Double-column circulating chromatography system and use method thereof

Abstract

The invention discloses a double-column circulating chromatographic system which comprises an isocratic elution device, a gradient elution device, a pre-column valve, a chromatographic column device, a post-column valve, a detector device, a waste liquid tank, a fraction collection device, a reflux device and a connecting pipeline, the isocratic elution device and the gradient elution device are connected with the pre-column valve, the chromatographic column device and the post-column valve through connecting pipelines after being connected in parallel, the post-column valve is connected with the detector device and the waste liquid tank which are arranged in parallel through connecting pipelines, and the detector device is connected with the fraction collector through a connecting pipeline. The method has the beneficial effects that (1) not only can the purification efficiency and yield be improved, but also stable peak appearance time and a highly consistent purification spectrum can be obtained, so that stable impurity spectrum distribution is obtained, and continuous automatic production is realized; and (2) multi-time multi-system automatic circulation purification of the same system is realized, the purification efficiency is remarkably improved, human intervention and operation errors are reduced, the pollution risk is reduced, and industrialization of continuous flow purification is realized.

Description

Technical Field

[0001] The present invention relates to a chromatographic system, and particularly to a dual-column circulating chromatographic system and a method for using the same. Background Art

[0002] As the core means of drug analysis, separation and purification at present, chromatographic separation technology has significant advantages in separation efficiency and selectivity. However, due to factors such as low sample loading capacity of the chromatographic column and long operation cycle, traditional chromatographic systems generally have a production capacity bottleneck and are only applied to high-value-added products. Currently, the single-cycle chromatographic systems widely used in the market usually adopt a single chromatographic column to perform an intermittent operation process of "equilibration - sample loading - elution - regeneration". This operation mode has two significant technical defects: on the one hand, it cannot achieve automatic continuous operation, resulting in difficult improvement of production efficiency; on the other hand, due to only being equipped with one chromatographic column, its purification efficiency is relatively low, making it difficult to meet the increasingly strict drug quality requirements and the actual needs of continuously reducing production costs. In order to overcome these technical bottlenecks, some multi-column chromatographic circulation systems have also emerged one after another. CN202411504307.5 discloses an automated chromatographic system with multiple columns in parallel. By arranging multiple chromatographic columns in parallel and sharing a set of mobile phase device, detector device and fraction collector device, this invention successfully realizes the simultaneous operation of multiple chromatographic columns. However, this chromatographic system still has certain limitations. It cannot achieve multiple purifications of samples, which to a certain extent affects the purification efficiency. In addition, the tailings cannot be directly recycled, which also has a certain negative impact on the yield. Patent CN201310087690.4 discloses a dual-column circulating chromatographic system. Through pipeline design, this system realizes multiple adsorption and desorption of unseparated components to improve the purification effect. However, this system uses multiple sets of the same equipment, such as detection equipment, resulting in high manufacturing costs. At the same time, this system fails to effectively solve the problems of unstable peak emergence time and inconsistent peak shapes in the preparation chromatogram, which to a certain extent limits its application in actual production. Summary of the Invention

[0003] In view of this, in order to overcome the deficiencies of the prior art, the present invention provides a dual-column circulating chromatography system, which is configured with two or more chromatographic columns arranged in parallel, and all chromatographic columns share a set of core components such as an elution device, a detection device, a fraction collection device, and a reflux device. This system enables the sample to achieve multiple purification operations within the same system. On the one hand, it effectively avoids potential risks such as environmental pollution and air oxidation of the sample after it flows out of the chromatographic column, ensuring the purity and quality stability of the sample; on the other hand, it greatly reduces the labor intensity of production personnel, shortens the time required for separation and purification, and significantly improves production efficiency. The present invention also has the function of recycling and purifying tailings. This system can eliminate problems such as time loss, solvent waste, and increased labor costs caused by secondary treatment of tailings. Moreover, recycling and purifying tailings can significantly improve the yield and productivity of a single purification, further improving production benefits. In addition, this system realizes the functions of automatic operation and automatic sample collection, and can intelligently combine and collect qualified parts. This automated operation not only improves the accuracy and reliability of production, but also reduces the analysis and detection costs; at the same time, the highly integrated design of the system effectively reduces the occupancy of plant space by equipment and reduces the overall operating costs.

[0004] To achieve the above object, the present invention provides a solution: a dual-column circulating chromatography system, including an isocratic elution device, a gradient elution device, a pre-column valve 6, a chromatographic column device, a post-column valve 8, a detector device, a waste liquid tank 10, a fraction collection device, a reflux device, and connecting pipelines; the isocratic elution device includes at least 3 isocratic liquid storage tanks 1 arranged in parallel, a multi-way solvent selection valve 2, and an isocratic chromatographic pump 3. The isocratic liquid storage tanks 1 are connected to the multi-way solvent selection valve 2 → isocratic chromatographic pump 3 through connecting pipelines; the chromatographic column device includes 2 chromatographic columns 7 arranged in parallel; the detector device includes at least 1 detector 9; the fraction collection device includes 1 multi-channel fraction collector 11, at least 2 fraction collection tanks 12, and 1 waste collection tank 13; the reflux device includes at least 1 reflux pipeline 14 and at least 1 flow path control valve 15;

[0005] Among them, after the isocratic elution device and the gradient elution device are connected in parallel, they are connected to the pre-column valve 6 → chromatographic column device → post-column valve 8 through connecting pipelines. The post-column valve 8 is respectively connected to the detector device and the waste liquid tank 10 through connecting pipelines. The detector device is connected to the fraction collector 11 through a connecting pipeline. The fraction collector (11) is respectively connected to the fraction collection tank 12 and the waste collection tank 13 through connecting pipelines. One end of the reflux pipeline 14 is connected between the multi-channel fraction collector and one of the fraction collection tanks 12 through a flow path control valve 15. The other end of the reflux pipeline 14 is connected between the pre-column valve 6 and the chromatographic column device through a flow path control valve 15, or the other end of the reflux pipeline 14 is directly connected between the pre-column valve 6 and the chromatographic column device.

[0006] The present invention provides another solution: a dual-column recycling chromatography system, comprising an isocratic elution device, a gradient elution device, a pre-column valve 6, a chromatographic column device, a post-column valve 8, a detector device, a waste liquid tank 10, a fraction collection device, a reflux device and connecting pipelines; the isocratic elution device includes at least three isocratic liquid storage tanks 1 arranged in parallel, a multi-way solvent selection valve 2, and an isocratic chromatographic pump 3, and the isocratic liquid storage tanks 1 are connected to the multi-way solvent selection valve 2 → the isocratic chromatographic pump 3 through connecting pipelines; the chromatographic column device includes two chromatographic columns 7 arranged in parallel; the detector device includes at least one detector 9; the fraction collection device includes a multi-channel fraction collector 11, at least two fraction collection tanks 12 and a waste collection tank 13; the reflux device includes at least one reflux pipeline 14 and at least one flow path control valve 15, and at least one of the flow path control valves 15 is a two-position six-way valve;

[0007] Among them, after the isocratic elution device and the gradient elution device are connected in parallel, they are connected to the pre-column valve 6 → the chromatographic column device → the post-column valve 8 through connecting pipelines. The post-column valve 8 is respectively connected to the detector device and the waste liquid tank 10 through connecting pipelines. The detector device is connected to the fraction collector 11 through a connecting pipeline. The fraction collector 11 is respectively connected to at least one fraction collection tank 12 and the waste collection tank 13 through connecting pipelines. At the same time, the fraction collector 11 is connected to the flow path control valve 15 through a connecting pipeline and then respectively connected to at least one fraction collection tank 12 and one end of the reflux pipeline 14. The other end of the reflux pipeline 14 is connected between the pre-column valve 6 and the chromatographic column device through the flow path control valve 15.

[0008] The present invention provides a third solution: a dual-column recycling chromatography system, comprising an isocratic elution device, a gradient elution device, a pre-column valve 6, a chromatographic column device, a post-column valve 8, a detector device, a waste liquid tank 10, a fraction collection device, a reflux device and connecting pipelines; the isocratic elution device includes at least three isocratic liquid storage tanks 1 arranged in parallel, a multi-way solvent selection valve 2, and an isocratic chromatographic pump 3, and the isocratic liquid storage tanks 1 are connected to the multi-way solvent selection valve 2 → the isocratic chromatographic pump 3 through connecting pipelines; the chromatographic column device includes two chromatographic columns 7 arranged in parallel; the detector device includes at least one detector 9; the fraction collection device includes a multi-channel fraction collector 11, at least one fraction collection tank 12 and a waste collection tank 13; the reflux device includes at least one reflux pipeline 14 and at least one flow path control valve 15;

[0009] Among them, after the isocratic elution device and the gradient elution device are connected in parallel, they are connected to the pre-column valve 6 → chromatographic column device → post-column valve 8 through a connecting pipeline. The post-column valve 8 is respectively connected to the detector device and the waste liquid tank 10 through a connecting pipeline. The detector device is connected to the fraction collector 11 through a connecting pipeline. The fraction collector 11 is respectively connected to the fraction collection tank 12 and the waste collection tank 13 through a connecting pipeline. One end of the reflux pipeline 14 is connected to the fraction collector 11, and the other end of the reflux pipeline 14 is connected between the pre-column valve 6 and the chromatographic column device through a flow path control valve 15.

[0010] The isocratic elution device is generally used for four operations: equilibration, sample loading, column washing, and dilution, and can have four isocratic infusion tanks; according to the different properties of the sample, the equilibration solution can also be used for dilution, or only three isocratic infusion tanks can be used.

[0011] The parallel chromatographic columns can use the same or different chromatographic columns. When the front and rear peak tailings are recycled and reused, the same chromatographic column is used. When the sample is purified multiple times, the same or different chromatographic columns can be used.

[0012] The function of the flow path control valve 15 is to control the fraction to flow into the reflux pipeline and flow into the chromatograph through the reflux pipeline, and at the same time, it can prevent the fraction from flowing back, avoiding problems such as diffusion.

[0013] According to a preferred embodiment, the gradient elution device includes 2 groups of gradient storage tanks 4 and gradient chromatographic pumps 5, and the number of gradient storage tanks in each group = 1. Each group of gradient storage tanks 4 and gradient chromatographic pumps 5 are connected in series through a connecting pipeline as a gradient elution unit, and 2 groups of gradient elution units are connected in parallel to form a gradient elution device.

[0014] According to another preferred embodiment, the gradient elution device includes 2 groups of gradient storage tanks 4, multi-channel solvent selection valves 16, and gradient chromatographic pumps 5, and the number of gradient storage tanks in each group > 1. Each group of gradient storage tanks 4, multi-channel solvent selection valves 16, and gradient chromatographic pumps 5 are connected in series in sequence through a connecting pipeline to form a gradient elution unit, and 2 groups of gradient elution units are connected in parallel to form a gradient elution device.

[0015] According to the third preferred embodiment, the gradient elution device includes 2 groups of gradient storage tanks 4, proportioning valves 17, and also includes 1 gradient chromatographic pump 5, and the number of gradient storage tanks in each group = 1. Each group of gradient storage tanks 4 and proportioning valves 17 are connected in series through a connecting pipeline as a gradient elution unit. After 2 groups of gradient elution units are connected in parallel, they are then connected to the gradient chromatographic pump 5 through a connecting pipeline to form a gradient elution device.

