A system and method for integrated preparation of capillary chromatographic column

Abstract

The present invention discloses a system and method for preparing a capillary chromatographic column in an integrated manner, wherein the system for preparing a capillary chromatographic column in an integrated manner is disclosed, including an injection system and a connecting system provided with a connecting station, the connecting station is used to connect the capillary, the injection system includes a plurality of low-pressure injection pumps, a high-pressure injection pump, and a quantitative ring; the connecting system includes a connecting pipeline and a multi-way valve, each high-pressure injection pump, a low-pressure injection pump, and a quantitative ring are connected to the connecting pipeline, and the multi-way valve is adjusted to switch the connection status between each high-pressure injection pump, a low-pressure injection pump, a quantitative ring, and a capillary connected to the connecting station; the low-pressure injection pump is used to inject a chromatographic filler-organic solvent suspension or a high-concentration protein sample into the quantitative ring, and the high-pressure injection pump is used to press the chromatographic filler-organic solvent suspension or the high-concentration protein sample in the quantitative ring into the capillary connected to the connecting station. The present invention also discloses a method for preparing a capillary chromatographic column using the aforementioned system.

Description

Technical Field

[0001] The invention relates to the field of capillary chromatographic column preparation, and in particular to a system and method for integrated preparation of a capillary chromatographic column. Background Art

[0002] Proteins are the direct executors of cell functions. Qualitative and quantitative analysis of trace proteome samples such as the proteome of single cells, trace body fluids or micro-regional tissues can provide a more accurate assessment of the detailed physiological state of an organism, and can also help doctors conduct targeted personalized precision treatments, improve the success rate of disease treatment, and help researchers further understand the mechanism of action of the immune system and the occurrence of tumors, and reveal the detailed differentiation mechanisms at different stages of embryonic development. However, since the protein content in the above trace samples is extremely low and cannot be amplified, powerful analytical methods are required.

[0003] In recent years, with the continuous improvement of the sensitivity of mass spectrometry technology and the pressure resistance of liquid chromatographs, the bottom-up "shotgun method" based on liquid chromatography-mass spectrometry has become one of the most mainstream analytical techniques for studying trace sample proteomics. Since the composition of proteome samples is extremely complex and the dynamic range is extremely wide, extremely high requirements are placed on the core component of liquid chromatography separation and analysis - the liquid chromatography column. At present, most commercial chromatographic columns on the market have an inner diameter of hundreds of microns, which are easy to adsorb samples, and the corresponding dead volume of the flow path is large, which is not suitable for the separation of trace proteome samples. Therefore, most researchers in this field currently use homemade capillary filling columns to achieve separation functions.

[0004] The complete operation steps for making a typical integrated needle capillary column include: using a laser drawing machine to prepare a quartz capillary with an integrated needle, dispersing the chromatographic filler, filling the chromatographic filler into the quartz capillary, gradually increasing the pressure to compact the capillary column bed, saturating the column with a high concentration of protein sample, repeatedly washing the column with a high organic phase, and balancing the column with water.

[0005] An existing device for preparing a capillary chromatographic column, such as a packed chromatographic column preparation device disclosed in a Chinese patent with publication number CN209076099U, comprises a vacuum box and a vacuum pump arranged on one side of the vacuum box, a vacuum port is arranged on one side of the vacuum box, the vacuum pump is connected to the vacuum port on the vacuum box through a connecting pipe, a base is arranged at the bottom of the vacuum box, a vibrator is arranged in the base, a feeding funnel is arranged above the vacuum box, the small-diameter end of the feeding funnel is connected to the interior of the vacuum box, a drainage port 1 and a drainage port 2 are arranged on the outer side wall of the vacuum box, the drainage port 1 and the drainage port 2 are connected through a connecting pipe, a chromatographic column is arranged in the vacuum box, reducers are arranged at both ends of the chromatographic column, one end of the chromatographic column is connected to the feeding funnel, and the other end of the chromatographic column is connected to the drainage port 1, a pressure relief valve is arranged on the vacuum box, and a pressure gauge for detecting the internal pressure is arranged on one side of the vacuum box.

