CN118707009B

CN118707009B - A fully automatic chromatography purification system

Info

Publication number: CN118707009B
Application number: CN202411203653.XA
Authority: CN
Inventors: 冯俊雄; 王洪宇; 周丽; 汪群杰
Original assignee: Tianjin Boyun Purification Equipment Material Technology Co ltd
Current assignee: Tianjin Boyun Purification Equipment Material Technology Co ltd
Priority date: 2024-08-30
Filing date: 2024-08-30
Publication date: 2025-01-03
Anticipated expiration: 2044-08-30
Also published as: CN118707009A

Abstract

The present invention proposes a fully automatic chromatography purification system, including: a sample tank, a loading system, an eluent tank, an infusion pump, a chromatographic column, an online detection device with a full-wavelength UV-visible detector and an online ultra-high performance liquid chromatograph, a target pure product tank, an impure product recovery tank and a solvent mixing tank; the data detected by the online detection device is transmitted to the control cabinet computer to control the opening and closing of multiple valves. The closed-loop fully automatic chromatography purification system proposed in the present invention can accurately determine the purity of the fraction through online UV-visible detection and online UHPLC detection, and realize automatic determination and automatic collection of target pure products, impure products (recoverable for secondary purification) and waste liquid, which not only improves efficiency and reduces the space occupied by the collection tank, but also improves the yield; the system can also realize automatic solvent recovery and automatic liquid preparation, thereby realizing the fully automatic operation of the entire chromatography purification process.

Description

Full-automatic chromatographic purification system

Technical Field

The invention relates to the technical field of devices and methods for purifying polypeptides, in particular to a high performance liquid chromatography device and a method for purifying polypeptides.

Background

The high performance liquid chromatography is an effective separation and analysis means and also an important high-efficiency preparation and separation technology. The high performance liquid chromatograph generally consists of a liquid reservoir, a pump, a sample injector, a chromatographic column, a detector and a recorder. The mobile phase in the liquid storage device is pumped into the system by a high-pressure pump, the sample solution enters the mobile phase through the sample injector, the mobile phase is loaded into the chromatographic column (stationary phase), and as each component in the sample solution has different distribution coefficients in two phases, when the two phases do relative motion, the components are separated into single components which flow out from the column in sequence after the distribution process of adsorption-desorption is repeated for a plurality of times, and when the sample solution passes through the detector, the concentration of the sample is converted into an electric signal to be transmitted to the recorder, and data are printed in a pattern form.

Firstly, as shown in fig. 1, the existing high performance liquid chromatograph detects fractions by adopting an ultraviolet single-wavelength or multi-wavelength detection mode, because the sample injection amount of the preparative chromatographic separation is large, the ultraviolet-visible detector presents a whole peak which cannot be completely separated, and the content or concentration of the target and the impurity in each time period cannot be determined, so that the fractions can only be completely collected by adopting a pot-separated collection mode, the content and the concentration of the target in the fractions collected in each pot are detected off-line, whether the fractions collected in each pot are qualified is determined, and the fractions in the pot with the detection result within the qualified range are combined. The fraction is collected by the fraction collector in two modes, one is that the collector moves linearly under the drive of a motor to enable each fraction collecting tank to move to a fraction outlet conduit and collect a certain amount of fraction (US 8858899B2 and US 4422151A), and the other is that the fraction outlet conduit is arranged on a rotating arm and can move back and forth along the rotating arm, and the rotating arm can move rotationally under the drive of the motor through the rotating movement of the rotating arm and the forward and backward movement of the fraction outlet conduit along the rotating arm to enable the fraction outlet conduit to sequentially pass through the inlet of each fraction collecting tank and inject the fraction into the collecting tank (US 11686712B2, liquid fraction collector for liquid chromatography system).

The problems with the current on-line detection and fraction collection devices and techniques are that ①, in order to avoid mixing the acceptable fraction with the unacceptable fraction, the time period required for fraction collection needs to be shortened as much as possible, so that a lot of collection tanks are required, and not only a lot of space is occupied, but also a lot of post-treatment (off-line detection, merging and cleaning of collection tanks, etc.) work and product losses caused by post-treatment (residues of products in the tanks, product deterioration, risks of miscibility and cross contamination, etc.) are caused, and if the collection frequency is too low, the yield of acceptable products is reduced, and when the fraction collector is used for collecting fractions, a ② fraction outlet conduit and a fraction collection pipe need to be corresponded one by using a linear motor or a rotary arm driving motor, and the mechanical transmission has high failure rate and uncertainty in continuous long-term operation, so that the loss of collected products, especially for high-value products, is caused. Therefore, the method for collecting the qualified products in a sectional manner, detecting the qualified products offline and recombining the qualified products is large in workload, low in efficiency and easy to cause errors and loss.

And secondly, separating and purifying all the fractions obtained by the existing high performance liquid chromatograph, wherein no target substance or fraction with low target substance content is directly treated as waste liquid. The general treatment mode of the waste liquid is to combine all the sections of fractions which are not necessary for purification, concentrate the fractions, give the solid as hazardous chemical substances to a hazardous chemical substance treatment mechanism with qualification for innocent treatment, measure the proportion of the liquid, and then transfer the liquid into a mobile phase for recycling after being configured to be qualified. The waste liquid generated during the separation and purification of the target object by utilizing the high performance liquid chromatography technology increases the occupied space due to the storage problem on one hand, and increases the labor and equipment investment due to the waste liquid treatment on the other hand, and increases the waste liquid treatment cost due to the treatment of the dangerous chemical treatment mechanism. Thus, there is a need for further improvements in waste recovery systems.

Disclosure of Invention

In order to solve the problems, the invention provides a closed-loop full-automatic chromatographic purification system which integrates a chromatographic system main body device, an on-line detection device, a sample automatic collection device and a control system, wherein the control system is connected with the devices, and the connection comprises strong electric connection and weak electric connection.

(1) The chromatographic system main body device comprises a sample tank, a sample loading pump, an eluent tank, an infusion pump and a chromatographic column. Wherein the sample tank and the eluent tank are respectively connected with a chromatographic column liquid inlet through pipelines, a chromatographic column liquid outlet is connected with an on-line detection device and a sample automatic collection device, a full-automatic chromatographic purification system is controlled by a control system,

(2) The on-line detection device comprises a full-wavelength ultraviolet-visible detector, an on-line ultra-high performance liquid chromatograph, an automatic sampling device, a data processing and transmitting device,

(3) An automatic sample collecting device controlled by the on-line detection result,

(4) An automatic solvent recovery device comprises solvent recovery and online liquid preparation,

(5) The control system comprises a control cabinet for carrying out data acquisition, analysis and integrated control on the whole system and a remote control center controller. The remote control center is in communication connection with the control cabinet, and is in data transmission and instruction transmission with the control cabinet, and the control cabinet is connected with all devices to realize data acquisition and electric control.

The purification system provided by the invention is shown in fig. 2:

(1) The chromatographic column inlet is respectively connected with a sample loading device and a high-pressure eluting device through a switching valve, the sample loading device comprises a sample tank and a sample loading pump behind the sample tank, the high-pressure eluting device comprises an eluent tank and a high-pressure constant flow pump (the withstand voltage is not lower than 2 megapascals) behind the sample tank, the high-pressure constant flow pump and the switching valve are controlled by a central controller and a control cabinet, the eluent can be prepared on line, so that gradient elution is realized, and the specific implementation mode is that the eluting solvents A and B are respectively arranged in the eluent tanks A and B, and a high-pressure constant flow pump A and a high-pressure constant flow pump B are arranged behind each eluent tank.

(2) The chromatographic column outlet is connected with an on-line detection device. The on-line detection device comprises a full-wavelength ultraviolet-visible detector, an automatic sampling device, an on-line ultra-high performance liquid chromatograph and a data processing and data transmission device. The method comprises the steps of scanning fractions prepared by high-efficiency phase chromatography by a full-wavelength ultraviolet-visible detector, detecting whether the fractions have chromatographic peaks containing target objects, collecting fractions containing no chromatographic peaks of the target objects, entering a solvent recovery tank, alternately collecting the fractions containing the chromatographic peaks of the target objects, entering a first collecting buffer tank or a second collecting buffer tank, an online sampling device comprising the first collecting buffer tank, the second collecting buffer tank, an analysis sample sampling device, an online diluter and a plurality of control valves, alternately sampling the first collecting buffer tank and the second collecting buffer tank by the online sampler through the control valves, analyzing and detecting the analysis sample after being diluted online, entering the ultra-high-efficiency liquid chromatography, measuring the purity of the target objects and the content of impurities in corresponding fraction solutions, transmitting detected data to a control center after data processing, controlling a switching valve of an automatic fraction collecting device through a control system, and respectively transferring the corresponding fractions in the buffer tanks into a target pure product tank, a non-pure product recovery tank and a waste liquid tank according to a preset purity standard.

The first collecting buffer tank and the second collecting buffer tank can also be directly connected with the chromatographic column outlet through a valve, and the online ultra-high performance liquid chromatograph can still achieve the purposes of detecting the purity of the fraction and controlling the fraction collection under the configuration, but the invention has the following two defects that firstly, all the liquid flowing out of the chromatographic column is detected by the online ultra-high performance liquid chromatograph, the workload of the high performance liquid chromatograph is increased, and secondly, all the front and back fractions, including the fractions with chromatographic peaks and the fractions without chromatographic peaks, pass through the buffer tank and the fraction collecting device, so that the whole device is easy to be cross-polluted, therefore, the invention preferably uses two detectors of the full-wavelength ultraviolet-visible detector and the online ultra-high performance liquid chromatograph in series at the same time. The time bottleneck of the on-line ultra-high performance liquid chromatograph determines the width of the fraction segments, thereby affecting the purity and yield of the fraction, so the adopted liquid chromatograph needs to ensure that the time of each analysis is not more than 10 minutes, and the preferable time of each analysis is within 5 minutes. A liquid chromatograph satisfying the above requirements is defined as ultra-high performance liquid chromatograph. In order to achieve the purpose, the invention provides a method for carrying out rapid analysis and detection by adopting a core-shell type analysis chromatographic column, which not only ensures the rapid analysis result, but also can avoid using an ultra-high pressure pump.

