Column for chromatography

Abstract

To provide a chromatography analysis which can acquire a symmetric peak regardless of the time of elution on a chromatogram and can increase the number of theoretical stages.SOLUTION: In a column for chromatography, there is formed a flow rate control mechanism formed of a joint having one of a flow rate control plate and a flow rate control trench on the exit side of the column.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a chromatography column, more specifically to the configuration of an inlet / outlet portion within a chromatography column, and more particularly to the configuration of an inlet / outlet portion within a column used for liquid chromatography, gas chromatography, or supercritical chromatography. [Background technology]

[0002] Chromatography is an analytical method in which a mixture of samples is injected into a chromatograph, mixed with a mobile phase of gas, liquid, or supercritical fluid, and then passed through a column containing a stationary phase such as a packing material, where the components are separated and detected through interactions with the stationary phase. Chromatography is widely used in the fields of chemical industry, food, environment, medicine, etc. to determine the amount of specific components contained in a sample.

[0003] In order to reproducibly induce interactions between the sample components and the stationary phase, which is composed of a packing material packed in a chromatography column, it is necessary to uniformly introduce the sample mixture into the stationary phase in the column.

[0004] In liquid chromatography, the injected sample solution to be analyzed is introduced into the liquid chromatography column through a piping after merging with the mobile phase delivery line. If the sample is highly diffused in the piping in front of the column, the sample will spread back and forth when injected into the column, so a thin and short piping is used. However, with such a configuration, the piping becomes thin relative to the cross section of the column, and the sample is introduced into the center of the column, so that the sample in the center of the column advances in the column and the sample advances more slowly toward the outside of the column. The sample continues to spread as it moves through the column, so that if it is extremely delayed, the peak of the chromatogram will split or tail, resulting in a poor peak shape. Therefore, it is desirable for the sample to advance through the column while being positioned on the same plane as the column cross section, in other words, the cross section perpendicular to the liquid delivery direction.

[0005] For this reason, conventional high performance liquid chromatography (HPLC) columns are configured to have a space, a so-called countersink, between the joint and the filter on the inlet side of the column so that the sample spreads evenly in the cross-sectional direction of the column and moves forward at the same time.

[0006] However, even if the countersunk hole is formed, in the case of a preparative column in which the inner diameter of the column is large compared to the inner diameter of the pipe connected to the column inlet side, when a large amount of sample is introduced into the column, the sample cannot spread completely uniformly in the cross-sectional direction of the column, and the sample is distributed unevenly in the liquid delivery direction, so that the component concentration in the center of the column cross-section is high, and the moving speed becomes slower as it moves away from the center and toward the outside, so that the sample in the center of the column cross-section advances ahead. Since the packing material is packed closely inside the column, the spread of the sample band is suppressed compared to inside the pipe, but the state in which the sample in the center advances ahead immediately after injection into the column is not improved and the sample is eluted from the column outlet as it is. Therefore, the sample in the center of the cross-section of the column is eluted first, and the samples in the surrounding areas are eluted later, so that the peak shape of the chromatogram is largely tailed and there is a risk of overlapping with the next peak, and in the case of an extremely delayed peak, the peak may be split.

[0007] However, since the purpose of chromatography is to separate individual components, it is required to obtain peaks that are as symmetrical as possible.

[0008] Therefore, in addition to the countersinking, various techniques have been proposed to spread the sample uniformly over the cross section of the column, including providing a ball at the center of the inlet side of the column to restrict the movement of the central part, providing a baffle plate at the inlet side of the column to diffuse the sample in the cross section, providing a permeable diffusion plate at the inlet side of the column, combining a porous body and a diffusion plate at the inlet side of the column to diffuse the sample while restricting the flow, gradually spreading the sample through multiple spaces at the inlet side of the column, stacking two disks with an opening or multiple small holes at the inlet side of the column to form a diffusion mechanism, and forming a thin film on a filter at the inlet side of the column to restrict the flow at the center and spread the sample toward the periphery.

[0009] In particular, in a liquid chromatography preparative column having a relatively large inner diameter and using a slow-diffusing liquid as the mobile phase, a dispersion plate is provided between the inlet nut or joint and the filter in order to uniformly diffuse the injected sample mixture in the cross-sectional direction of the column. In addition to providing a dispersion plate on the inlet side, a configuration has also been proposed in which the same dispersion plate is provided on the outlet side in order to collect and elute the sample diffused in the cross-sectional direction (Patent Document 1). [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Actual development Showa 62-71560 Summary of the Invention [Problem to be solved by the invention]

[0011] However, the conventional technology is basically a configuration and technology at the sample introduction side to the column, and it is necessary to provide the configuration at the inlet side of the column, which is the upstream side of the stationary phase in the column. Specifically, a diffusion mechanism such as a counterbore, a baffle plate, a dispersion plate, a diffusion plate, a multi-stage structure, etc. is installed at the inlet part of the column, so that a large space is generated at the inlet part of the column, and so-called dead volume is generated. Since a peak that is eluted late on a chromatogram is not easily affected by the dead volume at the inlet side of the column, a peak with good symmetry is obtained, but for a peak that is eluted early, there is a problem that the sample band is broadened, the peak shape is deteriorated, tailing occurs, and the theoretical plate number is reduced due to the dead volume at the inlet side of the column.

[0012] For this reason, in the past, when using large-diameter preparative or purification columns for liquid chromatography, the peak shape of peaks that elute early on the chromatogram had to be given up.In addition, large-diameter columns could not be used as fast LC columns (first columns) that require a short analysis time per run.

[0013] Thus, uniform introduction of the sample into the column in the cross-sectional direction of the column and suppression of the broadening of the sample band within the column are contradictory.

[0014] On the other hand, with the trend towards faster analysis, higher resolution, and smaller amounts of sample, there is an increasing demand to reduce the broadening of sample bands in the column and to make chromatogram peaks sharper. The broadening of a sample band in a column is expressed by the following formula, σcol 2 = σ jointi 2 +σ filteri 2 +σ sp 2 +σ pspace 2 +σ jointo 2 +σ filtero 2As shown above, it is the sum of the diffusion in the inlet joint, the diffusion in the inlet filter, the diffusion in the stationary phase, the diffusion in the space between the stationary phase (diffusion in the space between the packing materials), the diffusion in the outlet joint, and the diffusion in the outlet filter.

[0015] Therefore, an object of the present invention is to diffuse an introduced sample in the cross-sectional direction within a column in a chromatographic analysis, thereby introducing the sample uniformly, and to suppress the broadening of the sample band within the column.

[0016] Another object of the present invention is to obtain a well-symmetrical peak in a chromatographic analysis, regardless of the elution time on a chromatogram, and to increase the number of theoretical plates. [Means for solving the problem]

[0017] The present invention, as a means for solving the above problems, is a column in which a flow rate control mechanism is provided on the outlet side within the column, more specifically, at the outlet portion downstream of the stationary phase within the column.

[0018] Specifically, it is a chromatography column equipped with a flow rate control mechanism on the outlet side of the column.

[0019] The chromatography column is also provided with a flow rate control mechanism on the outlet side of the column, and a diffusion mechanism on the inlet side of the column.

[0020] The column is also a chromatography column having a flow control mechanism on the outlet side of the column and a diffusion mechanism on the inlet side of the column, the spatial volume on the inlet side of the column being smaller than the spatial volume on the outlet side of the column.