[0006] However, the chromatographic column preparation device disclosed in the above patent can only complete the filling of the chromatographic column filler. After the filling of the filler is completed, the capillary needs to be removed and then connected to a high-pressure injection pump such as a liquid chromatograph for subsequent cleaning and saturation. In other words, the device cannot complete the entire preparation process of the chromatographic column. The use of the device to prepare the chromatographic column is cumbersome and the column bed is easily loosened by recoil during the pressure relief process. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the present invention provides a system and method for preparing a capillary chromatographic column in an integrated manner.

[0008] An integrated system for preparing a capillary chromatographic column comprises an injection system and a connecting system provided with a connecting station, wherein the connecting station is used to connect a capillary.

[0009] The injection system includes a plurality of low-pressure injection pumps, a high-pressure injection pump, and a quantitative ring;

[0010] The communication system includes a plurality of communication pipes and a multi-way valve, each high-pressure injection pump, a low-pressure injection pump and a quantitative ring are connected to the communication pipe, and the communication status among each high-pressure injection pump, a low-pressure injection pump, a quantitative ring and a capillary connected to the connection station is switched by adjusting the multi-way valve;

[0011] The low-pressure injection pump is used to inject the chromatographic filler-organic solvent suspension or high-concentration protein sample into the quantitative loop, and the high-pressure injection pump is used to press the chromatographic filler-organic solvent suspension or high-concentration protein sample in the quantitative loop into the capillary connected to the connection station.

[0012] Specifically, the low-pressure injection pumps, the high-pressure injection pumps and the quantitative loop are connected by a connecting pipe, and the connection between the low-pressure injection pumps, the high-pressure injection pumps, the quantitative loop and the capillary is switched by adjusting the multi-way valve, and the low-pressure injection pump is filled with a chromatographic filler-organic solvent suspension or a high-concentration protein sample. That is, all processes of making a capillary chromatographic column can be completed in the same system by adjusting the multi-way valve, and the whole process will not experience a pressure relief process, ensuring that the chromatographic column will not be dispersed by recoil;

[0013] At the same time, a quantitative ring structure is also provided. The chromatographic filler - organic solvent suspension and high-concentration protein sample are first injected into the quantitative ring for metering and then pressed into the capillary via a high-pressure injection pump, further ensuring the quality of the produced chromatographic column.

[0014] Preferably, the injection system further comprises an ultrasonic cleaning device for uniformly distributing the chromatographic filler in the chromatographic filler-organic solvent suspension in the quantitative loop in the organic solvent.

[0015] Specifically, after the chromatographic filler-organic solvent suspension enters the quantitative loop, the chromatographic filler is prone to sedimentation, so it is necessary to set up an ultrasonic cleaning device to ensure that the chromatographic filler is evenly distributed in the organic solvent.

[0016] Preferably, the organic solvent in the chromatographic filler-organic solvent suspension is isopropanol.

[0017] Specifically, isopropanol has a suitable viscosity, can effectively reduce the sedimentation velocity of the chromatographic filler, and is conducive to the uniform distribution of the chromatographic filler. It is necessary to select isopropanol as the dispersing organic solvent of the chromatographic filler.

[0018] Preferably, the injection system further comprises a waste liquid pool for collecting waste liquid, and the waste liquid pool is connected to the connecting pipe.

[0019] Specifically, in the process of making the chromatographic column, it is inevitable to produce excess liquid. In order to prevent the excess liquid from flowing back into the low-pressure injection pump or the high-pressure injection pump, it is appropriate to set up a waste liquid pool to collect the excess liquid.

[0020] Preferably, a spray needle is provided at one end of the capillary tube, and the end of the capillary tube not provided with the spray needle is connected to a connecting pipe.

