, and separate first and second sample loops, forming flow channels that can be alternately positioned in fluidic connection with either the second outlet partial stream or the second chromatographic column. Each of the first and second sample loops defines a first and second sample loop volume, respectively. The second outlet partial stream's flow rate allows the second outlet partial stream to fill a corresponding volume of the first and second sample loops within a defined time interval, which is the sum of the analysis time and the equilibration time of the second chromatographic column. BRIEF DESCRIPTION OF THE DRAWINGS Fig. Figure 1 is a schematic representation of a multidimensional liquid analysis system of the present invention; Fig. Figure 2A is a schematic representation of a flow metering device of the present invention; Fig. Figure 2B is a schematic representation of a flow metering device of the present invention; Fig. Figure 3 is a view of a part of a flow metering device of the present invention, broken down into individual parts; Fig. Figure 4 is an isolation view of a part of a flow metering device of the present invention; Fig. 5 an isolation view of part of a flow metering device of the present invention; Fig. Figure 6 is a schematic diagram of a flow metering device of the present invention; Fig. Figure 7A is a schematic representation of an injection valve of the present invention; and Fig. Figure 7B is a schematic representation of an injection valve of the present invention. DETAILED DESCRIPTION

[0013] To achieve a constant division of the outflow from the first-dimensional analysis system, a flow metering device is used to selectively close or open a pressurized fluid flow path exiting an outlet of a flow divider, thereby controlling the flow from such an outlet to a defined rate. The resulting flow from a second outlet of the flow divider is also controlled in this way. Such control requires that the flow rate at both outlets of the flow divider be known.

[0014] A first schematic representation of an arrangement of the present invention is shown in Fig. 1. A multidimensional liquid analysis system 10 comprises a first-dimensional analysis system 12 and a second-dimensional analysis system 14, wherein a mobile phase is passed through a first-dimensional separation column 16 by a first-dimensional pump 18, guided through a first-dimensional injection valve 19. The first separation column 16 chromatographically separates the liquid mobile phase into a first-dimensional outflow 20 with a first-dimensional pressure and outflow rate. The first-dimensional outflow 20 can be delivered directly to a first-dimensional detector 22 or can first be divided by a flow divider 24. The first-dimensional outflow rate into the flow divider 24 is controlled by the first-dimensional pump 18, which defines the flow rate of the mobile phase through the first-dimensional separation column 16.The pump of first dimension 18 further defines the first pressure of the outflow of first dimension 20 downstream of the separation column of first dimension 16. The first pressure of the outflow of first dimension 20 is preferably at least one kilopascal and is typically between 1 and 10,000 kilopascals. Preferably, the first pressure of the outflow of first dimension 20 is sufficient to establish a positive fluid pressure at both the detector of first dimension 22 and the analysis system of second dimension 14.

[0015] The flow divider 24 can include a T-shaped connection port with a first inlet and a first and second outlet, such as the one available from Kinesis-USA as the "Micro-Splitter Valve 10-32 / 6-32 Port 55 Needle (EA)". In the Fig. In the arrangement shown in Figure 1, a first outlet partial flow 26 from the flow divider 24 can be directed to the first-dimensional detector 22, and a second outlet partial flow 28 from the flow divider 24 can be directed to the second-dimensional analysis system 14 at a second pressure between 1 and 10,000 kilopascals. To assist in maintaining the second pressure at a setpoint between 1 and 10,000 kilopascals, a flow restrictor 27 can be positioned to limit the first outlet partial flow 26.

[0016] The analysis system 10 performs a chemical analysis of a liquid sample that is pumped into the columns of the first and second dimensions 16, 34. For the purposes of this invention, the first and second dimension “columns” can be generally designed to include analytical modalities that do not necessarily involve a column. For example, one or more of the dimensions can include liquid chromatography, HPLC, preparative-scale liquid chromatography, supercritical fluid analysis, gel permeation chromatography, mass spectrometry, other spectrometry or chromatographic analysis, and combinations thereof. In a particular application, the first and second dimensions are each chromatographic columns for evaluating a liquid sample.In some embodiments, such liquid chromatography may be “high-performance liquid chromatography” or “high-efficiency liquid chromatography” (HPLC), which is a general technique for performing chromatographic separations of solutions of compounds. These compounds are dispensed by a pump to an injection valve or autosampler for injection into the chromatographic column. Liquids and liquid mixtures used to transport the compounds are referred to herein as the “mobile phase.” The “stationary phase” of the liquid chromatography typically consists of the packing materials within the separation columns 16 and 34.

