Nano-liter-scale capillary chromatographic column filling instrument and filling method

Abstract

The invention discloses a nanoliter-scale capillary chromatographic column filling instrument and a filling method, and relates to the technical field of chromatographic column filling, the nanoliter-scale capillary chromatographic column filling instrument comprises a base, a liquid adding device body and a plugging connector, the liquid adding device body is connected to the base, a containing groove is formed in the upper end of the liquid adding device body, the containing groove is used for storing filler, and the plugging connector is used for plugging the filler. A stirring device used for stirring the filler is arranged in the containing groove, a pressurizing opening is formed in the side wall of the containing groove, and the pressurizing opening is connected with a liquid chromatograph so as to inject liquid into the containing groove; one end of the plugging joint is in threaded connection with the interior of the accommodating groove so as to seal an opening in the upper end of the accommodating groove, a mounting hole is formed in the plugging joint, and a capillary chromatographic column is arranged in the mounting hole in a penetrating manner. According to the invention, the filling efficiency of the capillary chromatographic column is obviously improved.

Description

Technical Field

[0001] The invention relates to the technical field of chromatographic column filling, and in particular to a nanoliter capillary chromatographic column filling instrument and a filling method. Background Art

[0002] In the research field of proteins, peptides, small molecule compounds, etc. based on nanoliter liquid chromatography-mass spectrometry tandem technology, nano-level reverse chromatographic separation is one of the most commonly used and most critical technologies, which directly depends on the performance of capillary chromatographic columns. Compared with conventional chromatographic columns, not only are the inner and outer diameters of the capillary chromatographic column at the micron level, which are significantly smaller than the inner / outer diameters of conventional chromatographic columns, but the back pressure of the chromatographic column is also higher, and the application pressure is usually above 300 bar, which is significantly higher than the operating pressure environment of conventional chromatographic columns. Therefore, the filling technology of capillary chromatographic columns is more sophisticated and more difficult. The existing conventional method of filling capillary chromatographic columns is based on the suspension filling method, that is, the reverse chromatographic filler is suspended in a solvent medium, an empty capillary is inserted below the liquid surface of the filler suspension, and the filler suspension is pressed into the capillary with pressure provided by a high-pressure steel cylinder to make a capillary column. This method is simple and crude to operate and does not require complex equipment. However, the maximum filling pressure of a capillary column based on the cylinder pressure supply can only be ~100 bar, and the column filling speed is slow. It usually takes at least 30 minutes to fill a 20 cm capillary column with a particle size of 3 μm. For submicron diameter (particle size less than 2 μm) fillers, the filling speed is even slower. It takes 2 hours or even longer to fill a 20 cm capillary column with submicron diameter fillers, and the filling may even fail. In addition, because the gas is compressed in the filler column bed of the chromatographic column, when the chromatographic column is filled and the pressure is released, the chromatographic filler will loosen rapidly, the filler column bed will break, and there may even be a situation where the filler recoils out of the capillary, which directly affects the column efficiency of the chromatographic column. Summary of the invention

[0003] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a nanoliter capillary chromatographic column filling instrument and filling method, which can significantly improve the filling efficiency of the capillary chromatographic column.

[0004] According to the first aspect of the present invention, a nanoliter capillary chromatography column filling instrument and filling method include: a base, a liquid adding body, and a plugging joint, wherein the liquid adding body is connected to the base, a receiving groove is provided at the upper end of the liquid adding body, the receiving groove is used to store filler, a stirring device for stirring the filler is arranged in the receiving groove, a pressurizing port is provided on the side wall of the receiving groove, and the pressurizing port is connected to a liquid chromatograph to inject liquid into the receiving groove; one end of the plugging joint is threadedly connected to the receiving groove to seal the upper end opening of the receiving groove, and a mounting hole is provided on the plugging joint, and a capillary chromatography column is passed through the mounting hole.

[0005] A nanoliter capillary chromatographic column filling instrument and filling method according to an embodiment of the present invention have at least the following beneficial effects: the filling instrument uses a stirring device to stir the filler steadily and evenly, combined with the stable supply pressure of the liquid chromatograph, to significantly improve the filling efficiency of the capillary chromatographic column, especially the filling efficiency and success rate of the submicron diameter capillary chromatographic column. The filling instrument is used in conjunction with the liquid chromatograph (500), and can directly judge the performance parameters such as back pressure and flow rate of the capillary chromatographic column in the actual liquid chromatography-mass spectrometry tandem system application according to the supply pressure, flow rate and other values ​​of the liquid chromatograph, without the need for subsequent testing.

