Microcolumns containing packing material

Abstract

To provide a new microcolumn that contains a packing material and can be connected to a capillary tube.SOLUTION: A connection structure provided in one aspect includes a connector or a microcolumn and a plurality (for example, two) of capillary tubes. A connection structure in one embodiment is configured such that the microcolumn (or connector) and the capillary tube are in fluid communication, and a connection structure in one embodiment is configured to be liquid-tight (to prevent liquid from flowing out from the inside of a structure to the outside).SELECTED DRAWING: None

Description

[Technical Field]

[0001] This disclosure relates to microcolumns that can be used in capillary electrophoresis and high-performance liquid chromatography (HPLC). [Background technology]

[0002] In analytical techniques using capillary tubes, such as capillary electrophoresis and HPLC, various types of capillary tubes are used, including functional capillary tubes such as chromatography columns, and capillary tubes for piping that connect to valves and samplers to form a series of fluid-communicating channels. For example, these capillary tubes are connected by attaching a sleeve member to the end of an unconnected capillary tube to expand its outer diameter, inserting the capillary tube and sleeve member together into a ferrule member (often integrated with a set screw), and pushing them into a union. This fixes one capillary tube to the union, and the other capillary tube is similarly fixed to the same union, thus maintaining liquid-tightness in the piping. In this way, multiple members are used to connect capillary tubes, and the connection part often occupies a large volume (Patent Document 1, etc.). Furthermore, when multiple members are attached to the connection part, a certain length is required for the capillary tube to accommodate these members, and functional capillary tubes such as very short chromatography columns may be difficult to connect in the first place. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] International Publication No. 2009 / 088663 [Overview of the project] [Means for solving the problem]

[0004] As a result of diligent research, the inventors have developed a microcolumn that can be connected to existing capillary tubes used in capillary electrophoresis and liquid chromatography-mass spectrometry (LC-MS). In one aspect, the microcolumn of this disclosure is a connector for capillary tubes. In another aspect, the microcolumn of this disclosure is a small, functional component with functions such as separation. This disclosure provides a microcolumn and a method for producing the same that can be usefully used in analytical techniques using capillary tubes, such as capillary electrophoresis and LC-MS.

[0005] Therefore, this disclosure provides the following: (Item 1) A microcolumn for use in connection structures, The aforementioned connection structure comprises the microcolumn and two capillary tubes. The microcolumn comprises a column tube, a packing material filled inside the column tube, and a porous member positioned inside the column tube in contact with both ends of the packing material. The microcolumn is a microcolumn having openings at both ends for receiving the capillary tube. (Item 2) The aforementioned pore member is any of the aforementioned microcolumns, comprising a porous material. (Item 3) The aforementioned microcolumn is any of the above-mentioned microcolumns, wherein the porous member contains a fibrous material. (Item 4) The aforementioned microcolumn comprises a capillary tube fragment, as described above. (Item 5) The microcolumn described above, wherein the pore member comprises a porous material and a capillary tube fragment, and the porous material is arranged to be in contact with the packing material. (Item 6) The microcolumn described above, wherein the pore member has a length of 40% or less of the length of the microcolumn. (Item 7) The aforementioned microcolumn, wherein the packing material has a length of 5% or more of the length of the microcolumn. (Item 8) Any of the above-mentioned microcolumns, wherein the inner diameter of the column tube is substantially the same as the outer diameter of the capillary tube. (Item 9) The aforementioned microcolumn is any of the above-mentioned microcolumns, wherein the packing material is a particulate packing material. (Item 10) The aforementioned microcolumn, wherein the opening is the portion of the column tube. (Item 11) A microcolumn of any of the aforementioned types, having a length of approximately 1 to 50 mm. (Item 12) The aforementioned column tube is a microcolumn made of a fluororesin material, as described above. (Item 13) Any of the above-mentioned microcolumns, wherein the capillary tube is provided with positioning means for defining the position in which it is received by the column tube. (Item 14) The positioning means defines a position such that the end of the capillary tube contacts the end of the pore member, any of the aforementioned microcolumns. (Item 15) The aforementioned microcolumn, wherein the column tube includes an inner column tube, the inner diameter of the column tube is substantially the same as the outer diameter of the inner column tube, and the inner column tube contains the packing material inside. (Item 16) The above-mentioned microcolumn wherein the internal column tube contains the pore member inside. (Item 17) Any of the above-mentioned microcolumns, wherein the pore member is positioned in contact with the internal column tube. (Item 18) A microcolumn as described above, wherein the internal column tube is made of glass material. (Item 19) The aforementioned column tube is a microcolumn consisting of a single tube, as described above. (Item 20) The microcolumn according to any one of the preceding items, wherein the column tube is constituted by a plurality of tubes, and the plurality of tubes are connected via the inner column tube. (Item 21) The microcolumn according to any one of the preceding items, wherein the microcolumn further comprises a tubular member covering the column tube. (Item 22) The microcolumn according to any one of the preceding items, wherein the column tube is constituted by three tubes including a central tube and tubes at both ends, and the central tube contains the filler. (Item 23) The microcolumn according to any one of the preceding items, wherein an inner diameter of the central tube is larger than an inner diameter of the tubes at both ends. (Item 24) The microcolumn according to any one of the preceding items, wherein the tubes at both ends contain the porous member. (Item 25) The microcolumn according to any one of the preceding items, wherein the tubular member is constituted of a heat-shrinkable material. (Item 26) The microcolumn according to any one of the preceding items, wherein the tubular member partially covers end portions of the column tube. (Item 27) further comprising a force applying means that applies an external force to the tubular member, The microcolumn according to any one of the preceding items, wherein the tubular member is deformed by application of the external force from the force applying means, and is configured to expand or contract a space between the central tube and the tubular member. (Item 28) The force applying means comprises a pressure control member, The microcolumn according to any one of the preceding items, wherein the pressure control member is disposed around at least a portion of the tubular member that covers the central tube, such that a pressure adjustment space is formed between the pressure control member and the tubular member. (Item 29) The microcolumn according to any one of the preceding items, wherein the pressure control member is configured to expand the space between the central tube and the tubular member by reducing pressure in the pressure adjustment space, and contract the space between the central tube and the tubular member by increasing pressure in the pressure adjustment space. (Item 30) The aforementioned microcolumn, wherein the central tube includes the pore member. (Item 31) Any of the above-mentioned microcolumns, wherein at least the portion of the tubular member covering the central tube is made of a flexible material. (Item 32) The aforementioned flexible material is a silicone material, one of the aforementioned microcolumns. (Item 33) One of the aforementioned microcolumns, wherein the outer surface of the capillary tube is coated. (Item 34) One of the microcolumns mentioned above, The aforementioned capillary tube, Positioning means for defining the position in which the capillary tube is received by the microcolumn A microcolumn kit including this item. (Item 35) The positioning means is either a stopper attached to the capillary tube or an indicator provided on the capillary tube, in either of the aforementioned microcolumn kits. (Item 36) A method for measuring a sample, The process involves connecting one of the aforementioned microcolumns to a capillary tube to form a connection structure, The process of flowing the sample through the aforementioned connection structure, A step of measuring the sample that has passed through the aforementioned connection structure. Methods that include... (Item 37) Any of the aforementioned methods, which include capillary electrophoresis, LC-MS, or capillary electrophoresis-MS. (Item 38) A method for manufacturing any of the aforementioned microcolumns, A step of filling the inside of the column tube with the packing material, The process of inserting the porous member into the column tube and bringing it into contact with the packing material, Methods that include...

