Method for operating a fluid system, fluid system, and computer program product

Abstract

The present invention relates to a method of operating a fluid system, wherein the fluid system comprises a fluid resistance element, and wherein the method comprises a step of defining a volumetric flow rate, in which a defined volume of fluid is forced out of the fluid resistance element, and wherein the defined volume is the fluid flowing out of the fluid resistance element within a first time interval defined by a time t 开始 and a time t 结束 The step of defining a volumetric flow rate comprises: at a time t 开始 , switching the system from a first operating state to a second operating state so that the pressure in the fluid resistance element changes from a first pressure value to a second pressure value, the second pressure value exceeding the first pressure value, and at a time t 开始 after and not later than a time t 结束 of t 减少 , switching the system to a third operating state so that the pressure in the fluid resistance element becomes a third pressure value, the third pressure value being lower than the second pressure value. The present invention also relates to a corresponding system and a corresponding computer program product.

Description

Technical Field

[0001] The present invention generally relates to the operation of fluid systems, and more particularly to fluid systems including fluid resistance elements such as chromatography columns. More specifically, the present invention relates to loading such fluid resistance elements with fluid.

[0002] Particular embodiments of the present invention are directed to the field of chromatography, particularly high performance liquid chromatography (HPLC). However, while the present invention will be described with reference to HPLC, it should be understood that the present invention is not limited thereto and is also applicable in other fields where fluid resistance elements are loaded with fluid. Particularly with respect to HPLC, it should be understood that HPLC is designed to improve the performance of chromatographic separations, with particular emphasis on improving the reproducibility, accuracy, and throughput of chromatographic separations. In addition, embodiments of the present invention are particularly focused but not limited to the field of low flow HPLC. Background Art

[0003] In low flow applications, a mass spectrometer can be used to detect analytes. Particularly for such a very complex detector, a very high utilization time (i.e., data acquisition) is advantageous for the effective operation of the system. Data is only acquired during the gradient step of the chromatographic separation. Therefore, high MS utilization can only be achieved if the time for transferring the analytical sample to the analytical column (column loading) and column equilibration is as short as possible. Since the gradient step is typically carried out at a relatively low flow rate and low pressure to improve separation efficiency, the entire pressure footprint of the system can be utilized to accelerate column loading and column equilibration by using an increased flow rate.

[0004] However, the volume delivered during these two steps is advantageously accurate and precise to achieve reproducible results. Reproducibility is advantageous in HPLC because identification and quantification are achieved by comparing a sample containing the analyte of interest with a reference of known composition and concentration. Therefore, reproducibility of the column loading process is also advantageous. Incorrect and imprecise column loading can have a significant adverse impact on the analytical performance. For example, if the volume actually loaded onto the analytical column during the column loading step is lower than expected, the corresponding chromatogram in the downstream detection process will correspondingly show peaks with incorrect peak heights and peak areas. Therefore, the compound of interest will be incorrectly quantified as being lower than the actual concentration.

[0005] In contrast, in the case where the loaded volume is higher than expected, early eluting compounds may be inadvertently eluted. Therefore, these compounds will either be completely absent or represented by peaks with incorrect low peak areas and heights. Therefore, the identification and quantification of these compounds are incorrect.

[0006] For column equilibration, a reproducible delivery volume is also beneficial since column equilibration directly affects the next gradient separation. If the delivery volume is too low, an excessive amount of organic solvent remains on the column and the separation efficiency will be significantly reduced. Unpredictable changes in retention times will occur and co-elution of peaks may be observed, which again results in incorrect identification and quantification of these compounds. If the delivered volume is too large, the equilibration time will increase and the MS utilization time will decrease.

[0007] EP 1918705 A1 discloses an apparatus and method for loading a sample into a trapping device. Although this technique disclosed in EP1918705 A1 may be satisfactory in some respects, it has some drawbacks and limitations, particularly in terms of the precision and efficiency of the loading process.

[0008] US2011 / 0005304 A1 also discloses a system and method for controlling fluid flow in a liquid chromatography device. SUMMARY OF THE INVENTION

[0009] In view of the above, an object of the present invention is to provide a technique that allows a defined volume to be loaded into a fluid resistance element, such as into a chromatography column. In particular, the technique should be relatively accurate and time-efficient.

[0010] These objects are achieved by the present invention.

[0011] In a first aspect, the present invention relates to a method of operating a fluid system, wherein the fluid system comprises a fluid resistance element, and wherein the method comprises a defined volume flow step, wherein, in the defined volume flow step, a defined volume of fluid is forced out of the fluid resistance element, wherein the defined volume is the fluid that flows out of the fluid resistance element within a first time interval defined by time t 开始 and time t 结束 wherein the defined volume flow step comprises: at time t 开始 , switching the system from a first operating state to a second operating state so that the pressure in the fluid resistance element changes from a first pressure value to a second pressure value, the second pressure value exceeding the first pressure value, at a time after time t 开始 and not later than time t 结束 at time t 减少, the system is switched to a third operating state to bring the pressure in the fluid resistance element to a third pressure value, which is lower than the second pressure value. Hereinafter, the operating state can be understood as the configuration of the fluid system, which can include, for example, the operating pressure and a specific configuration of the fluid connections between the various parts of the fluid system. An advantage of the method described above can be, for example, allowing the loading of fluid onto the trapping column at a pressure close to the maximum pressure footprint of the fluid system, which can make this loading process more efficient compared to, for example, a flow-controlled loading process.

[0012] The third pressure value can be lower than, equal to, or exceed the first pressure value. For example, the third pressure value can correspond to the pressure for injecting a sample into the separation column, the second pressure can be the pressure for loading the fluid resistance element, and the first pressure can be the pressure during equilibration. If the loading and / or equilibration should be accelerated, a pressure higher than the third pressure (for injecting the sample into the column) can be used. In this case, the third pressure value can be lower than the first pressure value for equilibration. If only the loading phase (e.g., by pressure-controlled operation) is accelerated, the equilibration and gradient phases will have approximately the same pressure level - at least during the start of a possible gradient operation. This is because typically gradient start conditions are used for the equilibration of the column. Thus, in such cases, the first pressure and the third pressure will be at least approximately equal to each other. However, it is also possible to perform the equilibration at a pressure lower than the pressure during sample injection, such that the third pressure value can also exceed the first pressure value.

[0013] The fluid system can additionally include a high-pressure section upstream of the fluid resistance element.

[0014] The method can include maintaining the system in the second operating state from t 开始 to t 减少 .

[0015] The system can include a pump system upstream of the high-pressure section. For example, maintaining the system in the second operating state from t 开始 to t 减少 can include maintaining the operating pressure of the pump system at the second pressure value.

[0016] Switching the system from the first operating state to the second operating state can include switching the pump system. This can involve, for example, switching the operating pressure of the pump system to the second pressure value. Generally, switching the pump system should be understood to include changing the pump operating parameters (e.g., pressure or its flow rate) or the composition of the solvent mixture delivered by the pump.

[0017] Switching the system to the third operating state can also include switching the pump system.

[0018] Switching the system to the third operating state can include switching the high-pressure section to the third pressure value at time t 减少 .

[0019] Switching the system to a third operating state may additionally include at time t 减少 Switching the pump system to zero flow. This may include actively regulating the operating pressure of the pump to match the pressure downstream of the pump. Switching the pump to zero flow can be advantageous when ensuring the delivery of an accurate volume of fluid to the fluid resistance element, as the measurement of the volume flowing out of the pump during the switch to a lower pressure can be complex and may require additional sensors.

[0020] Switching the system to a third operating state includes venting the high-pressure section.

[0021] The pump system may additionally be configured to supply at least one solvent.

[0022] Additionally, the pump system may be configured to supply a solvent mixture of a first solvent and a second solvent at different mixing ratios, wherein the pump system may include a first pump configured to supply the first solvent, a second pump configured to supply the second solvent, and a mixing unit downstream of the first pump and the second pump. In cases where the fluid system is used for gradient separation processes, the solvent mixture can have particular advantages. Here, the composition of the solvent mixture can vary continuously, typically where the concentration of the stronger solvent increases over time, in order to allow compounds with higher solubility to be eluted first, followed by compounds that may be more difficult to dissolve in the solvent mixture with a higher concentration of the strong solvent.

[0023] The mixing unit may be a mixing T. The mixing T may include a mixer with robust performance for various solvent ratios, solvent miscibility, and flow rates. Therefore, it may be provided with a mechanism for assisting the mixing process. For example, it may have a Dionex SpinFlow mixing design, including both radial and longitudinal mixing paths, and may operate at pressures up to 103 MPa.

[0024] time t 开始 and time t 减少 may define a second time interval.

[0025] The pump system may supply the solvent mixture within the second time interval, wherein the first solvent exceeds 20 vol-% of the solvent mixture, preferably exceeds 50 vol-%.

[0026] The pump system can supply a solvent mixture within a second time interval, where the first solvent constitutes 100% of the solvent mixture. For example, the first solvent can be a loading solvent for loading a sample onto a fluid resistance element, and the fluid resistance element can be a trapping column. In this case, it may be a strong solvent, so that all samples are dissolved in the loading flow. For example, due to a zero error of the flow rate sensor of the second solvent pump, some volume of the first solvent may also flow into the fluid channel of the second solvent. Then a cleaning or rinsing step may be required to ensure that the fluid channel of the second solvent reliably contains only the second solvent.

[0027] The above method can additionally include: at time t 减少 after and time t 结束 before time t 清洗 , switching the pump system, and the pump system supplies a solvent mixture after t 清洗 , where the second solvent exceeds 20 vol-% of the solvent mixture, and the second solvent preferably constitutes 100% of the solvent mixture after t 清洗 . For example, the second solvent can be a weak solvent and the subsequent gradient separation step may require a starting composition of a solvent mixture including a high concentration of the weak solvent. If the fluid channel containing solvent B before the mixing unit is not rinsed off, the resulting mixture downstream of the mixing unit will contain a higher concentration of the first (stronger) solvent and will have an adverse effect on the separation process.

[0028] The method can additionally include a pump system that supplies a cleaning volume V 清洗 of a solvent mixture within a cleaning time interval Δt 清洗 starting from time t B_清洗 . The exact time interval Δt 清洗 can depend on the relative concentrations of the first solvent and the second solvent delivered during the second time interval. A higher relative concentration of the first solvent will require a longer cleaning duration Δt 清洗 .

[0029] The above fluid system can additionally include a sample storage section. For example, the sample storage section can be a sample loop. The sample loop can have the advantage of reliably providing a defined storage volume. Knowing this defined volume can allow the sample loop to also be used for parking effluents, for example, in the above cleaning step.

[0030] The sample storage section can be fluidly connected to the pump system and downstream of the pump system within a time interval defined by time t 开始 and time t 减少 . This can be a preferred embodiment when the fluid resistance element is a trapping column and the above method is used to load a sample onto the trapping column.

[0031] The sample storage section may also be fluidly connected to the pump system and downstream of the pump system within a time interval starting from time t 清洗 This may occur, for example, when the sample storage section is used to park the effluent during a cleaning step.

[0032] The sample storage section may alternatively be fluidly connected to the pump system and downstream of the pump system within a time interval defined by t 减少 and t 清洗 This can help reduce the pressure in the sample loop and prevent, for example, cross-flow between two solvent channels of the pump system and sudden pressure changes when, for example, the sample loop needs to be disconnected from the analysis path after loading within a second time interval. The reduction in pump pressure can help accelerate the decompression process of the sample storage section.

[0033] Alternatively, within a time interval defined by t 减少 and t 清洗 the sample storage section may not be fluidly connected to the pump system.

