Multi-channel liquid chromatography separation device

Abstract

The invention provides a multichannel liquid chromatography separation device, which comprises a sample accommodating unit, a plurality of independent liquid path channels and a control unit, the sample accommodating unit is used for accommodating various reagents, and each independent liquid path channel respectively comprises a sample introduction unit, a chromatography unit, a detection unit and a collection unit according to the flow direction of a liquid path. The multi-channel liquid chromatogram separation device has a first working mode and a second working mode, and in the first working mode, the independent liquid path channels can perform liquid chromatogram separation in parallel; and in the second working mode, at least one independent liquid path channel can be used for re-sampling the samples collected by the collecting units in other independent liquid path channels, so that secondary liquid chromatography separation treatment is carried out.

Description

Technical Field

[0001] The present application relates to the technical field of separation and purification, and in particular to a multi-channel liquid chromatography separation device. Background Art

[0002] Liquid chromatography (LC), a core technology in modern analytical chemistry, is widely used in fields such as drug development, environmental monitoring, and food safety. Traditional single-channel LC systems separate components using a single mobile phase and stationary phase, but their limitations, such as low analytical throughput and long analytical times, have become increasingly prominent. Multichannel LC separation devices, through parallel design, can significantly improve separation efficiency and have become a key development direction in chromatographic technology in recent years.

[0003] Complex sample systems often contain a vast array of components, encompassing not only a wide variety of compounds but also highly variable concentrations. For example, in traditional Chinese medicine extracts, multiple chemical components, such as alkaloids, flavonoids, and terpenes, may coexist, with concentrations ranging from tens of percent of the primary component to trace amounts of a few parts per million. Environmental water samples may also contain a variety of organic pollutants, heavy metal ions, and microbial metabolites. Comprehensive and accurate analysis of such complex samples requires technologies and devices capable of high-resolution, high-throughput separation of all components. Multidimensional liquid chromatography (MDLC) has emerged as a key development in rapid chromatographic separation technology. By combining chromatographic columns with different separation mechanisms, it effectively increases peak capacity and significantly improves the resolution of complex sample components, becoming a key development in rapid chromatographic separation technology. Two-dimensional liquid chromatography (2DLC), a representative example of MDLC, involves injecting the eluate from the first-dimension column into a second-dimension column for secondary separation. This leverages two distinct separation mechanisms to achieve orthogonal separation of the sample, significantly enhancing the separation capability of complex samples.

[0004] Currently, common two-dimensional liquid chromatography separation systems are mainly of two types: online storage coil type and capture column type. Although online storage coil type two-dimensional liquid chromatography systems have certain advantages in separation speed, the components separated by the first-dimensional liquid chromatography are stored in the sample loop coil and then enter the second-dimensional liquid chromatography for separation. However, this system has a significant drawback: the separation process of the second-dimensional liquid chromatography is severely restricted by the first-dimensional liquid chromatography, requiring the elution solvents of the first-dimensional chromatography and second-dimensional chromatography to be fully compatible, which greatly limits its application areas. For example, in certain sample separations with stringent solvent compatibility requirements, this system often fails to meet the requirements.

[0005] The trapping column two-dimensional liquid chromatography system first runs the first-dimensional liquid chromatography system to enrich the separated components in the trapping column. After the first-dimensional chromatographic separation is completed, the trapping column is eluted and the sample is loaded into the second-dimensional liquid chromatography system for second-dimensional chromatographic separation. However, the existing trapping column two-dimensional liquid chromatography systems are mostly single-channel separation systems, that is, the first-dimensional chromatography corresponds to a single second-dimensional chromatography. This single-channel design is only suitable for the separation of a single target component. In the actual industrial production process, facing the complex and diverse separation systems and separation targets, its production efficiency is extremely low. Taking the pharmaceutical industry as an example, in the process of new drug research and development, it is necessary to separate and analyze multiple active ingredients or impurities at the same time. The single-channel separation system cannot meet the needs of simultaneous separation of multiple target components, resulting in extended research and development cycles and increased costs. In large-scale production, the single-channel system is also difficult to meet the needs of cyclic separation of multiple target components, which seriously affects production efficiency and economic benefits.

[0006] Furthermore, traditional liquid chromatography separation devices face challenges with complex method development and low analytical throughput when dealing with a wide variety of sample types. Different sample types often require the development of specific analytical methods, and switching between methods is complex, making efficient and rapid sample analysis difficult. Furthermore, as analytical demands continue to increase, the throughput requirements for liquid chromatography separation devices are also increasing, and traditional devices are no longer able to meet the growing demand for high-throughput analysis. Summary of the Invention

[0007] In order to overcome the above-mentioned deficiencies of the prior art, the present invention proposes a multi-channel liquid chromatography separation device, which can provide multi-channel automatic liquid chromatography separation processing, and can perform automatic liquid chromatography separation processing in a multi-channel parallel or multi-channel serial manner, thereby significantly improving the efficiency of liquid chromatography separation.

[0008] The object of the present invention is to provide a multi-channel liquid chromatography separation device, which includes a reagent holding module, a plurality of independent liquid channels and a control module, wherein the reagent holding module is used to hold a plurality of reagents including samples, carrier liquids and stabilizers, and each of the independent liquid channels includes an injection unit, a fluid delivery unit, a chromatography unit, a detection unit and a collection unit according to the flow direction of the liquid channel, and the control module is used to control each unit in the independent liquid channels to realize automatic processing; the injection unit includes an injection needle for injecting the sample or reagent held by the reagent holding module; the fluid delivery unit is used to deliver mobile phase to the chromatography unit; the chromatography unit includes a chromatography column, for separating the components contained in the sample; the detection unit is used to detect the components in the sample separated from the chromatography column; the collection unit is used to collect the target components in the sample separated by the chromatography unit; the multi-channel liquid chromatography separation device has a first working mode and a second working mode, wherein, in the first working mode, each of the independent liquid channels can separate samples in parallel; in the second working mode, the injection needle of one independent liquid channel among the multiple independent liquid channels can perform a second injection on the eluate containing the target component collected by the collection unit in another independent liquid channel, thereby realizing serial separation processing of multiple independent liquid channels.

[0009] Furthermore, the collection unit includes a collection rack, on which are provided a plurality of collection tubes arranged in the length direction of the collection rack, the collection racks of each of the independent liquid channels are arranged in a direction perpendicular to the length direction of the collection rack, and the injection needle is capable of moving in the arrangement direction of the collection rack; when the multi-channel liquid chromatography separation device is in the first working mode, the injection needle of each of the independent liquid channels is aligned with the corresponding collection rack in the length direction of the collection rack; when the multi-channel liquid chromatography separation device is in the second working mode, the injection needle of one of the multiple independent liquid channels moves in the arrangement direction of the collection rack to be aligned with the collection rack of another independent liquid channel in the length direction of the collection rack.

[0010] Furthermore, the type of the chromatography column corresponding to one independent liquid channel is the same as or different from the type of the chromatography column corresponding to another independent liquid channel.

[0011] Furthermore, the number of chromatography columns corresponding to one independent liquid channel is the same as or different from the number of chromatography columns corresponding to another independent liquid channel.

[0012] Furthermore, the chromatography unit in each of the independent liquid channels includes one or more chromatography columns.

[0013] Furthermore, the eluate containing the target component collected by the collecting unit of the one independent liquid channel after the chromatography treatment by the chromatography unit of the one independent liquid channel among the multiple independent liquid channels after the secondary injection can be injected again.

[0014] Furthermore, before the second injection, a stabilizer may be applied to the eluate containing the target component collected by the collection unit through injection.

[0015] Furthermore, the liquid channel for re-injection is the independent liquid channel.

[0016] Furthermore, the liquid channel for re-injection is another independent liquid channel different from the one independent liquid channel and the other independent liquid channel.

[0017] The multi-channel liquid chromatography separation device proposed in the present invention includes multiple independent liquid channels for performing liquid chromatography separation operations respectively. It can not only perform liquid chromatography separation in parallel through multiple liquid channels, but also perform secondary liquid chromatography separation operations on processed samples of other liquid channels, realizing fully automatic serial operation of multi-step separation (such as affinity chromatography → ion exchange chromatography), thereby improving the separation efficiency of the device and realizing more complex liquid chromatography separation processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope.