[0006] According to some embodiments of the present invention, the stirring device includes a stirrer and a magnetic driving device, the stirrer is placed in the receiving tank, the magnetic driving device is connected to the lower end of the base, and the magnetic driving device can drive the stirrer to rotate in the receiving tank through a changing magnetic field.

[0007] According to some embodiments of the present invention, a sealing ring is sleeved on the sealing joint, and the sealing ring is used to seal the gap between the sealing joint and the liquid charger body.

[0008] According to some embodiments of the present invention, a threaded hole is formed at an upper end of the plugging joint, and the capillary chromatographic column is threadedly connected in the threaded hole.

[0009] According to some embodiments of the present invention, the pressurizing port is detachably connected to a connector, and the base is provided with a connector temporary storage slot for storing the connector.

[0010] According to some embodiments of the present invention, the outer peripheral wall of one end of the sealing joint away from the liquid charger body is a regular hexagon.

[0011] The present invention also provides a nanoliter capillary chromatographic column filling method, which utilizes the above-mentioned nanoliter capillary chromatographic column filling instrument, and the steps include: S1: heating the capillary until the surface layer of the polyimide is burnt, wiping the capillary with cotton paper dipped in methanol to remove the burnt material, and placing the capillary with the polyimide coating removed into a laser needle drawing instrument to draw the capillary chromatographic column;

[0012] S2: connecting the liquid charger body to the base, connecting the liquid chromatograph pipeline to the pressurizing port, adding filler to the containing tank and starting the stirring device to stir the filler;

[0013] S3: connecting the capillary chromatographic column to the plugging joint, and then threading the plugging joint into the containing groove;

[0014] S4: Start the liquid chromatograph to inject liquid into the containing tank, and observe whether the filler fills the capillary chromatographic column.

[0015] According to some embodiments of the present invention, the liquid chromatograph in S4 is flushed with an organic solvent and then vented before startup.

[0016] According to some embodiments of the present invention, the concentration of the solid matter in the filler in S2 is 100 mg / mL.

[0017] According to some embodiments of the present invention, the pressure at which the liquid chromatograph injects liquid into the containing tank in S4 is 30 MPa.

[0018] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0020] Figure 1 A schematic diagram of an installation structure of an embodiment of the present invention;

[0021] Figure 2 A schematic diagram of a plugging joint according to an embodiment of the present invention;

[0022] Figure 3 The figure is a schematic diagram of a liquid charger body according to an embodiment of the present invention.

[0023] Figure Number:

[0024] Base 100, joint temporary storage slot 110;

[0025] Liquid charger body 200;

[0026] Accommodating groove 300, pressurizing port 310, joint 311;

[0027] A stirring device 400, a stirring rod 410, and a magnetic driving device 420;

[0028] Liquid chromatograph 500;

[0029] Sealing joint 600, mounting hole 610, sealing ring 620, threaded hole 630;

[0030] Capillary Column 700. DETAILED DESCRIPTION

[0031] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0032] In the description of the present invention, it should be understood that descriptions involving orientation, such as orientation or positional relationship indicated as up, down, etc., are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present invention.

[0033] In the description of the present invention, "a plurality" means more than two. If there is a description of "first" or "second", it is only used for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0034] In the description of the present invention, unless otherwise clearly defined, terms such as setting, installing, connecting, etc. should be understood in a broad sense, and technicians in the relevant technical field can reasonably determine the specific meanings of the above terms in the present invention based on the specific content of the technical solution.

[0035] Reference Figures 1 to 3As shown, a nanoliter capillary chromatography column filling instrument and filling method according to an embodiment of the present invention include: a liquid adding body 200 is connected to a base 100, a positioning groove is provided on the base 100 for the lower end of the liquid adding body 200 to be embedded, and a receiving groove 300 is provided at the upper end of the liquid adding body 200. The cross section of the receiving groove 300 is circular so as to facilitate the processing of threads on the side wall of the receiving groove 300. The volume of the receiving groove 300 does not exceed 1mL, so that the filler can be quickly supplied with liquid to fill the entire filling pool, thereby increasing the filling speed. The receiving groove 300 is used to store the filler, and the filler is added to the receiving groove from the opening at the upper end of the receiving groove 300. The filler is composed of solid reverse chromatographic filler particles suspended in a solvent medium. A stirring device 400 for stirring the filler is provided in the receiving tank 300, and a pressurizing port 310 is provided on the side wall of the receiving tank 300. The pressurizing port 310 is connected to a liquid chromatograph 500 to inject high-pressure liquid into the receiving tank 300, thereby increasing the pressure in the receiving tank 300; the specific structure of the liquid chromatograph 500 is prior art and will not be described in detail. One end of the plugging joint 600 is threadedly connected in the receiving tank 300 to seal the upper end opening of the receiving tank 300, and a mounting hole 610 is provided on the plugging joint 600, and a capillary chromatographic column 700 is passed through the mounting hole 610. The specific structure of the capillary chromatographic column 700 is prior art and will not be described in detail. The lower end of the capillary chromatographic column 700 extends into the receiving tank 300 and is immersed in the filler, and the filler enters the capillary chromatographic column 700 under the pressure difference between the receiving tank 300 and the outside. The filling instrument uses the stirring device 400 to stir the filler steadily and evenly, and combines with the stable pressure supply of the liquid chromatograph 500 to significantly improve the filling efficiency of the capillary, especially the filling efficiency and success rate of the capillary chromatographic column filled with submicron diameter filler. The filling instrument is used in conjunction with the liquid chromatograph 500, and can directly judge the back pressure, flow rate and other performance parameters of the capillary chromatographic column 700 in the actual liquid chromatography-mass spectrometry tandem system application according to the pressure supply, flow rate and other values ​​of the liquid chromatograph 500, without the need for subsequent testing.

