A multi-channel two-dimensional liquid chromatography separation system

Abstract

The invention relates to a multi-channel two-dimensional liquid chromatography separation system. The multi-channel two-dimensional liquid chromatography separation system comprises a first-dimensional chromatography system, a main pipeline and a second-dimensional chromatography system. The second-dimensional chromatography system comprises a second-dimensional pump system, a second-dimensional detector, a collector system, a split flow switching valve, a confluence switching valve and at least two second-dimensional chromatography branches. Each second-dimensional chromatography branch is provided with a capture column and a second-dimensional chromatography column. The second-dimensional pump system is used for pumping eluent to the capture column. The inlet of each second-dimensional chromatography branch is connected to the main pipeline through the split flow switching valve. The outlet of each second-dimensional chromatography branch in the same second-dimensional chromatography system is connected to the inlet of the second-dimensional detector through the confluence switching valve. The outlet of the second-dimensional detector is connected to the collector system. The separation process of different flow fractions can be partially performed synchronously. The working efficiency is high and the system is suitable for rapid separation and preparation of multi-target component samples.

Description

Technical Field

[0001] The invention relates to the technical field of liquid chromatography separation, and in particular to a multi-channel two-dimensional liquid chromatography separation system. Background Art

[0002] With the rapid development of separation technology, exploring and separating components in complex sample systems has become a hot research area. Multidimensional liquid chromatography technology effectively improves the separation of complex sample components by increasing peak capacity, and has become the development direction of rapid chromatographic separation technology. In order to be able to perform automated, large-scale, and systematic separation and purification of complex samples with uneven content and unknown components, and to quickly perform high-resolution and high-throughput separation of all components, it is necessary to establish a multidimensional liquid chromatography separation system and multidimensional separation method.

[0003] Two-dimensional liquid chromatography is a liquid chromatography technique that injects the eluent of the first-dimensional chromatographic column of the sample into the second-dimensional chromatographic column for further separation. This separation technology can use two chromatographic columns with different separation mechanisms to perform orthogonal separation of samples. Common two-dimensional liquid chromatography separation systems mainly include online storage coil two-dimensional liquid chromatography systems and trapping column two-dimensional liquid chromatography systems.

[0004] For the online storage coil two-dimensional liquid chromatography system, the components separated by the first-dimensional liquid chromatography are stored in the quantitative loop coil and then enter the second-dimensional liquid chromatography for separation. Although the separation speed of this method is relatively fast, the separation of the second-dimensional liquid chromatography is severely restricted by the first-dimensional liquid chromatography. The elution solvents of the first-dimensional chromatography and the second-dimensional chromatography must be compatible, and the application field is subject to certain restrictions.

[0005] For a trapping column type two-dimensional liquid chromatography system, it is necessary to first run the first-dimensional liquid chromatography system, enrich the components separated by the first-dimensional liquid chromatography system in the trapping column, and after the first-dimensional chromatographic separation is completed, elute the trapping column and load the sample into the second-dimensional liquid chromatography system, and then start the second-dimensional chromatographic separation. For example, a two-dimensional liquid chromatography device disclosed in the Chinese utility model patent document with the authorization announcement number CN215768412U includes a first-dimensional chromatographic system, a switching valve, a second-dimensional chromatographic system, and a waste liquid pool. The first-dimensional chromatographic system includes a first pump (first-dimensional pump), a first-dimensional chromatographic column, and a first detector. The second-dimensional chromatographic system includes a second pump (second-dimensional pump), a trapping column, a second-dimensional chromatographic column, a second detector, and a fraction collector, which are switched to different positions by the switching valve to perform first-dimensional chromatographic separation and second-dimensional chromatographic separation, respectively.

[0006] Although the above-mentioned trapping column two-dimensional liquid chromatography system avoids the phenomenon that the second-dimensional liquid chromatography is restricted by the first-dimensional liquid chromatography, since its first-dimensional chromatography corresponds to a single second-dimensional chromatography, that is, it is a single-channel separation system, it is only suitable for the separation of a single target component. In the actual industrial production process, the separation system and separation targets are often complex and diverse. The use of the existing single-channel separation system will result in low production efficiency and it is difficult to adapt to the needs of simultaneous separation of multiple target components or cyclic separation of multiple target components. Summary of the invention

[0007] The object of the present invention is to provide a multi-channel two-dimensional liquid chromatography separation system to solve the problem that the existing two-dimensional liquid chromatography separation system is difficult to adapt to the separation of multiple target components.