[0034] For example, the sample storage section may also be fluidly connected to a metering device within a time interval defined by t 减少 and t 清洗 Switching the system to the third operating state may additionally include switching the sample storage section from a second pressure value to a third pressure value at time t

[0035] The fluid system may additionally include a metering device, and the method may additionally include using the metering device to switch the sample storage section from a second pressure value to a third pressure value. This can be done with the sample storage section fluidly disconnected from the analysis flow path; otherwise, the fluid resistance of the system would be very large and more energy would be required to decompress the sample storage section using the metering device. 减少 The fluid system may alternatively include a pump, and the method may include using the pump to switch the sample storage section from a second pressure value to a third pressure value.

[0036] Alternatively, the method may include venting the sample storage section to switch the sample storage section from a second pressure value to a third pressure value.

[0037] Switching the above system to the third operating state may include switching the pump system to operate at a third pressure value. This may cause reverse flow in the pump system for a short period of time, which must be measured to accurately determine the volume of fluid delivered to the fluid resistance element.

[0038] The method may include, after time t

[0039] 减少

[0040] and before time t 减少 ​结束 Prior to t 对准 , the pump system is switched to supply a solvent mixture having a defined mixing ratio. For example, as described above, at t 对准 it may be advantageous to change the composition to a high concentration of weak solvent and a low concentration of strong solvent, while at t 开始 it may be preferred to have different high concentrations of weak solvent and low concentrations of weak solvent to allow for rapid loading and controlled gradient separation. The alignment phase can then assist in achieving the preferred mixing ratio.

[0041] The method may additionally include the pump system supplying an alignment volume V 对准 of solvent mixture within an alignment time interval Δt 对准 starting from time t 对准 , where the alignment time interval Δt 对准 preferably ends at an end time t 结束 . The length of the time interval Δt 对准 can be determined by the relative concentrations of the strong and weak solvents required for the subsequent gradient separation step. For example, a higher concentration of strong solvent at the start of the gradient separation step may result in a longer Δt 对准 .

[0042] The time t 对准 can be after time t 清洗 to ensure that any one of the pumps in the pump system connected to the fluid channels of the mixing unit is reliably filled with the pumped solvent and to compensate for any cross-flow that may have occurred in the previous stages.

[0043] The sample storage section as described above can be fluidly connected to the pump system within the alignment time interval Δt 对准 . This can be done again to park the effluent from the pump system in the sample storage section.

[0044] The sample storage section can remain fluidly connected to the pump system until time t 结束 .

[0045] The defined mixing ratio as described above can be supplied within the alignment time interval Δt 对准 . For example, the defined mixing ratio can include increasing the concentration of the strong solvent within the time interval Δt 对准 , e.g., from 0 vol-% strong solvent to 2 vol-% strong solvent (and 98 vol-% weak solvent), where a solvent mixture comprising 100 vol-% weak solvent may have been delivered in the cleaning phase prior to t 对准 .

[0046] The fluid resistance element can be a chromatographic column, preferably a separation column.

[0047] The fluid system can be a liquid chromatography system and is preferably a high performance liquid chromatography system.

[0048] The above-defined volumetric flow rate step can additionally include loading a sample onto the chromatography column.

[0049] The method can additionally include a separation step starting at time t 结束 wherein the solvent mixture can be supplied to the chromatography column by a pump system.

[0050] At time t 结束 the supplied solvent mixture can include an initial separation mixing ratio.

[0051] The solvent mixture can have a mixing ratio that varies over time after time t 结束 For example, a typical mixing ratio can start at 2 vol-% strong solvent (and 98 vol-% weak solvent) at t 结束 and go to 40 vol-% strong solvent at the end.

[0052] The above-defined mixing ratio can be the same as the initial separation mixing ratio. The defined mixing ratio can be the mixing ratio at the end of the alignment phase.

[0053] The above sample storage section can be not fluidly connected to the chromatography column during the separation step.

[0054] The fluid system can include at least one flow rate sensor, and the method can include using the flow rate sensor to measure the flow rate of the fluid flowing through the fluid resistance element.

[0055] The fluid system can additionally include at least one flow rate sensor upstream of the fluid resistance element, and the method can additionally include using the flow rate sensor to measure the pumping flow rate of the fluid discharged by the pump system. The two above flow rate measurements can be compared to determine an equilibrium state in which the flow rate of the fluid discharged by the pump system is the same as the flow rate of the fluid flowing through the fluid resistance element. This can be used to define time t a .

[0056] The method can additionally include using the pumping flow rate to determine the total volume of fluid discharged by the pump system up to a given time.

[0057] The method can include a pressurization time interval Δt 开始 between a start time t a and time t 累积 wherein the pressure in the fluid resistance element rises from a first pressure value to a second pressure value. The method can additionally include determining the cumulative volume V 累积 of the fluid flowing out of the fluid resistance element during the pressurization time interval Δt 累积 .

[0058] The method may include at time t a and t 减少 a constant pressure time interval Δt 恒定加载 , wherein the pressure in the fluid resistance element is at a second pressure value during the constant pressure time interval Δt 恒定加载 . The method may additionally include determining a constant pressure volume V of the fluid flowing out of the fluid resistance element within the constant pressure time interval Δt 恒定加载 . 恒定加载

[0059] The method may include a pressure reduction time interval Δt starting from time t 减少 , wherein the pressure is reduced from the second pressure value to a third pressure value. The method may additionally include determining a reduced volume V of the fluid flowing out of the fluid resistance element within the pressure reduction time interval Δt 减少 . 减少 减少

[0060] The method may additionally include determining t by considering any one of the cumulative volume V 累积 , the constant pressure volume V 恒定加载 , the constant reduction volume V 减少 , the cleaning volume V B_清洗 and the alignment volume V 对准 . Once the sum of the volumes V 减少 , V 累积 , V 恒定加载 , V 减少 , V B_清洗 and V 对准 is equal to a defined volume of the fluid to be forced out of the fluid resistance element, the loading process may end.

[0061] The third pressure value may be the same as the pressure in the separation step. This may be the case when the separation step is to be performed after the loading step. Alternatively, for example, when the above method is used to clean and balance the trapping column, the third pressure value may be selected to be equal to the equilibrium pressure value.

[0062] The pressure in the separation step may be between 20 bar and 1500 bar, preferably between 200 bar and 1500 bar.

[0063] The first pressure value may be between 1 bar and 1500 bar.

[0064] The second pressure value may be between 200 bar and 2000 bar, preferably between 800 bar and 1500 bar.

[0065] The third pressure value may be between 20 bar and 1500 bar.

[0066] At least one of the at least one solvent described above may be any one of an organic solvent, an inorganic solvent, a polar solvent, and a nonpolar solvent.​​​

[0067] According to a second aspect, the present invention relates to a fluid system, wherein the fluid system includes a fluid resistance element, wherein the fluid system includes a control unit, and wherein the fluid system is configured to perform any one of the above methods.

[0068] The fluid system may include a high-pressure section upstream of the fluid resistance element.

[0069] The fluid system may include a pump system upstream of the high-pressure section.

[0070] The pump system may be configured to supply at least one solvent.

[0071] The pump system may be configured to supply a solvent mixture of a first solvent and a second solvent at different mixing ratios, wherein the pump system includes a first pump configured to supply the first solvent, a second pump configured to supply the second solvent, and a mixing unit downstream of the first pump and the second pump.

[0072] The mixing unit may be a mixing tee.

[0073] The fluid system may include a sample storage section.

[0074] The sample storage section may be a sample loop.

[0075] The fluid resistance element may be a chromatographic column, preferably a separation column.

[0076] The fluid system may be a liquid chromatography system and preferably a high-performance liquid chromatography system.

[0077] The system may include at least one flow rate sensor.

[0078] The system may additionally include a bypass line, and the system may be configured to take: a first configuration, wherein the pump system, the sample storage section, and the fluid resistance element are fluidly connected to each other; and a second configuration, wherein the pump system and the fluid resistance element are fluidly connected to each other by means of the bypass line, while the sample storage section is not fluidly connected to the fluid resistance element.

[0079] The pump system may include two pumps, each pump being configured to supply a different solvent.

[0080] According to a third aspect, the present invention relates to a method of using the above system to perform any one of the above methods.

[0081] According to a fourth aspect, the present invention relates to a computer program product comprising instructions which are configured to cause a fluid system to perform the method according to any one of the foregoing method embodiments when run on a control unit of the fluid system. For example, such a program may include instructions for changing the operating pressure of a pump upstream of a fluid resistance element based on the volume of fluid that has been conveyed through the fluid resistance element. In embodiments where active adjustment of the operating pressure may be required, it may be particularly advantageous to adjust the flow rate downstream of the pump to zero at time t 减少 when the flow rate downstream of the pump is adjusted to zero at time t

[0082] The present invention also relates to the following numbered embodiments.

[0083] Below, method embodiments will be discussed. These embodiments are indicated by the letter M followed by a number. Whenever a method embodiment is referred to herein, these embodiments are meant.

[0084] M1. A method of operating a fluid system, wherein the fluid system includes a fluid resistance element,

[0085] wherein the method includes a step of defining a volumetric flow rate, in which step a defined volume of fluid is forced out of the fluid resistance element, where the defined volume is the fluid that flows out of the fluid resistance element within a first time interval defined by time t 开始 and time t 结束 The fluid flowing out of the fluid resistance element within the defined first time interval,

[0086] wherein the step of defining a volumetric flow rate includes

[0087] At time t 开始 the system is switched from a first operating state to a second operating state to cause the pressure in the fluid resistance element to change from a first pressure value to a second pressure value, the second pressure value exceeding the first pressure value,

[0088] At time t 开始 after and not later than time t 结束 at time t 减少 the system is switched to a third operating state to cause the pressure in the fluid resistance element to become a third pressure value, the third pressure value being lower than the second pressure value.

[0089] M2. The method according to the foregoing embodiment, wherein the third pressure value exceeds the first pressure value.

[0090] M3. The method according to any one of the foregoing embodiments, wherein the fluid system includes a high-pressure section upstream of the fluid resistance element.

[0091] M4. The method according to any one of the foregoing embodiments,

[0092] wherein the method includes from t 开始 to t减少 Maintain the system in the second operating state.

[0093] M5. The method according to any one of the foregoing embodiments having the features of embodiment M3, wherein the system comprises a pump system upstream of the high-pressure section.

[0094] M6. The method according to the foregoing embodiment, wherein switching the system from the first operating state to the second operating state comprises switching the pump system.

[0095] M7. The method according to any one of the foregoing two embodiments, wherein switching the system to the third operating state comprises switching the pump system.

[0096] M8. The method according to the foregoing embodiment and having the features of embodiment M3, wherein switching the system to the third operating state comprises, at time t 减少 Switch the high-pressure section to a third pressure value.

[0097] M9. The method according to the penultimate embodiment, wherein switching the system to the third operating state comprises, at time t 减少 Switch the pump system to zero flow.

[0098] M10. The method according to any one of the foregoing embodiments having the features of embodiment M3, wherein switching the system to the third operating state comprises venting the high-pressure section.

[0099] M11. The method according to any one of the foregoing embodiments having the features of embodiment M5, wherein the pump system is configured to supply at least one solvent.

[0100] M12. The method according to the foregoing embodiment, wherein the pump system is configured to supply a solvent mixture of a first solvent and a second solvent at different mixing ratios, wherein the pump system comprises a first pump configured to supply the first solvent, a second pump configured to supply the second solvent, and a mixing unit downstream of the first pump and the second pump.