[0021] The column is also a chromatography column that is equipped with a flow control mechanism on the outlet side of the column and a diffusion mechanism on the inlet side of the column, and the combined spatial volume of the outlet side spatial volume and the inlet side spatial volume of the column is less than one-tenth of the system spatial volume.

[0022] The chromatography column is also provided with a flow control mechanism on the outlet side of the column and a diffusion mechanism on the inlet side of the column, wherein the spatial volume on the inlet side of the column is smaller than the spatial volume on the outlet side of the column, and the combined spatial volume of the inlet side spatial volume and the outlet side spatial volume is less than one-tenth of the system spatial volume.

[0023] In the above-mentioned chromatography column, the flow rate control mechanism is a chromatography column configured to include a joint having a flow rate control plate or a flow rate control groove.

[0024] The above-mentioned chromatography column is a chromatography column selected from a preparative column for liquid chromatography, a semi-microcolumn for liquid chromatography, an analytical column for liquid chromatography, a preparative column for supercritical chromatography, a semi-microcolumn for supercritical chromatography, and an analytical column for supercritical chromatography.

[0025] The present invention also provides a chromatograph comprising the above-mentioned chromatography column.

[0026] Also, the chromatograph is a liquid chromatograph or a supercritical chromatograph.

[0027] The present invention also provides a chromatographic analysis method using the above-mentioned chromatography column.

[0028] Also, the chromatography is a chromatographic analysis method which is liquid chromatography or supercritical chromatography.

[0029] The present invention also provides a method for improving the peak shape of a chromatogram obtained by chromatographic analysis using the above-mentioned chromatography column.

[0030] Further, the above-mentioned improvement of the peak shape is a method for improving the peak shape of a chromatogram by suppressing tailing of the peak.

[0031] The present invention also provides a method for manufacturing a chromatography column, in which a flow rate control mechanism is provided on the outlet side of the column.

[0032] Also, the present invention relates to a method for manufacturing a chromatography column, in which a flow control mechanism is provided on the outlet side of the column and a diffusion mechanism is provided on the inlet side of the column. Effect of the Invention

[0033] According to the present invention as described above, by providing a flow control mechanism at the outlet side of the column, more specifically at the outlet section on the downstream side of the column, it is possible to control and suppress the flow rate and flow velocity within the column, particularly at the center part of the column, thereby making it possible to move the sample introduced at the center uniformly in the cross-sectional direction and suppress the broadening of the sample band within the column. It is also possible to obtain a column with good symmetry even for peaks that elute early on the chromatogram, and it is possible to obtain sharp peaks with good symmetry and no tailing even for components that elute early, thereby improving the precision of separation analysis.

[0034] In addition, by providing a flow control mechanism on the outlet side of the column, specifically at the outlet, the sample flows uniformly through the stationary phase in the column, and a sharp peak with good symmetry can be obtained in the chromatogram even without providing a dispersion mechanism such as a diffusion plate or a dispersion plate at the inlet side of the column. In addition, by not providing a dispersion mechanism such as a diffusion plate or a dispersion plate at the inlet side of the column, the dead volume is reduced, so that even for components that elute quickly, a peak with good symmetry can be obtained in the chromatogram, and high separation can be achieved. Furthermore, since there is no dead volume at the inlet side of the column, it can be used as a high-speed analysis column for fine particles that are easily affected by dead volume, a supercritical chromatography column that is more easily diffused than a liquid and is more easily affected by dead volume, a semi-micro column with a narrow inner diameter, a gas chromatography column, etc.

[0035] In addition, by making the dead volume of the dispersion mechanism such as the diffusion plate or dispersion plate at the inlet side of the column smaller than the spatial volume of the flow control mechanism installed at the outlet side, it is possible to suppress the spread of the sample band over the entire column. Furthermore, by installing a dispersion mechanism at the inlet side of the column, it is possible to obtain a peak with good symmetry on the chromatogram and to increase the theoretical plate number even when a sample liquid that does not mix well with the mobile phase or a large amount of sample liquid is injected.

[0036] In addition, the total volume of the inlet and outlet side spatial volumes of the column is less than one tenth of the spatial volume from the injector to the detector cell (hereinafter also referred to as the "system spatial volume"), so that there is no significant effect on the peak shape of the chromatogram. The system spatial volume is the total volume of the injector internal spatial volume, the piping joint internal spatial volume, the column internal spatial volume, and the detector cell internal spatial volume, and can generally be substituted by V0 (hold-up volume), which is the retention time of components not retained by the packing material, which is the stationary phase, multiplied by the flow rate. The column internal spatial volume includes the inlet side spatial volume and the outlet side spatial volume of the column, and is composed of the internal volumes of the inlet joint, the inlet filter, the stationary phase, the space between the stationary phases, the outlet filter, and the outlet joint. [Brief description of the drawings]

[0037] [Figure 1] Cross-sectional view of one embodiment of the present invention [Diagram 2] An exploded perspective view of an embodiment of the present invention [Diagram 3] Partial view of an embodiment of the present invention equipped with a flow control plate [Figure 4] Partial view of an embodiment of the present invention having a joint with a flow control groove. [Diagram 5] Cross-sectional view of another embodiment of the present invention [Figure 6] Exploded perspective view of another embodiment of the present invention [Figure 7] Exploded perspective view of another embodiment of the present invention [Figure 8] Cross-sectional view of another embodiment of the present invention [Figure 9] Exploded perspective view of another embodiment of the present invention [Figure 10] Cross-sectional view of another embodiment of the present invention [Figure 11] Exploded perspective view of another embodiment of the present invention [Figure 12] Front view of an embodiment of a flow control plate [Figure 13] Chromatogram of Example 1 of the present invention [Figure 14] Graph showing peak symmetry plot for Example 1 of the present invention [Figure 15] Chromatogram of Example 2 of the present invention [Figure 16] Graph showing peak symmetry plot for Example 2 of the present invention [Figure 17] Chromatogram of Example 3 of the present invention [Figure 18] Graph showing peak symmetry plot for Example 3 of the present invention [Figure 19] A graph plotting the theoretical plate number of Example 3 of the present invention. [Figure 20] Chromatogram of Example 4 of the present invention [Figure 21] Graph showing peak symmetry plot for Example 4 of the present invention [Figure 22] A graph plotting the theoretical plate number of Example 4 of the present invention. [Diagram 23] Chromatogram of Example 5 of the present invention [Figure 24] Graph showing peak symmetry plot for Example 5 of the present invention [Diagram 25] A graph plotting the theoretical plate number of Example 5 of the present invention. [Figure 26] Chromatogram of Example 6 of the present invention [Figure 27] Chromatogram of Example 7 of the present invention [Figure 28] Graph showing peak symmetry plot for Example 7 of the present invention [Figure 29] A graph plotting the theoretical plate number of Example 7 of the present invention. [Diagram 30] Chromatogram of Example 8 of the present invention [Diagram 31]Graph showing peak symmetry plot for Example 8 of the present invention [Diagram 32] A graph plotting the theoretical plate number of Example 8 of the present invention. [Diagram 33] Chromatogram of Example 9 of the present invention DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] The chromatography column of the present invention is provided with a flow rate control mechanism at the outlet side of the column, more specifically, at the outlet portion downstream of the stationary phase in the column. Hereinafter, an embodiment of the present invention will be described with reference to the drawings. The outlet side of the column means the downstream side of the stationary phase in the column, and the inlet side of the column means the upstream side of the stationary phase in the column.