(3) The automatic sample collecting device connected to the on-line detector includes target pure product tank, non-pure product recovering tank, waste liquid tank and switching valve controlled by the control system based on the analysis data of the on-line super high performance liquid phase.

(4) The automatic solvent recovery and online liquid preparation device can be used as an option, can be embedded into the whole system, thereby realizing full-automatic recycling of the solvent, and can also be used independently in an off-line mode, and then the recovered solvent is added into an eluent tank after being configured according to the required proportion. The automatic solvent recovery device comprises a solvent mixing tank, a solvent buffer tank and a solvent tank set, wherein the input end of the solvent buffer tank is connected with a target pure product tank, a non-pure product recovery tank and a waste liquid tank through pipelines, pumps and valves are arranged on the pipelines, the output end of the solvent buffer tank is connected with the first input end of the solvent mixing tank through a pipeline, a high-precision flowmeter is arranged on the pipeline, the solvent tank set is connected with the second input end of the solvent mixing tank through a pipeline, the pump and the valve are arranged on the pipeline, the solvent mixing tank is also connected with an eluent tank through a pipeline, decompression distillation devices are respectively arranged on the connecting pipelines of the target pure product tank, the non-pure product recovery tank and the waste liquid tank and the solvent buffer tank, the solvent recovery to the solvent buffer tank is realized through on-line vacuum decompression distillation, the proportion of different solvents in a solution is detected and calculated through a mass flowmeter in the process of transferring to the solvent mixing tank, and a proper amount of specific solvent is added from the solvent tank set to the solvent mixing tank in real time to a set proportion. A solvent recovery tank is also provided in parallel with the first and second collection buffer tanks, behind the full-wavelength uv-vis detector, for collecting solvent or fractions that do not contain the target. The outlet end of the solvent recovery tank is respectively connected with the solvent buffer tank and the eluent tank through a switching valve, wherein a reduced pressure distillation device is arranged on a pipeline connected with the solvent buffer tank, when the solvent components and the proportion in the solvent recovery tank are completely the same as those in the eluent tank and no impurity is contained, the solvent can be directly conveyed to the eluent tank, and when the solvent in the solvent recovery tank is impure or the solvent components in the eluent tank are inconsistent in proportion, the solvent is input into the solvent buffer tank through reduced pressure distillation.

The invention adopts the following technical means, and by using the device, more accurate and highly-automatic chromatographic separation and purification are realized.

On the one hand, a full-automatic chromatographic purification system is provided, which comprises an automatic control device, a sample loading device, a high-pressure elution device, a chromatographic column, an on-line detection device and an automatic fraction collection device, wherein a sample tank and an eluent tank are respectively connected with the inlet end of the chromatographic column through a pipeline and a switching valve, a sample pump and a high-pressure infusion pump are respectively arranged on the pipeline, the chromatographic column is preferably dynamically and axially pressurized to improve the separation effect, a chromatographic stationary phase, preferably a chromatographic stationary phase of a spherical silica gel matrix, is filled in the chromatographic column, the filling length of the chromatographic column is 20cm-100cm, further, 2 or more silanes (except for a tail sealing agent, the total carbon content of which is not more than 5) are selected for bonding modification (not including an alkyl silica group for tail sealing), one of the two silanes is polar silane, the other is nonpolar silane, or two types of nonpolar silane are different.

The sample solution to be purified enters the chromatographic column from the sample tank through the sample injection pump, then the switching valve is controlled by the automatic control device to switch the high-pressure eluting device to be connected with the chromatographic column, the eluent in the eluent tank is continuously pumped into the chromatographic column, and the sample is continuously and dynamically exchanged between the eluent and the stationary phase in the process of continuously flowing the eluent in the chromatographic column, so that gradual separation is realized.

Furthermore, a protective column can be arranged in front of the chromatographic column, and then a filler with larger particle size can be filled in the protective column so as to prevent the colloidal precipitation in the sample from polluting the chromatographic column, and simultaneously prevent the back pressure of the protective column and the back pressure of the whole chromatographic system from being too high during the colloidal precipitation, and further can alternately enter sample solution and pure water to reduce sample diffusion.

The chromatographic column fraction outlet is provided with a main pipeline and three branches formed by the tail end of the main pipeline, the three branches are respectively connected with a solvent recovery tank, a first collecting buffer tank and a second collecting buffer tank, and valves are arranged on the three branches.

The fraction solution flowing out from the lower end of the chromatographic column enters a full-wavelength ultraviolet-visible detector, all the solution flowing through the full-wavelength ultraviolet-visible detector enters a solvent recovery tank before the chromatographic peak containing the target object is not found, and the fraction of the solution containing the target object is detected to alternately enter a first collecting buffer tank and a second collecting buffer tank.

Further, the online detection device further comprises an online ultra-high performance liquid chromatograph, and the online ultra-high performance liquid chromatograph is connected with the first collection buffer tank and the second collection buffer tank and is used for detecting the purity of the target object in the fractions of the first collection buffer tank and the second collection buffer tank.

Further, the online ultra-high performance liquid chromatograph is electrically connected with a control cabinet, and the control cabinet is configured to control the opening and closing of the valves of the first branch, the second branch, the third branch, the fourth branch, the fifth branch and the sixth branch according to the result of the online ultra-high performance liquid chromatograph;

The first branch is a connecting pipeline of the first collecting buffer tank and the target pure product tank, the second branch is a connecting pipeline of the first collecting buffer tank and the non-pure product recovery tank, the third branch is a connecting pipeline of the second collecting buffer tank and the target pure product tank, the fourth branch is a connecting pipeline of the second collecting buffer tank and the non-pure product recovery tank, the fifth branch is a connecting pipeline of the first collecting buffer tank and the waste liquid tank, and the sixth branch is a connecting pipeline of the second collecting buffer tank and the waste liquid tank.

The on-line sampling device alternately samples from the first collecting buffer tank and the second collecting buffer tank, and the fractions are quantitatively diluted on line and then enter an ultra-high performance liquid chromatograph to measure the purity of the fractions, when the purity of the fractions meets the preset standard, a valve connected with the first or third branch is opened, the fractions meeting the purity requirement are automatically collected and enter a target pure product tank, when the purity of the fractions does not meet the purity standard but the concentration and the purity meet the recovery and repurification standard, the fractions enter a non-pure product recovery tank through the second or fourth branch, and when the concentration or the purity of the fractions is lower than the preset recovery standard, the fractions enter a waste liquid tank through the fifth or sixth branch.

Further, one output end of the target pure product tank is connected with the post-treatment tank, the concentrated solution is crystallized or freeze-dried in the post-treatment tank to obtain target pure product polypeptide, one output end of the non-pure product recovery tank is connected with the sample tank, and the fraction is concentrated and then enters the sample tank for further purification.

Furthermore, the invention also comprises an on-line solvent recovery and on-line liquid preparation system which can be used in combination with the chromatographic automation system or can be used independently off line, and the core of the system comprises a solvent recovery buffer tank, a solvent mixing tank and a solvent tank group. The solvent recovery buffer tank is respectively connected with the target pure product tank, the non-pure product recovery tank, the waste liquid tank and the solvent recovery tank, and the connecting pipelines are respectively provided with a reduced pressure distillation device, and the solvents in the 4 tanks are subjected to reduced pressure distillation, cooled and collected and then enter the solvent recovery buffer tank. The high-precision mass flowmeter is arranged on a pipeline connecting the solvent recovery buffer tank and the solvent mixing tank, the solvent component proportion entering the solution mixing tank can be obtained after measurement and calculation through the flowmeter, and the solvent component and the mass of the solvent tank group entering the mixing tank are adjusted and controlled according to the difference value of the solvent component proportion in the solution mixing tank and the solvent component proportion in the preset eluent tank.

Compared with the application method of the existing chromatographic purification system for purifying organic molecules, the invention has the following advantages and beneficial effects:

1. The closed-loop full-automatic chromatographic purification system integrating the high-pressure chromatographic separation device, the high-performance liquid chromatography on-line detection and fraction automatic collection device and the on-line solvent automatic recovery and liquid preparation device not only improves the purification yield of target products, improves the purification operation efficiency, reduces the operation space and the personnel cost and the solvent loss cost, but also further reduces the workload because the fraction off-line analysis waiting process and the fraction merging process are eliminated, further reduces the loss caused by the residues when the target products are deteriorated and merged, also reduces the environmental protection and safety risks caused by the discharge of solvents in the process, and further eliminates human error factors through whole-process automation, so that the whole process is more stable and more reliable.

Specific:

(1) The invention provides an on-line ultra-high performance liquid chromatograph, which comprises a buffer tank switch and an on-line ultra-high performance liquid chromatograph, wherein an accurate and quick on-line detection device is firstly established to ensure that the purity of the fraction purified by a chromatographic column can be accurately detected in time, and an automatic sample collection device is controlled to realize automatic collection, and because the accurate and quick detection of the on-line ultra-high performance liquid chromatograph can compress the detection interval of each sampling to 1-5 minutes, the detection interval is not more than 10 minutes at most, thereby ensuring the accuracy of the purity of the collected product, and reducing the yield loss caused by the collection volume of a single fraction to the minimum, thereby improving the purification yield of the target product.

(2) The invention directly controls the fractions to enter the target pure product collecting tank, the non-pure product collecting tank (used for secondary purification) and the waste liquid tank respectively by detecting the purity of each fraction on line, thereby not only reducing the space, but also eliminating the waiting process of the analysis of the fraction separation and the merging process of the fractions, further reducing the workload, reducing the loss caused by residues when the target products are degenerated and merged, reducing the environmental protection and safety risks caused by the discharge of the solvent in the process, and eliminating the human error factors by whole-course automation.

(3) Through online solvent intelligent recovery and liquid preparation system, realize the furthest recovery and the utilization to the solvent in the fraction, automatic recovery to the solvent that does not have the chromatographic peak, carry out automatic ratio recovery to the solvent that has the chromatographic peak as required to reduce the waste of solvent. The intelligent automatic solvent recovery device reduces labor cost and equipment, shortens recovery time, improves efficiency and reduces waste liquid treatment cost.

