Double-column circulating chromatographic system

Abstract

The utility model 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 collecting 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 device disclosed by the utility model has the beneficial effects that (1) not only can the purification efficiency and the yield be improved, but also stable peak appearance time and a highly consistent purification spectrum can be obtained, stable impurity spectrum distribution can be obtained, and automatic production can be 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 utility model relates to a kind of chromatography systems, in particular to a kind of double-column circulating chromatography system. BACKGROUND

[0002] As the core means of current drug analysis separation and purification, the separation efficiency and selectivity advantage of chromatographic separation technology are remarkable. However, the traditional chromatography system is generally bottlenecked in production capacity due to low sample loading capacity of chromatographic column and long running cycle, and is only applied to high value-added products. At present, the single circulating chromatography system widely used in the market usually adopts a single chromatographic column to perform the intermittent operation process of "equilibrium-sample loading-elution-regeneration". This operation mode has two significant technical defects: on the one hand, it cannot realize automatic continuous operation, resulting in difficulty in improving production efficiency; on the other hand, due to the provision of only one chromatographic column, its purification efficiency is relatively low, which is difficult to meet the actual needs of increasingly strict drug quality requirements and continuous compression of production cost. In order to overcome these technical bottlenecks, some multi-column chromatography circulating systems have also been introduced. CN202411504307.5 discloses an automatic chromatography system with multiple columns in parallel. The utility model realizes the simultaneous operation of multiple chromatographic columns by arranging multiple chromatographic columns in parallel and sharing a set of mobile phase device, detector device and fraction collector device. However, this chromatography system still has certain limitations, as it cannot realize multiple purification of samples, which affects the purification efficiency to some extent. In addition, the tailings cannot be directly used, which has a certain negative impact on the yield. CN201310087690.4 discloses a double-column circulating chromatography system. The system realizes multiple adsorption and desorption of undetached components through pipeline design, aiming to improve the purification effect. However, the system adopts multiple sets of the same equipment, such as detection equipment, resulting in high manufacturing cost. At the same time, the system fails to effectively solve the problems of unstable peak time and inconsistent peak shape of preparation spectrum, which limits its application in actual production to some extent. SUMMARY

[0003] In view of this, in order to overcome the deficiencies of the prior art, the utility model provides a kind of double column circulating chromatography system, the system is equipped with two and more parallelly arranged chromatographic column, and all chromatographic columns share a set of elution device, detection device, fraction receiving device and reflux device and other core components.The system enables sample to realize multiple purification operation in the same system, on the one hand, effectively avoids potential risks such as environmental pollution and air oxidation of sample after flowing out of chromatographic column, ensures sample purity and quality stability;On the other hand, it greatly reduces the labor intensity of production personnel, while shortening the time required for separation and purification, greatly improving production efficiency.The utility model also has the function of tailing material reuse purification, and the system can eliminate the problems such as time loss, solvent waste and increase of labor cost caused by secondary processing of tailing material.Not only that, tailing material reuse purification can significantly improve the yield and yield of single purification, further improve production efficiency.In addition, the system realizes automatic operation and automatic sample collection function, and can intelligently combine and collect qualified parts.This automatic operation not only improves the accuracy and reliability of production, but also reduces the cost of analysis and detection;At the same time, the highly integrated design of the system effectively reduces the occupation of equipment to workshop space, reduces the overall operating cost.

[0004] To achieve the above object, the utility model provides a kind of scheme: a kind of double column circulating chromatography system, including isocratic elution device, gradient elution device, pre-column valve 6, chromatographic column device, post-column valve 8, detector device, waste tank 10, fraction collection device, reflux device and connecting pipeline;Isocratic elution device includes at least 3 parallelly arranged isocratic liquid storage tank 1 and multi-pass solvent selection valve 2, isocratic chromatographic pump 3, isocratic liquid storage tank 1 is connected with multi-pass solvent selection valve 2→isocratic chromatographic pump 3 through connecting pipeline;The chromatographic column device includes 2 parallelly arranged chromatographic columns 7;Detector device includes at least 1 detector 9;Fraction collection device includes 1 multi-channel fraction collector 11 and at least 2 fraction collection tanks 12 and 1 waste collection tank 13;Reflux device includes at least 1 reflux pipeline 14 and at least 1 flow control valve 15;

[0005] Wherein, isocratic elution device and gradient elution device are connected with pre-column valve 6→chromatographic column device→post-column valve 8 through connecting pipeline, post-column valve 8 is connected with detector device and waste tank 10 through connecting pipeline respectively, detector device is connected with fraction collector 11 through connecting pipeline, fraction collector (11) is connected with fraction collection tank 12 and waste collection tank 13 through connecting pipeline respectively, one end of reflux pipeline 14 is connected between multi-channel fraction collector and one of fraction collection tank 12 through flow control valve 15, the other end of reflux pipeline 14 is connected between pre-column valve 6 and chromatographic column device through flow control valve 15 or the other end of reflux pipeline 14 is directly connected between pre-column valve 6 and chromatographic column device.

[0006] The utility model provides another scheme: a kind of double column circulating chromatography system, including isocratic elution device, gradient elution device, pre-column valve 6, chromatographic column device, post-column valve 8, detector device, waste tank 10, fraction collection device, reflux device and connecting pipeline;Isocratic elution device includes at least 3 parallelly arranged isocratic liquid storage tank 1 and multi-pass solvent selection valve 2, isocratic chromatographic pump 3, isocratic liquid storage tank 1 is connected with multi-pass solvent selection valve 2→isocratic chromatographic pump 3 by connecting pipeline;Chromatographic column device includes 2 parallelly arranged chromatographic column 7;Detector device includes at least 1 detector 9;Fraction collection device includes 1 multi-channel fraction collector 11 and at least 2 fraction collection tank 12 and 1 waste collection tank 13;Reflux device includes at least 1 reflux pipeline 14 and at least 1 flow control valve 15, and at least one of flow control valve 15 is two six-way valve;

[0007] Among them, isocratic elution device and gradient elution device are connected with pre-column valve 6→chromatographic column device→post-column valve 8 after parallel connection by connecting pipeline, post-column valve 8 is connected with detector device and waste tank 10 respectively by connecting pipeline, detector device is connected with fraction collector 11 by connecting pipeline, fraction collector 11 is connected with at least one fraction collection tank 12, waste collection tank 13 respectively by connecting pipeline, and fraction collector 11 is connected with at least one fraction collection tank 12, one end of reflux pipeline 14 respectively after being connected with flow control valve 15 by connecting pipeline, the other end of reflux pipeline 14 is connected between pre-column valve 6 and chromatographic column device by flow control valve 15.

[0008] The utility model provides third scheme: a kind of double column circulating chromatography system, including isocratic elution device, gradient elution device, pre-column valve 6, chromatographic column device, post-column valve 8, detector device, waste tank 10, fraction collection device, reflux device and connecting pipeline;Isocratic elution device includes at least 3 parallelly arranged isocratic liquid storage tank 1 and multi-pass solvent selection valve 2, isocratic chromatographic pump 3, isocratic liquid storage tank 1 is connected with multi-pass solvent selection valve 2→isocratic chromatographic pump 3 by connecting pipeline;Chromatographic column device includes 2 parallelly arranged chromatographic column 7;Detector device includes at least 1 detector 9;Fraction collection device includes 1 multi-channel fraction collector 11 and at least 1 fraction collection tank 12 and 1 waste collection tank 13;Reflux device includes at least 1 reflux pipeline 14 and at least 1 flow control valve 15;

[0009] The isocratic elution device and the gradient elution device are connected in parallel, connected with the pre-column valve 6, the chromatographic column device and the post-column valve 8 through the connecting pipeline, the post-column valve 8 is connected with the detector device and the waste tank 10 through the connecting pipeline, the detector device is connected with the fraction collector 11 through the connecting pipeline, the fraction collector 11 is connected with the fraction collection tank 12 and the waste collection tank 13 through the connecting pipeline, one end of the reflux pipeline 14 is connected with 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.

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

[0011] The parallel chromatographic columns can adopt the same or different chromatographic columns, when the peak front and peak tail recovery is used, the same chromatographic column is adopted, and when the sample is purified for multiple times, the same or different chromatographic columns can be adopted.

[0012] The flow path control valve 15 controls the fraction flow into the reflux pipeline and the chromatographic column through the reflux pipeline, and can also make the fraction not produce reflux, so as to avoid diffusion and other problems.

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

[0014] According to another preferred embodiment, the gradient elution device includes two 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 is greater than 1, each group of gradient liquid storage tanks 4, the multi-channel solvent selection valve 16 and the gradient chromatographic pump 5 are connected in series through the connecting pipeline to form a gradient elution unit, and two groups of gradient elution units are connected in parallel to form the gradient elution device.

[0015] According to a third preferred embodiment, the gradient elution device includes two groups of gradient liquid storage tanks 4 and a proportional valve 17, and further includes a gradient chromatographic pump 5, and the number of gradient liquid storage tanks in each group is 1, each group of gradient liquid storage tanks 4 and the proportional valve 17 are connected in series through the connecting pipeline to form a gradient elution unit, and two groups of gradient elution units are connected in parallel, and then connected with the gradient chromatographic pump 5 through the connecting pipeline to form the gradient elution device.