[0016] According to the fourth preferred embodiment, the gradient elution device includes two groups of gradient reservoirs 4, a multi-channel solvent selection valve 16, a proportional valve 17, and also includes one gradient chromatography pump 5. And for each group of gradient reservoirs, the number is greater than 1. Each group of gradient reservoirs 4, the multi-channel solvent selection valve 16, and the proportional valve 17 are connected in series in sequence through connecting pipes as a gradient elution unit. After the two groups of gradient elution units are connected in parallel, they are then connected to the gradient chromatography pump 5 through connecting pipes to form a gradient elution device.

[0017] Among them, the number of the two groups of gradient reservoirs can be the same or different.

[0018] Preferably, the flow path control valve 15 is selected from one or more of a stop valve, a three-way valve, a two-position six-way valve, and a check valve. The more integrated channels there are in valves such as a three-way valve and a two-position six-way valve, the fewer control points there are. The use of a check valve can not only reduce the dead volume but also reduce the automatic control points.

[0019] Preferably, the chromatographic column 7 is selected from a normal-phase chromatographic column, a reversed-phase chromatographic column, an ion-exchange column, and a gel chromatographic column.

[0020] Preferably, the detector 9 is selected from an ultraviolet detector, a conductivity detector, a diode array detector, an evaporative light scattering detector, a mass spectrometry detector, and a Raman detector.

[0021] In addition, the present invention provides a method for operating the dual-column circulating chromatography system described in the present invention for a purification process. The purification process includes at least two different stages (A, B): at least one stage A, one end of the reflux pipe 14 is connected to the multi-channel fraction collector 11, and the other end of the reflux pipe 14 is connected to the chromatographic column device 7 so that the fractions are re-purified; and at least one stage B, the reflux pipe 14 is not connected to the multi-channel fraction collector 11 to collect the fractions. Wherein the operation method includes at least the following steps: Ⅰ. Conduct a sample test before the purification process to determine the purity of the fractions at different time periods; Ⅱ. Switch different stages of the purification process through the running time and / or absorption value.

[0022] The parallel chromatographic columns adopt the same operation steps, use exactly the same elution device, detection device, fraction receiving device, reflux device, and equal-length connecting pipelines, which can significantly improve the reproducibility of the purification results. Specifically, such a highly identical configuration not only ensures the stability of the peak emergence time between the parallel chromatographic columns but also effectively solves the high consistency of the peak shapes. On this basis, by accurately switching different stages according to the time parameters and / or absorption value, the error can be controlled within an extremely small range.

[0023] The technical solutions described in the present invention show a series of significant advantages compared with the prior art, specifically reflected in:

[0024] (1) The reuse of tailings can not only improve the purification efficiency and yield, but also ensure a stable peak elution time and highly consistent purification chromatograms, thereby obtaining a stable impurity spectrum distribution. When operating automatically, it can ensure reliable product quality and achieve continuous automated production; (2) Realize multiple automatic cyclic purifications of multiple systems in the same system, significantly improving the purification efficiency: reducing human intervention and operation errors, reducing the risk of contamination, and realizing the industrialization of continuous flow purification. Description of the Drawings

[0025] Figure 1 It is a schematic structural diagram of the dual-column cyclic chromatography system described in Example 1;

[0026] Figure 2 It is a schematic structural diagram of the dual-column cyclic chromatography system described in Example 2;

[0027] Figure 3 It is a schematic structural diagram of the dual-column cyclic chromatography system described in Example 3;

[0028] Figure 4 It is a schematic structural diagram of the dual-column cyclic chromatography system described in Example 4;

[0029] Figure 5 It is a schematic structural diagram of the dual-column cyclic chromatography system described in Examples 5 and 6 when the two-position six-way valve is in state ⅰ;

[0030] Figure 6 It is a schematic structural diagram of the dual-column cyclic chromatography system described in Examples 5 and 6 when the two-position six-way valve is in state ⅱ;

[0031] Figure 7 It is an enlarged view of the dual-column cyclic chromatography system described in Examples 5 and 6 when the flow path control valve 15-2 is in state ⅰ;

[0032] Figure 8 It is an enlarged view of the dual-column cyclic chromatography system described in Examples 5 and 6 when the flow path control valve 15-1 is in state ⅰ;

[0033] Figure 9 It is an enlarged view of the dual-column cyclic chromatography system described in Examples 5 and 6 when the flow path control valve 15-2 is in state ⅱ;

[0034] Figure 10 It is an enlarged view of the dual-column cyclic chromatography system described in Examples 5 and 6 when the flow path control valve 15-1 is in state ⅱ;

[0035] Description of reference numerals in the figure: 1-1, the first isocratic liquid storage tank; 1-2, the second isocratic liquid storage tank; 1-3, the third isocratic liquid storage tank; 1-4, the fourth isocratic liquid storage tank; 2, the first multi-way solvent selection valve; 3, the isocratic chromatography pump; 4-1, the first gradient liquid storage tank; 4-2, the second gradient liquid storage tank; 4-3, the third gradient liquid storage tank; 4-4, the fourth gradient liquid storage tank; 4-5, the fifth gradient liquid storage tank; 5-1, the first gradient chromatography pump; 5-2, the second gradient chromatography pump; 6, the pre-column valve; 7-1, the first chromatographic column; 7-2, the second chromatographic column; 8, the post-column valve; 9, the detector; 10, the waste liquid tank; 11, the multi-channel fraction collector; 12-1, the first fraction collection tank; 12-2, the second fraction collection tank; 12-3, the third fraction collection tank; 13, the waste liquid collection tank; 14, the reflux pipeline; 15-1, the first flow path control valve; 15-2, the second flow path control valve; 15-3, the third flow path control valve; 15-4, the fourth flow path control valve; 15-5, the fifth flow path control valve; 15-6, the sixth flow path control valve; 16, the second multi-channel solvent selection valve; 17-1, the first proportional valve; 17-2, the second proportional valve; 1#, 2#, 3#, 4#, 5#, 6# are the connection holes on the two-position six-way valve; 11-1, 11-2, 11-3, 11-4 are the outlets of the multi-channel fraction collector. Detailed implementation manners

[0036] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention. For example, replacing the types and quantities of valves to achieve the same effects are all within the scope.

[0037] Embodiment 1

[0038] As Figure 1 shown, a double-column system includes the first isocratic liquid storage tank 1-1, the second isocratic liquid storage tank 1-2, the third isocratic liquid storage tank 1-3, the first multi-way solvent selection valve 2, the isocratic chromatography pump 3, the first gradient liquid storage tank 4-1, the second gradient liquid storage tank 4-2, the first gradient chromatography pump 5-1, the second gradient chromatography pump 5-2, the pre-column valve 6, the first chromatographic column 7-1, the second chromatographic column 7-2, the post-column valve 8, the detector 9, the waste liquid tank 10, the multi-channel fraction collector 11, the first fraction collection tank 12-1, the second fraction collection tank 12-2, the waste liquid collection tank 13, the reflux pipeline 14, the first flow path control valve 15-1, and the second flow path control valve 15-2.

[0039] The first isocratic liquid storage tank 1-1, the second isocratic liquid storage tank 1-2, and the third isocratic liquid storage tank 1-3 are respectively connected to the multi-way solvent selection valve 2 → isocratic chromatographic pump 3 → pre-column valve 6 through connecting pipelines; the first gradient liquid storage tank 4-1 and the first gradient chromatographic pump 5-1 are connected in series as the first gradient elution unit, the second gradient liquid storage tank 4-2 and the second gradient chromatographic pump 5-2 are connected in series as the second gradient elution unit, the first gradient elution unit and the second gradient elution unit are connected in parallel to form a gradient elution device, and the gradient elution device is connected to the pre-column valve 6 through a connecting pipeline; the pre-column valve 6 is connected to the chromatographic columns 7-1 and 7-2 → post-column valve 8 arranged in parallel through a connecting pipeline; the post-column valve 8 is respectively connected to the detector 9 and the waste liquid tank 10 through connecting pipelines; the detector 9 is connected to the multi-channel fraction collector 11 through a connecting pipeline, and the outlets 11-1, 11-2, and 11-3 of the multi-channel fraction collector 11 are respectively connected to the first fraction collection tank 12-1, the second fraction collection tank 12-2, and the waste liquid collection tank 13 through connecting pipelines; one end of the reflux pipeline 14 is connected between the multi-channel fraction collector and the first fraction collection tank 12-1 through the first flow path control valve 15-1, and the other end of the reflux pipeline 14 is connected between the pre-column valve and the first chromatographic column 7-1.

[0040] Among them: the first isocratic liquid storage tank 1-1, the second isocratic liquid storage tank 1-2, and the third isocratic liquid storage tank 1-3 are respectively used to store the equilibration or dilution solution, the column washing solution, and the sample solution; the first multi-way solvent selection valve 2 is a three-channel solvent selection valve and is used to select and transport different solutions; the isocratic chromatographic pump 3 is used for solution transportation; the first gradient liquid storage tank 4-1 and the second gradient liquid storage tank 4-2 are used to store the gradient elution solution; the first gradient chromatographic pump 5-1 and the second gradient chromatographic pump 5-2 are used to transport the elution solution, and the two cooperate to form a gradient program; the pre-column valve 6 and the post-column valve 8 are used to select different liquid flow directions; the detector 9 is used to monitor the absorption value; the waste liquid tank 10 is used to receive the waste liquid during the processes of equilibration, sample loading, dilution, column washing, etc.; the multi-channel fraction collector 11 is used to select the fractions at different time periods to enter different collection tanks or waste collection tanks; the first fraction collection tank 12-1 is used to receive the effluent from the second chromatographic column 7-2, and the second fraction collection tank 12-2 is used to receive the qualified fractions flowing out of the first chromatographic column 7-1; the waste collection tank 13 is used to receive the fractions during testing and the unqualified waste fractions during automatic operation; the reflux pipeline 14 is used for fraction reflux; the first flow path control valve 15-1 is used to select whether the fraction is connected to the first fraction collection tank 12-1 or the reflux pipeline 14.

[0041] Status description:

[0042] When the status of the pre-column valve 6 is a, the isocratic chromatographic pump 3 is connected to the first chromatographic column 7-1 through the pre-column valve 6, and at the same time, the first gradient chromatographic pump 5-1 and the second gradient chromatographic pump 5-2 connected in parallel are connected to the second chromatographic column 7-2 through the pre-column valve 6;

[0043] When the state of the pre-column valve 6 is b, the isocratic chromatography pump 3 is connected to the second chromatographic column 7-2 through the pre-column valve 6. At the same time, the parallel first gradient chromatography pump 5-1 and the second gradient chromatography pump 5-2 are connected to the first chromatographic column 7-1 through the pre-column valve 6;

[0044] When the state of the post-column valve 8 is a, the first chromatographic column 7-1 is connected to the waste liquid tank 10 through the post-column valve, and at the same time, the second chromatographic column 7-2 is connected to the detector 9 through the post-column valve;

[0045] When the state of the post-column valve 8 is b, the first chromatographic column 7-1 is connected to the detector 9 through the post-column valve, and at the same time, the second chromatographic column 7-2 is connected to the waste liquid tank 10 through the post-column valve.

[0046] The initial states of the pre-column valve and the post-column valve are both a.

[0047] Before the fractions flow out during the target time period, the multi-channel fraction collector 11 is connected to the waste collection tank 13.

[0048] The working process of this chromatographic column system includes the following steps:

[0049] 1. Fraction testing of different chromatographic columns at different time periods

[0050] Using the traditional single-column mode, the second chromatographic column 7-2 is balanced, loaded, and eluted. The fractions are collected, and the time period that meets the requirements of secondary purification is detected and determined. After the target fractions in this time period are combined, they are loaded onto the first chromatographic column 7-1, eluted, and the time period when the qualified fractions flow out from the first chromatographic column 7-1 is detected and determined.

