JP6221422B2 - Liquid chromatography column - Google Patents

Description

  The present invention relates to a liquid chromatography column.

  The liquid chromatograph is configured by injecting a sample into a mobile phase and connecting functional elements for separating and detecting each component by piping. As shown in FIG. 1, there are connected portions such as an eluent 1, a pump 2, a sample injection valve 3 having a sample loop 4, a column 5, and a detector 7. In FIG. 1, white circles are not frequently attached / detached, and black circles are places where the user frequently performs attachment / detachment work. Of these, column attachment / detachment is particularly frequent.

  There are various connection methods for the piping connection of each part, and the standards differ depending on the apparatus and the column supply company. For low pressure connection with a working pressure of about 0.1 MPa, a method is generally used in which the tip of the resin pipe is flanged or flared and connected with a dedicated nut (see FIGS. 2c and d). For high-pressure connection with a working pressure of several MPa or more, a method of connecting the metal pipe 14 with a push screw 13 and a ferrule 15 is generally used (see FIG. 2a).

  On the other hand, there is known a connecting member 16 made of resin, which eliminates the need for fixing the ferrule and integrates the ferrule and the push screw (see FIG. 2b). When this is used, a pipe is passed through the connector 16, inserted into the threaded portion of the column end connector 12, and lightly tightened by hand or with a tool. In this case, the pipe and the connection tool 16 can be detached from each other. Even when different columns are used, or even when the same column is used, it is necessary to check the protruding length of the pipe every time. There is a risk of misalignment and a liquid pool space (see FIG. 3). Moreover, since the material of the connection tool 16 is resin, it is necessary to pay attention to the breakage of the screw part due to overtightening.

  Column shapes having various shapes are commercially available depending on the purpose and intended use. As shown in FIG. 4, the most common column used in high-pressure liquid chromatography is a metal column tube 8 having ferrules 15 and nuts 10 at both ends thereof, and an end connector 12 incorporating a filter. It is a fixed format. This type of column can be freely selected by changing the pipe length. In addition, there is a type in which a screw is cut at the tip of the column tube 60 and directly fixed to the end connector 61 by screwing (see FIG. 5). In the former type, a column having a short length cannot be produced because of the physical external dimensions of the nut 10 at both ends and the end connector 12. In general, columns of 50 mm or less cannot be produced in this format. Unlike the former, the latter type does not use a nut, so that a shorter column can be produced. However, a column of 35 mm or less cannot be produced because of the physical outer dimensions of the end connector 61.

  For a shorter column, the column tube 58 is machined as shown in FIG. 6 to provide a cavity filled with gel at one end and fixed with the nut 10 / ferrule 15 and the end connector 12, while the other end is fixed. The thing of the type used as the piping connection part for connecting with the push screw 13 is also commercialized. With this type of column, a column of about 10 mm can be produced. In this type of column, the filter 11 on the downstream side needs to be inserted from the opening of the column tube and securely fixed at the deepest part before the gel is filled, so that a column with a thick inner diameter can be easily manufactured. However, there is a manufacturing problem that it is difficult to insert / fix the filter in a column having a small inner diameter (see FIG. 7).

  As a low-pressure liquid chromatography column, Patent Document 1 discloses a column in which a gel is packed in a cylindrical container having a luer-shaped tip (FIG. 8). It is composed of an injection tube-shaped column tube 80 having a male luer at the tip, an outer insertion tube 87 for holding the column tube 80, and inlet / outlet side fixing devices 86 and 84 for connecting inlet / outlet pipes. An outlet side fixing device 84 capable of filling a syringe 82 column tube 80 having an outlet side filter 81 with a gel 82 and inserting the gel 82 into an outer insertion tube 87 to fix the male luer shape at the tip of the column tube, an inlet side filter It fixes and uses with the entrance side fixing tool 86 provided with 83. FIG. The column having such a shape has an advantage that only the gel portion inserted into the extratubation tube can be easily replaced, but has a disadvantage that it is difficult to manufacture a capillary tube column.

JP 2005-114460 A

  An object of the present invention is to provide a column for liquid chromatography suitable for medium-low pressure high-performance liquid chromatography and capable of narrowing the inner diameter and shortening the length. Another object of the present invention is to provide the above-mentioned column for liquid chromatography that is easy for the manufacturer to manufacture. Still another object of the present invention is to provide the above-mentioned column for liquid chromatography that can be easily attached and detached for the user.

