Preparative high performance liquid chromatography pre-column and column packing method

Abstract

The invention belongs to the technical field of chromatographic separation, and particularly relates to a preparative high performance liquid chromatography pre-column and a packing method.The pre-column comprises an upper sieve plate, a column tube, a lower sieve plate and a column foot, the middle column tube is located between the upper sieve plate and the lower sieve plate and connected with the upper sieve plate and the lower sieve plate in a sealed mode, and a space for containing chromatographic packing is formed in the column tube; a sealing gasket is arranged between the upper sieve plate and the column pipe and is positioned above the space, and the top surface of the sealing gasket is higher than the upper surface of the upper sieve plate; the upper sieve plate is provided with a liquid inlet communicated with the space, the lower sieve plate is provided with a liquid outlet communicated with the space, and the lower sieve plate is further connected with detachable column feet. The pre-column can protect the main column of the high-performance preparative liquid chromatographic column from being polluted by impurities, and the service life of the high-performance preparative liquid chromatographic column is prolonged; the column packing method disclosed by the invention is simple and practical, does not need a column packing machine, can be repeatedly used for column packing, can resist the maximum pressure of 20Mpa, is used in series with the main column, and does not influence the column efficiency, peak shape and separation degree of the main column.

Description

Technical Field

[0001] This invention belongs to the field of chromatographic separation technology, specifically a preparative high-performance liquid chromatography pre-column and column packing method. Background Technology

[0002] High-performance liquid chromatography (HPLC) is a separation technique characterized by high column efficiency, large sample throughput, and low solvent usage; however, HPLC columns are generally expensive, especially those with small particle size packing. When using HPLC for separation, purification, or preparation experiments, the material composition is complex. As the material is separated and purified, the packing material inside the column gradually becomes contaminated, leading to increased column pressure, decreased resolution, and reduced adsorption capacity. To reduce contamination of the column packing and extend its lifespan, a short pre-column is typically added at the front end of the column. This pre-column acts as a filter to protect the column without affecting peak shape or separation performance.

[0003] Since the function of the pre-column is to protect the main column, it is generally connected to the front end of the main column. The presence of the pre-column should not affect the peak shape or resolution of the main column. Therefore, the pre-column height is generally shorter, and its pressure resistance must be good, especially in the field of high-efficiency preparative chromatography columns, where the pre-column needs to be able to operate normally at 10 MPa, and some even need to operate normally at 20 MPa. To ensure that the pre-column does not affect the peak shape and separation effect of the main column, the corresponding pre-column should also be a high-pressure pre-column, and its column bed should be able to remain stable and not collapse under the impact of high-pressure mobile phase; otherwise, it will affect the peak shape and separation effect of the main column. For pre-columns with shorter column heights, to ensure column bed stability under high pressure, the pre-column should be subjected to corresponding pressure during packing. However, obtaining sufficient pressure for the pre-column bed has always been a challenge, especially for packing materials with particle sizes below 10 μm. If the packing is not tight, the pre-column bed can easily collapse under the impact of the high-pressure mobile phase, forming cavities within the pre-column and thus affecting the peak shape and separation performance of the main chromatographic column.

[0004] One existing technology involves a core-type pre-column. The principle is to press a filter sieve plate into the pre-column tube using a column packer to form a core, which is then sealed with an outer shell to create the pre-column. While this method allows for high-pressure packing and the pre-column bed can withstand high pressure after packing, it requires precise calculation of the packing material amount beforehand. Too much packing material will prevent the sieve plate from being fully pressed into the column tube, while too little will result in internal cavities or excessive dead volume, affecting peak shape and separation efficiency of the main column. The conditions for successful packing are extremely stringent. Furthermore, the core of this type of pre-column cannot be repeatedly packed; each core can only be packed with one type of packing material, and packing material cannot be changed. This makes it extremely inflexible in practical use, especially in drug development applications where the demand for pre-columns is often diverse and immediate. Each new sample requires a new core, easily leading to core waste and increased separation costs. Summary of the Invention