[0051] 2. Equilibration of the second chromatographic column 7-2

[0052] The first multi-way solvent selection valve 2 is connected to the first isocratic liquid storage tank 1-1. The states of the pre-column valve 6 and the post-column valve 8 are both b, and the isocratic elution device is operated to equilibrate the second chromatographic column 7-2;

[0053] 3. Loading of the second chromatographic column 7-2

[0054] The first multi-way solvent selection valve 2 is connected to the third isocratic liquid storage tank 1-3. The states of the pre-column valve 6 and the post-column valve 8 are both b, and the isocratic elution device is operated to load the second chromatographic column 7-2;

[0055] 4. Elution of the second chromatographic column 7-2; equilibration and dilution loading of the first chromatographic column 7-1

[0056] The states of the pre-column valve 6 and the post-column valve 8 are both a. The gradient elution device is operated to perform gradient elution on the second chromatographic column 7-2. Before the sample flows out during the target time period, the fractions of the second chromatographic column 7-2 pass through the detector 9 → the multi-channel fraction collector 11 → the waste collection tank 13, and before the fractions flow out during the target time period, the first chromatographic column 7-1 is balanced. The first multi-way solvent selection valve 2 is used to connect the first isocratic liquid storage tank 1-1, and the isocratic elution device is operated to balance the first chromatographic column 7-1. When the fractions of the second chromatographic column 7-2 flow out during the target time period, the multi-channel fraction collector 11 is switched to the outlet 11-1, and the first flow path control valve 15-1 is switched so that the fractions during this time period flow into the first chromatographic column 7-1 through the reflux pipeline 14. At the same time, the first multi-way solvent selection valve 2 is used to connect the first isocratic liquid storage tank 1-1, and the isocratic elution device is operated to dilute the sample flowing into the first chromatographic column 7-1.

[0057] 5. Elution of the first chromatographic column 7-1; Washing, balancing, and sample loading of the second chromatographic column 7-2

[0058] The states of the pre-column valve 6 and the post-column valve 8 are both b. The gradient elution device is operated to perform gradient elution on the first chromatographic column 7-1. Before the fractions flow out during the target time period, the fractions of the first chromatographic column 7-1 pass through the detector 9 → the multi-channel fraction collector 11 and are received into the waste collection tank 13. When the fractions flow out during the target time period, the multi-channel fraction collector 11 is switched to the second fraction collection tank 12-2 to collect the fractions. At the same time, the first multi-way solvent selection valve 2 is used to connect the second isocratic liquid storage tank 1-2, the first isocratic liquid storage tank 1-1, and the third isocratic liquid storage tank 1-3 in sequence according to the requirements of washing, balancing, and sample loading time, and the isocratic elution device is operated to wash, balance, and load the sample on the second chromatographic column 7-2.

[0059] 6. Elution of the second chromatographic column 7-2; Washing, balancing, dilution, and sample loading of the first chromatographic column 7-1

[0060] The states of the pre-column valve 6 and the post-column valve 8 are both a. The first gradient chromatographic pump 5-1 and the second gradient chromatographic pump 5-2 are operated to perform gradient elution on the second chromatographic column 7-2. Before the fractions flow out during the target time period, the fractions of the second chromatographic column 7-2 pass through the detector 9 → the multi-channel fraction collector 11 and are received into the waste collection tank 13. At the same time, according to the requirements of washing and balancing time, the second isocratic liquid storage tank 1-2 and the first isocratic liquid storage tank 1-1 are connected in sequence through the first multi-way solvent selection valve 2, and the isocratic elution device is operated to wash and balance the first chromatographic column 7-1. When the sample flows out during the target time period, the multi-channel fraction collector 11 is switched to the outlet 11-1, and the first flow path control valve 15-1 is switched so that the sample during this time period flows into the first chromatographic column 7-1. At the same time, the first multi-way solvent selection valve 2 is used to connect the first isocratic liquid storage tank 1-1, and the isocratic elution device is operated to dilute the sample flowing into the first chromatographic column 7-1.

[0061] 7. Cycle

[0062] Repeat the above steps 5 and 6 to continuously purify the sample so that it undergoes two purifications to obtain a fraction meeting the purity requirements.

[0063] Example 2

[0064] As Figure 2 shown, a dual-column circulating chromatography system includes a first isocratic liquid storage tank 1-1, a second isocratic liquid storage tank 1-2, a third isocratic liquid storage tank 1-3, a fourth isocratic liquid storage tank 1-4, a first multi-way solvent selection valve 2, an isocratic chromatography pump 3, a first gradient liquid storage tank 4-1, a second gradient liquid storage tank 4-2, a third gradient liquid storage tank 4-3, a fourth gradient liquid storage tank 4-4, a first gradient chromatography pump 5-1, a second gradient chromatography pump 5-2, a pre-column valve 6, a first chromatographic column 7-1, a second chromatographic column 7-2, a post-column valve 8, a detector 9, a waste liquid tank 10, a multi-channel fraction collector 11, a first fraction collection tank 12-1, a second fraction collection tank 12-2, a waste liquid collection tank 13, a reflux pipeline 14, a first flow path control valve 15-1, a second multi-way solvent selection valve 16-1, and a third multi-way solvent selection valve 16-2.

[0065] The first isocratic liquid storage tank 1-1, the second isocratic liquid storage tank 1-2, the third isocratic liquid storage tank 1-3, and the fourth isocratic liquid storage tank 1-4 are connected to the convenient multi-way solvent selection valve 2 → isocratic chromatography pump 3 → pre-column valve 6 through connecting pipelines; the first gradient liquid storage tank 4-1 and the second gradient liquid storage tank 4-2 are respectively connected to the second multi-way solvent selection valve 16-1 → first gradient chromatography pump 5-1 through connecting pipelines as the first gradient elution unit; the third gradient liquid storage tank 4-3 and the fourth gradient liquid storage tank 4-4 are respectively connected to the third multi-way solvent selection valve 16-2 → second gradient chromatography pump 5-2 through connecting pipelines as the second gradient elution unit; the first gradient elution unit and the second gradient elution unit are connected in parallel to form a gradient elution device, and the gradient elution device is connected to the pre-column valve 6 through a connecting pipeline; the pre-column valve 6 is connected to the chromatographic columns 7-1 and 7-2 → post-column valve 8 arranged in parallel through a connecting pipeline; the post-column valve 8 is respectively connected to the detector 9 and the waste liquid tank 10 through a connecting pipeline; the detector 9 is connected to the multi-channel fraction collector 11 through a connecting pipeline, and the outlets 11-1, 11-2, and 11-3 of the multi-channel fraction collector 11 are respectively connected to one end of the reflux pipeline 14, the first fraction collection tank 12-1, and the waste liquid collection tank 13; the other end of the reflux pipeline 14 is connected between the pre-column valve 6 and the first chromatographic column 7-1 through the first flow path control valve 15-1.

[0066] Among them: the first isocratic liquid storage tank 1-1, the second isocratic liquid storage tank 1-2, the third isocratic liquid storage tank 1-3, and the fourth isocratic liquid storage tank 1-4 are respectively used to store balance, dilution solution, column washing solution, and sample solution; the first multi-way solvent selection valve 2 is a four-channel solvent selection valve, used to select and transport different solutions; the isocratic chromatography pump 3 is used for solution transportation; the first gradient liquid storage tank 4-1, the second gradient liquid storage tank 4-2, the third gradient liquid storage tank 4-3, and the fourth gradient liquid storage tank 4-4 are used to store gradient elution solutions; the second multi-way solvent selection valve 16-1 and the third multi-way solvent selection valve 16-2 are two-channel solvent selection valves, used to select and transport different solutions; the first gradient chromatography pump 5-1 and the second gradient chromatography pump 5-2 are used to transport elution solutions, and the two cooperate to form a gradient program; the pre-column valve 6 and the post-column valve 8 are used to select different liquid flow directions; the detector 9 is used to detect the absorbance of the sample; the waste liquid tank 10 is used to receive waste liquid during processes such as balance, sample loading, dilution, and column washing; the multi-channel fraction collector 11 is used to select fractions at different time periods to enter different fraction collection tanks or waste collection tanks; the first fraction collection tank 12-1 is used to receive qualified fractions; the waste collection tank 13 is used to receive fractions during testing and unqualified waste fractions during automatic operation; the reflux pipeline 14 is used for fraction reflux; the first flow path control valve 15-1 is a one-way valve used to inject the target time period of the second chromatographic column 7-2 into the first chromatographic column 7-1 and prevent the liquid from flowing back into the reflux pipeline 14.

[0067] Status description:

[0068] When the pre-column valve 6 is in state a, the isocratic chromatography pump 3 is connected to the first chromatographic column 7-1 through the pre-column valve 6, and at the same time, the parallel first gradient chromatography pump 5-1 and second gradient chromatography pump 5-2 are connected to the second chromatographic column 7-2 through the pre-column valve 6;

[0069] When the pre-column valve 6 is in state b, the isocratic chromatography pump 3 is connected to the second chromatographic column 7-2 through the pre-column valve 6, and at the same time, the parallel first gradient chromatography pump 5-1 and second gradient chromatography pump 5-2 are connected to the first chromatographic column 7-1 through the pre-column valve 6;

[0070] When the post-column valve 8 is in state a, the first chromatographic column 7-1 is connected to the waste liquid tank 10 through the post-column valve, and at the same time, the second chromatographic column 7-2 is connected to the detector 9 through the post-column valve;

[0071] When the post-column valve 8 is in state b, the first chromatographic column 7-1 is connected to the detector 9 through the post-column valve, and at the same time, the second chromatographic column 7-2 is connected to the waste liquid tank 10 through the post-column valve.

[0072] The initial states of the pre-column valve and the post-column valve are both a.

[0073] Before the fractions flow out during the target time period, the multi-channel fraction collector 11 is connected to the waste collection tank 13.

[0074] The working process of this chromatographic column system includes the following steps:

[0075] 1. Fraction testing at different time periods

[0076] Using the traditional single-column mode, balance, load samples, and elute the second chromatographic column 7-2, collect fractions, detect and determine the time period that meets the requirements of secondary purification. After merging the target fractions in this time period, load them onto the first chromatographic column 7-1, elute, and detect and determine the time period when the qualified fractions flow out from the first chromatographic column 7-1.

[0077] 2. Balancing of the second chromatographic column 7-2

[0078] The first multi-way solvent selection valve 2 is connected to the first isocratic liquid storage tank 1-1, and the states of the pre-column valve 6 and the post-column valve 8 are both b. Operate the isocratic elution device to balance the second chromatographic column 7-2;

[0079] 3. Loading samples onto the second chromatographic column 7-2

[0080] The first multi-way solvent selection valve 2 is connected to the fourth isocratic liquid storage tank 1-4, and the states of the pre-column valve 6 and the post-column valve 8 are both b. Operate the isocratic elution device to load samples onto the second chromatographic column 7-2;

[0081] 4. Elution of the second chromatographic column 7-2; balancing and diluting the sample loading of the first chromatographic column 7-1

[0082] The states of the pre-column valve 6 and the post-column valve 8 are both a. Switch the second multi-way solvent selection valve 16-1 to be connected to the first gradient liquid storage tank 4-1, and the third multi-way solvent selection valve 16-2 to be connected to the third gradient liquid storage tank 4-3. Operate the gradient elution device to perform gradient elution on the second chromatographic column 7-2. Before the fractions in the target time period flow out, the fractions of the second chromatographic column 7-2 pass through the detector 9 → the outlet 11-3 of the multi-channel fraction collector 11 → the waste collection tank 13. And before the fractions in the target time period flow out, use the first multi-way solvent selection valve 2 to connect to the first isocratic liquid storage tank 1-1, and operate the isocratic elution device to balance the first chromatographic column 7-1 to complete the balance procedure of the first chromatographic column 7-1. When the fractions in the target time period of the second chromatographic column 7-2 flow out, switch the outlet of the multi-channel fraction collector to 11-1, so that the fractions in this time period pass through the reflux pipeline 14 and the first flow path control valve 15-1 and flow into the first chromatographic column 7-1. At the same time, use the first multi-way solvent selection valve 2 to connect to the second isocratic liquid storage tank 1-2, and operate the isocratic elution device to dilute the sample flowing into the first chromatographic column 7-1.