[0008] The technical solution of the multi-channel two-dimensional liquid chromatography separation system of the present invention is:

[0009] A multi-channel two-dimensional liquid chromatography separation system comprises a first-dimensional chromatography system and a second-dimensional chromatography system, and also comprises a main pipeline, wherein the first-dimensional chromatography system is connected to the main pipeline, and the second-dimensional chromatography system is provided with at least one, the second-dimensional chromatography system comprises a second-dimensional pump system, a second-dimensional detector, a collector system, a shunt switching valve, a confluence switching valve and at least two second-dimensional chromatography branches, each second-dimensional chromatography branch is provided with a capture column and a second-dimensional chromatography column, the capture column is connected to the second-dimensional pump system, the second-dimensional pump system is used to pump eluent to the capture column, the inlet of each second-dimensional chromatography branch is connected to the main pipeline through a shunt switching valve, the shunt switching valve has a shunt switching position, each shunt switching position is used to connect the main pipeline with the second-dimensional chromatography branch in the corresponding second-dimensional chromatography system, the outlet of each second-dimensional chromatography branch in the same second-dimensional chromatography system is connected to the inlet of the second-dimensional detector through a confluence switching valve, the confluence switching valve has a confluence switching position, each confluence switching position is used to connect the corresponding second-dimensional chromatography branch with the second-dimensional detector, and the outlet of the second-dimensional detector is connected to the collector system.

[0010] Beneficial effect: by setting at least one second-dimensional chromatography system, each second-dimensional chromatography system is provided with at least two second-dimensional chromatography branches, so that the first-dimensional chromatography can correspond to the second-dimensional chromatography in different second-dimensional chromatography branches respectively, forming a multi-channel design, so that after the sample to be separated is separated by the first-dimensional chromatography system, the different split switching positions are switched in sequence by the split switching valve, so that the first-dimensional chromatography system is connected with different second-dimensional chromatography branches through the trunk pipeline, and the flow separated by the first-dimensional chromatography can be enriched in the capture columns of each second-dimensional chromatography branch in sequence; at the same time, after the enrichment of the previous capture column is completed, the enrichment of the next capture column is carried out. During collection, the required eluent can be input through the second-dimensional pump system to elute the capture column that has completed enrichment first. The target flow after elution is switched to the corresponding confluence switching position through the confluence switching valve to enter the second-dimensional detector and then enter the collector system. The multi-channel design can meet the needs of separating different components in a complex mixture system. While one component is enriched, another component can be eluted. The separation process of different components can be partially carried out simultaneously. Different target components are detected by the same second-dimensional detector. It is easy to operate, the system structure layout is flexible, and the work efficiency is high. It is suitable for rapid industrial separation and preparation of multi-target component samples.

[0011] Furthermore, the second dimension pump system includes a pump liquid pipeline, an inlet pipeline, and an inlet switching valve. The inlet pipeline includes inlet branches. The number of inlet branches is equal to the number of second dimension chromatographic branches in the same second dimension pump system. The capture column on the second dimension chromatographic branch is connected to the outlet end of the corresponding inlet branch. The inlet end of each inlet branch is connected to the pump liquid pipeline through the inlet switching valve. The inlet switching valve has an inlet switching position. Each inlet switching position is used to connect the selected inlet branch to the pump liquid pipeline. A second dimension pump is provided on the pump liquid pipeline. The second dimension pump is used to pump eluent to the corresponding capture column through the inlet branch.

[0012] Beneficial effect: By setting each liquid inlet branch, the liquid inlet branch corresponds to the second-dimensional chromatographic branch one by one, and each liquid inlet branch is connected to the pump liquid pipeline through the liquid inlet switching valve, which is convenient for pumping eluent to different second-dimensional chromatographic branches and helps to reduce costs.

[0013] Furthermore, the pump liquid pipeline includes a pump branch and an intermediate pipeline, each pump branch is connected to the inlet end of the intermediate pipeline, the liquid inlet switching valve is arranged at the outlet end of the intermediate pipeline, and each pump branch is provided with a second dimension pump.

[0014] Beneficial effect: By setting up each pump branch, it is suitable for mixing more than two solvents into an elution solvent of a multi-solvent system. The flow rate of each second-dimensional pump pumping solvent can be changed as needed to change the elution solvent ratio, making it convenient to pump a suitable eluent to the capture column.

[0015] Furthermore, a waste liquid outlet of the pump system is provided on the intermediate pipeline.

[0016] Beneficial effect: the waste liquid outlet is arranged on the middle pipeline, and each pump branch can share one waste liquid outlet, which is conducive to reducing costs.

[0017] Furthermore, each pump branch is connected to a solvent input pipeline.

[0018] Beneficial effect: Each pump branch has an independent solvent input pipeline, which is convenient for control and avoids misoperation.

[0019] Furthermore, a three-way valve is provided on the upstream of the capture column in the second-dimension chromatography branch, and the liquid inlet branch is connected to the second-dimension chromatography branch through the three-way valve.

[0020] Beneficial effect: By setting a three-way valve, three ports, two inlets and one outlet, are formed. When one of the inlets is connected to the outlet, a passage is formed in the part of the second-dimensional chromatographic branch located upstream of the capture column. When the other inlet is connected to the outlet, the inlet end of the capture column is connected to the liquid inlet branch, which facilitates the control of the on-off of the pipeline.