[0019] Figure 1 This is a perspective schematic diagram of an embodiment of the multi-channel liquid chromatography separation device disclosed in the present invention as viewed from the front;

[0020] Figure 2 This is a perspective schematic diagram of an embodiment of the multi-channel liquid chromatography separation device disclosed in the present invention as viewed from the right side;

[0021] Figure 3 This is a schematic diagram of a preferred embodiment of a chromatography unit structure of the multi-channel liquid chromatography separation device disclosed in the present invention;

[0022] Figure 4 This is a schematic diagram of the sample injection of the multi-channel liquid chromatography separation device disclosed in the present invention for aspirating the sample contained in the reagent containing module;

[0023] Figure 5 This is a schematic diagram of the multi-channel liquid chromatography separation device disclosed in the present invention for aspirating the carrier liquid contained in the reagent containing module;

[0024] Figure 6 This is a schematic diagram of a fluid delivery unit in a first working state in one embodiment of the multi-channel liquid chromatography separation device disclosed in the present invention;

[0025] Figure 7 This is a schematic diagram of a fluid delivery unit in a second working state in one embodiment of the multi-channel liquid chromatography separation device disclosed in the present invention;

[0026] Figure 8 A schematic diagram of a fluid path for transporting mobile phase solvents in mobile phase containers by a fluid transport unit in one embodiment of a multi-channel liquid chromatography separation device disclosed in the present invention;

[0027] Figure 9 It is a schematic diagram of multiple transfer and positioning mechanisms and collection racks of the multi-channel liquid chromatography separation device disclosed in the present invention in a parallel working mode;

[0028] Figure 10 It is a schematic diagram of multiple transfer and positioning mechanisms and collection racks of the multi-channel liquid chromatography separation device disclosed in the present invention in an independent working mode;

[0029] Figure 11 A schematic diagram of the working principle of a collection unit of the multi-channel liquid chromatography separation device disclosed in the present invention in the first collection mode;

[0030] Figure 12 A schematic diagram of the working principle of a collection unit of the multi-channel liquid chromatography separation device disclosed in the present invention in the second collection mode;

[0031] Figure 13 This is a schematic diagram of the multi-channel liquid chromatography separation device disclosed in the present invention performing fluid aspiration with an injection needle in the first working mode;

[0032] Figure 14 This is a schematic diagram of the multi-channel liquid chromatography separation device disclosed in the present invention performing fluid aspiration with an injection needle in the second working mode;

[0033] Figure 15 It is a schematic diagram of the state of absorbing stabilizer in the stabilizer applying step of the multi-channel liquid chromatography separation device disclosed in the present invention;

[0034] Figure 16 This is a schematic diagram of the stabilizer adding state in the stabilizer applying step of the multi-channel liquid chromatography separation device disclosed in the present invention;

[0035] Figure 17-18 This is a schematic diagram of the working principle of the first embodiment of the pre-stabilizer addition mode of the multi-channel liquid chromatography separation device disclosed in the present invention;

[0036] Figure 19-20 This is a schematic diagram of the working principle of the second embodiment of the pre-stabilizer addition mode of the multi-channel liquid chromatography separation device disclosed in the present invention;

[0037] Figure 21 This is a schematic diagram of the working principle of the first embodiment of the post-stabilizer addition mode of the multi-channel liquid chromatography separation device disclosed in the present invention;

[0038] Figure 22-24 This is a schematic diagram of the working principle of the second embodiment of the post-stabilizer addition mode of the multi-channel liquid chromatography separation device disclosed in the present invention;

[0039] Figures 25-26 This is a schematic diagram of the working principle of the third embodiment of the post-stabilizer addition mode of the multi-channel liquid chromatography separation device disclosed in the present invention;

[0040] Figures 27-29 This is a schematic diagram of the working principle of the fourth embodiment of the multi-channel liquid chromatography separation device disclosed in the present invention in the post-stabilizer mode;

[0041] Figure 30 This is a schematic diagram of the working principle of the first embodiment of the multi-channel liquid chromatography separation device disclosed in the present invention under the first and second injection modes;

[0042] Figures 31-32 This is a schematic diagram of the working principle of the second embodiment of the multi-channel liquid chromatography separation device disclosed in the present invention under the first and second injection modes;

[0043] Figure 33 This is a schematic diagram of the working principle of the third embodiment of the multi-channel liquid chromatography separation device disclosed in the present invention in the first and second injection modes;

[0044] Figures 34-35 This is a schematic diagram of the working principle of the fourth embodiment of the multi-channel liquid chromatography separation device disclosed in the present invention in the first and second injection modes;

[0045] Figure 36 This is a schematic diagram of the working principle of the fifth embodiment of the multi-channel liquid chromatography separation device disclosed in the present invention in the first and second injection modes;

[0046] Figure 37 This is a schematic diagram of the injection principle in the second secondary injection mode of the multi-channel liquid chromatography separation device disclosed in the present invention;

[0047] Figure 38 This is a schematic diagram of the working principle of an embodiment of the multi-channel liquid chromatography separation device disclosed in the present invention under the second secondary injection mode;

[0048] Figure 39This is a schematic diagram of the working principle of an embodiment of the multi-channel liquid chromatography separation device disclosed in the present invention under the second secondary injection mode;

[0049] Figure 40 This is a schematic diagram of the working principle of an embodiment of the multi-channel liquid chromatography separation device disclosed in the present invention under the second secondary injection mode.

[0050] Reference numerals:

[0051] 100 multi-channel liquid chromatography separation device;

[0052] 10 reagent holding modules; 20 injection units; 30 fluid delivery units; 40 chromatography units; 50 detection units; 60 collection units;

[0053] 21 injector; 211 injection needle; 212 injection drive mechanism (injection arm); 22 switching valve; 23 metering pump; 24 sample loop;

[0054] 31 inlet valve; 32 system pump; 33 static mixer; 34 pressure sensor; 35 bubble sensor;

[0055] 41 column valve; 42 chromatography column;

[0056] 61 transfer and positioning mechanism; 62 collection needle; 63 collection solenoid valve; 64 collection rack. DETAILED DESCRIPTION

[0057] In the description of the embodiments of the present application, it should be noted that the indicated orientation or positional relationship is based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship in which the application product is usually placed when in use, or the orientation or positional relationship commonly understood by those skilled in the art. It is only for the convenience of describing the present application and simplifying the description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present application. In addition, the terms "first", "second", "third", etc. are only used to distinguish the description and cannot be understood as indicating or implying relative importance. The term "connected" in the text can be a direct or indirect connection. In addition, the X-axis direction defined in the present invention is the direction along the length of the multi-channel liquid chromatography separation device, the Y-axis direction is the direction perpendicular to the X-axis direction in the horizontal direction, and the Z-axis direction is the direction perpendicular to the X-axis direction in the vertical direction.

[0058] The embodiments of the present invention will be further explained below with reference to the accompanying drawings.

[0059] The multi-channel liquid chromatography separation device 100 provided by the present invention includes a reagent containing module 10, a plurality of independent liquid channels and a control module.

[0060] <Reagent Containing Module>

[0061] like Figure 1 As shown, in one embodiment, the reagent containing module 10 is disposed on the inner base of the multi-channel liquid chromatography separation device 100 .

[0062] The reagent storage module 10 is used to accommodate various reagents, including samples, carrier fluids, and stabilizers. It can include multiple modular sample boxes and reagent racks. The multiple sample boxes can accommodate different-sized injection tubes, and the multiple reagent racks can accommodate different-sized reagent kits, meeting the batch processing requirements of samples of different specifications (volumes) and different reagents. By replacing the sample boxes and reagent racks, support for new consumables (samples, reagents, etc.) can be expanded in the future, reducing system upgrade costs.

[0063] In a preferred embodiment, the reagent holding module 10 may include two standard 96-well plate-sized reagent kits, support 96-well deep-well plates (1-2 mL / well), and reagent racks of various specifications such as 5 mL reagent racks, 15 mL reagent racks and 50 mL reagent racks to meet the batch processing requirements of samples of different volumes.

[0064] In addition, by replacing sample boxes and reagent racks, support for new consumables (samples, reagents, etc.) in the future can be expanded, thereby reducing system upgrade costs.

[0065] In a preferred embodiment, the reagent holding module 10 is also integrated with a semiconductor refrigeration device (not shown in the figure), and the temperature control range is preferably 4-25°C, which can ensure the stability of the sample before storage and injection and prevent protein denaturation or degradation.

[0066] <Independent fluid channels>

[0067] In a preferred embodiment, the present invention includes a plurality of independent fluid channels, each of which can independently and automatically implement the injection, separation, collection of target components, and application of stabilizers for each sample.

[0068] like Figure 1-2 As shown, each independent fluid channel includes, according to the flow direction of the fluid channel, a sampling unit 20, a fluid delivery unit 30, a chromatography unit 40, a detection unit 50, and a collection unit 60. The following is a detailed description of the sampling unit 20, the fluid delivery unit 30, the chromatography unit 40, the detection unit 50, and the collection unit 60 in each independent fluid channel.