[0006] In this disclosure, the one or more of the above features are intended to be provided in combinations other than those explicitly stated. Further embodiments and advantages of this disclosure will be apparent to those skilled in the art, by reading and understanding the detailed description below as necessary. [Effects of the Invention]

[0007] This disclosure provides a microcolumn that can offer benefits such as compact connection of capillary tubes, provision of a micro-separation system applicable to trace samples, and / or fine control of the flow in the capillary tube system. Furthermore, since the microcolumn of this disclosure can be installed by inserting it into the middle of a capillary tube on existing piping (for example, by inserting it at a cut point in the capillary tube), it can be used without changing the established instrument configuration. [Brief explanation of the drawing]

[0008] [Figure 1A] Exemplary embodiments of the microcolumns and connection structures of this disclosure are shown. [Figure 1B] Exemplary embodiments of the microcolumns and connection structures of this disclosure are shown. [Figure 1C] Exemplary embodiments of the microcolumns and connection structures of this disclosure are shown. [Figure 1D] Exemplary embodiments of the microcolumns and connection structures of this disclosure are shown. [Figure 2] Exemplary embodiments of the microcolumns and connection structures of this disclosure are shown. [Figure 3] Exemplary embodiments of the microcolumns and connection structures of this disclosure are shown. [Figure 4] Figure 3 is a schematic diagram of the operation of the microcolumn. [Figure 5]This graph shows affinity chromatography of the antibody drug cetuximab using a microcolumn with protein G as the affinity ligand. The horizontal axis represents time (minutes), and the vertical axis represents fluorescence intensity (solid line) or electrical conductivity (dotted line). [Modes for carrying out the invention]

[0009] The present disclosure is described below in best form. Throughout this specification, singular expressions should be understood to include the concept of their plural form unless otherwise specified. Accordingly, articles used with singular forms (e.g., "a," "an," "the" in English) should be understood to include the concept of their plural form unless otherwise specified. Furthermore, terms used herein should be understood to have the meaning commonly used in the art unless otherwise specified. Accordingly, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. In case of any conflict, this specification (including definitions) shall prevail.

[0010] The following provides definitions of terms used specifically in this specification and / or basic technical concepts as appropriate.

[0011] (Definition, etc.) In this specification, “connection structure” refers to the combined portion of the microcolumn (or connector) and the structure to be connected to the microcolumn (or connector) (such as a capillary tube). When the connection structure occupies a small proportion of the overall structure, such as when it is incorporated into part of the piping of an LC system, the term “connection structure” may be used herein to refer to the portion of the structure located between the two ends of the microcolumn (or connector).

[0012] In this specification, "connector" refers to a structure (including the state in which multiple capillary tubes are connected) that can connect multiple capillary tubes to one another.

[0013] In this specification, “microcolumn” refers to a tubular structure having a small size (e.g., a length of 5 cm or less) and comprising at least a column tube and packing material, which is used in connection with a capillary tube and may have functionalities such as separation. If an additional structure, such as a tubular member covering the microcolumn described herein, is further attached to the tubular structure, the portion including this additional structure in addition to the tubular structure may be referred to as the microcolumn. In one embodiment, a microcolumn can be formed by including packing material inside a connector.

[0014] In this specification, "column tube" refers to a cylindrical or tubular structure capable of holding packing material inside.

[0015] In this specification, "packing material" refers to a substance that is held inside a column tube and, by coming into contact with the fluid passing through the column tube, affects the behavior of the analyte in the fluid.

[0016] In this specification, “porous member” refers to a member that is held inside a column tube and positioned in contact with the packing material. Porous members include porous materials having multiple pores (also called frit) and capillary tube segments having a single pore (also called end pieces). Porous members can prevent the loss (flow) of packing material and / or fix the position of the packing material.

[0017] In this specification, "internal column tube" refers to a cylindrical or tubular structure provided inside a column tube.

[0018] In this specification, "capillary tube" refers to a hollow tube with a small inner diameter (typically about 0.01 to 1 mm). Capillary tubes used in capillary electrophoresis and HPLC are often made of fused silica or glass, but the material of the capillary tube is not particularly limited. Typically, capillary tubes are liquid-tight except at their ends. Capillary tubes may also be coated on the outside with polyimide or other materials, and are often used with the coating attached unless it interferes with optical detection.

[0019] In this specification, when describing tubular structures, the term "length" may be used with respect to the direction in which the hollow space extends, and the terms "circumference," "outer diameter," and "inner diameter" may be used with respect to the direction perpendicular to the direction in which the hollow space extends.

[0020] In this specification, “kit” means a unit in which the parts to be provided (e.g., microcolumns, capillary tubes, etc.) are provided, usually divided into two or more compartments. It is advantageous that the kit preferably includes instructions or manuals describing how to use or operate the provided parts, etc.

[0021] In this specification, "length" for elongated members such as capillary tubes, column tubes, and pore members means the length of the member in the longitudinal direction.

[0022] In this specification, the term "approximately" refers to plus or minus 10% of the indicated value unless otherwise specified. When "approximately" is used for temperature, it refers to plus or minus 5°C of the indicated temperature; when "approximately" is used for pH, it refers to plus or minus 0.5 of the indicated pH.

[0023] In this specification, the term “substantially identical” means that two values ​​are within a range of plus or minus 10% difference from each other. More preferably, two substantially identical values ​​are within a range of plus or minus 5% difference from each other.

[0024] (Preferred embodiment) Preferred embodiments of the Disclosure are described below. The embodiments provided below are provided for a better understanding of the Disclosure, and it will be understood that the scope of the Disclosure should not be limited to the descriptions below. Accordingly, it will be obvious that those skilled in the art can make appropriate modifications within the scope of the Disclosure, taking into consideration the descriptions herein. It will also be understood that the embodiments of the Disclosure below can be used individually or in combination.