[0017] The second dimension analysis system 14 contains a second separation column 34 for chromatographic separation of the second outlet partial stream 28 into a second dimension efflux 38 for analysis at a second dimension detector 39, which may, for example, include a mass spectrometer.

[0018] As described above, flow control can be applied to the second outlet partial stream 28 using a flow metering device 30, which is operable to selectively close or open the outlet flow path 29 of the second outlet partial stream 28. A flow metering device 30 can operate in response to a control signal 72 from a metering controller 74, which is programmed to allow a second outlet partial stream 28 from the flow divider 24 at a predetermined second partial outlet flow rate, as determined by a volume of the second outlet partial stream 28 that is allowed by the flow metering device 30 along the outlet flow path 29 within a defined time interval.

[0019] The metering controller 74 can receive data inputs, such as fluid pressure, flow rate, temperature, and the like, from the first-dimensional flow, including the first-dimensional outlet 20 and a first-dimensional inlet 15. Together with the input system information, the metering controller 74 can send a control signal 72 to operate the flow metering device 30 in order to open or close the outlet flow path 29 at desired time intervals, thereby regulating the second partial outlet flow rate over a defined period. The flow metering device 30 also controls a flow rate from the first outlet 26 of the flow divider 24.

[0020] The flow metering device 30 can include a valve capable of selectively opening or closing the outlet flow path 29 at desired time intervals. In some embodiments, the flow metering device 30 can include a valve capable of moving separate partial volumes of liquid across a discontinuity in the flow path. The movement of the separate partial volume of liquid represents a "flow" of the volume along the flow path or a crossing of a discontinuity in the flow path. Thus, the flow metering device 30 receives the second partial outlet flow 28 and discharges it along the outlet flow path 29, the flow metering device 30 representing a discontinuity in the outlet flow path 29 that can be bridged discontinuously by separate partial volumes of liquid.The rate at which the discontinuity in the outlet flow path 29 is bridged by the flow metering device 30, as well as the volume of each separate liquid subset, determines the second partial outlet flow rate enabled by the flow metering device 30.

[0021] A number of valve structures are considered useful for the flow metering device 30. Some exemplary conventional mechanisms for bridging a flow discontinuity by transferring a separate partial volume of liquid are described in US patents 6,890,489 B2 and 7,575,723 B2. Another exemplary mechanism for a flow metering device 30 is described in US patent application 2014 / 0373605 A1. In general, such a valve mechanism includes a boat for receiving a partial volume of liquid of known volume from the second outlet partial stream 28 at an inlet station and moving the liquid-filled boat across the discontinuity to an outlet station, where the partial volume of liquid is discharged from the boat, for example, into a waste container 31.The flow metering device 30 therefore moves the boat between at least two separate boat stations to allow separate volumes of the second outlet partial flow 28 to flow along the outlet flow path 29 at a desired rate. The flow rates of both the first and second outlet partial flows 26, 28 can be operatively controlled. If the boat does not receive the second outlet partial flow 28 at its inlet station, the flow metering device 30 blocks the second outlet partial flow 28 upstream of the flow metering device 30. The valve-limited flow rate of the second outlet partial flow 28, together with the fluid pressure of the first-dimension outlet flow 20, forces a known fluid flow rate through the first outlet leg of the flow divider 24 to the first outlet partial flow 26.

[0022] A schematic representation of an exemplary embodiment for a flow metering device 30 is shown in Fig. 2A shown. According to the invention, the flow metering device 30 comprises a stator 112 with a first stator inlet passage 114, which extends along the outlet flow path 29 through the stator 112 and opens through a first stator opening 118 into a stator surface 116. In the Fig. In the embodiment shown in Figure 2A, the stator 112 comprises a first and second discharge passage 120, 122, wherein the first discharge passage 120 extends along the outlet flow path 29 through the stator 112 and opens into a stator surface 116 through a second stator opening 124, which is spaced apart from the first stator opening 118. The illustrated embodiment of the flow metering device 30 further comprises a rotor 128 with a rotor surface 130, which is in fluid-tight contact with the stator surface 116 at an interface. The rotor surface 130 includes a first boat 132, which is designed to receive a partial quantity of liquid in fluid-technical contact with the interface.The rotor 128 is preferably rotatable about a rotational axis 136 with respect to a stator 112 in order to move a first boat 132 successively into several stations spaced apart about the axis, where a first station 138 aligns the first boat 132 in fluid connection with the outlet flow path 29 at a first stator opening 118. A second station 140 aligns the first boat 132 in fluid connection with the outlet flow path 29 at a second stator opening 124.