(4) The eluent infusion pump with precise control is adopted, the flow error is ensured to be within 1.5 percent, the flow is precisely measured on line through the high-precision mass flowmeter, and when the flow fluctuates, the precise adjustment of the flow is immediately realized through the control cabinet, so that the precise and stable separation effect is further realized, and the yield of a target product is improved.

(5) The invention creatively realizes the system integration of 1) ultraviolet visible on-line monitoring, 2) UHPLC on-line detection, 3) fraction collection automatic control, 4) solvent recovery control and 5) on-line liquid preparation based on accurate density monitoring, realizes closed-loop and accurate intelligent control, and realizes the accuracy, high efficiency and stability of organic compound chromatographic purification. The closed loop system of the invention enables the whole purification to realize continuous operation including solvent recycling, thereby not only improving the efficiency, but also reducing the pollution possibly introduced in the off-line recovery process, and reducing the problems of loss, air pollution, safety risk and the like caused by off-line recovery.

2. Aiming at the polypeptide with larger molecular weight above 2000D, the target object has the characteristics of high similarity with impurities, difficult separation, easy tailing formation, self aggregation formation and the like, and in order to realize the accurate separation of the polypeptide with large molecular weight (2000D-6000D), the invention also adopts the following key innovation from chromatographic columns and chromatographic packing:

(1) The invention further adopts 2 or more silanes for mixed bonding, and utilizes the difference of different silanes with different actions from the target and impurities, namely the actions of polar silanes with the target and impurities and the actions of nonpolar silanes with the target and impurities, the actions of nonpolar silanes with the impurities are different, the separation effect can be improved after the actions are added, the selectivity can be improved, and the separation of chromatographic packing stationary phase relative to macromolecular polypeptides and other mixtures with very similar compositions and structures can be further improved. The silanes described herein do not include no more than 5 carbon capping agents for capping the tail.

(2) The invention also effectively reduces tailing and improves peak shape and separation effect through controlling consistency and accuracy of chromatographic column temperature and eluent temperature (the temperature is selectable between 10 ℃ and30 ℃ and the error is within +/-3 ℃).

(3) The sample injection end is designed to alternately enter sample solution and pure water to reduce sample diffusion, and meanwhile, a large-particle-size filled protective column is adopted to keep strong adsorbed impurities, colloid in the solution and possibly a small amount of precipitated solids in the protective column, so that excessive column pressure and filler pollution of the separation and purification chromatographic column are avoided.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 is a schematic diagram of a conventional preparative liquid chromatography system;

FIG. 2 is a schematic diagram of a fully automated chromatographic purification system in accordance with one embodiment of the present invention;

FIG. 3 is a polypeptide separation ultraviolet on-line detection profile;

FIG. 4 is a schematic illustration of a cylinder-free chromatography column;

FIG. 5 is a cross-sectional view of a cylinder-free chromatographic column A-A;

FIG. 6 is a chromatogram of comparative example 3 BS AQ C18 10 μm 120A isolated hydrophobic hexapeptide preparation;

FIG. 7 is a chromatogram of comparative example 1 BS AQ C18/C8 10 μm 120A isolated hydrophobic hexapeptide preparation;

FIG. 8 is a chromatogram of comparative example 2 BS AQ C8 10 μm 120A isolated crude hydrophobic decapeptides;

FIG. 9 is a chromatogram of the crude preparation of 10 μm 120A isolated hydrophobic decapeptides from example 2 BS AQ C8/C12 (12.7% C12).

Wherein 1, an eluent tank; sample tank, 3, infusion pump, 4, sample loading pump, 5, chromatographic column, 6, UV detection, 7, fraction collection, 8, waste liquid, 9, on-line detection device, 10, target pure product tank, 11, non-pure product recovery tank, 12, solvent mixing tank, 13-1, valve one, 13-2, valve two, 13-3, valve three, 13-4, valve four, 13-5, valve five, 14, full-wavelength ultraviolet-visible detector, 15, first collection buffer tank, 16, second collection buffer tank, 17, solvent recovery tank, 18, on-line ultra-high performance liquid chromatograph, 19, post-treatment tank, 20, waste liquid tank, 21, solvent tank set, 22-1, high-precision flow regulating valve one, 22-2, high-precision flow regulating valve two, 22-4, high-precision flow regulating valve four, 22-5, high-precision flow regulating valve five, 23-1, multistage variable frequency centrifugal pump one, 23-2, multistage variable frequency centrifugal pump two, 23-3, multistage variable frequency centrifugal pump three, 18-4, multistage variable frequency centrifugal pump five, multistage variable frequency centrifugal pump two, 23-5, high-precision variable frequency centrifugal pump five, high-precision flow rate pump five. 25. A solvent buffer tank.

Detailed Description

Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.

As a basic implementation mode of the invention, the embodiment provides a full-automatic chromatographic purification system, as shown in figure 2, which comprises a chromatographic system main body device, an on-line detection device, a sample automatic collection device and a control system, wherein the control system is connected with the devices, the connection comprises strong electric connection and weak electric connection,

The control system consists of a remote control center and a control cabinet, and realizes data transmission and electric control, wherein the remote control center is in communication connection with the control cabinet, and realizes data transmission and instruction transmission with the control cabinet.

The chromatographic system main body device comprises a sample tank 2, a loading pump 4, an eluent tank 1, an infusion pump 3 and a chromatographic column 5. The sample tank 2 and the eluent tank 1 are respectively connected with a liquid inlet of the chromatographic column 5 through pipelines, a liquid outlet of the chromatographic column 5 is connected with an on-line detection device and a sample automatic collection device, and the full-automatic chromatographic purification system is controlled by a control system;

the online detection device comprises an online Ultra High Performance Liquid Chromatograph (UHPLC), an automatic sampling device and a data processing and data transmission device, wherein the automatic sampling device comprises a first collecting buffer tank 15, a second collecting buffer tank 16, an analysis sample sampler, online quantitative dilution and a plurality of control valves;

The outlet of the chromatographic column 5 is provided with a main pipeline, and two branches formed by the tail end of the main pipeline are respectively connected with a first collecting buffer tank 15 and a second collecting buffer tank 16, and the two branches are provided with valves;

The first collecting buffer tank 15 and the second collecting buffer tank 16 are also connected with an analysis sample sampler through pipelines, the other end of the analysis sample sampler is connected with an online ultra-high performance liquid chromatograph 18, the online ultra-high performance liquid chromatograph 18 is used for detecting the purity of a target object in fractions of the first collecting buffer tank 15 and the second collecting buffer tank 16, the online detection device is also electrically connected with a control cabinet and provides detection data for a control system, online quantitative sampling and quantitative dilution can be realized in such a way that a valve III 13-3 is arranged on a lower end pipeline of an electrically controlled injection pump, the valve III 13-3 is respectively connected with an analysis chromatographic column of the first collecting buffer tank 15, the second collecting buffer tank 16 and a diluent tank, a sample injection ring is connected with the online ultra-high performance liquid chromatograph 18 through a sample injection ring, a certain amount of sample fraction and a certain amount of diluent are quantitatively extracted into the sample injection ring through the injection pump and the valve switch, and then the sample solution diluted in the sample ring is sent to a column head of the analysis chromatographic column after the sample solution is switched to a passage connected with the analysis chromatographic column.

The automatic sample collecting device comprises a switching valve at the lower end outlet of a first collecting buffer tank 15 and a second collecting buffer tank 16, a target pure product tank 10, a non-pure product recovery tank 11 and a waste liquid tank 20, wherein a control system controls a valve IV 13-4 at the lower end of the first collecting buffer tank 15 and a valve V13-5 at the lower end of the second collecting buffer tank 16 according to detected target purity data in the first collecting buffer tank 15 and the second collecting buffer tank 16, and respectively sends qualified target pure products, unqualified products and waste liquid to the corresponding target pure product tank 10, the non-pure product recovery tank 11 and the waste liquid tank 20;

The control system controls the valve switches of the first branch, the second branch, the third branch, the fourth branch, the fifth branch and the sixth branch according to the detection result of the online ultra-high performance liquid chromatograph 18, and the automatic fraction collection comprises the collection of target pure products, non-pure products capable of being purified secondarily and waste liquid;

The first branch is a connecting pipeline of the first collecting buffer tank 15 and the target pure product tank 10, the second branch is a connecting pipeline of the first collecting buffer tank 15 and the non-pure product recovery tank 11, the third branch is a connecting pipeline of the second collecting buffer tank 16 and the target pure product tank 10, the fourth branch is a connecting pipeline of the second collecting buffer tank 16 and the non-pure product recovery tank 11, the fifth branch is a connecting pipeline of the first collecting buffer tank 15 and the waste liquid tank 20, and the sixth branch is a connecting pipeline of the second collecting buffer tank 16 and the waste liquid tank 20.

It can be understood that the device comprises a sample tank 2, an eluent tank 1, a chromatographic column 5, a first collecting buffer tank 15, a second collecting buffer tank 16, an online ultra-high performance liquid chromatograph 18, a target pure product tank 10, an impure product recovery tank 11, a solvent mixing tank 12, a waste liquid tank 20 and a solvent tank group 21, wherein the sample tank 2 and the eluent tank 1 are respectively connected with one end of the chromatographic column 5 through pipelines, pumps are arranged on the pipelines, a main pipeline is arranged at a fraction outlet of the chromatographic column 5, two branches formed by tail ends of the main pipeline are respectively connected with the first collecting buffer tank 15 and the second collecting buffer tank 16, and valves are arranged on the two branches.

The target pure product tank 10, the non-pure product recovery tank 11 and the waste liquid tank 20 are respectively connected with the first collecting buffer tank 15 and the second collecting buffer tank 16 through pipelines, valves are arranged on the connecting pipelines, the first collecting buffer tank 15 and the second collecting buffer tank 16 are also sequentially connected with an analysis sample sampler and an online ultra-high performance liquid chromatograph 18, the other ends of the target pure product tank 10, the non-pure product recovery tank 11 and the waste liquid tank 20 are respectively connected with a solvent mixing tank 12 through pipelines, and the solvent mixing tank 12 is connected with an eluent tank 1 through pipelines.