[0025] In one aspect, the present disclosure provides a connection structure comprising a connector or microcolumn and a plurality (e.g., two) capillary tubes. In one embodiment, the connection structure may be configured to allow fluid communication between the microcolumn (or connector) and the capillary tubes. In one embodiment, the connection structure may be configured to be liquid-tight (to prevent the outflow of liquid from the inside to the outside of the structure). In one embodiment, the connection structure of the present disclosure may also perform functions other than connecting a plurality of capillary tubes (e.g., functions of microcolumns described herein, such as separation and flow control).

[0026] The capillary tubes described herein may be commercially available, for example, those sold as tubing for capillary electrophoresis or HPLC. Capillary tubes may have outer diameters such as approximately 0.05–5 mm, 0.05 mm, 0.1 mm, 0.18 mm, 0.2 mm, 0.36 mm, 0.5 mm, 1 mm, 2 mm, or 5 mm. Capillary tubes may have inner diameters such as approximately 0.01–1 mm, 0.01 mm, 0.02 mm, 0.05 mm, 0.1 mm, 0.2 mm, 0.5 mm, or 1 mm. Capillary tubes may be made of, for example, fused silica or glass. The outside of the capillary tube may be coated with polyimide or the like. The performance (pressure resistance, etc.) of the connection structure disclosed herein may depend not only on the physical strength of the capillary tube itself, but also on the interaction of the interface where the microcolumn and the capillary tube come into contact. Therefore, it may be advantageous for the capillary tube to have a coating (such as polyimide) that is suitable for the material of the microcolumn (such as Teflon®).

[0027] In one aspect, the present disclosure provides a microcolumn (or connector) or a combination of a microcolumn (or connector) and a capillary tube (e.g., as a kit) for use in a connection structure. In one embodiment, the microcolumn of the present disclosure includes or consists of a connector and a column tube. In one embodiment, the microcolumn (or connector) has a plurality of openings, each receiving another capillary tube. The microcolumn (or connector) may receive capillary tubes having the same outer diameter or capillary tubes having different outer diameters, and the size of the openings may be adjusted according to the outer diameter of the capillary tubes.

[0028] In one embodiment, the microcolumn (or connector) of the Disclosure may have a length of about 0.5 mm, about 1 mm, about 2 mm, about 5 mm, about 10 mm, about 20 mm, about 50 mm, or in a range between any two of these, for example, about 0.5 to 50 mm, about 1 to 50 mm, about 1 to 20 mm, or about 1 to 10 mm. The microcolumn (or connector) of the Disclosure may be easy to connect and may require a small width for mounting various components, as fewer components may be needed to form the connection structure (for example, only the microcolumn and the capillary tube, or only the connector and the capillary tube). In one embodiment, the portion of the microcolumn (or connector) of the Disclosure that receives the capillary tube may have an inner diameter that is the same as or about 80% or more, about 90% or more, about 95% or more, about 98% or more, or about 99% or more of the outer diameter of the capillary tube.

[0029] The packing material of the present invention may be a particulate packing material or a monolithic packing material. In one embodiment, the microcolumn (or connector) of the present disclosure includes a particulate packing material. Typically, the microcolumn may also include a pore member to hold the particulate packing material within the microcolumn. Particulate packing materials are generally preferred because they can achieve a higher binding capacity compared to monolithic packing materials.

[0030] In another embodiment, the microcolumn (or connector) of the present disclosure includes a monolithic packing material. When a monolithic packing material is used, a pore member for holding the packing material in the microcolumn may not be necessary, which is preferable.

[0031] In one embodiment, the column tube of the present disclosure may have an outer diameter of about 0.1 mm, about 0.2 mm, about 0.5 mm, about 1 mm, about 2 mm, about 5 mm, about 10 mm, or in a range between any two of these, for example, about 0.1 to 10 mm, about 0.5 to 10 mm, or about 0.5 to 5 mm. If no further members are attached around the column tube, the outer diameter of the column tube may become the outer diameter of the connecting structure, so that the connecting structure of the present disclosure can be formed in a very small space and may allow for highly flexible piping. In one embodiment, the column tube of the present disclosure may have an inner diameter of about 0.05 mm, about 0.1 mm, about 0.2 mm, about 0.5 mm, about 1 mm, about 2 mm, about 5 mm, or in a range between any two of these, for example, about 0.05 to 5 mm, about 0.1 to 5 mm, or about 0.2 to 2 mm.

[0032] In one embodiment, the column tube of the present disclosure comprises an opening for directly receiving a capillary tube. This opening may be an opening located on the outside in a microcolumn. In one embodiment, the opening of the column tube may be adjusted to have an inner diameter equal to or greater than the outer diameter of the capillary tube to facilitate insertion of the capillary tube. For example, the opening of the column tube may be widened in a trumpet shape. In one embodiment, the column tube (the portion that receives the capillary tube) may have an inner diameter equal to or smaller than the outer diameter of the capillary tube, and approximately 70% or more, approximately 80% or more, approximately 90% or more, approximately 95% or more, approximately 98% or more, or approximately 99% or more of the outer diameter of the capillary tube. In one aspect, if the microcolumn includes a tubular member, the column tube may be a means for adjusting the outer diameter of the capillary tube so that the tubular member can receive the capillary tube.

[0033] In one embodiment, the column tube of the present disclosure may be made of materials such as fluororesins (e.g., polytetrafluoroethylene (PTFE), perfluoroalkoxyalkanes (PFA), fluorinated ethylene propylene (FEP), ethylene tetrafluoroethylene (ETFE), TEFLON®, TEFZEL®, DELRIN®), polyether ether ketone (PEEK), polyetherimide (PEI), sulfurized polyphenylene (PPS), polypropylene, sulfone polymer, polyolefin, polyimide, polyaryl ether ketone, and polyoxymethylene (POM). Preferably, the column tube of the present disclosure is made of a fluororesin with flexibility and rigidity, such as TEFLON®, and becomes liquid-tight when a capillary tube is received.

