JP5283263B2 - High-speed counter-current chromatograph - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a high-speed countercurrent chromatographic apparatus having higher separation efficiency. <P>SOLUTION: The high-speed countercurrent chromatographic apparatus includes a coiled column. The coiled column rotates around an axis of rotation while revolving around an axis of revolution. The coiled column is disposed so that the axis of rotation is twisted against the axis of revolution. The coiled column includes a hub, flow tube, and the like. A coiled part of the flow tube coiled around a drum part of the hub uses a locular tube 2. The inside of the locular tube 2 is formed by a plurality of compartments 4 connected in series. The locular tube 2 is made by forming a narrowed part 6 at constant intervals d with, for example, forceps. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to a high-speed countercurrent chromatograph in which a tube in which a plurality of compartments are formed in communication is used as a sample separation column.

In order to efficiently separate the target substance from the sample solution, there is a high-speed counter-current chromatograph that separates the target substance from the sample solution by using the difference in partition coefficient between the solvent as the stationary phase and the solvent as the mobile phase. It is developing. The high-speed counter-current chromatograph apparatus has a coiled column in which a tube into which a solvent as a stationary phase and a solvent as a mobile phase are introduced is spirally wound, and the coiled column revolves around a revolution axis. At the same time, it rotates around the rotation axis. The high-speed counter-current chromatograph apparatus is a type in which the rotation axis of the coiled column described in Patent Document 1 is located at a twisted position with respect to the revolution axis (cross-axis type high-speed counter-current chromatography apparatus), There is a type in which the axis is substantially parallel to the revolution axis. Using these high-speed counter-current chromatographs, a target substance such as a protein is introduced into one of a solvent introduced as a stationary phase and a solvent introduced as a mobile phase from a sample solution introduced into a coiled column. Could be eluted.
JP 2006-064533 A

  However, the conventional high-speed counter-current chromatograph apparatus may take a long time to separate the target substance from the sample solution, and the high-speed counter-current chromatograph apparatus that can separate the target substance in a shorter time Was demanded.

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a high-speed counter-current chromatograph apparatus that can perform separation of a target substance from a sample solution in a shorter period of time.

As a result of intensive studies to solve the above problems, the present inventor has separated a tube for forming a coiled column by using a tube having a tube in which a plurality of compartments communicated in series. It has been found that the efficiency is improved, and the present invention has been completed.
That is, the present invention is a high-speed counter-current chromatograph apparatus provided with a coiled column in which tubes are arranged in a coil shape, wherein the tubes are formed between a plurality of compartments and compartments. The communication openings having a cross section smaller than the cross section are alternately formed in the pipe along the length direction.
An inner diameter of the communication opening is made smaller than an inner diameter of the compartment, and a cross section of the communication opening is made smaller than a section of the compartment.

  In the high-speed countercurrent chromatograph apparatus of the present invention, it is preferable that the communication openings are formed at regular intervals in the length direction.

  In the high-speed counter-current chromatograph apparatus of the present invention, it is preferable to arrange the coiled column so that the rotation axis is located at a twisted position with respect to the revolution axis.

  In the high-speed counter-current chromatograph of the present invention, the coiled column is formed by alternately forming a plurality of compartments having a large cross section and a communication opening having a small cross section formed between the compartments along the length direction in the tube. It is preferable that the tube is disposed in a coil shape around the rotation axis.

  Furthermore, the present invention is a method for separating a target substance, comprising a step of processing a sample using the high-speed countercurrent chromatograph apparatus.

  By implementing the present invention, the number of theoretical plates of the coiled column can be increased. As a result, it is possible to expect a reduction in the period required for separation of the target substance from the sample solution.

  The high-speed countercurrent chromatograph apparatus of the present invention will be described below. In the present invention, a coiled column used in a high-speed counter-current chromatograph apparatus is produced using a “local tube”. This "Lucral tube" has a plurality of compartments and communication openings formed between the compartments, each having a cross section smaller than the cross section of the compartments, formed alternately in the tube along the length direction. It is. The constituent material, the inner diameter, the outer diameter, the interval between the narrowed portions, the cross-sectional shape, and the like are not limited to the following, and may be appropriately changed. For example, as shown in FIG. 1A, the tubular tube 2 has a tube wall 3, a compartment 4, a narrowed portion 6, and a communication opening 8. A plurality of compartments 4 are formed along the length direction X. The communication openings 8 are formed between the compartments 4, whereby the compartments 4 and the communication openings 8 are alternately formed in the pipe 2 a along the length direction X.