[0083] 5. Elution of the first chromatographic column 7-1; washing, balancing, and loading samples onto the second chromatographic column 7-2

[0084] The states of the pre-column valve 6 and the post-column valve 8 are both b. Switch the second multi-way solvent selection valve 16-1 to the second gradient liquid storage tank 4-2, and the third multi-way solvent selection valve 16-2 to the fourth gradient liquid storage tank 4-4. Operate the gradient elution device to perform gradient elution on the first chromatographic column 7-1. Before the fraction of the target time period flows out, the fraction of the first chromatographic column 7-1 passes through the detector 9 → the multi-channel fraction collector outlet 11-3 → the waste collection tank 13. When the fraction of the target time period flows out, switch the multi-channel fraction collector outlet to 11-2 to connect to the first fraction collection tank 12-1 to collect the sample. During the elution, use the first multi-way solvent selection valve 2 to connect the third isocratic liquid storage tank 1-3, the first isocratic liquid storage tank 1-1, and the fourth isocratic liquid storage tank 1-4 in sequence according to the requirements of column washing, equilibration, and sample loading time. Operate the isocratic elution device to wash, equilibrate, and load the sample onto the second chromatographic column 7-2.

[0085] 6. Elution of the second chromatographic column 7-2; washing, equilibration, and dilution of sample loading on the first chromatographic column 7-1

[0086] The states of the pre-column valve 6 and the post-column valve 8 are both a. Switch the second multi-way solvent selection valve 16-1 to the first gradient liquid storage tank 4-1, and the third multi-way solvent selection valve 16-2 to the third gradient liquid storage tank 4-3. Operate the gradient elution device to perform gradient elution on the second chromatographic column 7-2. Before the fraction of the target time period flows out, the fraction of the second chromatographic column 7-2 passes through the detector 9 → the multi-channel fraction collector outlet 11-3 → the waste collection tank 13. And before the fraction of the target time period flows out, use the first multi-way solvent selection valve 2 to connect the third isocratic liquid storage tank 1-3 and the first isocratic liquid storage tank 1-1 in sequence according to the time program. Operate the isocratic elution device to wash and equilibrate the first chromatographic column 7-1 to complete the washing and equilibration procedures of the first chromatographic column 7-1. When the fraction of the target time period of the second chromatographic column 7-2 flows out, switch the multi-channel fraction collector outlet to 11-1, so that the fraction of this time period passes through the reflux pipeline 14 and the first flow path control valve 15-1 and flows into the first chromatographic column 7-1. At the same time, use the first multi-way solvent selection valve 2 to connect the second isocratic liquid storage tank 1-2, and operate the isocratic elution device to dilute the sample flowing into the first chromatographic column 7-1.

[0087] 7. Circulation

[0088] Circulate the above steps 5 and 6 to continuously purify the sample so that it undergoes two purifications to obtain fractions meeting the purity requirements.

[0089] Example 3

[0090] As Figure 3A double-column circulating chromatography system shown includes a first isocratic liquid storage tank 1-1, a second isocratic liquid storage tank 1-2, a third isocratic liquid storage tank 1-3, a first multi-way solvent selection valve 2, an isocratic chromatography pump 3, a first gradient liquid storage tank 4-1, a second gradient liquid storage tank 4-2, a third gradient liquid storage tank 4-3, a fourth gradient liquid storage tank 4-4, a gradient chromatography pump 5, a pre-column valve 6, a first chromatographic column 7-1, a second chromatographic column 7-2, a post-column valve 8, a detector 9, a waste liquid tank 10, a multi-channel fraction collector 11, a first fraction collection tank 12-1, a second fraction collection tank 12-2, a third fraction collection tank 12-3, a waste liquid collection tank 13, a first reflux pipeline 14-1, a second reflux pipeline 14-2, a first flow path control valve 15-1, a second flow path control valve 15-2, a third flow path control valve 15-3, a fourth flow path control valve 15-4, a fifth flow path control valve 15-5, a sixth flow path control valve 15-6, a seventh flow path control valve 15-7, an eighth flow path control valve 15-8, a second multi-way solvent selection valve 16-1, a third multi-way solvent selection valve 16-2, a first proportional valve 17-1, and a second proportional valve 17-2.

[0091] The first isocratic liquid storage tank 1-1, the second isocratic liquid storage tank 1-2, and the third isocratic liquid storage tank 1-3 are respectively connected to the multi-way solvent selection valve 2 → the isocratic chromatographic pump 3 → the pre-column valve 6 through connecting pipelines; the first gradient liquid storage tank 4-1 and the second gradient liquid storage tank 4-2 are respectively connected to the second multi-way solvent selection valve 16-1 → the first proportional valve 17-1 through connecting pipelines, serving as the first gradient elution unit; the third gradient liquid storage tank 4-3 and the fourth gradient liquid storage tank 4-4 are respectively connected to the third multi-way solvent selection valve 16-2 → the second proportional valve 17-2 through connecting pipelines, serving as the second gradient elution unit; after the first gradient elution unit and the second gradient elution unit are connected in parallel, they are connected to the gradient chromatographic pump 5 to form a gradient elution device, and the gradient elution device is connected to the pre-column valve 6 through a connecting pipeline; the pre-column valve 6 is connected to the chromatographic columns 7-1 and 7-2 arranged in parallel → the post-column valve 8 through a connecting pipeline; the post-column valve 8 is respectively connected to the detector 9 and the waste liquid tank 10 through connecting pipelines; the detector 9 is connected to the multi-channel fraction collector 11 through a connecting pipeline, and the outlets 11-1, 11-2, 11-3, and 11-4 of the multi-channel fraction collector are respectively connected to the first fraction collection tank 12-1, the second fraction collection tank 12-2, the third fraction collection tank 12-3, and the waste liquid collection tank 13; one end of the first reflux pipeline 14-1 is connected between the outlet 11-1 of the multi-channel fraction collector and the first fraction collection tank 12-1 through the first flow path control valve 15-1 and the second flow path control valve 15-2, and the other end is connected to the fifth flow path control valve 15-5 and the sixth flow path control valve 15-6 connected in parallel, and is connected between the pre-column valve 6 and the first chromatographic column 7-1 through the fifth flow path control valve 15-5, and is connected between the pre-column valve 6 and the second chromatographic column 7-2 through the sixth flow path control valve 15-6; one end of the second reflux pipeline 14-2 is connected between the outlet 11-3 of the multi-channel fraction collector and the third fraction collection tank 12-3 through the third flow path control valve 15-3 and the fourth flow path control valve 15-4, and the other end is connected to the seventh flow path control valve 15-7 and the eighth flow path control valve 15-8 connected in parallel, and is connected between the pre-column valve 6 and the first chromatographic column 7-1 through the seventh flow path control valve 15-7, and is connected between the pre-column valve 6 and the second chromatographic column 7-2 through the eighth flow path control valve 15-8.

[0092] Among them: the first isocratic liquid storage tank 1-1, the second isocratic liquid storage tank 1-2, and the third isocratic liquid storage tank 1-3 are respectively used to store the equilibration and dilution solution, the column washing solution, and the sample solution; the first multi-way solvent selection valve 2 is a three-channel solvent selection valve, which is used to select and transport different solutions; the isocratic chromatography pump 3 is used for solution transportation; the first gradient liquid storage tank 4-1, the second gradient liquid storage tank 4-2, the third gradient liquid storage tank 4-3, and the fourth gradient liquid storage tank 4-4 are used to store the gradient elution solution; the second multi-way solvent selection valve 16-1 and the third multi-way solvent selection valve 16-2 are two-channel solvent selection valves, which are used to select and transport different solutions; the first proportional valve 17-1 and the second proportional valve 17-2 are connected in parallel and cooperate with each other to form a gradient program with the gradient chromatography pump 5; the pre-column valve 6 and the post-column valve 8 are used to select different liquid flow directions; the detector 9 is used to detect the sample absorption value; the waste liquid tank 10 is used to receive the waste liquid during the processes of equilibration, sample loading, dilution, column washing, etc.; the multi-channel fraction collector 11 is used to select the fractions at different time periods to enter different collection tanks or the waste collection tank; the first fraction collection tank 12-1 is used to receive the pre-peak fractions with recovery value, the second fraction collection tank 12-2 is used to receive the qualified fractions, and the third fraction collection tank 12-3 is used to receive the post-peak fractions with recovery value; the waste collection tank 13 is used to receive the unqualified waste fractions; the first reflux pipeline 14-1 is used to reflux the pre-peak fractions with recovery value, and the second reflux pipeline 14-2 is used to reflux the post-peak fractions with recovery value; the flow path control valves all adopt globe valves. The first flow path control valve 15-1 and the second flow path control valve 15-2 are used to control the fractions coming out of the multi-channel fraction collector to flow into the reflux pipeline 14-1 or the fraction collection tank 12-1; the third flow path control valve 15-3 and the fourth flow path control valve 15-4 are used to control the fractions coming out of the multi-channel fraction collector to flow into the reflux pipeline 14-2 or the fraction collection tank 12-3; the fifth flow path control valve 15-5 and the sixth flow path control valve 15-6 are used to control the fractions in the reflux pipeline 14-1 to enter the first chromatographic column 7-1 or the second chromatographic column 7-2; the seventh flow path control valve 15-7 and the eighth flow path control valve 15-8 are used to control the fractions in the reflux pipeline 14-2 to enter the first chromatographic column 7-1 or the second chromatographic column 7-2.

[0093] Status description:

[0094] When the status of the pre-column valve 6 is a, the isocratic chromatography pump 3 is connected to the first chromatographic column 7-1 through the pre-column valve 6, and at the same time, the parallel gradient chromatography pump 5 is connected to the second chromatographic column 7-2 through the pre-column valve 6;

[0095] When the status of the pre-column valve 6 is b, the isocratic chromatography pump 3 is connected to the second chromatographic column 7-2 through the pre-column valve 6, and at the same time, the parallel gradient chromatography pump 5 is connected to the first chromatographic column 7-1 through the pre-column valve 6;

[0096] When the state of the post-column valve 8 is a, the first chromatographic column 7-1 is connected to the waste liquid tank 10 through the post-column valve, and at the same time, the second chromatographic column 7-2 is connected to the detector 9 through the post-column valve;

[0097] When the state of the post-column valve 8 is b, the first chromatographic column 7-1 is connected to the detector 9 through the post-column valve, and at the same time, the second chromatographic column 7-2 is connected to the waste liquid tank 10 through the post-column valve.

[0098] The initial state of the flow path control valve is closed.