[0021] Furthermore, the diversion switching valve is a diversion three-way valve, the number of the diversion three-way valves is equal to the number of the second-dimensional chromatographic branches, and the second-dimensional chromatographic branches are connected to the main pipeline through the diversion three-way valves.

[0022] Beneficial effects: Each second-dimensional chromatographic branch is connected to the main pipeline through the separately arranged diversion three-way valves, which facilitates the arrangement of the second-dimensional chromatographic branch.

[0023] Furthermore, two second-dimensional chromatography systems are provided.

[0024] Beneficial effects: The two second-dimensional chromatography systems have four second-dimensional chromatography branches, forming a four-channel design, which improves the adaptability to the preparation and separation of multi-target component samples.

[0025] Furthermore, a detection end waste liquid outlet is provided between the second dimension detector and the collector system.

[0026] Beneficial effect: The waste liquid outlet is arranged between the second-dimensional detector and the collector, so as to facilitate the discharge of the waste liquid.

[0027] Furthermore, the collector system includes collectors, the number of which is equal to the number of second-dimensional chromatographic branches, and in the same second-dimensional chromatographic system, the outlet of the second-dimensional detector is connected to each collector in a branching manner.

[0028] Beneficial effects: By setting collectors corresponding to the second-dimensional chromatographic branches one by one, it is convenient to collect different flow fractions through valve control, and it is easy to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1Schematic diagram of the structure of the multi-channel two-dimensional liquid chromatography separation system in Example 1;

[0030] Figure 2 It is a schematic diagram of the operation process of enriching the flow fraction by the first trapping column of the multi-channel two-dimensional liquid chromatography separation system;

[0031] Figure 3 It is a schematic diagram of the process of simultaneously operating the elution fraction of the second trapping column and the enrichment fraction of the third trapping column of the multi-channel two-dimensional liquid chromatography separation system;

[0032] Figure 4 It is a one-dimensional chromatogram of the first-dimension chromatographic column separation in Example 1;

[0033] Figure 5 The chromatogram is a two-dimensional column separation of the one-dimensional flow segmented by Trap1 in Example 1;

[0034] Figure 6 The chromatogram is a two-dimensional column separation of the one-dimensional flow segmented by Trap2 in Example 1;

[0035] Figure 7 The chromatogram is a two-dimensional column separation of the one-dimensional flow segmented by Trap3 in Example 1;

[0036] Figure 8 This is a chromatogram of two-dimensional column separation of one-dimensional flow segmentation captured by Trap4 in Example 1.

[0037] In the figure: 1, pump No. 1; 2, 1D column; 3, first detector; 4, first waste liquid discharge pipeline; 5, first diversion switching valve; 6, first capture column; 7, second waste liquid discharge pipeline; 8, first 2D column; 9, first confluence switching valve; 10, second detector; 11, first collector; 12, second collector; 13, third waste liquid discharge pipeline; 14, pump No. 2; 15, pump No. 3; 16, first liquid inlet switching valve; 17, second diversion switching valve; 18, first Three-way flow switching valve; 19, second capturing column; 20, third capturing column; 21, second 2D column; 22, fourth waste liquid discharge pipeline; 23, third 2D column; 24, second confluence switching valve; 25, third detector; 26, third collector; 27, No. 4 pump; 28, No. 5 pump; 29, second liquid inlet switching valve; 30, fourth collector; 31, fifth waste liquid discharge pipeline; 32, fourth 2D column; 33, sixth waste liquid discharge pipeline; 34, fourth capturing column. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention, that is, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. The components of the embodiments of the present invention described and shown in the drawings herein can be arranged and designed in various different configurations.

[0039] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention.

[0040] It should be noted that the terms that may appear in the specific embodiments of the present invention, such as relational terms such as "first" and "second", are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms that may appear, such as "include", "comprise" or any other variants thereof, are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements that are not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the possible statements "including one...", etc., do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0041] In the description of the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected" and "connected" that may appear should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For those skilled in the art, the specific meanings of the above terms in the present invention can be understood by specific circumstances.

[0042] In the description of the present invention, unless otherwise clearly specified and limited, the term "provided with" that may appear should be understood in a broad sense. For example, the object "provided with" may be a part of the body, or may be arranged separately from the body and connected to the body, and the connection may be a detachable connection or an inseparable connection. For those skilled in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0043] The present invention is described in further detail below in conjunction with embodiments.