[0039] 1 and 2, a chromatography column (hereinafter also simply referred to as "column") 1 of the present invention is provided with a flow control mechanism 3 at outlet 22 in column 1 for controlling and suppressing the flow rate and flow velocity of a mobile phase containing a sample in column 1, particularly in the central portion of column 1. Also, as shown in Figures 10 and 11, a chromatography column of the present invention is provided with a flow control mechanism 3 at outlet 22 in column 15, and a diffusion mechanism 4 for uniformly diffusing the sample in the cross-sectional direction of column 15 at inlet 21, which is on the inlet side of column 15 and upstream of the stationary phase in column 15.

[0040] Therefore, the column of the present invention may be configured as shown in FIGS. 1 and 2, in which a flow control mechanism 3 is installed only at the outlet portion 22 in the column 1, while no diffusion mechanism is installed at the inlet portion 21 in the column 1, or as shown in FIGS. 10 and 11, in which a diffusion mechanism 4 is installed at the inlet portion 21 in the column 11, and a flow control mechanism 3 is installed at the outlet portion 22.

[0041] As shown in Figures 1 and 2, the outlet section 22 of the column 1 can be configured on the outlet side within the column 1, downstream of the downstream filter 52 and upstream of the outlet hole 621 of the downstream joint 62, i.e., the part between the downstream filter 52 and the outlet hole 621 of the downstream joint 62, and the inlet section 21 of the column 1 can be configured on the inlet side within the column 1, upstream of the upstream filter 51 and downstream of the inlet hole 611 of the upstream joint 61, i.e., the part between the upstream filter 51 and the inlet hole 611 of the upstream joint 61.

[0042] The configuration of the flow control mechanism 3 at the outlet of the column 1 is not particularly limited, but the symmetry of each peak in the chromatogram should be less than 1.5, and preferably 1.25 or less, and the number of theoretical plates should satisfy a standard value depending on the type of packing material. For example, in an ODS silica gel packed column, the theoretical plate numbers should be 150,000 plates / m or more and 68,000 plates / m or more for 3 μm and 5 μm particles, respectively.

[0043] The flow control mechanism 3 is installed at the outlet 22 in the column 1, and controls and suppresses the flow rate and flow velocity in the column 1, especially in the central part of the column 1, so that the diffusion of the sample in the flow direction becomes uniform in any part of the cross section in the upstream part of the stationary phase of the column 1, and a uniform flow rate is obtained in any part of the column cross section, and the sample is uniformly led to the column bed (layer composed of packing material). Therefore, even peaks that elute early on the chromatogram are sharp without tailing.

[0044] The structure of the flow control mechanism can be configured according to the type of filter in the column, the joint structure, etc., and is not limited thereto, but the flow control mechanism can be configured with a flow control plate 31 installed on the bottom surface 620 of a joint 62 as shown in Fig. 3, or with a joint 63 having a flow control groove 32 on the bottom surface 630 as shown in Fig. 4. In addition, the diffusion mechanism can be configured using known structures and members such as a countersink, a baffle plate, a permeable diffusion plate, a combination of a porous body and a diffusion plate, a multi-stage space on the inlet side of the column, a stack of two disks with an opening or a plurality of small holes, a dispersion plate, a diffusion plate, etc.

[0045] Here, column 1 is configured with nuts 71, 72, joints 61, 62 for introducing liquid, filters 51, 52 for stopping the filler, a tube body 70, and a flow control mechanism 3. The joints 61, 62 are of a nut-separate type configured separately from the nuts 71, 72, but as shown in Figures 5 and 6, column 12 can use nut-integrated joints 710, 720, etc., in which the joint is configured integrally with the nut.

[0046] Next, the configuration of the chromatography column of the present invention will be specifically described. The column 1 is configured to have a flow control mechanism 3 only at the outlet section 22 in the column 1, and is configured to have an upstream nut 71, an upstream joint 61, an upstream filter 51, a tube body 70, a downstream filter 52, a downstream joint 62, a flow control plate 31 as a flow control mechanism 3 installed between the downstream filter 52 and the downstream joint 62, i.e., at the outlet section 22, and a downstream nut 72, as shown in Figures 1 and 2. Note that the upstream joint 61 is not provided with a diffusion mechanism such as a countersink.

[0047] 5 and 6, the column 12 is configured to have a flow control mechanism 3 only at the outlet portion 22 in the column 12, and is configured to have an upstream nut-integrated joint 710, an upstream filter 51, a pipe main body 70, a downstream filter 52, and a downstream nut-integrated joint 720 equipped with the flow control mechanism 3. A flow control groove 32 is formed in the bottom surface 630 of the downstream nut-integrated joint 720 as the flow control mechanism 3. The upstream nut-integrated joint 710 is not provided with a diffusion mechanism such as a countersink.

[0048] 7, the column 13 is configured to have a flow control mechanism only at the outlet portion 22 in the column 13, and is configured by replacing the nut-integrated joint having the flow control groove 32 of the column 12 shown in Figures 5 and 6, and is configured to have an upstream nut-integrated joint 710, an upstream filter 51, a pipe body 70, a downstream filter 52, a downstream nut-integrated joint 720, and a flow control plate 31 as the flow control mechanism 3 installed between the downstream filter 52 and the downstream nut-integrated joint 720. The upstream nut-integrated joint 710 is not provided with a diffusion mechanism such as a countersink.

[0049] Furthermore, as shown in Figures 8 and 9, the column 14 is configured by installing a joint 63 having a flow control groove 32 as a flow control mechanism 3 as shown in Figure 4, instead of the downstream joint 62 and the flow control plate 31 of the column 1 shown in Figures 1 and 2.

[0050] Moreover, the column 15 is configured to include a flow control mechanism 3 at the outlet portion 22 within the column 15, and a diffusion mechanism 4 at the inlet portion 21, and is configured to include an upstream nut 71, an upstream joint 61, an upstream filter 51, a diffusion plate 41 as the diffusion mechanism 4 installed between the upstream joint 61 and the upstream filter 51, i.e., at the inlet portion 21, a pipe body 70, a downstream filter 52, a downstream joint 62, a flow control plate 31 as the flow control mechanism 3 installed between the downstream filter 52 and the downstream joint 62, i.e., at the outlet portion 22, and a downstream nut 72, as shown in Figures 10 and 11.

[0051] Although not shown, the column 15 shown in Figures 10 and 11 may be configured with a nut-integrated joint instead of the separated nut and joint. Furthermore, although not shown, the column 15 shown in Figures 10 and 11 may be configured with a joint having a flow control groove on the bottom surface instead of the joint and flow control plate. Furthermore, although not shown, the column 15 shown in Figures 10 and 11 may be configured with a nut-integrated joint having a flow control groove on the bottom surface instead of the nut, joint and flow control plate. Furthermore, although not shown, a countersink may be provided instead of or together with the diffusion plate.

[0052] The flow control plate 31 is a plate body that controls and suppresses the flow in the downstream direction in the column 1 and guides the flow to the outlet. As shown in FIG. 3, the circular plate body has a through hole 310 in the center that communicates with the outlet hole 621 of the downstream joint 62, and the flow passage 311 that communicates with the through hole 310 is formed radially outward from the through hole 310. The flow passage 311 is configured to penetrate the plate body, but may be formed as a groove that does not penetrate the plate body formed on the upstream side surface of the plate body. Although not shown, one or more through holes that communicate with the outlet hole of the downstream joint may be provided in places other than the center of the plate body. The downstream joint 63 with the flow control groove 32 is a joint that controls and suppresses the flow in the downstream direction in the column and guides the flow to the outlet. As shown in FIG. 4, the bottom surface 630 of the joint 63 has a groove 32 as a flow passage that communicates with the outlet hole 621 formed radially outward from the outlet hole 621.