[0099] The initial states of the pre-column valve and the post-column valve are both a.

[0100] Before the fractions flow out during the target time period, the multi-channel fraction collector 11 is connected to the waste collection tank 13.

[0101] The working process of this chromatographic column system includes the following steps:

[0102] 1. Fraction testing of different chromatographic columns at different time periods

[0103] Using the traditional single-column method, balance, sample loading, and elution are carried out on the first chromatographic column 7-1 and the second chromatographic column 7-2 respectively. Samples are collected from any of the first fraction collection tank 12-1, the second fraction collection tank 12-2, the third fraction collection tank 12-3, and the waste collection tank 13 to determine the time periods of the pre-peak fractions with recovery value, the qualified fraction time periods, and the post-peak fractions with recovery value.

[0104] 2. Equilibration and sample loading of the first chromatographic column 7-1

[0105] According to the time period requirements, the first multi-way solvent selection valve 2 is sequentially switched to the first isocratic liquid storage tank 1-1 and the third isocratic liquid storage tank 1-3, and the isocratic chromatographic pump 3 is operated to balance and load the sample on the first chromatographic column 7-1.

[0106] 3. Elution of the first chromatographic column 7-1, column washing, equilibration, and sample loading of the second chromatographic column 7-2

[0107] The states of the pre-column valve 6 and the post-column valve 8 are both switched to b. The gradient elution device is used to perform gradient elution on the first chromatographic column 7-1. The elution is divided into two stages: in the first stage, the first gradient liquid storage tank 4-1 and the third gradient liquid storage tank 4-3 are used, and the ratios are adjusted through the first proportional valve 17-1 and the second proportional valve 17-2 respectively, and the gradient chromatographic pump 5 is operated to elute the first chromatographic column 7-1; in the second stage, the second gradient liquid storage tank 4-2 and the fourth gradient liquid storage tank 4-4 are used, and the ratios are adjusted through the first proportional valve 17-1 and the second proportional valve 17-2 respectively, and the gradient chromatographic pump 5 is operated to elute the first chromatographic column 7-1.

[0108] Before the time period of the pre-peak fraction with recovery value, according to the time period requirements, sequentially switch through the first multi-way solvent selection valve 2 to the second isocratic liquid storage tank 1-2 and the first isocratic liquid storage tank 1-1, and operate the isocratic chromatographic pump 3 to complete the column washing and equilibration procedures for the second chromatographic column 7-2. When the time period of the pre-peak fraction with recovery value appears, switch the outlet of the multi-channel fraction collector to 11-1, open the second flow path control valve 15-2 and the sixth flow path control valve 15-6, so that the pre-peak fraction enters the second chromatographic column 7-2 through the first return pipeline 14-1. At the same time, start the isocratic chromatographic pump 3, switch the first multi-way solvent selection valve 2 to the first isocratic liquid storage tank 1-1, and use this solution to dilute the fraction entering the second chromatographic column 7-2; when the time period of the pre-peak fraction with recovery value is completed, switch the outlet of the multi-channel fraction collector to 11-2 to communicate with the second fraction collection tank 12-2 to collect the qualified fraction. At the same time, start the isocratic chromatographic pump 3, switch the first multi-way solvent selection valve 2 to the third isocratic liquid storage tank 1-3, and load the sample onto the second chromatographic column 7-2; when the time period of the post-peak fraction with recovery value appears, switch the outlet of the multi-channel fraction collector to 11-3 to communicate with the third fraction collection tank 12-3, open the fourth flow path control valve 15-4 and the eighth flow path control valve 15-8, so that the fraction enters the second chromatographic column 7-2 through the second return pipeline 14-2. At the same time, start the isocratic chromatographic pump 3, switch the first multi-way solvent selection valve 2 to the first isocratic liquid storage tank 1-1, and use this solution to dilute the fraction entering the second chromatographic column 7-2;

[0109] 4. Elution of the second chromatographic column 7-2, column washing, equilibration, and sample loading of the first chromatographic column 7-1

[0110] Switch the states of the pre-column valve 6 and the post-column valve 8 to a respectively. Use the gradient elution device to perform gradient elution on the second chromatographic column 7-2. The elution is divided into two stages: In the first stage, use the first gradient liquid storage tank 4-1 and the third gradient liquid storage tank 4-3, adjust the ratio through the first proportional valve 17-1 and the second proportional valve 17-2, and operate the gradient chromatographic pump 5 to elute the second chromatographic column 7-2; in the second stage, use the second gradient liquid storage tank 4-2 and the fourth gradient liquid storage tank 4-4, adjust the ratio through the first proportional valve 17-1 and the second proportional valve 17-2, and operate the gradient chromatographic pump 5 to elute the second chromatographic column 7-2. Before the time period of the peak front fraction with recovery value, sequentially switch through the first multi-way solvent selection valve 2 to the second isocratic liquid storage tank 1-2 and the first isocratic liquid storage tank 1-1, and operate the isocratic chromatographic pump 3 to complete the column washing and balancing procedures of the first chromatographic column 7-1. When the time period of the peak front fraction with recovery value appears, switch the outlet of the multi-channel fraction collector to 11-1, open the second flow path control valve 15-2 and the fifth flow path control valve 15-5, so that the peak front fraction enters the first chromatographic column 7-1 through the first return pipeline 14-1. At the same time, start the isocratic chromatographic pump 3, switch the first multi-way solvent selection valve 2 to the first isocratic liquid storage tank 1-1, and use this solution to dilute the peak front fraction entering the first chromatographic column 7-1; when the time period of the peak front fraction with recovery value is completed, switch the outlet of the multi-channel fraction collector to 11-2 to communicate with the second fraction collection tank 12-2 to collect the sample. At the same time, start the isocratic chromatographic pump 3, switch the first multi-way solvent selection valve 2 to the third isocratic liquid storage tank 1-3, and load the sample onto the first chromatographic column 7-1; when the time period of the peak back fraction with recovery value appears, switch the outlet 11-3 of the multi-channel fraction collector to the third fraction collection tank 12-3, open the fourth flow path control valve 15-4 and the seventh flow path control valve 15-7, so that the fraction enters the first chromatographic column 7-1 through the second return pipeline 14-2. At the same time, start the isocratic chromatographic pump 3, switch the first multi-way solvent selection valve 2 to the first isocratic liquid storage tank 1-1, and use this solution to dilute the fraction entering the first chromatographic column 7-1;

[0111] 5. Circulation

[0112] Repeat the above steps 3-4 continuously to purify the sample and obtain qualified fractions.

[0113] Example 4

[0114] As Figure 4A dual-column circulating chromatography system as shown includes a first isocratic liquid storage tank 1-1, a second isocratic liquid storage tank 1-2, a third isocratic liquid storage tank 1-3, a fourth isocratic liquid storage tank 1-4, a first multi-way solvent selection valve 2, an isocratic chromatography pump 3, a first gradient liquid storage tank 4-1, a second gradient liquid storage tank 4-2, a gradient chromatography pump 5, a pre-column valve 6, a first chromatographic column 7-1, a second chromatographic column 7-2, a post-column valve 8, a detector 9, a waste liquid tank 10, a multi-channel fraction collector 11, a first fraction collection tank 12-1, a second fraction collection tank 12-2, a third fraction collection tank 12-3, a waste liquid collection tank 13, a reflux pipeline 14, a first flow path control valve 15-1, a second flow path control valve 15-2, a third flow path control valve 15-3, a fourth flow path control valve 15-4, a first proportional valve 17-1, and a second proportional valve 17-2.

[0115] The first isocratic liquid storage tank 1-1, the second isocratic liquid storage tank 1-2, the third isocratic liquid storage tank 1-3, and the fourth isocratic liquid storage tank 1-4 are connected to the multi-way solvent selection valve 2 → isocratic chromatography pump 3 → pre-column valve 6 through connecting pipelines; the first gradient liquid storage tank 4-1 is connected to the first proportional valve 17-1 through a connecting pipeline as the first gradient elution unit; the second gradient liquid storage tank 4-2 is connected to the second proportional valve 17-2 through a connecting pipeline as the second gradient elution unit; after the first gradient elution unit and the second gradient elution unit are connected in parallel, a gradient elution device is formed through the gradient chromatography pump 5, and the gradient elution device is connected to the pre-column valve 6 through a connecting pipeline; the pre-column valve 6 is connected to the chromatographic columns 7-1 and 7-2 → post-column valve 8 arranged in parallel through a connecting pipeline; the post-column valve 8 is connected to the detector 9 and the waste liquid tank 10 arranged in parallel through a connecting pipeline; the detector 9 is connected to the multi-channel fraction collector 11 through a connecting pipeline, and the outlets 11-1, 11-2, 11-3, and 11-4 of the multi-channel fraction collector 11 are respectively connected to the first fraction collection tank 12-1, the second fraction collection tank 12-2, the third fraction collection tank 12-3, and the waste liquid collection tank 13; one end of the reflux pipeline 14 is connected between the outlet 11-1 of the multi-channel fraction collector and the first fraction collection tank 12-1 through the first flow path control valve 15-1, and the other end of the reflux pipeline 14 is connected to the second flow path control valve 15-2 and then connected to the third flow path control valve 15-3 and the fourth flow path control valve 15-4 connected in parallel, and is connected between the pre-column valve 6 and the first chromatographic column 7-1 through the third flow path control valve 15-3, and is connected between the pre-column valve 6 and the second chromatographic column 7-2 through the fourth flow path control valve 15-4.

[0116] Among them: the first isocratic liquid storage tank 1-1, the second isocratic liquid storage tank 1-2, the third isocratic liquid storage tank 1-3, and the fourth isocratic liquid storage tank 1-4 are respectively used to store balance solution, dilution solution, column washing solution, and sample solution; the first multi-way solvent selection valve 2 is a four-channel solvent selection valve used to select and transport different solutions; the isocratic chromatographic pump 3 is used for solution transportation; the first gradient liquid storage tank 4-1 and the second gradient liquid storage tank 4-2 are used to store gradient elution solutions; the first proportional valve 17-1 and the second proportional valve 17-2 are connected in parallel and cooperate with the gradient chromatographic pump 5 to form a gradient program; the pre-column valve 6 and the post-column valve 8 are used to select different liquid flow directions; the detector 9 is used to detect the absorbance of the sample; the waste liquid tank 10 is used to receive waste liquid during processes such as balancing, sample loading, dilution, and column washing; the multi-channel fraction collector 11 is used to select fractions with different absorbance values at different time periods to enter different collection tanks or the waste collection tank; the first fraction collection tank 12-1 is used to receive fractions and pre-peak fractions, and the second fraction collection tank 12-2 and the third fraction collection tank 12-3 are used to receive fractions; the waste collection tank 13 receives unqualified waste fractions; the reflux pipeline 14 is used to reflux fractions; the first flow path control valve 15-1 and the second flow path control valve 15-2 are both three-way valves. The first flow path control valve 15-1 controls the flow direction of the fraction to the reflux pipeline 14 or the first fraction collection tank 12-1, and the second flow path control valve 15-2 controls the flow direction of the fraction to the third flow path control valve 15-3 or the fourth flow path control valve 15-4. The third flow path control valve 15-3 and the fourth flow path control valve 15-4 are one-way valves that control the one-way flow of liquid to the chromatographic column 7-1 or 7-2 to prevent liquid backflow and contamination.