  In the tubular tube 2, for example, the outer diameter of the tube wall 3 is 2.5 mm, the inner diameter is 1.5 mm, and the communication opening 8 is 1.0 mm. The constituent material of the tubular tube 2 is preferably made of fluororesin from the viewpoint of durability, corrosion resistance, chemical resistance, etc., but may be changed as appropriate. Moreover, the space | interval d between each constriction part 6 is 10-50 mm, for example, Preferably it is 20-40 mm. The compartments 4 are regularly formed in the tube 2a. In the narrowed portion 6, the tube wall 3 protrudes inward, whereby a communication opening 8 that partitions the compartment 4 communication is formed in the tube 2 a. The inner diameter of the communication opening 8 is smaller than the inner diameter of the compartment 4, so that the cross-sectional area A 2 of the communication opening 8 is smaller than the cross-sectional area A 1 of the compartment 4. As a result, the communication opening 8 can function as a partition that partitions the compartments 4. There is no restriction | limiting in particular in the internal diameter of the communication opening 8, It is good to adjust suitably. By providing the compartments 4 and the communication openings 8 alternately in the tube 2a, it is considered that the tubular tube 2 can easily retain the sample solution in the compartment 4 compared to a normal tube having a constant outer diameter and inner diameter. It is done. Moreover, as shown in FIG.1 (b), the internal diameter of the compartment 4 can be made smaller than the thing of Fig.1 (a), or the space | interval of a constriction part can be made shorter than FIG.1 (a). For example, the local tube 10 having an outer diameter of 2.0 mm, an inner diameter of 1.0 mm, and an interval d between the constricted portions 6 of 20 mm may be manufactured.

  Examples of the method for producing the tubular tube 2 include a method in which a normal tube having a constant outer diameter and an inner diameter is sandwiched with forceps at regular intervals to form a narrowed portion 6 in which the inner tube 2a is narrowed. By clamping the normal tube with forceps at regular intervals and then releasing the clamp, the narrowed portion 6 is repeatedly formed at regular intervals, and the communication opening 8 is formed in the tube 2a. By forming the communication openings 8 at regular intervals, the compartment 4 can be formed between the communication openings 8.

  In the above description, the normal tube is clamped with forceps at regular intervals to produce the tubular tube 2 having a tube in which a plurality of compartments 4 are arranged in series. However, the method of producing the tubular tube is not limited to this. . For example, when it is desired to mass-produce a tubular tube more efficiently, the tubular tube can be produced by using an extruder having a nozzle capable of changing the wall thickness of the tube. As shown in FIG. 2, the extrusion molding machine 14 includes a nozzle 16 that can change the wall thickness without changing the outer diameter, and the tubular tube 23 in which the wall portion 18 is formed at regular intervals by the nozzle 16. Is extruded. Thin portions 19 are formed between the thick portions 18, whereby the compartments 21 and the communication openings 22 are alternately formed in the tube 23 a. By controlling the thickness of the extruded cylindrical tube 23 in this way, the thick portion 18 and the thin portion 19 can be formed in the tube, and the thick portion 18 partitions each compartment 21. Functions as a partition. By using the thus-produced molecular tube 23, the user can save time and effort to form a constricted portion in the normal tube with forceps, and more easily form a coiled column. In the above description, only one communication opening is formed between the compartments. However, the number of communication openings between the compartments may be plural, and the shape is also arbitrary. However, it is necessary to make the cross-sectional area of the compartments larger than the total area of all the communication openings formed between the adjacent compartments so that the communication openings function as partitions for partitioning the compartments.

  Moreover, in said embodiment, although the narrow part 6 was provided for every fixed interval and the several compartment 4 was formed in the pipe | tube, the formation aspect of a compartment is not restricted to this. For example, a plurality of compartments are formed in the tube by periodically changing the formation interval of the stenosis portion in order of 40 mm, 30 mm, and 20 mm, or a plurality of compartments having different capacities by randomly changing the formation interval of the stenosis portion. You may form in a tube.