[0053] On the other hand, the diffusion plate 41 is a plate for diffusing the sample in the cross-sectional direction of the column.

[0054] The flow control plate 31 is installed on the bottom surface 620 of the downstream joint 62, but when a joint with a counterbore is used, it is installed by fitting it into the counterbore, making it possible to eliminate the counterbore and install the flow control mechanism. The thickness of the flow control plate is not particularly limited, but can be 0.1 to 10 mm.

[0055] In the flow control mechanism 3, the configuration of the flow path 311 of the flow control plate 31 is not particularly limited as long as it communicates with the through hole 310 that communicates with the outlet hole 621 of the joint 62, and the configuration of the flow control groove 32 is not particularly limited as long as it communicates with the outlet hole 621 of the joint 63. In the case of the flow control plate 31, as shown in FIG. 12, a shape that branches radially from the center is preferable, and it is also preferable that it is symmetrical. Also, the flow path may be curved as shown in FIG. 12(a), or may be linear as shown in FIG. 12(b) or FIG. 12(c). Also, as shown in FIG. 12(a) or FIG. 12(b), one flow path may branch into two, or as shown in FIG. 12(c), three or more paths may branch.

[0056] Furthermore, although not shown, the flow control mechanism may be configured by combining a plurality of flow control plates, or by combining a flow control plate with a joint having a flow control groove.

[0057] The materials for the flow control plate and the joint with the flow control groove are preferably materials that have been conventionally used in HPLC, such as resins that are resistant to the eluent used in HPLC, such as polytetrafluoroethylene, polyethylene, and polyether ether ketone (PEEK), and metals with low corrosivity, such as stainless steel and titanium. If they are made of metal, they may be subjected to a surface deactivation treatment. The materials for the flow control plate and the joint with the flow control groove installed on the inlet side are preferably pressure-resistant PEEK, metal, etc.

[0058] The method for producing the flow control plate and the joint having the flow control groove is not limited as long as it is a practical method that can be adopted as a product, such as etching, laser processing, lathe processing, etc.

[0059] In order to clean the liquid-contacting surfaces of the flow control plate and the joint equipped with the flow control groove, it is preferable to perform degreasing, ultrasonic cleaning, drying under reduced pressure, etc. This is because if oil or grease remains on the surface or processing residue remains on the surface, the sample will be adsorbed to those parts. In addition, since burrs on corners and the like can also cause adsorption, it is preferable to chamfer the edges, and if necessary, it is more preferable to perform C chamfering of 0.5 or more.

[0060] In HPLC analysis, the initial mobile phase is recommended as the dissolving solution for the sample components. When the sample is dissolved in the initial mobile phase or when the injection amount of the sample containing the solvent is small, about 1% or less of the system space volume, it is sufficient to provide a flow control mechanism only on the outlet side to control the flow rate in the column. However, when the sample does not mix well with the initial mobile phase or when the injection amount of the sample containing the solvent is large, 1% or more of the system space volume, it becomes necessary to thoroughly mix the injected sample with the initial mobile phase before introducing it into the column bed (layer composed of packing material). For this reason, in addition to flow control on the outlet side, a diffusion mechanism may be provided on the inlet side of the column to thoroughly mix the sample solution and the mobile phase.

[0061] In the case of a configuration in which a flow control mechanism is provided on the outlet side of the column and a diffusion mechanism is provided on the inlet side, if the inlet-side spatial volume of the inlet side of the column is larger than the outlet-side spatial volume of the outlet side of the column, the effect of the flow control mechanism installed on the outlet side will be weakened due to the influence of diffusion due to the inlet-side spatial volume, and the peak shape will be worse than when the inlet-side spatial volume is smaller, so it is preferable that the inlet-side spatial volume is smaller than the outlet-side spatial volume. Furthermore, taking into account the variation error, it is more preferable that the inlet-side spatial volume is 80% or less of the outlet-side spatial volume. Here, the inlet side spatial volume is the total volume from the inlet side orifice on the inlet side in the column to the filter for fixing the packing, i.e., the total volume including the volume of the space of the inlet side orifice, the volume of the space of the diffusion mechanism such as the countersink or diffusion plate, and the volume of the space of the upstream filter, and the outlet side spatial volume is the total volume from the downstream side filter for fixing the packing on the outlet side in the column to the outlet side orifice, i.e., the total volume including the volume of the space of the downstream side filter, the volume of the space of the flow control mechanism, the volume of the space of the countersink, etc., and the volume of the space of the outlet side orifice. The space of the inlet side orifice or the space of the outlet side orifice is the remaining space when a screw, ferrule, etc. for piping connection is inserted into the space of the inlet hole 611 or the outlet hole 621.

[0062] In addition, the total spatial volume of the inlet spatial volume and the outlet spatial volume is preferably less than 10% of the system spatial volume. If the ratio of the total spatial volume to the system spatial volume increases, the peak width will increase in proportion to the volume, the number of theoretical plates will decrease, the degree of resolution will deteriorate, and quantification will be affected. Furthermore, in order to suppress the variation in quantification to the preferred level of less than 10%, the ratio of the total spatial volume to the system spatial volume must be less than 10%.

[0063] In recent years, the demand for first columns, which are short columns packed with small particle size packing materials, has been rapidly increasing because of the ability to shorten analysis time. However, the method of installing a dispersion mechanism such as a dispersion plate on the inlet side of the column cannot be applied to first columns, which have a short analysis time. On the other hand, the configuration of the present invention, in which a flow control mechanism is installed only on the outlet side, is optimal for first columns, since no space is generated on the inlet side of the column.

[0064] In addition, in columns with a large cross-sectional area, such as an inner diameter of 10 mm or more, the sample does not flow uniformly in the cross-sectional direction, and the peak shape and theoretical plate number are often poor due to the influence of the sample components added in large quantities and the pressure shock caused by each injection. In particular, short columns are prone to column deterioration. However, when the present invention is used, the sample components are introduced uniformly across the column cross section, so the impact of the injection amount and pressure shock on the inlet bed is reduced.

[0065] The chromatography column of the present invention can be used as, but is not limited to, a preparative column for liquid chromatography, a semi-microcolumn for liquid chromatography, an analytical column for liquid chromatography, a preparative column for supercritical chromatography, a semi-microcolumn for supercritical chromatography, or an analytical column for supercritical chromatography.

[0066] Although not shown, a chromatograph can be constructed by including the chromatography column of the present invention. The chromatograph including the chromatography column of the present invention may be, but is not limited to, a liquid chromatograph or a supercritical chromatograph.

[0067] Furthermore, the chromatography column of the present invention can be used to perform chromatographic analysis using known methods and devices. The chromatographic analysis using the chromatography column of the present invention may be, but is not limited to, liquid chromatography or supercritical chromatography. Such chromatographic analysis using the chromatography column of the present invention can be used as a method for improving the peak shape of a chromatogram and a method for suppressing peak tailing.