[0101] M13. The method according to the foregoing embodiment, wherein the mixing unit is Mixing T.

[0102] M14. The method according to any one of the foregoing embodiments, wherein time t 开始 and time t 减少 Define a second time interval.

[0103] M15. The method according to the foregoing embodiment and having the features of embodiment M13, wherein within the second time interval, the pump system supplies a solvent mixture, wherein the first solvent exceeds 20 vol-%E of the solvent mixture, preferably exceeds 50%.

[0104] M16. The method according to the foregoing embodiment, wherein during the second time interval, the pump system supplies a solvent mixture, wherein the first solvent constitutes 100% of the solvent mixture.

[0105] M17. The method according to any one of the foregoing embodiments having the features of embodiment M12, wherein the method further comprises

[0106] at time t 减少 after and time t 结束 before time t 清洗 , switching the pump system, and the pump system supplies a solvent mixture after t 清洗 , wherein the second solvent exceeds 20 vol-% of the solvent mixture, and the second solvent preferably constitutes 100% of the solvent mixture after t 清洗 .

[0107] M18. The method according to the foregoing embodiment, wherein the method further comprises the pump system supplying a cleaning volume V 清洗 of a solvent mixture during a cleaning time interval Δt 清洗 starting from time t B_清洗 .

[0108] M19. The method according to any one of the foregoing embodiments,

[0109] wherein the fluid system includes a sample storage section.

[0110] M20. The method according to the foregoing embodiment, wherein the sample storage section is a sample loop.

[0111] M21. The method according to any one of the foregoing two embodiments having the features of embodiment M12, wherein the sample storage section is fluidly connected to the pump system and downstream of the pump system during a time interval defined by time t 开始 and time t 减少 .

[0112] M22. The method according to any one of the foregoing embodiments having the features of embodiments M12, M17, and M19, wherein the sample storage section is fluidly connected to the pump system and downstream of the pump system during a time interval starting from time t 清洗 .

[0113] M23. The method according to any one of the foregoing embodiments having the features of embodiments M12, M17, and M19,

[0114] wherein the sample storage section is fluidly connected to the pump system and downstream of the pump system during a time interval defined by t 减少 and t 清洗 .

[0115] M24. A method according to any one of the preceding embodiments other than embodiment M23 and having the features of embodiments M12, M17, and M19,

[0116] wherein the sample storage section is not fluidly connected to the pump system within a time interval defined by t 减少 and t 清洗

[0117] M25. A method according to the preceding embodiment and having the features of embodiment M21, wherein switching the system to the third operating state further includes, at time t 减少 switching the sample storage section from a second pressure value to a third pressure value.

[0118] M26. A method according to the preceding embodiment, wherein the fluid system further includes a metering device, and wherein the method further includes using the metering device to switch the sample storage section from a second pressure value to a third pressure value.

[0119] M27. A method according to the penultimate embodiment, wherein the fluid system further includes a pump, and wherein the method further includes using the pump to switch the sample storage section from a second pressure value to a third pressure value.

[0120] M28. A method according to any one of the preceding embodiments and having the features of embodiment M25, wherein the method further includes venting the sample storage section to switch the sample storage section from a second pressure value to a third pressure value.

[0121] M29. A method according to any one of the preceding embodiments having the features of embodiment M5, wherein switching the system to the third operating state includes switching the pump system to operate at a third pressure value.

[0122] M30. A method according to any one of the preceding embodiments and having the features of embodiment M12, wherein the method includes:

[0123] at a time t 减少 after and at a time t 结束 before, switching the pump system to supply a solvent mixture having a defined mixing ratio. 对准

[0124] M31. A method according to the preceding embodiment, wherein the method further includes the pump system supplying an alignment volume V 对准 within an alignment time interval Δt 对准 starting from time t 对准 of the solvent mixture, wherein the alignment time interval Δt 对准 is preferably at the end time t 结束 ​​End.

[0125] M32. A method according to the penultimate embodiment and having the features of embodiment M18, wherein the time t 对准 At the time t 清洗 Afterwards.

[0126] M33. A method according to any one of the foregoing 3 embodiments and having the features of embodiments M12 and M19,

[0127] wherein the sample storage section is fluidly connected to the pump system during the alignment time interval Δt 对准 inside.

[0128] M34. A method according to the foregoing embodiment, wherein the sample storage section remains fluidly connected to the pump system until the time t 结束 .

[0129] M35. A method according to any one of the foregoing embodiments and having the features of embodiment M30, wherein a defined mixing ratio is supplied during the alignment time interval Δt 对准 inside.

[0130] M36. A method according to any one of the foregoing embodiments, wherein the fluid resistance element is a chromatographic column, preferably a separation column.

[0131] M37. A method according to any one of the foregoing embodiments, wherein the fluid system is a liquid chromatography system and preferably a high performance liquid chromatography system.

[0132] M38. A method according to any one of the foregoing embodiments having the features of embodiment M36, wherein the step of defining the volume flow rate includes loading the sample onto the chromatographic column.

[0133] M39. A method according to any one of the foregoing embodiments having the features of embodiments M12 and M36, wherein the method further comprises

[0134] a separation step starting from the time t 结束 wherein the solvent mixture is supplied to the chromatographic column by the pump system.

[0135] M40. A method according to the foregoing embodiment, wherein the solvent mixture supplied at the time t 结束 includes the starting separation mixing ratio.

[0136] M41. A method according to the foregoing embodiment, wherein the solvent mixture has a mixing ratio that varies with time after the time t 结束 .

[0137] M42. A method according to any one of the preceding 2 embodiments and having the features of embodiment M30, wherein the mixing ratio is defined to be the same as the starting separation mixing ratio.

[0138] M43. A method according to any one of the preceding 4 embodiments and having the features of embodiment M19,

[0139] wherein the sample storage section is not fluidly connected to the chromatographic column during the separation step.

[0140] M44. A method according to any one of the preceding embodiments, wherein the fluid system includes at least one flow rate sensor, and wherein

[0141] the method further includes using the flow rate sensor to measure the flow rate of the fluid flowing through the fluid resistance element.

[0142] M45. A method according to any one of the preceding embodiments and having the features of embodiment M5, wherein the fluid system further includes at least one flow rate sensor, and wherein

[0143] the method further includes using the flow rate sensor to measure the pumping flow rate of the fluid discharged by the pump system.

[0144] M46. A method according to the preceding embodiment, wherein the method further includes using the pumping flow rate to determine the total volume of the fluid discharged by the pump system up to a given time.

[0145] M47. A method according to any one of the preceding embodiments,

[0146] wherein the method includes a pressurization time interval Δt 开始 between a start time t a and a time t 累积 , wherein the pressure in the fluid resistance element rises from a first pressure value to a second pressure value, and wherein the method includes determining the cumulative volume V 累积 of the fluid flowing out of the fluid resistance element within the pressurization time interval Δt 累积 .

[0147] M48. A method according to the preceding embodiment,

[0148] wherein the method includes a constant pressure time interval Δt a between a time t 减少 and t 恒定加载 , wherein the pressure in the fluid resistance element is at the second pressure value during the constant pressure time interval Δt 恒定加载 , and wherein the method includes determining the constant pressure volume V 恒定加载 of the fluid flowing out of the fluid resistance element within the constant pressure time interval Δt恒定加载 。

[0149] M49. The method according to any one of the foregoing embodiments,

[0150] wherein the method comprises a decompression time interval Δt starting from time t 减少 during which the pressure decreases from a second pressure value to a third pressure value, and wherein the method comprises determining the reduced volume V of the fluid flowing out of the fluid resistance element during the decompression time interval Δt 减少 。 减少 。 减少 。

[0151] M50. The method according to any one of the foregoing embodiments, wherein the method further comprises determining t 减少 。

[0152] M51. The method according to the foregoing embodiment having the features of embodiment M47, wherein determining t 减少 comprises taking into account the cumulative volume V 累积 。

[0153] M52. The method according to any one of the foregoing embodiments having the features of embodiments M50 and M48, wherein determining t 减少 comprises taking into account the constant pressure volume V 恒定加载 。

[0154] M53. The method according to any one of the foregoing embodiments having the features of embodiments M50 and M49, wherein determining t 减少 comprises taking into account the constant reduced volume V 减少 。

[0155] M54. The method according to any one of the foregoing embodiments having the features of embodiments M50 and M18, wherein determining t 减少 comprises taking into account the cleaning volume V B _ 清洗 。

[0156] M55. The method according to any one of the foregoing embodiments having the features of embodiments M50 and M31, wherein determining t 减少 comprises taking into account the alignment volume V 对准 。

[0157] M56. The method according to any one of the foregoing embodiments having the features of embodiment M39, wherein the third pressure value is the same as the pressure in the separation step.

[0158] M57. A method according to any one of the foregoing embodiments and having the features of embodiment M39, wherein the pressure in the separation step is between 20 bar and 1500 bar, preferably between 200 bar and 1500 bar.

[0159] M58. A method according to any one of the foregoing embodiments, wherein the first pressure value is between 1 bar and 1500 bar.

[0160] M59. A method according to any one of the foregoing embodiments, wherein the second pressure value is between 200 bar and 2000 bar, preferably between 800 bar and 1500 bar.

[0161] M60. A method according to any one of the foregoing embodiments, wherein the third pressure value is between 20 bar and 1500 bar.

[0162] M61. A method according to any one of the foregoing embodiments and having the features of embodiment M11, wherein at least one of the at least one solvent is an organic solvent.

[0163] M62. A method according to any one of the foregoing embodiments and having the features of embodiment M11, wherein at least one of the at least one solvent is an inorganic solvent.

[0164] M63. A method according to any one of the foregoing embodiments and having the features of embodiment M11, wherein at least one of the at least one solvent is a polar solvent.

[0165] M64. A method according to any one of the foregoing embodiments and having the features of embodiment M11, wherein at least one of the at least one solvent is a non-polar solvent.

[0166] M65. A method according to any one of the foregoing embodiments and having the features of embodiment M9, wherein switching the pump system to zero flow includes actively adjusting the operating pressure of the pump to match the pressure downstream of the pump.

[0167] M66. A method according to any one of the foregoing embodiments that does not have the features of embodiment M2, wherein the third pressure value is lower than the first pressure value.

[0168] M67. A method according to any one of the foregoing embodiments that does not have the features of embodiments M2 and M66, wherein the third pressure value is the same as the first pressure value.

[0169] Next, system embodiments will be discussed. These embodiments are indicated by the letter S followed by a number. Whenever system embodiments are referred to herein, these embodiments are meant.

[0170] S1. A fluid system, wherein the fluid system includes a fluid resistance element, wherein the fluid system includes a control unit, and wherein the fluid system is configured to perform the method according to any one of the foregoing method embodiments.

[0171] S2. The system according to the foregoing embodiment, wherein the fluid system includes a high-pressure section upstream of the fluid resistance element.

[0172] S3. The system according to the foregoing embodiment, wherein the system includes a pump system upstream of the high-pressure section.

[0173] S4. The system according to the foregoing embodiment, wherein the pump system is configured to supply at least one solvent.

[0174] S5. The system according to the foregoing embodiment, wherein the pump system is configured to supply a solvent mixture of a first solvent and a second solvent at different mixing ratios, wherein the pump system includes a first pump configured to supply the first solvent, a second pump configured to supply the second solvent, and a mixing unit downstream of the first pump and the second pump.

[0175] S6. The system according to the foregoing embodiment, wherein the mixing unit is a mixing tee.

[0176] S7. The system according to any one of the foregoing system embodiments, wherein the fluid system includes a sample storage section.