[0021] Preferably, the system is provided with two connection stations, two high-pressure injection pumps, two low-pressure injection pumps, and three quantitative loops; the chromatographic filler-organic solvent suspension and the high-concentration protein sample are respectively injected into different quantitative loops through different low-pressure injection pumps and pressed into the capillary by different high-pressure injection pumps.

[0022] Specifically, under this structure, two capillary chromatographic columns can be prepared simultaneously.

[0023] A method for preparing a capillary chromatographic column in an integrated manner, using the system for preparing a capillary chromatographic column in an integrated manner as described in any one of the above items, comprising the following steps:

[0024] Step 1, respectively filling the chromatographic filler-organic solvent suspension and the high-concentration protein sample into different low-pressure injection pumps, and connecting the capillary to the connection station;

[0025] Step 2, adjusting the multi-way valve to connect the low-pressure injection pump filled with the chromatographic filler-organic solvent suspension to the quantitative loop, and injecting the chromatographic filler-organic solvent suspension into the quantitative loop after connection;

[0026] Step 3, adjusting the multi-way valve to connect the high-pressure injection pump, the quantitative loop filled with the chromatographic filler-organic solvent suspension, and the capillary in sequence, and starting the high-pressure injection pump to press the chromatographic filler in the quantitative loop into the capillary;

[0027] Step 4: After the chromatographic filler in the capillary is filled to a specified length, the high-pressure injection pump is pressurized to compact the chromatographic column bed formed by the accumulation of the chromatographic filler in the capillary;

[0028] Step 5, adjusting the multi-way valve to connect the low-pressure injection pump filled with the high-concentration protein sample to the quantitative loop, and injecting the high-concentration protein sample into the quantitative loop after the connection;

[0029] Step 6, adjusting the multi-way valve to connect the high-pressure injection pump, the quantitative loop filled with the high-concentration protein sample, and the capillary filled with the chromatographic filler in sequence, starting the high-pressure injection pump to press the high-concentration protein sample in the quantitative loop into the capillary filled with the chromatographic filler, and starting the gradient elution to saturate and clean the chromatographic column bed formed by the accumulation of the chromatographic filler. After the saturation and cleaning are completed, the chromatographic column preparation is completed.

[0030] Specifically, after the chromatographic filler-organic solvent suspension enters the quantitative loop, an ultrasonic cleaner may be turned on to make the chromatographic filler evenly distributed in the organic solvent.

[0031] Preferably, when two capillaries are connected to the communication system, the two capillaries are connected to different low-pressure injection pumps, high-pressure injection pumps, and quantitative loops by adjusting the multi-way valve, so that the two capillary chromatographic columns are in different preparation steps.

[0032] Compared with the prior art, the present invention is beneficial in that:

[0033] The low-pressure injection pumps, the high-pressure injection pumps and the quantitative loop are connected by a connecting system, and the connection status among the low-pressure injection pumps, the high-pressure injection pumps, the quantitative loop and the capillary is switched by a multi-way valve, so that the preparation of the entire capillary chromatographic column can be completed on the same system. The entire process does not experience a pressure relief process, which ensures that the chromatographic column will not be dispersed by recoil, thereby improving the preparation quality of the chromatographic column. At the same time, the integrated system for preparing the capillary chromatographic column provided by the present invention has greater flexibility. By adjusting the multi-way valve, multiple capillary chromatographic columns can be prepared at the same time, thereby improving the preparation efficiency of the capillary chromatographic columns. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 A schematic diagram of an integrated system for preparing a capillary chromatographic column provided by the present invention. DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0036] like Figure 1 As shown, the system for preparing the capillary chromatographic column in an integrated manner includes an injection system and a communication system having two connecting stations, wherein the connecting station is used to connect the capillary 70, and the two connecting stations are respectively a first connecting station 41 and a second connecting station 42.