[0068] The chromatography column of the present invention can be manufactured using a known column manufacturing method and by installing a flow control mechanism 3 at the outlet side of column 1, as shown in Figures 1 and 2, or by installing a flow control mechanism 3 at the outlet side of column 15 and a diffusion mechanism 4 at the inlet side of column 15, as shown in Figures 10 and 11. EXAMPLES

[0069] ODS gel (ODS A filler surface area 450m 2 HPLC analysis was performed using an HPLC column in which a stainless steel column with an inner diameter of 20 mm and a length of 250 mm was packed with 15% carbon (10 nm pore size, 15%). As Example 1, a column 1 (system space volume: 43.8 mL) equipped with an outlet side flow control mechanism 3 (31) as shown in Figures 1 and 2, which is equipped with a flow control plate 31 as a flow control mechanism 3 only on the outlet side, was compared with a column (system space volume: 42.4 mL) not equipped with a diffusion mechanism such as a countersink on the inlet side and not equipped with a flow control mechanism on the outlet side as Comparative Example 1, using the results of chromatograms.

[0070] The HPLC analysis conditions were: Mobile phase 65% acetonitrile Flow rate 20mL / min Column temperature: 40℃ Detection UV 254nm, Sample: Peak 1: uracil, Peak 2: acetophenone, Peak 3: benzene, Peak 4: toluene, Peak 5: naphthalene mixed sample Sample injection volume: 120μL

[0071] The chromatogram of the result is shown in FIG. 13. Also, a graph in which the peak symmetry is plotted on the vertical axis against the retention time of each peak is shown in FIG. 14. The peak symmetry of 1.00 is a symmetrical peak, and ideally, if it is less than 1.5, it is considered to be a column that can be generally used. In Comparative Example 1, as shown in FIG. 13(b) and FIG. 14, the earlier the retention time of the peak, the greater the peak symmetry and the more tailing there is. In the actual chromatogram, the tailing of the second peak of the conventional column can be confirmed. On the other hand, in Example 1 of the present invention, by simply installing a flow control mechanism on the outlet side, the symmetry of the first peak is improved from 1.78 to 1.19, and the symmetry of the second peak is improved from 1.59 to 1.10, as shown in FIG. 13(a) and FIG. 14. In the method of the present invention, the tailing of the second peak is eliminated on the actual chromatogram, and is greatly improved. In the column of Example 1 of the present invention, by providing a flow control mechanism on the outlet side, the system spatial volume of the column increases by 1.4 mL. However, the combined spatial volume of the inlet side spatial volume and the outlet side spatial volume is 6% of the system spatial volume, which is within the acceptable range of less than 10%. EXAMPLES

[0072] ODS gel (ODS B filler surface area 350m 2The HPLC analysis was carried out using an HPLC column in which a stainless steel column having an inner diameter of 20 mm and a length of 250 mm was packed with 100g of 100g of carbon (pore size 10 nm, carbon amount 8%). As Example 2-1, column 1 (system space volume: 40.3 mL, total space volume of inlet side space volume and outlet side space volume: 1.34 mL, total space volume / system space volume: 3.3%) shown in FIG. 1 and FIG. 2 was used, which was equipped with a flow control plate 31 as a flow control mechanism 3 only on the outlet side. As Example 2-2, column 15 (system space volume: 40.34 mL, total space volume of inlet side space volume and outlet side space volume: 1.38 mL, total space volume / system space volume: 3.4%, ratio of inlet side space volume to outlet side space volume: 86%) shown in FIG. 10 and FIG. 11 was used, which was equipped with a diffusion plate 41 as a diffusion mechanism 4 on the inlet side and a flow control plate 31 as a flow control mechanism 3 on the outlet side. As Comparative Example 2, a column equipped with a diffusion plate as a diffusion mechanism on the inlet side and outlet side (system space volume 38.5 mL) was used, and the results of the chromatograms were evaluated and compared.

[0073] The HPLC analysis conditions were: Mobile phase 65% acetonitrile Flow rate 20mL / min Column temperature: 40℃ Detection UV 254nm, Sample: Peak 1: uracil, Peak 2: acetophenone, Peak 3: benzene, Peak 4: toluene, Peak 5: naphthalene mixed sample Injection volume 120μL

[0074] Example 2-1 of the present invention is an example of analysis using a novel preparative column of the present invention in which a diffusion plate is not installed at the inlet side of the column, and a flow control plate with a volume of 140 μL is installed at the outlet. Example 2-2 of the present invention is an example of analysis using a preparative column of the present invention in which a diffusion plate with a volume of 40 μL is installed at the inlet side of the column, and the same flow control plate as in Example 2-1 is installed at the outlet side.

[0075] The chromatogram of the result is shown in FIG. 15. Also, a graph in which the peak symmetry is plotted on the vertical axis against the retention time of each peak is shown in FIG. 16. The evaluation method of plotting the peak symmetry against the elution time is correct for evaluating the column, and the peak symmetry of 1.00 is a symmetrical peak, which is ideal. Although it varies depending on the manufacturer, it is generally considered that a column with a peak symmetry of less than 1.5 can be generally used. In the present invention, the goal is an even better peak symmetry of 1.25 or less. In Example 2-2 of the present invention, in which a flow control plate is provided on the outlet side and a diffusion plate as the diffusion mechanism 4 on the inlet side, the peak symmetry at a retention time of 2 minutes is improved to 1.2 as shown in FIG. 15(b) and FIG. 16, and in Example 2-1 of the present invention, in which a flow control plate is provided only on the outlet side, it is improved to 1.17 as shown in FIG. 15(a) and FIG. 16.

[0076] In addition, in Comparative Example 1, as shown in Figures 13(b) and 14, the symmetry of Peak 1, which elutes earliest, was 1.78, whereas in Comparative Example 2, as shown in Figures 15(c) and 16, the symmetry was 1.45. It was thus confirmed that even in a configuration in which a diffuser plate is installed on the inlet side, the peak symmetry is better than in a configuration in which no diffuser plate is installed.

[0077] In addition, it was confirmed that even if a diffusion mechanism is provided on the inlet side, when the spatial volume on the inlet side is smaller than the spatial volume on the outlet side, the same level of performance can be obtained as in a configuration in which a diffusion mechanism is not provided on the inlet side and a flow control mechanism is provided only on the outlet side. Furthermore, by comparing Example 1 and Example 2, it was proven that the same effect can be obtained even if the type of packing material is changed, and the present invention can be applied to liquid chromatography using various columns. EXAMPLES

[0078] Using an HPLC column filled with the same ODS gel packing as in Example 2-1, HPLC analysis was performed under the same HPLC analysis conditions as in Example 2. As Example 3 of the present invention, column 1 shown in Figures 1 and 2, which is provided with a flow control plate as a flow control mechanism only on the outlet side of the column, was used. Also, as comparative examples, Comparative Example 3-1, in which the flow control plate was removed from the configuration of column 1 and countersunk joints were formed on the upstream and downstream sides, Comparative Example 3-2, in which the flow control plate was removed from the configuration of column 1, countersunk joints were formed on the upstream and downstream sides, and a diffuser plate was installed only on the inlet side, and Comparative Example 3-3, in which the flow control plate was removed from the configuration of column 1, countersunk joints were formed on the upstream and downstream sides, and a diffuser plate was installed on both the inlet and outlet sides, were evaluated and compared based on the results of chromatograms.