[0177] S8. The system according to the foregoing embodiment, wherein the sample storage section is a sample loop.

[0178] S9. The system according to any one of the foregoing system embodiments, wherein the fluid resistance element is a chromatographic column, preferably a separation column.

[0179] S10. The system according to any one of the foregoing system embodiments, wherein the fluid system is a liquid chromatography system and preferably a high-performance liquid chromatography system.

[0180] S11. The system according to any one of the foregoing embodiments, wherein the system includes at least one flow rate sensor.

[0181] S12. The system according to any one of the foregoing system embodiments having the features of embodiments S3 and S7, wherein the system further includes a bypass line, and wherein the system can be configured to adopt: a first configuration, wherein the pump system, the sample storage section, and the fluid resistance element are fluidly connected to each other; and a second configuration, wherein the pump system and the fluid resistance element are fluidly connected to each other by means of the bypass line, while the sample storage section is not fluidly connected to the fluid resistance element.

[0182] S13. The system according to any of the foregoing system embodiments having the features of embodiment S3, wherein the pump system includes two pumps, each pump being configured to supply a different solvent.

[0183] U1. Use of the system according to any of the foregoing system embodiments, the system being for performing the method according to any of the foregoing method embodiments.

[0184] P1. A computer program product comprising instructions, wherein the instructions are configured to cause a fluid system to perform the method according to any of the foregoing method embodiments when run on a control unit of the fluid system. Description of the Drawings

[0185] The present invention will now be described with reference to the drawings, which are to be illustrative only and not to limit the scope of the present invention.

[0186] Figure 1 An operating phase in a liquid chromatography system is depicted;

[0187] Figure 2 A flow rate signal varying with time is depicted;

[0188] Figures 3(A) and 3(B) respectively depict a pressure and a flow rate signal varying with time;

[0189] Figure 4 A signal related to the solvent composition in a liquid chromatography program gradient scheme varying with time is depicted;

[0190] Figure 5 Variations of the flow rate, pressure, flow volume, and valve position signals with time are depicted; and

[0191] Figure 6 An exemplary fluid system in different operating modes is depicted. Detailed Description

[0192] Figure 1 A typical gradient separation is shown. In this regard, it should be noted that embodiments of the present technology relate to fluid systems, such as fluid systems for liquid chromatography (LC) such as high performance liquid chromatography (HPLC). An exemplary fluid system 100 that can be used for LC is in Figure 6Depicted in. The fluid system 100, also simply referred to as system 100, includes two pumps 12, 14 that can supply different solvents. Each of the pumps 12, 14 is connected to respective channels 22, 24, and the channels 22, 24 are joined to each other at a mixing unit 26 (e.g., a mixing tee). Downstream of the mixing unit 26, two flow paths 30, 40 are provided, where one of the flow paths 30 includes a sample storage section 32 (e.g., a sample loop 32), and the other flow path 40 can be referred to as a bypass line. The system 100 also includes a valve 34 for selecting which of the flow paths 30, 40. That is, the system 100 is adapted to (e.g., by means of at least one valve) select which of the flow paths 30, 40 is fluidly connected to the mixing unit 26. The flow paths 30, 40 can be joined to each other, and further downstream, a separation column 50 (as an example of a fluid resistance element 50) can be provided.

[0193] In addition, as Figure 6 shown in A) of, the system 100 may also include a controller 60, which can also be referred to as a control unit 60. For ease of illustration, this controller 60 is only depicted in Figure 6 A) of. However, it should be understood that this controller 60 also exists in the Figure 6 states depicted in B) to D) of. The controller 60 is operably connected to other components, as Figure 6 shown by the dashed lines in. More specifically, the controller 60 is operably connected to the pumps 12, 14 and the valve 34.

[0194] The controller 60 may include a data processing unit and may be configured to control the system and perform specific method steps. The controller can send or receive electronic signals of instructions. The controller can also be referred to as a microprocessor. The controller can be contained on an integrated circuit chip. The controller can include a processor with a memory and associated circuitry. A microprocessor is a computer processor that combines the functions of a central processing unit on a single integrated circuit (IC), or sometimes on multiple integrated circuits such as eight integrated circuits. The microprocessor can be a multi-purpose, clock-driven, register-based digital integrated circuit that accepts binary data as input, processes it according to instructions stored in its memory, and provides a result (also in binary form) as output. The microprocessor can contain both combinational logic and sequential digital logic. The microprocessor operates on numbers and symbols represented in the binary number system.

[0195] In addition, it should be understood that the system can be configured to measure pressures at different locations of the system. For example, the system can include multiple pressure sensors. For example, a first pressure sensor can be located in pump 12, while a second pressure sensor can be located in pump 14. These pressure sensors can also be operatively connected to controller 60, and controller 60 can use the readings of these pressure sensors when controlling the operation of the system. The pressure sensors can be configured to directly measure pressure. However, it should be understood that other parameters can also be measured and other parameters can be used to determine the corresponding pressure (and such processes should also be understood as pressure measurement and the components involved should be understood as pressure sensors). For example, it should be understood that when analyzing the power consumption of pumps 12, 14 supplying solvent at a certain flow rate, the power consumption of pumps 12, 14 will also depend on the pressure at which they operate - the higher the operating pressure, the higher the power consumption. Therefore, for example, the power consumption of pumps 12, 14 can also be used to deduce the pressure present at pumps 12, 14. Thus, system 100 can generally be configured to measure the pressures present at different locations of system 100.

[0196] Generally speaking, as Figure 6 shown, the operation of system 100 can be separated into different stages (see also Figure 1 ).

[0197] One of the stages can be called sample injection: In this stage, a specified injection volume of the sample to be analyzed is drawn from the corresponding vial and introduced into the high-pressure flow path.

[0198] Another stage can be called column loading: The sample (which can also be called the sample plug) is transferred from the sample loop 32 to the separation column 50, which can also be called the analytical column 50. Column 50 typically includes a stationary phase. The compound(s) of interest interact with the stationary phase of analytical column 50 and bind temporarily and reversibly. This results in the accumulation and enrichment of the analyte(s) of interest on analytical column 50.

[0199] Another stage can be called gradient separation: During the gradient execution, the reversibly bound analytes are gradually removed from analytical column 50 and detected by a detector located downstream of column 50, for example, by mass spectrometry. With particular reference to gradient separation, it should be understood that in particular the composition of the solvent can change over time. In this regard, Figure 6 pumps 12, 14 in Figure 1 can be operated in a manner such that the amount of solvent B in a mixture of, for example, A and B (e.g., continuously) increases (also refer to

[0200] where the y-axis depicts the concentration of B in the mixture). Such a scheme can be advantageous because it can improve the decomposition rate as different compounds of the sample can be eluted at different solvent compositions.A further additional stage may be termed washing and equilibration: after separating the compound of interest, the analytical column 50 is washed with a solvent (e.g., with a high organic solvent), and then re-equilibrated to the starting conditions for the next injection.

[0201] Overall, the fluid system 100 can thus operate according to a gradient separation scheme, and Figure 1 depicts the typical stages of the above-mentioned gradient separation. Data is only acquired during the actual gradient separation step, as Figure 1 shown, where data acquisition occurs only during the gradient step.

[0202] Embodiments of the present technology thus particularly relate to the description of the separation column 50 in the fluid system 100. However, while embodiments of the present technology may be particularly described with reference to an LC system and a loaded separation column 50, it should be understood that the described technology is not limited to LC systems and the loading of separation columns 50, but can actually be employed whenever a defined volume of fluid is desired to flow through a fluid resistance element.

[0203] In this specification, the term "loading" generally refers to the delivery of a defined loading volume onto the analytical column 50, which applies to column loading and column equilibration. In the following, the present technology will be particularly described with reference to column loading, and it should be understood that, particularly for column loading, it is generally desirable to supply a defined quantity of fluid to the column. However, corresponding considerations can also apply to column equilibration - again here, a defined quantity of fluid may need to be supplied to the column. Similarly, it should generally be understood that the described technology can be used whenever a controlled quantity of liquid is desired to be supplied to a fluid resistance element.

[0204] In the following, concepts that contribute to the understanding of the present technology will be described before more specific embodiments of the present technology are described in more detail.

[0205] Generally speaking, a fluid resistance element such as the separation column 50 can be loaded in different ways.

[0206] One way of loading a fluid resistance element (i.e., allowing a certain quantity of liquid to flow into the element, and it should be understood that typically the same quantity of liquid will also flow out of the element) is referred to as flow-controlled loading.

[0207] For example, loading a sample onto an analytical column typically occurs in a flow-controlled manner. Thus, the sample is delivered to the column at a set constant flow rate f 加载 . For example, the pressure can then be set in such a way as to achieve the flow rate. Thus, at time t 加载 after volume V 加载 has been delivered, the loading is complete, where

[0208] t 加载 = V 加载 / f 加载 .

[0209] The advantage of this loading method is that the time required for loading is predictable and thus can be well planned. Therefore, it can be widely used. However, flow-controlled loading may have several disadvantages.

[0210] It may be inefficient: If the entire flow / pressure footprint of the chromatographic system is used, frequent loading may be significantly accelerated. In other words, in flow-controlled loading, the flow rate is set, for example, at 1 ml / min (although this is just an example), and in response, the pump operates at a pressure of, for example, 100 bar. If 1 ml is desired for loading, this loading takes 60 seconds. However, the system can also be operated at a higher pressure, for example, at a pressure of 1,000 bar. If such high pressure is used, in principle, a higher flow rate, for example, 10 ml / min, can also be achieved, so that the loading will be achieved in as little as 6 seconds. Therefore, with flow-controlled loading, the system can, in some cases, operate at a pressure substantially lower than the pressure at which the system can in principle operate, thus resulting in a longer sample loading time than actually required.

[0211] In addition, flow-controlled loading may be inaccurate: If the loading parameters are not adequately selected, the effective loading volume may be significantly different from the expected volume. Especially at elevated pressures, this may be related to the compressibility of the fluid. Therefore, compressing the fluid at the expense of the energy available for transporting the volume through the analytical column consumes a large amount of energy.

[0212] Flow-controlled loading may also be error-prone: In addition to the above problems related to insufficient loading parameters, the presence of air or changes in the backpressure of the analytical column are typically not detected, thus potentially having a direct adverse effect on the loading performance.

[0213] Another way to load a fluid resistance element (e.g., a separation column) is called pressure-controlled loading.

[0214] Pressure-controlled loading achieves loading at a constant pressure. That is, the pressure is set and the pump(s) operate at the flow rate generated at the set pressure. Thus, the loading volume V 加载 is measured either using a flow sensor or by volume displacement (i.e., the position of the pump piston). Therefore, the loading flow rate f 加载 varies depending on the backpressure of the fluid system, especially the backpressure of the analytical column. Therefore, the loading time t 加载 varies accordingly:

[0215] t 加载 = V 加载 / f 加载 .

[0216] This must be taken into account during the planning of the chromatography workflow, as there is no constant and predictable / predefined loading time when a defined loading volume is desired.

[0217] However, pressure-controlled loading has several advantages compared to flow-controlled loading. In particular, it can be more efficient as the loading can be carried out at the maximum pressure of the fluid system (similar to the considerations provided above).

[0218] Currently, pressure-controlled loading is rarely employed. An example is the ThermoFisher EASY-nLC nano-HPLC system. One drawback of the EASY-nLC loading mechanism is inaccurate volume measurement. It is assumed that loading starts once the loading pressure is established. However, during pressurization, flow is delivered through the column. As a result, the actual loading volume is greater than the expected (set) loading volume. Additionally, this difference depends on the loading pressure, the compressibility of the fluid, and the volume of the conduits that need to be compressed.

