JP6897052B2 - Column filter - Google Patents

Description

The present invention relates to a column filter characterized by being a sintered body composed of spherical powder.

In analysis by liquid chromatography, a column filter may be provided to prevent the column packing material from flowing out of the column. As a column filter for liquid chromatography for measuring a biological sample, a stainless steel filter that is inactive to some extent with respect to the sample is used.

As a specific example of such a stainless steel filter, a stainless steel short fiber or fine powder sintered under high temperature and high pressure is used. Conventional stainless steel filters use irregularly shaped powders such as short fibers or fine powders whose size and shape are not strictly constant, but since they do not have a constant unit structure, they are filled in the column on the column outlet side. It led to the accumulation and aggregation of substances other than the agent.

Further, a filter using a sintered spherical metal powder has also been proposed (see, for example, Patent Documents 1 to 3), but the particle size of the column filler is not sufficiently considered and is used. The average particle size of the metal powder to be used was not in an appropriate range.

Japanese Unexamined Patent Publication No. 8-89731 Japanese Unexamined Patent Publication No. 10-237504 JP-A-2004-195464

An object of the present invention is to promote the outflow of aggregates formed by non-specific adsorption of lipids and proteins in a biological sample in the analysis column without staying in the column in the analysis of the biological sample by liquid chromatography. However, it is an object of the present invention to provide a column filter capable of improving the life of the column.

As a result of diligent studies, the present inventor has found that by using a spherical powder sintered filter made of a sintered body formed of spherical powder, deterioration of column durability due to an increase in pressure can be suppressed. The invention was completed.

Hereinafter, the present invention will be described in detail.

The present invention is a sintered body characterized in that a spherical powder having a minimum particle size of 30 μm or more and a maximum particle size of 50 μm or less is sintered and the particles are maintained as spherical particles. It relates to a column filter for liquid chromatography having a thickness of 1.5 mm or more.

As the spherical powder having a minimum particle size of 30 μm or more and a maximum particle size of 50 μm or less, there is no problem if a powder classified within the range of 30 to 50 μm is used. The minimum particle size of 30 μm or more is assumed to be about 6 to 12 times the particle size of the column packing material, and the maximum particle size of 50 μm or less is about 10 to 20 times the particle size of the column packing material. Is assumed. The effect of the present invention can be expected to suppress an increase in column pressure regardless of the particle size of the column packing material. However, since the pressure increase usually becomes a problem in a column having a small particle size of the column packing material, the column packing material A large effect can be expected with a column having a particle size of 5 μm or less.

The present invention needs to be composed of a sintered body characterized in that the particles are maintained as spherical particles, but the sintering method is particularly limited as long as the particles are maintained as spherical particles. However, for example, there is no problem if the particles are filled and held in a container without pressure and sintered with an inert gas or vacuum under no pressure.

Further, the present invention needs to have a thickness of 1.5 mm or more, preferably 1.5 mm or more and 2.5 mm or less. If the thickness is less than 1.5 mm, the column filler may leak due to insufficient strength of the filter due to deformation from external pressure or deterioration of filter performance. On the other hand, if the thickness exceeds 2.5 mm, the dead volume tends to increase, and the performance of the column tends to decrease, for example, the number of theoretical plates of the measurement result decreases.

Further, the filtration accuracy of the column filter is preferably about 0.1 to 1.0 times the average value of the particle size distribution of the column packing material. If the filtration accuracy is too small, the pressure loss of the filter itself becomes large, and the outflow effect from the column filter may not be expected. If the column filtration accuracy is too large, the column packing material may leak.

The conventional deformed powder sintered filter has a high density, and as shown in FIG. 1, some of the flow paths have a dead end shape, so that the pressure loss of the filter itself is large. On the other hand, in the spherical powder sintered filter, since the spheres are in contact with each other, the density is smaller than the filtration accuracy, and the unit structure thereof is easily maintained. Therefore, as shown in FIG. 2, the flow path is not easily blocked and the filter is filtered. The pressure loss of itself can be reduced. By reducing the density, each substance containing impurities with aggregation that exists in the column can flow out without staying at the contact surface between the column packing material on the column outlet side and the filter or inside the column filter. Life can be improved. The spherical powder sintered filter can be used on the inlet side of the column, but it is effective to apply this effect to the outlet side (outflow side) in the liquid feeding direction.

As the spherical powder in the present invention, glass or engineering plastic resin can also be applied, but spherical metal powder which is inactive to some extent with respect to the biological sample as described above and has high strength is preferable, and stainless steel is particularly preferable. preferable.

It is an enlarged photograph of a deformed powder sintered filter. An enlarged photograph of a spherical powder sintered filter is shown. The schematic diagram of a column and a column filter is shown. The system outline of the liquid chromatograph apparatus is shown. The chromatogram obtained by the apparatus of FIG. 4 is shown. The graph which shows the relationship between the number of injections of a sample and the pressure applied to a column is shown.

Hereinafter, the present invention will be described in detail by giving examples and comparative examples of the present invention, but the present invention is not limited thereto.

(Example 1)
Spherical powder sintering filter (φ3.1 mm x thickness 2.0 mm, filtration accuracy) formed by sintering spherical powder made of stainless steel, which has a particle size classified within the range of 30 to 50 μm. 1 μm) was provided by Tomoe Seisakusho Co., Ltd.

This spherical powder sintered filter is applied to the outlet side column filter of "TSKgel Lipopak-AEXII" (manufactured by Tosoh Corporation), which is a dedicated column for an automatic lipoprotein analyzer (manufactured by Tosoh Corporation, trade name "HLC-729LPII"). did.