[0034] In one embodiment, the packing material may be packed over lengths of approximately 0.5 mm, 1 mm, 2 mm, 5 mm, 10 mm, 20 mm, 50 mm, or any two of these ranges, for example, approximately 0.5–50 mm, 1–50 mm, 1–20 mm, or 1–10 mm of the column tube (or internal column tube). In one embodiment, the packing material may be packed over lengths of approximately 3% or more, approximately 5% or more, approximately 10% or more, approximately 20% or more, approximately 40% or more, approximately 60% or more, or approximately 80% or more of the length of the microcolumn. Since the volume of packing material that can be held increases with the length of the column tube, it may be advantageous to configure the column tube to be somewhat longer, for example, to improve separation performance. However, since the longer the distance over which the packing material is present, the more the flow pressure needs to be increased to pass through it, it may be important to adjust this length to be within a pressure range that can maintain a liquid-tight connection structure.

[0035] The packing material can be a column packing structure used in HPLC systems, such as a particulate packing material or a monolithic packing material. Because the length of the microcolumn of this disclosure is short, it can be operated at low pressure even when packed with particulate packing material, and additional connecting members may not be required when connecting the microcolumn to a capillary tube. Furthermore, the packing material can be a column packing material used in affinity chromatography, ion exchange chromatography, reversed-phase chromatography, normal-phase chromatography, etc., and similar separation modes can be implemented. Examples of target molecule / affinity ligand combinations in affinity chromatography include biotin-binding proteins such as avidin and streptavidin / biotin, maltose-binding proteins / maltose, G proteins / guanine nucleotides, oligohistidine peptides / metal ions such as nickel or cobalt, glutathione-S-transferase / glutathione, DNA-binding proteins / DNA, antibodies / antigen molecules (epitopes), antigen molecules (epitopes) / antibodies, antibodies / protein A, antibodies / protein G, antibodies / protein L, lectins / sugars, calmodulin / calmodulin-binding peptides, ATP-binding proteins / ATP, or estradiol receptor proteins / estradiol.

[0036] In one embodiment, the pore member is positioned inside the column tube (or internal column tube) in contact with both ends of the packing material. Attaching the pore member can suppress movement, leakage, and / or deformation of the packing material. In one embodiment, the pore member has a length of about 50% or less, about 40% or less, about 20% or less, about 10% or less, about 8% or less, about 6% or less, about 4% or less, about 2% or less, or about 1% or less of the length of the microcolumn. In one embodiment, the pore member may be a porous material (also called frit), a capillary tube fragment (also called an end piece), or both. If the pore member includes both a porous material and a capillary tube fragment, the porous material may be positioned in contact with the packing material. In one embodiment, the pore member is not fixed to the column tube (or internal column tube) but exists in a state where it can move by pressure (including insertion of a capillary tube).

[0037] In one embodiment, the porous material includes fibrous materials (such as cotton, asbestos, or quartz fibers), but is not particularly limited as long as it is a material through which liquid can pass. Excessive compression of the porous material can lead to an increase in the back pressure of the microcolumn, so it is preferable to avoid pressing by the capillary tube using positioning means or the like. In one embodiment, the capillary tube fragment has the same outer and inner diameter as the capillary tube connected to the microcolumn, but may have different outer and inner diameters. In embodiments in which the capillary tube fragment is in direct contact with the packing material, the inner diameter of the capillary tube fragment and the structure and size of the packing material are selected so that the packing material does not cause clogging of the capillary tube fragment.

[0038] In one embodiment, the capillary tube is provided with positioning means for defining the position in which it is received in the microcolumn. If the capillary tube is inserted too deeply into the microcolumn, it may push the pore members, packing material, and / or the internal column tube, causing these members to shift their positions within the microcolumn or resulting in excessive compression of the packing material and / or pore members. Conversely, if the insertion is too shallow, a dead volume is created between the pore members and the capillary end, resulting in a substantial decrease in column performance. It is preferable to have positioning means to avoid this. In one embodiment, the positioning means is a flange-shaped stopper attached to the capillary tube (for example, a short (e.g., about 1 mm) section of a tubular structure having an inner diameter that can receive the capillary tube, glued in the appropriate position), or an indicator provided on the capillary tube (for example, a line, recess, or protrusion on the capillary surface).

[0039] In one embodiment, an internal column tube is placed inside a column tube. In one embodiment, the internal column tube may be made of glass, but is not limited to any particular material. In one embodiment, the internal column tube contains a packing material. Since the internal column tube can be protected by the column tube, it does not require particular strength, and the inner diameter of the internal column tube may be about 95%, 90%, 80%, 70%, 60%, 50%, etc., of the inner diameter of the column tube. In one embodiment, the inner diameter of the column tube may be configured to be substantially the same as the outer diameter of the internal column tube (for example, a difference of about 10% or less, about 5% or less, about 2% or less, or about 1% or less). If there is an internal column tube having an outer diameter substantially the same as the inner diameter of the column tube, the entire structure can be liquid-tight if only the ends of the internal column tube are covered by the column tube. In one embodiment, the column tube does not need to cover the central portion of the internal column tube; that is, the column tube may be composed of multiple tubes connected via the internal column tube.

[0040] In one embodiment, the microcolumn (or connector) of the present disclosure comprises a tubular member on the outside of the column tube. In this embodiment, the column tube may consist of multiple tubes, for example, three tubes: two at each end and a central tube, because it is fixed by the tubular member. In one embodiment, the central tube contains a packing material, and the inner diameter of the central tube is configured to be larger. In one embodiment, a pore member is positioned inside and / or in contact with the central tube. Since the inside of the central tube may contain a packing material, it may be preferable to increase the inner diameter of the central tube to improve the adsorption and separation performance of the packing material and / or to suppress the increase in pressure required for flow due to the presence of the packing material. Therefore, in one embodiment, the inner diameter of the central tube may be configured to be larger than the inner diameters of the tubes at both ends of the column tube and / or the capillary tubes. In this embodiment, the capillary tubes may be inserted inside the central tube. Therefore, in one embodiment, the capillary tube is provided with positioning means (as described above) that define the position in which it is received by the column tube, and is configured so that the capillary tube does not reach the central tube, thereby preventing the pore member and packing material from being pushed in by the capillary tube.

[0041] In one embodiment, the tubular member of the Disclosure is the same length as the microcolumn (or connector) of the Disclosure. In one embodiment, at least a portion of the column tube may protrude from the end of the tubular member. In one embodiment, the inner diameter of the tubular member (when undeformed, if deformable) may be configured to be approximately equal to the outer diameter of the column tube (e.g., a difference of about 10% or less, about 5% or less, about 2% or less, or about 1% or less). In one embodiment, the tubular member of the Disclosure may have an outer diameter of about 0.1 mm, about 0.2 mm, about 0.5 mm, about 1 mm, about 2 mm, about 5 mm, about 10 mm, or in a range between any two of these, e.g., about 0.1 to 10 mm, about 0.5 to 10 mm, or about 0.5 to 5 mm. If no further members are attached around the tubular member, the outer diameter of the tubular member may become the outer diameter of the connecting structure, so that the connecting structure of the Disclosure can be formed in a very small space and may allow for highly flexible piping. In one embodiment, the tubular member of the present disclosure may have an inner diameter of about 0.05 mm, about 0.1 mm, about 0.2 mm, about 0.5 mm, about 1 mm, about 2 mm, about 5 mm, or in a range between any two of these, for example, about 0.05 to 5 mm, about 0.1 to 5 mm, and about 0.2 to 2 mm.