[0069] <Injection Unit>

[0070] like Figure 1-2 ,as well as Figure 6-7As shown, in a preferred embodiment, the injection unit 20 includes an injector 21, a switching valve 22, a metering pump 23, and a sample loop 24, wherein the injector includes an injection needle 211 and an injection drive mechanism (injection arm) 212. The injection needle 211, metering pump 23, and sample loop 24 constitute an independent injection channel, and the metering pump 23 allows the injection needle to absorb the sample and transfer the sample to the sample loop.

[0071] The injection drive mechanism 212 can drive the injection needle 211 to move in the horizontal plane, and can also drive the injection needle 211 to move up and down in the vertical direction perpendicular to the horizontal plane to move closer to or away from the reagent holding module 10. The injection needle 211 is used to absorb the sample or reagent in the reagent holding module 10; the sample ring 24 can receive the sample or reagent absorbed by the injection needle 211; the switching valve 22, when it is switched to Figure 6-7 When in the position shown, the sample or reagent sucked by the injection needle 211 can be injected into the flow path to inject the reagent or sample in the reagent holding module 10; the metering pump 23 is used to transport the sample or reagent in the injection needle 211 to the sample ring 24.

[0072] In one embodiment of the present invention, the injection needle 211 can utilize a high-precision plunger pump and a corrosion-resistant injection needle, achieving microliter-level liquid pipetting accuracy (error <1%), ensuring repeatability of operations such as sample loop loading and stabilizer addition. In the first injection position, the injection needle 211 can be inserted into the reagent holding module 10 to aspirate reagents (e.g., stabilizer) and sample. Furthermore, as described in detail below, in the stabilizer addition position, the injection needle 211 can be inserted into a collection tube in the collection rack 64 to perform a secondary injection of the target component-containing eluate in the collection tube, and can also be inserted into a collection tube in the collection rack 64 to add a reagent to the target component-containing eluate in the collection tube, thereby completing the stabilizer application operation for the target component-containing eluate.

[0073] like Figure 4-5 As shown, in a specific embodiment of the present invention, the sampling drive mechanism 212 is composed of a motor (not shown in the figure) and a driving arm, and the driving arm can move along the X-axis, Y-axis and Z-axis under the control of the motor. When the sampling drive mechanism drives the sampling needle 211 to move along the X-axis and Y-axis, the sample or reagent in the different sampling tubes (reagent tubes) in the reagent holding module 10 is absorbed; when the sampling drive mechanism 212 drives the sampling needle 211 to move along the Z-axis, the sampling needle 211 is inserted into the sampling tube (reagent box) in the reagent holding module 10 at the first sampling position to absorb the sample or reagent. Furthermore, the sampling drive mechanism 212 can also drive the sampling needle 211 to perform fine-tuning along the Y-axis direction, so that the spacing between multiple sampling needles 211 can be adjusted to adapt to the spacing between sampling tubes of different specifications.

[0074] When the switching valve is in Figure 6 In the illustrated position, one end of sample loop 24 is connected to injection needle 211, and the other end is connected to metering pump 23. This allows for further precise control of sample injection, ensuring consistent analysis volume between analyses and avoiding manual errors. The multiple sample loops 24 can have varying capacities, including large-capacity sample loops (e.g., with a maximum capacity of 5 ml) and small-capacity sample loops. By switching the valve position, the sample can be selectively fed into either the large-capacity or small-capacity sample loop, thereby meeting the requirements for loading samples of varying volumes.

[0075] The switching valve 22 selectively switches the sample loop 24 of the injection unit to the injection channel or the fluid delivery channel by switching the valve position. Figure 6 As shown, the multiple ports of the switching valve 22 are connected to the injection needle 211, the sample loop 24, the system pump 32 and the metering pump 23 respectively. When the sample loop 24 is switched to the injection channel, the injection needle 211 performs injection and the sample is delivered to the sample loop 24 through the metering pump 23. Figure 7 As shown, multiple ports of the switching valve 22 are respectively connected to the sample loop 24 and the system pump 32 . At this time, the sample in the sample loop 24 is transported to the chromatography column 42 through the system pump 32 .

[0076] The metering pump 23 not only enables the injection needle 211 to aspirate samples and reagents, but also allows for secondary injection of the target component-containing eluate from the collection tube during secondary injection and also delivers the reagents to the collection tube during stabilizer application. Parameters such as the aspiration volume and injection speed can be controlled by the electronic control and software system to control the metering pump's operation. The metering pump 23 is preferably a plunger pump.

[0077] In a preferred embodiment, when the sample and the carrier liquid are successively aspirated through the injection needle 211 , an air barrier can be formed between the sample and the carrier liquid to prevent the sample from diffusing into the carrier liquid.

[0078] Furthermore, as described in detail below, during secondary injection, the injection drive mechanism 212 can move the injection needle 211, which has completed the injection operation for a particular channel, to the second injection position to perform the secondary injection. At the second injection position, the injection needle 211 can be inserted into the collection tube to aspirate the eluate containing the target component in the collection tube for the secondary injection. The injection drive mechanism 212 drives the injection needle 211 to move along the X-axis, allowing the injection needle 211 to switch back and forth between the first injection position and the secondary injection position.

[0079] During the stabilizer application operation, the injection drive mechanism 212 can also drive the injection needle 211 to the stabilizer application position to add the stabilizer to the target component-containing eluate in the collection tube, thereby completing the stabilizer application operation for the target component-containing eluate. When the injection needle 211 is in the stabilizer application position, the injection needle 211 is located on a side of the collection needle 62 that is close to the reagent holding module 10 and is adjacent to the collection needle 62.

[0080] In a preferred embodiment, the second injection position of the injection needle 211 and the stabilizer application position may be the same position.

[0081] It should be noted that the secondary injection and stabilizer application operation procedures will be introduced in detail later.

[0082] <Fluid Transport Unit>

[0083] like Figure 6-8 As shown, in one embodiment, the fluid delivery unit includes an inlet valve 31 and a system pump 32 .

[0084] Each inlet of the inlet valve 31 is respectively connected to a mobile phase container containing a mobile phase such as a buffer solution / eluent, and is used to dynamically switch the various mobile phase solvents (such as samples, equilibrium buffer solutions, eluents, regeneration solutions, etc.) contained in the mobile phase container to adapt to the different stages of liquid chromatography separation and complex gradient elution requirements. The valve position selection of the inlet valve 31 enables automatic switching of multiple solvents, reduces manual intervention, and ensures that the purification process is carried out efficiently. The inlet valve 31 can be, for example, a 10-position valve, but is not limited to 10 positions. In addition, one or more inlet valves 31 can be included, and multiple inlet valves 31 can be connected in series or in parallel to each other to adapt to the number of different mobile phase types.

[0085] like Figure 6-7 As shown, according to the switching of the switching valve 22, the fluid delivery unit 30 has a first working state and a second working state. When the fluid delivery unit 30 is in the first working state, that is, the 1st and 6th ports of the switching valve 22 are connected, the 2nd and 3rd ports are connected, and the 4th and 5th ports are connected (see Figure 6 As shown), the system pump 32 delivers the solvent or sample contained in the mobile phase container to the chromatography unit 40 through the inlet valve 31; when the fluid delivery unit 30 is in the second working state, that is, when the 5th and 6th ports of the switching valve 22 are connected, the 1st and 2nd ports are connected, and the 3rd and 4th ports are connected (see Figure 7 As shown in FIG, the system pump 32 delivers the reagent or sample in the sample loop 24 to the chromatography unit 40.

[0086] In addition, if Figure 8As shown, one end of the system pump 32 is connected to the mobile phase container through the inlet valve 31, and the other end is connected to the chromatography unit 40 through the switching valve 22. It transports various mobile phase solvents contained in the mobile phase container to the chromatography unit 40 through the inlet valve 31.

[0087] In a preferred embodiment, the system pump 32 may be a valveless ceramic pump, which can cover a flow range of 0.1 to 30 mL / min, achieve low-pulsation delivery, and is suitable for high-viscosity protein samples.

[0088] like Figure 2 In another preferred embodiment shown, the flow path of the fluid delivery unit may further include a static mixer 33, a pressure sensor 34, and a bubble sensor 35. The bubble sensor 35 is disposed upstream of the system pump. The bubble sensor 35 can detect bubbles in the liquid path in real time, triggering a pause or exhaust procedure to prevent bubbles from interfering with the separation efficiency of the chromatography column or distorting the detector signal. The static mixer 33 and the pressure sensor 34 are disposed in the liquid path between the system pump 32 and the injection unit 20. The static mixer 33 ensures uniform mixing of the buffer gradient to avoid a decrease in separation efficiency due to flow rate changes. The pressure sensor 34 monitors the liquid path pressure in real time to prevent overpressure damage to the chromatography column and optimize flow rate stability.