[0219] Embodiments of the present technology relate to loading a defined volume of fluid into a fluid resistance element in a reliable and preferably relatively fast manner. That is, a limited volume of fluid is forced into the fluid resistance element, and it should be understood that the same amount of fluid is thus also forced out of the fluid resistance element.

[0220] More specifically, in embodiments of the present technology, fast loading is achieved by pressure-controlled loading under high-pressure conditions, for example, at the maximum operating conditions (typically the maximum rated pressure) of the fluid system to obtain maximum efficiency.

[0221] Accuracy and precision can be achieved through a loading control algorithm that takes into account volume losses during loading due to the compressibility of the fluid and the volume expansion of the fluid system. When the present technology is employed in an LC system, a smooth transition to a subsequent gradient phase (i.e., flow-controlled operation) can be achieved towards the end of a loading program that can be carried out in a (constant) pressure-controlled mode.

[0222] Additionally, the loading control mechanism can also provide means for diagnosing the loading process such as incorrect fluid, separation column, fluid connections, or leaks.

[0223] In embodiments of the present technology, the compressibility of the fluid(s) is taken into account to accurately deliver a compressible fluid volume across the fluid resistance. It should be understood that whenever such fluid is discharged from a stationary state, compression work is consumed, which results in a pressure build-up in the conduit upstream of the fluid resistance element (which can also be referred to as a "resistor").

[0224] This can be referred to Figure 6 in A) of Figure 2 and further understood. Figure 6A) generally depicts loading a sample present in the sample storage section 32 into the separation column 50. To this end, a limited amount of solvent is supplied, and this limited amount (i.e., volume) of solvent flows into the separation column 50, and the same amount (i.e., volume) also flows out of the separation column 50.

[0225] Figure 2 depicts the volumetric flow rate in a fluid system as a function of time, as Figure 6 depicted in A) above. More specifically, Figure 2 contains two curves, one related to the displacement flow rate f 排出 and the other related to the flow rate f 出 actually delivered through a flow resistor (e.g., the separation column 50). Considering that at the beginning, the pump does not provide flow rate and pressure (see Figure 6 12) in A) above), and the pump 12 starts operating in a flow rate controlled manner at time t1 and stops its operation again at time t2.

[0226] During the time interval defined by t1 and t2, there will be a constant displacement flow rate f 排出 . However, this will not exactly correspond to the flow rate through the resistor 50. Instead, when the pump 12 starts operating, the fluid upstream of the resistor 50 will be compressed, and this compression explains the difference between the displacement flow rate f 排出 of the pump and the actual flow rate f 出 flowing through the resistor 50. At a certain time (indicated by t a ), the pressure upstream of the resistor 50 will be so high that the flow rate f 出 through the resistor will be substantially equal to the displacement flow rate f 排出 , thus reaching equilibrium.

[0227] Also consider that at time t2, the flow rate is shut off so that the pump does not provide additional fluid flow rate, i.e., f 排出 becomes 0. However, at t2, the fluid upstream of the resistor 50 is still pressurized and there is still a flow rate f 出 flowing through the resistor. The more fluid flows through and out of the resistor, the lower the pressure in the section upstream of the resistor 50. Therefore, this flow rate f 出 decreases (exponentially) until there is no overpressure upstream of the resistor. Then the corresponding process can also be repeated, as shown by the times t1', t a , t2' corresponding to the times t1, t a shown above.

[0228] Therefore, during the interval defined by t1 and ta, the volumetric displacement flow rate f 排出 of the fluid upstream of the resistor 50 is greater than the corresponding flow rate f 加载 (and thus downstream of the column).. Once the equilibrium state is reached (at time t a ), this difference between f 排出 and f 加载 disappears. Then the pressure is constant and corresponds to the backpressure value for the given flow rate f 排出 . Now f 排出 = f 加载 (see Figure 2 ). During this stage, the difference between the corresponding volumes V 排出 discharged and loaded and V 加载 is the compression volume V 压缩 . Thus, V 压缩 can be determined by pressurizing the system to prevent outlet blockage, i.e., f 加载 = 0.

[0229] Once the driving of the fluid stops, i.e., f 排出 = 0 (see t2), no additional energy is added to the system. The energy stored in the compressed fluid is released through the flow rate f 出 that exponentially decays through the restrictor 50. The volume delivered during this last stage is the same as the compression volume V 压缩 required to initially compress the system. Thus, the loading volume is equal to the discharge volume.

[0230] During this process, the duration of compression / decompression depends on the volume of the conduit / fluid being compressed, its compressibility, and the fluid resistance. This is analogous to the charging / discharging of a resistor and capacitor in an electrical circuit.

[0231] Thus, depending on the volume and fluid resistance, the periods of compression and decompression have a significant impact on the total duration of the loading process. In particular, the decompression phase may be time-limited because compression can occur quite rapidly with sufficient fluid driving. For example, compression can be achieved quite rapidly by a piston of a pump pushing the fluid into the fluid resistance element. However, decompression by, for example, the reverse displacement of a piston may cause fluid backflow, which may change the loading volume (requiring additional measurement of the volume of the backflowing fluid) or may contaminate the fluid in the pump or the storage section that can be connected between the pump and the fluid resistance element. An accurate but rapid method of loading and decompression is described below.

[0232] Again, generally referring to Figure 6 , it is desired to have a defined volume of fluid flow into the restrictor 50, and it should be understood that a substantially corresponding amount of fluid also flows out of the restrictor 50. Generally, it is desired that this amount be well-defined, and embodiments of the present technology additionally seek to reduce the time during which this defined amount of fluid is forced into the restrictor 50.

[0233] Thus, embodiments of the present technology relate to a method that includes a step of defining a volumetric flow rate. In the step of defining a volumetric flow rate, a defined volume of fluid is forced out of a fluid resistance element 50 (also referred to as a restrictor 50), such as a column 50, and it should be understood that this corresponds to the same volume of fluid flowing into the fluid resistance element 50. The defined volume is the fluid that flows out of the restrictor 50 within a first time interval defined by a time t 开始 (which may also be referred to as the start time) and a time t 结束 (which may also be referred to as the end time). In this regard, reference may also be made to Figure 5 .

[0234] Figure 5 is a schematic illustration of a loading process. Figure 5 depicts different characteristics over time. More specifically, Figure 5 A) of 排出 depicts the displacement flow rate f 加载 caused by the displacement of the pump (which may therefore also be referred to as the pumping flow rate) and the loading flow rate f

[0235] Figure 5 through the restrictor 50 over time. Similarly, it will be understood that the loading flow rate through the restrictor 50 is generally (at least substantially) equal to the flow rate into the restrictor 50 and the flow rate out of the restrictor 50. Figure 5 B) of

[0236] Figure 5 depicts the pressure during loading. Similarly, Figure 5 B) of depicts the pressure changing over time. More specifically, the pressure in the restrictor 50 is depicted as changing over time.

[0236] Figure 5 Figure 5 C) of

[0237] is an illustration of the loading volume changing over time, which may also include the orifice alignment volume and the cleaning volume. Again, it will be understood that this is the volume of fluid flowing into (and thus also out of) the fluid resistance element 50 over time. It will also be understood that this volume is the integral of the loading flow rate f 加载 depicted in A) of Figure 5 over time.

[0237] Figure 5 In addition, Figure 5 D) of

[0238] is an illustration of the position of an injection valve during the loading process according to some embodiments of the present technology.

[0238] Generally speaking, at time t 开始 , the system can switch from a first operating state to a second operating state. More specifically, the pump 12 can switch from operating at a first relatively low pressure to operating at a second higher pressure. Thus, the pressure in the fluid resistance element 50 can change from a first pressure value P 平衡 to a second pressure value P 加载 (see Figure 5 B) of Figure 5 ), where the second pressure value P 加载 exceeds the first pressure value P平衡 .

[0239] At a later time t 减少 (at time t 开始 after and before time t 结束 or at time t 结束 ), the system can switch to a third operating state to make the pressure in the damper 50 become lower than the second pressure value P 加载 to a third pressure value P 目标 (see Figure 5 B)).

[0240] Referring to Figure 5 , in particular Figure 5 B) and Figure 5 D) of, the different times indicated are valid for all Figures A) to D). It is understood that the switching time does not necessarily coincide with the pressure presenting the corresponding pressure state in the fluid damper (see Figure 5 B)). For example, the system switches at time t 开始 and this switching causes the pressure in the damper 50 to reach pressure P 加载 . However, pressure P 加载 is not established at the instant of switching at time t 开始 , but reaches the corresponding pressure P 累积 after a time interval of Δt 加载 . However, it is still the switching at time t 开始 that causes the pressure in the damper 50 to present this pressure. Correspondingly, the switching at time t 减少 does not necessarily cause the pressure in the fluid damper 50 to instantaneously present pressure P 目标 , but there may again be a time lag between the two. However, similarly, the switching at time t 减少 is causally related to the damper 50 presenting pressure P 结束 at time t 目标 (i.e., it is at least one cause).

[0241] Hereinafter, more specific embodiments of the present technology will be described. Similarly, it should be understood that the embodiments of the present technology relate to causing a defined volume of fluid to flow into the fluid resistance element 50 (and thus also causing a corresponding volume to flow out of this element 50). In other words, the embodiments of the present technology relate to a defined loading process of such a fluid resistance element 50 (e.g., separation column 50). This loading process can have different steps or stages, which will be discussed below.

[0242] The loading process can have an equilibration phase. Referring to Figure 5 , this phase is defined by a time interval Δt 平衡Identification. During this phase, the backpressure of the restrictor 50 (e.g., the analytical column 50) can be determined.

[0243] More specifically, before loading, the analytical column 50 is equilibrated. This step is typically carried out under isocratic conditions, i.e., the total flow rate as well as the solvent composition can be constant. More particularly, the solvent composition is typically the same as the solvent composition used for subsequent loading. Thus, this equilibration phase represents a quasi-equilibrium state. This helps to determine the backpressure (fluid resistance) R of the system (analytical column) 系统 :

[0244] R 系统 = P 平衡 / f 平衡 .

[0245] Here, P 平衡 is the system pressure, and f 平衡 is the flow rate during the equilibration of the analytical column.

[0246] At this point, reference can also be made again to Figure 5 , where during the equilibration phase Δt 平衡 , Figure 5 A) depicts the equilibration flow rate f 平衡 , while Figure 5 B) depicts the equilibration pressure present in the restrictor 50.

[0247] After the equilibration phase can be the pressure build-up phase, starting at time t 开始 and lasting for a period of time Δt 累积 (see Figure 5 ). During this phase, the pressure is built up until the loading volume V 加载 (which has been loaded during the pressure build-up phase) is loaded into the restrictor 50, or until the loading pressure P 加载 is reached. The flow rate f 加载 effectively delivered through the restrictor 50 at a given time point t can be calculated using R 系统 and the corresponding pressure P(t) in the restrictor 50 at this time point,

[0248] f 加载 (t) = P(t) / R 系统 .

[0249] If the loading amount required during the pressure build-up phase has been loaded into the restrictor 50, the system switches to a state that allows for a rapid reduction in the pressure upstream of the restrictor 50, thereby rapidly reducing the flow rate into and out of the restrictor 50. For example, in such cases, the pump causing the fluid flow can be switched to a pressure equal to the pressure measured downstream of the restrictor 50. As the pump 50 typically operates at a higher pressure prior to this switch, this corresponds to an active pressure reduction in the section upstream of the fluid restrictor 50, effectively resulting in a rapid cessation of the additional flow. However, as will be understood by those skilled in the art, this may not represent a typical scenario and may indicate an inadequate selection of the loading method parameters. Such a selection may additionally have an adverse impact on the results of the separation process after loading.