[0117] Status description:

[0118] When the pre-column valve 6 is in state a, the isocratic chromatographic pump 3 is connected to the first chromatographic column 7-1 through the pre-column valve 6, and at the same time, the parallel gradient chromatographic pump 5 is connected to the second chromatographic column 7-2 through the pre-column valve 6;

[0119] When the pre-column valve 6 is in state b, the isocratic chromatographic pump 3 is connected to the second chromatographic column 7-2 through the pre-column valve 6, and at the same time, the parallel gradient chromatographic pump 5 is connected to the first chromatographic column 7-1 through the pre-column valve 6;

[0120] When the post-column valve 8 is in state a, the first chromatographic column 7-1 is connected to the waste liquid tank 10 through the post-column valve, and at the same time, the second chromatographic column 7-2 is connected to the detector 9 through the post-column valve;

[0121] When the post-column valve 8 is in state b, the first chromatographic column 7-1 is connected to the detector 9 through the post-column valve, and at the same time, the second chromatographic column 7-2 is connected to the waste liquid tank 10 through the post-column valve.

[0122] The initial states of the pre-column valve and the post-column valve are both a.

[0123] Before the fractions flow out during the target time period, the multi-channel fraction collector 11 is connected to the waste collection tank 13 all the time.

[0124] The working process of this chromatographic column system includes the following steps:

[0125] 1. Testing of fractions in different intervals of different chromatographic columns

[0126] Using the traditional single-column method, balance, sample loading, and elution are carried out on the first chromatographic column 7-1 and the second chromatographic column 7-2 respectively. Fractions are collected from any of the first fraction collection tank 12-1, the second fraction collection tank 12-2, the third fraction collection tank 12-3, and the waste collection tank 13 to determine the pre-peak fractions and qualified fraction intervals with recovery value. This interval is determined by combining time and absorption value.

[0127] 2. Balancing and sample loading of the first chromatographic column 7-1

[0128] According to the time period requirements, sequentially switch through the first multi-way solvent selection valve 2 to the first isocratic liquid storage tank 1-1 and the fourth isocratic liquid storage tank 1-4, and operate the isocratic chromatographic pump 3 to balance and load the sample on the first chromatographic column 7-1.

[0129] 3. Elution of the first chromatographic column 7-1, washing, balancing, and sample loading of the second chromatographic column 7-2

[0130] Switch the states of the pre-column valve 6 and the post-column valve 8 to b respectively. Use the gradient elution device to perform gradient elution on the first chromatographic column 7-1. Before the interval of the pre-peak fractions with recovery value, sequentially switch through the first multi-way solvent selection valve 2 to the third isocratic liquid storage tank 1-3 and the first isocratic liquid storage tank 1-1, and operate the isocratic chromatographic pump 3 to complete the column washing and balancing procedures for the second chromatographic column 7-2. When the interval of the pre-peak fractions with recovery value appears, switch the outlet of the multi-channel fraction collector to 11-1, switch the first flow path control valve 15-1 to connect to the reflux pipeline 14, switch the second flow path control valve 15-2 and connect to the fourth flow path control valve 15-4, so that the fraction enters the second chromatographic column 7-2 through the flow path. At the same time, start the isocratic chromatographic pump 3, switch the first multi-way solvent selection valve 2 to the second isocratic liquid storage tank 1-2, and use this solution to dilute the fraction entering the second chromatographic column 7-2; when the interval of the pre-peak fractions with recovery value is completed, switch the outlet 11-2 or 11-3 of the multi-channel fraction collector 11 to connect to the second fraction collection tank 12-2 or the third fraction collection tank 12-3, collect fractions according to the parameters of the qualified fraction interval. At the same time, start the isocratic chromatographic pump 3, switch the first multi-way solvent selection valve 2 to the fourth isocratic liquid storage tank 1-4, and load the sample on the second chromatographic column 7-2;

[0131] 4. Elution of the second chromatographic column 7-2, washing, balancing, and sample loading of the first chromatographic column 7-1

[0132] Switch the states of the pre-column valve 6 and the post-column valve 8 to a respectively. Use the gradient elution device to perform gradient elution on the second chromatographic column 7-2. Before the peak front fraction interval with recovery value, sequentially pass through the first multi-way solvent selection valve 2, switch to the third isocratic liquid storage tank 1-3 and the first isocratic liquid storage tank 1-1, and operate the isocratic chromatographic pump 3 to complete the column washing and equilibration procedures for the first chromatographic column 7-1. When the peak front fraction interval with recovery value appears, switch the outlet 11-1 of the multi-channel fraction collector, switch the first flow path control valve 15-1 to connect to the reflux pipeline 14, and switch the second flow path control valve 15-2 to connect to the third flow path control valve 15-3, so that the fraction enters the first chromatographic column 7-1 through the flow path. At the same time, start the isocratic chromatographic pump 3, switch the first multi-way solvent selection valve 2 to connect to the second isocratic liquid storage tank 1-2, and dilute the fraction entering the first chromatographic column 7-1; when the peak front fraction interval with recovery value is completed, switch the outlet 11-2 or 11-3 of the multi-channel fraction collector to the second fraction collection tank 12-2 or the third fraction collection tank 12-3, collect the fraction according to the qualified fraction interval parameters. At the same time, start the isocratic chromatographic pump 3, switch the first multi-way solvent selection valve 2 to connect to the fourth isocratic liquid storage tank 1-4, and load the sample onto the first chromatographic column 7-1;

[0133] 5. Circulation

[0134] Repeat the above steps 3 and 4 to continuously purify the sample and obtain qualified fractions.

[0135] Example 5

[0136] As Figure 5 shown, a dual-column system includes a first isocratic liquid storage tank 1-1, a second isocratic liquid storage tank 1-2, a third isocratic liquid storage tank 1-3, a fourth isocratic liquid storage tank 1-4, a first multi-way solvent selection valve 2, an isocratic chromatographic pump 3, a first gradient liquid storage tank 4-1, a second gradient liquid storage tank 4-2, a third gradient liquid storage tank 4-3, a fourth gradient liquid storage tank 4-4, a fifth gradient liquid storage tank 4-5, a first gradient chromatographic pump 5-1, a second gradient chromatographic pump 5-2, a pre-column valve 6, a first chromatographic column 7-1, a second chromatographic column 7-2, a post-column valve 8, a detector 9, a waste liquid tank 10, a multi-channel fraction collector 11, a first fraction collection tank 12-1, a second fraction collection tank 12-2, a third fraction collection tank 12-3, a waste liquid collection tank 13, a reflux pipeline 14, a first flow path control valve 15-1, a second flow path control valve 15-2, a third flow path control valve 15-3, a fourth flow path control valve 15-4, a fifth flow path control valve 15-5, a sixth flow path control valve 15-6, a second multi-way solvent selection valve 16-1, and a third multi-way solvent selection valve 16-2.

[0137] The first isocratic liquid storage tank 1-1, the second isocratic liquid storage tank 1-2, the third isocratic liquid storage tank 1-3, and the fourth isocratic liquid storage tank 1-4 are connected to the multi-way solvent selection valve 2 → the isocratic chromatography pump 3 → the pre-column valve 6 through connecting pipelines; the first gradient liquid storage tank 4-1, the second gradient liquid storage tank 4-2, and the third gradient liquid storage tank 4-3 are respectively connected to the second multi-way solvent selection valve 16-1 → the first gradient chromatography pump 5-1 through connecting pipelines as the first gradient elution unit; the fourth gradient liquid storage tank 4-4 and the fifth gradient liquid storage tank 4-5 are respectively connected to the third multi-way solvent selection valve 16-2 → the second gradient chromatography pump 5-2 through connecting pipelines as the second gradient elution unit; the first gradient elution unit and the second gradient elution unit are connected in parallel to form a gradient elution device, and the gradient elution device is connected to the pre-column valve 6 through a connecting pipeline; the pre-column valve 6 is connected to the chromatographic columns 7-1 and 7-2 arranged in parallel → the post-column valve 8 through a connecting pipeline; the post-column valve 8 is respectively connected to the detector 9 and the waste liquid tank 10 through connecting pipelines; the detector 9 is connected to the multi-channel fraction collector 11 through a connecting pipeline, and the fraction collector 11

[0138] The outlet 11-1 of the multi-channel fraction collector is connected to the first fraction collector 12-1 through the first flow path control valve 15-1, the outlet 11-2 of the multi-channel fraction collector is connected to the second fraction collection tank 12-2 through a connecting pipeline, the outlet 11-3 of the multi-channel fraction collector is connected to the third fraction collector 12-3 through the first flow path control valve 15-1, and the outlet 11-4 of the multi-channel fraction collector is connected to the waste liquid collection tank 13 through a connecting pipeline. One end of the first return pipeline 14-1 is connected to the outlet 11-1 of the multi-channel fraction collector through the first flow path control valve 15-1, and the other end is successively connected to the third flow path control valve 15-3 and the fourth flow path control valve 15-4 connected in parallel through the second flow path control valve 15-2, and is connected between the pre-column valve 6 and the second chromatographic column 7-2 through the third flow path control valve 15-3, and is connected between the pre-column valve 6 and the first chromatographic column 7-1 through the fourth flow path control valve 15-4. One end of the second return pipeline 14-2 is connected to the outlet 11-3 of the multi-channel fraction collector through the first flow path control valve 15-1, and the other end is successively connected to the fifth flow path control valve 15-5 and the sixth flow path control valve 15-6 connected in parallel through the second flow path control valve 15-2, and is connected between the pre-column valve 6 and the first chromatographic column 7-1 through the fifth flow path control valve 15-5, and is connected between the pre-column valve 6 and the second chromatographic column 7-2 through the sixth flow path control valve 15-6.

[0139] Among them: the first isocratic liquid storage tank 1-1, the second isocratic liquid storage tank 1-2, the third isocratic liquid storage tank 1-3, and the fourth isocratic liquid storage tank 1-4 are respectively used to store balance solution, dilution solution, column washing solution, and sample solution; the first multi-way solvent selection valve 2 is a four-channel solvent selection valve, used to select and transport different solutions; the isocratic chromatography pump 3 is used for solution transportation; the first gradient liquid storage tank 4-1, the second gradient liquid storage tank 4-2, the third gradient liquid storage tank 4-3, the fourth gradient liquid storage tank 4-4, and the fifth gradient liquid storage tank 4-5 are used to store gradient elution solutions; the second multi-way solvent selection valve 16-1 is a three-channel solvent selection valve, and the third multi-way solvent selection valve 16-2 is a two-channel solvent selection valve, used to select and transport different solutions; the first gradient chromatography pump 5-1 and the second gradient chromatography pump 5-2 are used to transport elution solutions, and the two work together to form a gradient program; the pre-column valve 6 and the post-column valve 8 are used to select different liquid flow directions; the detector 9 is used to detect the absorbance of the sample; the waste liquid tank 10 is used to receive waste liquid during processes such as balancing, loading, dilution, and column washing; the multi-channel fraction collector 11 is used to switch different outlets; the first fraction collection tank 12-1, the second fraction collection tank 12-2, and the third fraction collection tank 12-3 are used to receive fractions; the waste collection tank 13 is used to receive unqualified waste fractions; the reflux pipeline is used for fraction reflux; the first flow path control valve 15-1 is a two-position six-way valve, controlling the fraction to enter the reflux pipeline or enter the first fraction collection tank 12-1 and the third fraction collection tank 12-3; the second flow path control valve 15-2 is a two-position six-way valve, controlling the fraction to enter different chromatographic columns; the third flow path control valve 15-3, the fourth flow path control valve 15-4, the fifth flow path control valve 15-5, and the sixth flow path control valve 15-6 are all one-way valves to prevent liquid backflow. The first reflux pipeline 14-1 and the second reflux pipeline 14-2 are used to transport liquids.