[0079] The resulting chromatogram is shown in Figure 17. Figure 18 is a graph in which the peak symmetry is plotted on the vertical axis against the retention time of each peak, and Figure 19 is a graph in which the theoretical plate number is plotted on the vertical axis. Peak symmetry of 1.00 is ideal, with a bilaterally symmetrical peak, and a number less than 1.5 is considered to be a column that can be generally used. The higher the theoretical plate number, the sharper the peak will be, and in the case of 5 μm particles, a column with 17,000 plates or more (68,000 plates / m) is considered to be usable.

[0080] In the column of Example 3 of the present invention, as shown in Figs. 17(a), 18, and 19, a high theoretical plate number of 21,900 or more is obtained even for a peak with early retention that elutes in less than 6 minutes, and the peak symmetry is good and less than 1.25. In the column of Comparative Example 3-1, as shown in Figs. 17(b), 18, and 19, a plate number of 17,000 or more is obtained for a peak that elutes in 8 to 9 minutes, and sufficient performance is obtained. However, for a peak that elutes early, which elutes by 7 minutes, the theoretical plate number is reduced, and the peak symmetry is 1.5 or more, causing tailing and poor peak shape. In the column of Comparative Example 3-2, as shown in Figs. 17(c), 18, and 19, sufficient performance is obtained for a peak that elutes after 6 minutes. However, for a peak that elutes early, which elutes by 5.5 minutes, the theoretical plate number is reduced, and the peak symmetry is 1.5 or more, causing tailing and poor peak shape. The column of Comparative Example 3-3 had the same performance as the column of Comparative Example 3-2, as shown in Figures 17(d), 18, and 19. With the conventional diffusion plate installed on the inlet side, the effect on the outlet side was cancelled out, as the sample was diffused in the inlet side space due to the effect of the spatial volume on the inlet side, and no improvement was obtained.

[0081] Thus, in the comparative example, the first peak, uracil, was not retained and did not undergo reverse-phase partitioning, and therefore no theoretical plate number was obtained. However, in Example 3 of the present invention, in which a flow control plate was installed on the outlet side, the components were uniformly introduced into the column, and therefore the peak symmetry was less than 1.5, and high performance was obtained. EXAMPLES

[0082] HPLC analysis was performed using an HPLC column in which a spherical 5 μm ODS packing material InertSustain (registered trademark) C18, particle size 5 μm (manufactured by GL Sciences Inc.) was packed into a stainless steel column with an inner diameter of 20 mm and a length of 250 mm, and the peak shape of the chromatogram was confirmed.

[0083] As Example 4-1, a column 13 was used in which a flow control plate 31 made of SUS316 material and having a thickness of 0.5 mm was installed on a nut-integrated joint 720 as shown in FIG. 7, which was provided only on the outlet side of the column 13 as a flow control mechanism 3. As Example 4-2, a column 12 was used in which a nut-integrated joint 720 was installed on the outlet side of the column 12 as shown in FIG. 5 and FIG. 6, which was provided with a flow control groove 32 having a depth of 0.5 mm. In Example 4-2, the volume of the flow control groove 32 was 61 μL, the volume of the downstream filter 52 and the outlet side orifice was a total of 534 μL, the outlet side spatial volume was 595 μL, and the total spatial volume of 1.129 mL obtained by adding the inlet side spatial volume including the inlet side orifice volume, the space, and the upstream side filter volume to this was 2.8% of the system spatial volume of 40.3 mL, which is less than 10%. The HPLC analysis conditions were the same as in Example 1.

[0084] The chromatogram of the result is shown in FIG. 20. The shapes of five peaks eluted with retention coefficients (k) of 0 to 4 were examined. FIG. 21 shows a graph in which the retention time of each peak is plotted against the peak symmetry on the vertical axis, and FIG. 22 shows a graph in which the theoretical plate number is plotted on the vertical axis. The peak symmetry was 1.25 or less in both Example 4-1 shown in FIG. 20(a) and Example 4-2 shown in FIG. 20(b), and a peak shape with good symmetry was obtained. In addition, the theoretical plate number was 20,000 or more, except for the Uracil peak (theoretical plate number 17,000 or more) that was not distributed and retained in the ODS layer, and a sharp peak of 80,000 or more per m was obtained. In addition, instead of the 0.5 mm thick flow control plate made of SUS316 material and shaped as shown in Fig. 12(a) in Example 4-1, a 0.5 mm thick flow control plate made of SUS316 material and shaped as shown in Fig. 12(b) and Fig. 12(c) was installed in the joint and HPLC analysis was performed under the same conditions, with the same results. It was demonstrated that the performance of the column can be maximized by installing the flow control mechanism on the outlet side, resulting in a high-performance column. EXAMPLES

[0085] The change in the peak shape of the chromatogram due to the change in the inlet side space volume relative to the outlet side space volume was confirmed. HPLC analysis was performed using an HPLC column packed with spherical 5 μm ODS packing material InertSustain (registered trademark) C18, particle size 5 μm (manufactured by GL Sciences Inc.) in a stainless steel column with an inner diameter of 50 mm and a length of 250 mm, and the peak shape of the chromatogram was confirmed.

[0086] A joint 63 having a flow control groove 32 of 5 mm depth was installed on the outlet side of the column 14 as shown in Figures 8 and 9, and a flow control mechanism 3 was installed on the outlet side of the column 14. The inlet side of the column was equipped with a diffusion plate made of SUS316 material with different thicknesses, and four types of HPLC columns with different ratios of the inlet side spatial volume to the outlet side spatial volume were prepared and used for HPLC analysis.

[0087] In Example 5-1, no diffusion plate was installed on the inlet side of column 14, and in Examples 5-2 to 5-4, diffusion plates with thicknesses of 1 mm (Example 5-2), 4 mm (Example 5-3), and 8 mm (Example 5-4) were installed in the joint on the inlet side of column 14 shown in Figure 9. In Example 5-1, the inlet side spatial volume was 2.5 mL, and the inlet side spatial volume ratio to the outlet side spatial volume was 23% (the combined spatial volume of the inlet side spatial volume and the outlet side spatial volume was 5.1% of the system spatial volume), in Example 5-2, the inlet side spatial volume was 3.3 mL, and the inlet side spatial volume ratio to the outlet side spatial volume was 30% (the combined spatial volume of the inlet side spatial volume and the outlet side spatial volume was 5.5% of the system spatial volume), in Example 5-3, the inlet side spatial volume was 5.7 mL, and the inlet side spatial volume ratio to the outlet side spatial volume was 52% (the combined spatial volume of the inlet side spatial volume and the outlet side spatial volume was 6.4% of the system spatial volume), and in Example 5-4, the inlet side spatial volume was 8.9 mL, and the inlet side spatial volume ratio to the outlet side spatial volume was 81% (the combined spatial volume of the inlet side spatial volume and the outlet side spatial volume was 7.6% of the system spatial volume). As Comparative Example 5, a conventional commercially available HPLC column was prepared in which diffusion plates were installed on both the outlet and inlet sides of the column, and the inlet side spatial volume was 100% relative to the outlet side spatial volume.