[0042] In one embodiment, the tubular member of the disclosure may be made of a heat-shrinkable material. Examples of heat-shrinkable materials include, but are not limited to, polyethylene, vinyl acetate polymer, ethylene methacrylate copolymer, and polypropylene, and any known heat-shrinkable material can be used. As a heat-shrinkable material, for example, a material can be used that shrinks to about 90% or less, about 80% or less, or about 70% or less in length when a rod-shaped molded product with a diameter of 1 mm is heated at 150°C for 10 minutes. In one embodiment, the tubular member of the disclosure may be made of a flexible material. Examples of flexible materials include, but are not limited to, silicone material, fluororubber, urethane rubber, butadiene rubber, natural rubber, and elastomer, and any known flexible material can be used. As a flexible material, for example, a material with a shrinkage of about 0.02 kgf / mm² at 25°C can be used. 2 , about 0.05kgf / mm 2 Approximately 0.1 kgf / mm² 2, about 0.2 kgf / mm 2 , about 0.4 kgf / mm 2 , about 0.6 kgf / mm 2 , about 0.8 kgf / mm 2 , about 1 kgf / mm 2 , or a range between any two of these, for example, about 0.02 to 1 kgf / mm 2 , about 0.05 to 0.6 kgf / mm 2 , about 0.1 to 0.4 kgf / mm 2 A material having a tensile modulus of can be used. As the flexible material, for example, a material that exhibits a volume increase rate of about 20% or less, about 10% or less, or about 5% or less when a square molded product having a thickness of 1 mm and a side length of 1 cm is immersed in acetonitrile, ethyl acetate, ethanol, methanol and water at 50° C. for 100 hours may be preferable.

[0043] In one embodiment, the microcolumn (or connector) of the present disclosure further comprises force-applying means (e.g., around the tubular member) for applying an external force to a tubular member, the application of the external force by the force-applying means causing the tubular member to deform and the space between the central tube and the tubular member to expand or contract. For example, the expansion or contraction of the space between the central tube and the tubular member may be accompanied by deformation in a direction perpendicular to the axial direction of the microcolumn. In this embodiment, at least the porous member is located inside the central tube. The external force applied by the force-applying means may be a mechanical tensile force or a force that utilizes the pressure difference generated inside and outside the tubular member through pressure manipulation around the tubular member. For example, the force-applying means may include a member attached to the tubular member for mechanically pulling the tubular member, a member attached around the tubular member to create an airtight space around the tubular member, and a member (such as a cylinder or pump connected in communication with the pressure adjustment space) for increasing or decreasing the air pressure in this airtight space (pressure adjustment space). In this embodiment, the tubular member may be made of the flexible material described above, such as silicone. By changing the volume of the space between the central tube and the tubular member, a new flow path can be formed between the central tube and the annular member while maintaining a liquid-tight state, thereby expanding the possibilities for controlling the speed and direction of flow inside and outside the capillary tube and the central tube.

[0044] When the space between the central tube and the tubular member is expanded, a gap is created between the tubular member and the central tube. Since there is no packing material in this gap, the resistance is low, and the flow can preferentially flow through this gap. For example, in a system in which a solid-phase extraction column (central tube) placed on the anode side is directly connected to isoelectric focusing electrophoresis separation, if a voltage is applied to both ends of the device while the solid-phase extraction column is immersed in acidic anodic solution, electroosmotic flow toward the anode occurs within the column, causing the sample and separation solution to be drawn out of the isoelectric focusing capillary toward the anode. However, by using the flow control described above, if a gap is created on the outside of the column tube after eluting the protein from the solid-phase extraction column but before applying the voltage, the liquid flow due to electroosmosis can reflux on the outside of the column, preventing flow from occurring in the downstream isoelectric focusing capillary. In this embodiment, it is preferable that a flow path is formed at the boundary between the central tube and the tubes at both ends. Therefore, the length of the central tube is made shorter than the length between the tubes at both ends, or grooves (for example, radial grooves extending from the center outward) are provided on the end faces of the tubes at both ends facing the central tube, thereby forming a flow path between the central tube and the tubes at both ends. Alternatively, by contracting the space between the central tube and the tubular member, the gap between the tubular member and the central tube disappears, the flow path outside the central tube is closed, and the flow path is switched to inside the central tube. This state may also be utilized. In one preferred embodiment, sulfonic acid groups can be bonded to the outer wall of the central tube to direct the electroosmotic flow toward the cathode under acidic conditions.

[0045] In one embodiment, the force-applying means may include a pressure control member, which may be positioned around at least the portion of the tubular member covering the central pipe so as to form a pressure adjustment space between itself and the tubular member. The pressure control member may expand the space between the central pipe and the tubular member by reducing the pressure adjustment space, and contract the space between the central pipe and the tubular member by increasing the pressure adjustment space. In particular, the portion of the tubular member covering the central pipe may be made of a flexible material.

[0046] In one embodiment, the microcolumn (or connector) of the present disclosure does not include any additional members (e.g., members that press the tubular member from the outside) around the tubular member. In another embodiment, the microcolumn (or connector) of the present disclosure may include additional members (e.g., members that press the tubular member from the outside) around the tubular member, thereby potentially improving liquid-tightness. In this embodiment, the tubular member may be made of the heat-shrinkable material described above. In one embodiment, the tubular member may be configured to partially cover the end of the column tube (by heat shrinkage), thereby potentially improving the pressure resistance of the microcolumn. For example, such a structure may be formed by inserting various members into a long tubular member, heat-shrinking the tubular member, and then cutting the tubular member at a position away from the end of the column tube.

[0047] While not intended to be limiting, specific embodiments of this disclosure will be described below with reference to the drawings for the sake of clarity.