[0023] The flow metering device 30 can include a second boat 134 on the rotor surface 130 at a location on the rotor surface 130 spaced relative to the axis, such that when the first boat 132 is in fluid communication with the outlet flow path 29 at the first stator opening 118, the second boat 134 is in fluid communication with the outlet flow path 29 at the second stator opening 124. In this way, the flow metering device 30 can simultaneously receive a second partial quantity of liquid from the second outlet partial flow 28, while a first, previously received partial quantity of liquid from the second boat 134 can be discharged through the first discharge passage 120. It is considered that one or more boats can be provided on the rotor surface 130 to accommodate different target points for the second partial outlet flow rate.

[0024] According to the invention, the flow metering device 30 includes a second dispensing passage 122, which extends along a dispensing path 33 through a stator 112 and opens through a third stator opening 126 into a stator surface 116. In this arrangement, according to the invention, the second and third stator openings 124, 126 are fluidically connected to each other, at least when the first or second boat 132, 134 is positioned at a second station 140. The second dispensing passage 122 can be provided for receiving pressurized gas, which is supplied by a pressurized gas source 82 via a dispensing path 33. The supplied pressurized gas can displace the liquid portion from the first or second boat 132, 134 when it is positioned at the second station 140.Such displacement forces the liquid portion outwards through the second stator opening 124 and through the first discharge passage 120 along the outlet flow path 29.

[0025] In another embodiment, the second station 140 of the rotor 128 can position the first or second boat 132, 134 in fluidic connection with the second stator opening 124 in order to discharge the partial quantity of liquid along the outlet flow path 29 through the first discharge passage 120. A reduced pressure can be applied through the first discharge passage 120 by a vacuum pump 84 to discharge the partial quantity of liquid from the corresponding first or second boat 132, 134. The vacuum force generated in the outlet flow path 29 between the second stator opening 124 and the vacuum pump 84 operatively evacuates the partial quantity of liquid from the first or second boat 132, 134 when it is positioned at the second station 140.In some embodiments, applying a vacuum force to dispense the liquid portion from the corresponding boat may be preferred over other dispensing means for the liquid portion, since the reduced pressure environment remaining in the boat as it moves to the first station 138 can help to fill the boat with the second outlet partial flow 28 at the first station 138. That is, the reduced pressure in the boat generates a higher differential pressure (Δρ) between the second outlet partial flow 28 and the "empty volume" of the first or second boat 132, 134, with the increased Δρ helping to drive the second outlet partial flow 28 into the first or second boat 132, 134.

[0026] As previously described, the second outlet partial flow 28 is possible when a boat 132, 134 is positioned at the first station 138. Typically, a flow metering device 30 is programmed to allow a rotor dwell time at the first station 138 and second station 140 sufficient to fill or unload the boats 132, 134 as needed. After the rotor dwell time has elapsed, the metering control 74 signals the flow metering device 30 to rotate the rotor 128 about the axis 136 to bring the first boat 132 to the second station 140. During this transition period, the rotor surface 130 and stator surface 116 are in fluid-tight contact, thus closing the outlet flow path 29.As soon as the first boat 132 reaches the second station 140, the rotor 128 is stopped again for a suitable filling / discharging time, during which period the outlet flow path 29 is open, thus enabling a second outlet partial flow 28. A flow cycle can be defined by the sum of the inlet / discharging rotor dwell time and the boat transition rotor rotation time span. The flow rate for the second outlet partial flow 28 is therefore determined by the boat volume and the flow cycle time. The rotor cycle time can be controlled to a certain extent by the metering control 74, but may be limited by operational constraints in the rotor speed as well as fluid flow characteristics for the inlet and discharge of liquid, which are controlled by Δρ at both the first and second stations 138, 140, and by physical fluid properties such as viscosity, surface tension, and the like.Upstream flow pressures can be measured and the resulting data can be sent to the metering control 74, so that the flow metering device 30 can be operated at desired parameters to control the flow rates of both the first and second outlet partial streams 26, 28.