[0037] The injection system includes a first low-pressure injection pump 11, a second low-pressure injection pump 12, a first high-pressure injection pump 21, a second high-pressure injection pump 22, a first quantitative ring 31, a second quantitative ring 32, and a third quantitative ring 33;

[0038] The communication system includes a plurality of communication pipes and a multi-way valve, wherein the multi-way valve includes a six-way valve 51 and a twelve-way valve 52. The first low-pressure injection pump 11, the first high-pressure injection pump 21 and the first quantitative ring 31 are directly connected to the six-way valve 51 through a connecting channel, the second low-pressure injection pump 12, the second high-pressure injection pump 22, the second quantitative ring 32 and the third quantitative ring 33 are directly connected to the twelve-way valve 52 through a connecting pipe, and the second quantitative ring 32 is close to the first connecting station 41, and the third quantitative ring 33 is close to the second connecting station 42. The six-way valve 51 and the twelve-way valve 52 are respectively connected to a waste liquid pool 61, and the first connecting station 41 and the second connecting station 42 are arranged between the six-way valve 51 and the twelve-way valve 52.

[0039] By adjusting the six-way valve 51 or the twelve-way valve 52, the communication status among the first low-pressure injection pump 11, the second low-pressure injection pump 12, the first high-pressure injection pump 21, the second high-pressure injection pump 22, the first quantitative ring 31, the second quantitative ring 32, the third quantitative ring 33, and the capillary 70 connected to the connection station is switched;

[0040] The first low-pressure injection pump 11 or the second low-pressure injection pump 12 is used to inject the chromatographic filler-organic solvent suspension or the high-concentration protein sample into the quantitative loop, and the first high-pressure injection pump 21 or the second high-pressure injection pump 22 is used to press the chromatographic filler-organic solvent suspension or the high-concentration protein sample in the quantitative loop into the capillary 70 connected to the connecting station.

[0041] The low-pressure injection pumps, the high-pressure injection pumps and the quantitative loop are connected by a connecting pipe, and the connection between the low-pressure injection pumps, the high-pressure injection pumps, the quantitative loop and the capillary 70 is switched by adjusting the multi-way valve, and the low-pressure injection pump is filled with a chromatographic filler-organic solvent suspension or a high-concentration protein sample. That is, all processes of making a capillary chromatographic column can be completed in the same system by adjusting the multi-way valve, and the whole process will not experience a pressure relief process, ensuring that the chromatographic column will not be dispersed by recoil;

[0042] At the same time, a quantitative ring structure is also provided, and the chromatographic filler-organic solvent suspension and high-concentration protein sample are first injected into the quantitative ring for measurement and then pressed into the capillary 70 via a high-pressure injection pump, further ensuring the quality of the produced chromatographic column.

[0043] The injection system further comprises an ultrasonic cleaning device 62 for uniformly distributing the chromatographic filler in the chromatographic filler-organic solvent suspension in the quantitative loop in the organic solvent.

[0044] After the chromatographic filler-organic solvent suspension enters the quantitative loop, the chromatographic filler is prone to sedimentation, so it is necessary to provide an ultrasonic cleaning device 62 to ensure that the chromatographic filler is evenly distributed in the organic solvent.

[0045] The organic solvent in the chromatographic filler-organic solvent suspension is isopropanol.

[0046] The injection system further includes a waste liquid pool 61 for collecting waste liquid, and the waste liquid pool 61 is connected to the communication pipeline.

[0047] In the process of making the chromatographic column, it is inevitable that excess liquid will be produced. In order to prevent the excess liquid from flowing back into the low-pressure injection pump or the high-pressure injection pump, it is appropriate to set up a waste liquid pool 61 to collect the excess liquid.

[0048] One end of the capillary tube 70 is provided with a spray needle, and the end of the capillary tube 70 not provided with the spray needle is communicated with the communication pipe.