[0048] In Figures 1A to 1D, a packing material 33 is packed into a column tube 30, a pore member 31 is inserted to form a microcolumn 20, and then a capillary tube 22 is inserted to form a connection structure 10. The column tube 30 acts as a connector. The presence of a positioning means 23 prevents the capillary tube 22 from pushing the pore member 31, thus protecting the packing material 33. In Figure 1A, the column tube 30 is composed of a single tube. In Figure 1B, the pore member includes a porous material 31a and a capillary tube fragment 31b. In Figure 1C, an inner column tube is inserted into the column tube, with the packing material and pore material 31a placed inside the inner column tube, and the capillary tube fragment 31b placed outside the inner column tube. In Figure 1D, the column tube 30 is composed of two tubes, with the inner column tube connecting the two column tubes.

[0049] In Figure 2, a column tube 30 (two tubes 30a at both ends and a central tube 30b) is inserted into a tubular member 35 (heat-shrinkable material), a packing material 33 is filled into the central tube 30b, a pore member 31 is inserted into the two tubes 30a at both ends, heat is applied to shrink the tubular member 35 to form a microcolumn 20, and then a capillary tube 22 equipped with a positioning means 23 is inserted to form a connection structure 10. The tubular member 35 and the two tubes 30a at both ends constitute a connector. The presence of the positioning means 23 prevents the capillary tube 22 from pushing in the pore member 31, protecting the packing material 33. Due to the heat shrinkage of the tubular member 35, the tubular member 35 partially covers the ends of the two tubes 30a at both ends.

[0050] In Figure 3, the microcolumn 20 is first formed by inserting the tubes 30a at both ends and the central tube 30b (including the packing material 33 and the pore member 31) into the tubular member 35 (flexible material) (a). Then, the connection structure 10 is formed by inserting the capillary tube 22 equipped with the positioning means 23 (b). Next, the pressure control member 40, O-ring 42 and set screw 41 are attached around the tubular member 35 to form the microcolumn 20 equipped with the pressure control means (c). The tubular member 35 and the tubes 30a at both ends constitute a connector. Due to the presence of the positioning means 23, the capillary tube 22 does not penetrate into the interior of the central tube 30b and does not push in the pore member 31. The pressure control member 40, O-ring 42 and set screw 41 constitute a force-applying means. A pressure control space is formed between the pressure control member 40 and the tubular member 35, and by controlling the pressure, the volume around the central tube 30b increases or decreases by deforming the tubular member 35 made of flexible material. A flow path is formed between the central pipe 30b and the pipes 30a at both ends by radial grooves provided at the ends of these pipes.

[0051] Refer to Figure 4 for further details on the behavior of the microcolumn under pressure control shown in Figure 3. Here, we assume that an isoelectric focusing capillary tube is connected downstream of the microcolumn (top of the figure). (A) When the pressure control space is pressurized, only a channel is formed near the central tube 30b that passes through the central tube 30b. By flowing the sample with a pump or the like, the target substance (protein, etc.) can be trapped in the packing material 33 of the central tube 30b. (B) When the pressure control space is depressurized, the tubular member 35 covering the central tube 30b expands, creating a gap between the tubular member 35 and the central tube 30b. This gap, along with the radial grooves provided on the end faces of the tubes 30a at both ends, forms a new channel. After flowing the anodic acid through the central tube 30b, the pressure is reduced at the start of electrophoresis. When a voltage is applied in this state, the electroosmotic flow generated in the central tube 30b toward the anode flows back into the central tube 30b through the groove on the anode side, to the outside of the central tube 30b, and then through the groove on the cathode side. In this way, the influence of the electroosmotic flow on the isoelectric focusing capillary on the cathode side can be eliminated. To further enhance this situation, it may be desirable to bond sulfonic acid groups to the outer wall of the central tube 30b to generate electroosmotic flow toward the cathode under acidic conditions.

[0052] (Manufacturing method) In one aspect, the present disclosure provides a method for manufacturing a microcolumn (or connector) of the present disclosure. In one embodiment, the manufacturing method may include a step of filling the inside of a column tube with a packing material. In one embodiment, the step of filling the inside of a column tube with a packing material may include inserting an internal column tube filled with the packing material into a column tube or filling the inside of an internal column tube with a packing material. In one embodiment, the manufacturing method may include a step of inserting a porous member into a column tube and bringing it into contact with the packing material. In one embodiment, the step of inserting a porous member into a column tube and bringing it into contact with the packing material may include inserting an internal column tube containing the packing material and the porous member into a column tube or inserting a porous member into an internal column tube. In one embodiment, the manufacturing method may include a step of inserting an internal column tube into a column tube. For example, packing of the packing material may be carried out by first inserting one of the two porous members to be inserted into the column tube (or internal column tube), then injecting a solution containing the packing material into the column tube (or internal column tube) under pressure, and finally inserting the other porous member into the column tube (or internal column tube). When manufacturing the microcolumn (or connector) of this disclosure, it may be preferable to appropriately select the inner / outer diameters of the column tube, capillary tube, internal column tube, annular member, etc., to ensure that the overall structure is liquid-tight. Since the inner / outer diameter of a tubular structure can change slightly due to heat and pressure, heat and pressure may be applied to the tubular structure when manufacturing the microcolumn (or connector) of this disclosure. For example, when inserting the column tube into the annular member, or when inserting the internal column tube into the column tube, the opening of the annular member or column tube may be widened, and / or the annular member or column tube may be heated to facilitate insertion. In one embodiment, when a microcolumn (or connector) of the present disclosure is manufactured using a column tube made of a heat-shrinkable material, the method for manufacturing the microcolumn (or connector) may include a step of heating the column tube. The heating temperature may be, for example, about 100°C, about 150°C, about 200°C, about 300°C, about 400°C, about 500°C, or in a range between any two of these, for example, about 100°C to 500°C, but can be appropriately set by those skilled in the art depending on the type of heat-shrinkable material.

[0053] (Application) The microcolumn (or connector) of this disclosure can be used in connection with a capillary tube used in capillary electrophoresis, LC-MS, etc. The microcolumn (or connector) of this disclosure may be easy to connect because it does not require the use of a clamping mechanism or other pressing means when connecting to a capillary tube. The microcolumn of this disclosure may contain an adsorbent and / or a separating agent as a packing material, so that specific substances can be separated or isolated, and can be suitably used in combination with various analytical means. The microcolumn of this disclosure may be easy to connect because it does not require the use of a clamping mechanism or other pressing means when connecting to a capillary tube. In one embodiment, the microcolumn of this disclosure may be used under relatively low pressure conditions such as about 1 MPa or less, about 0.5 MPa or less, about 0.2 MPa or less, about 0.1 MPa or less, about 0.05 MPa or less, about 0.02 MPa or less, and about 0.01 MPa or less.

[0054] (Note) In this specification, "or" is used when "at least one" of the items listed in the text can be adopted. The same applies to "or else". In this specification, when it is specified that "within the range" of "two values", that range includes the two values ​​themselves.