[0140] Status description:

[0141] When the status of the pre-column valve 6 is a, the isocratic chromatography pump 3 is connected to the first chromatographic column 7-1 through the pre-column valve 6, and at the same time, the parallel first gradient chromatography pump 5-1 and the second gradient chromatography pump 5-2 are connected to the second chromatographic column 7-2 through the pre-column valve 6;

[0142] When the status of the pre-column valve 6 is b, the isocratic chromatography pump 3 is connected to the second chromatographic column 7-2 through the pre-column valve 6, and at the same time, the parallel first gradient chromatography pump 5-1 and the second gradient chromatography pump 5-2 are connected to the first chromatographic column 7-1 through the pre-column valve 6;

[0143] When the status of the post-column valve 8 is a, the first chromatographic column 7-1 is connected to the waste liquid tank 10 through the post-column valve, and at the same time, the second chromatographic column 7-2 is connected to the detector 9 through the post-column valve;

[0144] When the post-column valve 8 is in state b, the first chromatographic column 7-1 is connected to the detector 9 through the post-column valve, and at the same time, the second chromatographic column 7-2 is connected to the waste liquid tank 10 through the post-column valve.

[0145] The first flow path control valve 15-1 and the second flow path control valve 15-2 adopt two-position six-way valves, each having six connection holes numbered 1#, 2#, 3#, 4#, 5#, and 6#.

[0146] The first flow path control valve 15-1 has two states: In state ⅰ, 1#-2#, 3#-4#, and 5#-6# are connected; in state ⅱ, 2#-3#, 4#-5#, and 6#-1# are connected. When running the continuous purification program and needing to circulate the fractions, state ⅰ is used: The outlet 11-1 of the multi-channel fraction collector is switched to be connected to the first reflux pipeline 14-1, and the outlet 11-3 of the multi-channel fraction collector is switched to be connected to the second reflux pipeline 14-2. When needing to collect the fractions of the first purification or the second purification, state ⅱ is used: The outlet 11-1 of the multi-channel fraction collector is switched to be connected to the first fraction collection tank 12-1, and the outlet 11-3 of the multi-channel fraction collector is switched to be connected to the third fraction collection tank 12-3.

[0147] The second flow path control valve 15-2 has two states: In state ⅰ, 1#-2#, 3#-4#, and 5#-6# are connected; in state ⅱ, 2#-3#, 4#-5#, and 6#-1# are connected. When running the continuous purification program and needing to introduce the fractions into the first chromatographic column 5-1, state ⅰ is used. At this time, the first reflux pipeline 14-1 is connected to the first chromatographic column 7-1 through the second flow path control valve 15-2 → the fourth flow path control valve 15-4 →, and the second reflux pipeline 14-2 is connected to the first chromatographic column 7-1 through the second flow path control valve 15-2 → the fifth flow path control valve 15-5 →. If needing to introduce the sample into the second chromatographic column 5-2, state ⅱ is used: The first reflux pipeline 14-1 is connected to the second chromatographic column 7-2 through the second flow path control valve 15-2 → the third flow path control valve 15-3 →, and the second reflux pipeline 14-2 is connected to the second chromatographic column 7-2 through the second flow path control valve 15-2 → the sixth flow path control valve 15-6 →.

[0148] The initial states of the pre-column valve and the post-column valve are both a.

[0149] Before the fractions flow out during the target time period, the multi-channel fraction collector 11 is connected to the waste collection tank 13.

[0150] The working process of this chromatographic column system includes the following steps:

[0151] 1. Testing of fractions in different intervals of different chromatographic columns

[0152] Using the traditional single-column method, the first chromatographic column 7-1 is equilibrated, loaded, and eluted. Fractions are collected from any of the first fraction collection tank 12-1, the second fraction collection tank 12-2, the third fraction collection tank 12-3, and the waste collection tank 13 to determine the qualified interval for primary purification, that is, the fraction interval to be subjected to secondary purification. This interval is determined by combining time and absorbance values. The samples in this interval are combined, loaded onto the second chromatographic column 7-2, and after elution, the final qualified sample interval is determined, which is determined by combining time and absorbance values.

[0153] 2. Equilibration and loading of the first chromatographic column 7-1

[0154] According to the time period requirements, sequentially switch through the first multi-way solvent selection valve 2 to the first isocratic liquid storage tank 1-1 and the fourth isocratic liquid storage tank 1-4, and operate the isocratic chromatographic pump 3 to equilibrate and load the first chromatographic column 7-1.

[0155] 3. Elution of the first chromatographic column 7-1, washing, equilibration, and loading of the second chromatographic column 7-2

[0156] Switch the states of both the pre-column valve 6 and the post-column valve 8 to b. Use the gradient elution device to perform gradient elution on the first chromatographic column 7-1, switch the second multi-way solvent selection valve 16-2 and the third multi-way solvent selection valve 16-3 to connect the first gradient liquid storage tank 4-1 and the fourth gradient liquid storage tank 4-4, and operate the corresponding first gradient chromatographic pump 5-1 and the second gradient chromatographic pump 5-2 to elute the first chromatographic column 7-1; before the qualified interval for primary purification appears, sequentially switch through the first multi-way solvent selection valve 2 to the third isocratic liquid storage tank 1-3 and the first isocratic liquid storage tank 1-1, and operate the isocratic chromatographic pump 3 to complete the column washing and equilibration procedures for the second chromatographic column 7-2. When the qualified interval for primary purification appears, switch the outlet 11-1 of the multi-channel fraction collector, switch the state of the first flow path control valve 15-1 to i and the state of the second flow path control valve 15-2 to ii, so that the fraction passes through the multi-channel fraction collector 11 → the first reflux pipeline 14-1 and enters the second chromatographic column 7-2. At the same time, start the isocratic chromatographic pump 3, switch the first multi-way solvent selection valve 2 to the second isocratic liquid storage tank 1-2, and use this solution to dilute the fraction entering the second chromatographic column 7-2;

[0157] 4. Column washing, equilibration, and loading of the first chromatographic column 7-1, elution of the second chromatographic column 7-2

[0158] Switch the states of the pre-column valve 6 and the post-column valve 8 to a respectively. Use the gradient elution device to perform gradient elution on the second chromatographic column 7-2. The elution is divided into two stages: In the first stage, switch the second multi-way solvent selection valve 16-1 and the third multi-way solvent selection valve 16-3 to use the second gradient liquid storage tank 4-2 and the fifth gradient liquid storage tank 4-5, and respectively elute the second chromatographic column 7-2 through the first gradient chromatographic pump 5-1 and the second gradient chromatographic pump 5-2; in the second stage, switch the second multi-way solvent selection valve 16-1 and the third multi-way solvent selection valve 16-3 to use the third gradient liquid storage tank 4-3 and the fifth gradient liquid storage tank 4-5, and respectively elute the second chromatographic column 7-2 through the first gradient chromatographic pump 5-1 and the second gradient chromatographic pump 5-2; when the qualified interval of secondary purification appears, switch the outlet 11-2 of the multi-channel fraction collector to communicate with the second fraction collection tank 12-2 to collect the sample. While performing gradient elution on the second chromatographic column 7-2, use the first multi-way solvent selection valve 2 to sequentially connect the third isocratic liquid storage tank 1-3, the first isocratic liquid storage tank 1-1, and the fourth isocratic liquid storage tank 1-4 according to the requirements of column washing, equilibration, and sample loading time, and perform column washing, equilibration, and sample loading on the first chromatographic column 7-1 through the isocratic elution device.

[0159] 5. Circulation

[0160] Repeat the above steps 3 and 4 to continuously purify the sample to obtain a sample meeting the purity requirements.

[0161] Example 6

[0162] Adopt the dual-column circulating chromatographic system described in Example 5. Another working process of this system includes the following steps:

[0163] 1. Fraction testing of different chromatographic columns at different time periods

[0164] Adopt the traditional single-column method to respectively perform equilibration, sample loading, and elution on the first chromatographic column 7-1 and the second chromatographic column 7-2, and collect samples from any of the first fraction collection tank 12-1, the second fraction collection tank 12-2, the third fraction collection tank 12-3, and the waste collection tank 13 to determine the time periods of the pre-peak fractions with recovery value, the qualified fraction time periods, and the post-peak fractions with recovery value.

[0165] 2. Equilibration and sample loading of the first chromatographic column 7-1

[0166] According to the time period requirements, sequentially switch to the first isocratic liquid storage tank 1-1 and the fourth isocratic liquid storage tank 1-4 through the first multi-way solvent selection valve 2, and operate the isocratic chromatographic pump 3 to perform equilibration and sample loading on the first chromatographic column 7-1.

[0167] 3. Elution of the first chromatographic column 7-1, column washing, equilibration, and sample loading of the second chromatographic column 7-2

[0168] Switch the states of the pre-column valve 6 and the post-column valve 8 to b respectively. Use the gradient elution device to perform gradient elution on the first chromatographic column 7-1. The elution is divided into two stages: In the first stage, switch the second multi-way solvent selection valve 16-1 and the third multi-way solvent selection valve 16-3 to use the first gradient liquid storage tank 4-1 and the third gradient liquid storage tank 4-3, and operate the corresponding first gradient chromatographic pump 5-1 and the second gradient chromatographic pump 5-2 to elute the first chromatographic column 7-1; In the second stage, switch the second multi-way solvent selection valve 16-1 and the third multi-way solvent selection valve 16-2 to use the second gradient liquid storage tank 4-2 and the fourth gradient liquid storage tank 4-4, and operate the corresponding first gradient chromatographic pump 5-1 and the second gradient chromatographic pump 5-2 to elute the first chromatographic column 7-1. Before the time period of the peak front fraction with recovery value, according to the time period requirements, sequentially pass through the first multi-way solvent selection valve 2 and switch to the third isocratic liquid storage tank 1-3 and the first isocratic liquid storage tank 1-1, and operate the isocratic chromatographic pump 3 to complete the column washing and equilibration procedures for the second chromatographic column 7-2. When the time period of the peak front fraction with recovery value appears, switch the outlet 11-1 of the multi-channel fraction collector, switch the state of the first flow path control valve 15-1 to i and the state of the second flow path control valve 15-2 to ii, so that this fraction passes through the first return pipeline 14-1 and the third flow path control valve 15-3 into the second chromatographic column 7-2. At the same time, start the isocratic chromatographic pump 3, switch the first multi-way solvent selection valve 2 to the second isocratic liquid storage tank 1-2, and use this solution to dilute the fraction entering the second chromatographic column 7-2. When the time period of the peak front fraction with recovery value is completed, switch the outlet 11-2 of the multi-channel fraction collector to the second fraction collection tank 12-2 to collect the fraction. At the same time, start the isocratic chromatographic pump 3, switch the first multi-way solvent selection valve 2 to the fourth isocratic liquid storage tank 1-4, and load the sample onto the second chromatographic column 7-2. When the time period of the peak back fraction with recovery value appears, switch the outlet 11-3 of the multi-channel fraction collector, switch the state of the first flow path control valve 15-1 to i and the state of the second flow path control valve 15-2 to ii, so that this fraction passes through the second return pipeline 14-2 → the sixth flow path control valve 15-6 → the second chromatographic column 7-2. At the same time, start the isocratic chromatographic pump 3, switch the first multi-way solvent selection valve 2 to the second isocratic liquid storage tank 1-2, and use this solution to dilute the fraction entering the second chromatographic column 7-2;