  As a result of intensive studies on the above problems, the present inventor has reached the present invention. That is, the present invention is as follows.

(1) a column body having a packing space for storing a packing material for liquid chromatography, an inlet through which liquid flows in, and an outlet through which liquid flows out;
An inlet-side connector for connecting to the inlet of the column body to connect with a liquid inflow connector;
An outlet-side connector for connecting to an outlet of the column body and connecting with a liquid outlet connector;
A column for liquid chromatography comprising
The packing space of the column main body extends from a predetermined site inside the column main body to the outlet, and the outside of the column main body has a male luer shape that tapers from the predetermined site to the outlet,
The liquid chromatography column, wherein the outlet-side connector has a female luer-shaped portion whose base end portion is closely fitted to the male luer shape of the column main body.

  (2) The liquid chromatography column according to claim 1, wherein the outlet-side connector is provided with a column filter in front of the female luer-shaped portion.

  (3) The column for liquid chromatography according to claim 1 or 2, wherein a column filter is interposed between the column main body and the inlet side connector.

(4) In any one of claims 1 to 3, the inlet-side connector is adapted to a liquid inflow connector having a means for reversibly pressing a member holding the liquid inflow pipe, and maintains a liquid tight state. Having liquid shape and locking means,
The outlet side connector is a column for liquid chromatography having a male luer-shaped portion adapted to the liquid outflow connector.

  The packing space in the column body extends to the outlet of the column body, and the outside of the column body has a male luer shape that tapers to the outlet, so the outside of the packing space can be connected to the outlet side connector. The length of the column body can be shortened.

  In addition, it is preferable that the column filter is mounted in front of the female luer portion of the outlet side connector so that the column filter can be easily mounted regardless of the inner diameter of the packing space of the column body. This configuration is advantageous in terms of manufacturing as compared with the configuration (FIG. 7) in which a filter is inserted in the deepest part of the packing space of the column body.

  Furthermore, the inlet side connector has a liquid contact shape having a means for reversibly pressing a member that holds the liquid inflow pipe and a liquid contact shape that holds a liquid tight state and a locking means, and a locking means. In addition, by adopting a male luer shape suitable for the liquid outflow connector as the outlet side connector, even an unfamiliar user can achieve a reliable column connection operation with a simple operation.

It is the figure which showed the flow-path system of the general liquid chromatograph apparatus. The circles in the figure indicate the joints, the white circles indicate the parts that are not frequently removed, and the black circles indicate the parts that are frequently removed. It is the figure which showed typically the connection method of a general column-piping. It is the figure which showed typically the disorder | damage | failure at the time of the connection of piping by a push screw and a ferrule integrated nut. It is the figure which showed the shape of the general column. It is the figure which showed the shape of the general screw-in type column. It is the figure which showed the shape of the general short column. It is the figure which showed typically insertion of the filter in a short column. It is the figure which showed the shape of the column of a general low pressure column. It is the figure which showed an example of the structure of the column for liquid chromatography of this invention. It is the figure which showed an example of the structure of the part which connects the column inlet_port | entrance and piping of this invention. It is the figure which showed the procedure of the gel packing to the column of this invention. It is the figure which showed typically the gel packing method to the column of this invention. It is the figure which showed typically the gel packing method to the column of this invention. It is the figure which showed the 1st connection form of the column and detector cell of this invention. It is the figure which showed the 2nd connection form of the column and detector cell of this invention. It is the figure which showed the structure of the column used in Example 1,2. 1 is a flow chart of a system used in Example 1. FIG. FIG. 3 is a diagram showing the results of a pressure resistance test in Example 1. It is a figure which shows the relationship between the elapsed time of Example 1, and a pressure. FIG. 6 is a flow chart of the system used in Example 2. 2 is a chromatogram showing separation in Example 2. FIG.