[0055] The present disclosure will be described below based on examples, but the above description and the following examples are provided for illustrative purposes only and not to limit the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but is limited only by the claims. [Examples]

[0056] (Example 1: Microcolumn using particulate packing material) The microcolumn shown in the schematic diagram of Figure 1 was prepared according to the following procedure.

[0057] 1.1 Filling of particulate fillers A cotton plug (1 mm long) was inserted as a pore-filling component approximately 8 mm from one end of a Teflon® column tube (0.35 mm inner diameter, 1.5 mm outer diameter, 30 mm length). A 0.35 mm outer diameter capillary tube, connected to a negative pressure generator, was inserted 10 mm into the same end of the column tube. As the capillary tube was inserted, the cotton plug moved within the column tube while in contact with the end of the capillary tube. While operating the negative pressure generator at atmospheric pressure minus 0.5 atmospheres, the other end of the column tube was immersed in a suspension of agarose gel particles (average particle size 0.034 mm) under a microscope, and a predetermined amount of gel particles were aspirated into the column tube. The gel particles accumulated in front of the cotton plug. The pressure of the negative pressure generator was set to atmospheric pressure, and another cotton plug was inserted until it touched the rear end of the aspirated and accumulated gel particles. The column tube was cut 10 mm from the end of this cotton plug that was not in contact with the gel particles.

[0058] 1.2 Affinity chromatography using microcolumns Agarose gel particles immobilized with protein G as an affinity ligand were packed into a Teflon® column tube (inner diameter 0.35 mm, outer diameter 1.5 mm, length 26 mm) in the same manner as described above. The length of the packing material was 4 mm, and a 1 mm long cotton plug was used as a pore-forming member. The distance between the pore-forming member and the end of the column tube was 10 mm. A fused silica capillary (with polyimide sheath) (Molex LLC, USA) with an inner diameter of 0.05 mm and an outer diameter of 0.36 mm was used as a capillary to connect to the column. A PEEK tube (Upchurch Scientific Inc., USA) with an inner diameter of 0.4 mm, an outer diameter of 1.5 mm, and a length of 1 mm was bonded to the end of the capillary 10 mm from the end of the capillary as a stopper, so that the optimal insertion depth could be obtained when the capillary was pushed into the microcolumn. The length of the inlet capillary was 80 mm, and the length of the outlet capillary was 340 mm. The polyamide coating was removed from the outlet capillary at a position 220 mm from the microcolumn, and a fluorescence detector was placed there to detect fluorescence (excitation light 280 nm, detection light 340 nm) originating from tryptophan residues of the protein inside the capillary. In addition, a non-contact conductivity detector was placed at a position 180 mm from the microcolumn to detect changes in electrical conductivity. When physiological phosphate buffer (PBS, pH 7.3) was delivered at atmospheric pressure + 0.5 atm, the flow rate was 1.1 μL / min. On the other hand, the flow rate without the microcolumn connected was 1.3 μL / min.

[0059] In the following experiment (results shown in Figure 5), the fluid was delivered at 0.5 atmospheres and at room temperature. A solution of the antibody drug cetuximab dissolved in PBS at a concentration of 500 ng / μL was injected into the above microcolumn, which had been equilibrated with PBS, for 3 minutes (0-3 minutes; it took approximately 1 minute for the injected solution to reach the detection point). Next, PBS was injected for 3 minutes to wash away any substances not bound to protein G (3-6 minutes). Then, 0.2 M iminodiacetic acid (pH 2.25) was passed through for 3 minutes (6-9 minutes), and then PBS was passed through for 3 minutes to return the column to neutral conditions (9-12 minutes).

[0060] As seen in the chromatogram in Figure 5, almost no protein was detected at the detection point during sample addition (1-3 minutes) and column washing with PBS (3-6 minutes). This indicates that almost all of the added cetuximab was strongly captured by the Protein G column (solid line). On the other hand, when an acidic 0.2 M iminodiacetic acid solution was passed through (6-9 minutes), protein elution was observed. A decrease in electrical conductivity was observed with the detection of the protein peak (dotted line), which is because iminodiacetic acid has lower electrical conductivity than PBS, indicating that the replacement of the liquid in the column with iminodiacetic acid caused the elution of cetuximab.

[0061] Thus, the microcolumn of this disclosure can be easily incorporated into a system and can achieve good separation in a small space. When using particulate packing material, a higher binding capacity can be easily achieved than when using monolithic packing material, which may allow for miniaturization of the microcolumn and reduction of flow resistance. Furthermore, since the microcolumn of this disclosure can achieve separation at extremely low flow rates, it is suitable for analysis using a mass spectrometer as a detector. In addition, the microcolumn of this disclosure can be effectively used in isoelectric focus variant analysis of specific proteins in biological samples using a direct coupled analytical method of solid-phase extraction and capillary isoelectric focusing.

[0062] (Example 2: Microcolumn equipped with a tubular component for micro-separation) A microcolumn similar to the one shown in the schematic diagram of Figure 2 was prepared according to the following procedure. However, although the following is an example of a microcolumn using a monolithic packing material, a microcolumn using a particulate packing material can be prepared using the same procedure.

[0063] 2.1 Preparation of glycol monolith packed column tubes (inner diameter 0.8 mm) A glycol monolith was formed inside a glass capillary tube (Fuji Rika Kogyo Co., Ltd., Osaka) with an outer diameter of 1.6 mm and an inner diameter of 0.8 mm. The above glycol monolith-embedded capillary tube was cut to a length of approximately 3 mm, and both ends were polished with 400-grit waterproof sandpaper until the length was 2.0 mm, completing the glycol monolith-packed column tube to be used as the central tube of the column.

[0064] 2.2 Preparation of microcolumns and connection structures To form the tubes at both ends of the column, Teflon® tubing with an outer diameter of 1.6 mm and an inner diameter of 0.35 mm was cut to a length of 9.5 mm, and one of the openings was widened into a trumpet shape by pushing a heated needle into it. One of the tubes at both ends, the central tube of the column, and the other of the tubes at both ends were inserted into a tubular component of heat shrink tubing (Sumitube A, Sumitomo Electric Industries, Ltd., Osaka City) with an inner diameter of 2.1 mm, a wall thickness of 0.2 mm, and a length of 30 mm, in the following order. At this time, the widened trumpet-shaped ends of the tubes were positioned facing outwards. The three components were placed in close contact within the heat shrink tubing, and the entire assembly was placed in a 100-degree Celsius airflow for approximately 30 seconds to shrink the tubing. After cooling, both ends of the heat shrink tubing were cut so that they were approximately 0.5 mm longer than the ends of the tubes at both ends, completing the microcolumn. The connection structure was completed by connecting 0.36mm outer diameter fused silica capillary tubes (with polyimide sheaths) (Molex LLC, USA) with stoppers fixed 10mm from the end to both sides of the microcolumn. The stoppers were attached by cutting a 1mm length of 1.6mm outer diameter, 0.4mm inner diameter PEEK tubing (Upchurch Scientific Inc., USA) and passing the capillary tube through it, then securing it with super glue.