The on-line detection device 9 is also electrically connected with the control cabinet to control the opening and closing of a plurality of valves.

The control cabinet comprises a signal receiver and a signal transmitter, functions of related equipment are controlled according to signals fed back by external equipment, the principles of signal receiving and controlling belong to the prior art, and all control cabinets capable of realizing related functions can be used as the control cabinet of the embodiment, and details are omitted here.

It will be appreciated that the sample tank 2 is used to hold a crude solution to be purified which is flowed into the column 5 during purification. In the purification process, crude solution in a sample tank 2 and eluent in an eluent tank 1 are pumped into a chromatographic column 5 by a pump respectively, after the purity of fractions flowing out of the chromatographic column 5 is detected by an online detection device 9, a control cabinet controls the opening and closing of valves on different pipelines according to the purity of the fractions, so that the fractions with the purity reaching a preset standard enter a target pure product tank 10, the fractions with the purity not reaching the preset standard enter a non-pure product recovery tank 11, the fractions in the target pure product tank 10 and the non-pure product recovery tank 11 are subjected to vacuum volatilization in a closed system and then are cooled, and the solvents are separated from the fractions and are led into a solvent mixing tank 12, and then enter the eluent tank 1.

In this embodiment, the chromatographic column 5 is a dynamic axial compression chromatographic column (DAC), and the chromatographic column is made of stainless steel.

The packed length of the column was 40cm.

The eluent is connected with a dynamic axial pressurization chromatographic column (DAC) through a constant flow mode infusion pump 3.

The eluent tank 1, chromatographic column 5 and other parts of the closed-loop full-automatic chromatographic purification system are subjected to temperature control through a fluid coil, and the temperature ranges from 20 ℃ to 23 ℃.

As shown in fig. 2, the on-line detection device is an on-line ultra-high performance liquid chromatograph 18, and the on-line ultra-high performance liquid chromatograph 18 is connected to the first collection buffer tank 15 and the second collection buffer tank 16, and is used for detecting the purity of the target in the fractions of the first collection buffer tank 15 and the second collection buffer tank 16.

Further, the online ultra high performance liquid chromatograph 18 is electrically connected to a control cabinet configured to control the opening and closing of the valves of the first branch, the second branch, the third branch, the fourth branch, the fifth branch, and the sixth branch of the first collecting buffer tank 15 and the second collecting buffer tank 16 according to the result of the online ultra high performance liquid chromatograph 18.

The on-line ultra-high performance liquid chromatograph 18 can perform on-line and on-site process analysis on the fractions and provide real-time data, so that the control cabinet can control the flow direction of the fractions at different stages in real time.

The on-line ultra high performance liquid chromatograph 18 detects the purity of the target in the fraction of the first collection buffer tank 15 or the second collection buffer tank 16 by means of on-line sampling. The online ultra-high performance liquid chromatograph 18 is electrically connected with the control cabinet to realize that the control cabinet controls the branch valve according to the detection result of the online ultra-high performance liquid chromatograph 18.

The mode of on-line sampling is that an on-line ultra-high performance liquid chromatograph 18 alternately extracts 10 mu l of fractions from a first collecting buffer tank 15 and a second collecting buffer tank 16 by an analysis sample sampler, and the fractions enter the on-line ultra-high performance liquid chromatograph 18 after on-line dilution to detect the purity of a target object. The purity of the target can be set to 98.0% -99.9% according to the requirement.

When the full-automatic chromatographic purification system of the embodiment is used for purifying the crude polypeptide, the method comprises the following steps:

S1, a chromatographic column 5 filled with spherical mesoporous silica gel matrix reverse phase chromatographic packing is adopted, the packing height of the column is 40 cm, the total content of other metals except silicon element in the packing is not more than 100 mug/g, the average pore diameter range is 90A-150A, the porosity is 0.8-1.0cm3/g, the specific surface area is 280-380m ²/g, the average particle diameter range is 10 microns, the micropore ratio of pore diameter range below 70A is less than 15%, and the micropore ratio below 40A is less than 5%;

s2, preparing a crude polypeptide solution to be purified, placing the crude polypeptide solution into a sample tank 2, preparing an eluent, and placing the eluent into an eluent tank 1 (preferably an aqueous solution containing acetonitrile);

S3, quantitatively pumping a sample solution into the chromatographic column 5, then pumping a mobile phase solution in the eluent tank 1 into the chromatographic column 5, controlling the temperature of the eluent and the chromatographic column 5 through the column and a liquid coil outside the tank, wherein the temperature range is 20-23 ℃, and the temperature difference between the eluent and the chromatographic column 5 is within +/-3 ℃.

S4, collecting fractions, detecting the fractions by using an online detection device, and enabling the fractions to enter different tank bodies according to detection results of the fractions;

The method comprises the following steps:

S411, after the valve of the first collecting buffer tank 15 is opened, the fraction enters the first collecting buffer tank 15, and the first collecting buffer tank 15 collects the fraction, and at this time, the second collecting buffer tank 16 is in a vacant state. After the fraction collected in the first collection buffer tank 15 reaches a predetermined volume or a predetermined time has elapsed, the fraction is controlled by a valve to switch flow into the second collection buffer tank 16.

Of course, the fraction may first flow into the second collection buffer tank 16, and for this embodiment, after the valve of the second collection buffer tank 16 is opened, the fraction enters the second collection buffer tank 16, and the second collection buffer tank 16 collects the fraction, and the first collection buffer tank 15 is empty. After the fraction collected in the second collection buffer tank 16 reaches a predetermined volume or a predetermined time has elapsed, the fraction is controlled by a valve to switch flow into the first collection buffer tank 15.

S412, the first collecting buffer tank 15 and the second collecting buffer tank 16 circularly perform fraction collecting and fraction detecting work until the sample is purified.

S42, automatically sampling and detecting the purity of the target object from the first collecting buffer tank 15 or the second collecting buffer tank 16 by using an online ultra-high performance liquid chromatograph 18, and transferring the fraction with the purity reaching the preset standard of the target object to the target pure product tank 10 according to the detection result;

S5, transferring the concentrated solution in the target pure product tank 10 to a post-treatment tank 19 for crystallization or freeze drying to obtain a target polypeptide pure product, wherein the concentrated solution is obtained by volatilizing fractions in the target pure product tank 10 in vacuum;

Further, as another embodiment, the on-line detection device further includes a full-wavelength uv-vis detector 14 (as shown by the yellow dotted line in fig. 2), where the full-wavelength uv-vis detector 14 is disposed on the main line of the eluent outlet of the chromatographic column 5, and is used for detecting whether the eluent in the main line has a chromatographic peak containing the target, and the end of the main line of the eluent outlet of the chromatographic column 5 forms a seventh branch in addition to the two branches, and the seventh branch is connected to the solvent recovery tank 17.

As an embodiment, when the full-automatic chromatographic purification system of the present embodiment purifies a polypeptide sample, acetonitrile-water is used as an eluent, as shown in fig. 3, the absorption threshold of the full-wavelength uv-vis detector 14 is set to 35mAU, after sample injection, the absorption value is lower than 35mAU and is kept at a stable absorption value for 0-4.7min, and no chromatographic peak is generated, during this period, the branch of the control cabinet control valve 13-1 leading to the solvent recovery tank 17 is opened, the fraction enters the solvent recovery tank 17, and during this period, the chromatographic peak detected by the full-wavelength uv-vis detector 14 contains a polypeptide target, so that the branch of the control cabinet control valve 13-1 leading to the first collecting buffer tank 15 and the second collecting buffer tank 16 is opened, the fraction enters the first collecting buffer tank 15 or the second collecting buffer tank 16, that is, the full-automatic chromatographic purification system enters the sample UHPLC on-line detection and automatic fraction collection mode;

during the treatment process, the control cabinet control valve II 13-2 opens a valve leading to the branch of the first collecting buffer tank 15 or the branch of the second collecting buffer tank 16, and the first collecting buffer tank 15 or the second collecting buffer tank 16 collects fractions with chromatographic peaks.

The first collecting buffer tank 15 and the second collecting buffer tank 16 collect the fractions with chromatographic peaks, the full-wavelength ultraviolet-visible detector 14 cannot distinguish the content or concentration of the target object from the impurity due to the large sample injection amount of the preparative chromatographic separation, so the fractions entering the first collecting buffer tank 15 and the second collecting buffer tank 16 may be the fractions with the target object purity reaching the preset standard or the fractions with the target object purity not reaching the preset standard, the fractions with the target object purity reaching the preset standard need to be collected by using the target pure product tank 10, the fractions with the target object purity not reaching the preset standard need to be collected by using the non-pure product recovery tank 11, and the fractions with lower purity and content enter the waste liquid tank 20. Therefore, it is necessary to connect the output ends of the first collecting buffer tank 15 and the second collecting buffer tank 16 to the target pure product tank 10, the non-pure product recovery tank 11, and the waste liquid tank 20, so that the corresponding fractions are collected using the respective tanks according to the purity.

The specific method comprises the steps of firstly, opening a valve of a first collecting buffer tank 15, enabling the fraction to enter the first collecting buffer tank 15, collecting the fraction by the first collecting buffer tank 15, enabling a second collecting buffer tank 16 to be in an empty state at the moment, and switching the fraction to flow into the second collecting buffer tank 16 under the control of the valve after the first collecting buffer tank 15 is completely collected.

Of course, the fraction may first flow into the second collection buffer tank 16, the second collection buffer tank 16 collects the fraction, and the first collection buffer tank 15 is empty, and after the second collection buffer tank 16 collects the fraction, the fraction is controlled by the valve to switch to flow into the first collection buffer tank 15.