[0005] In view of the above-mentioned problems of existing pre-columns, the purpose of this invention is to provide a preparative high-performance liquid chromatography pre-column and a packing method. The pre-column of this invention can achieve repeated packing, and its column bed can withstand high pressure after packing. Moreover, the packing conditions are simple and can meet the requirements of high-performance chromatography columns for continuous use of pre-columns.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] The preparative high-performance liquid chromatography pre-column of the present invention includes an upper sieve plate, a column tube, a lower sieve plate, and column feet. The column tube is located between the upper sieve plate and the lower sieve plate and is sealed to both the upper sieve plate and the lower sieve plate respectively. The column tube has a space for accommodating chromatographic packing material. A sealing gasket is provided between the upper sieve plate and the column tube. The sealing gasket is located above the space, and the top surface of the sealing gasket is higher than the upper surface of the upper sieve plate. The upper sieve plate has an inlet communicating with the space, and the lower sieve plate has an outlet communicating with the space. A detachable column foot is also connected to the lower sieve plate.

[0008] Wherein: a through hole is opened in the middle of the column tube, which is the space for accommodating the chromatographic packing material; an inner groove is opened on the upper surface of the column tube, and the sealing gasket is accommodated in the inner groove; multiple threaded holes A are evenly opened along the circumferential direction on the outer edge of the column tube for connecting with the upper sieve plate and the lower sieve plate.

[0009] The inner diameter of the sealing gasket is the same as the diameter of the through hole, and the thickness of the sealing gasket is greater than the groove depth of the inner groove.

[0010] The outer edges of the upper and lower sieve plates are uniformly provided with threaded holes B along the circumferential direction. The threaded holes B and threaded holes A are connected by screws. The number of threaded holes B on the upper sieve plate is the same as the number of threaded holes B on the lower sieve plate, and both are even numbers. The number of threaded holes A is equal to the sum of the number of threaded holes B on the upper and lower sieve plates. The threaded holes A on the column tube that connect to the threaded holes B on the upper sieve plate and those that connect to the threaded holes B on the lower sieve plate are spaced apart.

[0011] The upper screen plate includes a cover plate, a distributor, a screen, and a sealing ring. The cover plate has a groove along its thickness on the lower surface facing the column tube. The distributor and the screen are respectively housed in the groove from top to bottom. The distributor is mounted on the cover plate, and the screen is mounted on the distributor. The sealing ring is located on the lower surface of the cover plate facing the column tube and is in contact with the sealing gasket. The liquid inlet is opened on the cover plate and communicates with the groove.

[0012] The lower sieve plate includes a cover plate, a distributor, a screen, and a sealing ring. The cover plate has a groove along its thickness on the upper surface facing the column tube. The distributor and the screen are respectively housed in the groove from bottom to top. The distributor is mounted on the cover plate, and the screen is mounted on the distributor. The sealing ring is located at the position where the upper surface of the cover plate faces the column tube and contacts the column tube. The lower surface of the cover plate has a column foot hole for connecting the column foot. The liquid outlet is opened on the cover plate and communicates with the groove.

[0013] The present invention discloses a pre-column packing method for preparative high-performance liquid chromatography (HPLC). This method employs a wet packing process using a homogenizer, combined with negative pressure suction to remove the homogenizer solvent. This allows the chromatographic packing material to form a column bed layer by layer from the bottom. After the column bed is filled in the pre-column, the pre-column's structural design ensures high pressure. Specifically:

[0014] First, the chromatographic packing material to be packed into the column is ultrasonically homogenized with solvent. Then, the lower sieve plate, column tube, and column feet of the preparative high-performance liquid chromatography pre-column are installed, and a sealing gasket is installed at the upper end of the column tube. The outlet of the lower sieve plate is connected to a vacuum pump via a pipeline. The homogenized solution is gradually poured into the space inside the column tube where the chromatographic packing material is placed. The vacuum pump is turned on, creating negative pressure in the pipeline connecting the outlet, which draws out the homogenized solvent. The packing material accumulates at the bottom of the column tube, forming a column bed. The vacuum pump is then turned off. The homogenized solution is poured in again, and the vacuum pump is turned on again to remove the solvent. This process is repeated until the packing layer fills the column tube and reaches the height of the sealing gasket. Then, a straight scraper is used to scrape off the excess packing material, making the packing layer flush with the sealing gasket. The upper sieve plate is then placed on top and tightened gradually with screws until the upper sieve plate exerts sufficient pressure on the packing material.

[0015] Wherein: the amount of homogenizing solvent used is more than 3 times the mass of the filler, and the ultrasonic time of the homogenizing solvent is more than 5 minutes.