  Next, a description will be given of a coiled column in which a tubular tube is disposed in a coil shape. In the high-speed countercurrent chromatograph described later, a plurality of four coiled columns are mounted. These coiled columns have the same structure. As shown in FIGS. 3A and 3B, the coiled column 31 includes a hub 40 formed in a reel shape, and the cylindrical tube 2 is wound around a body portion 41 (see FIG. 8) of the hub 40. ing. A flange 42 is provided at one end and the other end of the hub 40, and a horizontal shaft (spinning shaft) 50 is formed on each flange 42. The horizontal shaft 50 is pivotally supported by the holding frame 35 (see FIG. 7), and the rotation axis S 1 of the coiled column 31 passes through the center of the horizontal shaft 50.

  Between one flange 42 and the other flange 42, for example, a columnar body 41 having a diameter of 30 mm and a distance of 50 mm is formed. A coil-shaped portion 72 is formed on the outer periphery of the body portion 41 in which the tubular tube 2 is wound in a coil shape from one flange 42 toward the other flange 42. The coil-shaped portion 72 is formed, for example, in a five-layer structure, and a multi-layered coil-shaped column 31 provided with such a multilayer-structured coil-shaped portion 72 is disposed around a revolution axis described later.

  In the above description, the multi-layered coiled column 31 in which the tubular tube 2 is wound around the outer periphery of the body part 41 of the hub 40 is illustrated, but the form of the coiled column is not limited to this and may be changed as appropriate. . For example, as shown in FIG. 4, an eccentric type column in which a coiled column in which a tubular tube is wound in a coil shape is arranged around a predetermined point so that through-shafts passing through the coil are substantially parallel to each other. May be produced. As shown in FIG. 5, a toroidal column in which a plurality of coiled columns formed in a ring shape are overlapped may be manufactured. Furthermore, as shown in FIG. 6, a spiral column in which a plurality of spiral columns wound in multiple layers from the inside to the outside may be stacked.

  7 and 8 show an embodiment of the column unit 60 including four coiled columns 31 to 34. FIG. As shown in FIGS. 7 and 8, the column unit 60 includes first to fourth coiled columns 31 to 34, a holding frame 35 for rotatably holding the coiled columns 31 to 34, and the like. The revolution axis R passes through the center of the vertical shaft 62, and the first to fourth coiled columns 31 to 34 are sequentially arranged around the vertical shaft 62. Each of the coiled columns 31 to 34 is arranged such that each rotation axis S1 to S4 is located at a twisted position with respect to the revolution axis R, and a plane including the rotation axis S1 to S4 is substantially the same as the revolution axis R. Intersects vertically.

  The vertical shaft 62 is hollow and both ends thereof are open, and a flow tube 65 integrally formed with the molecular tube 2 can be inserted into the hollow portion 62 a of the vertical shaft 62. The vertical shaft 62 has a first perforation 67 for leading the flow tube 65 in the hollow portion 62a to the outside of the vertical shaft, and a second perforation 68 for introducing the flow tube from the outside of the vertical shaft into the hollow portion 62a. Is formed.

  As shown in FIG. 8, there are a liquid feeding section 70 that is connected to a pump (not shown) and feeds a two-phase solvent and a sample solution, and a flow path that is wound in a coil shape to flow the two-phase solvent and the sample solution. The four coil-shaped parts 72 to be formed, the relay part 74 connecting the coil-shaped parts 72 of the first to fourth coiled columns 31 to 34, and the liquid from the coil-shaped part 72 of the fourth coiled column 34 The drainage part 76 for discharging is integrally formed by the flow tube 65. As the flow tube 65, for example, the coil-shaped portion 72 can be formed using a Teflon (registered trademark) flow tube having an inner diameter of 1.0 mm.

  The liquid feeding part 70 of the flow tube 65 is guided from one end of the vertical shaft 62 to the hollow part 62 a and passed through the hollow part 62 a to the first perforation 67. The flow tube 65 led out from the first perforations 67 is formed into a coil shape to form the coil shape portion 72 of the first to fourth coiled columns 31 to 34. Each coil shape part 72 is connected by the relay part 74, and, thereby, each coil shape part 72 is connected in series. The drainage portion 76 of the flow tube 65 extending from the fourth coiled column 34 is introduced into the hollow portion 62a through the second perforation 68, and the bottom of the apparatus main body 82 (see FIG. 9) from the opening at the other end of the vertical shaft 62. Derived towards 88. The drainage part 76 led out from the other end of the vertical shaft 62 is guided to the outside of the high-speed countercurrent chromatograph 80 via the bottom part 88 and connected to, for example, a detector (not shown).