It will be appreciated that after the operation is stable, when the first collection buffer tank 15 collects the fraction, the on-line ultra-high performance liquid chromatograph 18 detects the purity of the target object in the fraction from the second collection buffer tank 16, when the purity of the target object in the fraction reaches the preset standard, the control cabinet controls the third branch valve to open, the fraction flows to the target pure product tank 10, when the purity of the target object in the fraction does not reach the preset standard, the control cabinet controls the fourth branch valve to open, the fraction flows to the non-pure product recovery tank 11, and when the purity of the target object in the fraction is lower (lower than 50%), the fraction enters the waste liquid tank 20.

When the second collecting buffer tank 16 collects fractions, the online ultra-high performance liquid chromatograph 18 detects the purity of the target object in the fractions of the first collecting buffer tank 15, when the purity of the target object in the fractions reaches a preset standard, the control cabinet controls the first branch valve to open, the fractions flow to the target pure product tank 10, when the purity of the target object in the fractions does not reach the preset standard, the control cabinet controls the second branch valve to open, the fractions flow to the non-pure product recovery tank 11, and when the purity of the target object in the fractions is lower (lower than 50%), the fractions flow to the waste liquid tank 20.

Therefore, the first collection buffer tank 15 and the second collection buffer tank 16 are circulated to perform the fraction collection and detection operation, and the fraction having the chromatographic peak containing the target substance can be collected at a predetermined purity by using only 3 tanks. The fraction of the target object with the purity reaching the preset standard is collected from the target pure object tank 10, the solid product is obtained by freeze drying or other modes after further concentration, the fraction of the target object with the purity not reaching the preset standard is collected from the non-pure object recovery tank 11 so as to be further recovered and then purified again, and the fraction with lower purity is collected from the waste liquid tank 20 without recovery of the purified fraction.

Still further, a further embodiment of the present invention also includes an automatic solvent recovery device (as shown in phantom in FIG. 2) to form a complete fully automatic continuous cycle purification system. The front end of the automatic solvent recovery device is respectively connected with the solvent recovery tank 17, the target pure product tank 10, the non-pure product recovery tank 11 and the waste liquid tank 20, and the rear end is connected with the eluent tank 1. The automatic solvent recovery device comprises a solvent mixing tank 12, a solvent buffer tank 25, a solvent tank group 21, a matched switching valve and a pump, and further comprises a reduced pressure distillation and cooling collection device arranged among the solvent buffer tank 25, a solvent recovery tank 17, a target pure product tank 10, an impure product recovery tank 11 and a waste liquid tank 20, wherein the input end of the solvent buffer tank 25 is connected with the target pure product tank 10, the impure product recovery tank 11 and the waste liquid tank 20 through pipelines, the pumps and the valves are arranged on the pipelines, the output end of the solvent buffer tank 25 is connected with the first input end of the solvent mixing tank 12 through a pipeline, the pipeline is provided with a high-precision flowmeter, the solvent tank group 21 is connected with the second input end of the solvent mixing tank 12 through a pipeline, the pipeline is provided with the pump and the valve, and the solvent mixing tank 12 is further connected with the eluent tank 1 through a pipeline.

It will be appreciated that by using the full-wavelength uv-vis detector 14 and the on-line ultra-high performance liquid chromatograph 18 at the same time, the fraction flowing out of the chromatographic column 5 can be intelligently divided into four parts, namely, a fraction without target object (a fraction entering the solvent recovery tank 17), a target pure fraction (i.e., a fraction flowing into the target pure tank 10), a non-pure fraction (i.e., a fraction flowing into the non-pure recovery tank 11) and a waste liquid (i.e., a fraction flowing into the waste liquid tank 20), then the solvent in the fraction is recovered to the greatest extent by the reduced pressure distillation device, then the component proportion of the recovered solvent can be detected by the high-precision mass flowmeter between the solvent buffer tank 25 and the solvent blending tank 12, finally, the corresponding solvent component is provided by the solvent tank group 21 as required, so that on-line automatic liquid blending is realized, and the component proportion identical to that of the eluent is obtained.

As an example, when the closed-loop full-automatic chromatographic purification system of the present embodiment is used to purify crude polypeptide, acetonitrile-water is used as eluent, as shown in fig. 3, the absorption threshold of the full-wavelength uv-vis detector 14 is set to 35mAU, after sample injection, the absorption value is lower than 35mAU and is kept at a stable absorption value for 0-4.7min, and no chromatographic peak is generated, during this period, the control cabinet controls the branch of the valve 13-1 leading to the solvent recovery tank 17 to be opened, the fraction enters the solvent recovery tank 17, and then enters the eluent tank 1 through the solvent recovery tank 17 to realize direct circulation of the solvent, and during the period between 4.7-7.5min and 17.5-30min, the full-wavelength uv-vis detector 14 detects chromatographic peaks which do not contain targets, and the fraction also enters the solvent recovery tank 17, and then the solvent is distilled and cooled for recovery by reduced pressure distillation, and enters the solvent buffer tank 25.

In the range of 7.5-17.5 min, the chromatographic peak detected by the full-wavelength uv-vis detector 14 contains the polypeptide target, so that the branch of the control cabinet control valve one 13-1 leading to the first collecting buffer tank 15 and the second collecting buffer tank 16 is opened, and the fraction enters the first collecting buffer tank 15 or the second collecting buffer tank 16, i.e. the closed-loop full-automatic chromatographic purification system enters the sample UHPLC on-line detection and fraction automatic collection mode. The separation period was calculated to be 35.5 minutes and the ratio of recovered acetonitrile was approximately 90%, with about 15% of the fraction directly used without compounding by distillation under reduced pressure for the presence of chromatographic peaks containing the target.

Whereas conventional liquid chromatography requires the collection of fractions of the entire flat head peak. In order to improve the precision of fraction collection, small-volume fraction collection pipes are needed, in other words, the higher the precision is, the smaller the fraction volume collected by each fraction collection pipe is, the more number of fraction collection pipes are needed, the more working table space is needed to be occupied, and then the content and concentration of target substances in the fractions of each collection pipe are detected off-line, and the qualified range of the collected fractions is determined for pipe combination treatment.

The solvent in the fraction of the target pure product tank 10 is in a closed system, is volatilized in vacuum, is cooled and is drained into the solvent blending tank 12.

It will be appreciated that the target purity tank 10 is a concentrating tank during the vacuum evaporation while cooling operation. The concentration kettle has a double-layer structure, adopts heat conduction oil to heat, drives a stirring paddle to stir the reaction kettle through a mechanical pump to uniformly heat, is connected with the reaction kettle through a vacuum pump to reduce the vacuum degree in the kettle, can reduce the boiling point of a solvent through the reduction of the vacuum degree, can evaporate the solvent at a lower heating temperature, and can improve the concentration efficiency of target polypeptide. The distilled solvent is condensed by a glass condenser to be converted into liquid from gas, and is drained into a solvent buffer tank 25 to complete the recycling of the solvent.

The other output end of the target pure product tank 10 is connected with the post-treatment tank 19, and after the solvent in the fraction of the target pure product tank 10 is removed by vacuum volatilization in a closed system, the residual concentrated solution remains in the target pure product tank 10 and finally enters the post-treatment tank 19 through a pipeline.

Further, as shown in FIG. 2, the concentrated solution is crystallized or freeze-dried in a post-treatment tank 19 to obtain the target polypeptide.

It will be appreciated that the post-treatment tank 19 is a reaction vessel with high and low temperatures where the crystallization is carried out, and that the crystallization is carried out directly in the reaction vessel. Specifically, the sample is dissolved in a hot solvent such as 60 ℃ ethanol, then gradually cooled to room temperature, the target substance is supersaturated and separated out due to the solubility difference, and the impurity content is low, so that the target substance can be left in the solution.

It will be appreciated that the post-treatment tank 19 is a lyophilization bottle or tray when the lyophilization operation is performed. The method comprises the steps of removing an organic solvent from a sample by reduced pressure rotary evaporation, and then placing the sample aqueous solution in a freeze-drying bottle or a freeze-drying disc, freezing the sample aqueous solution into ice by a refrigerator or liquid nitrogen, and connecting the freeze-drying bottle with a freeze-dryer pipeline through a bottle opening or placing the freeze-drying disc in the freeze dryer for freeze-drying operation. The freeze dryer is in a high vacuum environment, so that water is continuously reduced to be completely removed by sublimation, and the residual solid is purified target polypeptide.

Solvent in the fractions of the non-pure product recovery tank 11 and the waste liquid tank 20 is also introduced into the solvent buffer tank 25 after the vacuum evaporation and re-cooling process similar to that described above in a closed system.

After the solvent in the fraction of the non-pure product recovery tank 11 is removed by vacuum volatilization in a closed system, the residual concentrated solution remains in the non-pure product recovery tank 11, enters the sample tank 2 under the control of a control cabinet, and is used as a sample to pass through the chromatographic column 5 again for purification operation.

The residues generated by the fractions in the waste liquid tank 20 are cleaned periodically in such a way that the residues after the concentration of the waste liquid tank 20 are cleaned and discharged at intervals.

With the above embodiment, since the vacuum evaporation and cooling operation is performed on the solvent in the fractions of the target pure product tank 10, the non-pure product recovery tank 11 and the waste liquid tank 20, and the solvent obtained after the operation is drained into the solvent buffer tank 25, the solvent collected in the solvent buffer tank 25 contains the solvent volatilized from the purifiable fraction and also contains the solvent volatilized from the non-pure product fraction, and the original solvent is provided for the subsequent automatic liquid preparation process.

Further, as shown in fig. 2, a first output end of the target pure product tank 10 is connected with a solvent buffer tank 25 through a pipeline, and a high-precision flow regulating valve I22-1 and a multistage variable frequency centrifugal pump I23-1 are arranged on the pipeline;

the first output end of the non-pure product recovery tank 11 is connected with a solvent buffer tank 25 through a pipeline, and a high-precision flow regulating valve II 22-2 and a multistage variable frequency centrifugal pump II 23-2 are arranged on the pipeline.

As a possible implementation, as shown in fig. 2, the waste liquid tank 20 is connected to the solvent buffer tank 25 through a pipeline, and a high-precision flow regulating valve five 22-5 and a multistage variable frequency centrifugal pump five 23-5 are arranged on the pipeline.