[0016] The upper screen plate is tightened by screws in a diagonal manner, and the upper screen plate must be kept horizontal during the tightening process.

[0017] The advantages and positive effects of this invention are as follows:

[0018] 1. The upper and lower sieve plates and column tube of the pre-column of this invention are designed to be detachable, making them flexible and convenient to use. The design of the sieve plate adds a distributor, which can effectively solve the problem of uneven distribution of mobile phase caused by the scale-up from analytical column to preparative column. Thus, the pre-column can be used in conjunction with the high-performance chromatography column without affecting the peak shape and separation effect of the high-performance chromatography column.

[0019] 2. The column tube structure of the present invention, combined with the shrinkage of the sealing gasket, conveniently and effectively solves the problem of how to obtain high pressure in the column bed inside the column tube without damaging the sealing performance of the pre-column, and how to keep the column bed stable, when the pre-column column tube is generally short and has no piston.

[0020] 3. The pre-column of the present invention has significant advantages over the core-type pre-column of the prior art. It can be repeatedly loaded with columns, and the loading method is simple and practical, without the need for a professional column loading machine. The entire pre-column can be used flexibly and in various ways, which increases efficiency and reduces costs. The pre-column of the present invention for preparing high-performance liquid chromatography has a simple structure, is easy to operate and practical, and has great potential for commercial promotion. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the three-dimensional structure of the pre-column for high performance liquid chromatography of the present invention;

[0022] Figure 2 This is a cross-sectional view of the internal structure of the pre-column for high-performance liquid chromatography (HPLC) of this invention.

[0023] Figure 3 for Figure 1 A schematic diagram of the three-dimensional structure after removing the upper sieve plate;

[0024] Figure 4 This is a three-dimensional structural diagram of the upper sieve plate in the pre-column of the preparative high-performance liquid chromatography of the present invention;

[0025] Figure 5 This is a three-dimensional structural diagram of the column tube in the preparative high-performance liquid chromatography pre-column of the present invention;

[0026] Figure 6 This is a cross-sectional view of the internal structure of the column tube in the pre-column of the pre-column for high performance liquid chromatography of the present invention;

[0027] Figure 7This is a schematic diagram of the internal structure of the lower sieve plate in the pre-column of the preparative high performance liquid chromatography of the present invention;

[0028] Figure 8 This is a spectrum of the individual column efficiency test of the C18-5μm packing pre-column of the present invention;

[0029] Figure 9 This is a test spectrum of the column efficiency of the C18-5μm packing pre-column tandem main column of the present invention;

[0030] Figure 10 This is the column efficiency test spectrum of the C18HCE-5μm packing pre-column tandem main column of the present invention;

[0031] Wherein: 1 is the upper sieve plate, 2 is the column tube, 3 is the lower sieve plate, 4 is the column foot, 5 is the screw, 6 is the sealing gasket, 7 is the liquid inlet, 8 is the liquid outlet, 9 is the cover plate, 10 is the distributor, 11 is the screen, 12 is the sealing ring, 13 is the inner groove, 14 is the threaded hole A, 15 is the column foot hole, 16 is the threaded hole B, and 17 is the through hole. Detailed Implementation

[0032] The invention will now be described in further detail with reference to the accompanying drawings.

[0033] like Figures 1-7 As shown, the preparative high-performance liquid chromatography pre-column of the present invention includes an upper sieve plate 1, a column tube 2, a lower sieve plate 3, and column feet 4. The column tube 2 is located between the upper sieve plate 1 and the lower sieve plate 3 and is sealed to both the upper sieve plate 1 and the lower sieve plate 3. The column tube 2 has a space for accommodating chromatographic packing material. A sealing gasket 6 is provided between the upper sieve plate 1 and the column tube 2. The sealing gasket 6 is located above the space, and the top surface of the sealing gasket 6 is higher than the upper surface of the upper sieve plate 1. The upper sieve plate 1 has an inlet 7 communicating with the space, and the lower sieve plate 3 has an outlet 8 communicating with the space. The lower sieve plate 3 is also connected to a detachable column foot 4.