[0044] Embodiment 1 of the multi-channel two-dimensional liquid chromatography separation system of the present invention:

[0045] like Figure 1 and Figure 2 As shown, the multi-channel two-dimensional liquid chromatography separation system includes a first-dimensional chromatography system, a main pipeline and a second-dimensional chromatography system. The first-dimensional chromatography system is connected to the main pipeline. The second-dimensional chromatography system is provided with two, the second-dimensional chromatography system includes a second-dimensional pump system, a second-dimensional detector, a collector system, a diversion switching valve, a confluence switching valve and two second-dimensional chromatography branches. Each second-dimensional chromatography branch is provided with a capture column and a second-dimensional chromatography column. The capture column is connected to the second-dimensional pump system. The second-dimensional pump system is used to pump eluent to the capture column. The inlet of each second-dimensional chromatography branch is connected to the main pipeline through a diversion switching valve. The diversion switching valve has a diversion switching position. Each diversion switching position is used to connect the main pipeline with the second-dimensional chromatography branch in the corresponding second-dimensional chromatography system. The outlet of each second-dimensional chromatography branch in the same second-dimensional chromatography system is connected to the inlet of the second-dimensional detector through a confluence switching valve. The confluence switching valve has a confluence switching position. Each confluence switching position is used to connect the corresponding second-dimensional chromatography branch with the second-dimensional detector. The outlet of the second-dimensional detector is connected to the collector system.

[0046] The second dimension pump system includes a pump liquid pipeline, a liquid inlet pipeline, and a liquid inlet switching valve. The liquid inlet pipeline includes a liquid inlet branch. The number of liquid inlet branches is equal to the number of second dimension chromatographic branches in the same second dimension pump system. The capture column on the second dimension chromatographic branch is connected to the outlet end of the corresponding liquid inlet branch. The inlet end of each liquid inlet branch is connected to the pump liquid pipeline through the liquid inlet switching valve. The liquid inlet switching valve has a liquid inlet switching position. Each liquid inlet switching position is used for connecting the selected liquid inlet branch with the pump liquid pipeline. The pump liquid pipeline is provided with a second dimension pump. The second dimension pump is used to pump eluent to the corresponding capture column through the liquid inlet branch. The pump liquid pipeline includes a pump branch and an intermediate pipeline. There are two pump branches. Each pump branch is connected to the inlet end of the intermediate pipeline. The liquid inlet switching valve is set at the outlet end of the intermediate pipeline. Each pump branch is provided with a second dimension pump. The intermediate pipeline is provided with a waste liquid outlet of the pump system, and each pump branch is connected to a solvent input pipeline. The eluent is a binary solvent system formed by two second-dimensional pumps each pumping a solvent. The flow rate of the solvent pumped by the two second-dimensional pumps is changed as needed to change the solvent ratio.

[0047] In the entire multi-channel two-dimensional liquid chromatography separation system, the corresponding pipelines and components are connected by three-way valves. The three-way valve is a pneumatic three-way valve with three connection sites and two switching paths. There are 80 three-way valves in total, which are named V1, V2, V3, ..., V80, and are used in the following description of the three-way valve.

[0048] The first-dimension chromatography system includes a first-dimension pump system, a 1D column 2, a first detector 3 and V4. The 1D column 2 is the first-dimension chromatography column. The first-dimension pump system includes a regeneration liquid storage tank, an eluent storage tank, a sample storage tank, V1, V2, V3, and pump No. 1. The regeneration liquid storage tank, the eluent storage tank, and the sample storage tank are all connected to the inlet of pump No. 1. V1, V2, and V3 are respectively arranged on the connecting pipeline between the regeneration liquid storage tank, the eluent storage tank, the sample storage tank and pump No. 1. The outlet of pump No. 1 is connected to the inlet of 1D column 2, and the outlet of 1D column 2 is connected to the inlet of the first detector 3. The first detector 3 is UV1. The outlet of the first detector 3 is connected to the main pipeline and the first waste liquid discharge pipeline 4 through V4.

[0049] In the two second-dimensional chromatography systems, one is upstream of the main pipeline, i.e., the first second-dimensional chromatography system, and the other is downstream of the main pipeline, i.e., the second second-dimensional chromatography system. In the first second-dimensional chromatography system, there are two second-dimensional chromatography branches, one is upstream of the main pipeline, i.e., the first second-dimensional chromatography branch, and the other is downstream of the main pipeline, i.e., the second second-dimensional chromatography branch. In the second second-dimensional chromatography system, there are two second-dimensional chromatography branches, one is upstream of the main pipeline, i.e., the third second-dimensional chromatography branch, and the other is downstream of the main pipeline, i.e., the fourth second-dimensional chromatography branch.

[0050] The diversion switching valve is a diversion three-way valve, the number of the diversion three-way valves is equal to the number of the second-dimensional chromatographic branches, and the second-dimensional chromatographic branches are connected to the main pipeline through the diversion three-way valves.

[0051] The inlet end of the first second-dimensional chromatographic branch is connected to the main pipeline through the first diversion switching valve 5, the first diversion switching valve 5 is V5, and the first second-dimensional chromatographic branch is provided with V9, the first capture column 6, V13, and the first 2D column 8. V9 is located upstream of the first capture column 6, and the first 2D column 8 is located downstream of the first capture column 6. The first 2D column 8 is a second-dimensional chromatographic column, that is, Figure 2 The 2D column 1 in the embodiment, the first trapping column 6 is Trap1, V13 is arranged between the first trapping column 6 and the first 2D column 8, the inlet of V13 is connected to the first trapping column 6, one outlet is connected to the first 2D column 8, and the other outlet is connected to the second waste liquid discharge pipeline 7.