[0089] <Chromatography Unit>

[0090] The chromatography unit 40 is used to separate and purify target components in the input sample mixture, such as Figure 3 As shown, each chromatography unit may include a column valve 41 and one or more chromatography columns 42 connected to the column valve. The column valve 41 has multiple positions, which can selectively connect one of the multiple chromatography columns to the liquid circuit, or not connect the chromatography column to the liquid circuit, that is, the bypass position.

[0091] When the chromatography unit 40 has multiple chromatography columns 42, the types of the multiple chromatography columns 42 can be the same or different. When the types of the multiple chromatography columns 42 are different, by switching the valve position of the column position valve 41, the eluate containing the target component that has been initially purified by one chromatography column 42 of the chromatography unit in an independent liquid channel can be re-injected into the independent liquid channel and passed through another chromatography column, thereby achieving multiple purifications in series and improving the purification purity. Based on this, the type of chromatography column corresponding to the secondary injection can be the same as or different from the type of chromatography column corresponding to the first injection.

[0092] Furthermore, the number of chromatography columns 42 can be one or more, such as two. In one embodiment, each chromatography unit 40 can be equipped with a three-position valve, which can simultaneously accommodate two 1ml-5ml chromatography columns (e.g., affinity chromatography columns, ion exchange columns) and a bypass position. By switching the three-position valve, the eluate containing the target component from the initial separation can be automatically re-injected into a new chromatography column, achieving multiple purifications in series.

[0093] It should be noted that after the secondary injection and secondary purification process, multiple injections may be performed, such as further injections, and those skilled in the art may expand the process according to actual needs.

[0094] Through the design of the above-mentioned chromatography column, the present invention can perform chromatography treatments of various complexities, thereby greatly improving the use function of the multi-channel liquid chromatography separation device.

[0095] <Detection Unit>

[0096] The detection unit 50 is disposed between the chromatography unit 40 and the collection needle 62 and is used to dynamically monitor and measure the concentrations of the components in the eluate flowing from the chromatography column and the properties of the eluate, such as pH and conductivity, to ensure the quality controllability of the target product. In the present invention, each of the detection units 50 can optionally include an optical detector, a conductivity detector, and a pH detector.

[0097] The optical detector can be, for example, a dual-wavelength detector, with each channel capable of simultaneously monitoring both the protein concentration at 280 nm and the nucleic acid concentration at 260 nm, allowing for real-time determination of elution peak purity and identification of the target protein elution peak to trigger target collection signals. It should be noted that the 280 nm and 260 nm wavelengths used in this invention are merely exemplary and may be adapted by those skilled in the art as needed.

[0098] The flow path where the detection unit is located constitutes a detection channel. Each detection channel is connected to the chromatography unit in the corresponding independent liquid channel, so that the eluate purified by chromatography on the chromatography column of the chromatography unit passes through the detection channel.

[0099] The conductivity detector monitors the salt concentration by changes in conductivity, helping to determine whether the elution conditions are appropriate. It can also help identify the elution peak of the target protein (usually eluted when the salt concentration changes).

[0100] The pH detector monitors the elution conditions by pH changes to ensure the stability of the elution process. It can also assist in identifying the elution peak of the target protein (usually eluted when the pH changes).

[0101] <Collection Unit>

[0102] like Figure 1-2 、 Figure 9-10 As shown, each collection unit 60 can perform collection operations in parallel or asynchronously, allowing samples at different purification stages to be processed simultaneously, thereby improving throughput and purification efficiency. Each independent collection unit can initiate a collection process based on real-time detection signals from the corresponding detection unit (e.g., triggered by a dual-wavelength detector), sorting the eluate containing the target component into a collection tube, thereby avoiding cross-contamination and achieving process flexibility.

[0103] In one embodiment, each collection unit 60 includes a transfer and positioning mechanism 61, a collection needle 62, a collection solenoid valve 63, and a collection rack 64. The transfer and positioning mechanism is used to drive the collection rack to move along the X-axis; the collection needle is used to input the eluate containing the target component separated by the chromatography unit into the collection tube on the collection rack, and can be started based on the protein concentration peak detected by the above-mentioned real-time detection unit, or the timing of starting to transport the eluate containing the target component according to the preset time of protein purification; the collection solenoid valve is provided on the pipeline between the collection needle and the detection unit, and is used to switch the mobile phase flowing through the detection unit to flow to the collection needle or to switch it to flow to the waste liquid collector; the collection rack is used to carry a plurality of arranged collection tubes, and the collection tubes are used to collect the eluate containing the target component transported from the collection needle.

[0104] like Figure 9-10 As shown, the transfer and positioning mechanism 61 is fixedly mounted on the base of the device, located below the collection needle 62 and spaced apart along the Y direction, to support the collection rack 64. Each transfer and positioning mechanism is equipped with an independent motor and motion guide rail, allowing for parallel or asynchronous collection operations, supporting the simultaneous processing of samples at different purification stages and improving throughput efficiency. The transfer and positioning mechanism 61 is used to drive the collection rack 64 along the X direction, allowing multiple collection tubes to reach the collection position below the collection needle 62 one by one to receive the target component-containing eluate delivered by the collection needle 62.

[0105] Specifically, the collection rack 64 is movable between a first position close to the reagent holding module and a second position away from the reagent holding module. The collection rack 64 has a first end close to the reagent holding module and a second end away from the reagent holding module. The collection rack can accommodate a plurality of collection tubes arranged in a row between the first end and the second end along the length direction of the collection rack. Figure 17 As shown, when the collection rack 64 is in the first position, the plurality of collection tubes are all located on the side of the collection needle close to the reagent holding module, and the plurality of collection tubes are all located on the side of the injection needle at the stabilizer application position close to the reagent holding module. Figure 25 As shown, when the collection rack 64 is located at the second position, the plurality of collection tubes are all located on the other side of the collection needle away from the reagent containing module.

[0106] In one embodiment, the transfer and positioning mechanism 61 further includes a positioning mechanism that can accurately position the collection tube to the collection position. The positioning mechanism can be implemented by controlling the displacement distance or an optical sensor, which is not specifically limited in the present invention.

[0107] In one embodiment, the transfer and positioning mechanism includes a guide rail, a servo motor, a lead screw, a slider, and a clamp. The servo motor drives the lead screw, causing the slider and clamp to move along the guide rail, thereby driving the collection rack on the clamp. The clamp is a telescopic structure, including two jaws, one or both of which can be extended and retracted (for example, in the X-axis) to adjust the spacing between the two jaws to accommodate collection racks of different sizes.

[0108] The collection unit includes a collection rack 64, on which are arranged a plurality of collection tubes in the longitudinal direction of the collection rack. The collection racks of the independent liquid channels are arranged in a direction perpendicular to the longitudinal direction of the collection rack, and the injection needle can move in the arrangement direction of the collection rack; Figure 13 As shown, when the multi-channel liquid chromatography separation device is in the first working mode, the injection needle of each independent liquid channel is aligned with the corresponding collection rack in the length direction of the collection rack; Figure 14 As shown, when the multi-channel liquid chromatography separation device is in the second working mode, the injection needle of one independent liquid channel among the multiple independent liquid channels moves in the arrangement direction of the collection rack to be aligned with the collection rack of another independent liquid channel in the length direction of the collection rack.

[0109] A plurality of collection tubes arranged along the X direction are placed on the collection rack 64. The specifications of the collection tubes can be the same or different. In order to be compatible with collection tubes of different specifications, there are three different collection racks. In a preferred embodiment, the collection rack can include three collection racks for centrifuge tubes with collection volumes of 1.5ml / 5ml / 15ml respectively.

[0110] <Control Module>

[0111] Electronic control system:

[0112] This high-throughput protein purification system achieves data communication with the computer system through CAN bus and USB interface, ensuring precise control and convenient operation.

[0113] System architecture: The control system of the system is mainly composed of the following modules: computer system, CAN to USB interface, network switch and serial port server, various sensors and actuators, control circuit board and logic control unit.

[0114] Main Components and Functions: The computer system, as the core of the entire control system, is responsible for processing data, issuing control commands, and communicating with downstream modules via a CAN-to-USB interface. The CAN-to-USB interface module facilitates data exchange between the computer system and field control modules. Network ports and switches enable centralized management of multiple CAN buses. Network switches and 4-to-1 and 8-to-1 serial port servers expand the number of interfaces, ensuring that multiple sensors and actuators can be connected simultaneously to the control system.

[0115] Software system:

[0116] The high-throughput protein purification system software is Windows-based and PC-operated, featuring a simple, stylish interface and convenient operation. It automatically runs pre-set protein purification processes, supports user-defined purification processes for storage and recall, and enables real-time communication between the PC and liquid chromatography separation device via a built-in wireless network. The software controls the high-throughput protein purification system's accessories and liquid chromatography separation device to achieve separation and purification. The software also supports integration with external information management systems and databases, supports uploading process data, and can read specific data from databases to generate action sequences based on this data, enabling seamless integration with various devices.