  The coiled columns 31 to 34 are connected in series in the order of the first coiled column 31, the second coiled column 32, the third coiled column 33, and the fourth coiled column 34 from the liquid feeding side. The winding direction of the flow tube 65 on the outer periphery of the body portion 41 of each hub 40 is not limited to the following mode, and may be changed as appropriate. For example, the first coiled column 31 is wound clockwise from the supply side to the discharge side, similarly, the second coiled column 32 is counterclockwise wound, the third coiled column 33 is wound clockwise, The 4-coiled column 34 was left-handed. Each coil-shaped part 72 is formed in, for example, a five-layer structure, and the multi-layered first to fourth coiled columns 31 to 34 having such a multilayered coil-shaped part 72 are arranged around the revolution axis R. They are arranged symmetrically. The coil-shaped portions 72 of the coiled columns 31 to 34 have the same number of turns (the number of pitches), and the weights of the coiled columns 31 to 34 are adjusted to be the same in advance when the column unit 60 is assembled. Has been.

  For example, when the column unit 60 rotates counterclockwise, the first to fourth coiled columns 31 to 34 rotate in the clockwise direction from the supply side to the discharge side. . Thereby, each coiled column 31-34 can rotate, revolving, without the flow tube 65 being twisted. The rotation direction of the column unit 60 and the first to fourth coiled columns 31 to 34 is not limited to the above, but the column unit 60 is rotated clockwise and the first to fourth coiled columns 31 to 34 are supplied to the supply side. May be rotated counterclockwise from the outlet toward the discharge side.

  As shown in FIG. 9, the high-speed counter-current chromatograph apparatus 80 includes an apparatus main body 82 and a column unit 60, and the upper part of the apparatus main body 82 is covered with a lid 85. The apparatus main body 82 has an accommodating space for accommodating the column unit 60 therein, and the column unit 60 is accommodated in this space. The column unit 60 has a vertical shaft 62 that passes through the center of gravity, and upper and lower ends of the vertical shaft 62 are pivotally supported by the apparatus main body 82. Thereby, the vertical shaft 62 functions as a revolution axis, and the column unit 60 can be rotated around the revolution axis R passing through the center of the vertical shaft 62.

  The apparatus main body 82 includes a side wall 86 and a bottom portion 88 connected to the side wall 86, and the side wall 86 and the bottom portion 88 form a storage space having a substantially circular cross section. A pillar 90 is provided in the accommodation space, and the pillar 90 is fixed to the bottom portion 88. The pillar 90 supports a frame 95 for stably holding the column unit 60 rotatably.

  The bottom 88 includes a power supply unit 91, and the power supply unit 91 includes a motor and a motor controller that controls driving of the motor. With such a configuration, the power supply unit 91 can generate power for rotating the column unit 60 around the revolution axis R and power for rotating each coiled column described later. Although not shown, an operation unit for adjusting the rotation speed of the motor is provided outside the apparatus main body 82, and the user operates the operation unit to revolve the column unit 60 via the motor controller. Speed etc. can be adjusted.

  The lid 85 is attached to the upper part of the apparatus main body 82 so as to be freely opened and closed. The lid 85 includes an introduction hole 93 for introducing the flow tube 65 into the high-speed counter-current chromatograph apparatus 80, an air hole for sending air into the accommodation space of the apparatus main body 82, and the like, and is accommodated in the accommodation space. The column unit 60 is configured to be suitable for driving.

  Next, the operation of the present invention will be described. In the flow tube 65 of the high-speed counter-current chromatograph 80, the liquid feeding unit 70 is connected to a pump, and the drainage unit 76 is connected to a detector. One of the upper and lower layers constituting the two-phase solvent such as an aqueous system is filled in the flow tube 65 as a stationary phase.