[0016] According to the fourth preferred embodiment, the gradient elution device comprises two sets of gradient liquid storage tanks 4, a multi-channel solvent selection valve 16, and a proportional valve 17, further comprises one gradient chromatography pump 5, and each set of gradient liquid storage tanks is greater than one, and the two sets of gradient liquid storage tanks 4, the multi-channel solvent selection valve 16, and the proportional valve 17 are connected in series through connecting pipes as a gradient elution unit, and the two sets of gradient elution units are connected in parallel and then connected with the gradient chromatography pump 5 through connecting pipes, thereby forming the gradient elution device.

[0017] Preferably, the number of the two sets of gradient liquid storage tanks 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 one-way valve. The more channels integrated in the three-way valve, the two-position six-way valve, and the like, the fewer control points. The use of the one-way valve can not only reduce the dead volume, but also reduce the self-control point.

[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 spectrometer detector, and a Raman detector.

[0021] The parallel chromatographic columns adopt the same operation steps, use the same elution device, detection device, fraction receiving device, reflux device, and equal-length connecting pipelines, and can significantly improve the reproducibility of the purification result. Specifically, the highly identical configuration not only ensures the stability of the peak time between the parallel chromatographic columns, but also effectively solves the high consistency of the peak shape. On this basis, by accurately switching different stages according to the time parameter and / or the absorption value, the error can be controlled within a very small range.

[0022] Compared with the prior art, the technical scheme described in the utility model exhibits a series of significant advantages, which are embodied in the following aspects:

[0023] (1) The tailing material is reused, which can not only improve the purification efficiency and yield, but also ensure the stable peak time and highly consistent purification spectrum, thereby obtaining a stable impurity spectrum distribution, ensuring the reliable product quality during automatic operation, and realizing continuous automatic production; (2) The automatic cyclic purification of multiple systems is realized in the same system, which significantly improves the purification efficiency, reduces human intervention and operation errors, reduces the pollution risk, and realizes the industrialization of continuous flow purification. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 FIG. 1 is a structural schematic diagram of a double-column cyclic chromatography system according to an embodiment of the utility model;

[0025] FIG. 2 is a structural schematic diagram of a double-column cyclic chromatography system according to another embodiment of the utility model.Figure 2 Structure diagram of the double column circulation chromatography system described in Example 2;

[0026] Figure 3 Structure diagram of the double column circulation chromatography system described in Example 3;

[0027] Figure 4 Structure diagram of the double column circulation chromatography system described in Example 4;

[0028] Figure 5 Structure diagram of the double column circulation chromatography system described in Example 5, 6 when the two-position six-way valve is in state i;

[0029] Figure 6 Structure diagram of the double column circulation chromatography system described in Example 5, 6 when the two-position six-way valve is in state ii;

[0030] Figure 7 Enlarged diagram of the flow path control valve 15-2 in the double column circulation chromatography system described in Example 5, 6 when the valve is in state i;

[0031] Figure 8 Enlarged diagram of the flow path control valve 15-1 in the double column circulation chromatography system described in Example 5, 6 when the valve is in state i;

[0032] Figure 9 Enlarged diagram of the flow path control valve 15-2 in the double column circulation chromatography system described in Example 5, 6 when the valve is in state ii;

[0033] Figure 10 Enlarged diagram of the flow path control valve 15-1 in the double column circulation chromatography system described in Example 5, 6 when the valve is in state ii;

[0034] Explanation of the labels in the diagram: 1-1, First isocratic reservoir; 1-2, Second isocratic reservoir; 1-3, Third isocratic reservoir; 1-4, Fourth isocratic reservoir; 2, First multi-port solvent selection valve; 3, Isocratic pump; 4-1, First gradient reservoir; 4-2, Second gradient reservoir; 4-3, Third gradient reservoir; 4-4, Fourth gradient reservoir; 4-5, Fifth gradient reservoir; 5-1, First gradient pump; 5-2, Second gradient pump; 6, Pre-column valve; 7-1, First column; 7-2, Second column; 8, Post-column valve; 9, Detector; 10, Waste tank; 11, Multi-channel fraction collector; 12-1, First... 12-2, Second fraction collection tank; 12-3, Third fraction collection tank; 13, Waste liquid collection tank; 14, Reflux pipe; 15-1, First flow path control valve; 15-2, Second flow path control valve; 15-3, Third flow path control valve; 15-4, Fourth flow path control valve; 15-5, Fifth flow path control valve; 15-6, Sixth flow path control valve; 16, Second multi-channel solvent selection valve; 17-1, First proportional valve; 17-2, Second proportional valve; 1#, 2#, 3#, 4#, 5#, 6# are connection holes on two-position six-way valves; 11-1, 11-2, 11, 3, 11-4 are outlets of the multi-channel fraction collector. Detailed Implementation

[0035] The technical solutions of the present utility model will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are only some embodiments of the present utility model, and not all embodiments. Based on the embodiments of the present utility model, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present utility model. For example, changing the type or quantity of valves to achieve the same effect is also within the scope.

[0036] Example 1

[0037] like Figure 1 The dual-column system shown includes a first isocratic reservoir 1-1, a second isocratic reservoir 1-2, a third isocratic reservoir 1-3, a first multi-port solvent selection valve 2, an isocratic chromatographic pump 3, a first gradient reservoir 4-1, a second gradient reservoir 4-2, 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 waste liquid collection tank 13, a reflux pipe 14, a first flow path control valve 15-1, and a second flow path control valve 15-2.

[0038] 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 connected with the multi-way solvent selection valve 2, the isocratic chromatographic pump 3 and the 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 a 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 a 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 with the pre-column valve 6 through a connecting pipeline; the pre-column valve 6 is connected with the chromatographic column 7-1 and the chromatographic column 7-2 which are connected in parallel, and then connected with the post-column valve 8 through a connecting pipeline; the post-column valve 8 is connected with the detector 9 and the waste liquid tank 10 through connecting pipelines; the detector 9 is connected with the multi-channel fraction collector 11 through a connecting pipeline; the outlet 11-1, 11-2 and 11-3 of the multi-channel fraction collector 11 are connected with the first fraction collection tank 12-1, the second fraction collection tank 12-2 and the waste liquid collection tank 13 through connecting pipelines respectively; 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.

[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 used for storing balance or dilution solution, column washing solution and sample solution; the first multi-way solvent selection valve 2 is a three-way solvent selection valve used for selecting different solutions; the isocratic chromatographic pump 3 is used for solution delivery; the first gradient liquid storage tank 4-1 and the second gradient liquid storage tank 4-2 are used for storing gradient elution solution; the first gradient chromatographic pump 5-1 and the second gradient chromatographic pump 5-2 are used for delivering elution solution, and the two work together to form a gradient program; the pre-column valve 6 and the post-column valve 8 are used for selecting different liquid path directions; the detector 9 is used for monitoring absorbance; the waste liquid tank 10 is used for receiving waste liquid in the processes of balance, sample loading, dilution and column washing; the multi-channel fraction collector 11 is used for selecting different time period fractions to enter different collection tanks or waste collection tanks; the first fraction collection tank 12-1 is used for receiving the outflow liquid of the second chromatographic column 7-2, and the second fraction collection tank 12-2 is used for receiving the qualified fraction outflow from the first chromatographic column 7-1; the waste collection tank 13 is used for receiving fractions during testing and receiving unqualified waste fraction during automatic operation; the reflux pipeline 14 is used for fraction reflux; and the first flow path control valve 15-1 is used for selecting fraction access to the first fraction collection tank 12-1 or the reflux pipeline 14.

[0040] State description:

[0041] When the pre-column valve 6 is in state a, the isocratic chromatographic pump 3 communicates with the first chromatographic column 7-1 through the pre-column valve 6, and the first gradient chromatographic pump 5-1 and the second gradient chromatographic pump 5-2 communicate with the second chromatographic column 7-2 through the pre-column valve 6 in parallel;

[0042] When the pre-column valve 6 is in state b, the isocratic chromatographic pump 3 communicates with the second chromatographic column 7-2 through the pre-column valve 6, and the first gradient chromatographic pump 5-1 and the second gradient chromatographic pump 5-2 are in parallel and communicate with the first chromatographic column 7-1 through the pre-column valve 6;

[0043] The post-column valve 8 is in state a, the first chromatographic column 7-1 communicates with the waste tank 10 through the post-column valve, and the second chromatographic column 7-2 communicates with the detector 9 through the post-column valve;

[0044] The post-column valve 8 is in state b, the first chromatographic column 7-1 communicates with the detector 9 through the post-column valve, and the second chromatographic column 7-2 communicates with the waste tank 10 through the post-column valve.

[0045] The initial states of the pre-column valve and the post-column valve are both a.

[0046] Before the target time period of the fraction flow, the multi-channel fraction collector 11 communicates with the waste collection tank 13.

[0047] The working process of the chromatographic column system includes the following steps:

[0048] 1. Different chromatographic column different time period fraction test

[0049] In the traditional single column mode, the second chromatographic column 7-2 is balanced, loaded, eluted, and the fraction is collected and detected to determine the time period that meets the secondary purification requirements. The target fraction of the time period is combined and loaded into the first chromatographic column 7-1, eluted, and the qualified fraction is detected to determine the time period that the qualified fraction flows out of the first chromatographic column 7-1.