[0052] The inlet end of the second second-dimensional chromatographic branch is connected to the main pipeline through the second diversion switching valve 17, the second diversion switching valve 17 is V6, one outlet of V5 is connected to the inlet of V6, and the second second-dimensional chromatographic branch is provided with V10, a second capture column 19, V14, and a second 2D column 21. V10 is located upstream of the second capture column 19, and the second 2D column 21 is located downstream of the second capture column 19. The second 2D column 21 is a second-dimensional chromatographic column, that is, Figure 2 The 2D column 2 in the middle, the second trapping column 19 is Trap2, V14 is arranged between the second trapping column 19 and the second 2D column 21, the inlet of V14 is connected to the second trapping column 19, one outlet is connected to the second 2D column 21, and the other outlet is connected to the fourth waste liquid discharge pipeline 22.

[0053] The outlet of the first 2D column 8 and the outlet of the second 2D column 21 are connected to the inlet of the second detector 10 through the first confluence switching valve 9, the first confluence switching valve 9 is V21, the second detector 10 is UV2, the second detector 10 is a second-dimensional detector, the outlet of the second detector 10 is connected to the collector system, the number of collectors is equal to the number of second-dimensional chromatographic branches, the collector system of the first second-dimensional chromatographic system includes two collectors, namely the first collector 11 and the second collector 12, and V23 is provided between the second detector 10 and the collector system, and V23 One outlet forms the waste liquid outlet of the detection end and is connected to the third waste liquid discharge pipeline 13, and the other outlet of V23 is connected to the first collector 11 and the second collector 12 through a switching valve. The switching valve can be a common three-way valve, one of the inlets is connected to the outlet of V23, and the two outlets are connected to the first collector 11 and the second collector 12 respectively. By controlling the switching of the passage in the three-way valve to allow the outlet of V23 to be connected to the first collector 11 or the second collector 12, the outlet of the second-dimensional detector is connected to each collector in a branching manner in the same second-dimensional chromatography system. The first collector 11 includes V33-V44, and the second collector 12 includes V45-V56.

[0054] The second dimension pump system of the first second dimension chromatography system has two pump branches and two liquid inlet branches, and pump No. 2 14 and pump No. 3 15 are respectively provided on the two pump branches, and both pump No. 2 14 and pump No. 3 15 are second dimension pumps. A solvent storage tank 1 and a solvent storage tank 2 are provided upstream of pump No. 2 14, and the solvent storage tanks 1 and 2 are respectively connected to the inlet of pump No. 2 14 through solvent input pipelines, and V25 and V26 are respectively provided on the solvent input pipelines between the solvent storage tanks 1 and 2 and pump No. 2 14, and a solvent storage tank 3 and a solvent storage tank 4 are provided upstream of pump No. 3 15, and the solvent storage tanks 3 and 4 are respectively connected to the inlet of pump No. 3 15 through solvent input pipelines, and V27 and V28 are respectively provided on the solvent input pipelines between the solvent storage tanks 3 and 4 and pump No. 3 15, and the outlets of pump No. 2 14 and pump No. 3 15 are connected to the inlets of the corresponding intermediate pipelines, and V17 is provided on the intermediate pipeline, and one outlet of V17 forms a waste liquid outlet of the pump system. The inlets of the two liquid inlet branches in the first second-dimensional chromatography system are connected to the corresponding intermediate pipelines through V19, which is the first liquid inlet switching valve 16. The outlets of the two liquid inlet branches are connected to V9 and V10 respectively.

[0055] The inlet end of the third second-dimensional chromatographic branch is connected to the main pipeline through the third diversion switching valve 18, the third diversion switching valve 18 is V7, and the third second-dimensional chromatographic branch is provided with V11, a third capture column 20, V15, and a third 2D column 23, V11 is located upstream of the third capture column 20, and the third 2D column 23 is located downstream of the third capture column 20, and the third 2D column 23 is a second-dimensional chromatographic column, that is, Figure 2 The 2D column 3 in the figure, the third trapping column 20, namely Trap3, V15 is arranged between the third trapping column 20 and the third 2D column 23, the inlet of V15 is connected to the third trapping column 20, one outlet is connected to the third 2D column 23, and the other outlet is connected to the fourth waste liquid discharge pipeline 22.

[0056] The inlet end of the fourth second-dimensional chromatographic branch is connected to the main pipeline through the fourth shunt switching valve, the fourth shunt switching valve is V8, an outlet of V7 is connected to the inlet of V8, and the fourth second-dimensional chromatographic branch is provided with V12, a fourth capture column 34, V16, and a fourth 2D column 32. V12 is located upstream of the fourth capture column 34, and the fourth 2D column 32 is located downstream of the fourth capture column 34. The fourth 2D column 32 is a second-dimensional chromatographic column, that is, Figure 2 The 2D column 4 in the figure, the fourth trapping column 34, namely Trap4, V16 is arranged between the fourth trapping column 34 and the fourth 2D column 32, the inlet of V16 is connected to the fourth trapping column 34, one outlet is connected to the fourth 2D column 32, and the other outlet is connected to the sixth waste liquid discharge pipeline 33.