  A typical example of the structure of the column of the present invention is shown in FIG. In this embodiment (FIG. 9), an outlet-side connector 35 into which the outlet-side column filter 25 is inserted, a column body 36 filled with a packing material, an inlet-side column filter 30, and a pipe connection for introducing an eluent / sample. It is comprised with the inlet side connection tool 37 which is a part. One end (A1 end) of the outlet-side connector 35 is an eluent outlet and is connected to the detector, and the other end (A2 end) is connected to the column body 36. The A1 portion has a male luer shape 26 that is generally used in medical instruments, and may be provided with a screw lock mechanism by the cap portion 26A. The other end (A2 end) has a female luer-shaped portion 24 that fits closely with the male luer-shaped portion 27 (B1 end) of the column body 36. A column filter 25 that dams up the filler is built in front of the female luer-shaped portion 24. When the column main body 36 and the outlet-side connector 35 are closely fitted together, an adhesive may be applied and bonded between the female luer-shaped portion 24 and the male luer-shaped portion 27, or after the fitting. It can also be provided by heat fusion.

  The column main body 36 has a packing space 29 for storing the packing material. The end B1 is a joint with the outlet side connector 35 as described above, and the outer shape has a male luer shape 27. The B2 end is a joint portion with the inlet side connection tool 37, and may have a screw-in shape. The filling space is divided into a portion having a constant inner diameter and a portion extending in a tapered shape from the B1 end to the B2 end. The filler is preferably filled from a portion having a constant inner diameter to a portion expanding in a tapered shape. This means that the surface area of the front surface portion of the filler that contacts the sample flow is increased, which is effective in suppressing clogging of the microcolumn and extending the column life.

  The inlet side connection tool 37 plays a role of fixing the inlet side column filter 30 and connecting a pipe to the column. The inlet side column filter 30 may be inserted into the column main body after being filled with the packing material and then fastened and fixed by the inlet side connector 37, or may be fitted and fixed in advance to the C1 end of the inlet side connector 37. May be. An O-ring 32 is inserted into the C1 end, which is a joint with the column main body 36, and the filter 30 is fixed and liquid-tight by, for example, screwing.

  The C2 end that is a connection port of the inlet side connection tool 37 with the liquid inflow connection tool is compatible with a liquid inflow connection tool having means for reversibly pressing a member that holds the liquid inflow pipe and maintains a liquid tight state. It preferably has a liquid shape and locking means. FIG. 10 shows an example of a connection method between the C2 end (column inlet 33) of the inlet side connector and the external piping. This connection method incorporates a mechanism of a connector for connecting a fine flow path and a liquid feeding tube disclosed in Japanese Patent Application Laid-Open No. 2010-137143. A liquid inflow connector 78 for connecting the external pipe to the column inlet 33 of the inlet side connector is a member for holding the liquid inflow pipe, one end is flat and the other end is a tapered ferrule 64, and one end is tapered. A ferrule 65 having a shape, a sleeve 66, and a spring 67, which is a means for reversibly pressing the member, and an outer tube 68 for storing them are configured. A fixing projection (pin) 70 is provided at the lower portion of the outer insertion tube 68. When the pin 70 is fitted in the notch 71 provided in the column inlet 33 and pressed against the flow path end of the column inlet 33 on the receiving side of the ferrule 64, the taper surface of the ferrule 64 is The taper surface of the ferrule 65 is pressed, and internal stress is applied to the ferrule 64, and the liquid tightness between the pipe 69 passing through the center and the ferrule 64, and the liquid tightness between the flat surface of the ferrule 64 and the column inlet 33 on the receiving side. Secured. When the outer tube 68 is rotated by a certain angle in the pushed state, the pin 70 of the outer tube is caught on the receiving side, and the spring is held in a pressed state, so that the liquid tightness is maintained in both the pipe and the column. By further rotating the extratubation by a certain angle, the hook is released and the extratubation can be easily removed.

  The column outlet 34 (A1 end of the outlet side connector) is connected to a detector, and two types of methods are possible, for example. The first method is a method of connecting a commonly used PEEK (polyetheretherketone resin) pipe 49 to the union 43 (see FIG. 14), and the second method is to use the pipe 49. First, it is a method of connecting directly to the joint 44, for example, screwed to the detector cell (see FIG. 15). Alternatively, although not shown, the cell block itself may be processed into the same shape as the receiving portion of the joint 44. Regardless of the first method or the second method, the outlet side connector preferably has a male luer shape adapted to the liquid outflow connector, and the connecting joint 44 has a shape adapted thereto.