[0250] Generally, during the pressure build-up phase, the volume V that has been conveyed through the restrictor 50 (which can be an analytical column) 加载 By starting from the pressure build-up phase t 开始 until the time point t for f 加载 is obtained by integration:

[0251]

[0252] It should be noted that the flow rate f determined by a flow sensor (which can measure the displacement flow rate caused by the displacement of the pump upstream of the restrictor 50) during this phase 排出 may not represent the flow rate through the column, i.e., f 排出 ≠f 加载 . This may be due to a portion f 排出 in f 压缩 that contributes to the compression of the fluid volume upstream of the flow restrictor 50. Therefore, f 加载 = f 排出 - f 压缩 . Thus, the compressed volume V 压缩 can be determined:

[0253] V 压缩 (t) = V 排出 (t) - V 加载 (t).

[0254] Here, V 排出 (t) is the volume determined by the flow sensor from time t 开始 to time t,

[0255]

[0256] Note that after time t a , when the displacement flow rate becomes equal to the flow rate measured downstream of the restrictor 50, the fluid is not additionally compressed (see Figure 5A)). At this point, a new "equilibrium state" is reached. Thus, it may be sufficient to determine the volume of the fluid compressed at this time. V 压缩 The determination can be beneficial to ensure the accuracy of the volume of fluid delivered through the restrictor 50 during the pressure-controlled loading process. In an embodiment of the present technology, the compressed volume V 压缩 is determined to improve the accuracy of loading a defined volume into the restrictor 50 (e.g., column 50). Then, this compressed volume V 压缩 can be considered. In particular, it can be used to determine V 压缩2 , i.e., the volume of fluid delivered to the restrictor 50 during the decompression phase (see below; also see Figure 5 C)), which can be beneficial when determining t 减少 , i.e., the time at the end of the constant-pressure loading phase.

[0257] After the pressure accumulation phase, there can be a constant-pressure loading phase with a duration of Δt 恒定加载 (see Figure 5 ). Once the pressure P 加载 is reached, loading can continue at the constant pressure P = P 加载 until a volume V 当前 = V 加载 - V 压缩2 – V B_清洗 - V 对准 of fluid has been loaded.

[0258] Here, V 压缩2 is the volume that can be loaded during the decompression phase (see below), V B_清洗 is the volume that can be loaded during the flushing of the channel 24 of the pump 14 (see the flushing phase below, also see Figure 6 ), and V 对准 is the volume that may be loaded when aligning the solvent composition for starting the gradient separation (see the gradient alignment phase below, also see Figure 6 ).

[0259] Although in this specification, it will be described to consider all volumes V 压缩2 , V B_清洗 and V 对准 to determine the time t 减少 , it should be understood that this is merely exemplary, and in embodiments of the present technology, only some of these volumes are considered.

[0260] P 加载 being higher than P 平衡 can be preferred. It can be additionally preferred to select a P 加载 equal to or at least substantially equal to the maximum pressure footprint of the system 100. This can allow effectively forcing the fluid through the restrictor 50. In other words, P 加载A relatively high pressure that can be selected to be at or near the maximum pressure rating of system 100. In particular, it should be understood that by selecting a relatively high pressure, such as above 500 bar or even above 1,000 bar, the loading process can be accelerated, thereby reducing the time of the loading process.

[0261] At the end of the loading phase, the back pressure R of the system can be determined again 系统2 . This can be done to account for potential changes in the back pressure of the restrictor 50 (possibly a separation column) during the loading process. Similar to before, R 系统2 can be obtained as R 系统2 = P 加载 / f 加载 . Here, f 加载 is the flow rate during the constant pressure loading phase.

[0262] During this phase, the loading flow rate may be equal to the displacement flow rate, i.e., f 加载 = f 排出 , because the compression is complete, so f 压缩 = 0. This is depicted in Figure 5 A) as reaching equilibrium between the displacement flow rate and the loading flow rate at time t a . Therefore, assuming the system is sealed, the following condition can also be satisfied: f 排出 = f 加载 . Therefore, f 排出 can be used as an alternative to f 加载 to determine the volume loaded during this phase. If there is a difference between the two flow rates (i.e., f 排出 - f 加载 ≠ 0), this may indicate a high-pressure leak in the system. Similarly, a significant difference between R 系统2 and R 系统 (especially when R 系统2 is significantly smaller) may also indicate a leak at elevated pressure. Therefore, these parameters can be used for system health monitoring purposes. Using R 系统2 , the target pressure P 目标 of the subsequent gradient phase (see the gradient phase below) can be calculated as P 目标 = f 梯度 * R 系统2 , where f 梯度 is the target flow rate during the gradient phase. P 目标 may be higher than P 平衡 .

[0263] The decompression phase can follow the constant pressure loading phase. Referring to Figure 5 , this phase is identified by the time interval Δt 减少 . During this phase, the pressure is reduced from the loading pressure P 加载 to the pressure P目标 .

[0264] During this phase, the analytical pump is operable such that the net flow rate at the pump outlet is adjusted to zero flow (i.e., f 排出 = 0). This is described by the displacement flow curve in A) of Figure 5 becoming zero at time t 减少 . The gradient composition can be maintained simultaneously. The volume that can be loaded during this phase is V 压缩2 . Similar to the pressure build-up phase, where V 压缩 is the volume that may have been compressed during system pressurization, V 压缩2 is the volume that may be delivered during depressurization.

[0265] In different embodiments, this pressure reduction from P 加载 to P 目标 can be achieved differently.

[0266] In one embodiment, the analytical pump can actively reduce the pressure by reverse piston displacement. In this regard, reference can also be made to Figure 5 B). It should be understood that during the pressure reduction interval Δt 减少 , the pressure decays exponentially. Similarly, in the depicted embodiment, this is achieved by switching the pump to zero flow. However, since the fluid upstream of the restrictor 50 is compressed, the energy resulting from the compression remains stored in the system, and this energy is dissipated by the fluid flowing into and then out of the restrictor 50 until a further equilibrium state is reached.

[0267] That is, the pump is actively adjusted to zero flow. Thus, no flow leaves the pump. However, there is still fluid flowing through the fluid restrictor (e.g., the analytical column) downstream of the pump. This results in pressure decay in the system. In addition, there is a pressurized volume inside the analytical pump. Therefore, the pump also needs to follow the pressure decay to maintain zero flow. This can be achieved by retracting the piston(s) of the pump accordingly.

[0268] However, in embodiments of the present technology, this step can be accelerated by not interrupting the flow caused by the pump to zero, but by actually switching the pump to a new pressure. For example, if the loading pressure is 700 bar and the target pressure P 目标 is 300 bar, the pump can be switched to the target pressure of 300 bar at time t 减少 . Thus, the remaining pressure in the high-pressure section (between the pump and the fluid restrictor) not only dissipates slowly as shown in B) of Figure 5 , but actually reduces to P 目标 much faster.

[0269] That is to say, in such operations, the pump operates not in a flow-controlled mode but in a pressure-controlled mode. This means that the pressure is actively reduced to a lower target value, which may result in a reverse flow into the pump. For example, in order to ensure that the gradient composition is not completely compromised (i.e., cross-flow between channel A and channel B), the gradient composition can be maintained during this process.

[0270] This allows for a rapid and effective pressure reduction. Similarly, during reverse displacement, a net (reflux) flow into the pump may occur. During this process, the solvent composition in the A / B solvent channels (see channels 22 and 24 in Figure 6 ) of pumps 12 and 14 may be compromised, i.e., cross-flow may occur between the two channels. Such active pressure reduction may compromise the solvent composition during subsequent gradient phases and ultimately affect chromatographic performance.

[0271] At this point, reference can be made to Figure 6 A). In this figure, pump 12 with solvent A can be used to push the sample from the sample loop 32 into the fluid resistor 50. Thus, during the constant loading phase Δt 恒定加载 , pump 12 operates and causes the fluid to flow through the sample loop 32 under elevated pressure and into the fluid resistor 50. It should be understood that in Figure 6 , solvent A is depicted as a solid black line, while solvent B is depicted as an unfilled black line. These solvents can be mixed at the mixing unit 26.

[0272] However, if pump 12 is used for loading, then this pump 12 supplies solvent A at high pressure, which may cause solvent A to also flow into channel 24, as shown in Figure 6 A).

[0273] Accordingly, when the pressure is actively reduced, for example, by the piston retraction time t 减少 of pump 12, this may cause the solvent in channel 24 to also flow into channel 22, or vice versa, the solvent in channel 22 to flow into channel 24, potentially compromising the solvent composition.

[0274] At this point, reference can also be made to Figure 4 . Figure 4 depicts the set solvent composition (see dashed line) and the actual solvent composition delivered during the experiment (see solid line). Referring again to Figure 6 A), during the previous phase, it may occur that solvent A is actually pushed into channel 24 (see also the following description in Figure 6 at this point). Thus, when this effect is not considered and the pump is operated as if only solvent B were present in channel 24, this may result in less solvent B being delivered during the initial gradient phase, as shown in Figure 4As shown, when the solvent plug 25 of solvent A present in channel 24 is removed, the actual gradient provided only corresponds to the set gradient after approximately 10 minutes.

[0275] For example, to counteract such detrimental effects, an additional gradient alignment step can be used as described below.

[0276] In another embodiment of the decompression phase, the sample loop 32 - which can hold the maximum pressurized fluid volume downstream of the pump - can be switched out of the flow path (as shown in D of Figure 6 ), but it should be noted that another step is depicted in this figure). Then the flow of the pump can be stopped (e.g., actively regulated to f 排出 = 0) and the flow path can be decompressed through the separation column. At the same time, loop 32 can be decompressed separately to P 目标 (e.g., using a metering device (not shown)).

[0277] Based on the actual experimental data of different columns, the effect of offline switching of the loop to accelerate decompression is shown in FIGS. 3(A) and 3(B). FIGS. 3(A) and 3(B) depict the actual measured data of column loading experiments.

[0278] FIG. 3(A) depicts a comparison of the loading processes. Exemplary data of the pressure signal of an HPLC gradient pump (e.g., a UHPLC pump) are shown. More specifically, FIG. 3(A) depicts two pressure signals varying with time, where the solid line is the pressure signal of an embodiment of the present invention with "active decompression", and the dashed line is a comparative example without such active decompression.

[0279] In these two signals, at time t = 1.5 minutes, sample pick-up is completed and the loading process starts. Thus, the pressure is accumulated. At t = 3 minutes, the set pressure for loading (1200 bar) is reached, and loading continues at a constant flow rate or pressure until t = 5 minutes. It should be understood that the two signals correspond to each other at this point.

[0280] At this time (i.e., t = 5 minutes), the decompression phase, also known as decompression, starts. In the case of decompression with the loop switched offline (which is one way to perform active decompression), decompression to the target pressure (250 bar) is completed at t = 5.5 minutes (black, solid line). In the case of decompression with the loop remaining online and without performing active decompression, decompression to the target pressure is completed at t = 8.75 minutes. At t = 10 minutes, loading is completed, the loop sample loop is switched offline, and the gradient phase starts.

[0281] When comparing the solid line (i.e., with active decompression) and the dashed line (without active decompression), it should be understood that the total time to reach the target pressure is significantly reduced, thereby reducing the total time of the analytical run and thus improving efficiency.