[0057] The outlet of the third 2D column 23 and the outlet of the fourth 2D column 32 are connected to the inlet of the third detector 25 through the second confluence switching valve 24, the second confluence switching valve 24 is V22, the third detector 25 is UV3, the third detector 25 is a second-dimensional detector, the outlet of the third detector 25 is connected to the corresponding collector system, the number of collectors is equal to the number of corresponding second-dimensional chromatographic branches, the collector system of the second second-dimensional chromatographic system includes two collectors, namely the third collector 26 and the fourth collector 30, V24 is provided between the third detector 25 and the collector system, one outlet of V24 forms the detection end waste liquid outlet and is connected to the fifth waste liquid discharge pipeline 31, and the other outlet of V24 is connected to the third collector 26 and the fourth collector 30 through the switching valve, so that in the same second-dimensional chromatographic system, the outlet of the second-dimensional detector is connected to each collector in a branching manner. The third collector 26 includes V57-V68, and the fourth collector 30 includes V69-V80.

[0058] The second dimension pump system of the second second dimension chromatography system has two pump branches and two liquid inlet branches. The two pump branches are respectively provided with pump No. 4 27 and pump No. 5 28. Pump No. 4 27 and pump No. 5 28 are both second dimension pumps. A solvent storage tank No. 5 and a solvent storage tank No. 6 are provided upstream of pump No. 4 27. The solvent storage tank No. 5 and the solvent storage tank No. 6 are respectively connected to the inlet of pump No. 4 27 through solvent input pipelines, and V29 and V30 are respectively provided on the solvent input pipeline between the solvent storage tank No. 5 and the solvent storage tank No. 6 and pump No. 4 27. , a solvent tank 7 and a solvent tank 8 are provided upstream of the No. 5 pump 28, and the solvent tank 7 and the solvent tank 8 are connected to the inlet of the No. 5 pump 28 through the solvent input pipeline, and V31 and V32 are provided on the solvent input pipeline between the solvent tank 7 and the solvent tank 8 and the No. 5 pump 28, respectively. The outlets of the No. 4 pump 27 and the No. 5 pump 28 are connected to the inlet of the corresponding intermediate pipeline, and the intermediate pipeline is provided with V18, and one outlet of V18 forms the waste liquid outlet of the pump system of the second second-dimensional chromatography system. The inlet of the two liquid inlet branches in the second second-dimensional chromatography system is connected to the corresponding intermediate pipeline through V20, and V20 is the second liquid inlet switching valve 29, and the outlets of the two liquid inlet branches are connected to V11 and V12 respectively.

[0059] When separating and preparing different components in tobacco extract, the multi-channel two-dimensional liquid chromatography separation system operates as follows:

[0060] The first-dimension pump system and the second-dimension pump system are medium-pressure elution pump systems with a flow rate of 0.5-5L / min; the first-dimension chromatographic column is a gel permeation chromatographic column in the prior art, with a specification of 100×180mm, and the elution mobile phase is an aqueous solution; the second-dimension chromatographic column is a conventional C18 chromatographic column, with a specification of 70×180mm, and the elution mobile phase A is ethanol (solvent one), and the mobile phase B is water (solvent three), and the elution gradient is: 0-70min, phase A 10%-100%; the capture column system is a conventional C18 chromatographic column, with a specification of 60×70mm.

[0061] Working status:

[0062] (1) In the one-dimensional separation state, V3 is connected to pump 1 for automatic sample loading. After the sample loading is completed, Figure 2 As shown, V3 is switched to the stop state, V2 is opened and connected to pump No. 1, entering the first dimension elution state, when the switching valve enters the V4>V5>V9>V13>waste liquid connection state, Trap1 is in enriched fraction 1; when the switching valve enters the V4>V5>V6>V10>V14>waste liquid connection state, Trap2 is in enriched fraction 2; when the switching valve enters the V4>V5>V6>V7>V11>V15>waste liquid connection state, Trap3 is in enriched fraction 3; when the switching valve enters the V4>V5>V6>V7>V8>V12>V16>waste liquid connection state, Trap4 is in enriched fraction 4.

[0063] (2) In the two-dimensional separation state, Figure 3 As shown, Trap3 is in the enriched state, V25 and V27 are connected to pump No. 2 and pump No. 3 respectively, and when V17>V19>V10>V14>V21>V23>the collectors are connected, the flow fraction on Trap2 enters the second dimension of separation.