[0169] 4. Elution of the second chromatographic column 7-2, column washing, equilibration, and sample loading of the first chromatographic column 7-1

[0170] Switch the states of the pre-column valve 6 and the post-column valve 8 to a respectively. Use the gradient elution device to perform gradient elution on the second chromatographic column 7-2. The elution is divided into two stages: In the first stage, switch the second multi-way solvent selection valve 16-1 and the third multi-way solvent selection valve 16-2 to use the first gradient liquid storage tank 4-1 and the third gradient liquid storage tank 4-3, and operate the corresponding first gradient chromatographic pump 5-1 and the second gradient chromatographic pump 5-2 to elute the second chromatographic column 7-2; in the second stage, switch the second multi-way solvent selection valve 16-1 and the third multi-way solvent selection valve 16-2 to use the second gradient liquid storage tank 4-2 and the fourth gradient liquid storage tank 4-4, and operate the corresponding first gradient chromatographic pump 5-1 and the second gradient chromatographic pump 5-2 to elute the second chromatographic column 7-2. Before the time period of the pre-fraction of the peak with recovery value, according to the time period requirements, sequentially switch through the first multi-way solvent selection valve 2 to the third isocratic liquid storage tank 1-3 and the first isocratic liquid storage tank 1-1, and operate the isocratic chromatographic pump 3 to complete the column washing and balancing procedures of the first chromatographic column 7-1. When the time period of the pre-fraction of the peak with recovery value appears, switch the outlet 11-1 of the multi-channel fraction collector, switch the state of the first flow path control valve 15-1 to i, and the state of the second flow path control valve 15-2 to i, so that the fraction passes through the first return pipeline 14-1 → the fourth flow path control valve 15-4 → the first chromatographic column 7-1. At the same time, start the isocratic chromatographic pump 3, switch the first multi-way solvent selection valve 2 to the second isocratic liquid storage tank 1-2, and use this solution to dilute the fraction entering the first chromatographic column 7-1; when the time period of the pre-fraction of the peak with recovery value is completed, switch the outlet 11-2 of the multi-channel fraction collector to connect to the second fraction collection tank 12-2 to collect the sample. At the same time, start the isocratic chromatographic pump 3, switch the first multi-way solvent selection valve 2 to the fourth isocratic liquid storage tank 1-4, and load the first chromatographic column 7-1; when the time period of the post-fraction of the peak with recovery value appears, switch the outlet 11-3 of the multi-channel fraction collector, switch the state of the first flow path control valve 15-1 to i, and the state of the second flow path control valve 15-2 to i, so that the fraction passes through the second return pipeline 14-2 → the fifth flow path control valve 15-5 → the first chromatographic column 7-1. At the same time, start the isocratic chromatographic pump 3, switch the first multi-way solvent selection valve 2 to the second isocratic liquid storage tank 1-2, and use this solution to dilute the fraction entering the second chromatographic column 7-2;

[0171] 5. Cycle

[0172] Cycle the above steps 3 and 4 to continuously purify the sample to obtain a sample meeting the purity requirements.

Claims

1. A dual-column circulating chromatography system, comprising an isocratic elution device, a gradient elution device, a pre-column valve (6), a chromatographic column device, a post-column valve (8), a detector device, a waste liquid tank (10), a fraction collection device, a reflux device and connecting pipelines; characterized in that: The isocratic elution device includes at least 3 isocratic liquid storage tanks (1) arranged in parallel, a multi-way solvent selection valve (2), and an isocratic chromatographic pump (3). The isocratic liquid storage tanks (1) are connected to the multi-way solvent selection valve (2) → isocratic chromatographic pump (3) through connecting pipelines; the chromatographic column device includes 2 chromatographic columns (7) arranged in parallel; the detector device includes at least 1 detector (9); The fraction collection device includes 1 multi-channel fraction collector (11), at least 2 fraction collection tanks (12), and 1 waste collection tank (13); the reflux device includes at least 1 reflux pipeline (14) and at least 1 flow path control valve (15); Among them, after the isocratic elution device and the gradient elution device are connected in parallel, they are connected to the pre-column valve (6) → chromatographic column device → post-column valve (8) through connecting pipelines. The post-column valve (8) is respectively connected to the detector device and the waste liquid tank (10) through connecting pipelines. The detector device is connected to the fraction collector (11) through a connecting pipeline. The fraction collector (11) is respectively connected to the fraction collection tank (12) and the waste collection tank (13) through connecting pipelines. One end of the reflux pipeline (14) is connected between the multi-channel fraction collector and one of the fraction collection tanks (12) through the flow path control valve (15), and the other end of the reflux pipeline (14) is connected between the pre-column valve (6) and the chromatographic column device through the flow path control valve (15) or the other end of the reflux pipeline (14) is directly connected between the pre-column valve (6) and the chromatographic column device.

2. A dual-column cyclic chromatography system, comprising an isocratic elution device, a gradient elution device, a pre-column valve (6), a chromatographic column device, a post-column valve (8), a detector device, a waste liquid tank (10), a fraction collection device, a reflux device and connecting pipelines; characterized in that: The isocratic elution device includes at least 3 isocratic liquid storage tanks (1) arranged in parallel, a multi-way solvent selection valve (2), and an isocratic chromatographic pump (3). The isocratic liquid storage tanks (1) are connected to the multi-way solvent selection valve (2) → isocratic chromatographic pump (3) through connecting pipelines; the chromatographic column device includes 2 chromatographic columns (7) arranged in parallel; the detector device includes at least 1 detector (9); The fraction collection device includes 1 multi-channel fraction collector (11), at least 2 fraction collection tanks (12), and 1 waste collection tank (13); the reflux device includes at least 1 reflux pipeline (14) and at least 1 flow path control valve (15), and at least one of the flow path control valves (15) is a two-position six-way valve; Among them, after the isocratic elution device and the gradient elution device are connected in parallel, they are connected to the pre-column valve (6) → chromatographic column device → post-column valve (8) through connecting pipelines. The post-column valve (8) is respectively connected to the detector device and the waste liquid tank (10) through connecting pipelines. The detector device is connected to the fraction collector (11) through a connecting pipeline. The fraction collector (11) is respectively connected to at least one fraction collection tank (12) and the waste collection tank (13) through connecting pipelines. At the same time, the fraction collector (11) is connected to the flow path control valve (15) through a connecting pipeline and then respectively connected to at least one fraction collection tank (12) and one end of the reflux pipeline (14). The other end of the reflux pipeline (14) is connected between the pre-column valve (6) and the chromatographic column device through the flow path control valve (15).

3. A dual-column circulating chromatography system, comprising an isocratic elution device, a gradient elution device, a pre-column valve (6), a chromatographic column device, a post-column valve (8), a detector device, a waste liquid tank (10), a fraction collection device, a reflux device and connecting pipelines; characterized in that: The isocratic elution device includes at least 3 isocratic liquid storage tanks (1) arranged in parallel, a multi-way solvent selection valve (2), and an isocratic chromatographic pump (3). The isocratic liquid storage tanks (1) are connected to the multi-way solvent selection valve (2) → isocratic chromatographic pump (3) through connecting pipelines; the chromatographic column device includes 2 chromatographic columns (7) arranged in parallel; The detector device includes at least 1 detector (9); The fraction collection device includes 1 multi-channel fraction collector (11), at least 1 fraction collection tank (12) and 1 waste collection tank (13); the reflux device includes at least 1 reflux pipeline (14) and at least 1 flow path control valve (15); Among them, after the isocratic elution device and the gradient elution device are connected in parallel, they are connected to the pre-column valve (6) → chromatographic column device → post-column valve (8) through connecting pipelines. The post-column valve (8) is respectively connected to the detector device and the waste liquid tank (10) through connecting pipelines. The detector device is connected to the fraction collector (11) through a connecting pipeline. The fraction collector (11) is respectively connected to the fraction collection tank (12) and the waste collection tank (13) through connecting pipelines. One end of the reflux pipeline (14) is connected to the fraction collector (11), and the other end of the reflux pipeline (14) is connected between the pre-column valve (6) and the chromatographic column device through the flow path control valve (15).

4. The dual-column circulating chromatography system according to claims 1 to 3, characterized in that: The gradient elution device includes 2 groups of gradient liquid storage tanks (4) and a gradient chromatographic pump (5), and the number of gradient liquid storage tanks in each group = 1. Each group of gradient liquid storage tanks (4) and the gradient chromatographic pump (5) are connected in series through connecting pipelines as a gradient elution unit, and 2 groups of gradient elution units are connected in parallel to form a gradient elution device.

5. The dual-column recycling chromatography system according to claims 1-3, characterized in that: The gradient elution device includes 2 groups of gradient liquid storage tanks (4), a multi-channel solvent selection valve (16), and a gradient chromatographic pump (5), and the number of gradient liquid storage tanks in each group > 1. Each group of gradient liquid storage tanks (4), the multi-channel solvent selection valve (16), and the gradient chromatographic pump (5) are sequentially connected in series through connecting pipelines to form a gradient elution unit, and 2 groups of gradient elution units are connected in parallel to form a gradient elution device.

6. The dual-column circulating chromatography system according to claims 1-3, characterized in that: The gradient elution device includes 2 groups of gradient liquid storage tanks (4), a proportional valve (17), and further includes 1 gradient chromatographic pump (5), and the number of gradient liquid storage tanks in each group = 1. Each group of gradient liquid storage tanks (4) and the proportional valve (17) are connected in series through connecting pipelines as a gradient elution unit. After 2 groups of gradient elution units are connected in parallel, they are then connected to the gradient chromatographic pump (5) through a connecting pipeline to form a gradient elution device.

7. The dual-column recycling chromatography system according to claims 1-3, wherein: The gradient elution device includes two groups of gradient liquid storage tanks (4), a multi-channel solvent selection valve (16), and a proportional valve (17), and further includes a gradient chromatography pump (5). Each group of gradient liquid storage tanks has a quantity greater than 1. Each group of gradient liquid storage tanks (4), the multi-channel solvent selection valve (16), and the proportional valve (17) are connected in series in sequence through connecting pipes as a gradient elution unit. After the two groups of gradient elution units are connected in parallel, they are then connected to the gradient chromatography pump (5) through connecting pipes to form a gradient elution device.

8. The dual-column recycling chromatography system according to claims 1 to 3, wherein: The flow path control valve (15) is selected from one or more of a globe valve, a three-way valve, a two-position six-way valve, and a check valve.

9. The dual-column recycling chromatography system according to claims 1-3, characterized in that: The chromatographic column (7) is selected from a normal-phase chromatographic column, a reversed-phase chromatographic column, an ion exchange column, and a gel chromatographic column.

10. The dual-column cyclic chromatography system according to claims 1-3, characterized in that: The detector (9) is selected from an ultraviolet detector, a conductivity detector, a diode array detector, an evaporative light scattering detector, a mass spectrometry detector, and a Raman detector.

11. A method for purifying a process by operating the bi-column recycling chromatography system according to any one of claims 1-10, characterized in that, The purification process includes at least two different stages (A, B): at least one stage A, one end of the reflux pipe (14) is communicated with the multi-channel fraction collector (11), and the other end of the reflux pipe (14) is communicated with the chromatographic column device (7) so that the fractions are re-purified; and at least one stage B, the reflux pipe (14) is not communicated with the multi-channel fraction collector (11) to collect the fractions. The method includes at least the following steps: I. Conduct a sample test before the purification process to determine the purity of the fractions at different time periods; II. Switch different stages of the purification process by running time and / or absorption value.

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