  After filling with the stationary phase solvent, the revolution and rotation of the first to fourth coiled columns 31 to 34 are started to fix the stationary phase solvent in the coil-shaped portion 72. After rotating each of the coiled columns 31 to 34, a sample solution containing a biological substance (target substance) or the like is sent into the coil shape part 72 of the first to fourth coiled columns 31 to 34 together with the mobile phase solvent. Liquid. The coil-shaped part 72 is produced by the cylindrical tube 2, and the theoretical plate number of the 1st-4th coiled columns 31-34 is raised by this. In addition, although the mechanism by which the number of theoretical plates is increased by the tubular tube has not been completely elucidated, it is related to the use of the tubular tube 2 to increase the agitation of the liquid in the coil-shaped portion 72. I think that the. The rotation speed, rotation direction, rotation direction, and rotation time of rotation and revolution can be set arbitrarily. (See, for example, Japanese Patent Application Laid-Open No. 2006-066453 etc.) For example, biological substances in the sample solution introduced into the coil-shaped portion 72 are distributed, separated, and moved between the stationary phase and the mobile phase. Elute with phase solvent. The mobile phase discharged through the drainage unit 76 is sent to the detector, and the biological substance is detected. Examples of the biological substance include proteins, amino acids, nucleic acids such as DNA and RNA, enzymes and the like, but are not particularly limited.

  In the above embodiment, the high-speed counter-current chromatograph apparatus 80 in which the respective coiled columns 31 to 34 are arranged so that the rotation axis is located at a twisted position with respect to the revolution axis is exemplified. Not exclusively. For example, as shown in FIG. 10, it may be used in a high-speed countercurrent chromatograph apparatus 100 in which a coiled column is arranged so that each rotation axis S is positioned in parallel to the revolution axis R.

  Separation and purification of biological materials from sample solutions using a high-speed counter-current chromatograph (see FIGS. 7 to 9) equipped with a coiled column (see FIG. 3) produced using the cylindrical tube described in FIG. An experiment was conducted.

Experiment 1
Production of a tubular tube After clamping a Teflon (registered trademark) tube with an outer diameter of 2.5 mm and an inner diameter of 1.5 mm with forceps at intervals of 40 mm, the clamp is released, and a plurality of compartments are arranged in series in the tube. Arranged local tubes were made. Similarly, after clamping a tube made of Teflon (registered trademark) having an outer diameter of 2.5 mm and an inner diameter of 1.5 mm with forceps at intervals of 20 mm, the clamp is released, and a plurality of compartments are arranged in series in the tube. A tubular tube was prepared.

Production of Coiled Column A produced multi-layered five-layer coiled column was produced by rotating the produced tubular tube around the outer periphery of the body 41 of the hub 40 five times. The produced coiled column was attached to the apparatus main body 82 to produce a high-speed countercurrent chromatograph apparatus 80.

Experimental conditions Sample: cytochrome C (2 mg), myoglobin (8 mg), lysozyme (10 mg) as standard protein
Aqueous two-phase solvent: 12.5% (w / w) polyethylene glycol 1000-12.5% (w / w) K ₂ HPO ₄ aqueous solution Mobile phase: Lower layer Rotation (revolution) Speed: 1000 rpm,
Revolution direction: Counterclockwise Flow rate: 0.4ml / min Detection: UV280nm

Results The chromatogram obtained by the experiment is shown in FIG. 11, and the obtained numerical data is shown in Table 1 below.

Experiment 2
Preparation of a tubular tube After clamping a Teflon (registered trademark) tube having an outer diameter of 2.0 mm and an inner diameter of 1.0 mm with forceps at intervals of 40 mm, the clamp is released, and a plurality of compartments are arranged in series in the tube. Arranged local tubes were made. Similarly, a tube made of Teflon (registered trademark) having an outer diameter of 2.0 mm and an inner diameter of 1.0 mm is clamped with forceps at intervals of 20 mm, and then the clamp is released, so that a plurality of compartments are arranged in series in the tube. Arranged local tubes were made.

Production of Coiled Column The produced circular tube was made to circulate around the outer periphery of the body part 41 of the hub 40 five times to produce a 5-layer coiled column. The produced coiled column was attached to the apparatus main body 82, and the high-speed counter-current chromatograph apparatus 80 was created.

Experimental conditions Sample: Myoglobin (8 mg), lysozyme (10 mg) as standard protein
Aqueous two-phase solvent: 12.5% (w / w) polyethylene glycol 1000-12.5% (w / w) K ₂ HPO ₄ aqueous solution Mobile phase: upper layer Rotation (revolution) speed: 1000 rpm,
Rotation direction: clockwise Flow rate: 0.4 ml / min Detection: UV 280 nm

Results The chromatogram obtained by the experiment is shown in FIG. 12, and the obtained numerical data is shown in Table 2 below.

