An ultra-high performance liquid preparative chromatography column

Abstract

The application discloses an ultra-high performance liquid preparation chromatographic column and relates to the field of liquid chromatography, which comprises a column tube, a liquid inlet and a liquid outlet are arranged on the column tube, a pushing assembly, a piston and a threaded tube above the piston, an upper pressing push rod is arranged above the threaded tube, a disc spring assembly is arranged on the threaded tube, a bottom flange assembly is arranged at the bottom of the column tube, the bottom flange assembly, the column tube and the piston form a cavity, the cavity is filled with packing to form a column bed, a piston distributor is arranged below the liquid outlet, and a piston sieve plate is arranged below the piston distributor. The ultra-high performance liquid preparation chromatographic column can guarantee the realization of 200,000 theoretical plates per meter of the ultra-high performance preparation column. The column is filled with 1-5 micrometers of silica gel packing, an external ultra-high pressure disc spring group and 60 MPa pressure-resistant column tube, and thus 200,000 theoretical plates per meter of the ultra-high performance preparation column are realized.

Description

Technical Field

[0001] This invention relates to the field of liquid chromatography, specifically to an ultra-high performance liquid chromatography preparative column. Background Technology

[0002] Since Svictor first disclosed adsorption chromatography columns in 1903, liquid chromatography column technology has become one of the indispensable and efficient separation techniques in modern analytical chemistry through continuous development. It is widely used in the separation and analysis of active ingredients in natural products, pharmaceutical intermediates, biological products, and fine chemical excipients.

[0003] Conventional preparative chromatographic columns are packed with 10-micron particle size packing material to achieve 40,000 theoretical plates per meter. The column tube is pressure resistant to 10 MPa. As described in Liu Renmin's "Research Progress in Preparative Chromatography Technology and Equipment", high-performance liquid chromatography has the characteristics of high column efficiency, high flow rate and short separation time. Its column efficiency can reach 40,000 to 60,000 per meter of column bed.

[0004] Waters achieved a column packing density of 80,000 theoretical plates per meter on a 30 mm diameter preparative column using 5-micron packing material. However, due to the lag in hardware development, such as the pressure resistance of the column tube and the uniformity of sieve plate distribution, it is far from reaching the level of 200,000 theoretical plates per meter for ultra-high efficiency analytical columns. This cannot meet the growing purification needs of complex samples such as traditional Chinese medicine, especially the application requirements of ultra-high efficiency analytical columns with small particle size packing material below 5 microns being scaled up to preparative columns. Summary of the Invention

[0005] The purpose of this invention is to at least solve one of the technical problems existing in the background art, and to provide an ultra-high performance liquid chromatography (UHPLC) preparative chromatography column. Specifically, addressing the problems in the background art, this invention designs a 316L stainless steel column tube with an overall sealed pressure resistance of 60 MPa and an inner wall roughness of 0.2 micrometers; the multi-partitioned oblique H-tree type distributor with 138,500 distribution points per square meter provides better distribution uniformity and effect, and the external disc spring assembly continuously pushes to maintain the compactness of the packed column bed, avoiding column efficiency decline and separation loss caused by collapse, maintaining long-term separation capability and service life. The theoretical plate count per meter can reach 200,000, meeting the equivalent scale-up preparation needs of complex samples such as traditional Chinese medicine from ultra-high performance analytical columns to preparative columns.

[0006] The technical solution of the present invention is as follows:

[0007] An ultra-high performance liquid chromatography column for preparative chromatography includes a column tube with an inlet and an outlet.

[0008] A pressing assembly includes a piston and a threaded tube located above the piston, with an upper pressing rod disposed above the threaded tube;

[0009] The disc spring assembly is nested on the upper push rod and is fixed by bolts to the upper flange, middle flange and column tube upper flange;

[0010] The bottom flange assembly, located at the bottom of the column tube, together with the column tube and piston, forms a cavity wall filled with filler to form a column bed;

[0011] Piston distributor, which is embedded in the bottom of the piston;

[0012] A piston screen plate is embedded in the bottom of the piston and located below the piston distributor; the bottom flange assembly contains, from top to bottom, a lower screen plate and a lower distributor.

[0013] As a preferred embodiment of the present invention, the piston distributor and the lower distributor have at least 10,000 distribution points per square meter.

[0014] As a preferred embodiment of the present invention, one surface of the piston distributor and the lower distributor is a multi-equally divided oblique H-tree shape, and the other surface is a uniformly distributed flower-shaped distribution hole.

[0015] As a preferred embodiment of the present invention, the diameter of the column tube is 20-100mm.

[0016] As a preferred embodiment of the present invention, the height of the column bed is 100-400mm.