[0027] In the Fig. In the schematic arrangement shown in Figure 1, the flow metering device 30 is operably positioned downstream of a second-dimensional injection valve 32. However, it is clear that the flow metering device 30 can also be operably positioned between the flow divider 24 and the second-dimensional injection valve 32 along the outlet flow path 29, such that the portion of the liquid delivered through the first delivery channel 120 is directed towards the second-dimensional injection valve 32.In such an embodiment, a flow metering discharge pump (not shown), which pumps a liquid mobile phase of the second dimension from a liquid source, pumps such liquid mobile phase through the second discharge channel 122 to discharge the liquid portion of the second partial outflow 28 contained in the corresponding boat 132, 134 at the second station 140, and through the first discharge channel 120 along the outlet flow path 29 to the outside. The liquid mobile phase of the second dimension could be the same as the liquid mobile phase of the first dimension. To receive the refill of the boats 132, 134 with the second outlet partial flow 28 at the first station 138, at least a third station is desirable for evacuating or gas discharge of the liquid mobile phase of the second dimension through the boats 132, 134 at the second station 140.

[0028] An embodiment of the flow metering device 30, which contains more than two rotor stations, is described in Fig. Figures 3-6 show the flow metering device 230 comprising a stator 232 and a rotor 234, which is rotatable about an axis of rotation 236 with respect to the stator 232. The rotor 234 is arranged in the flow metering device 230 in fluid-tight contact with the stator 232 at an interface 238, so that fluid can pass between the stator 232 and rotor 234 without leaking from the flow metering device 230. The stator 232 includes a stator surface 242 designed for sealing engagement with the rotor surface 244 of the rotor 234. In some embodiments, the stator surface 242 and rotor surface 244 can be substantially planar and placed in sealing engagement with each other by means of an external mounting assembly (not shown).

[0029] The stator 232 also includes a stator inlet passage 246, which extends along the outlet flow path 29 through the stator 232 and opens into the stator surface 242 through the first stator opening 250. A secondary stator inlet passage 252 extends along a secondary path 254 through the stator 232 and opens into the stator surface 242 through a second stator opening 256. A secondary stator outlet passage 258 extends along the secondary path 254 through the stator 232 and opens into the stator surface 242 through a third stator opening 260. An inlet discharge passage 262 extends along a discharge path 264 through the stator 232 and opens into the stator surface 242 through a fourth stator opening 266. An outlet discharge passage 268 extends along the discharge path 264 through the stator 232 and opens into the stator surface 242 through a fifth stator opening 270.In some embodiments, the outlet discharge passage 268 extends along the outlet flow path 29, as described above. In other embodiments, an inlet conveying passage 272 extends along a conveying path 274 through the stator 232 and opens into the stator surface 242 through a sixth stator opening 276. An outlet conveying passage 278 can extend along the outlet flow path 29 through the stator 232 and can open into the stator surface 242 through a seventh stator opening 280.

[0030] The passages described above are fluid passages that facilitate the passage of fluids through the stator 232. Such passages can be provided in sets, such as in groups of at least two, with an inlet passage and an outlet passage grouped together to guide a corresponding fluid. However, it is considered that at least the stator inlet passage 246 can be provided in a set of at least one passage, without the need for separate inlet and outlet passages for a particular fluid conveyance.

[0031] The sets of fluid passages are coordinated with one or more boats 282 in the rotor surface 244 of the rotor 234 to receive a partial quantity of fluid in fluid flow communication with the interface 238. The rotor 234 is rotatable about the axis of rotation 236 with respect to the stator 232 in order to move boats 282 sequentially into several stations spaced about the axis in fluid flow alignment with corresponding sets of fluid passages. A primary stator set 284 can include a stator inlet passage 246 and optionally a primary outlet stator passage 286, which extends along an optional primary path 248 through the stator 232 and opens into the stator surface 242 through an optional primary path opening 288.In some embodiments, the primary path 248 can be plugged to prevent an unwanted passage of the second outlet partial flow 28 through a boat 282 in the case of a fluid-technical connection with the stator inlet passage 246.