[0050] 2. The second chromatographic column 7-2 is balanced

[0051] The first multi-channel solvent selection valve 2 communicates with the first isocratic liquid storage tank 1-1, the pre-column valve 6 and the post-column valve 8 are both in state b, and the isocratic elution device is operated to balance the second chromatographic column 7-2;

[0052] 3. The second chromatographic column 7-2 is loaded

[0053] The first multi-channel solvent selection valve 2 communicates with the third isocratic liquid storage tank 1-3, the pre-column valve 6 and the post-column valve 8 are both in state b, and the isocratic elution device is operated to load the second chromatographic column 7-2;

[0054] 4. The second chromatographic column 7-2 is eluted; the first chromatographic column 7-1 is balanced and diluted and loaded

[0055] Pre-column valve 6, post-column valve 8 state is a. Running gradient elution device for the second chromatographic column 7-2 gradient elution. In the target period before the sample out, the second chromatographic column 7-2 fraction through the detector 9 to multi-channel fraction collector 11 to waste collection tank 13, and in the target period before the fraction out, the first chromatographic column 7-1 equilibrium, using the first multi-pass solvent selection valve 2 connected to the first isocratic reservoir 1-1, running isocratic elution device for the first chromatographic column 7-1 equilibrium.

[0056] 5. The first chromatographic column 7-1 elution; second chromatographic column 7-2 column washing, equilibrium, sample

[0057] Pre-column valve 6, post-column valve 8 state is b. Running gradient elution device for the first chromatographic column 7-1 gradient elution. In the target period before the fraction out, the first chromatographic column 7-1 fraction through the detector 9 to multi-channel fraction collector 11 to waste collection tank 13, and in the target period before the fraction out, the multi-channel fraction collector 11 to the second fraction collection tank 12-2 to collect the fraction. At the same time using the first multi-pass solvent selection valve 2 according to the column washing, equilibrium, sample time requirements are connected in turn the second isocratic reservoir 1-2, the first isocratic reservoir 1-1, the third isocratic reservoir 1-3, running isocratic elution device for the second chromatographic column 7-2 column washing, equilibrium, sample.

[0058] 6. The second chromatographic column 7-2 elution; first chromatographic column 7-1 column washing, equilibrium, dilution sample

[0059] Pre-column valve 6, post-column valve 8 state is a. Running the first gradient chromatographic pump 5-1, the second gradient chromatographic pump 5-2 for the second chromatographic column 7-2 gradient elution. In the target period before the fraction out, the second chromatographic column 7-2 fraction through the detector 9 to multi-channel fraction collector 11 to waste collection tank 13. At the same time according to the column washing, equilibrium time requirements are connected in turn through the first multi-pass solvent selection valve 2 the second isocratic reservoir 1-2, the first isocratic reservoir 1-1, running isocratic elution device for the first chromatographic column 7-1 column washing, equilibrium. When the target period sample out, the multi-channel fraction collector 11 to the outlet 11-1, and switch the first flow control valve 15-1, so that the time period sample into the first chromatographic column 7-1, and at the same time using the first multi-pass solvent selection valve 2 connected to the first isocratic reservoir 1-1, running isocratic elution device for the first chromatographic column 7-1 sample dilution.

[0060] 7. Loop

[0061] Repeat steps 5 and 6 above to continuously purify the sample, performing purification twice to obtain a fraction that meets the purity requirements.

[0062] Example 2

[0063] like Figure 2 The dual-column circulating chromatography system shown includes a first isocratic reservoir 1-1, a second isocratic reservoir 1-2, a third isocratic reservoir 1-3, a fourth isocratic reservoir 1-4, a first multi-port solvent selection valve 2, an isocratic chromatographic pump 3, a first gradient reservoir 4-1, a second gradient reservoir 4-2, a third gradient reservoir 4-3, a fourth gradient reservoir 4-4, 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 waste liquid collection tank 13, a reflux pipe 14, a first flow path control valve 15-1, a second multi-port solvent selection valve 16-1, and a third multi-port solvent selection valve 16-2.

[0064] The first isocratic reservoir 1-1, the second isocratic reservoir 1-2, the third isocratic reservoir 1-3, and the fourth isocratic reservoir 1-4 are connected to the multi-port solvent selection valve 2 → isocratic chromatographic pump 3 → pre-column valve 6 via connecting pipes; the first gradient reservoir 4-1 and the second gradient reservoir 4-2 are connected to the second multi-port solvent selection valve 16-1 → first gradient chromatographic pump 5-1 via connecting pipes, serving as the first gradient elution unit; the third gradient reservoir 4-3 and the fourth gradient reservoir 4-4 are connected to the third multi-port solvent selection valve 16-2 → second gradient chromatographic pump 5-2 via connecting pipes, serving as the second gradient elution unit; the first gradient elution unit and the second gradient elution unit... The units are connected in parallel to form a gradient elution device, which is connected to the pre-column valve 6 via a connecting pipe. The pre-column valve 6 is connected to the parallel-connected chromatographic columns 7-1 and 7-2 → post-column valve 8 via a connecting pipe. The post-column valve 8 is connected to the detector 9 and the waste liquid tank 10 via connecting pipes. The detector 9 is connected to the multi-channel fraction collector 11 via a connecting pipe. The outlets 11-1, 11-2, and 11-3 of the multi-channel fraction collector 11 are connected to one end of the reflux pipe 14, the first fraction collection tank 12-1, and the waste liquid collection tank 13, respectively. The other end of the reflux pipe 14 is connected between the pre-column valve 6 and the first chromatographic column 7-1 via the first flow path control valve 15-1.

[0065] Wherein: the first isocratic liquid tank 1-1, the second isocratic liquid tank 1-2, the third isocratic liquid tank 1-3, the fourth isocratic liquid tank 1-4 are used to store the balance, dilution solution, column washing solution, sample solution; the first multi-pass solvent selection valve 2 is suitable for four-channel solvent selection valve, used to select the delivery of different solutions; isocratic chromatographic pump 3 is used for solution delivery; the first gradient liquid tank 4-1, the second gradient liquid tank 4-2, the third gradient liquid tank 4-3, the fourth gradient liquid tank 4-4 are used to store gradient elution solution; the second multi-pass solvent selection valve 16-1, the third multi-pass solvent selection valve 16-2 are suitable for two-channel solvent selection valve, used to select the delivery of different solutions; the first gradient chromatographic pump 5-1, the second gradient chromatographic pump 5-2 are used to deliver elution solution, both work together to form a gradient program; pre-column valve 6, post-column valve 8 are used to select different liquid path flow direction; detector 9 is used to detect sample absorbance; waste tank 10 is used to receive the waste liquid in the process of balance, sample loading, dilution, column washing; multi-channel fraction collector 11 is used to select different time period fraction into different fraction collection tank or waste collection tank; the first fraction collection tank 12-1 is used to receive qualified fraction; waste collection tank 13 is used to receive fraction during testing, and receive unqualified waste fraction during automatic operation; reflux pipe 14 is used for fraction reflux; the first flow control valve 15-1 adopts one-way valve to inject the target time period of the second chromatographic column 7-2 into the first chromatographic column 7-1, and prevent liquid from flowing back to the reflux pipe 14.

[0066] State description:

[0067] When the pre-column valve 6 is in state a, the isocratic chromatographic pump 3 communicates with the first chromatographic column 7-1 through the pre-column valve 6, and the first gradient chromatographic pump 5-1 and the second gradient chromatographic pump 5-2 in parallel communicate with the second chromatographic column 7-2 through the pre-column valve 6;

[0068] When the pre-column valve 6 is in state b, the isocratic chromatographic pump 3 communicates with the second chromatographic column 7-2 through the pre-column valve 6, and the first gradient chromatographic pump 5-1 and the second gradient chromatographic pump 5-2 in parallel communicate with the first chromatographic column 7-1 through the pre-column valve 6;

[0069] When the post-column valve 8 is in state a, the first chromatographic column 7-1 communicates with the waste tank 10 through the post-column valve, and the second chromatographic column 7-2 communicates with the detector 9 through the post-column valve;

[0070] When the post-column valve 8 is in state b, the first chromatographic column 7-1 communicates with the detector 9 through the post-column valve, and the second chromatographic column 7-2 communicates with the waste tank 10 through the post-column valve.

[0071] The initial state of the pre-column valve and the post-column valve is a.

[0072] Before the target time period fraction flows out, the multi-channel fraction collector 11 is connected to the waste collection tank 13.

[0073] The working flow of the chromatographic column system includes the following steps:

[0074] 1. Different time period fraction test

[0075] In the traditional single column mode, the second chromatographic column 7-2 is equilibrated, loaded, eluted, and the time period reaching the secondary purification requirement is determined by collecting and testing the target fraction. The target fraction of the time period is combined and loaded into the first chromatographic column 7-1, eluted, and the time period of the qualified fraction flowing out of the first chromatographic column 7-1 is determined.

[0076] 2. Equilibrium of the second chromatographic column 7-2

[0077] The first multi-pass solvent selection valve 2 is connected to the first isocratic liquid tank 1-1, the pre-column valve 6 and the post-column valve 8 are both in state b, and the isocratic elution device is operated to equilibrate the second chromatographic column 7-2.

[0078] 3. Loading of the second chromatographic column 7-2

[0079] The first multi-pass solvent selection valve 2 is connected to the fourth isocratic liquid tank 1-4, the pre-column valve 6 and the post-column valve 8 are both in state b, and the isocratic elution device is operated to load the second chromatographic column 7-2.