[0017] As a preferred embodiment of the present invention, the disc spring is one or more of the following: stainless steel disc spring, carbon steel disc spring, chromium-manganese alloy disc spring, chromium-vanadium alloy disc spring, silicon-manganese alloy disc spring, spring steel disc spring, or a variety of alloy disc springs.

[0018] As a preferred embodiment of the present invention, the filler is one or more of silica gel filler, polymer filler, and agarose filler.

[0019] As a preferred embodiment of the present invention, the particle size of the filler is 1-5 μm.

[0020] As a preferred embodiment of the present invention, the aperture of the piston sieve plate and the lower sieve plate is 0.5-2μm.

[0021] As a preferred embodiment of the present invention, the roughness of the inner wall of the column tube, the piston sieve plate, the lower sieve plate, the piston distributor, and the lower distributor is 0.1-0.5μm.

[0022] The beneficial effects of this invention are:

[0023] (1) The present invention provides an ultra-high efficiency liquid chromatography column that ensures the realization of an ultra-high efficiency theoretical plate number of 200,000 per meter: The present invention adopts hardware innovations such as 1-5 micron silica gel packing, external ultra-high pressure disc spring assembly and 60MPa pressure-resistant column tube to achieve a column packing of 200,000 ultra-high efficiency theoretical plate number of 200,000 per meter.

[0024] (2) Effective solution to the problem of equivalent scale-up preparation of ultra-high efficiency chromatographic columns in this invention: This study achieves a breakthrough in the technology of ultra-high efficiency preparative chromatographic columns with diameters of 30 mm, 50 mm and 100 mm, providing a solution for the realization of equivalent scale-up of small particle size packings from analytical chromatography to preparative chromatography, which is conducive to promoting the development of industrial preparative chromatography. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the overall structure of the ultra-high performance liquid chromatography column of the present invention;

[0026] Figure 2 This is a diagram showing the connection structure of the ultra-high efficiency liquid phase preparation column of the present invention, including the upper pressure push rod, upper flange, disc spring assembly, disc spring flange, threaded pipe, and piston.

[0027] Figure 3 The diagram shows a column assembly consisting of a middle flange, fixing bolts, column tube flange, column tube, lower flange, column bottom outlet, and flange distributor, which is the ultra-high efficiency liquid chromatography preparation column of the present invention.

[0028] Figure 4 The diagram shows the structure of the piston in the ultra-high efficiency liquid chromatography column of this invention, including the piston outlet, fixing bolts, piston flange, piston distributor, and piston sieve plate.

[0029] Figure 5 The layout of the distribution points of the piston distributor and flange distributor for the 50mm diameter ultra-high efficiency liquid chromatography column of the present invention;

[0030] Figure 6 This is a front view of the piston distributor for the 50mm diameter ultra-high efficiency liquid chromatography column of the present invention.

[0031] Figure 7 This is a reverse structural diagram of the piston distributor for the 50mm diameter ultra-high efficiency liquid chromatography column of the present invention.

[0032] Figure 8 To illustrate the column efficiency evaluation chromatogram of a 250mm high chromatographic column filled with 5-micron C18HD silica gel packing using the 50mm diameter column of this invention, Example 1 is provided.

[0033] Figure 9 Example 2 shows a chromatogram evaluating the column efficiency of a 250mm high chromatographic column filled with 5-micron C18HCE silica gel packing material, prepared using the 50mm diameter column of this invention.

[0034] Figure 10 Example 3 shows a chromatogram evaluating the column efficiency of a 250mm high chromatographic column filled with 5-micron C18HD silica gel packing, prepared using the 30mm diameter column of this invention.

[0035] Figure 11 Example 4: chromatogram of column efficiency evaluation for a 100mm high chromatographic column packed with 3.5-micron C18HCE silica gel filler, prepared using the 30mm diameter column of this invention.

[0036] 1-Column head inlet pipe, 201-Upper flange, 202-Middle flange, 203-Column tube upper flange, 204-Column tube lower flange, 205-Lower flange, 206-Disc spring flange, 207-Piston flange, 3-Disc spring assembly, 4-Threaded pipe, 5-Piston, 6-Column tube, 7-Column bed, 8-Upper pressure push rod, 901-First fixing bolt, 902-Fixing screw, 903-Second fixing bolt, 904-Third fixing bolt, 11-Column bottom outlet, 12-Piston distributor, 1201-Distribution point, 13-Piston sieve plate, 1301-Slanted H-tree shape, 1302-Flower-shaped distribution hole structure, 14-Piston outlet. Detailed Implementation

[0037] This section will describe in detail specific embodiments of the present invention. Preferred embodiments of the present invention are shown in the accompanying drawings. The purpose of the drawings is to supplement the textual description with graphics, so that people can intuitively and vividly understand each technical feature and overall technical solution of the present invention, but they should not be construed as limiting the scope of protection of the present invention.