[0117] The software of this system is divided into two parts: instrument control and instrument management. The instrument control part can realize instrument control, method editing, standard curve, data processing and reporting through shortcut icons, and can quickly open the corresponding interface.

[0118] <Multi-channel fluid path>

[0119] It will be appreciated that the above description is directed to a liquid chromatography separation device having a single fluid channel. In a preferred embodiment, the liquid chromatography separation device disclosed herein includes multiple independent fluid channels, each of which has the same structure as described above and will not be further described here. It should be emphasized that when multiple independent fluid channels are included, the number of chromatography columns in the chromatography unit of each channel may be the same or different.

[0120] When the liquid chromatography separation device disclosed in the present invention has multiple liquid channels, each of the liquid channels can perform separation processing in parallel and synchronously, or can perform separation processing independently (e.g., Figure 9-10 As shown, each channel can operate independently without interfering with each other. Based on this, the multi-channel liquid chromatography separation device disclosed in the present invention can complete the separation, collection, and stabilizer addition of multiple samples simultaneously or in a time-sharing manner, thereby significantly improving processing efficiency.

[0121] The following describes the basic action processes of a single fluid channel in combination with the above modules.

[0122] The core steps of liquid chromatography separation and purification include: injection (binding the target component in the sample to the chromatography column filler) → washing (using washing liquid to remove impurities that are not bound or weakly bound to the chromatography column filler) → elution (using eluent to release the target component from the chromatography column) → detection and analysis (detecting the components in the eluate) → collection (collecting the portion of the eluate containing the target component), among which washing and elution are chromatography steps.

[0123] <Injection Steps>

[0124] In the present invention, a single independent liquid channel has two basic working modes: small-dose injection and large-dose injection, which are described in detail below.

[0125] 1. Small dose injection

[0126] like Figure 6 As shown, when performing a small-dose injection, the injection drive mechanism drives the injection needle to move, and the injection needle draws the sample to be purified and the carrier liquid loaded on the reagent holding module. At this time, the switching valve switches the sample loop to the injection channel between the injection needle and the metering pump. The metering pump controls the injection needle to draw the small-dose sample and the carrier liquid, and uses the carrier liquid to deliver the small-dose sample to the sample loop. It should be noted that during the process of delivering the small-dose sample and the carrier liquid to the sample loop by the metering pump, the system pump and the chromatography unit are in a connected state. The mobile phase contained in the mobile phase container connected to the inlet valve can be continuously delivered to the chromatography unit by the system pump to prepare for the subsequent chromatography steps.

[0127] like Figure 7 As shown, after the sample is delivered to the sample loop, the switching valve switches the sample loop to between the system pump and the chromatography unit, and the small-dose sample in the sample loop is delivered to the chromatography unit by the system pump, thereby completing the small-dose injection.

[0128] 2. Large dose injection

[0129] like Figure 8 As shown, in a specific embodiment of the present invention, large-dose injection can also be achieved through the use of an inlet valve and a system pump. In this case, the inlet valve is connected to a sample container containing a large-dose sample. Specifically, during large-dose injection, a switching valve switches the system pump to an independent fluid channel, thereby establishing communication between the system pump and the chromatography unit. The system pump then transfers the large-dose sample contained in the mobile phase container to the chromatography unit through the selection of the inlet valve, thereby preparing for the subsequent chromatography steps.

[0130] <Chromatography steps>:

[0131] After the sample is added to the chromatography column, the flow rate and pressure of the buffer solution are adjusted to separate the sample in the chromatography column. Proteins will be separated into different peaks according to their size, charge, affinity and other characteristics. The selection valve cuts the system pump into an independent liquid circuit, and at the same time, the inlet valve switches the buffer container to connect with the system pump, and the system pump delivers the buffer solution in the buffer container to the chromatography column to clean the chromatography column. In addition, according to the type of chromatography, the inlet valve can be switched to select the elution solution or solvent to elute the target component from the chromatography column; in addition, the balancing and cleaning steps of the chromatography column are the same as the elution action, the only difference is that the inlet valve switches the corresponding mobile phase solvent to the liquid circuit. Since the chromatography method is not the inventive point of the present invention, it will not be described here.

[0132] <Test steps>:

[0133] During the chromatography process, the detection unit continuously monitors and measures the concentration and characteristics of each component in the eluate exiting the column in real time, thereby confirming the protein separation. Based on the signal generated by the detection unit, the control unit controls the collection unit to collect the eluate containing the target component when predetermined conditions are met.

[0134] <Collection steps>:

[0135] When a predetermined target component is detected, the control unit controls the collecting unit to collect the eluate containing the target component.

[0136] During collection, the collecting needle cannot move along the X-axis. At this time, the collecting rack can be driven to move along the X-axis by the transfer and positioning mechanism. The collecting rack can move between a first position close to the reagent holding module and a second position away from the reagent holding module, and align with the collecting needle in a stationary state, thereby enabling the deliquescence flowing out of the collecting needle to be injected into each collection tube in the collecting rack.

[0137] Furthermore, in a preferred embodiment, the collection unit of the present invention has two operating modes: (a) a first collection mode in which the collection rack collects the eluate containing the target component while moving away from the reagent holding module; and (b) a second collection mode in which the collection rack collects the eluate containing the target component while moving toward the reagent holding module. The two operating modes are described in detail below.

[0138] 1. First collection mode

[0139] In a specific embodiment, the transfer and positioning mechanism drives the collection rack to move from the leftmost position (first position) to the rightmost position (second position) of the travel, and the collection tubes located at the second end of the collection rack to the collection tube located at the first end of the collection rack are sequentially positioned under the collection needle to collect the eluate containing the target component transported from the collection needle. For example, Figure 11As shown, the collection rack first positions the rightmost collection tube 1 below the collection needle to collect the eluate containing the target component delivered by the collection needle. It then moves one tube position to the right to collect the eluate from the second collection tube 2 from the right, and so on to the leftmost collection tube N. It can be seen that after collecting the eluate containing the target component in the first collection mode, the collection rack is at the rightmost position of the travel range.

[0140] 2. Second collection mode

[0141] In a specific embodiment, the collection rack first moves from the default position, i.e., the leftmost position of the travel, to the rightmost position of the travel. During the movement from the rightmost position to the leftmost position of the travel, the collection rack collects, and the collection tubes located at the first end of the collection rack to the collection tubes located at the second end of the collection rack among the multiple collection tubes are sequentially positioned under the collection needle to collect the eluate containing the target component transported by the collection needle. For example, Figure 12 As shown, the collection rack first positions the leftmost collection tube N below the collection needle for collection, then moves one tube position to the left to collect the second collection tube N-1 from the left, and so on to the rightmost collection tube 1. It can be seen that after collecting the eluate containing the target component in the second mode, the collection rack is at the leftmost position of the travel range.

[0142] <Stabilizer application steps>:

[0143] In the present invention, a stabilizer application step is further included. For various types of chromatography (such as affinity chromatography and ion exchange), it is necessary to elute the target component from the solid phase carrier of the chromatography column by adjusting the pH value of the buffer or the ion (Nacl) strength of the eluent, and extreme pH values ​​or high ionic strengths will affect the stability of the target component (such as protein). Therefore, it is necessary to modify the eluent (stabilizer application), that is, by adding a modified treatment liquid (i.e., a stabilizer) to the eluent containing the target component to neutralize the pH value of the eluent containing the target component or dilute the ionic strength of the eluent. The stabilizer can be a reagent placed in a reagent tray that can change the pH value or ionic strength. By applying the stabilizer, a closed loop of the "collection-processing-storage" process is achieved. In the stabilizer application step, the injection unit can apply a stabilizer to the collection tube of the collection rack.

[0144] like Figure 15-16 As shown, the present invention drives the injection needle to absorb the stabilizer from the reagent holding module at the first injection position through the injection drive mechanism, and then moves to the stabilizer application position. At this time, the transfer and positioning mechanism drives the collection rack with multiple collection tubes to move under the injection needle and pass under the stabilizer application position in turn. The injection needle adds stabilizer to the multiple collection tubes one by one.

[0145] The stabilizer application step proposed in the present invention includes two modes: pre-stabilizer application and post-stabilizer application. These two stabilizer application modes are described in detail below.

[0146] 1. Pre-stabilizer mode

[0147] In the pre-stabilizer mode, the sampling unit pre-applies stabilizer to the collection tube before collecting the target component-containing eluate delivered by the collection needle. Specifically, before collecting the target component-containing eluate, the sampling drive mechanism moves the sampling needle in the sampling unit to the stabilizer application position. The transfer and positioning mechanism then drives the collection rack, carrying multiple collection tubes, to pass under the sampling needle in sequence below the stabilizer application position. The sampling needle then adds stabilizer to each of the collection tubes one by one.