[0034] In this embodiment, the column tube 2 is a cylindrical tube used to pack chromatographic packing material. The chromatographic packing material in this embodiment has a particle size of 3 to 100 micrometers. A through hole 17 is provided in the middle of the column tube 2, which is the space for accommodating the chromatographic packing material. An inner groove 13 is provided on the upper surface of the column tube 2, and the sealing gasket 6 is accommodated in the inner groove 13. The inner diameter of the sealing gasket 6 is the same as the diameter of the through hole 17, and the thickness of the sealing gasket 6 is greater than the groove depth of the inner groove 13. In this embodiment, the height of the column tube 2 is no higher than 30mm, the groove depth of the inner groove 13 is less than 3mm, and the sealing gasket 6 is an O-ring sealing gasket with a thickness at least 0.2mm thicker than the groove depth of the inner groove 13. The function of the sealing gasket 6 is to ensure that the packing material is slightly higher than the column tube 2 during column loading, while preventing the slightly higher section of packing material from collapsing under pressure. During column loading, the sealing gasket 6 is compressed by the upper sieve plate 1, and the contractility of the sealing gasket 6 causes the slightly higher section of packing material to also be subjected to corresponding pressure, while preventing the packing material from collapsing, thereby keeping the packing material in the column tube 2 under high pressure. The outer edge of the column tube 2 is evenly provided with multiple threaded holes A14 for connecting with the upper sieve plate 1 and the lower sieve plate 3 along the circumferential direction.

[0035] In this embodiment, the upper screen plate 1 is disc-shaped and includes a cover plate 9, a distributor 10, a screen 11, and a sealing ring 12. The cover plate 9 has a groove along the thickness direction on the lower surface facing the column tube 2. The distributor 10 and the screen 11 are respectively housed in the groove from top to bottom. The distributor 10 is embedded in the cover plate 9, and the screen 11 is embedded in the distributor 10. The sealing ring 12 is located on the lower surface of the cover plate 9 facing the column tube 2 and is in contact with the sealing gasket 6. The liquid inlet 7 is opened in the middle of the cover plate 9, extending from the upper surface of the upper screen plate 1 to the top of the groove, and is connected to the groove.

[0036] In this embodiment, the lower sieve plate 3 is disc-shaped and includes a cover plate 9, a distributor 10, a screen 11, and a sealing ring 12. The upper surface of the cover plate 9 facing the column tube 2 has a groove along the thickness direction. The distributor 10 and the screen 11 are respectively housed in the groove from bottom to top. The distributor 10 is embedded in the cover plate 9, and the screen 11 is embedded in the distributor 10. The sealing ring 12 is located at the position where the upper surface of the cover plate 9 facing the column tube 2 contacts the column tube 2. The lower surface of the cover plate 9 has a column foot hole 15 for connecting the column foot 4. The liquid outlet 8 is opened in the middle position of the cover plate 9, extending from the lower surface of the lower sieve plate 3 to the bottom of the groove and communicating with the groove.

[0037] The distributor 10 in this embodiment is existing technology and can be a distributor produced by Changzhou Ruixi Biotechnology Co., Ltd. The function of the distributor 9 is to make the mobile phase evenly distributed on the cross-section of the pre-column bed to reduce the impact of uneven distribution of the mobile phase between the center and the edge of the pre-column.

[0038] In this embodiment, the screen 11 is a stainless steel sintered plate or other types of porous filter plate with a filtration accuracy of 0.2 to 5 micrometers. The function of the screen 11 is to block the packing material and allow the sample and mobile phase to pass through.

[0039] In this embodiment, the sealing ring 12 is an O-ring made of polytetrafluoroethylene. The function of the sealing ring 12 is to seal the connection between the column tube 2 and the upper screen plate 1 and the connection between the column tube 2 and the lower screen plate 3.

[0040] The outer edges of the upper sieve plate 1 and the lower sieve plate 3 are each uniformly provided with threaded holes B16 along the circumferential direction. The threaded holes B16 and A14 are connected by screws 5. The number of threaded holes B16 on the upper sieve plate 1 is the same as the number of threaded holes B16 on the lower sieve plate 3, and both are even numbers. The number of threaded holes A14 is equal to the sum of the number of threaded holes B16 on the upper sieve plate 1 and the number of threaded holes B16 on the lower sieve plate 3. The threaded holes A14 connected to the threaded holes B16 on the upper sieve plate 1 and the threaded holes A14 connected to the threaded holes B16 on the lower sieve plate 3 are spaced apart. In this embodiment, the outer edge of the column tube 2 has 12 threaded holes A uniformly provided along the circumferential direction, and the outer edges of the upper sieve plate 1 and the lower sieve plate 3 each have 6 screw holes B16 uniformly provided along the circumferential direction.