[0080] 4. Elution of the second chromatographic column 7-2; equilibrium and dilution loading of the first chromatographic column 7-1

[0081] The pre-column valve 6 and the post-column valve 8 are both in state a. The second multi-pass solvent selection valve 16-1 is connected to the first gradient liquid tank 4-1, the third multi-pass solvent selection valve 16-2 is connected to the third gradient liquid tank 4-3, and the gradient elution device is operated to perform gradient elution on the second chromatographic column 7-2. Before the target time period fraction flows out, the fraction of the second chromatographic column 7-2 passes through the detector 9→ the multi-channel fraction collector 11 outlet 11-3→ the waste collection tank 13, and before the target time period fraction flows out, the first multi-pass solvent selection valve 2 is connected to the first isocratic liquid tank 1-1, and the isocratic elution device is operated to equilibrate the first chromatographic column 7-1, completing the first chromatographic column 7-1 equilibrium program. When the target time period fraction of the second chromatographic column 7-2 flows out, the multi-channel fraction collector outlet is switched to 11-1, so that the fraction of the time period flows into the first chromatographic column 7-1 through the reflux pipe 14 and the first flow path control valve 15-1, and at the same time, the first multi-pass solvent selection valve 2 is connected to the second isocratic liquid tank 1-2, and the isocratic elution device is operated to dilute the sample flowing into the first chromatographic column 7-1.

[0082] 5. Elution of the first chromatographic column 7-1; washing, equilibration, and loading of the second chromatographic column 7-2

[0083] Pre-column valve 6, post-column valve 8 state is b. Switch the second multi-pass solvent selection valve 16-1 to the second gradient reservoir 4-2, the third multi-pass solvent selection valve 16-2 to the fourth gradient reservoir 4-4, run the gradient elution device to gradient elute the first chromatographic column 7-1. Before the target time period fraction flows out, the first chromatographic column 7-1 fraction passes through the detector 9→ multi-channel fraction collector outlet 11-3→ waste collection tank 13, and when the target time period fraction flows out, switch the multi-channel fraction collector outlet to 11-2 to connect the first fraction collection tank 12-1 to collect the sample. At the same time of elution, use the first multi-pass solvent selection valve 2 to connect the third isocratic reservoir 1-3, the first isocratic reservoir 1-1, and the fourth isocratic reservoir 1-4 in turn according to the column washing, equilibration, and sample loading time requirements, and run the isocratic elution device to wash, equilibrate, and load the sample on the second chromatographic column 7-2.

[0084] 6. Elution of the second chromatographic column 7-2; column washing, equilibration, and sample loading dilution of the first chromatographic column 7-1

[0085] Pre-column valve 6, post-column valve 8 state is a. Switch the second multi-pass solvent selection valve 16-1 to the first gradient reservoir 4-1, the third multi-pass solvent selection valve 16-2 to the third gradient reservoir 4-3, and run the gradient elution device to gradient elute the second chromatographic column 7-2. Before the target time period fraction flows out, the second chromatographic column 7-2 fraction passes through the detector 9→ multi-channel fraction collector outlet 11-3→ waste collection tank 13, and before the target time period fraction flows out, use the first multi-pass solvent selection valve 2 to connect the third isocratic reservoir 1-3, the first isocratic reservoir 1-1 in turn according to the time program, and run the isocratic elution device to wash and equilibrate the first chromatographic column 7-1, completing the column washing and equilibration program of the first chromatographic column 7-1. When the target time period fraction 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 flows into the first chromatographic column 7-1 through the reflux pipe 14 and the first flow control valve 15-1, and at the same time, use the first multi-pass solvent selection valve 2 to connect the second isocratic reservoir 1-2, and run the isocratic elution device to dilute the sample flowing into the first chromatographic column 7-1.

[0086] 7. Cycle

[0087] Cycle the above steps 5 and 6 to continuously purify the sample, so that it is purified twice to obtain a fraction meeting the purity requirements.

[0088] Example 3

[0089] As Figure 3A dual column recycling chromatography system is shown, including a first isocratic reservoir 1-1, a second isocratic reservoir 1-2, a third isocratic reservoir 1-3, a first multi-port solvent selection valve 2, an isocratic pump 3, a first gradient reservoir 4-1, a second gradient reservoir 4-2, a third gradient reservoir 4-3, a fourth gradient reservoir 4-4, a gradient pump 5, a pre-column valve 6, a first chromatography column 7-1, a second chromatography column 7-2, a post-column valve 8, a detector 9, a waste reservoir 10, a multi-port fraction collector 11, a first fraction collection reservoir 12-1, a second fraction collection reservoir 12-2, a third fraction collection reservoir 12-3, a waste collection reservoir 13, a first return line 14-1, a second return line 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-port solvent selection valve 16-1, a third multi-port solvent selection valve 16-2, a first proportioning valve 17-1, a second proportioning valve 17-2.

[0090] 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 connected with 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 second gradient liquid storage tank 4-2 are connected with the second multi-way solvent selection valve 16-1→first proportional valve 17-1 through connecting pipelines, serving as a first gradient elution unit; the third gradient liquid storage tank 4-3 and the fourth gradient liquid storage tank 4-4 are connected with the third multi-way solvent selection valve 16-2→second proportional valve 17-2 through connecting pipelines, serving as a 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 with the gradient chromatographic pump 5, and the gradient elution device is connected with the pre-column valve 6 through a connecting pipeline; the pre-column valve 6 is connected with the parallelly arranged chromatographic column 7-1, chromatographic column 7-2→post-column valve 8 through a connecting pipeline; the post-column valve 8 is connected with the detector 9 and waste liquid tank 10 through connecting pipelines; the detector 9 is connected with the multi-channel fraction collector 11 through a connecting pipeline, and the multi-channel fraction collector outlets 11-1, 11-2, 11-3, 11-4 are connected with 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 respectively; one end of the first reflux pipeline 14-1 is connected between the multi-channel fraction collector outlet 11-1 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 with the fifth flow path control valve 15-5 and the sixth flow path control valve 15-6 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 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 multi-channel fraction collector outlet 11-3 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 with the seventh flow path control valve 15-7 and the eighth flow path control valve 15-8 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 between the pre-column valve 6 and the second chromatographic column 7-2 through the eighth flow path control valve 15-8.

[0091] Wherein: the first isocratic liquid tank 1-1, the second isocratic liquid tank 1-2, the third isocratic liquid tank 1-3 are used to store the balance and dilution solution, column washing solution, sample solution; the first multi-pass solvent selection valve 2 is suitable for three-channel solvent selection valve, used for selecting to transport different solution; isocratic chromatographic pump 3 is used for solution delivery; the first gradient liquid tank 4-1, the second gradient liquid tank 4-2, the third gradient liquid tank 4-3, the fourth gradient liquid tank 4-4 are used to store gradient elution solution; the second multi-pass solvent selection valve 16-1, the third multi-pass solvent selection valve 16-2 are suitable for two-channel solvent selection valve, used for selecting to transport different solution; the first proportional valve 17-1 and the second proportional valve 17-2 are connected in parallel, and the two cooperate with the gradient chromatographic pump 5 to form a gradient program; the pre-column valve 6, the post-column valve 8 are used to select different liquid path flow direction; the detector 9 is used for detecting sample absorption value; the waste liquid tank 10 is used to receive the waste liquid in the process of balance, sample loading, dilution, column washing and the like; the multi-channel fraction collector 11 is used to select different time period fraction into different collection tank or waste collection tank; the first fraction collection tank 12-1 is used to receive the peak front fraction with recycling value, the second fraction collection tank 12-2 is used to receive qualified fraction, the third fraction collection tank 12-3 is used to receive the peak rear fraction with recycling value; the waste collection tank 13 is used to receive unqualified waste fraction; the first reflux pipe 14-1 is used to reflux the peak front fraction with recycling value, the second reflux pipe 14-2 is used to reflux the peak rear fraction with recycling value; the flow path control valve is all stop valve, the first flow path control valve 15-1, the second flow path control valve 15-2 are used to control the fraction flowing out of the multi-channel fraction collector to flow into the reflux pipe 14-1 or the fraction collection tank 12-1; the third flow path control valve 15-3, the fourth flow path control valve 15-4 are used to control the fraction flowing out of the multi-channel fraction collector to flow into the reflux pipe 14-2 or the fraction collection tank 12-3; the fifth flow path control valve 15-5, the sixth flow path control valve 15-6 are used to control the fraction in the reflux pipe 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, the eighth flow path control valve 15-8 are used to control the fraction in the reflux pipe 14-2 to enter the first chromatographic column 7-1 or the second chromatographic column 7-2.

[0092] State description:

[0093] When the pre-column valve 6 is in state a, the isocratic chromatographic pump 3 communicates with the first chromatographic column 7-1 through the pre-column valve 6, and the parallel gradient chromatographic pump 5 communicates with the second chromatographic column 7-2 through the pre-column valve 6;

[0094] When the pre-column valve 6 is in state b, the isocratic chromatographic pump 3 communicates with the second chromatographic column 7-2 through the pre-column valve 6, and the parallel gradient chromatographic pump 5 communicates with the first chromatographic column 7-1 through the pre-column valve 6;

[0095] When the post-column valve 8 is in state a, the first chromatographic column 7-1 is connected to the waste tank 10 through the post-column valve, and the second chromatographic column 7-2 is connected to the detector 9 through the post-column valve.

[0096] 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 the second chromatographic column 7-2 is connected to the waste tank 10 through the post-column valve.

[0097] The initial state of the flow path control valve is closed.

[0098] The initial state of the pre-column valve and the post-column valve is a.

[0099] Before the target time period of the fraction flow, the multi-channel fraction collector 11 is connected to the waste collection tank 13.

[0100] The working process of the chromatographic column system includes the following steps:

[0101] 1. Different time period fraction test of different chromatographic columns

[0102] In the traditional single column mode, the first chromatographic column 7-1 and the second chromatographic column 7-2 are respectively balanced, loaded, and eluted, and 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 peak pre-fraction time period with recovery value, the qualified fraction time period, and the peak post-fraction time period with recovery value.