This "HLC-729LPII" contains high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL) in serum based on the anion exchange chromatography method. Five lipoproteins can be separated and measured in 5.2 minutes per sample. A schematic diagram of the column and the column filter is shown in FIG. A deformed powder sintered filter was applied as the inflow side column filter (002), and a spherical powder sintered filter was applied as the outflow side column filter (004). There is a column filler (003) between them, and each filter is fitted in a Teflon sleeve (001). Liquid feeding lines (005) connecting to the system are connected to both ends of the column. The outline of the system is shown in FIG. "TSKgel Lipopak-AEXII" is an anion exchange column having a DEAE group which is an anion exchange group and having a packing material having a particle size of 2.5 μm as a center value. As the eluate, the eluent 1st solution (100) (Tris buffer), the eluent 2nd solution (101) (Tris buffer, 250 mM sodium perchlorate), and the eluent 3rd solution, which are the eluents dedicated to the above apparatus. (102) (Tris buffer, 400 mM sodium perchlorate) is used. Anion exchange columns (108) with column filters (109), fed by step gradients with varying salt concentrations by pumps (103, 104 and 105) corresponding to each eluent, mixed by mixer (106). ) Separates and elutes to fractionate the lipoprotein. The measurement sample was automatically injected with a sampler (107) using serum. Each lipoprotein separated was cholesterol-stained by a cholesterol reaction solution (110) and a reaction solution pump (111), and detected by a detector (112) at a wavelength of 590 nm. In addition, a pressure sensor (113) was provided between the pump and the column. An example of the chromatogram obtained by this system is shown in FIG. Cholesterol in HDL (201), LDL (202), IDL (203), VLDL (204), Other (205), which are lipoproteins in human serum (Other includes chylomicrons and Lipoprotein (a)) Was measured. A pool sample of a healthy person was used as a serum sample. This serum sample was automatically injected with 1.0 μL per measurement by the above device without pretreatment or dilution. The measurement was carried out for 50 to 100 samples per batch of measurement until a significant increase in pressure was confirmed. This measurement was performed on three columns manufactured in the same way. The number of measurement injections and the results of the column pressure {measured by the pressure sensor (113)} are shown in FIG. In each column, the system pressure reached 10 MPa by measuring 450 samples or more.

(Comparative Example 1)
The measurement was carried out in the same manner as in Example 1 except for the outlet side column filter of "TSKgel Lipopak-AEXII" (manufactured by Tosoh Corporation). As the outlet side filter, a deformed powder sintered filter (φ3.5 mm × thickness 1.0 mm, filtration accuracy 1 μm) formed by sintering a deformed powder made of stainless steel was used. This deformed powder sintered filter was provided by Tomoe Seisakusho Co., Ltd. This measurement was performed on three columns manufactured in the same way. The number of measurement injections and the results of the column pressure {measured by the pressure sensor (113)} are shown in FIG. In each column, the system pressure reached 10 MPa in the measurement of 300 samples or less.

In Example 1, the pressure rise was gradual in each of the columns as compared with the column of Comparative Example 1. This is derived from blood deposited or aggregated inside the column by adopting a spherical powder sintered filter formed by sintering stainless steel spherical powder as the outlet side column filter in Example 1. It was suggested that the material was promoted outflow without staying in the outlet column filter.

(Comparative Example 2)
Spherical powder sintering filter (φ3.1 mm x thickness 2.0 mm, filtration accuracy) formed by sintering spherical powder made of stainless steel, which has a particle size classified within the range of 50 to 100 μm. 2 μm) was provided by Tomoe Seisakusho Co., Ltd.

This spherical powder sintered filter is applied to the outlet side column filter of "TSKgel Lipopak-AEXII" (manufactured by Tosoh Corporation), which is a dedicated column for an automatic lipoprotein analyzer (manufactured by Tosoh Corporation, trade name "HLC-729LPII"). did. When the column was filled with a filler having a DEAE group which is an anion exchange group and having a particle size of 2.5 μm as the center value, gel leakage occurred in this example. This is because in a spherical powder sintering filter formed by sintering spherical powder made of stainless steel having a particle size of 50 μm or more, the main role of the column is to prevent the column filler from flowing out of the column. The result was that the function of the filter was not fulfilled.

(001) Column filter sleeve (002) Inflow side (inlet side) deformed powder sintered column filter (003) Column filler (004) Outflow side (outlet side) spherical powder sintered column filter (005) Liquid feed line (100) Eluent 1
(101) Eluent 2
(102) Eluent 3
(103) Pump 1
(104) Pump 2
(105) Pump 3
(106) Mixer (107) Auto Sampler (108) Column (109) Column Filter (110) Cholesterol Reaction Solution (111) Pump for Feeding Cholesterol Reaction Solution (112) Detector (113) Pressure Sensor (201) HDL Cholesterol Peak (202) LDL Cholesterol Peak (203) IDL Cholesterol Peak (204) VLDL Cholesterol Peak (205) Other Cholesterol Peak

Claims (2)

A sintered body characterized in that spherical powder having a minimum particle size of 30 μm or more and a maximum particle size of 50 μm or less is sintered and the particles are maintained as spherical particles (however, ceramic baking). A column filter for liquid chromatography with a thickness of 1.5 mm or more on the outlet side in the liquid feeding direction, which is composed of (excluding batters). The column filter for liquid chromatography according to claim 1, wherein the sintered body is stainless steel.

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