[0148] In such Figure 17-18 In one embodiment shown, when the injection unit applies a stabilizer to the collection tube before the collection tube collects the eluate containing the target component delivered by the collection needle, the injection needle is moved to the stabilizer application position after drawing the stabilizer from the reagent holding module at the first injection position. In the first collection mode, during the movement of the collection rack from the first position to the second position, since the injection needle is located at the stabilizer application position, the injection needle is located on the side of the collection needle close to the reagent holding module and is adjacent to the collection needle, so the collection tubes located at the second end of the collection rack to the collection tubes located at the first end of the collection rack are sequentially passed through Passing under the injection needle and under the collection needle, when the collection tube is first located under the injection needle, the injection needle adds a stabilizer to the collection tube. When the collection tube with the stabilizer added is subsequently located under the collection needle, the eluate containing the target component transported by the collection needle is collected. That is, after adding the post-processing stabilizer in this mode, the collection tube can directly collect the eluate containing the target component, thereby realizing the pre-application of the stabilizer, so that the pH value of the collected eluate can be directly neutralized or the ionic strength of the eluate can be directly diluted, which can maximize the reduction of the impact of extreme pH value or ionic strength on the target component and save more time. It should be emphasized that in this embodiment, after adding the stabilizer to a single collection tube, the collection tube can immediately collect the eluate.

[0149] In such Figure 19-20In another embodiment shown, when the injection unit pre-applies stabilizer to the collection tube before the collection tube collects the eluate containing the target component delivered by the collection needle, the injection needle draws stabilizer from the reagent holding module at the first injection position and then moves to the stabilizer application position. The collection rack moves from the first position to the second position, and the collection tubes located at the second end of the collection rack and the collection tubes located at the first end of the collection rack sequentially pass under the injection needle. When the collection tube is first positioned under the injection needle, stabilizer is added to the collection tube by the injection needle. The collection rack moves from the second position to the first position, and the collection tubes located at the first end of the collection rack and the collection tubes located at the second end of the collection rack are sequentially positioned under the collection needle to collect the eluate containing the target component delivered by the collection needle. That is, in this mode, stabilizer is added to all collection tubes before the collection rack collects the eluate containing the target component during its leftward movement, thereby achieving pre-application of stabilizer. It should be emphasized that in this embodiment, eluate is collected only after stabilizer is added to all collection tubes.

[0150] The pre-stabilizer mode is preferably suitable for the first collection mode ( Figure 17-18 (See the embodiment shown in the figure), which enables simultaneous application of stabilizer and collection of eluate. In this processing mode, because stabilizer can be added in advance, processing speed can be increased during multi-channel parallel processing. Furthermore, the collected eluate can be directly modified without waiting time, minimizing damage to the target components.

[0151] In addition, the pre-stabilizer mode is more suitable for isocratic elution processes with a constant mobile phase ratio because it cannot adjust the amount of stabilizer added in real time according to the test results.

[0152] 2. Post-stabilizer mode

[0153] After a collection tube has collected the eluate containing the target component, that is, after the eluate is collected, the transfer and positioning mechanism drives the collection rack to carry the collection tube away from the collection position to the predetermined position. At this predetermined position, the injection needle can add the required amount of stabilizer to the collection tube based on the test results.

[0154] In such Figure 21 In one embodiment shown, when the sampling unit collects the eluate containing the target component delivered from the collecting needle in the collecting tube, the collecting rack is moved from the first position to the second position, and the collecting tubes located at the second end of the collecting rack to the collecting tube located at the first end of the collecting rack among the multiple collecting tubes are sequentially positioned under the collecting needle to collect the eluate containing the target component delivered from the collecting needle (see Figure 11After all collection tubes have completed collection, the injection needle draws the stabilizer from the reagent holding module at the first injection position and then moves to the stabilizer application position, causing the collection rack to move from the second position to the first position. The multiple collection tubes, from the collection tube at the first end of the collection rack to the collection tube at the second end of the collection rack, pass under the injection needle in sequence, and the stabilizer is added by the injection needle, thereby achieving post-application of the stabilizer. It should be emphasized that in this embodiment, the stabilizer is added only after all collection tubes have completed collection.

[0155] In such Figure 22-24 In one embodiment shown, when the sampling unit collects the eluate containing the target component delivered from the collecting needle in the collecting tube, the collecting rack is moved from the first position to the second position, and the collecting tubes located at the second end of the collecting rack to the collecting tube located at the first end of the collecting rack among the multiple collecting tubes are sequentially positioned under the collecting needle to collect the eluate containing the target component delivered from the collecting needle (see Figure 11 After all the collection tubes have been collected, the collection rack is returned from the second position to the first position, that is, the collection rack needs to be reset by the optical coupler (from the rightmost position to the default position, i.e., the leftmost position) (e.g. Figure 22 After the injection needle absorbs the stabilizer from the reagent holding module at the first injection position, it moves to the stabilizer application position, causing the collection rack to move from the first position to the second position. The collection tubes located at the second end of the collection rack and the collection tubes located at the first end of the collection rack are sequentially positioned below the injection needle, so that the stabilizer is added by the injection needle, thereby achieving the post-application of the stabilizer. For example, Figure 23-24 As shown, the injection needle is fixed in the X-axis direction. The collection rack first positions the rightmost collection tube 1 below the injection needle to add stabilizer. Then, it moves one tube to the right to add stabilizer to the second collection tube 2 from the right. The stabilizer is then added to the leftmost collection tube N. It should be emphasized that in this embodiment, stabilizer is added after all collection tubes have been collected.

[0156] In such Figures 25-26In one embodiment shown, when the injection unit applies a stabilizer to the collecting tube after the collecting tube collects the eluate containing the target component delivered from the collecting needle, the injection needle absorbs the stabilizer from the reagent holding module at the first injection position and then moves to the stabilizer application position. In the above-mentioned second collection mode, during the movement of the collecting rack from the second position to the first position, since the injection needle is located on the side of the collecting needle close to the reagent holding module and adjacent to the collecting needle when it is located at the stabilizer application position, the collection tube located at the first end of the collecting rack to the collection tube located at the second end of the collecting rack among the multiple collection tubes successively pass under the collecting needle and under the injection needle. When the collection tube is first located under the collecting needle, the eluate containing the target component delivered from the collecting needle is collected. When the collection tube that has collected the eluate containing the target component is subsequently located under the injection needle, the injection needle adds the stabilizer to the collection tube, thereby realizing the post-application of the stabilizer. For example, Figures 25-26 As shown, the collection rack first positions the leftmost collection tube N under the collection needle to collect the target protein, and similarly the collection rack moves tube by tube to the rightmost collection tube 1 for collection. At the same time, after the injection needle has absorbed the stabilizer, it moves to the stabilizer application position, and starts adding stabilizer from the collection tube N on the leftmost side of the collection rack, and then moves one tube position to the left to the second collection tube N-1 from the left to add stabilizer, and similarly adds stabilizer to the rightmost collection tube 1. It should be emphasized that in this embodiment, after a single collection tube has collected, the collection tube can be immediately added with stabilizer. It can be seen that in this mode, the addition of stabilizer and collection can be carried out simultaneously, so that the pH value of the collected eluate can be directly neutralized or the ionic strength of the eluate can be quickly diluted, which can greatly reduce the impact of extreme pH value or ionic strength on the target component and save more time.

[0157] In such Figures 27-29 In one embodiment shown, Figure 27 As shown, when the sampling unit applies a stabilizer to the collection tube after the collection tube collects the eluate containing the target component delivered from the collection needle, the collection rack is moved from the second position to the first position, and the collection tubes located at the first end of the collection rack to the collection tube located at the second end of the collection rack pass under the collection needle in sequence. When the collection tube is first located under the collection needle, the eluate containing the target component delivered from the collection needle is collected. Figures 28-29 As shown, after all collection tubes have completed collection, the injection needle draws stabilizer from the reagent holding module at the first injection position and then moves to the stabilizer application position, causing the collection rack to move from the first position to the second position. The collection tubes, from the second end of the collection rack to the first end of the collection rack, are positioned sequentially below the injection needle, allowing stabilizer to be added by the injection needle, thereby achieving post-application of the stabilizer. It should be emphasized that in this embodiment, stabilizer is added after all collection tubes have completed collection.