[0032] A secondary stator passage set 290 comprises secondary stator inlet and outlet passages 252, 258 and is preferably spaced about the axis from the primary stator passage set 284 on the stator surface 242. A discharge passage set 292 may comprise inlet and outlet discharge passages 262, 268 and may be spaced about the axis from the primary and secondary stator passage sets 284, 290 on the stator surface 242. A conveying passage set 294 may comprise inlet and outlet conveying passages 272, 278 and may be spaced about the axis from each primary stator passage set 284, secondary stator passage set 290, and discharge passage set 292 on the stator surface 242. The positions of each of the through sets 284, 290, 292, 294 can preferably define a station on the stator surface 242, wherein boats 282 can be moved from one orientation to the next by the rotation of the rotor 234.In some embodiments, the rotor 234 is rotatable 360° about the axis of rotation 236, so that it is brought into axial alignment successively with each of the stations defined by passage sets 284, 290, 292, 294 in the stator surface 242.

[0033] In some embodiments of the invention, the corresponding fluid passages of one or more of the passage sets 284, 290, 292, 294 can be fluidically connected to form a continuous fluid flow along the corresponding fluid passage through the stator 232 to the interface 238 along a first leg and then from the interface 238 through the stator 232 back along a second leg of the fluid path. Such a fluidic connection among the flow passages in a particular passage set enables a continuous fluid flow along the corresponding fluid paths when the stator surface 242 is sealed against the rotor surface 242, regardless of the position of the boat 282 about the axis. When the boat 282 is located between stations on the stator surface 242, the rotor surface 244 serves as a block for a fluid passage at the interface 238. An exemplary embodiment is shown in Fig. Figure 5 shows a primary bypass channel 298 arranged in the stator 232 to fluidically connect the stator inlet passage 246 with the primary stator outlet passage 286. A secondary bypass channel 300 is also provided in the stator 232 to form a fluidic connection between secondary stator inlet and outlet passages 252 and 258. A discharge bypass channel 302 may be provided in the stator 232 to form a fluidic connection between the inlet discharge passage 262 and the outlet discharge passage 268. In the illustrated embodiment, no bypass channel is provided for a conveying passage set 294. However, it is considered that a bypass channel 304 may be included for the conveying passage set 294 in order to form a flow-technical connection in the stator 232 between inlet and outlet conveying passages 272, 278.It is clear that one, all, or none of the fluid passages in one, all, or none of the passage sets 284, 290, 292, 294 can have a fluid connection in the stator 232, as with corresponding bypass channels 298-304. The purpose of bypass channels 298-304, as described above, is to establish a fluid connection between corresponding fluid passages in the stator 232. It is considered that the bypass channels 298-304 can be of any suitable size or configuration to expedite a bypass fluid flow between corresponding fluid passages and along a corresponding fluid path. Bypass channels 298-304 can, for example, be grooves in the stator surface 242 extending between corresponding openings of the fluid passages. In such an embodiment, the bypass channel to the interface 238 can be open, but enclosed by the rotor surface 244.In other embodiments, one or more of the bypass channels 298-304 can be completely enclosed in the stator 232. The channel widths “W” vary to different degrees. Fig. Figure 5 is shown, representing exemplary widths relative to corresponding openings on the stator surface 242.