[0041] The present invention discloses a pre-column packing method for preparative high-performance liquid chromatography (HPLC). This method employs a wet packing process using a homogenizer, combined with negative pressure suction to remove the homogenizer solvent. This allows the chromatographic packing material to form a column bed layer by layer from the bottom. After the column bed is filled in the pre-column, the pre-column's structural design ensures high pressure. Specifically:

[0042] First, the chromatographic packing material to be packed into the column is ultrasonically homogenized with solvent. Then, the lower sieve plate 3, column tube 2, and column foot 4 in the pre-column are installed. A sealing gasket 6 is installed in the inner groove 13 at the upper end of the column tube 2. The liquid outlet 8 at the bottom of the lower sieve plate 3 is connected to one end of the pipeline and connected to the vacuum pump. The homogenized solution is gradually poured into the space of the column tube 2 containing the chromatographic packing material. The vacuum pump is turned on to create negative pressure in the pipeline connected to the liquid outlet 8. The homogenized solvent will be quickly extracted, and the packing material will accumulate at the bottom of the column tube 2 to form a column bed. The vacuum pump is then turned off. The homogenized solution is poured in again, and the vacuum pump is turned on again to remove the solvent. This process is repeated until the packing layer fills the column tube 2 and reaches the height of the sealing gasket 6. Then, the excess packing material is scraped off with a straight scraper so that the packing layer is flush with the sealing gasket 6. The upper sieve plate 1 is then placed on top and the screws 5 are gradually tightened until the upper sieve plate 1 exerts sufficient pressure on the packing material.

[0043] The amount of homogenizing solvent used should be more than three times the mass of the filler. The ultrasonic treatment time for the homogenizing solvent should be more than 5 minutes.

[0044] The upper screen plate 1 is tightened gradually with screws 5 in a diagonal manner, and the upper screen plate 1 must be kept horizontal during the tightening process, and the final tightening pressure must reach the required pressure.

[0045] Experimental Example 1

[0046] Pre-column specifications: inner diameter 50mm, column height 10mm; inner groove 13, groove depth 1mm; sealing gasket 6, thickness 2mm.

[0047] Column packing material: C18CE, particle size 5μm.

[0048] Column packing: First, weigh 18g of C18CE packing material in a beaker, pour in 60mL of ethanol, and sonicate and stir for 5 minutes on an ultrasonicator. Attach the pre-column lower sieve plate 3 to the column legs 4 and place it on a horizontal experimental table. Then, attach the column tube 2 to the lower sieve plate 3 and the sealing gasket 6 to the column tube 2. Connect the outlet 8 at the bottom of the lower sieve plate 3 to the vacuum line, and connect the other end of the line to the vacuum pump. Carefully pour the homogenized slurry into the column tube 2, being careful not to let the slurry overflow. Turn on the vacuum pump to homogenize the slurry. The agent flows out quickly from the outlet 8, and the packing material is evenly piled up at the bottom of the column tube 2. Continue to pour in the homogenizing liquid so that the column bed is formed layer by layer from the bottom of the column tube 2 until the packing layer fills the column tube 2 and reaches the height of the sealing gasket 6. Turn off the vacuum pump and scrape off the excess packing material with a straight scraper so that the packing interface is flush with the sealing gasket 6. Cover with the upper screen plate 1, install the screws 5, and tighten the screws 5 diagonally step by step. During the tightening process, the upper screen plate 1 should always be kept in a horizontal position until the screws 5 can not be tightened any further.

[0049] The results of separate pre-column tests after column loading are as follows: Figure 8 As shown.

[0050] The peak shape symmetry of the standalone pre-column test is 1.16, the column efficiency is 65000 N / m, and the test pressure is 4 MPa.

[0051] The test was conducted by connecting the pre-column to the high-performance chromatography main column, and the test results are as follows: Figure 9 As shown.

[0052] The peak shape symmetry was 1.06, the high efficiency column efficiency was 83000 N / m, and the test pressure was 16.5 MPa.

[0053] Experimental Example 2

[0054] Pre-column specifications: inner diameter 30mm, column height 10mm; inner groove 13, groove depth 0.5mm; sealing gasket 6, thickness 2mm.