It will be appreciated that a multistage variable frequency centrifugal pump is used to deliver recovered solvent to the solvent buffer tank 25 and that a high precision flow control valve can precisely control the volume of solvent flowing in the pipeline. The pipeline between the solvent buffer tank 25 and the solvent mixing tank 12 is provided with a high-precision mass flowmeter 24-1, the high-precision mass flowmeter can detect the density of the recovered solvent, so that the solvent component proportion is calculated, the detection result is fed back to the control cabinet, and the control cabinet controls the opening and closing of the corresponding solvent tank valve of the solvent tank group 21 according to the detection result to convey the corresponding solvent, so that the same component proportion of the eluent is obtained through mixing.

As shown in fig. 2, the input of the solvent compounding tank 12 is connected to a solvent tank set 21. The solvent tank group 21 can be used for combining a plurality of solvent tanks according to the proportioning requirement of the eluent, for example, 1-3 solvent tanks can be set, wherein each solvent tank can be used for storing a proper solvent according to the requirement and can be water-soluble acid, water-soluble alkali or water-soluble salt or organic solvent. The solvent in the solvent tank set 21 may be pumped into the solvent compounding tank 12 according to the brewing requirements.

Further, the recovered solvent is measured and calculated by a mass flowmeter to obtain the corresponding component proportion, and the proportion is automatically adjusted to the proportion of the eluent in the mixing tank.

It can be understood that the high-precision mass flowmeter and the solvent mixing tank 12 are introduced into the chromatographic purification system, the high-precision mass flowmeter is used for detecting and comparing the density of the recovered solvent with the density of the eluent, the required solvent volume can be obtained by calculating according to the comparison result of the density of the required eluent and the density of the recovered solvent, and the control cabinet further regulates and controls the solvent tank group 21 to enable the solvent tank group 21 to add a certain volume of corresponding solvent into the solvent mixing tank 12, and the proportion of the solvent recovered in the solvent mixing tank 12 is adjusted to be the set proportion of the eluent.

The online liquid preparation device automatically detects the density of the recovered solvent through a high-precision mass flowmeter, compares the density with the density of the eluent, calculates the volume of the compensating solvent according to the density difference and the total volume, automatically supplements the corresponding solvent through online liquid preparation, then compares and calculates the density again, further supplements the corresponding solvent until the density of the solvent after liquid preparation is consistent with the density of the eluent, and further indicates that the proportion of the recovered solvent after liquid preparation and the proportion of the eluent are completely consistent at the moment, and the online liquid preparation device can be used as the eluent for recycling.

Further, as shown in fig. 2, the output end of the solvent mixing tank 12 is connected with the eluent tank 1, and the prepared solvent is input into the eluent tank 1, so that the solvent can be recycled. Thereby improving the utilization rate of the solvent and reducing the cost of waste liquid treatment.

Further, as shown in fig. 2, a first output end of the solvent recovery tank 17 is connected to the eluent tank 1 through a pipeline, and a second output end of the solvent recovery tank 17 is connected to the solvent buffer tank 25.

The solvent without chromatographic peak in the solvent recovery tank 17 can enter the eluent tank 1, and the solvent with chromatographic peak can be cooled and recovered pure solvent after reduced pressure distillation and enter the solvent buffer tank 25, which is detected by the full-wavelength ultraviolet-visible detector 14. The solvent in the solvent buffer tank 25 is subjected to solvent compensation by the detection result of automatic detection by the high-precision flowmeter, and is mixed in the mixing tank until the solvent proportion of the eluent is met, and then is drained into the eluent tank 1.

As a possible embodiment, the chromatographic column 5 is preferably a dynamic axial compression chromatographic column (DAC).

As a possible embodiment, the packing packed in the chromatographic column 5 is a spherical chromatographic packing of silica gel matrix.

Furthermore, the surface of the silica gel is modified by bonding more than two kinds of silane except for a tail sealing agent, wherein the proportion of any one of the two kinds of silane is not less than 10%, and one of the two kinds of silane is polar silane, the other kind of silane is nonpolar silane or the two kinds of silane are different nonpolar silanes.

As a possible embodiment, the eluent, the chromatographic column 5 is temperature controlled, the temperature being in the range of 10 ℃ to 30 ℃.

Furthermore, two eluent tanks 1 are respectively used for containing the eluents with different densities, high-pressure constant-flow pumps are respectively arranged on the connecting pipelines of the eluent tanks 1 and the chromatographic column 5, and the online gradient mixing of the eluents is realized by respectively controlling the flow ratio of the two high-pressure constant-flow pumps so as to carry out gradient elution on samples.

Further, the method is used for reversed-phase purification of organic matters such as polypeptide, macrolide, cyclosporin and the like.

The full-automatic chromatographic purification system of the invention realizes the whole automation of the whole chromatographic purification circulation process for the first time, not only improves the purification efficiency, reduces human errors and errors, saves the space of a working table, reduces the loss in the solvent circulation use, but also reduces the safety risk and the environmental pollution.

The automatic liquid preparation system can perform automatic mixing and preparation for the initial solvent and the recovered solvents of each section collected in a sectionalized manner according to a set proportion, adopts a multistage variable frequency centrifugal pump, a high-precision mass flowmeter, a high-precision flow regulating valve and other devices, realizes automatic liquid preparation under the calculation, correction and control of a liquid preparation workstation, avoids manual off-line operation, and is accurate, reliable and high in automation degree;

The detector and the tank body are matched in the processes of fraction collection, solvent recovery and automatic liquid preparation, so that a foundation is laid for the whole-course automation of the purification process.

Further, the chromatographic column filler is spherical mesoporous silica gel matrix reversed phase chromatographic filler, the column filling height is 20-100 cm, preferably 20-40 cm, the total content of other metals except silicon element in the filler is not more than 50 mug/g, the average pore diameter range is 90-150 a, the porosity is 0.8-1.0m ³/g, the specific surface area is 280-380m ²/g, and the average particle diameter range is 5-30 microns;

micropores with the pore diameter range below 70A account for less than 15%, and micropores with the pore diameter below 40A account for less than 5%;

The eluent for separation is aqueous solution containing water-soluble acid, alkali or salt or water-soluble organic solvent.

Further, the chromatographic packing has more than 2 kinds of silane groups bonded thereto, wherein the silane groups not containing the above polar groups contain not less than 5 carbon atoms, and wherein the lowest content of silane groups is not less than 10%.

Further, the silane group refers to an alkylsiloxane group containing a polar group and a non-polar group, and comprises a siloxane group modified by a polar group, and further comprises an octadecyldimethylsiloxane group, an octadecyldiisopropylsiloxane group, an octadecyldiisobutylsiloxane group, a hexadecyldimethylsiloxane group, a dodecyl dimethylsiloxane group, a decaneyldimethylsiloxane group, an octyl dimethylsiloxane group, a hexyl dimethylsiloxane group, a butyl dimethylsiloxane group, a propyl dimethylsiloxane group, an isopropyl dimethylsiloxane group, an isobutyl dimethylsiloxane group, an aminopropyl dimethylsiloxane group and an aminoethyldimethylsiloxane group.

Further, the high-precision mass flowmeter is used for accurately monitoring the flow in real time and feeding back the flow to the control cabinet, the control cabinet is used for immediately adjusting the flow to a set value so as to ensure the flow precision, the high-precision mass flowmeter is used for monitoring the density of the recovered solvent in real time and feeding back the density to the control cabinet, and the control cabinet is used for controlling the solvent tank to add the corresponding solvent with the required volume according to the density of the recovered solvent, so that the density of the mixed solvent reaches the set value of the density of the eluent.

It can be understood that after the density of the recovered solvent is measured by the high-precision mass flowmeter, the recovered solvent is compared with the density of the eluent and fed back to the control cabinet, the control cabinet calculates the volume of the solvent required according to the comparison result, the control cabinet controls the valve of the corresponding solvent tank of the solvent tank group 21 to be opened, and injects the corresponding solvent with the required volume into the solvent mixing tank 12, and the recovered solvent is transferred to the eluent tank 1 after being adjusted to the set proportion of the eluent, so that the solvent is recovered and reused.

Example 1 comparison of high precision flow Meter and common flow Meter in insulin purification separation

By introducing the high-precision flowmeter, the stability of the preparation and purification process is improved, so that the purity and yield of the purification are higher, and the variables introduced in the process are reduced.

Still further, the peak shape and separation effect were effectively improved by precise temperature control of the chromatographic column and eluent and consistent, as shown in example 2.

Further, the temperature of the chromatographic column and the eluent is controlled between 10 ℃ and 30 ℃, the chromatographic column and the eluent are kept constant and consistent, and the temperature difference between the eluent and the chromatographic column is within +/-3 ℃.

From example 2 (fig. 9), comparative example 1 (fig. 7), comparative example 2 (fig. 8) and comparative example 3 (fig. 6), it is understood that the variation in column temperature causes an increase in peak broadening. The mobile phase and the column temperature control need to be controlled accurately within a certain range to obtain optimal separation, the temperature of the column temperature or the eluent is increased to weaken the retention of a main peak, impurities in front of the peak cannot be separated effectively, the temperature of the column temperature or the eluent is reduced, the retention time is increased, the separation time is prolonged, the separation from the post-impurities is poor, and if the column is not at a proper constant temperature, a radial temperature gradient is formed in the column and the separation efficiency (theoretical plate number, N) and the retention of the analyte are influenced. When the eluent has a large temperature difference with the chromatographic column, the peak is deformed, and the separation degree is reduced.

Example 2, the main peak was separated from both the pre-and post-impurities when both the column and the eluent were maintained at 20 ℃.

In comparative example 1, when the temperature of both the column and the eluent was reduced to 10 ℃, the retention time of the sample was increased, and the pre-impurities were separated and the post-impurities were packed.

In comparative example 2, when the temperature of the chromatographic column and the eluent are raised to 40 ℃, the retention time of the sample is reduced, and the main peak and the front and rear impurities are not separated.

Comparative example 3, in which the eluent temperature was reduced to 5 ℃, the column was maintained at 20 ℃, and the sample retention time was increased, but the impurity separation was deteriorated before and after.