[0038] In the description of this invention, it should be understood that the orientation descriptions, such as up, down, front, back, left, right, etc., are based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting this invention.

[0039] In the description of this invention, "several" means one or more, "more than" means two or more, "greater than," "less than," and "exceeding" are understood to exclude the stated number, while "above," "below," and "within" are understood to include the stated number. The use of "first" and "second" in the description is merely for distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly indicating the number of indicated technical features, or implicitly indicating the order of the indicated technical features.

[0040] In the description of this invention, unless otherwise explicitly defined, terms such as "setting," "installing," and "connecting" should be interpreted broadly, and those skilled in the art can reasonably determine the specific meaning of the above terms in this invention in conjunction with the specific content of the technical solution.

[0041] Reference Figures 1 to 7 Preferred embodiments of the present invention:

[0042] An ultra-high performance liquid chromatography column includes a column tube 6, which is provided with an inlet and a bottom outlet 11.

[0043] The push assembly includes a piston 5 and a threaded tube 4 located above the piston 5, with an upper push rod 8 disposed above the threaded tube 4; more specifically, a column head liquid inlet pipe 1 is connected to the liquid inlet, and a piston outlet 14 is disposed on the piston, which is connected to the column head liquid inlet pipe 1 through the threaded tube 4.

[0044] The disc spring assembly 3 is nested on the upper push rod 8 and is fixed by bolts to the upper flange 201, the middle flange 202 and the upper flange 203 of the column tube. Specifically, the flanges are fixed by the first fixing bolt 901 and the fixing screw 902 respectively.

[0045] The bottom flange assembly is located at the bottom of the column tube 6. It, the column tube 6, and the piston 5 form a cavity, and the cavity is filled with filler to form a column bed 7.

[0046] Piston distributor 12, which is embedded in the bottom of piston 5;

[0047] Piston screen plate 13 is embedded in the bottom of piston 5 and located below piston distributor 12;

[0048] The bottom flange assembly contains, from top to bottom, a lower sieve plate and a lower distributor;

[0049] More specifically, the bottom of the column tube 6 is connected and fixed to the lower screen plate and the lower distributor through the lower flange 204, the lower flange 205 and the piston flange 207 of the column tube, respectively, and the flanges are fixed together by the first fixing bolt 903 and the second fixing bolt 904.

[0050] As a preferred embodiment of the present invention, it may also have the following additional technical features:

[0051] The piston distributor 12 and the lower distributor have at least 10,000 distribution points per square meter.

[0052] The piston distributor 12 and the lower distributor have a multi-equal-divided oblique H-tree structure on one surface and a uniformly distributed flower-shaped distribution hole structure on the other surface, with 10,000-15,000 distribution points per square meter.

[0053] The diameter of the column tube 6 is 20-100mm.

[0054] The height of the column bed 7 is 100-400mm.

[0055] The disc spring assembly is composed of one or more of the following: stainless steel disc spring, carbon steel disc spring, chromium-manganese alloy disc spring, chromium-vanadium alloy disc spring, silicon-manganese alloy disc spring, spring steel disc spring, or a variety of alloy disc springs.

[0056] The filler is one or more of silica gel filler, polymer filler, and agarose filler.

[0057] The particle size of the filler is 1-4 μm.

[0058] The aperture of the piston sieve plate 13 and the lower sieve plate is 0.5 to 2 μm;

[0059] The roughness of the inner wall of the column tube 6, the piston sieve plate 13, the lower sieve plate, the lower distributor, and the piston distributor 12 is 0.1-0.5 μm.

[0060] The column structure of this embodiment can continuously and stably maintain the packed column bed in a compact state, avoiding the decrease in resolution caused by the collapse of the packed column bed, and maintaining the long-term separation capability and service life of the chromatographic column;

[0061] The following describes specific embodiments and appendices. Figure 8-11 To further illustrate the present invention, it should be understood that the specific embodiments are for illustrative purposes only and are not intended to limit the invention.

[0062] Example 1

[0063] First, immerse the piston and lower flange sieve plate in an ethanol solution and sonicate for 5 minutes. Fix the upper flange to the column tube with bolts, with the column tube head facing upwards and fitted into the column loading machine's fixing groove. Fix the lower flange to the plug plate with bolts, and simultaneously seal the lower outlet with a plug. Connect the piston to the inlet pipe and fix it to the threaded pipe. Assemble the disc spring flange, disc spring, upper pressure push rod, and upper flange sequentially on the threaded pipe. Calculate the required amount of packing material based on the height of all column beds to be filled and the packing material's bulk density. For example, 320g of 5-micron C18HD silica gel packing material is used to prepare 640mL of isopropanol:ethanol homogenate with a volume ratio of 1:1. Add the packing material, stir, and sonicate for 10 minutes. Pour the spring column into the column, quickly place the piston and threaded pipe assembly into the column opening, press down the piston with a hydraulic cylinder at a pressure of 20MPa, tighten the screws, maintain the pressure for 30 minutes, tighten the screws again, raise the hydraulic cylinder, and remove the spring column. The column loading is complete.