[0055] Column packing material: C18HCE, particle size 5μm.

[0056] Column packing: First, weigh 5g of C18HCE packing material in a beaker, pour in 20mL of ethanol, and sonicate and stir for 8 minutes on an ultrasonicator. Attach the pre-column lower sieve plate 3 to the column legs 4 and place it on a horizontal experimental table. Then, attach the column tube 2 to the lower sieve plate 3 and the sealing gasket 6 to the column tube 2. Connect the outlet 8 at the bottom of the lower sieve plate 3 to the vacuum line, and connect the other end of the line to the vacuum pump. Carefully pour the homogenized slurry into the column tube 2, being careful not to let the slurry overflow. Turn on the vacuum pump to homogenize the slurry. The solvent flows out quickly from the outlet 8, and the packing material is evenly piled up at the bottom of the column tube 2. The homogenate is poured in continuously, so that the column bed is formed layer by layer from the bottom of the column tube 2 until the packing layer fills the column tube 2 and reaches the height of the sealing gasket 6. The vacuum pump is turned off, and the excess packing material is scraped off with a straight scraper so that the packing interface is flush with the sealing gasket 6. The upper sieve plate 1 is covered, and the screws 5 are installed. The screws 5 are tightened diagonally step by step. During the tightening process, the upper sieve plate 1 should always be kept in a horizontal position until the screws 5 can not be tightened any further.

[0057] The results of the pre-column connection to the high-performance chromatography main column after column packing are as follows: Figure 10 As shown.

[0058] After connection, the peak symmetry of the main column is 1.23, and the column efficiency is 95000 N / m.

[0059] Experiment Example 3

[0060] Pre-column specifications: inner diameter 50mm, column height 30mm; inner groove 13, groove depth 3mm; sealing gasket 6, thickness 4mm.

[0061] Column packing material: C18CE, particle size 5μm.

[0062] After the column was packed in the manner described in Experiment Example 1, its pressure resistance was tested. When the pressure reached 3500 ps i and the test lasted for 30 minutes (i.e., 24 MPa), the pre-column seal remained good and there was no leakage. After the test, the pre-column was disassembled and the column bed inside was still intact and there was no collapse.

[0063] This invention pertains to an auxiliary device for high-efficiency preparative liquid chromatography (HPLC) columns, providing pre-column protection. The pre-column protects the HPLC main column from contamination by impurities, thus extending its lifespan. The packing method of this invention is simple and practical, requiring no packing machine, allowing for repeated packing and reuse. It withstands a maximum pressure of 20 MPa and can be used in series with the main column without affecting its column efficiency, peak shape, or resolution.

Claims

1. A preparative high performance liquid chromatography pre-column, characterized by: The application relates to a chromatographic column, which comprises an upper sieve plate (1), a column tube (2), a lower sieve plate (3) and a column foot (4), wherein the column tube (2) is located between the upper sieve plate (1) and the lower sieve plate (3) and is in sealing connection with the upper sieve plate (1) and the lower sieve plate (3) respectively, a space for accommodating chromatographic filler is arranged in the column tube (2), a sealing gasket (6) is arranged between the upper sieve plate (1) and the column tube (2), the sealing gasket (6) is located above the space, the top surface of the sealing gasket (6) is higher than the upper surface of the upper sieve plate (1), a liquid inlet (7) is arranged on the upper sieve plate (1) and is in communication with the space, a liquid outlet (8) is arranged on the lower sieve plate (3) and is in communication with the space, and the lower sieve plate (3) is further connected with the detachable column foot (4).

2. The preparative high performance liquid chromatography precolumn of claim 1, wherein: A through hole (17) is arranged in the middle of the column tube (2), the through hole (17) is the space for accommodating chromatographic filler, an inner groove (13) is arranged on the upper surface of the column tube (2), and the sealing gasket (6) is accommodated in the inner groove (13); a plurality of screw holes A (14) for connecting the upper sieve plate (1) and the lower sieve plate (3) are uniformly arranged on the outer edge of the column tube (2) in the circumferential direction.

3. The preparative high performance liquid chromatography precolumn of claim 2, wherein: The inner diameter of the sealing gasket (6) is the same as the hole diameter of the through hole (17), and the thickness of the sealing gasket (6) is greater than the slotting depth of the inner groove (13).