[0034] Boats 282 can have the form of depressions in the rotor surface 244 and can have equal or unequal volumes. Exemplary volumes defined in the boats 282 can range from 10 to 1000 nanoliters, the shapes of which are suitable for effectively receiving and releasing partial quantities of fluid, as well as establishing and maintaining desired fluid flow characteristics when positioned at a corresponding station in alignment with a corresponding fluid passage set 284, 290, 292, 294 of the stator 232. It is considered that one or more of the boats 282 can be provided in the rotor surface 244. The one or more boats 282 are movable with the rotor 234 to several stations spaced about the axis by the rotation of the rotor 234 about the axis of rotation 236.A first station aligns a shuttle 282 at the first stator opening 250 in fluid-flow connection with the outlet flow path 29. A rotation of the rotor 230 by a predetermined amount about the axis of rotation 236 moves the shuttle 282 to a second station, which aligns the shuttle 282 in fluid-flow connection with the secondary path at the second and third stator openings 256, 260. A further rotation of the rotor 234 moves the shuttle 282 to a third station, which aligns the shuttle 282 in fluid-flow connection with the discharge path 264 at the fourth and fifth stator openings 266, 270. In some embodiments, a further rotation of the rotor 264 moves the boat 282 to a fourth station, which aligns the boat 282 in fluid-technical connection with the conveying path 274 at a sixth and seventh stator opening 276, 280.In some embodiments, the stations described above are each separated by a 90° rotation about the axis of rotation 236, so that the rotor 234 is rotated 360° about the axis of rotation 236 to allow the shuttle 282 to pass sequentially through the stations spaced about the axis by aligning itself with the respective sets of passages 284, 290, 292, 294. The cycle is repeatable by continuous rotation about the axis of rotation 236. Although only one shuttle 282 in the rotor area 244 can perform the necessary functions of a flow metering device 230, the desired rate for the second outlet flow 28 may be such that there is insufficient time for a single shuttle 282 to pass through each of the defined stations.In an exemplary situation where a partial quantity of liquid is taken per second from the second outlet partial stream 28, a single boat 282 must pass through each station in a total of one second, pausing at each station for a sufficient duration to exchange the correct fluids. The inherent limitations preclude a transition across the flow path discontinuity of one sample per second from the second outlet partial stream 28 using only a single boat 282. However, the rotational speed of the rotor 234 can be significantly reduced by providing additional boats 282. In the same example, with a sampling interval of one sample per second, a rotor area 244 with four boats 282 equally spaced around the axis could achieve the desired sampling rate at a rotational speed of one revolution every four seconds (15 rpm).The significantly lower rotational speed of rotor 234 allows for longer dwell times at each individual boat station in the transfer cycle.

[0035] In some embodiments, the second-dimensional injection valve 32 is a multi-way, multi-sampling-loop valve known in the prior art. An exemplary 10-way valve known in the prior art is described in Fig. 7A and Fig. Figure 7B shows a second-dimensional injection valve 32 in a first and second valve position. In particular, a second-dimensional injection valve 32 is shown in Fig. 7A is shown at a first valve position 35a, wherein fluid from the second outlet partial flow 28, which flows along the outlet flow path 29, is received at the inlet opening (1). In this embodiment, the second-dimensional injection valve 32 can establish two separate flow paths for use as a double-loop injection system. A first sample loop 36a comprises a flow passage extending between first sample loop openings (10, 7), wherein fluid is discharged from the inlet opening (1) and an excess flows from the outlet opening (6).In the first valve position 35a, the first sample loop 36a is fluidically coupled to the inlet port (1) and outlet port (6), while a second sample loop 36b connects the ports (2, 5) and is fluidically coupled to an injection path, driven by a second-dimensional pump 39, leading to the second-dimensional column 34. This injection path runs via a pump inlet port (9) through an injection port (4) containing ports (8, 3). Consequently, the first sample loop 36a can be filled with a second outlet partial flow 28 while the sample in the second sample loop 36b is analyzed in the second-dimensional column 34.

[0036] A second dimension 32 valve is in Fig.7B is shown in a second position 35b, where the second sample loop 36b can be filled while the sample in the first sample loop 36a is analyzed in the second-dimensional column 34. In this case, the second outlet partial flow can be received at the inlet port (1) at a flow rate controlled by the flow metering device 30 to pass through the second sample loop 36b at ports (2, 5) and out of the second-dimensional injection valve 32 at the outlet port (6). A second-dimensional pump 39 drives the sequential injection of a first sample loop 36a through ports (9, 10, 7, 8, 3, 4). A flow of the second outlet partial flow 28 can be directed alternately into the first or second sample loop 36a, 36b.The controlled flow rate of the second outlet partial stream 28 can be such that the filled sample loop volume represents a volume consumed over the entire time for the analysis and equilibration of the second-dimensional analysis. The first and second sample loops 36a, 36b can be filled alternately and injected into the second-dimensional column 34. An advantage of this technique is that each sample loop 36a, 36b is completely washed with the mobile phase of the second dimension over the entire analysis time to eliminate carryover. As described above, the first sample loop 36a is in fluid communication with the second separation column 34 when the second sample loop 36b is in fluid communication with the second outlet partial stream 28.