CONCLUSION Regardless of whether the upper layer or the lower layer is a mobile phase, the number of theoretical plates in the case of separation with a cylindrical tube is lower than that of separation with a normal tube having a constant outer diameter and inner diameter. Can be high. Thereby, compared with a normal tube, achievement of a higher degree of separation can be expected for a tubular tube.

It is a fragmentary sectional view of the cylindrical tube which forms a coil shape part. It is explanatory drawing explaining the principal part of the mass production method of a tubular tube. It is a fragmentary perspective view of the multilayer type | formula coiled column which wound the cylindrical tube around the hub outer periphery in the shape of a coil. FIG. 4 is a schematic perspective view of an eccentric column in which a plurality of coil-shaped portions each having a cylindrical tube wound in a coil shape are arranged around a predetermined point so that through axes passing through the coil are substantially parallel to each other. . It is a schematic perspective view of the toroidal type column which piled up the plurality of coiled columns formed in the shape of a ring. FIG. 3 is a schematic perspective view of a spiral column in which a plurality of spiral columns wound in multiple layers from the inside to the outside are overlaid. It is explanatory drawing which observed the column unit from the direction perpendicular | vertical to the plane containing rotation axis | shaft S1-S4. It is explanatory drawing of the column unit which showed the connection aspect of the 1st-4th coiled column roughly. It is the schematic of a high-speed countercurrent chromatograph apparatus. It is the schematic of the high-speed countercurrent chromatograph apparatus which has arrange | positioned the coiled column so that the autorotation axis | shaft of the coiled column produced using the cylindrical tube may be parallel to a revolution axis | shaft. 6 is a chart showing the results of a separation experiment conducted under the conditions of Experiment 1. 6 is a chart showing the results of a separation experiment conducted under the conditions of Experiment 2.

Explanation of symbols

2 Tubular tube 4 Compartment 6 Constriction 8 Communication opening 10 Tubular tube 31 First coiled column 32 Second coiled column 33 Third coiled column 34 Fourth coiled column 40 Hub 41 Body 42 Flange 50 Horizontal Shaft (Rotating shaft)
60 Column unit 62 Vertical shaft (revolving shaft)
62a Hollow part 65 Flow tube 70 Liquid feeding part 72 Coil-shaped part 74 Relay part 76 Drainage part 80 High-speed countercurrent chromatograph R Revolution axis S Rotation axis

Claims (9)

A high-speed counter-current chromatograph apparatus having a coiled column in which tubes are arranged in a coil shape,
The tube has a plurality of compartments and communication openings formed between the compartments and having a cross section smaller than the cross section of the compartments alternately formed in the pipe along the length direction. Flow chromatograph.   The high-speed countercurrent chromatograph according to claim 1, wherein the communication openings are formed at regular intervals in the length direction.   The high-speed countercurrent chromatograph device according to claim 1 or 2, wherein an inner diameter of the communication opening is smaller than an inner diameter of the compartment. The coiled column rotates while revolving,
The high-speed countercurrent chromatograph according to any one of claims 1 to 3, wherein the coiled column is arranged such that the rotation axis is located at a twisted position with respect to the revolution axis.   The coiled column includes a plurality of compartments and tubes formed between the compartments, each having a communication opening having a cross section smaller than the cross section of the compartments, formed alternately in the length direction. The high-speed countercurrent chromatograph device according to any one of claims 1 to 4, wherein the high-speed countercurrent chromatograph device is disposed in a coil shape around a rotation axis. A column used in a high-speed counter-current chromatograph apparatus, wherein a plurality of compartments and a communication opening formed between the compartments and having a cross section smaller than the cross section of the compartment are alternately arranged along the length direction. A column, characterized in that the tubes formed in the above are arranged in a coil shape.   A method for separating a target substance in a sample solution, comprising a step of treating the sample solution using the high-speed counter-current chromatograph device according to claim 1. The high-speed countercurrent chromatograph apparatus according to any one of claims 1 to 5, wherein a constriction is formed on an outer peripheral surface of the tube. The column according to claim 6, wherein a narrowed portion is formed on an outer peripheral surface of the tube.