[0158] The post-stabilizer mode is preferably suitable for the second collection mode ( Figures 25-26 In one embodiment shown in FIG. 1 , the eluent can be collected and the stabilizer can be applied simultaneously, thereby increasing the processing speed. That is, after each collection tube has collected the eluent containing the target component, the transfer and positioning mechanism drives the corresponding collection rack to move to the target position in the direction close to the injector, and the required amount of stabilizer can be added immediately. The waiting time is short, and the damage to the target component can be minimized. In this processing mode, since the corresponding amount of stabilizer is added in real time according to the test results, the modification process can be carried out more effectively. Since the post-stabilizer addition mode can adjust the amount of stabilizer added in real time according to the test results, it is suitable for gradient elution processes in which the mobile phase ratio changes.

[0159] In a preferred embodiment, after the stabilizer is added to the collection tube through the injection needle, the injection needle can mix the mixture of the stabilizer and the eluent through multiple suction and exhalation actions.

[0160] <Secondary Injection Steps>

[0161] After the first injection of the sample is separated and processed by the chromatography unit, it is transported by the collection needle to the collection tube on the collection rack. By utilizing the feature that the injection needle can be moved in the X direction, it can be moved to the second injection position. At this time, a second injection operation can also be performed, thereby achieving complex multidimensional chromatographic separation without increasing the complexity of the flow path.

[0162] Specifically, the injection needle in the injection unit can move successively along the Y-axis and the X-axis to move back and forth between the first injection position above the reagent disk module and the second injection position above the collection rack. The eluent containing the target component collected in each collection tube is respectively transported to the liquid channel again by the movement of the collection rack relative to the injection needle located at the second injection position. It should be noted that when performing the secondary injection operation, the eluent containing the target component in the collection tube is sucked up by the injection needle in the injection unit and reintroduced into the liquid channel. The subsequent chromatography step, detection step, collection step, and stabilizer application step are the same as the processing flow after the first injection described above, and therefore will not be repeated here.

[0163] In addition, in order to achieve a mixed-mode (multi-dimensional) chromatography purification operation, the chromatography unit performed by the second injection can also be different from the chromatography unit corresponding to the first injection. In other words, the chromatography unit performed by the second injection can also be the same as the chromatography unit corresponding to the first injection. In this case, when the chromatography performed by the first injection is of the same type as the chromatography performed by the second injection, multiple chromatography can be performed using the same type of chromatography unit through the second or multiple injections, thereby further improving the chromatographic purity; when the chromatography performed by the first injection is of a different type than the chromatography performed by the second injection, multiple chromatography can be performed using different types of chromatography units through the second or multiple injections, thereby further improving the chromatographic purity.

[0164] It should be noted that the above-mentioned secondary injection process can be performed multiple times according to needs, such as three injections, four injections, etc.

[0165] Therefore, as described above, the present invention has a first secondary injection mode (ie, single-channel secondary injection).

[0166] <First and second injection modes> The injection drive mechanism drives the injection needle of the injection unit in the first liquid channel to the second injection position of the collection unit in the first liquid channel, and the injection needle absorbs the eluent containing the target component in the first collection tube, that is, the eluent in the collection tube is twice in the same independent liquid channel.

[0167] In one embodiment, when the collection rack moves from the first position to the second position, the collection tubes located at the first end of the collection rack to the collection tubes located at the second end of the collection rack pass under the collection needle in sequence. When the collection tubes are located under the collection needle, the eluate containing the target component transported by the collection needle is collected (see Figure 11 ).like Figure 30 As shown, after all collection tubes have been collected, the injection needle moves to the second injection position, and the collection rack moves from the second position to the first position. The collection tubes, from the second end of the rack to the first end of the rack, are positioned sequentially below the injection needle, allowing the injection needle to draw the eluate containing the target component. It should be emphasized that in this embodiment, the injection needle performs a second injection after all collection tubes have been collected.

[0168] In one embodiment, when the collection rack moves from the first position to the second position, the collection tubes located at the first end of the collection rack to the collection tubes located at the second end of the collection rack pass under the collection needle in sequence. When the collection tube is first located under the collection needle, the eluate containing the target component transported by the collection needle is collected (see Figure 11After all the collection tubes have been collected, the collection rack is returned from the second position to the first position, that is, the collection rack needs to be reset by the optical coupler (from the rightmost position to the default position, i.e., the leftmost position) (e.g. Figure 31 As shown). Figure 32 As shown, after the collection rack is reset, the injection needle moves to the second injection position. The collection rack moves from the first position to the second position. The collection tubes, from the collection tube at the second end of the collection rack to the collection tube at the first end of the collection rack, are positioned sequentially below the injection needle, allowing the injection needle to aspirate the eluate containing the target component. It should be emphasized that in this embodiment, the injection needle performs a second injection after all collection tubes have collected.

[0169] In one embodiment, Figure 33 As shown, when the collection rack moves from the second position to the first position, the collection tubes located at the first end of the collection rack to the collection tubes located at the second end of the collection rack sequentially pass under the collection needle. When the collection tubes are first located under the collection needle, they collect the eluate containing the target component transported by the collection needle. In the above-mentioned second collection mode, during the movement of the collection rack from the second position to the first position, because when the injection needle is in the second injection position, the injection needle is located on the side of the collection needle close to the reagent holding module and adjacent to the collection needle, the collection tubes located at the first end of the collection rack to the collection tubes located at the second end of the collection rack sequentially pass under the collection needle and under the injection needle. When the collection tubes are first located under the collection needle, they collect the eluate containing the target component transported by the collection needle. When the collection tubes that have collected the eluate containing the target component are subsequently located under the injection needle, the injection needle aspirates the eluate containing the target component to achieve a second injection. It should be emphasized that in this embodiment, after a single collection tube has completed collection, the injection needle can directly perform a second injection. It can be seen that in this mode, collection and secondary injection can be performed simultaneously, so that the secondary injection can be performed as quickly as possible and more time is saved.

[0170] In one embodiment, Figure 34 As shown, when the collection rack moves from the second position to the first position, the collection tubes located at the first end of the collection rack to the collection tubes located at the second end of the collection rack pass under the collection needle in sequence. When the collection tube is first located under the collection needle, it collects the eluate containing the target component transported by the collection needle. Figure 35 As shown, after all collection tubes have been collected, the injection needle moves to the second injection position, and the collection rack moves from the first position to the second position. The collection tubes, from the collection tube at the second end of the rack to the collection tube at the first end of the rack, are positioned sequentially below the injection needle, allowing the injection needle to aspirate the eluate containing the target component. It should be emphasized that in this embodiment, the injection needle performs a second injection after all collection tubes have been collected.

[0171] In a preferred embodiment, in the post-stabilizer addition mode, after the stabilizer is added to the collection tube by the injection needle, the injection needle can deliver the mixture of the stabilizer and the eluent to the liquid channel again. That is, the stabilizer application step and the secondary injection step can be combined into one step and performed in tandem. For example, in Figures 25-26 In the embodiment shown, the collection tube is first positioned below the collection needle to collect the eluate containing the target component delivered by the collection needle. When the collection tube containing the eluate containing the target component is subsequently positioned below the injection needle, the injection needle adds a stabilizer to the collection tube. At this time, after the injection needle adds the stabilizer to the collection tube, the injection needle can immediately deliver the mixture of the stabilizer and the eluate to the fluid channel again, that is, a second injection is performed directly after the stabilizer is applied (see Figure 36 ).

[0172] It should be noted that in the first and second injection modes, when the chromatography unit includes multiple chromatography columns, the column position valve can be switched so that the eluate containing the target component that has been initially purified by a chromatography column of the chromatography unit in an independent liquid channel can be injected again into the independent liquid channel and pass through another chromatography column, thereby enabling the sample to achieve complex multidimensional chromatography.

[0173] In addition, when the liquid chromatography separation device disclosed in the present invention has multiple liquid channels, the above-mentioned injection step, chromatography step, and collection step all include further processing operations, which are introduced one by one below.

[0174] <Multi-channel injection steps>

[0175] In the present invention, the multi-channel liquid channel also has two basic working modes: small-dose injection and large-dose injection, which are described in detail below.

[0176] 1. Small dose injection

[0177] As described above, the small-dose injection process of a multi-channel liquid channel is the same as the small-dose injection process of a single liquid channel, but it should be emphasized that when the liquid chromatography separation device disclosed in the present invention has multiple channels, each liquid channel in the multiple channels can perform small-dose injection in parallel and synchronously, that is, the timing of small-dose injection of each liquid channel is the same, or can perform small-dose injection asynchronously, that is, each channel can perform small-dose injection independently without interfering with each other, and the timings can be different.

[0178] 2. Large dose injection

[0179] As described above, the large-dose injection process of a multi-channel liquid channel is the same as the large-dose injection process of a single liquid channel, but it should be emphasized that when the liquid chromatography separation device disclosed in the present invention has multiple channels, each liquid channel in the multiple channels can perform large-dose injection in parallel and synchronously, that is, the timing of large-dose injection of each liquid channel is the same, or can perform large-dose injection asynchronously, that is, each channel can perform large-dose injection independently without interfering with each other, and the timings can be different.