[0282] Figure 3(B) shows exemplary data of the signal of the flow sensor of channel A of an HPLC gradient pump (e.g., a UHPLC pump) during accelerated loading with active decompression (i.e., rapid decompression) by offline switching of the sample loop (see Figure 6 22 in

[0283] At time t = 1.5 minutes, the pressurization phase begins, resulting in an increase in flow. Subsequently, the flow gradually decreases to slowly approach the loading pressure. At t = 3 minutes, the pressurization is complete, and the loading of the fluid resistance element occurs at a constant flow rate of approximately 1500 nanoliters per minute between t = 3.0 minutes and t = 5.0 minutes. At t = 5 minutes, the decompression phase begins, which can also be referred to as the pressure reduction phase. The flow is set to zero; thus, the measured flow rapidly decays to zero flow. Subsequently, at t = 5.5 minutes, the flushing phase begins, which can also be referred to as the cleaning or purging step. In the depicted embodiment, the total flow is set to 300 nanoliters per minute and the volume percentages of A and B are 50%. Thus, the measured flow is approximately 150 nanoliters per minute. At t = 7.5 minutes, the alignment phase begins (total flow rate = 300 nanoliters per minute, 0% B), resulting in a flow value of approximately 300 nanoliters per minute. At t = 10 minutes, the loading is complete, the sample loop is switched offline, and the gradient phase begins.

[0284] During decompression, the influence of V 压缩2 is related to the total volume of the fluid downstream of the pump that includes the volume of the restrictor 50 (which can be a separation column). This volume is typically negligible. In the case where the loop is not switched offline, its volume may contribute significantly to V 压缩2 Similar to the pressure accumulation phase, where V 压缩 is the volume that may have been compressed during system pressurization, V 压缩2 is the volume that may be delivered during decompression. The compressed volume V 压缩 may be related to V 压缩2 , i.e., V 压缩2 = V 压缩 *f, where f is the ratio of the pressurization / decompression pressure difference

[0285] f = (P 加载 - P 目标 ) / (P 加载 - P 平衡 ).

[0286] This is based on an approximate linear relationship between the compressed volume and pressure, which is typically sufficient. Nevertheless, other more accurate means can be employed to relate the compressed volume and pressure. In many use cases, f is ≈ 1. Thus, the volume V 压缩2 that can be loaded during the decompression phase can be within V压缩 is determined with the help of the V 压缩 which may have been determined during the pressure build-up phase. A robust calculation of the volume V 压缩2 can be beneficial to ensure the accuracy of a defined volume of fluid delivered through the restrictor 50, especially for highly compressible fluids.

[0287] In yet another embodiment during the decompression phase, the pressure reduction can be achieved by actively venting the flow path to P using an additional valve upstream of the restrictor 50 (which can be a separation column). 环境 For example, this method can be used for EASY-nLC UHPLC products. This method can also allow for rapid decompression. However, a rather drastic pressure drop can impose significant stress on the restrictor 50 (alternatively, the separation column) and other fluids. Therefore, under such operations, the lifespan of the restrictor 50 (which can be a separation column) may be significantly reduced.

[0288] Also in such setups, the pressure does not drop instantaneously due to the resistance of the conduits and valves upstream of the column. Therefore, once the target pressure is reached, the venting can be controlled to stop. This is done, for example, in an EASY-nLC system.

[0289] Once P 目标 is reached, the loading can continue in a flow control mode of f 加载 = f 梯度 where f 梯度 can be the target flow rate for the gradient separation step.

[0290] During the previous phases identified by Figure 5 the duration Δt related to the equilibration interval in 平衡 the duration Δt related to the interval during pressure build-up 累积 the duration Δt related to the interval of constant pressure loading 恒定加载 and the duration Δt related to the pressure reduction 减少 there may have been minor cross-flow between two (or more) solvent channels of the pump (such as channel 22 and channel 24) (see Figure 6 A) of

[0291] such cross-flow may be particularly relevant for high-pressure gradient (HPG) pumps, i.e., pumps that can be used especially in HPLC applications where the solvent(s) may need to be pushed into the flow path under high pressure. As previously mentioned, this can lead to inaccurate solvent composition in the channels. Consequently, the gradient may not be accurately delivered in subsequent gradient phases, which can have a significant adverse impact on chromatographic performance (see Figure 4) This type of cross - flow typically occurs when the flow rate ratio between two (or more) channels of a pump is set such that effectively only one channel conveys the entire flow rate (e.g., 0% or 100% of a given solvent).

[0292] For a typical low - flow UHPLC (Ultra - High Performance Liquid Chromatography) protocol, this can be prominent (see Figure 4 ). The total flow rate can be set, for example, at 300 nanoliters per minute, where the solvent composition during the loading step is set, for example, at 0 vol-% of solvent B. This can allow for reliable detection of early - eluting compounds during the downstream gradient phase. Thus, the set flow rate of channel 22) in channel A ( Figure 6 of A) can be 300 nanoliters per minute, while the set flow rate of channel 24) in channel B ( Figure 6 of A) can be 0 nanoliters per minute. Flow rate regulation of such low - flow UHPLC pumps can rely on flow (or pressure) sensors to achieve accurate flow delivery. However, such sensors may exhibit non - ideal behavior such as offset flow, drift, noise, and precision deviation.

[0293] Assume that the flow rate sensor of pump 14) in solvent B pump ( Figure 6 of A) may show a small offset, for example, 5 nanoliters per minute, at an actual zero flow rate, which will effectively result in a net reverse flow of 5 nanoliters per minute of solvent A into channel 24), at the expense of the total flow rate delivered to column 50 (solvent A is only 295 nanoliters per minute). Thus, assume that this situation persists, for example, for equilibration and loading for 15 minutes, during which a total volume of 75 nanoliters of solvent plug A will flow into 25) in channel 24) (see Figure 6 of A). During the gradient phase, the negative impact of this situation will become imminent. For example, assume a typical 60 - minute gradient starting from 2 vol-% of solvent B and ending at 40 vol-% of solvent B, with a flow rate of, for example, 300 nanoliters per minute, which will result in no solvent B being introduced during the first 8 minutes of the 60 - minute period (see Figure 4 ). Flushing out a volume of 75 nanoliters from channel 24) will take approximately 8 minutes. During this period, the concentration of solvent B in the gradient mixture pushed into the analytical flow path will correspondingly increase to approximately 7 vol-% of solvent B. Thus, peaks that would normally elute gradually during this period will elute all at once with a sudden increase in the solvent B composition at t = 8 minutes. Therefore, the early stage of the chromatogram may be compromised.

[0294] For example, to avoid such negative impacts, channel 24) can be flushed at the end of the loading period before starting the gradient (see Figure 6 of B)). This stage can be referred to as the flushing stage of channel 24) in solvent line B ( Figure 6 of A), withFigure 5 The duration Δt in B_清洗 For this purpose, the solvent composition can be briefly increased to facilitate the flow of solvent B (for example, increased to 80 vol-% of solvent B). Then, a pre-defined volume V can be delivered B_冲洗 . V B_冲洗 can be chosen large enough to ensure that channel 24 is reliably filled only with solvent B.

[0295] During the flushing phase, it may be advantageous to protect the sample partially loaded into the separation column 50 in the previous step from the influence of solvent B (for example, it can be organic). That is, it may be advantageous that the solvent used during the flushing phase does not reach the fluid resistor 50 (for example, the separation column 50). For this purpose, the sample loop 32 that may have been individually depressurized to P during the decompression step 目标 can be switched back online. It can be used as a buffer in which V B_冲洗 can be stored. At the same time, the sample can continue to be loaded into the separation column 50. Therefore, during this phase, the loading amount may increase by V B_冲洗 .

[0296] For example, to ensure the correct solvent composition B 梯度、开始 , for example, can be 2 vol-% of solvent B and 98 vol-% of solvent A (although it should be understood that this is only exemplary). For starting the gradient in the next step, the fluid can be flushed to remove the (incorrect) solvent from the previous flushing phase (see Figure 6 C)). The solvent composition can be set to B 梯度、开始 , and a pre-defined volume V can be delivered during Δt 对准 (see 对准 ). V Figure 5 ). V 对准 can be chosen large enough to ensure that all the fluid between the pump outlet and the injection valve is filled with the solvent of composition B 梯度、开始 . At the same time, the sample can continue to be loaded into the separation column. Therefore, during this phase, the loading amount may increase by V 对准 . This phase can be called the gradient alignment phase, identified by the duration Δt in Figure 5 The duration Δt in 对准 is identified.

[0297] Once V 对准 has been delivered, the loading can be completed and the sample loop 32 holding the solvent plugs V B_冲洗 and V 对准 can be switched offline. It can be cleaned separately there (for example, using a metering device).

[0298] In a further step, this can be called the gradient phase, which is identified by the duration Δt in Figure 5 The duration Δt in梯度 is identified, the gradient can be delivered directly, i.e., by bypassing the sample loop 32, to the separation column (see Figure 6 of D)).

[0299] That is to say, Figure 6 is a schematic illustration of the fluid routing according to an embodiment of the present technique during the loading process: Figure 6 A) of depicts loading the sample from the sample loop into the separation column. This operation may cause a small amount of solvent A to flow back into the channel 24. Figure 6 B) of depicts flushing the channel 24. The solvent B plug is "parked" in the sample loop, thereby continuing to load the sample from the loop. Figure 6 C) of depicts the preparation (alignment) of the solvent composition for the start of the gradient phase. The alignment solvent plug is also "parked" in the sample loop. Thus, the sample continues to be loaded from the loop. In addition, Figure 6 D) of depicts the state when the loading is completed. The loop with the "parked" solvent plug is switched offline. The gradient is delivered via a loop that directly bypasses the column.

[0300] In other words, Figure 6 depicts different operating states of the fluid system 100 that can be used in embodiments of the present technique.

[0301] As discussed, embodiments of the present technique may involve delivering a defined volume of fluid (e.g., liquid) to a fluid resistor 50, such as to a separation column.

[0302] The system 100 can be switched to a state where the pressure in the fluid resistor 50 is increased from a first value to a second value that exceeds the first value. Referring again to Figure 5 , this can be done at time t 开始 completed. More specifically referring to Figure 6 A) of, for example, the sample can be located in the sample loop 32 and at time t 开始 , loading of the sample into the fluid resistor 50 can be started.

[0303] It should be understood that the system 100 can include a valve 34 that is configured to switch the pumps 12, 14 to be in fluid connection with the sample loop 32 (as shown in Figure 6 A) of), or to switch these pumps to be in fluid connection with the bypass line 40 (as shown in Figure 6 D) of).

[0304] When in the connection state as shown in Figure 6 A) of, the system 100 can be switched at time t 开始 to increase the pressure in the fluid resistor 50. As discussed, the increase in pressure in the fluid resistor 50 may not occur instantaneously, and there may be a certain time lag Δt before the pressure in the fluid resistor reaches the desired pressure累积 .

[0305] However, during a time interval Δt 累积 the liquid may flow to and into the fluid resistor 50, i.e., this fluid also contributes to loading the fluid resistor 50.

[0306] Generally, it may be necessary to cause a defined volume of fluid to flow into the resistor 50 during the loading step. In particular, in the case of the chromatographic application of the present technique, this may be desirable for reproducibility.

[0307] After loading is complete, the system 100 may assume a sample separation configuration. In the sample separation configuration (see Figure 6 D)), a solvent may be supplied to the resistor 50 and the components of the sample may be eluted from the resistor 50 and subsequently detected.

[0308] It can thus be understood that it may be necessary to load a defined volume into the resistor 50 before the separation step.