4. The preparative high performance liquid chromatography precolumn of claim 2, wherein: The outer edges of the upper sieve plate (1) and the lower sieve plate (3) are respectively uniformly provided with screw holes B (16) in the circumferential direction, the screw holes B (16) and the screw holes A (14) are connected through screws (5), the number of the screw holes B (16) on the upper sieve plate (1) is the same as that of the screw holes B (16) on the lower sieve plate (3) and is an even number, the number of the screw holes A (14) is equal to the sum of the number of the screw holes B (16) on the upper sieve plate (1) and the number of the screw holes B (16) on the lower sieve plate (3), and the screw holes A (14) connected with the screw holes B (16) on the upper sieve plate (1) and the screw holes A (14) connected with the screw holes B (16) on the lower sieve plate (3) are arranged at intervals.

5. The preparative high performance liquid chromatography precolumn of claim 1, wherein: The upper sieve plate (1) comprises a cover plate (9), a distributor (10), a sieve net (11) and a sealing ring (12), the lower surface of the cover plate (9) towards the column tube (2) is provided with a groove in the thickness direction, the distributor (10) and the sieve net (11) are accommodated in the groove from top to bottom, the distributor (10) is installed on the cover plate (9), the sieve net (11) is installed on the distributor (10), the sealing ring (12) is located at the position where the lower surface of the cover plate (9) towards the column tube (2) contacts with the sealing gasket (6), and the liquid inlet (7) is arranged on the cover plate (9) and is in communication with the groove.

6. The preparative high performance liquid chromatography precolumn of claim 1, wherein: The lower sieve plate (3) comprises a cover plate (9), a distributor (10), a sieve (11) and a sealing ring (12), the cover plate (9) is provided with a groove in the thickness direction towards the upper surface of the column tube (2), the distributor (10) and the sieve (11) are respectively accommodated in the groove from bottom to top, the distributor (10) is installed on the cover plate (9), the sieve (11) is installed on the distributor (10), and the sealing ring (12) is located at the position where the upper surface of the cover plate (9) contacts the column tube (2); the lower surface of the cover plate (9) is provided with a column foot hole (15) for connecting the column foot (4); and the liquid outlet (8) is provided on the cover plate (9) and communicates with the groove.

7. A method of packing a preparative high performance liquid chromatography pre-column, characterized by: The homogenate wet method is used to fill the column, and the homogenate solvent is pumped out by negative pressure, so that the chromatographic packing is formed into a column bed layer by layer from the bottom, and after the column bed is filled in the pre-column, the structure design of the pre-column is matched to obtain high pressure for the column bed.

8. The method of packing the preparative high performance liquid chromatography pre-column according to claim 7, characterized by: Specifically: First, the chromatographic packing to be filled in the column is ultrasonically homogenized with a solvent, then the lower sieve plate (3), the column tube (2) and the column foot (4) in the preparative high performance liquid chromatography pre-column of any one of claims 1 to 6 are installed, a sealing gasket (6) is installed on the upper end of the column tube (2), the liquid outlet (8) on the lower sieve plate (3) is connected with a filter vacuum pump through a pipeline; the homogenized homogenate solution is gradually poured into the space containing the chromatographic packing in the column tube (2), the filter vacuum pump is started to form negative pressure in the connecting pipeline of the liquid outlet (8), the homogenate solvent is pumped out, the packing is accumulated at the bottom of the column tube (2) and forms a layer of column bed, and the filter vacuum pump is turned off; The homogenate solution is continuously poured, and the filter vacuum pump is started again to pump out the solvent; the operation is repeated until the packing layer fills the column tube (2) and the packing layer reaches the height of the sealing gasket (6); then a flat scraper is used to scrape off the excess packing, so that the packing layer is flush with the sealing gasket (6); the upper sieve plate (1) is covered, and the upper sieve plate (1) is gradually tightened by screws (5) until the packing is subjected to sufficient pressure.

9. The method of packing a preparative high performance liquid chromatography pre-column according to claim 8, wherein: The amount of the homogenate solvent is more than 3 times the mass of the packing, and the ultrasonic time of the homogenate solvent is more than 5 minutes.

10. The method of packing a preparative high performance liquid chromatography pre-column according to claim 8, wherein: The upper sieve plate (1) is gradually tightened by screws (5) in a diagonal manner, and the upper sieve plate (1) is kept horizontal during the tightening process.

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