[0037] In some embodiments, the first and second sample loops 36a, 36b define a first and second sample loop volume, respectively, which is equal to or greater than the desired sample volume to be delivered to the second-dimensional column 34. The possible flow rate, as defined by the flow metering device 30, can be essentially equal to such a sample volume divided by the analysis time required for the second-dimensional column 34. In particular, the controlled flow rate is sufficient to allow the second outlet partial flow 28 to fill a corresponding portion of the first and second sample loop volumes within a defined time interval, which is the sum of the analysis time and the equilibration time of the second-dimensional column 34.Such a calculated flow rate of the second outlet partial stream 28 guarantees that a representative sample of the mobile phase passing through the flow divider 24 is delivered to a second dimension column 34.

[0038] The following is a ratio for an exemplary control scheme for a dosing control 74 to establish a suitable flow rate for the second outlet partial stream 28 in order to guarantee a complete chromatographic analysis of the mobile phase in the second dimension column 34: Fc≤VL / (T2a+T2e) where F c = controlled flow rate of the second outlet partial flow 28 V L = Volume of the sample loop that is filled T 2a = Analysis time of the second dimension column 34 T 2e = Equilibration time of the second-dimensional column 34

[0039] The second-dimensional equilibration time is the time required to flush a counterphase solvent from the second-dimensional column. For example, certain HPLC analyses are performed by first passing an aqueous phase through the column, followed by an organic phase, with the sample being injected into the aqueous and / or organic phase as appropriate. The sample is eluted through the chromatographic column by the succession of alternating aqueous / organic phases. Once the elution of the sample through the chromatographic column is complete, the column should be purged of any remaining aqueous / organic phase, which opposes the initial mobile phase in the subsequent sample analysis.Therefore, in the example of a sample that is first tested with an aqueous phase followed by an organic phase, such organic phase is preferably "flushed" from the column with an aqueous blank phase (such as water) before the subsequent sample sequence is initiated. This "flushing time" is the "equilibration time" used in the ratio above.

[0040] The invention has been described here in considerable detail to comply with patent regulations and to provide those skilled in the field with the information necessary to apply the novel principles and to design and use embodiments of the invention as required. However, it is clear that various modifications can be made without altering the scope of the invention as such.