[0064] Using 5-micron C18HD silica gel as the packing medium, and packing the column with a 50 mm diameter tube according to this invention, the column efficiency was evaluated using uracil and naphthalene. The results are as follows: Figure 8 As shown, the packed column has a column efficiency of 100,000 per meter, good symmetry, and its stability was verified by destructive testing using a large gradient methanol-water mobile phase.

[0065] Example 2

[0066] The difference from Example 1 is that 5-micron C18HCE silica gel packing material was used as the packing medium, and the column efficiency was evaluated using uracil and naphthalene. The results are as follows: Figure 9As shown, the packed column has a column efficiency of 100,000 per meter, good symmetry, and its stability was verified by destructive testing using a large gradient methanol-water mobile phase.

[0067] Example 3

[0068] The difference from Example 1 is that 5-micron silica gel was used as the packing medium, and a 30*250mm column was packed. The column efficiency was evaluated using uracil and naphthalene, and the results are as follows: Figure 10 As shown, the packed column has a column efficiency of 100,000 per meter, good symmetry, and its stability was verified by destructive testing using a large gradient methanol-water mobile phase.

[0069] Example 4

[0070] The difference from Example 1 is that 3.5-micron silica gel was used as the packing medium, and a 30*100mm column was packed. The column efficiency was evaluated using uracil and naphthalene, and the results are as follows. Figure 11 As shown, the packed column has a column efficiency of 200,000 per meter, good symmetry, and its stability was verified by destructive testing using a large gradient of methanol-water mobile phase.

[0071] In summary, by using 316L stainless steel columns with an overall pressure resistance of 60 MPa and an inner wall roughness of 0.2 micrometers, and a multi-equal-divided oblique H-tree type distributor with 13,852 distribution points per unit area, the preparation column packing can achieve a theoretical plate count of 200,000 per meter.

[0072] Experiments have demonstrated that the ultra-high performance liquid chromatography (UHPLC) preparative column of this invention exhibits high column efficiency, excellent resolution, superior stability, and long service life. It meets the application requirements for complex samples such as traditional Chinese medicine, which require equivalent scaling-up from UHPLC analytical columns to preparative columns, and will significantly promote the development of the preparative chromatography field.

[0073] Without causing conflict, those skilled in the art can freely combine and use the above-mentioned additional technical features.

[0074] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various other corresponding changes and modifications based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of the present invention.

Claims

1. A preparative ultra-high performance liquid chromatography column, characterized in that, The assembly includes a column tube (6) with an inlet and an outlet; a push assembly including a piston (5) and a threaded tube (4) above the piston (5), with an upper push rod (8) above the threaded tube (4); a disc spring assembly (3) nested on the upper push rod (8) and fixed by bolts to an upper flange (201), a middle flange (202) and a column tube upper flange (203); a bottom flange assembly located at the bottom of the column tube (6), which, together with the column tube (6) and the piston (5), forms a cavity, which is filled with packing to form a column bed (7); a piston distributor (12) embedded in the bottom of the piston (5); a piston sieve plate (13) embedded in the bottom of the piston (5) and located below the piston distributor (12); and the bottom flange assembly has a lower sieve plate and a lower distributor embedded inside from top to bottom. The filler is one or more of silica gel filler, polymer filler, and agarose filler; The particle size of the filler is 1–5 μm; The piston distributor (12) and the lower distributor have at least 10,000 distribution points per square meter; one surface of the piston distributor (12) and the lower distributor is a multi-equal-divided oblique H-tree structure, and the other surface is a uniformly distributed flower-shaped distribution hole structure; the pore diameter of the piston sieve plate (13) and the lower sieve plate is 0.5–2 μm. The roughness of the inner wall of the column tube (6), the piston sieve plate (13), the lower sieve plate, the piston distributor (12), and the lower distributor is 0.1~0.5 μm; The column tube (6) is a 60MPa pressure-resistant column tube; The disc spring assembly is composed of one or more of the following: stainless steel disc spring, carbon steel disc spring, chromium-manganese alloy disc spring, chromium-vanadium alloy disc spring, silicon-manganese alloy disc spring, spring steel disc spring, or a variety of alloy disc springs.

2. The ultra-high performance liquid chromatography column according to claim 1, characterized in that, The diameter of the column tube (6) is 30–200 mm.

3. The ultra-high performance liquid chromatography column according to claim 1, characterized in that, The height of the column bed (7) is 100–400 mm.

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