[0103] 2. Balancing and loading of the first chromatographic column 7-1

[0104] According to the time period requirement, the first multi-channel solvent selection valve 2 is sequentially switched to the first isocratic liquid reservoir 1-1 and the third isocratic liquid reservoir 1-3, and the isocratic chromatographic pump 3 is operated to balance and load the first chromatographic column 7-1.

[0105] 3. Elution of the first chromatographic column 7-1, column washing, balancing, and loading of the second chromatographic column 7-2

[0106] The states of the pre-column valve 6 and the post-column valve 8 are both switched to b. The first chromatographic column 7-1 is gradient eluted using the gradient elution device, which is divided into two stages: in the first stage, the first gradient liquid reservoir 4-1 and the third gradient liquid reservoir 4-3 are used, the proportions are adjusted through the first proportional valve 17-1 and the second proportional valve 17-2, and the gradient chromatographic pump 5 is operated to elute the first chromatographic column 7-1; in the second stage, the second gradient liquid reservoir 4-2 and the fourth gradient liquid reservoir 4-4 are used, the proportions are adjusted through the first proportional valve 17-1 and the second proportional valve 17-2, and the gradient chromatographic pump 5 is operated to elute the first chromatographic column 7-1.

[0107] Before the time period of pre-peak fraction with recycling value, according to the time period requirement, switch the first multi-pass solvent selection valve 2 to the second isocratic liquid storage tank 1-2 and the first isocratic liquid storage tank 1-1 in turn, and run the isocratic chromatographic pump 3 to complete the column washing and equilibrium program of the second chromatographic column 7-2. When the time period of pre-peak fraction with recycling value appears, switch the multi-pass fraction collector outlet 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 passes through the first reflux pipe 14-1 and enters the second chromatographic column 7-2, and at the same time, start the isocratic chromatographic pump 3, switch the first multi-pass solvent selection valve 2 to the first isocratic liquid storage tank 1-1, and use the solution to dilute the fraction entering the second chromatographic column 7-2; when the time period of pre-peak fraction with recycling value is completed, switch the multi-pass fraction collector outlet to 11-2 to communicate with the second fraction collection tank 12-2 to collect the fraction, and at the same time, start the isocratic chromatographic pump 3, switch the first multi-pass solvent selection valve 2 to the third isocratic liquid storage tank 1-3, and perform sample loading on the second chromatographic column 7-2; when the time period of post-peak fraction with recycling value appears, switch the multi-pass fraction collector outlet 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 passes through the second reflux pipe 14-2 and enters the second chromatographic column 7-2, and at the same time, start the isocratic chromatographic pump 3, switch the first multi-pass solvent selection valve 2 to the first isocratic liquid storage tank 1-1, and use the solution to dilute the fraction entering the second chromatographic column 7-2;

[0108] 4. Elution of the second chromatographic column 7-2, column washing, equilibrium and sample loading of the first chromatographic column 7-1

[0109] Switch the state of pre-column valve 6 and post-column valve 8 to a respectively. Use the gradient elution device to perform gradient elution on the second chromatographic column 7-2, which is divided into two stages: the first stage uses the first gradient liquid tank 4-1 and the third gradient liquid tank 4-3, adjusts the proportion through the first proportional valve 17-1 and the second proportional valve 17-2, and operates the gradient chromatographic pump 5 to elute the second chromatographic column 7-2; the second stage uses the second gradient liquid tank 4-2 and the fourth gradient liquid tank 4-4, adjusts the proportion through the first proportional valve 17-1 and the second proportional valve 17-2, and operates the gradient chromatographic pump 5 to elute the second chromatographic column 7-2. Before the time period of the pre-peak fraction with recycling value, switch the first multi-pass solvent selection valve 2 to the second isocratic liquid tank 1-2 and the first isocratic liquid tank 1-1 in turn, and operate the isocratic chromatographic pump 3 to complete the column washing and balancing program of the first chromatographic column 7-1. When the time period of the pre-peak fraction with recycling value appears, switch the multi-channel fraction collector outlet to 11-1, open the second flow path control valve 15-2 and the fifth flow path control valve 15-5, so that the pre-peak fraction enters the first chromatographic column 7-1 through the first reflux pipe 14-1, and at the same time, start the isocratic chromatographic pump 3, switch the first multi-pass solvent selection valve 2 to the first isocratic liquid tank 1-1, and use the solution to dilute the pre-peak fraction entering the first chromatographic column 7-1; when the time period of the pre-peak fraction with recycling value is completed, switch the multi-channel fraction collector outlet to 11-2 to communicate with the second fraction collection tank 12-2 to collect the sample, and at the same time, start the isocratic chromatographic pump 3, switch the first multi-pass solvent selection valve 2 to the third isocratic liquid tank 1-3, and load the sample into the first chromatographic column 7-1; when the time period of the post-peak fraction with recycling value appears, switch the multi-channel fraction collector outlet 11-3 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 reflux pipe 14-2, and at the same time, start the isocratic chromatographic pump 3, switch the first multi-pass solvent selection valve 2 to the first isocratic liquid tank 1-1, and use the solution to dilute the fraction entering the first chromatographic column 7-1;

[0110] 5. Cycle

[0111] Cycle the above steps 3-4 to continuously purify the sample and obtain qualified fractions.

[0112] Example 4

[0113] As Figure 4A double column circulation chromatography system is shown, including a first isocratic liquid tank 1-1, a second isocratic liquid tank 1-2, a third isocratic liquid tank 1-3, a fourth isocratic liquid tank 1-4, a first multi-pass solvent selection valve 2, an isocratic chromatography pump 3, a first gradient liquid tank 4-1, a second gradient liquid tank 4-2, a gradient chromatography pump 5, a pre-column valve 6, a first chromatography column 7-1, a second chromatography column 7-2, a post-column valve 8, a detector 9, a waste liquid tank 10, a multi-pass 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.

[0114] The first isocratic liquid tank 1-1, the second isocratic liquid tank 1-2, the third isocratic liquid tank 1-3, and the fourth isocratic liquid tank 1-4 are connected to the multi-pass solvent selection valve 2, the isocratic chromatography pump 3, and the pre-column valve 6 through connecting pipelines; the first gradient liquid tank 4-1 is connected to the first proportional valve 17-1 through a connecting pipeline, serving as a first gradient elution unit; the second gradient liquid tank 4-2 is connected to the second proportional valve 17-2 through a connecting pipeline, serving as a second gradient elution unit; the first gradient elution unit and the second gradient elution unit are connected in parallel, and then connected to the gradient chromatography pump 5, forming a gradient elution device; 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 parallelly arranged chromatography column 7-1 and chromatography column 7-2, and then connected to the post-column valve 8 through a connecting pipeline; the post-column valve 8 is connected to the parallelly arranged detector 9 and waste liquid tank 10 through a connecting pipeline; the detector 9 is connected to the multi-pass fraction collector 11 through a connecting pipeline; the outlet 11-1, 11-2, 11-3, and 11-4 of the multi-pass fraction collector 11 are 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, respectively; one end of the reflux pipeline 14 is connected between the outlet 11-1 of the multi-pass fraction collector and the first fraction collection tank 12-1 through the first flow path control valve 15-1; the other end of the reflux pipeline 14 is connected to the second flow path control valve 15-2, and then connected to the parallelly arranged third flow path control valve 15-3 and fourth flow path control valve 15-4, and then connected between the pre-column valve 6 and the first chromatography column 7-1 through the third flow path control valve 15-3, and connected between the pre-column valve 6 and the second chromatography column 7-2 through the fourth flow path control valve 15-4,

[0115] Wherein: the first isocratic liquid tank 1-1, the second isocratic liquid tank 1-2, the third isocratic liquid tank 1-3, the fourth isocratic liquid tank 1-4 are used to store the balance solution, dilution solution, column washing solution, sample solution respectively; the first multi-pass solvent selection valve 2 is suitable for four-channel solvent selection valve, used for selecting to transport different solutions; the isocratic chromatographic pump 3 is used for solution delivery; the first gradient liquid tank 4-1, the second gradient liquid tank 4-2 are used to store gradient elution solution; the first proportional valve 17-1 and the second proportional valve 17-2 are connected in parallel, and the two 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 path flow directions; the detector 9 is used to detect the sample absorption value; the waste liquid tank 10 is used to receive waste liquid in the processes of balance, sample loading, dilution, column washing and the like; the multi-channel fraction collector 11 is used to select different absorption value fractions in different time periods to enter different collection tanks or waste collection tanks; the first fraction collection tank 12-1 is used to receive fractions and pre-peak fractions, the second fraction collection tank 12-2, the third fraction collection tank 12-3 are used to receive fractions; the waste collection tank 13 receives unqualified waste fraction; the reflux pipeline 14 is used to return the fraction; the first flow path control valve 15-1, the second flow path control valve 15-2 are all three-way valves, the first flow path control valve 15-1 controls the fraction flow direction to the reflux pipeline 14 or the first fraction collection tank 12-1, the second flow path control valve 15-2 controls the fraction flow direction 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, the fourth flow path control valve 15-4 are selected to be one-way valves, which control the one-way flow direction of liquid to the chromatographic column 7-1 or 7-2, and prevent liquid backflow and pollution.