[0036] Reference Figures 1 to 3As shown, it is understandable that the stirring device 400 comprises a stirrer 410 and a magnetic drive device 420, and the stirrer 410 is placed in the receiving tank 300, and the material of the stirrer 410 is a material with magnetism. The specific structure of the magnetic drive device 420 is prior art, so it is not described in detail. The magnetic drive device 420 is connected to the lower end of the base 100, and the magnetic drive device 420 produces a variable magnetic field to drive the stirrer 410 to move and stir the filler, prevent the solid sedimentation in the filler, and make the filler entering the capillary chromatographic column 700 uniform. The magnetic drive device 420 can drive the stirrer 410 to rotate in the receiving tank 300 by the variable magnetic field. The relative magnetic permeability of the liquid adder body 200 material is less than 1 so that the magnetic field generated by the magnetic drive device 420 can pass through the liquid adder body 200. The receiving tank 300 cross section is circular, so that the stirrer 410 can stably and evenly stir the filler suspension.

[0037] Reference Figures 1 to 3 As shown, it can be understood that a sealing ring 620 is sleeved on the plugging joint 600, and the sealing ring 620 is used to seal the gap between the plugging joint 600 and the liquid charger body 200. It can be foreseen that a boss is connected to the upper end of the plugging joint 600, and the lower end of the boss abuts against the upper end of the liquid charger body 200. The lower end of the boss is provided with an annular groove for the sealing ring 620 to be embedded. The sealing ring 620 seals the gap between the lower end of the boss and the upper end of the liquid charger body 200 to prevent leakage of the packing.

[0038] Reference Figures 1 to 3 As shown, it can be understood that a threaded hole 630 is provided at the upper end of the plugging joint 600, and the capillary chromatographic column 700 is threadedly connected in the threaded hole 630. The capillary chromatographic column 700 is threadedly connected to the plugging joint 600, which has the advantage of being easy to install and not easy to leak.

[0039] Reference Figures 1 to 3 As shown, it can be understood that the pressurizing port 310 is threadedly connected with a connector 311, and the base 100 is provided with a connector temporary storage slot 110, and the shape and size of the connector temporary storage slot 110 match the connector 311 for the connector 311 to be inserted. The connector 311 is placed in the connector temporary storage slot 110 when not in use.

[0040] Reference Figures 1 to 3 As shown, it can be understood that the outer peripheral wall of the end of the plugging joint 600 away from the liquid charger body 200 is a regular hexagon. The plugging joint 600 can be conveniently tightened or loosened using a tool such as an adjustable wrench. It can be foreseen that the upper end of the plugging joint 600 can also be opened to rotate the plugging joint 600 using a tool such as an F-shaped wrench.

[0041] The present invention also provides a nanoliter capillary chromatographic column filling method, which uses the above-mentioned nanoliter capillary chromatographic column filling instrument and comprises the following steps:

[0042] S1: Heat the capillary with an alcohol lamp until the polyimide surface is burnt. Since the capillary surface is coated with polyimide for protection, the polyimide needs to be removed first. Wipe the capillary with cotton paper dipped in methanol to remove the burnt material. Put the capillary with the polyimide coating removed into a laser needle drawing instrument. The laser needle drawing instrument is a prior art and will not be described in detail. The laser needle drawing instrument draws the capillary into a capillary chromatographic column 700;

[0043] S2: insert the bottom end of the liquid charger body 200 into the positioning groove on the base 100, thread the pipeline of the liquid chromatograph 500 to the pressure port 310 through the joint 311, add filler into the containing groove 300 and start the stirring device 400 to stir the filler;

[0044] S3: Thread the capillary chromatographic column 700 onto the plugging connector 600, then thread the plugging connector 600 into the receiving groove 300, and tighten the plugging connector 600 using a tool such as an F-shaped wrench;

[0045] S4: Start the liquid chromatograph 500 to inject liquid into the containing tank 300, and observe whether the filler fills the capillary chromatographic column 700. If the filler is a semi-transparent color that is difficult to observe, a flashlight or other light source may be used to assist observation.