[0309] Similarly, in the configuration depicted in Figure 6 A), the pressure upstream of the resistor 50 may be increased during the interval Δt 累积 Subsequently, there may be a constant pressure loading phase during the interval Δt 恒定加载 .

[0310] During these intervals, a certain volume of fluid is delivered into the fluid resistor 50.

[0311] After that, at time t 减少 the system 100 may switch to another operating state to reduce the pressure in the fluid resistor 50, e.g., to reduce the pressure to the level that occurs during the separation step.

[0312] This concept relates to a first embodiment of the present technique. It is particularly noted that, thus that is to say, by switching the pressure upstream of the resistor 50 to a value higher than the value to be used later, a particularly high pressure can be used during loading, thus making the loading faster and thus more time-saving.

[0313] An additional concept of an embodiment of the present technique relates to determining the time t 减少 and taking into account additional effects that may adversely affect the operation of the system if not considered.

[0314] In particular, it should be understood that after switching the system 100 to another operating state at t 减少 an additional amount of fluid will still be loaded into the resistor. Embodiments of this embodiment take into account these amounts (and the amounts loaded during compression, i.e., during the time interval Δt 累积 ) to accurately set the time t减少 .

[0315] In addition, as also mentioned previously, operation of the system may cause the plug of solvent A to be located in solvent line 24 (see Figure 6 A)), which may compromise reproducibility.

[0316] To counteract such effects, there may be a flushing or "purge channel" step as shown in Figure 6 B). During this step, valve 34 may have the same configuration as in Figure 6 A). However, during this step, pumps 12, 14 may be operated such that any residual solvent B, A that is not intended to be in the respective channels 22, 24 is removed from the respective channels 22, 24. Thus, after this step, channel 22 is (at least substantially) free of solvent B (depicted in white) and channel 24 is (at least substantially) free of solvent A (depicted in black).

[0317] Referring to Figure 5 (in particular Figure 5 C)), it will be understood that this cleaning or purge step occurs during the interval Δt B_清洗 during which it should also be understood that this may also include cleaning of line 22. Referring to Figure 5 C), it should also be understood that this results in an additional volume V B清洗 flowing into fluid resistor 50.

[0318] After this cleaning step, the solvent composition present in mixing unit 26 may not be ideal for the subsequent separation step. Therefore, after this step, there may be a solvent alignment step as shown in Figure 6 C). Similarly, system valve 34 may assume the same positions as discussed above in connection with Figure 6 A) and B). During this solvent alignment step, pumps 12, 14 may be operated such that the solvent composition between mixing unit 26 and valve 34 corresponds to the one for the start of the separation step.

[0319] Similarly, this step may take a certain amount of time, referable to the solvent alignment time interval Δt 对准 (see Figure 5 ), and during this step, additional solvent may be loaded into the resistor, the volume of which is V 对准 (see Figure 5 C)).

[0320] For example, after solvent alignment is complete, valve 34 may be switched such that bypass line 40 is connected to pumps 12, 14 (see Figure 6 D)) and separation may be started.

[0321] It should be understood that during the above process, the damper 50 is loaded during the pressure accumulation Δt before the separation step, during the constant loading phase Δt, during the pressure reduction phase Δt, during the cleaning phase Δt, and during the alignment phase Δt. 累积 period, during the constant loading phase Δt 恒定加载 period, during the pressure reduction phase Δt 减少 period, during the cleaning phase Δt B_清洗 period, and during the alignment phase Δt 对准 period.

[0322] It should be further understood that the decompression phase, the cleaning phase, and the alignment phase occur after the system is switched to reduce the pressure so that the fluid damper 50 exhibits the target pressure P, where the switching occurs at t. 目标 after, where the switching occurs at t 减 less.

[0323] Embodiments of the present technology consider the volume delivered after the switch at t. For example, considering that the total loading volume should be 10 μl, and the system is set to a desired decompression volume V of 0.5 μl, a cleaning volume V of 0.3 μl, and an alignment volume V of 0.2 μl. Thus, the total volume contributing to the loading after t is 1 μl. In such cases, the system will determine that t is the time when 9 μl has been loaded. 减少 after the switch at t. For example, considering that the total loading volume should be 10 μl, and the system is set to a desired decompression volume V 压缩 of 0.5 μl, a cleaning volume V B_清洗 of 0.3 μl, and an alignment volume V 对准 of 0.2 μl. Thus, the total volume contributing to the loading after t 减少 is 1 μl. In such cases, the system will determine that t 减少 is the time when 9 μl has been loaded.

[0324] In summary, embodiments of the present technology thus allow a defined volume of fluid to be loaded into the fluid resistance element in a fast, reliable, and reproducible manner.

[0325] Whenever relative terms such as "about", "substantially", or "approximately" are used in this specification, such terms should also be interpreted as including the exact term. That is, for example, "substantially straight" should be interpreted as also including "(exactly) straight".

[0326] Whenever steps are recited in the foregoing or also in the appended claims, it should be noted that the order in which the steps are recited herein may be accidental. That is to say, unless otherwise stated or unless it is clear to the person skilled in the art, the order of the recited steps may be accidental. That is to say, when this document states, for example, that a method comprises step (A) and step (B), this does not necessarily mean that step (A) is before step (B), but it is also possible that step (A) (at least partially) is carried out simultaneously with step (B), or that step (B) is before step (A). Furthermore, when step (X) is said to be before another step (Z), this does not mean that there are no steps between step (X) and step (Z). That is to say, step (X) before step (Z) covers the case where step (X) is carried out directly before step (Z), and also covers the case where (X) is carried out before one or more steps (Y1) ……, followed by step (Z). Corresponding considerations apply when using terms such as "after" or "before".

[0327] Although the preferred embodiments have been described above with reference to the accompanying drawings, those skilled in the art will understand that this embodiment is provided for illustrative purposes only and should in no way be construed as limiting the scope of the invention, which is defined by the claims.

Claims

1. A method of operating a fluid system, wherein, The fluid system includes a fluid resistance element, Wherein, the method includes a step of defining a volumetric flow rate, wherein, in the step of defining a volumetric flow rate, a fluid of a defined volume is forced out of the fluid resistance element, and wherein the defined volume is the fluid that flows out of the fluid resistance element within a first time interval defined by a start time t 开始 and an end time t 结束 and flows out of the fluid resistance element within a first time interval defined by a start time t wherein the step of defining a volumetric flow rate includes At the start time t 开始 , the fluid system is switched from a first operating state to a second operating state so that the pressure in the fluid resistance element changes from a first pressure value to a second pressure value, the second pressure value exceeding the first pressure value. At the start time t 开始 and no later than the end time t 结束 of the time t 减少 , switch the fluid system to a third operating state so that the pressure in the fluid resistance element becomes a third pressure value, which is lower than the second pressure value. Wherein, the method includes at the start time t 开始 and the time t a between the pressurization time interval Δt 累积 , wherein the pressure in the fluid resistance element rises from the first pressure value to the second pressure value, and wherein the method includes determining the cumulative volume V of the fluid flowing out of the fluid resistance element within the pressurization time interval Δt 累积 , 累积 , wherein, the method further includes determining t 减少 , and wherein, determining t 减少 includes considering the cumulative volume V 累积 .

2. The method according to claim 1, wherein, the fluid system includes a high-pressure section upstream of the fluid resistance element, and wherein the fluid system includes a pump system upstream of the high-pressure section, and wherein switching the fluid system from the first operating state to the second operating state includes switching the pump system.

3. The method according to claim 2, wherein Switching the fluid system to the third operating state includes at the time t 减少 Switching the high-pressure section to the third pressure value.

4. The method according to claim 2, wherein Switching the fluid system to the third operating state includes at time t 减少 Switch the pump system to zero flow rate.

5. The method according to claim 2, wherein The pump system is configured to supply a solvent mixture of a first solvent and a second solvent at different mixing ratios, wherein the pump system includes a first pump configured to supply the first solvent, a second pump configured to supply the second solvent, and a mixing unit downstream of the first pump and the second pump, wherein the pump system supplies the solvent mixture, wherein the first solvent exceeds 20 vol-% of the solvent mixture, and wherein the method further includes At the time t 减少 after and before the end time t 结束 at the time t 清洗 , switch the pump system, and the pump system supplies a cleaning volume V B_清洗 of the solvent mixture, wherein the second solvent exceeds 20 vol-% of the solvent mixture.

6. The method according to claim 5, wherein, The first solvent constitutes 100% of the solvent mixture within the time interval defined by the start time t 开始 and the time t 减少 .

7. The method according to claim 5, wherein The second solvent constitutes 100% of the solvent mixture after time t 清洗 ​ 8. The method according to any one of claims 5 to 7, wherein the method includes At the time t 清洗 and before the end time t 结束 at the time t 对准 , switch the pump system to supply the solvent mixture having a defined mixing ratio, wherein the method further includes the pump system supplying an alignment volume V 对准 of the solvent mixture having the defined mixing ratio within an alignment time interval Δt 对准 starting from the time t 对准 .

9. The method according to claim 8, wherein The alignment time interval Δt 对准 at the end time t 结束 ends.

10. The method according to any one of claims 1 to 7, Among them, The method further includes at the time t a and t 减少 a constant pressure time interval Δt therebetween 恒定加载 wherein the pressure in the fluid resistance element is at the second pressure value during the constant pressure time interval Δt 恒定加载 and wherein the method includes determining a constant pressure volume V of the fluid flowing out of the fluid resistance element within the constant pressure time interval Δt 恒定加载 and wherein the method further includes 恒定加载 ​ from the time t 减少 of a decompression time interval Δt 减少 starting therefrom, wherein the pressure is reduced from the second pressure value to the third pressure value, and wherein the method comprises determining a constant reduced volume V of fluid flowing out of the fluid resistance element within the decompression time interval Δt 减少 thereof 减少 .

11. The method according to any one of claims 5 to 7, wherein, Determine t 减少 including considering the constant pressure volume V 恒定加载 , the constant reduction volume V 减少 , the cleaning volume V B_清洗 and the alignment volume V 对准 among any of them, wherein the method includes at the time t 清洗 after and before the end time t 结束 for a time t 对准 , switching the pump system to supply the solvent mixture having a defined mixing ratio, wherein the method further includes the pump system supplying the alignment volume V 对准 of the solvent mixture having the defined mixing ratio within an alignment time interval Δt 对准 starting from the time t 对准 . wherein, the method further includes at the time t a and t 减少 a constant pressure time interval Δt therebetween 恒定加载 , wherein the pressure in the fluid resistance element is at the second pressure value during the constant pressure time interval Δt 恒定加载 , and wherein the method includes determining a constant pressure volume V of the fluid flowing out of the fluid resistance element within the constant pressure time interval Δt 恒定加载 , 恒定加载 , And wherein, the method further includes from the time t 减少 starting decompression time interval Δt 减少 , wherein the pressure is reduced from the second pressure value to the third pressure value, and wherein the method includes determining the constant reduced volume V of the fluid flowing out of the fluid resistance element during the decompression time interval Δt 减少 within 减少 .

12. The method according to any one of claims 1 to 7, wherein the fluid system is a liquid chromatography system.

13. The method according to claim 12, wherein, The fluid system is a high performance liquid chromatography system.

14. A fluid system, wherein, The fluid system includes a fluid resistance element, wherein the fluid system includes a control unit, and wherein the fluid system is configured to perform the method according to any one of the preceding method claims.

15. A computer program product comprising instructions, wherein, The instructions are configured to cause the fluid system to perform the method according to any one of the preceding method claims when run on a control unit of the fluid system.

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