Further, as shown in fig. 4-7, the dynamic axial compression column employs a cylinder-free column.

It can be understood that the purpose of separation and purification is to obtain the target polypeptide with high purity, and the oil-free cylinder type chromatographic column can effectively avoid pollution to the separated sample caused by oil leakage.

Further, when the column separation is performed by eluting the mixture on the chromatographic column with an eluent, the eluent is used as a reference and is monitored by a full-wavelength ultraviolet-visible detector 14, when no target polypeptide is present, the collected solution enters an eluent recovery tank, when the target polypeptide is found to be present, the fraction is collected and enters a first collection buffer tank 15 or a second collection buffer tank 16, the target purity is detected by automatically sampling from the first collection buffer tank 15 or the second collection buffer tank 16 by an on-line ultra-high performance liquid chromatograph 18, the fraction of which the target purity reaches a preset standard is transferred to a target purity tank 10 according to the detection result, the fraction of which the target purity does not reach the preset standard but exceeds the preset 'recoverable fraction purity' is transferred to a non-pure recovery tank 11, the fraction in the non-pure recovery tank 11 is concentrated and then enters a sample tank 2 to be secondarily purified as a sample, and the fraction of which the target purity is even lower than the 'recoverable fraction purity' is transferred to a waste liquid tank 20.

Further, the fractions are collected and detected by an on-line detection device, and the fractions are made to enter different tank bodies according to the detection result of the fractions, wherein the on-line detection device comprises a full-wavelength ultraviolet-visible detector 14 and an on-line ultra-high performance liquid chromatograph 18, and the specific steps comprise:

S41, scanning the fraction prepared by high-efficiency phase chromatography by using a full-wavelength ultraviolet-visible detector 14, detecting whether the fraction has chromatographic peaks, collecting the fraction without the chromatographic peaks into a solvent recovery tank 17, collecting the fraction with the chromatographic peaks into a first collecting buffer tank 15 or a second collecting buffer tank 16, wherein,

S411, after the valve of the first collecting buffer tank 15 is opened, the fraction enters the first collecting buffer tank 15, the first collecting buffer tank 15 collects the fraction, and the second collecting buffer tank 16 is empty, after the fraction collected by the first collecting buffer tank 15 reaches a preset volume or a preset time, the fraction is controlled by the valve to flow into the second collecting buffer tank 16, or

The fraction flows into the second collecting buffer tank 16 firstly, after the valve of the second collecting buffer tank 16 is opened, the fraction enters the second collecting buffer tank 16, and the second collecting buffer tank 16 collects the fraction, and at the moment, the first collecting buffer tank 15 is in an empty state;

S412, the first collecting buffer tank 15 and the second collecting buffer tank 16 are used for circularly collecting fractions and detecting the purity of the fractions through UHPLC, the fractions reach the preset standard, the fractions are collected to the target pure product tank 10 through valve control, the fractions which do not reach the purity standard but exceed the recoverable fraction purity are collected to the non-pure product collecting tank for re-purification, the fractions which are even lower than the recoverable fraction purity are collected to the waste liquid tank 20, and the cycle is repeated until the content of the target substances is lower than the set value.

It will be appreciated that the chromatographic peak in S41 may also be characterized by the target, i.e. the fraction prepared by high performance phase chromatography is scanned by the full wavelength uv-vis detector 14, and the presence or absence of the target is detected, and the fraction without the target is collected into the solvent recovery tank 17, and the fraction with the target is collected into the first collection buffer tank 15 or the second collection buffer tank 16.

It will be appreciated that the fraction without chromatographic peaks can be regarded as essentially an eluent without impurities mixed into the sample, and that this fraction can be recovered for reuse. The bypass valve of the solvent recovery tank 17 is controlled to be opened by a control cabinet during the flow of the partial fraction, so that the partial fraction enters the solvent recovery tank 17.

Whereas for fractions with chromatographic peaks, it is considered that there may be targets to be purified, this fraction needs further treatment. During the treatment process, the control cabinet control valve II 13-2 opens a valve leading to the branch of the first collecting buffer tank 15 or the branch of the second collecting buffer tank 16, and the first collecting buffer tank 15 or the second collecting buffer tank 16 collects fractions with chromatographic peaks.

The purity of the target is automatically sampled and detected from the first collecting buffer tank 15 or the second collecting buffer tank 16 by an on-line ultra high performance liquid chromatograph 18, and according to the detection result, the fraction of the target having a purity reaching a preset standard is transferred to the target pure product tank 10, and the fraction of the target having a purity not reaching the preset standard is transferred to the non-pure product recovery tank 11.

Further, the preset volume is 3-10L, and the preset time is 1-10min.

The target object is collected in a sectional collection mode by utilizing the preset volume of the collection buffer tank. The preset volume can be 3-10L according to the needs, the smaller the preset volume is, the shorter the segmentation interval of fraction collection is, the loss can be reduced, and the yield can be improved on the premise of ensuring the purity, but the shorter the segmentation interval of fraction collection is, the higher the frequency of opening and closing a pump valve is, the higher the failure rate of the device can be increased, and the maintenance cost of the device is improved.

Too long a fraction collection interval may result in insufficient or even no fraction collection for the target content to meet the predetermined criteria.

In addition, the selection of the preset time is determined according to the nature of the purified substance, and different times are adopted for different polypeptides, and in this embodiment, 5L is preferable for polypeptide purification in combination with maintenance problems of the device and content requirements of the target product.

In addition, on the basis of fixed flow rate, time can be used for monitoring whether the preset volume is reached. The collection time can be set to 1-10min according to the preset volume. The time is used for representing whether the preset volume is reached, so that the equipment setting can be simplified, the risk of human error is avoided, and the quality of the fraction is ensured.

Further, the method comprises a solvent recycling step, and specifically comprises the following steps:

The solvent recycling includes two paths, the solvent recycling of the first path is the solvent recycling of the fraction in the solvent recycling tank 17, and the solvent recycling of the second path is the solvent recycling of the fractions in the target pure product tank 10, the non-pure product recycling tank 11 and the waste liquid tank 20.

The first path is the solvent recycling of the fraction in the solvent recycling tank 17, specifically, after the proportion of the fraction in the solvent recycling tank 17 is confirmed, the valve of the connecting pipeline of the solvent recycling tank 17 and the eluent tank 1 is opened for the fraction which accords with the set proportion of the eluent, and the fraction enters the eluent tank 1 to realize the recycling of the solvent in the fraction;

And for the fractions which do not accord with the setting proportion of the eluent, comparing the density with the density of the eluent after measuring the density by a high-precision mass flowmeter, calculating the volume of the required solvent according to the comparison result, controlling the opening of the valve of the corresponding solvent tank of the solvent tank group 21 by the control cabinet, injecting the corresponding solvent with the required volume into the solvent mixing tank 12, adjusting the proportion of the recovered fraction into the setting proportion of the eluent, and transferring the fraction to the eluent tank 1 to realize the recovery and reutilization of the solvent in the fraction.

The solvent recycling of the second path comprises the following steps:

S61, performing vacuum volatilization on fractions of the target pure product tank 10 and the non-pure product recovery tank 11 in a closed system, performing vacuum volatilization separation on solvents in the fractions, and condensing the solvents into a liquid solvent mixture after cooling;

S62, after measuring and calculating the density of the solvent mixture condensed into liquid through a high-precision mass flowmeter, comparing the density with the density of the eluent, calculating the volume of the required solvent according to the comparison result, controlling the opening of a valve of a corresponding solvent tank of the solvent tank group 21 by a control cabinet, injecting the corresponding solvent with the required volume into the solvent mixing tank 12, adjusting the proportion of the recovered solvent mixture into the set proportion of the eluent, and transferring the solvent mixture to the eluent tank 1 to realize the recovery and reutilization of the solvent in the fraction.

Further, preparing a saturated solution of the crude polypeptide to be purified by dissolving with an eluent, placing the prepared eluent in an eluent tank 1, wherein the eluent adopts a homogeneous solution containing a volatile organic solvent and water, and the organic solvent is methanol, ethanol, acetonitrile, acetone or tetrahydrofuran.

Furthermore, the eluent adopts pure water or aqueous solution, and acid, alkali or salt is added into the pure water or aqueous solution, wherein the acid, alkali or salt is formic acid, acetic acid, trifluoroacetic acid, ammonia water, triethylamine, ammonium acetate, ammonium carbonate or triethylamine acetate.

Furthermore, the control system is used for carrying out linkage control on the sample injection pump, the infusion pump and the switching valve in front of the column, so that the rapid alternate entry of the sample and the eluent is realized. The sample loading device also comprises a short column filled with chromatographic packing with large particle size, the particle size of the chromatographic packing is 20-100 mu m, the short column is arranged between the axial pressurizing column and the sample injection pump, the front end and the rear end of the short column are both provided with multi-channel switching valves, the switching valves are connected with the control device through electric control lines, the switching valves are in data transmission with the control device, and the switching valves are controlled by the control software through the control device. The stub length is between 1-10 cm.

Further, the separated target product fraction with qualified purity enters a target pure product tank 10, the fraction with impurities exceeding the recovery standard enters a non-pure product recovery tank 11, and the solvent of the fraction of the target pure product tank 10 is removed by adopting a vacuum freeze drying mode to obtain solid.

It can be understood that when the freeze-drying operation is performed, the sample is subjected to reduced pressure rotary evaporation to remove the organic solvent, and then the aqueous solution of the sample is remained, and then the aqueous solution of the sample is placed in a freeze-drying bottle or a freeze-drying tray, frozen into ice by a refrigerator or liquid nitrogen, and then the freeze-drying bottle is connected with a freeze-dryer pipeline through a bottle opening or the freeze-drying tray is placed in the freeze dryer for the freeze-drying operation. The freeze dryer is in a high vacuum environment, so that water is continuously reduced to be completely removed by sublimation, and the residual solid is purified target polypeptide.

Micropores with pore size range below 40 a account for less than 5%.

Further, the device for confirming the solvent ratio in the solvent recovery tank 17 is a high-precision mass flowmeter. A high accuracy mass flow meter is provided on the line between the solvent recovery tank 17 and the compounding tank.