[0049] The method for preparing a capillary chromatographic column by integration is provided in this embodiment. It is a method for preparing two capillary chromatographic columns at the same time by using the above-mentioned system for preparing a capillary chromatographic column by integration. The specific operation steps are as follows:

[0050] Step 1.1, filling the chromatographic filler-isopropanol suspension and the high-concentration protein sample into the first low-pressure injection pump 11 and the second low-pressure injection pump 12 respectively, and connecting the two capillaries 70 to the first connecting station 41 and the second connecting station 42 respectively;

[0051] Step 2.1, adjusting the six-way valve 51 to connect the first low-pressure injection pump 11 with the first quantitative loop 31, injecting the chromatographic filler-isopropanol suspension into the first quantitative loop 31 after the connection, and starting the ultrasonic cleaning instrument 62; at the same time, adjusting the twelve-way valve 52 to connect the second low-pressure injection pump 12 with the second quantitative loop 32, and injecting the high-concentration protein sample into the second quantitative loop 32 after the connection;

[0052] Step 3.1, adjusting the six-way valve 51 to connect the first high-pressure injection pump 21, the first quantitative ring 31, and the capillary 70 connected to the first connecting station 41 in sequence, starting the first high-pressure injection pump 21 to press the chromatographic filler in the first quantitative ring 31 into the capillary 70 connected to the first connecting station 41;

[0053] Step 4.1, after the chromatographic filler in the capillary 70 of the first connecting station 41 is filled to a specified length, the six-way valve 51 is adjusted so that the first high-pressure injection pump 21 is directly connected to the capillary 70 connected to the first connecting station 41, and the first low-pressure injection pump 11 is connected to the first quantitative loop 31, and then the first high-pressure injection pump 21 is pressurized to compress the column bed of the chromatographic filler in the capillary 70 connected to the first connecting station 41, and the first low-pressure injection pump 11 injects the chromatographic filler-isopropanol suspension into the first quantitative loop 31;

[0054] Step 5.1, adjusting the twelve-way valve 52 to connect the second high-pressure injection pump 22, the second quantitative loop 32, and the capillary 70 of the first connecting station 41, after which the high-concentration protein sample in the second quantitative loop 32 is pressed into the capillary 70 of the first connecting station 41, and then the second high-pressure injection pump 22 starts the gradient to saturate and clean the capillary 70 of the first connecting station 41. After the saturation and cleaning are completed, the prepared capillary chromatographic column is removed and replaced with a new capillary 70;

[0055] At the same time, the six-way valve 51 is adjusted so that the first high-pressure injection pump 21, the first quantitative ring 31, and the capillary 70 of the second connecting station 42 are connected in sequence, and the chromatographic filler-isopropanol suspension is pressed into the capillary 70 of the second connecting station 42. The six-way valve 51 is adjusted again so that the first high-pressure injection pump 21 is directly connected to the capillary 70 of the second connecting station 42 and continuously pressurized to compress the column bed of the capillary 70 of the second connecting station 42, and the first low-pressure injection pump 11 is connected to the first quantitative ring 31 again and the chromatographic filler-isopropanol suspension is injected into the first quantitative ring 31;

[0056] Step 6.1, adjusting the six-way valve 51 so that the first high-pressure injection pump 21, the first quantitative loop 31, and the newly replaced capillary 70 of the first connecting station 42 are connected in sequence, and the chromatographic column preparation steps of the capillary 70 of the first connecting station 41 are repeated;

[0057] At the same time, the twelve-way valve 52 is adjusted to connect the second high-pressure injection pump 22, the third quantitative loop 33, and the capillary 70 of the second connecting station 42, and the high-concentration protein sample is pressed into the capillary 70 of the second connecting station 42. The second high-pressure injection pump 22 starts gradient elution to saturate and clean the capillary chromatographic column of the second connecting station 42. After saturation and cleaning, the preparation of the capillary chromatographic column of the second connecting station 42 is completed, the prepared capillary chromatographic column is removed and replaced with a new one, and then the preparation steps of the capillary chromatographic column of the second connecting station 42 are repeated.

[0058] According to the above steps, the preparation of the capillary chromatographic column in the first connecting station 41 and the capillary chromatographic column in the second connecting station 42 can be performed simultaneously, and the preparation steps therebetween are staggered.