[0064] Preparation process:

[0065] (3) 5 kg of tobacco was crushed into powder, and the powder that passed through was sieved with a sieve. 15 L of 20% ethanol-water solution was added, heated at 60° C., refluxed 3 times, each time for 2 hours, filtered to remove the residue, and then filtered with a 50 μm filter membrane. The filtrate was decompressed and recovered to remove the ethanol solvent to obtain a tobacco crude extract.

[0066] (4) The switching valve puts the multi-channel two-dimensional liquid chromatography separation system into the first-dimension separation mode. After the sample is loaded, the separation begins. When the first target peak is observed on the first-dimension detector, the switching valve enters the V4>V5>V9>V13>waste liquid connection state, and Trap1 is in the enrichment fraction 1. The separation result is as follows: Figure 4 shown.

[0067] (5) After Trap1 is enriched, the switching valve enters the state of V4>V5>V6>V10>V14>waste liquid connection, and Trap2 is enriching fraction 2. At this time, the switching valve enters the state of V17>V19>V9>V13>V21>V23>collector passage, and the Trap1 fraction is eluted and loaded onto 2D column 1 to start the second dimension separation. The separation target peak is collected by the collector. The separation result is shown in FIG. Figure 5 shown.

[0068] (6) When Trap2 completes enrichment and the switching valve enters the state of V4>V5>V6>V7>V11>V15>waste liquid connection, Trap3 is enriching fraction 3.

[0069] (7) When Trap3 is enriched, the switching valve enters the state of V4>V5>V6>V7>V8>V12>V16>waste liquid connection, and Trap4 is enriching fraction 4. At this time, the switching valve enters the state of V18>V20>V11>V15>V22>V24>collector passage, and the Trap3 fraction is eluted and loaded onto 2D column 3, starting the second dimension separation. The separation target peak is collected by the collector. The separation result is shown in FIG. Figure 7 shown.

[0070] (8) When Trap1 completes the second-dimensional separation, the switching valve enters the V17>V19>V10>V14>V21>V23>collector state, and the Trap2 flow fraction is eluted and loaded onto 2D column 2 to start the second-dimensional separation. The separation target peak is collected by the collector. The separation results are shown in Figure 2. Figure 6 shown.

[0071] (9) When Trap3 completes the second-dimensional separation, the switching valve enters the V18>V20>V12>V16>V22>V24>collector connection state, and the Trap4 flow fraction is eluted and loaded onto 2D column 4 to start the second-dimensional separation. The separation target peak is collected by the collector. The separation results are shown in Figure 2. Figure 8 shown.

[0072] (10) Repeat the above steps (3) to (9) for 5 cycles.

[0073] (11) The effective components in the tobacco are collected separately, concentrated under reduced pressure, freeze-dried, and the alcohol and water are removed to obtain freeze-dried powder of a single tobacco fraction with a purity of more than 96% and a product yield of 80%.

[0074] In this way, the first-dimensional chromatogram can correspond to the second-dimensional chromatogram in different second-dimensional chromatographic branches respectively, forming a multi-channel design. After the sample to be separated is separated by the first-dimensional chromatographic system, the different split switching positions are switched in sequence through the split switching valve, so that the first-dimensional chromatographic system is connected to different second-dimensional chromatographic branches through the main pipeline, and the flow fractions separated by the first-dimensional chromatography can be enriched in the capture columns of each second-dimensional chromatographic branch in sequence; at the same time, after the enrichment of the previous capture column is completed and when the subsequent capture column is enriched, the required eluent can be input through the second-dimensional pump system to elute the capture column that has completed the enrichment in the previous one, and the target flow fraction after elution is switched to the corresponding one through the confluence switching valve. The corresponding confluence switching position can enter the second-dimensional detector and then enter the collector system; the multi-channel design can meet the needs of separating different components in a complex mixture system. While one component is enriched, another component can be eluted. The separation process of different components can be partially carried out simultaneously. Different target components are detected by the same second-dimensional detector. The operation is convenient, the system structure layout is flexible, the response is sensitive, the degree of automation is high, and the operation is convenient. Multiple channels can be switched and operated at the same time, which greatly improves the working efficiency of the preparative two-dimensional liquid chromatography system. It is suitable for the rapid industrial separation and preparation of multi-target component samples, and has great application potential in the direction of industrial preparative chromatography purification.

[0075] Example 2 of the multi-channel two-dimensional liquid chromatography separation system of the present invention:

[0076] The difference between this embodiment and embodiment 1 is that in embodiment 1, two second-dimensional chromatography systems are provided, while in this embodiment, only one first-dimensional chromatography system is provided.

[0077] Example 3 of the multi-channel two-dimensional liquid chromatography separation system of the present invention:

[0078] The difference between this embodiment and embodiment 1 is that in embodiment 1, each second-dimensional chromatography system has two second-dimensional chromatography branches. In this embodiment, each second-dimensional chromatography system has three second-dimensional chromatography branches, and the corresponding split flow switching valve and confluence switching valve are four-way valves.