  The first method has a feature that it is easy to control the temperature of only the column using the column oven 72 or the like because the detector cell and the column can be arranged at some distance from each other. Since the second method does not use piping between the column and the cell, the void capacity can be reduced and the column performance can be sufficiently exhibited. In this case, in order to control the temperature of the column, it is necessary to prepare a temperature control mechanism 73 including the detector 7.

  Next, a method for packing the filler gel into the column of the present invention will be described. The filling procedure is shown in FIG. First, the column outlet side connection tool 35 and the column main body 36 are closely fitted. Next, a certain amount of gel is packed into the column body 36. Next, the inlet side column filter 30 is inserted, and the inlet side connector 37 is screwed to fix the filter. The filling method of the filler gel may be suction filling or pressure filling according to the characteristics of the gel. In the case of a gel that can be easily packed, after the outlet side connector and the column body are closely fitted, the A1 end of the outlet side connector is connected to a vacuum device (such as an aspirator) via the vacuum adapter 39, A gel in a slurry state is dropped from the base end of the column body 36 and filled (see FIG. 13). For gels that are difficult to fill, a pressure filling device 38 called a packer can be used. After tightly fitting the outlet side connector and the column main body, the packer 38 is connected, and the filling solution can be fed from one end thereof to fill the gel (see FIG. 12).

[Example 1]
The pressure resistance performance of the column of the present invention was verified as follows. That is, the experiment was performed using a liquid chromatography column having the configuration of FIG. Specifically, TSKgel BORATE-5PW (manufactured by Tosoh Corporation) was sucked and filled into a column having an inner diameter of 1.5 mm with aspirator so as to have a packed portion length of 10 mm (see FIG. 16a). FIG. 17 shows a flow chart of the system used for the verification. An opening / closing valve 55 is arranged at the outlet of the column 75 so that nitrogen gas can be pumped to the column 75 at a constant pressure by the pressure regulator 52, and the column 75 is immersed in the water tank 57 so that the degree of leakage can be visually observed. . In addition, a pass-type pressure gauge 54 was disposed immediately before the column so that the actual column load pressure could be monitored.

  FIG. 18a is a diagram showing a change in pressure in front of the column (a signal from the pressure gauge 54) measured for a certain time (about 100 minutes) while changing the nitrogen gas pressure with the open / close valve 55 closed. FIG. 18 b is a diagram showing the relationship of the pressure gauge 54 arranged in front of the column with respect to the indicated pressure of the pressure regulator 52 (indicated value of the pressure gauge 53). From FIG. 18a, no pressure drop due to gas leakage was observed in the range from 25 kPa to 225 kPa, and gas leakage from all parts of the column could not be confirmed by visual observation. Further, the indicated pressure of the pressure regulator 52 and the value of the pressure gauge 54 arranged in front of the column are also in a linear relationship passing through the origin, suggesting that there is no leakage.

  Next, the pressure when the open / close valve 55 arranged downstream of the column is opened, the nitrogen gas pressure is adjusted to 225 kPa and nitrogen gas is pumped to the column for a while, and then the open / close valve 55 and the nitrogen gas pump is closed. A total of 54 changes were recorded (FIG. 19). While the pressure when the nitrogen gas pumping is in the closed state is 214.96 kPa, the column pressure after 1 hour can be maintained at 202.9 kPa and remains at a decrease rate of 5%. The above shows that the column of the present invention has sufficient pressure resistance in the range up to 225 kPa.