[0116] State description:

[0117] When the pre-column valve 6 is in state a, the isocratic chromatographic pump 3 communicates with the first chromatographic column 7-1 through the pre-column valve 6, and the parallel gradient chromatographic pump 5 communicates with the second chromatographic column 7-2 through the pre-column valve 6;

[0118] When the pre-column valve 6 is in state b, the isocratic chromatographic pump 3 communicates with the second chromatographic column 7-2 through the pre-column valve 6, and the parallel gradient chromatographic pump 5 communicates with the first chromatographic column 7-1 through the pre-column valve 6;

[0119] When the post-column valve 8 is in state a, the first chromatographic column 7-1 communicates with the waste liquid tank 10 through the post-column valve, and the second chromatographic column 7-2 communicates with the detector 9 through the post-column valve;

[0120] When the post-column valve 8 is in state b, the first chromatographic column 7-1 communicates with the detector 9 through the post-column valve, and the second chromatographic column 7-2 communicates with the waste liquid tank 10 through the post-column valve.

[0121] The initial states of the pre-column valve and the post-column valve are both a.

[0122] Before the target time period fraction flows out, the multi-channel fraction collector 11 is connected to the waste collection tank 13.

[0123] The working flow of the chromatographic column system includes the following steps:

[0124] 1. Different interval fraction test of different chromatographic columns

[0125] In the traditional single column mode, the first chromatographic column 7-1 and the second chromatographic column 7-2 are respectively balanced, 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 pre-peak fraction with recycling value and the qualified fraction interval. The interval is determined by combining time and absorption value.

[0126] 2. Balancing and loading of the first chromatographic column 7-1

[0127] According to the time period requirement, the first multi-pass solvent selection valve 2 is switched to the first isocratic liquid tank 1-1 and the fourth isocratic liquid tank 1-4 in turn, and the isocratic chromatographic pump 3 is operated to balance and load the first chromatographic column 7-1.

[0128] 3. Elution of the first chromatographic column 7-1, column washing, balancing, and loading of the second chromatographic column 7-2

[0129] The pre-column valve 6 and the post-column valve 8 are both switched to b. The first chromatographic column 7-1 is gradient eluted using the gradient elution device. Before the pre-peak fraction interval with recycling value appears, the first multi-pass solvent selection valve 2 is switched to the third isocratic liquid tank 1-3 and the first isocratic liquid tank 1-1 in turn, and the isocratic chromatographic pump 3 is operated to complete the column washing and balancing of the second chromatographic column 7-2. When the pre-peak fraction interval with recycling value appears, the multi-channel fraction collector outlet is switched to 11-1, the first flow path control valve 15-1 is switched to communicate with the reflux pipeline 14, the second flow path control valve 15-2 is switched to communicate with the fourth flow path control valve 15-4, and the fraction passes through the flow path to enter the second chromatographic column 7-2. At the same time, the isocratic chromatographic pump 3 is started, the first multi-pass solvent selection valve 2 is switched to the second isocratic liquid tank 1-2, and the fraction entering the second chromatographic column 7-2 is diluted using the solution. When the pre-peak fraction interval with recycling value is completed, the multi-channel fraction collector outlet 11-2 or 11-3 is switched to communicate with the second fraction collection tank 12-2 or the third fraction collection tank 12-3, and the fraction is collected according to the qualified fraction interval parameters. At the same time, the isocratic chromatographic pump 3 is started, the first multi-pass solvent selection valve 2 is switched to the fourth isocratic liquid tank 1-4, and the second chromatographic column 7-2 is loaded.

[0130] 4. Elution of the second chromatographic column 7-2, column washing, balancing, and loading of the first chromatographic column 7-1

[0131] Switch the states of both the pre-column valve 6 and the post-column valve 8 to state 'a'. Use a gradient elution device to perform gradient elution on the second chromatographic column 7-2. Before the pre-peak fraction with recovery value, sequentially switch to the third isocratic reservoir 1-3 and the first isocratic reservoir 1-1 through the first multi-port solvent selection valve 2, and run the isocratic chromatographic pump 3 to complete the washing and equilibration program of the first chromatographic column 7-1. When a pre-peak fraction with recoverable value appears, switch the multi-channel fraction collector outlet 11-1, switch the first flow path control valve 15-1 to connect to the reflux pipe 14, and switch the second flow path control valve 15-2 to connect to the third flow path control valve 15-3, allowing the fraction to enter the first chromatographic column 7-1 through the flow path. At the same time, start the isocratic chromatographic pump 3, and switch the first multi-port solvent selection valve 2 to connect to the second isocratic reservoir 1-2 to dilute the fraction entering the first chromatographic column 7-1. When the pre-peak fraction with recoverable value is completed, switch the multi-channel fraction collector outlet 11-2 or 11-3 to the second fraction collection tank 12-2 or the third fraction collection tank 12-3, collect the fraction according to the qualified fraction range parameters, and at the same time, start the isocratic chromatographic pump 3, and switch the first multi-port solvent selection valve 2 to connect to the fourth isocratic reservoir 1-4 to load the sample onto the first chromatographic column 7-1.

[0132] 5. Loop

[0133] Repeat steps 3 and 4 above to continuously purify the sample and obtain qualified fractions.

[0134] Example 5

[0135] like Figure 5 The illustrated dual-column system includes a first isocratic reservoir 1-1, a second isocratic reservoir 1-2, a third isocratic reservoir 1-3, a fourth isocratic reservoir 1-4, a first multi-port solvent selection valve 2, an isocratic chromatographic pump 3, a first gradient reservoir 4-1, a second gradient reservoir 4-2, a third gradient reservoir 4-3, a fourth gradient reservoir 4-4, a fifth gradient reservoir 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, and a post-column valve. Valve 8, Detector 9, Waste liquid tank 10, Multi-channel fraction collector 11, First fraction collection tank 12-1, Second fraction collection tank 12-2, Third fraction collection tank 12-3, Waste liquid collection tank 13, Reflux pipe 14, First flow path control valve 15-1, Second flow path control valve 15-2, Third flow path control valve 15-3, Fourth flow path control valve 15-4, Fifth flow path control valve 15-5, Sixth flow path control valve 15-6, Second multi-port solvent selection valve 16-1, Third multi-port solvent selection valve 16-2.

[0136] The first equal-graduation liquid storage tank 1-1, the second equal-graduation liquid storage tank 1-2, the third equal-graduation liquid storage tank 1-3, and the fourth equal-graduation liquid storage tank 1-4 are connected with the multi-way solvent selection valve 2→the equal-graduation chromatographic pump 3→the pre-column valve 6 through the connecting pipeline; 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 connected with the second multi-way solvent selection valve 16-1→the first gradient chromatographic pump 5-1 through the connecting pipeline respectively as the first gradient elution unit; the fourth gradient liquid storage tank 4-4 and the fifth gradient liquid storage tank 4-5 are connected with the third multi-way solvent selection valve 16-2→the second gradient chromatographic pump 5-2 through the connecting pipeline respectively as the second gradient elution unit; the first gradient elution unit and the second gradient elution unit are connected in parallel to constitute the gradient elution device, and the gradient elution device is connected with the pre-column valve 6 through the connecting pipeline; the pre-column valve 6 is connected with the chromatographic column 7-1 and the chromatographic column 7-2→the post-column valve 8 through the connecting pipeline and is arranged in parallel; the post-column valve 8 is connected with the detector 9 and the waste liquid tank 10 through the connecting pipeline respectively; the detector 9 is connected with the multi-channel fraction collector 11 through the connecting pipeline, and the fraction collector 11

[0137] The multi-channel fraction collector outlet 11-1 is connected to the first fraction collector 12-1 through the first flow path control valve 15-1, the multi-channel fraction collector outlet 11-2 is connected with the second fraction collector 12-2 through the connecting pipeline, the multi-channel fraction collector outlet 11-3 is connected to the third fraction collector 12-3 through the first flow path control valve 15-1, and the multi-channel fraction collector outlet 11-4 is connected with the waste liquid collector 13 through the connecting pipeline. One end of the first reflux pipeline 14-1 is connected with the multi-channel fraction collector outlet 11-1 through the first flow path control valve 15-1, the other end is sequentially connected with the third flow path control valve 15-3 and the fourth flow path control valve 15-4 which are 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 reflux pipeline 14-2 is connected with the multi-channel fraction collector outlet 11-3 through the first flow path control valve 15-1, the other end is sequentially connected with the fifth flow path control valve 15-5 and the sixth flow path control valve 15-6 which are 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.

[0138] Wherein: the first isocratic liquid tank 1-1, the second isocratic liquid tank 1-2, the third isocratic liquid tank 1-3, the fourth isocratic liquid tank 1-4 are used for storing the balance solution, dilution solution, column washing solution, sample solution respectively; the first multi-pass solvent selection valve 2 is suitable for four-channel solvent selection valve, used for selecting to transport different solutions; the isocratic chromatographic pump 3 is used for solution delivery; the first gradient liquid tank 4-1, the second gradient liquid tank 4-2, the third gradient liquid tank 4-3, the fourth gradient liquid tank 4-4, the fifth gradient liquid tank 4-5 are used for storing gradient elution solution; the second multi-pass solvent selection valve 16-1 is suitable for three-channel solvent selection valve, the third multi-pass solvent selection valve 16-2 is suitable for two-channel solvent selection valve, used for selecting to transport different solutions; the first gradient chromatographic pump 5-1, the second gradient chromatographic pump 5-2 are used for transporting elution solution, both work together to form a gradient program; the pre-column valve 6, the post-column valve 8 are used for selecting different liquid path flow direction; the detector 9 is used for detecting sample absorbance; the waste tank 10 is used for receiving waste liquid in the process of balance, sample loading, dilution, column washing; the multi-channel fraction collector 11 is used for switching different outlets; the first fraction collection tank 12-1, the second fraction collection tank 12-2, the third fraction collection tank 12-3 are used for receiving fractions; the waste material collection tank 13 is used for receiving unqualified waste material fractions; the reflux pipeline is used for fraction reflux; the first flow control valve 15-1 adopts two-position six-way valve, controls the fraction to enter the reflux pipeline or enter the first fraction collection tank 12-1, the third fraction collection tank 12-3; the second flow control valve 15-2 adopts two-position six-way valve, controls the fraction to enter different chromatographic columns; the third flow control valve 15-3, the fourth flow control valve 15-4, the fifth flow control valve 15-5, the sixth flow control valve 15-6 are all selected to use one-way valve, prevent liquid backflow. The first reflux pipeline 14-1, the second reflux pipeline 14-2 are used for transporting liquid.