[0088] The HPLC analysis conditions were: Mobile phase 65% acetonitrile Flow rate 125mL / min Column temperature: Room temperature 25℃ Detection UV 254nm, Sample: Peak 1: uracil, Peak 2: acetophenone, Peak 3: benzene, Peak 4: toluene, Peak 5: naphthalene mixed sample Sample injection volume: 500μL

[0089] The chromatogram of the result is shown in FIG. 23. Also, a graph in which the retention time of each peak is plotted against the peak symmetry on the vertical axis is shown in FIG. 24, and a graph in which the theoretical plate number is plotted on the vertical axis is shown in FIG. 25. In the columns of Examples 5-1 to 5-4 of the present invention, almost the same good results were obtained as shown in FIG. 23(a) to FIG. 23(d), FIG. 24, and FIG. 25. Specifically, in the columns of the examples of the present invention, the peak symmetry is less than 1.2, and the peaks are well symmetrical. In addition, a sharp peak with a theoretical plate number of about 20,000 is obtained for the peak after 4 minutes of reversed phase distribution. In contrast, in Comparative Example 5, as shown in FIG. 23(e), FIG. 24, and FIG. 25, the second and third peaks tail and have low peak heights. The symmetry of the elution peaks at 2 minutes, 4 minutes, and 6 minutes is 1.4, 1.4, and 1.3, respectively.

[0090] It has been demonstrated that in the column of the present invention in which a flow control mechanism is installed on both the outlet and inlet sides, if the spatial volume on the inlet side of the column is 0 to 81% of the spatial volume on the outlet side, a sharp peak with good peak symmetry can be obtained. EXAMPLES

[0091] Using the column of the present invention demonstrated in Example 5, a test was performed to confirm the peak shape of the chromatogram when a sample solvent that does not mix well with the mobile phase was used and the injection amount of the sample containing the solvent was increased. HPLC analysis was performed using an HPLC column packed with spherical 5 μm ODS packing material InertSustain (registered trademark) C18, particle size 5 μm (manufactured by GL Sciences Inc.) in a stainless steel column with an inner diameter of 50 mm and a length of 250 mm, and the peak shape of the chromatogram was confirmed.

[0092] A joint 63 having a flow control groove 32 with a depth of 5 mm, as shown in FIG. 4, was installed on the outlet side of the column 14, and a flow control mechanism 3 was installed on the outlet side of the column 14. As Example 6-1, the inlet side of the column 14 was configured such that no diffusion plate was installed on the inlet side of the column, and as Example 6-2, a diffusion plate made of SUS316 material with a thickness of 8 mm was installed on the upstream joint 61 on the inlet side of the column, and the inlet side spatial volume was 23% and 81% of the outlet side spatial volume, respectively, and two types of HPLC columns with different ratios of the inlet side spatial volume to the outlet side spatial volume were prepared. As Comparative Example 6, a diffusion plate with the same capacity was installed on the joint on the outlet side and inlet side of the column, and an HPLC column with the inlet side spatial volume being 100% of the outlet side spatial volume of the column was prepared. The HPLC analysis conditions were: Mobile phase 60% acetonitrile Flow rate 125mL / min Column temperature: Room temperature 25℃ Detection UV254nm Samples Benzene, toluene, and naphthalene were dissolved in isopropanol, which is a solvent that is poorly miscible with the mobile phase, and 5 mL (2% relative to the system space volume) was injected.

[0093] The resulting chromatograms are shown in FIG. 26. In the column of the present invention in Example 6-1, separation was ensured as shown in FIG. 26(a), and further improvement was achieved in the column of the present invention in Example 6-2 as shown in FIG. 26(b). In the conventional column of Comparative Example 6, separation was achieved as shown in FIG. 26(c), but the peaks were poorly shaped and tailed. It was confirmed that a column with a flow control mechanism installed on the outlet side was effective even for large-volume injection. Furthermore, it was demonstrated that the peak shape was further improved by installing a diffusion mechanism on the inlet side so that the volume of the inlet space was 81% or less of the volume of the outlet space. EXAMPLES

[0094] When a diffuser plate or the like is installed on the inlet side of the column, the spatial volume in the column increases, but generally, it is within the acceptable range if the total spatial volume of the columns is less than 10% of the system spatial volume. In recent years, the demand for first columns, which are short columns packed with a packing material having a small particle size, has been rapidly increasing because it can shorten the analysis time. However, the conventional method of using a diffuser plate on the inlet side cannot be applied to first columns with short peak elution times. On the other hand, the flow rate control on the outlet side of the column of the present invention is considered to be optimal for first columns because no space is generated on the inlet side.

[0095] Therefore, the application of the present invention to a short column was confirmed. A joint 63 having a flow control groove 32 with a depth of 0.5 mm, as shown in FIG. 4, was installed on the outlet side of the column. A packing material InertSustain (registered trademark) C18, particle size 5 μm (manufactured by GL Sciences Inc.) was packed in a stainless steel column with an inner diameter of 20 mm and a length of 50 mm to prepare a column of Example 7 of the present invention. The volume of the flow control groove 32 was 61 μL, the volume of the downstream filter and the outlet side orifice was 400 μL in total, the outlet side spatial volume was 461 μL, the inlet side spatial volume was 400 μL, and the combined spatial volume of the outlet side spatial volume and the inlet side spatial volume was 0.861 mL, which is 9.6% of the system spatial volume of 9 mL, which is less than 10%. As Comparative Example 7, a first column similar to that of Example 7 was prepared except that no flow control mechanism was provided on the outlet side, and HPLC analysis was performed, and the results of the chromatogram were evaluated and compared.

[0096] The HPLC analysis conditions were: Mobile phase 65% acetonitrile Flow rate 20mL / min Column temperature: 40℃ Detection UV 254nm, Sample: Peak 1: uracil, Peak 2: acetophenone, Peak 3: benzene, Peak 4: toluene, Peak 5: naphthalene mixed sample Injection volume 120μL

[0097] The resulting chromatogram is shown in Figure 27. Also, a graph in which the peak symmetry is plotted on the vertical axis against the retention time of each peak is shown in Figure 28, and a graph in which the theoretical plate number is plotted on the vertical axis is shown in Figure 29.

[0098] As shown in Figures 27(a), 28, and 29, the first column of the present invention has a peak symmetry of 1.25 or less for all five peaks, and the theoretical plate number is 90,000 plates / m for the third to fifth peaks. As shown in Figures 27(b), 28, and 29, the column of Comparative Example 7 has a tailing effect on the tail of the peaks that are retained earlier, but it was confirmed that the column of Example 7 of the present invention has improved tailing. In addition, the column of Comparative Example 7 has a symmetry of 1.3 even for the fifth peak, which has the slowest elution and has a retention factor (k) of 3, due to the influence of sample diffusion in the flow direction. Since the first column is short, it is more susceptible to the influence of sample diffusion in the flow direction than the 250 mm column, and tailing inevitably occurs in the first column of Comparative Example 7.

[0099] It was confirmed that the present invention is effective even for a short first column, and it was also demonstrated that the installation of the outlet flow control mechanism of the present invention can be applied to any column size. EXAMPLES

[0100] The experiment was conducted using hydrophilic interaction chromatography (HILIC). In reversed-phase partition columns such as ODS, sample components are concentrated at the inlet, which can improve tailing, so a demonstration experiment was conducted using a HILIC mode packing material that does not cause concentration.

[0101] A stainless steel column with an inner diameter of 20 mm and a length of 250 mm was filled with InertSustain (registered trademark) Amide, a carbamoyl group-bonded packing material, with a particle size of 5 μm (manufactured by GL Sciences Inc.), to prepare a HILIC column of Example 8 of the present invention, in which the total spatial volume of the inlet side and the outlet side spatial volume is 6% of the system spatial volume. As Comparative Example 8, a HILIC column similar to Example 8 was prepared except that no flow control mechanism was provided on the outlet side, and HPLC analysis was performed to evaluate and compare the results of the chromatogram. The case of the column of Example 8 is the same as that of Example 1 shown in FIG. 1 and FIG. 2.