[0180] <Multi-channel chromatography steps>:

[0181] The multi-channel chromatography steps are essentially the same as those described above for a single fluidic channel and will not be further described here. However, as is the inventive concept of the present invention, when multiple channels are present, the chromatography processes performed by the multiple channels can be performed simultaneously in parallel, or each fluidic channel can be independent and non-interfering, allowing for separate, sequential processes.

[0182] In addition, it should be emphasized that when there are multiple channels, when a second injection is performed across the channels, the number and type of chromatography columns used for the chromatography treatment may be the same as or different from those used for the first injection treatment.

[0183] <Multi-channel detection steps>:

[0184] The multi-channel detection steps are essentially the same as the chromatography steps described above for a single fluidic channel and will not be further described here. However, as is the inventive concept of the present invention, when multiple channels are present, the detection processing performed by these channels can be performed simultaneously in parallel, or each fluidic channel can be independent and non-interfering, allowing detection to be performed separately according to different time sequences.

[0185] <Multi-channel collection steps>:

[0186] The multi-channel collection process is essentially the same as the single-channel collection process described above and will not be further described here. However, as a key aspect of the present invention, when multiple channels are present, the collection process can be performed simultaneously or independently, with each channel independently processing at different time sequences.

[0187] <Multi-channel stabilizer application steps>:

[0188] The multi-channel collection process is essentially the same as the stabilizer application process described above for a single fluidic channel and will not be further described here. However, as a key aspect of the present invention, when multiple channels are present, the stabilizer application process can be performed simultaneously or independently, with each channel independently and in a different time sequence.

[0189] <Multi-channel secondary injection>

[0190] As described above, the secondary injection process of a multi-channel liquid channel is the same as the secondary injection process of a single liquid channel, but it should be emphasized that when the liquid chromatography separation device disclosed in the present invention has multiple channels, each liquid channel in the multiple channels can perform secondary injection in parallel and synchronously, that is, the timing of large-dose injection of each liquid channel is the same, or can perform secondary injection asynchronously, that is, each channel can perform secondary injection independently without interfering with each other, and the timings can be different.

[0191] In addition, if Figure 37 As shown, when the liquid chromatography separation device disclosed in the present invention has multiple fluid channels, in addition to the first and second injection modes described above, it further includes a second cross-channel injection mode, that is, the injection drive mechanism can move the injection needle that has completed the injection operation for a certain fluid channel to a second injection position in another fluid channel to achieve cross-channel secondary injection. In this second injection position, the injection needle of a certain fluid channel can be inserted into the collection tube on the collection rack of another fluid channel to absorb the eluate containing the target component in the collection tube for secondary cross-channel injection, that is, to achieve serial separation processing of multiple independent fluid channels. In other words, the liquid chromatography separation device disclosed in the present invention has a first operating mode and a second operating mode, wherein in the first operating mode, each of the independent fluid channels can perform sample separation in parallel; in the second operating mode, the injection needle of one of the multiple independent fluid channels can perform a second injection on the eluate containing the target component collected by the collection unit in another independent fluid channel, thereby achieving a series connection between the two independent fluid channels.

[0192] <Secondary Injection Mode> The injection drive mechanism drives the injection needle of the injection unit in the first fluidic channel to the secondary injection position of the collection unit in the second fluidic channel. The injection needle then draws the target component-containing eluate from the second collection tube in the second fluidic channel. The eluate in the collection tube then enters a different independent fluidic channel a second time. In other words, the target component-containing eluate collected in the collection tube on the collection rack of one fluidic channel can be drawn a second time by the injection needle of any fluidic channel.

[0193] like Figures 38-40 In the preferred cross-channel secondary injection embodiment shown, it can be seen that after the first injection needle in the first channel injects the sample from the reagent module and performs chromatography, the collection tube in the first collection rack collects the eluate containing the target component, and the first injection, chromatography and collection process are completed ( Figure 38 ).

[0194] When the eluate containing the target component collected in the first channel is to be sampled for a second time across channels, the second injection needle of the second channel is moved above the moving range of the first collection rack of the first channel, so that the eluate containing the target component in the collection tube of the first collection rack can be extracted one by one. After the second injection is completed, the eluate containing the target component is separated by the second chromatography unit and collected in the second collection rack ( Figure 39 ).

[0195] Of course, it is also possible to go a step further and inject the sample again from the second collection rack of the second channel through the third injection needle of the third channel, and after separation by the third chromatography unit, collect the eluate containing the target component into the third collection rack, thereby realizing the series connection from the first channel to the third channel ( Figure 40 ).

[0196] In addition, in another preferred embodiment, cross-channel injection can be performed multiple times on the eluate containing the target component collected in one channel. For example, after the eluate containing the target component in the 1st to 3rd collection tubes in the first collection rack in the first channel is injected twice through the second injection, the eluate containing the target component in the 4th to 6th collection tubes in the first collection rack in the first channel can be injected again through the third injection of the third channel.

[0197] The above examples are merely illustrative and do not have any specific limiting effect on the scope of protection of the present invention.

[0198] The present invention realizes cross-channel secondary injection by utilizing the characteristic that the injection needle can be moved in the Y direction, thereby being able to realize complex multidimensional chromatographic separation without increasing the complexity of the flow path.

[0199] The above are only preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may be subject to various modifications and variations.

Claims

1. A multi-channel liquid chromatography separation device, characterized in that: It includes a reagent holding module, multiple independent liquid channels and a control module. The reagent holding module is used to hold a variety of reagents including samples, carrier liquids, and stabilizers. Each of the independent liquid channels includes a sampling unit, a fluid delivery unit, a chromatography unit, a detection unit, and a collection unit according to the flow direction of the liquid channel. The control module is used to control each unit in each independent liquid channel to realize automatic processing; The sampling unit includes a sampling needle for injecting the sample or reagent contained in the reagent containing module; The fluid delivery unit is used to deliver the mobile phase to the chromatography unit; The chromatography unit includes a chromatography column for separating components contained in the sample; The detection unit is used to detect components in the sample separated from the chromatography column; The collecting unit is used to collect the target components in the sample separated by the chromatography unit; The multi-channel liquid chromatography separation device has a first working mode and a second working mode, wherein, in the first working mode, each of the independent liquid channels can separate samples in parallel; in the second working mode, the injection needle of an independent liquid channel among the multiple independent liquid channels can perform a secondary injection on the eluate containing the target component collected by the collection unit in another independent liquid channel, thereby realizing serial separation processing of multiple independent liquid channels.

2. The multi-channel liquid chromatography separation device according to claim 1, characterized in that: The collecting unit comprises a collecting rack, on which are provided a plurality of collecting tubes arranged in the longitudinal direction of the collecting rack. The collection racks of the independent liquid channels are arranged in a direction perpendicular to the length direction of the collection racks. The injection needle can move in the arrangement direction of the collection rack; When the multi-channel liquid chromatography separation device is in the first operating mode, the injection needle of each independent liquid channel is aligned with the corresponding collection rack in the length direction of the collection rack; When the multi-channel liquid chromatography separation device is in the second operating mode, the injection needle of one of the multiple independent liquid channels moves in the arrangement direction of the collection rack to be aligned with the collection rack of another independent liquid channel in the length direction of the collection rack.

3. The multi-channel liquid chromatography separation device according to claim 2, characterized in that: The type of the chromatography column corresponding to the one independent liquid channel is the same as or different from the type of the chromatography column corresponding to the other independent liquid channel.

4. The multi-channel liquid chromatography separation device according to claim 2, characterized in that: The number of chromatography columns corresponding to the one independent liquid channel is the same as or different from the number of chromatography columns corresponding to the other independent liquid channel.

5. The multi-channel liquid chromatography separation device according to any one of claims 2, characterized in that: The chromatography unit in each of the independent fluid channels includes one or more chromatography columns.

6. The multi-channel liquid chromatography separation device according to any one of claims 1 to 5, characterized in that: The eluate containing the target component collected by the collecting unit of the one independent liquid channel after the chromatography treatment by the chromatography unit of the one independent liquid channel among the multiple independent liquid channels after the secondary injection can also be injected again.

7. The multi-channel liquid chromatography separation device according to any one of claims 1 to 5, characterized in that: Before performing the second injection, a stabilizer may be applied to the eluate containing the target component collected by the collection unit through injection.

8. The multi-channel liquid chromatography separation device according to claim 6, characterized in that: The liquid channel for re-injection is the one independent liquid channel.

9. The multi-channel liquid chromatography separation device according to claim 8, characterized in that: The liquid channel for re-injection is another independent liquid channel different from the one independent liquid channel and the other independent liquid channel.

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