[0139] State description:

[0140] When the pre-column valve 6 is in state a, the isocratic chromatographic pump 3 communicates with the first chromatographic column 7-1 through the pre-column valve 6, and the first gradient chromatographic pump 5-1 and the second gradient chromatographic pump 5-2 in parallel communicate with the second chromatographic column 7-2 through the pre-column valve 6;

[0141] When the pre-column valve 6 is in state b, the isocratic chromatographic pump 3 communicates with the second chromatographic column 7-2 through the pre-column valve 6, and the first gradient chromatographic pump 5-1 and the second gradient chromatographic pump 5-2 in parallel communicate with the first chromatographic column 7-1 through the pre-column valve 6;

[0142] When the post-column valve 8 is in state a, the first chromatographic column 7-1 communicates with the waste tank 10 through the post-column valve, and the second chromatographic column 7-2 communicates with the detector 9 through the post-column valve at the same time;

[0143] When the post-column valve 8 is in state b, the first chromatographic column 7-1 is connected with the detector 9 through the post-column valve, and the second chromatographic column 7-2 is connected with the waste tank 10 through the post-column valve.

[0144] The first flow path control valve 15-1 and the second flow path control valve 15-2 are two-position six-way valves, each having six connection holes, i.e., 1#, 2#, 3#, 4#, 5# and 6#.

[0145] 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 a continuous purification program is run and the fractions need to be recycled, state i is used: the multi-channel fraction collector outlet 11-1 is connected with the first reflux pipeline 14-1, and the multi-channel fraction collector outlet 11-3 is connected with the second reflux pipeline 14-2. When the fractions of the first purification or the second purification need to be collected, state ii is used: the multi-channel fraction collector outlet 11-1 is connected with the first fraction collection tank 12-1, and the multi-channel fraction collector outlet 11-3 is connected with the third fraction collection tank 12-3.

[0146] 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 a continuous purification program is run and the fractions need to be recycled, state i is used: the multi-channel fraction collector outlet 11-1 is connected with the first reflux pipeline 14-1, and the multi-channel fraction collector outlet 11-3 is connected with the second reflux pipeline 14-2. When the fractions of the first purification or the second purification need to be collected, state ii is used: the multi-channel fraction collector outlet 11-1 is connected with the first fraction collection tank 12-1, and the multi-channel fraction collector outlet 11-3 is connected with the third fraction collection tank 12-3.

[0147] The pre-column valve and the post-column valve are both in state a initially.

[0148] Before the fractions flow out of the target time period, the multi-channel fraction collector 11 is connected with the waste collection tank 13.

[0149] The working process of the chromatographic column system includes the following steps:

[0150] 1. Different chromatographic column different interval fraction test

[0151] The first chromatographic column 7-1 is balanced, sample is loaded, and elution is performed, and the fraction is 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 a qualified interval of one-time purification, i.e. a fraction interval to be subjected to secondary purification, which is determined by combining time and absorption value. The sample in the interval is combined, and the sample is loaded into the second chromatographic column 7-2, and the final qualified sample interval is determined after elution, which is determined by combining time and absorption value.

[0152] 2. Balancing, sample loading of the first chromatographic column 7-1

[0153] According to the time period requirement, the first multi-pass solvent selection valve 2 is sequentially switched to the first isocratic liquid storage tank 1-1 and the fourth isocratic liquid storage tank 1-4, and the isocratic chromatographic pump 3 is operated to balance and load the sample into the first chromatographic column 7-1.

[0154] 3. Elution of the first chromatographic column 7-1, column washing, balancing, and sample loading of the second chromatographic column 7-2

[0155] The pre-column valve 6 and the post-column valve 8 are both switched to state b. The first chromatographic column 7-1 is gradient eluted using a gradient elution device, the second multi-pass solvent selection valve 16-2 and the third multi-pass solvent selection valve 16-3 are switched to connect the first gradient liquid storage tank 4-1 and the fourth gradient liquid storage tank 4-4, and the corresponding first gradient chromatographic pump 5-1 and second gradient chromatographic pump 5-2 are operated to elute the first chromatographic column 7-1. Before the qualified interval of one-time purification appears, the first multi-pass solvent selection valve 2 is sequentially switched to the third isocratic liquid storage tank 1-3 and the first isocratic liquid storage tank 1-1, and the isocratic chromatographic pump 3 is operated to complete the column washing and balancing of the second chromatographic column 7-2. When the qualified interval of one-time purification appears, the multi-pass fraction collector outlet 11-1 is switched, the first flow path control valve 15-1 is switched to state i, and the second flow path control valve 15-2 is switched to state ii, so that the fraction passes through the multi-pass fraction collector 11 to the first reflux pipe 14-1 and enters the second chromatographic column 7-2, and at the same time, the isocratic chromatographic pump 3 is started, the first multi-pass solvent selection valve 2 is switched to the second isocratic liquid storage tank 1-2, and the fraction entering the second chromatographic column 7-2 is diluted using the solution.

[0156] 4. Column washing, balancing, and sample loading of the first chromatographic column 7-1, and elution of the second chromatographic column 7-2

[0157] Switch the state of pre-column valve 6 and post-column valve 8 to a respectively. Use the gradient elution device to perform gradient elution on the second chromatographic column 7-2, which is divided into two stages: in the first stage, switch the second multi-pass solvent selection valve 16-1 and the third multi-pass solvent selection valve 16-3 to use the second gradient reservoir 4-2 and the fifth gradient reservoir 4-5, respectively, to 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-pass solvent selection valve 16-1 and the third multi-pass solvent selection valve 16-3 to use the third gradient reservoir 4-3 and the fifth gradient reservoir 4-5, respectively, to 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 the secondary purification appears, switch the multi-channel fraction collector outlet 11-2 to communicate with the second fraction collection tank 12-2 to collect the sample. At the same time of gradient elution of the second chromatographic column 7-2, use the first multi-pass solvent selection valve 2 to sequentially connect the third isocratic reservoir 1-3, the first isocratic reservoir 1-1, and the fourth isocratic reservoir 1-4 according to the column washing, equilibration, and sample loading time requirements, and use the isocratic elution device to wash, equilibrate, and load the sample on the first chromatographic column 7-1.

[0158] 5. Cycle

[0159] Cycle steps 3 and 4 above to continuously purify the sample and obtain a sample meeting the purity requirements.

[0160] Example 6

[0161] Another working process of the double-column cycle chromatography system described in Example 5 includes the following steps:

[0162] 1. Different time intervals of different chromatographic columns

[0163] Use the traditional single-column method to equilibrate, load, and elute the first chromatographic column 7-1 and the second chromatographic column 7-2 respectively, and collect the sample 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 interval of the pre-peak fraction with recycling value, the qualified fraction time interval, and the post-peak fraction with recycling value.

[0164] 2. Equilibrate and load the first chromatographic column 7-1

[0165] According to the time interval requirements, sequentially switch the first multi-pass solvent selection valve 2 to the first isocratic reservoir 1-1 and the fourth isocratic reservoir 1-4, and operate the isocratic chromatographic pump 3 to equilibrate and load the first chromatographic column 7-1.

[0166] 3. Elute the first chromatographic column 7-1 and wash, equilibrate, and load the second chromatographic column 7-2

[0167] Switch the state of pre-column valve 6 and post-column valve 8 to b respectively. Gradient elution is performed on the first chromatographic column 7-1 using the gradient elution device. The elution is divided into two stages: in the first stage, the second multi-pass solvent selection valve 16-1 and the third multi-pass solvent selection valve 16-3 are switched to use the first gradient reservoir 4-1 and the third gradient reservoir 4-3, and the corresponding first gradient chromatographic pump 5-1 and second gradient chromatographic pump 5-2 are operated to elute the first chromatographic column 7-1; in the second stage, the second multi-pass solvent selection valve 16-1 and the third multi-pass solvent selection valve 16-2 are switched to use the second gradient reservoir 4-2 and the fourth gradient reservoir 4-4, and the corresponding first gradient chromatographic pump 5-1 and second gradient chromatographic pump 5-2 are operated to elute the first chromatographic column 7-1. Before the time period of the pre-peak fraction with recycling value, the first multi-pass solvent selection valve 2 is switched to the third isocratic reservoir 1-3 and the first isocratic reservoir 1-1 in sequence according to the time period requirements, and the isocratic chromatographic pump 3 is operated to complete the column washing and balancing program of the second chromatographic column 7-2. When the time period of the pre-peak fraction with recycling value appears, the multi-channel fraction collector outlet 11-1 is switched, the state of the first flow path control valve 15-1 is switched to i, and the state of the second flow path control valve 15-2 is switched to ii, so that the fraction passes through the first reflux pipe 14-1 and the third flow path control valve 15-3 into the second chromatographic column 7-2, and at the same time the isocratic chromatographic pump 3 is started, the first multi-pass solvent selection valve 2 is switched to the second isocratic reservoir 1-2, and the solution is used to dilute the fraction entering the second chromatographic column 7-2. When the time period of the pre-peak fraction with recycling value is completed, the multi-channel fraction collector outlet 11-2 is switched to the second fraction collection tank 12-2 to collect the fraction, and at the same time the isocratic chromatographic pump 3 is started, the first multi-pass solvent selection valve 2 is switched to the fourth isocratic reservoir 1-4, and the second chromatographic column 7-2 is loaded. When the time period of the post-peak fraction with recycling value appears, the multi-channel fraction collector outlet 11-3 is switched, the state of the first flow path control valve 15-1 is switched to i, and the state of the second flow path control valve 15-2 is switched to ii, so that the fraction passes through the second reflux pipe 14-2→the sixth flow path control valve 15-6→the second chromatographic column 7-2, and at the same time the isocratic chromatographic pump 3 is started, the first multi-pass solvent selection valve 2 is switched to the second isocratic reservoir 1-2, and the solution is used to dilute the fraction entering the second chromatographic column 7-2;