[Example 2]
In order to verify the performance of the column of the present invention, an example is shown in which a standard sample is measured in the reverse phase mode. That is, the experiment was performed using a liquid chromatography column having the configuration of FIG. Specifically, TSKgel ODS-120A (5 μm) (manufactured by Tosoh Co., Ltd.) was sucked and used in a column having an inner diameter of 1.0 mm and a length of 15 mm shown in FIG. FIG. 20 shows a flow chart of the liquid chromatograph. The column 75 was installed in a column oven 72 at a constant temperature of 40 ° C. and connected to the cell portion 50 of the UV-visible detector through a union 43. A switching valve 90 is arranged on the outlet side of the pump 2 for feeding the eluent A (62), and the eluent B63 is introduced by a loop so that a step gradient is possible. The pump 2 uses a metering pump FMM20 manufactured by KNF, with a flow rate of 40 μL per minute, and the detector 7 including the cell unit 50, the light source 76 and the photosensor 77 is a UV-visible detector UV-8020 manufactured by Tosoh Corporation at a wavelength of 254 nm. Used in. The sample injection valve 3 was used by connecting a sample metering loop 4 (20 μL) to an automatic six-way switching valve manufactured by FLOM. A 5% acetonitrile aqueous solution was used as the eluent A (62), a 30% acetonitrile aqueous solution was used as the eluent B (63), and an aqueous p-hydroxybenzoate solution (0.01%) was used as the sample. After elution liquid A (62) is sent for a certain period of time and the column is initialized, the sample is injected for 0.05 minutes (sample injection volume is equivalent to 2 μL). After 0.07 minutes, the switching valve 90 is operated to operate the eluent liquid. Switched to B (63). Under these conditions, the liquid feeding pressure was about 150 kPa.

  Table 1 shows reproducibility (n = 10), and FIG. 21 shows the eluent switching pattern and chromatogram. Good reproducibility was obtained with a coefficient of variation Cv of elution time (retention time) of 1.12%, a peak height of 1.36%, and a peak area of 1.69%. It clearly states that there is no problem.

DESCRIPTION OF SYMBOLS 1 Eluent 2 Pump 3 Sample injection valve 4 Sample loop 5 Column 7 Detector 8 Column pipe 9 Resin piping 10 Nut 11 Filter 12 End connection tool 13 Set screw 14 Metal piping 15 Ferrule 16 Integrated resin connection tool 17 Flange nut 18 Flange 19 Resin piping 20 Flare 21 Flare nut 24. Female luer shape part 25. Column filter (outlet side)
26, 27 Male luer-shaped part 26A Cap part 28. Joint (female)
29. Filler filling space 30. Column filter (inlet side)
31. Joint (male)
32. O-ring 33. Column inlet 34. Column outlet 35. Outlet side connector 36. Column body 37. Inlet side connector 38. Packer 39. Vacuum adapter 40. Cell inlet 41. Detection optical path 42. Lens 43 Union 44 Joint 45, 46, 47 Connector 48 Cell outlet 49 Pipe 50 Cell 52 Pressure regulator 53 Pressure gauge (Built-in pressure regulator)
54 Pressure gauge 55 Open / close valve 57 Water tank 58, 60 Column pipe 61 End connector 62 Eluent A
63 Eluent B
64, 65 Ferrule 66 Sleeve 67 Spring 68 Extrapolation tube 69 Piping 70 Pin 71 Notch 72 Column oven 73 Temperature control mechanism 75 Column 76 Light source 77 Photo sensor 78 Liquid inflow connector (simple connector)
80 column tube 81 outlet side filter 82. Gel 83 Inlet side filter 84 Outlet side fixing device 85 O-ring 86 Inlet side fixing device 87 Extrapolation tube 88 Inlet side piping 89 Outlet side piping 90 Switching valve 91 Eluent B loop

Claims (4)

A column body having a packing space for storing a packing material for liquid chromatography, an inlet through which liquid flows, and an outlet through which liquid flows out;
An inlet-side connector for connecting to the inlet of the column body to connect with a liquid inflow connector;
An outlet-side connector for connecting to an outlet of the column body and connecting with a liquid outlet connector;
A column for liquid chromatography comprising
The packing space of the column main body extends from a predetermined site inside the column main body to the outlet, and the outside of the column main body tapers from the predetermined site to the outlet, and has a male luer shape without a screw , The column for liquid chromatography, wherein the outlet-side connector has a female luer-shaped portion without a screw , the base end portion of which is closely fitted to the male luer shape of the column main body. 2. The liquid chromatography column according to claim 1, wherein the outlet-side connector has a column filter mounted in front of the female luer-shaped portion. 3. The liquid chromatography column according to claim 1, wherein a column filter is interposed between the column main body and the inlet side connector. 4. The liquid contact shape according to claim 1, wherein the inlet side connector is adapted to a liquid inflow connector having means for reversibly pressing a member holding the liquid inflow pipe and maintains a liquid tight state. Having locking means,
The outlet side connector is a column for liquid chromatography having a male luer-shaped portion adapted to the liquid outflow connector.

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