[0065] The structure of the microcolumn fabricated in this embodiment allows for a larger amount of packing material to be retained compared to the microcolumn fabricated in Example 1. Therefore, it is thought that a larger amount of analytes can be separated, potentially increasing the variety of analytes that can be detected at levels exceeding the detection limit. Furthermore, because the inner diameter of the central tube is large, the pressure on the microcolumn can be reduced, potentially enabling analysis at higher flow rates and shorter timeframes.

[0066] (Example 3: Microcolumn with flow control function) A microcolumn similar to the one shown in the schematic diagram of Figure 3 was prepared according to the following procedure. However, although the following is an example of a microcolumn using a monolithic packing material, a microcolumn using a particulate packing material can be prepared using the same procedure.

[0067] 3.1 Preparation of glycol monolith packed column tubes Similar to Example 2, a glycol monolith packed column tube (outer diameter 1.6 mm, inner diameter 0.8 mm, length 2.0 mm) was prepared as the central tube among the column tubes.

[0068] 3.2 Preparation of microcolumns and connection structures For the two ends of the column tube, a Teflon® tube with an outer diameter of 1.6 mm and an inner diameter of 0.35 mm was cut to a length of 10 mm, and one of the openings was widened into a trumpet shape by pushing a heated needle into it. On the other end face, eight grooves with a depth of approximately 0.1 mm were cut to radiate from the center. One of the two end tubes, the central tube of the column tube, and the other end tube were inserted in that order into a tubular component of a silicone tube (Nagayanagi Kogyo Co., Ltd., Tokyo) with an outer diameter of 2 mm, an inner diameter of 1.5 mm, and a length of 22 mm to form a microcolumn. At this time, the trumpet-shaped ends of the two end tubes were positioned on the outside, and the three were tightly packed together inside the silicone tube. One end of the microcolumn was connected to a capillary tube with an outer diameter of 0.36 mm and an inner diameter of 0.15 mm (a fused silica capillary tube with a hydrophilic polymer coating on the inner wall and a polyimide coating on the outer wall), with a stopper (same as in Example 2) fixed 10 mm from the end. The other end of the tube was connected to an isoelectric focusing capillary tube with an outer diameter of 0.36 mm and an inner diameter of 0.05 mm (a fused silica capillary tube with a hydrophilic polymer coating on the inner wall and a polyimide coating on the outer wall), both with similar stoppers. An O-ring (NSA3 standard, wire diameter 1.5 mm, inner diameter 2.5 mm, outer diameter 5.5 mm) and a set screw (Miyamoto Resin Industry Co., Ltd., Fukushima City) were set into a pressure control member (made of PMMA) (Miyamoto Resin Industry Co., Ltd., Fukushima City) with an inner diameter of 2.7 mm in the center. Then, a microcolumn was inserted, the set screw was tightened to fix the column, and the microcolumn connection structure was completed by connecting it to a capillary tube for isoelectric focusing electrophoresis.

[0069] The use of small microcolumns allows for fine-tuning of the flow, addressing the electroosmotic flow problems that typically occur in downstream isoelectric focusing capillaries.

[0070] (Note) As described above, while the present disclosure has been illustrated using preferred embodiments thereof, it is understood that the scope of the present invention should be interpreted solely by the claims. Patents, patent applications and other documents cited herein should be incorporated herein by reference as if their contents were specifically described herein. [Industrial applicability]

[0071] This disclosure provides a novel microcolumn that can be used in analytical techniques using capillary tubes, such as capillary electrophoresis and LC-MS. [Explanation of Symbols]

[0072] 10: Connection structure 20: Microcolumn 22: Capillary tube 23: Positioning means 30: Column tube 30a: Tubes at both ends of the column tube 30b: The central tube of the column tube 31: Porous member 31a: Porous material 31b: Capillary tube fragment 32: Internal column tube 33: Filler 35: Tubular member 40: Pressure control component 41: Set screw 42: O-ring

Claims

1. A connection structure, The aforementioned connection structure comprises a microcolumn and two capillary tubes. The microcolumn comprises a column tube consisting of a single tube, a packing material filled inside the column tube, and a porous member positioned inside the column tube in contact with both ends of the packing material. The column tube has openings at both ends for receiving the capillary tube, The column tube is made of a flexible material and has an inner diameter that is the same as or smaller than the outer diameter of the capillary tube, and is connected to the capillary tube in such a way that it is liquid-tight with the capillary tube by receiving the capillary tube at the opening. A connection structure.

2. The connecting structure according to claim 1, wherein the column tube is made of a material consisting of fluororesin, polyetheretherketone (PEEK), polyetherimide (PEI), polyphenylene sulfide (PPS), polypropylene, sulfone polymer, polyolefin, polyimide, polyaryletherketone, polyoxymethylene (POM), or a combination thereof.

3. The connecting structure according to claim 1, wherein the porous member includes a porous material.

4. The connecting structure according to claim 1, wherein the porous member includes a fibrous material.

5. The connection structure according to claim 1, wherein the porous member includes a capillary tube fragment.

6. The connecting structure according to claim 1, wherein the porous member comprises a porous material and a capillary tube fragment, and the porous material is arranged to be in contact with the filler.

7. The connecting structure according to any one of claims 1 to 6, wherein the filler is a particulate filler.

8. The connection structure according to any one of claims 1 to 6, wherein the column tube is made of a fluororesin material.

9. The connection structure according to any one of claims 1 to 6, further comprising positioning means for defining the position in which the capillary tube is received by the column tube.

10. The connection structure according to claim 9, wherein the positioning means defines a position such that the end of the capillary tube contacts the end of the pore member.

11. The connection structure according to any one of claims 1 to 6, wherein the outer surface of the capillary tube is coated.

12. A microcolumn according to any one of claims 1 to 6, The aforementioned capillary tube, Positioning means for defining the position in which the capillary tube is received by the microcolumn A microcolumn kit including this item.

13. The microcolumn kit according to claim 12, wherein the positioning means is a stopper attached to the capillary tube or an indicator provided on the capillary tube.

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