[0168] 4. Elution of the second chromatographic column 7-2, column washing, balancing, and loading of the first chromatographic column 7-1

[0169] Switch the state of pre-column valve 6 and post-column valve 8 to a respectively. Gradient elution is performed on the second chromatographic column 7-2 using the gradient elution device, which is divided into two stages: the first stage, switch the second multi-pass solvent selection valve 16-1 and the third multi-pass solvent selection valve 16-2 to use the first gradient reservoir 4-1 and the third gradient reservoir 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; the second stage, switch the second multi-pass solvent selection valve 16-1 and the third multi-pass solvent selection valve 16-2 to use the second gradient reservoir 4-2 and the fourth gradient reservoir 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-peak fraction with recycling value, according to the time period requirements, switch the first multi-pass solvent selection valve 2 to the third isocratic reservoir 1-3 and the first isocratic reservoir 1-1, and operate the isocratic chromatographic pump 3 to complete the column washing and equilibration program of the first chromatographic column 7-1. When the time period of the pre-peak fraction with recycling value appears, switch the multi-channel fraction collector outlet 11-1, switch the state of the first flow control valve 15-1 to i and the state of the second flow control valve 15-2 to i, so that the fraction passes through the first reflux pipe 14-1→the fourth flow control valve 15-4→the first chromatographic column 7-1, and at the same time, start the isocratic chromatographic pump 3, switch the first multi-pass solvent selection valve 2 to the second isocratic reservoir 1-2, and use the solution to dilute the fraction entering the first chromatographic column 7-1; when the time period of the pre-peak fraction with recycling value is completed, switch the multi-channel fraction collector outlet 11-2 to connect the second fraction collection tank 12-2 to collect the sample, and at the same time, start the isocratic chromatographic pump 3, switch the first multi-pass solvent selection valve 2 to the fourth isocratic reservoir 1-4, and load the sample into the first chromatographic column 7-1; when the time period of the post-peak fraction with recycling value appears, switch the multi-channel fraction collector outlet 11-3, switch the state of the first flow control valve 15-1 to i and the state of the second flow control valve 15-2 to i, so that the fraction passes through the second reflux pipe 14-2→the fifth flow control valve 15-5→the first chromatographic column 7-1, and at the same time, start the isocratic chromatographic pump 3, switch the first multi-pass solvent selection valve 2 to the second isocratic reservoir 1-2, and use the solution to dilute the fraction entering the second chromatographic column 7-2;

[0170] 5. Cycle

[0171] Cycle the above steps 3 and 4 to continuously purify the sample and obtain a sample meeting the purity requirements.

Claims

1. A dual column recycling chromatography system comprising an isocratic elution device, a gradient elution device, a pre-column valve (6), a chromatography column device, a post-column valve (8), a detector device, a waste tank (10), a fraction collection device, a reflux device and connecting lines; characterized in that: The isocratic elution device comprises at least three isocratic liquid storage tanks (1) and a multi-way solvent selection valve (2) and an isocratic chromatographic pump (3), the isocratic liquid storage tank (1) is connected with the multi-way solvent selection valve (2) and the isocratic chromatographic pump (3) through a connecting pipeline; the chromatographic column device comprises two chromatographic columns (7) arranged in parallel; the detector device comprises at least one detector (9); The fraction collection device comprises a multi-channel fraction collector (11), at least two fraction collection tanks (12) and a waste collection tank (13); the reflux device comprises at least one reflux pipeline (14) and at least one flow path control valve (15); Wherein, the isocratic elution device and the gradient elution device are connected in parallel, and then connected with the pre-column valve (6) and the chromatographic column device and the post-column valve (8) through a connecting pipeline, the post-column valve (8) is connected with the detector device and the waste liquid tank (10) through a connecting pipeline, the detector device is connected with the fraction collector (11) through a connecting pipeline, the fraction collector (11) is connected with the fraction collection tank (12) and the waste collection tank (13) through a connecting pipeline, 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), 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 two-column recycling chromatography system comprising an isocratic elution device, a gradient elution device, a pre-column valve (6), a chromatography column device, a post-column valve (8), a detector device, a waste tank (10), a fraction collection device, a reflux device and connecting lines; characterized in that: The isocratic elution device comprises at least three isocratic liquid storage tanks (1) and a multi-way solvent selection valve (2) and an isocratic chromatographic pump (3), the isocratic liquid storage tank (1) is connected with the multi-way solvent selection valve (2) and the isocratic chromatographic pump (3) through a connecting pipeline; the chromatographic column device comprises two chromatographic columns (7) arranged in parallel; the detector device comprises at least one detector (9); The fraction collection device comprises a multi-channel fraction collector (11), at least two fraction collection tanks (12) and a waste collection tank (13); the reflux device comprises 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; Wherein, the isocratic elution device and the gradient elution device are connected in parallel, and then connected with the pre-column valve (6) and the chromatographic column device and the post-column valve (8) through a connecting pipeline, the post-column valve (8) is connected with the detector device and the waste liquid tank (10) through a connecting pipeline, the detector device is connected with the fraction collector (11) through a connecting pipeline, the fraction collector (11) is connected with the fraction collection tank (12) and the waste collection tank (13) through a connecting pipeline, and the fraction collector (11) is connected with at least one of the fraction collection tanks (12) and one end of the reflux pipeline (14) through a connecting pipeline and 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).

3. A double 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 tank (10), a fraction collection device, a reflux device and connecting pipelines; characterized in that: the isocratic elution device comprises at least three isocratic liquid storage tanks (1) and a multi-way solvent selection valve (2), an isocratic chromatographic pump (3), the isocratic liquid storage tanks (1) being connected to the multi-way solvent selection valve (2) and the isocratic chromatographic pump (3) through connecting pipelines; the chromatographic column device comprises two chromatographic columns (7) arranged in parallel; the detector device comprises at least one detector (9); the fraction collection device comprises a multi-channel fraction collector (11), at least one fraction collection tank (12) and one waste collection tank (13); the reflux device comprises at least one reflux pipeline (14) and at least one flow path control valve (15); wherein the isocratic elution device and the gradient elution device are connected in parallel through connecting pipelines to the pre-column valve (6), the chromatographic column device and the post-column valve (8), the post-column valve (8) is connected to the detector device and the waste tank (10) through connecting pipelines, the detector device is connected to the fraction collector (11) through connecting pipelines, the fraction collector (11) is 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 twin column recycling chromatography system of any one of claims 1-3, wherein: the gradient elution device comprises two groups of gradient liquid storage tanks (4) and a gradient chromatographic pump (5), and the number of gradient liquid storage tanks in each group is 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 two gradient elution units are connected in parallel to constitute the gradient elution device.

5. The twin column recycling chromatography system of any one of claims 1-3, wherein: the gradient elution device comprises two groups of gradient liquid storage tanks (4), a multi-way solvent selection valve (16) and a gradient chromatographic pump (5), and the number of gradient liquid storage tanks in each group is greater than 1, each group of gradient liquid storage tanks (4), the multi-way solvent selection valve (16) and the gradient chromatographic pump (5) are connected in series through connecting pipelines to constitute a gradient elution unit, and two gradient elution units are connected in parallel to constitute the gradient elution device.

6. The twin column recycling chromatography system of any one of claims 1-3, wherein: the gradient elution device comprises two groups of gradient liquid storage tanks (4) and a proportional valve (17), and further comprises a gradient chromatographic pump (5), and the number of gradient liquid storage tanks in each group is 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, two gradient elution units are connected in parallel, and then connected to the gradient chromatographic pump (5) through connecting pipelines to constitute the gradient elution device.

7. The twin column recycling chromatography system of any one of claims 1-3, wherein: The gradient elution device comprises two sets of gradient liquid storage tanks (4), a multi-channel solvent selection valve (16), a proportional valve (17), further comprises a gradient chromatographic pump (5), and each set of gradient liquid storage tank is greater than 1, the two sets of gradient liquid storage tanks (4), the multi-channel solvent selection valve (16) and the proportional valve (17) are connected by connecting pipes in sequence as a gradient elution unit, the two sets of gradient elution units are connected in parallel, then connected with the gradient chromatographic pump (5) through the connecting pipes, and the gradient elution device is formed.

8. The twin column recycling chromatography system of any one of claims 1-3, wherein: 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 one-way valve.

9. The twin column recycling chromatography system of any one of claims 1-3, wherein: 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 twin column recycling chromatography system of any one of claims 1-3, wherein: 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.

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