Claims

[1] Multidimensional liquid analysis system (10), comprising: an analysis system of first dimension (12) comprising a first separation column for the chromatographic separation of a liquid mobile phase into an efflux of first dimension (20) with an efflux rate of first dimension; a flow divider (24) for separating the outflow of first dimension (20) into a first outlet partial flow (26) and a second outlet partial flow (28) with a first pressure; a second-dimensional analysis system (14) comprising a second separation column (34) for chromatographically separating the second outlet partial stream (28) into a second-dimensional outflow (38) and an injection valve (32) for directing the samples from the second outlet partial stream (28) into the second separation column (34), wherein the injection valve (32) has an inlet opening (1) for receiving the second outlet partial stream (28), an outlet opening (6) and a first and second separate sample loop (36a, 36b), comprising flow channels that can be alternately positioned in fluidic connection with the second outlet partial stream (28) or the second separation column (34); and a flow metering device (30) for receiving the second exhaust partial flow (28) from the outlet opening (6) of the injection valve (32) along an exhaust flow path (29), wherein the flow metering device (30) comprises a valve for selectively closing or opening the exhaust flow path (29) in response to a control signal (72) from a metering control (74) programmed to allow the second exhaust partial flow (28) from the flow divider (24) at a predetermined second partial exhaust flow rate, wherein the second partial exhaust flow rate is determined by a volume of the second exhaust partial flow (28) enabled by the flow metering device (30) along the exhaust flow path (29) within a defined time interval. wherein the flow metering device (30) includes: (i) a stator (112) with a stator surface (116), a first stator inlet passage (114) extending along the outlet flow path (29) through the stator (112) and opening into the stator surface (116) through a first stator opening (118), a first discharge passage (120) extending along the outlet flow path (29) through the stator (112) and through a second stator opening (124) spaced apart from the first stator opening (118), and a second discharge passage (122) extending along a discharge path (33) through the stator (112) and opening into the stator surface (116) through a third stator opening (126); and (ii) a rotor (128) with a rotor surface (130) which is in fluid-tight contact with the stator surface (116) at an interface, wherein the rotor surface (130) includes a boat (132, 134) designed to receive a partial quantity of liquid in fluid-flow communication with the interface, wherein the rotor (128) is rotatable about an axis of rotation (136) with respect to the stator (112) in order to move the boat (132, 134) successively into several stations spaced apart about the axis, wherein a first station (138) aligns the boat (132, 134) in fluid-flow communication with the outlet flow path (29) at the first stator opening (118) and a second station (140) aligns the boat (132, 134) in fluid-flow communication with the outlet flow path (29) at the second aligns the stator opening (124), with the second and third stator openings (124, 126) being positioned at least when the boat (132, 134) is at the second station (140),are connected to each other in terms of fluid dynamics. [2] Multidimensional liquid analysis system (10) according to claim 1, comprising a dispensing means for dispensing the liquid subset from the boat (132, 134) through the first dispensing pass (120). [3] Multidimensional liquid analysis system (10) according to claim 1 wherein the first and second sample loop (36a, 36b) define a first and second sample loop volume respectively, which are equal to or greater than an injection volume of the second outlet partial stream (28) required for analysis by the second separation column (34). [4] Multidimensional liquid analysis system (10) according to claim 1, wherein the first pressure is at least one kilopascal. [5] Multidimensional liquid analysis system (10) according to claim 4, wherein the first pressure is between 1-10,000 kilopascals. [6] Multidimensional liquid analysis system (10) according to claim 1, wherein the first sample loop (36a) is in fluid-technical connection with the second separation column (34) when the second sample loop (36b) is in fluid-technical connection with the second outlet partial stream (28). [7] Multidimensional liquid analysis system (10) according to claim 1, wherein a first partial outlet flow rate from the flow divider (24) is defined as the difference between the first dimension outflow rate and the second partial outlet flow rate. [8] Method for analyzing a liquid sample with a multidimensional liquid analysis system according to any one of claims 1 to 7, comprising: (a) Pumping the liquid sample with a first pump (18) to a first-dimension analysis system (12) having a first chromatographic column (16) for identifying a chemical component of the liquid sample, wherein the first-dimension analysis system (12) provides a first-dimension liquid outflow (20) at a first-dimension outflow rate; (b) Separating the first dimension liquid outflow (20) into a first outlet partial flow (26) and a second outlet partial flow (28) with a flow divider (24) at a first pressure of at least one kilopascal, wherein each of the first and second outlet partial flows (26, 28) is motivated by the first pump from the flow divider (24); (c) Metering the second partial outlet flow (28) along an outlet flow path (29) to a second partial outlet flow rate by selectively closing or opening the outlet flow path (29) with a flow metering device (30); and(d) Directing the second outlet partial stream (28) through a second-dimensional analysis system (14) with a second chromatographic column (34) for identifying a chemical component of the liquid sample, wherein the second-dimensional analysis system (14) includes an injection valve (32) with an inlet port (1) for receiving the second outlet partial stream (28), an outlet port (6), and a first and second separate sample loop (36a, 36b) comprising flow passages that can be alternately positioned in fluidic connection with the second outlet partial stream (28) or the second chromatographic column (34), wherein the first and second sample loops (36a, 36b) contain a first and second sample loop, respectively.second sample loop volume defined, wherein the second partial outlet flow rate allows the second outlet partial flow (28) to fill a corresponding volume of the first and second sample loops within a defined time period, which is the sum of an analysis time and an equilibration time of the second chromatographic column (34). [9] Method according to claim 8, wherein the flow metering device (30) includes a rotor (128) with a boat (132, 134) designed to receive a partial quantity of liquid, wherein the rotor (128) is movable to move the boat (132, 134) to several spaced-apart stations, wherein a first station (138) aligns the boat (132, 134) to receive the partial quantity of liquid from the second outlet partial stream (28) along the outlet flow path (29) and a second station (140) aligns the boat (132, 134) to release the partial quantity of liquid to the second outlet partial stream (28) along the outlet flow path (29). [10] Method according to claim 9, wherein the second outlet partial flow (28) is discontinuously connected to the flow metering device (30) through the boat (132, 134). [11] Method according to claim 9, wherein the outlet flow path (29) is opened by positioning the boat (132, 134) at the first station (138). [12] Method according to claim 9, comprising directing the second outlet partial flow (28) from the outlet opening (6) of the injection valve (32) to the flow metering device (30). [13] Method according to claim 9, comprising dispensing the liquid portion from the boat (132, 134) by displacing the liquid portion with a gas. [14] Method according to claim 9, comprising displacing the liquid portion from the boat (132, 134) under an applied vacuum pressure.

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