[0046] It is understandable that, in S4, the liquid chromatograph 500 is flushed and vented with an organic solvent such as methanol or acetonitrile before startup to remove impurities in the liquid chromatograph 500 and improve the filling quality.

[0047] It can be understood that the concentration of the solid matter in the filler in S2 is 100 mg / mL.

[0048] It can be understood that the pressure at which the liquid is injected into the containing tank 300 by the liquid chromatograph 500 in S4 is 30 MPa.

[0049] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A nanoliter capillary chromatographic column filling instrument, characterized in that: include: Base (100); A liquid adding device body (200) is connected to the base (100); a receiving groove (300) is provided at the upper end of the liquid adding device body (200); the receiving groove (300) is used to store filler; a stirring device (400) is provided in the receiving groove (300) for stirring the filler; a pressurizing port (310) is provided on the side wall of the receiving groove (300); the pressurizing port (310) is connected to a liquid chromatograph (500) for injecting liquid into the receiving groove (300); A plugging joint (600) has one end threadedly connected in the accommodating groove (300) to seal the upper opening of the accommodating groove (300), and a mounting hole (610) is provided on the plugging joint 600, and a capillary chromatographic column (700) is passed through the mounting hole (610).

2. A nanoliter capillary chromatographic column filling instrument according to claim 1, characterized in that: The stirring device (400) comprises a stirring bar (410) and a magnetic driving device (420), wherein the stirring bar (410) is placed in the containing tank (300), and the magnetic driving device (420) is connected to the lower end of the base (100), and the magnetic driving device (420) can drive the stirring bar (410) to rotate in the containing tank (300) through a changing magnetic field.

3. A nanoliter capillary chromatographic column filling instrument according to claim 2, characterized in that: The plugging joint (600) is sleeved with a sealing ring (620), and the sealing ring (620) is used to seal the gap between the plugging joint (600) and the liquid charger body (200).

4. A nanoliter capillary chromatographic column filling instrument according to claim 3, characterized in that: A threaded hole (630) is formed at the upper end of the plugging joint (600), and the capillary chromatographic column (700) is threadedly connected in the threaded hole (630).

5. A nanoliter capillary chromatographic column filling instrument according to claim 4, characterized in that: The pressurizing port (310) is detachably connected to a joint (311), and the base (100) is provided with a joint temporary storage groove (110) for storing the joint (311).

6. A nanoliter capillary chromatographic column filling instrument according to claim 5, characterized in that: The outer peripheral wall of one end of the sealing joint (600) away from the liquid charger body (200) is in the shape of a regular hexagon.

7. A method for filling a nanoliter capillary chromatographic column, characterized in that: The nanoliter capillary chromatographic column filling instrument according to any one of claims 1 to 6 is used, and the specific steps are as follows: S1: heating the capillary until the surface layer of the polyimide is burnt, wiping the capillary with tissue paper dipped in methanol to remove the burnt material, placing the capillary with the polyimide coating removed into a laser needle drawing instrument, and drawing the capillary chromatographic column (700); S2: connecting the liquid charger body (200) to the base (100), connecting the pipeline of the liquid chromatograph (500) to the pressurizing port (310), adding filler to the containing tank (300) and starting the stirring device (400) to stir the filler; S3: Connecting the capillary chromatographic column (700) to the plugging joint (600), and then threading the plugging joint (600) into the receiving groove (300); S4: Start the liquid chromatograph (500) to inject liquid into the containing tank (300), and observe whether the filler fills the capillary chromatographic column (700).

8. A nanoliter capillary chromatographic column filling method according to claim 7, characterized in that: In S4, the liquid chromatograph (500) is flushed with an organic solvent and then vented before starting.

9. The method for filling a nanoliter capillary chromatographic column according to claim 7, characterized in that: The concentration of solid matter in the filler in S2 is 100 mg / mL.

10. The method for filling a nanoliter capillary chromatographic column according to claim 7, characterized in that: In S4, the pressure at which the liquid is injected into the containing tank (300) by the liquid chromatograph (500) is 30 MPa.

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