Claims

1. A system for preparing an integrated capillary chromatographic column, characterized in that: It includes an injection system and a communication system with a connection station, wherein the connection station is used to connect the capillary tube. The injection system includes a plurality of low-pressure injection pumps, a high-pressure injection pump, and a quantitative ring; The communication system includes a plurality of communication pipes and a multi-way valve, each high-pressure injection pump, a low-pressure injection pump and a quantitative ring are connected to the communication pipe, and the communication status among each high-pressure injection pump, a low-pressure injection pump, a quantitative ring and a capillary connected to the connection station is switched by adjusting the multi-way valve; The low-pressure injection pump is used to inject the chromatographic filler-organic solvent suspension or high-concentration protein sample into the quantitative loop, and the high-pressure injection pump is used to press the chromatographic filler-organic solvent suspension or high-concentration protein sample in the quantitative loop into the capillary connected to the connection station; The injection system also includes an ultrasonic cleaning device for uniformly distributing the chromatographic filler in the chromatographic filler-organic solvent suspension in the quantitative loop in the organic solvent; The system is provided with two connection stations, two high-pressure injection pumps, two low-pressure injection pumps and three quantitative loops. The chromatographic filler-organic solvent suspension and the high-concentration protein sample are respectively injected into different quantitative loops through different low-pressure injection pumps and pressed into the capillary by different high-pressure injection pumps.

2. The system for preparing an integrated capillary chromatographic column according to claim 1, characterized in that: The organic solvent in the chromatographic filler-organic solvent suspension is isopropanol.

3. The system for preparing an integrated capillary chromatographic column according to claim 1, characterized in that: The injection system also includes a waste liquid pool for collecting waste liquid, and the waste liquid pool is connected to the communication pipeline.

4. The system for preparing an integrated capillary chromatographic column according to claim 1, characterized in that: One end of the capillary is provided with a spray needle, and the end of the capillary not provided with the spray needle is communicated with a communication pipe.

5. A method for preparing an integrated capillary chromatographic column, characterized in that: The system for preparing an integrated capillary chromatographic column according to any one of claims 1 to 4 comprises the following steps: Step 1, respectively filling the chromatographic filler-organic solvent suspension and the high-concentration protein sample into different low-pressure injection pumps, and connecting the capillary to the connection station; Step 2, adjusting the multi-way valve to connect the low-pressure injection pump filled with the chromatographic filler-organic solvent suspension to the quantitative loop, and injecting the chromatographic filler-organic solvent suspension into the quantitative loop after connection; Step 3, adjusting the multi-way valve to connect the high-pressure injection pump, the quantitative loop filled with the chromatographic filler-organic solvent suspension, and the capillary in sequence, and starting the high-pressure injection pump to press the chromatographic filler in the quantitative loop into the capillary; Step 4, after the chromatographic filler in the capillary is filled to a specified length, the high-pressure injection pump is pressurized to compact the chromatographic column bed formed by the accumulation of the chromatographic filler in the capillary; Step 5, adjusting the multi-way valve to connect the low-pressure injection pump filled with the high-concentration protein sample to the quantitative loop, and injecting the high-concentration protein sample into the quantitative loop after the connection; Step 6, adjusting the multi-way valve to connect the high-pressure injection pump, the quantitative loop filled with the high-concentration protein sample, and the capillary filled with the chromatographic filler in sequence, starting the high-pressure injection pump to press the high-concentration protein sample in the quantitative loop into the capillary filled with the chromatographic filler, and starting the gradient elution to saturate and clean the chromatographic column bed formed by the accumulation of the chromatographic filler. After the saturation and cleaning are completed, the chromatographic column preparation is completed.

6. The method for integrated preparation of a capillary chromatographic column according to claim 5, characterized in that: When two capillaries are connected to the communication system, the two capillaries are connected to different low-pressure injection pumps, high-pressure injection pumps and quantitative loops by adjusting the multi-way valve, so that the two capillary chromatographic columns are in different preparation steps.

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