[0102] The HPLC analysis conditions were: Mobile phase 95% acetonitrile Flow rate 20mL / min Column temperature: 40℃ Detection UV254nm Sample: Peak 1: benzene, Peak 2: phenol, Peak 3: caffeine Injection volume 120μL

[0103] The resulting chromatogram is shown in Figure 30. Also, a graph in which the retention time of each peak is plotted against the vertical axis for peak symmetry is shown in Figure 31, and a graph in which the theoretical plate number is plotted against the vertical axis is shown in Figure 32.

[0104] As shown in Figures 30(a), 31, and 32, the HILIC column of Example 8 of the present invention had a peak symmetry of 1.25 or less and a theoretical plate number of 80,000 plates / m, and it was confirmed that sufficient performance was obtained for the HILIC mode. As shown in Figure 30(b), the column of Comparative Example 8 has a tendency to tailing overall, which is thought to be due to the sample not flowing uniformly across the cross section of the column. On the other hand, the column of Example 8 of the present invention has improved tailing because the sample flows uniformly across the cross section of the column.

[0105] It has been demonstrated that the column of the present invention exhibits sufficient effectiveness even in the HILIC mode, and that sufficient high performance can be obtained even when different packing materials or separation modes are used. EXAMPLES

[0106] The durability of the column was inspected. At the outlet side of the column, as shown in FIG. 1, a flow control plate 31 made of SUS316 material and having a thickness of 0.5 mm, as shown in FIG. 12(a), was installed at the downstream joint 62 to provide a flow control mechanism 3. The flow control mechanism 3 was provided by filling a packing material InertSustain (registered trademark) C18, particle size 5 μm (manufactured by GL Sciences Inc.) in a stainless steel column 1 having an inner diameter of 20 mm and a length of 50 mm. As a comparative example 9, a column similar to that of Example 9 was prepared except that no flow control mechanism was provided at the outlet side. A durability test was performed by injecting 100 μL of 20 mM propiophenone solution 500 times at 20 minute intervals with a 50% methanol eluent, which is the most highly pressured and susceptible to the effects of injection shock, at a flow rate of 20 mL / min and room temperature of 25° C. Then, HPLC analysis was performed under the following column inspection conditions, and the changes in the chromatogram before and after the durability test were confirmed and evaluated.

[0107] The column inspection conditions are: Mobile phase 65% acetonitrile Flow rate 20mL / min Column temperature: 40℃ Detection UV 254nm, Sample: Peak 1: uracil, Peak 2: acetophenone, Peak 3: benzene, Peak 4: toluene, Peak 5: naphthalene mixed sample Injection volume 120μL

[0108] Chromatogram results before and after the durability test for the column of Example 9 of the present invention and the column of Comparative Example 9 are shown in Figure 33. As shown in Figures 33(a) and 33(b), in the column of Example 9 of the present invention, the peak shape does not change before (Figure 33(a)) and after (Figure 33(b)) the 500-cycle durability test. In contrast, in the column of Comparative Example 9, as shown in Figures 33(c) and 33(d), a peak with little tailing was obtained in the initial state (Figure 33(c)), but tailing occurred after the 500-cycle durability test (Figure 33(d)).

[0109] In the column of the present invention, the sample is introduced uniformly in the cross-sectional direction of the column, so there is little injection shock or injection effect, and it has been demonstrated that the column has higher durability than conventional columns. [Industrial Applicability]

[0110] According to the present invention as described above, the sample flows uniformly through the stationary phase in the column, and sharp, well-symmetrical peaks can be obtained in the chromatogram. This improves the analytical ability of substances and makes the invention useful in research and applications in various fields. [Explanation of symbols]

[0111] 1 Column 21 Entrance 22 Exit section 3 Flow control mechanism 31 Flow control plate 32 Flow control groove 4. Diffusion Mechanism 51 Upstream filter 52 Downstream filter 61 Upstream joint 62 Downstream joint 621 Exit hole 70 Tube body 71 Upstream nut 72 Downstream nut

Claims

1. A chromatography column comprising a flow rate control mechanism at the outlet side of the column.

2. A chromatography column comprising a flow rate control mechanism at an outlet side of the column and a diffusion mechanism at an inlet side of the column.

3. 1. A chromatography column comprising: a flow rate control mechanism on an outlet side of the column; and a diffusion mechanism on an inlet side of the column, the spatial volume on the inlet side of the column being smaller than the spatial volume on the outlet side of the column.

4. 1. A chromatography column comprising a flow rate control mechanism on an outlet side of the column and a diffusion mechanism on an inlet side of the column, wherein a combined spatial volume of the outlet side of the column and the inlet side of the column is less than one-tenth of a system spatial volume.

5. 1. A chromatography column comprising: a flow rate control mechanism on an outlet side of the column; and a diffusion mechanism on an inlet side of the column; a spatial volume on the inlet side of the column is smaller than a spatial volume on the outlet side of the column; and a combined spatial volume of the inlet side and the outlet side spatial volume is less than one-tenth of a system spatial volume.

6. 6. The chromatography column according to claim 1, wherein the flow rate control mechanism comprises a flow rate control plate or a joint having a flow rate control groove.

7. 6. The chromatography column according to claim 1, wherein the chromatography column is a column selected from a preparative column for liquid chromatography, a semi-microcolumn for liquid chromatography, an analytical column for liquid chromatography, a preparative column for supercritical chromatography, a semi-microcolumn for supercritical chromatography, or an analytical column for supercritical chromatography.

8. A chromatograph comprising a chromatography column according to any one of claims 1 to 5.

9. A chromatograph comprising the chromatography column according to claim 6.

10. 9. The chromatograph of claim 8, wherein the chromatograph is a liquid chromatograph or a supercritical chromatograph.

11. 10. The chromatograph of claim 9, wherein the chromatograph is a liquid chromatograph or a supercritical chromatograph.

12. A chromatographic analysis method, comprising carrying out a chromatographic analysis using the chromatography column according to any one of claims 1 to 5.

13. A chromatographic analysis method, comprising carrying out a chromatographic analysis using the chromatography column according to claim 6.

14. 13. The chromatographic analysis method according to claim 12, wherein the chromatography is liquid chromatography or supercritical chromatography.

15. 14. The chromatographic analysis method according to claim 13, wherein the chromatography is liquid chromatography or supercritical chromatography.

16. A method for improving a peak shape of a chromatogram, comprising carrying out a chromatographic analysis using the chromatography column according to any one of claims 1 to 5.

17. A method for improving a peak shape of a chromatogram, comprising carrying out a chromatographic analysis using the chromatography column according to claim 6.

18. 17. The method for improving a peak shape of a chromatogram according to claim 16, wherein the improvement of the peak shape is suppression of tailing of the peak.

19. 18. The method for improving a peak shape of a chromatogram according to claim 17, wherein the improvement of the peak shape is suppression of tailing of the peak.

20. A method for manufacturing a chromatography column, comprising providing a flow rate control mechanism on the outlet side of the column.

21. A method for manufacturing a chromatography column, comprising providing a flow control mechanism on the outlet side of the column and providing a diffusion mechanism on the inlet side of the column.

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