[0079] Example 4 of the multi-channel two-dimensional liquid chromatography separation system of the present invention:

[0080] The difference between this embodiment and embodiment 1 is that in embodiment 1, the second dimension pump system includes two inlet branches and two pump branches, and the two inlet branches and the two pump branches are connected through an intermediate pipeline. In this embodiment, the second dimension pump system includes two independent infusion pipelines, and the infusion pipelines are connected to the corresponding second dimension chromatographic branches.

[0081] Example 5 of the multi-channel two-dimensional liquid chromatography separation system of the present invention:

[0082] The difference between this embodiment and embodiment 1 is that in embodiment 1, the second dimension chromatography system is provided with two split switching valves, which are split three-way valves, the number of which is equal to the number of the second dimension chromatography branches, and the second dimension chromatography branches are connected to the main pipeline through the split three-way valves. In this embodiment, the second dimension chromatography system is provided with one, and the inlets of the two second dimension chromatography branches are connected to the main pipeline through the same split three-way valve.

[0083] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions recorded in the aforementioned embodiments without creative work, or replace some of the technical features therein with equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A multi-channel two-dimensional liquid chromatography separation system, comprising a first-dimensional chromatography system and a second-dimensional chromatography system, characterized in that: It also includes a main pipeline, the first-dimensional chromatography system is connected to the main pipeline, and two second-dimensional chromatography systems are provided. The second-dimensional chromatography system includes a second-dimensional pump system, a second-dimensional detector, a collector system, a diversion switching valve, a confluence switching valve and at least two second-dimensional chromatography branches. Each second-dimensional chromatography branch is provided with a capture column and a second-dimensional chromatography column. The capture column is connected to the second-dimensional pump system. The second-dimensional pump system is used to pump eluent to the capture column. The inlet of each second-dimensional chromatography branch is connected to the main pipeline through a diversion switching valve. The diversion switching valve has a diversion switching position. Each diversion switching position is used to connect the main pipeline with the second-dimensional chromatography branch in the corresponding second-dimensional chromatography system. The outlet of each second-dimensional chromatography branch in the same second-dimensional chromatography system is connected to the inlet of the second-dimensional detector through a confluence switching valve. The confluence switching valve has a confluence switching position. Each confluence switching position is used to connect the corresponding second-dimensional chromatography branch with the second-dimensional detector. The outlet of the second-dimensional detector is connected to the collector system.

2. The multi-channel two-dimensional liquid chromatography separation system according to claim 1, characterized in that: The second dimension pump system includes a pump liquid pipeline, an inlet pipeline, and an inlet switching valve. The inlet pipeline includes inlet branches. The number of inlet branches is equal to the number of second dimension chromatographic branches in the same second dimension pump system. The capture column on the second dimension chromatographic branch is connected to the outlet end of the corresponding inlet branch. The inlet end of each inlet branch is connected to the pump liquid pipeline through the inlet switching valve. The inlet switching valve has an inlet switching position. Each inlet switching position is used for connecting the selected inlet branch with the pump liquid pipeline. A second dimension pump is provided on the pump liquid pipeline. The second dimension pump is used to pump eluent to the corresponding capture column through the inlet branch.

3. The multi-channel two-dimensional liquid chromatography separation system according to claim 2, characterized in that: The pump liquid pipeline includes a pump branch and an intermediate pipeline. Each pump branch is connected to the inlet end of the intermediate pipeline. The liquid inlet switching valve is arranged at the outlet end of the intermediate pipeline. Each pump branch is provided with a second dimension pump.

4. The multi-channel two-dimensional liquid chromatography separation system according to claim 3, characterized in that: A waste liquid outlet of the pump system is arranged on the intermediate pipeline.

5. The multi-channel two-dimensional liquid chromatography separation system according to claim 3, characterized in that: Each pump branch is connected with a solvent input pipeline.

6. The multi-channel two-dimensional liquid chromatography separation system according to claim 2, characterized in that: A three-way valve is arranged on the upstream of the trapping column in the second-dimension chromatographic branch, and the liquid inlet branch is connected with the second-dimension chromatographic branch through the three-way valve.

7. The multi-channel two-dimensional liquid chromatography separation system according to any one of claims 1 to 6, characterized in that: The diversion switching valve is a diversion three-way valve, the number of the diversion three-way valves is equal to the number of the second-dimensional chromatographic branches, and the second-dimensional chromatographic branches are connected to the main pipeline through the diversion three-way valves.

8. The multi-channel two-dimensional liquid chromatography separation system according to any one of claims 1 to 6, characterized in that: A detection end waste liquid outlet is provided between the second dimension detector and the collector system.

9. The multi-channel two-dimensional liquid chromatography separation system according to any one of claims 1 to 6, characterized in that: The collector system includes collectors, the number of which is equal to the number of second-dimensional chromatographic branches. In the same second-dimensional chromatographic system, the outlet of the second-dimensional detector is connected to each collector in a branching manner.

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