It can be understood that the full-wavelength ultraviolet-visible detector 14 and the online ultra-high performance liquid chromatograph 18 are used simultaneously, so that the fraction flowing out of the chromatographic column 5 can be intelligently divided into a fraction capable of directly recovering the solvent (i.e. the fraction which flows into the solvent recovery tank 17 and accords with the eluent setting proportion), and a fraction capable of being recovered after liquid preparation (i.e. the fraction which flows into the solvent recovery tank 17 and does not accord with the eluent setting proportion, the fraction which flows into the target pure product tank 10, the fraction of the non-pure product recovery tank 11 and the fraction which flows into the waste liquid tank 20), and the maximum recovery and utilization of the solvent in the fraction are realized, thereby reducing the waste of the solvent and the labor cost. The number of the collecting tank bodies can be reduced, and the space of the workbench surface is saved.

The solvent recovery efficiency can be improved, the solvent loss can be reduced, the existing solvent storage can be reduced, the equipment investment and the occupied area can be effectively reduced, and the safety and the environmental risk can be reduced by recovering the solvent in the solvent recovery tank 17, the non-pure product recovery tank 11 of the target pure product tank 10 and the waste liquid tank 20. Furthermore, the post-treatment volume of the solvent is reduced, so that the treatment cost of the waste solvent is reduced.

In order to realize the precise separation of the polypeptide with large molecular weight (2000D-6000D), the invention adopts the following key innovation from the purification device:

1. Because of the accurate and quick detection of the online UHPLC, the sampling detection interval of each time is shortest and can be compressed within 1 minute, thereby not only ensuring the accuracy of the purity of the collected product, but also reducing the yield loss caused by the collection volume of the single fraction to the minimum;

2. the eluent infusion pump with precise control is adopted, the flow error is ensured to be within 1.5 percent, the flow is precisely measured on line through the high-precision mass flowmeter, and the precise adjustment of the flow is immediately realized through the control cabinet when the flow fluctuates;

3. the invention also effectively reduces tailing and improves peak shape and separation effect through controlling the consistency and accuracy of the temperature of the chromatographic column and the temperature of the eluent (the temperature is selectable between 10 ℃ and 30 ℃ and the temperature difference between the eluent and the chromatographic column is within +/-3 ℃);

4. the invention adopts the axial pressurizing column without oil cylinder design, thereby thoroughly eliminating the risk of polluting the purified sample due to oil leakage;

5. the invention creatively realizes system integration of 1) ultraviolet visible on-line monitoring, 2) UHPLC on-line detection, 3) fraction collection automatic control, 4) solvent recovery control and 5) on-line liquid preparation based on accurate density monitoring, realizes closed-loop and accurate intelligent control, and realizes the accuracy, high efficiency and stability of polypeptide purification.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

It will be readily understood by those skilled in the art that the present invention, including any combination of parts described in the summary and detailed description of the invention above and shown in the drawings, is limited in scope and does not constitute a complete description of the various aspects of these combinations for the sake of brevity. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives, and variations may be made in the above embodiments by those skilled in the art without departing from the spirit and principles of the invention. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A full-automatic chromatographic purification system comprises a chromatographic system main body device, an on-line detection device, a sample automatic collection device and a control system, wherein the control system is connected with the devices and comprises strong electric connection and weak electric connection,

The control system consists of a remote control center and a control cabinet, and realizes data transmission and electric control;

The chromatographic system main body device comprises a sample tank, a sample loading pump, an eluent tank, an infusion pump and a chromatographic column, wherein the sample tank and the eluent tank are respectively connected with a liquid inlet of the chromatographic column through pipelines, a liquid outlet of the chromatographic column is connected with an online detection device and a sample automatic collection device, and the full-automatic chromatographic purification system is controlled by a control system;

The on-line detection device comprises an on-line ultra-high performance liquid chromatograph, an automatic sampling device and a data processing and data transmission device, wherein the automatic sampling device comprises a first collecting buffer tank, a second collecting buffer tank, an analysis sample sampler and a plurality of control valves;

The chromatographic column outlet is provided with a main pipeline, and two branches formed by the tail end of the main pipeline are respectively connected with a first collecting buffer tank and a second collecting buffer tank, and the two branches are provided with valves;

The device comprises a first collecting buffer tank, a second collecting buffer tank, an on-line detection device, a control cabinet, an analysis sample sampler, an on-line ultra-high performance liquid chromatograph, a control system and a control system, wherein the first collecting buffer tank and the second collecting buffer tank are also connected with the analysis sample sampler through pipelines, and the other end of the analysis sample sampler is connected with the on-line ultra-high performance liquid chromatograph which is used for detecting the purity of a target object in fractions of the first collecting buffer tank and the second collecting buffer tank;

the control system controls valves at the lower ends of the first collecting buffer tank and the second collecting buffer tank according to the detected target object purity data in the first collecting buffer tank and the second collecting buffer tank, and respectively sends qualified target pure objects, unqualified objects and waste liquid to corresponding target pure object tanks, non-pure object recycling tanks and waste liquid tanks;

The control system controls the valve switches of the first branch, the second branch, the third branch, the fourth branch, the fifth branch and the sixth branch according to the detection result of the online ultra-high performance liquid chromatograph, and the automatic collection of the fractions comprises the collection of target pure products, non-pure products capable of being purified secondarily and waste liquid;

The first branch is the connecting line of first collection buffer tank and the pure article jar of target, the second branch is the connecting line of first collection buffer tank and pure article recovery tank of non-, the third branch is the connecting line of second collection buffer tank and pure article jar of target, the fourth branch is the connecting line of second collection buffer tank and pure article recovery tank, the fifth branch is the connecting line of first collection buffer tank and waste liquid jar, the sixth branch is the connecting line of second collection buffer tank and waste liquid jar.

2. The full-automatic chromatographic purification system according to claim 1, wherein the on-line detection device further comprises a full-wavelength ultraviolet-visible detector, the full-wavelength ultraviolet-visible detector is arranged on a main pipeline of an eluent outlet of the chromatographic column and is used for detecting whether the eluent in the main pipeline has a chromatographic peak containing a target object, a seventh branch is formed at the tail end of the main pipeline of the eluent outlet of the chromatographic column except for the two branches, and the seventh branch is connected with a solvent recovery tank, and the other end of the solvent recovery tank is connected with a solvent buffer tank through a pipeline.

3. The full-automatic chromatographic purification system according to claim 1, further comprising an automatic solvent recovery device, thereby forming a complete full-automatic continuous circulation purification system, wherein the front end of the automatic solvent recovery device is respectively connected with a target pure product tank, an impure product recovery tank and a waste liquid tank, the rear end of the automatic solvent recovery device is connected with an eluent tank, the automatic solvent recovery device comprises a solvent mixing tank, a solvent buffer tank and a solvent tank group, the input end of the solvent buffer tank is connected with the target pure product tank, the impure product recovery tank and the waste liquid tank through pipelines, pumps, valves and a decompression distillation device are arranged on the pipelines, the output end of the solvent buffer tank is connected with the first input end of the solvent mixing tank through pipelines, a high-precision flowmeter is arranged on the pipelines, the solvent mixing tank is also connected with the eluent tank through pipelines, and the pump and the valves are arranged on the pipelines.

4. The fully automatic chromatographic purification system of claim 3, wherein the output end of the target pure product tank is connected with the solvent buffer tank through a pipeline, and the other output end of the target pure product tank is connected with the post-treatment tank.

5. The fully automatic chromatographic purification system of claim 4, wherein a reduced pressure distillation device is further provided between the target pure product tank and the solvent buffer tank.

6. The fully automatic chromatographic purification system according to claim 4, wherein the concentrate is crystallized or freeze-dried in a post-treatment tank to obtain the target pure product.

7. The fully automatic chromatographic purification system of claim 3, wherein the first output of the impure product recovery tank is connected to the solvent buffer tank by a conduit and the second output is connected to the sample tank by a conduit.

8. The fully automatic chromatographic purification system of claim 7, wherein a reduced pressure distillation device is further provided between the non-pure recovery tank and the solvent compounding tank.

9. A fully automatic chromatographic purification system in accordance with claim 3 wherein the input of the solvent compounding tank is connected to a solvent tank set which combines a plurality of solvent tanks according to the proportioning requirements of the eluent.

10. A fully automatic chromatographic purification system in accordance with claim 3 wherein the recovered solvent is measured and calculated by a mass flowmeter to obtain the corresponding component ratios and the ratios are automatically adjusted to the eluent ratios in the compounding tank.

11. The fully automatic chromatographic purification system of claim 3, wherein the first output of the solvent recovery tank is connected to an eluent tank line and the second output of the solvent recovery tank is connected to a solvent buffer tank.

12. The fully automated chromatographic purification system of claim 1, wherein the chromatographic column is a dynamic axial compression chromatographic column.

13. The fully automatic chromatographic purification system according to claim 1, wherein the packing material filled in the chromatographic column is spherical chromatographic packing material of silica gel matrix, and the packing length of the chromatographic column is 20cm-100cm.

14. The fully automatic chromatographic purification system of claim 13, wherein the silica gel surface has at least two silanes, one of which is polar silane and the other of which is non-polar silane, or two of which are different non-polar silanes, bonded to the silica gel surface in a proportion of not less than 10% other than the end capping agent.

15. The fully automatic chromatographic purification system according to claim 1, wherein the temperature of the eluent and the chromatographic column is controlled within a range of 10 ℃ to 30 ℃, and the temperature difference between the chromatographic column and the eluent is within + -3 ℃.

16. The full-automatic chromatographic purification system according to claim 1, wherein two eluent tanks are respectively used for containing eluents with different components or different component proportions, high-pressure constant-flow pumps are respectively arranged on connecting pipelines of the eluent tanks and the chromatographic column, and online gradient mixing of the eluents is realized by respectively controlling the flow ratio of the two high-pressure constant-flow pumps, so that the samples are subjected to gradient elution.

17. The fully automatic chromatographic purification system according to claim 1, for reversed phase purification of polypeptides, macrolides, cyclosporins organics.