JP2018059885A - Column filter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a column filter capable of extending life of a column by forcing aggregates, produced from lipids and proteins of a biological sample nonspecifically attracted in an analysis column, to flow out of the column without stagnating therein during a liquid chromatographic analysis of the biological sample.SOLUTION: A liquid chromatography column filter consists of a sintered product obtained by sintering spherical powder having a minimum particle diameter of 30 μm or greater and a maximum particle diameter of 50 μm or less to keep the particles spherical, and has a thickness no less than 1.5 mm.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a column filter that is a sintered body composed of spherical powder.

  In the 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 that uses a biological sample as a measurement object, a stainless steel filter that is inactive to some extent is used.

  As a specific example of such a stainless steel filter, a stainless steel short fiber or fine powder sintered at high temperature and high pressure is used. Conventional stainless steel filters used irregularly shaped powders such as short fibers or fine powders whose sizes and shapes are not strictly constant, but they do not have a fixed unit structure, so they are packed at the column outlet side. Substances other than the drug accumulated and led to aggregation.

  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 used. The average particle size of the metal powder was not in an appropriate range.

JP-A-8-89931 JP-A-10-237504 JP 2004-195464 A

  The purpose of the present invention is to promote the flow-out of aggregates generated by non-specific adsorption of lipids and proteins in a biological sample without remaining in the column in the analysis of the biological sample by liquid chromatography. The object of the present invention is to provide a column filter capable of improving the lifetime of the column.

  As a result of intensive studies, the present inventor has found that by using a spherical powder sintered filter made of a sintered body formed from a spherical powder, it is possible to suppress deterioration in the durability of the column due to an increase in pressure. The invention has been completed.

  Hereinafter, the present invention will be described in detail.

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

  A spherical powder having a minimum particle size of 30 μm or more and a maximum particle size of 50 μm or less has no problem if a powder classified in 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, and the maximum particle size of 50 μm or less is about 10 to 20 times that of the column packing. Is assumed. The effect of the present invention can be expected to suppress an increase in the column pressure regardless of the particle size of the column packing material. However, since the pressure increase is usually a problem in a column having a small column packing particle size, 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. For example, there is no problem if the container is filled and held without pressure and sintered under an inert gas or vacuum and no pressure.

  Moreover, this invention needs to be 1.5 mm or more in thickness, Preferably it is 1.5 mm or more and 2.5 mm or less. When the thickness is less than 1.5 mm, there is a possibility that the column filler may leak due to insufficient strength of the filter due to deformation from the external pressure or deterioration of the filter performance. On the other hand, when the thickness exceeds 2.5 mm, the dead volume increases, and the column performance tends to decrease, for example, the number of theoretical plates of the measurement result decreases.

  Furthermore, 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 filler. If the filtration accuracy is too small, the pressure loss of the filter itself is increased, and the outflow effect from the column filter may not be expected, and if the column filtration accuracy is too large, the column filler may leak.

  Since the conventional deformed powder sintered filter has a high density and a flow path has a bag-like shape as shown in FIG. 1, the pressure loss of the filter itself is large. On the other hand, since the spherical powder sintered filter is in contact with each other, the density is smaller than the filtration accuracy and the unit structure is easily maintained. Therefore, as shown in FIG. The pressure loss of itself can be reduced. By reducing the density, each substance that contains impurities with aggregation, which is present in the column, can flow out without staying on the column filler and filter contact surface on the column outlet side or inside the column filter. Can improve the service life. 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 be applied, but a spherical metal powder which is inactive to some extent as described above and has high strength is preferable, and particularly stainless steel. preferable.

The enlarged photograph of a deformed powder sintered filter is shown. The enlarged photograph of a spherical powder sintered filter is shown. The schematic of a column and a column filter is shown. The system outline | summary of a liquid chromatograph apparatus is shown. Fig. 5 shows a chromatogram obtained with the apparatus of Fig. 4. 3 is a graph showing the relationship between the number of injections of the specimen and the pressure applied to the column.

  EXAMPLES Hereinafter, although an Example and comparative example of this invention are given and this invention is demonstrated in detail, this invention is not limited to this.

Example 1
Spherical powder sintered filter (φ 3.1 mm × thickness 2.0 mm, filtration accuracy, formed by sintering stainless steel spherical powder with a particle size classified within a range of 30-50 μm in diameter. 1 μm) was provided by Sakai Manufacturing Co., Ltd.

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

  This “HLC-729LPII” includes high density lipoprotein (HDL), low density lipoprotein (LDL), and very low density lipoprotein (VLDL) as serum lipoproteins based on the principle of anion exchange chromatography. It is possible to separate and measure five lipoproteins in 5.2 minutes per sample. A schematic diagram of the column and column filter is shown in FIG. A deformed powder sintered filter was applied as the column filter (002) on the inflow side, and a spherical powder sintered filter was applied as the column filter (004) on the outflow side. Between them, there is a column filler (003), and each filter is fitted on a Teflon sleeve (001). A liquid feed line (005) connected to the system is connected to both ends of the column. The system outline is shown in FIG. “TSKgel Lipopropak-AEXII” is an anion exchange column having a packing having a particle size of 2.5 μm and having a DEAE group which is an anion exchange group. As eluents, eluent first liquid (100) (Tris buffer), eluent second liquid (101) (Tris buffer, 250 mM sodium perchlorate), eluent third liquid, which are eluents dedicated to the above apparatus (102) (Tris buffer, 400 mM sodium perchlorate) is used. The anion exchange column (108) having a column filter (109) fed by a step gradient with varying salt concentration by the pump (103, 104 and 105) corresponding to each eluent, mixed by a mixer (106), and having a column filter (109). ) To separate and elute and fractionate lipoproteins. The measurement sample was automatically injected with serum using a sampler (107). Each separated lipoprotein was stained with cholesterol by a cholesterol reaction solution (110) and a reaction solution pump (111), and detected by a detector (112) at a wavelength of 590 nm. A pressure sensor (113) was provided between the pump and the column. An example of a chromatogram obtained by this system is shown in FIG. Cholesterol in HDL (201), LDL (202), IDL (203), VLDL (204), Other (205) (others include chylomicron and Lipoprotein (a)) which are lipoproteins in human serum Was measured. A healthy human pool specimen was used as a serum sample. This serum sample was automatically injected with 1.0 μL per measurement by the above apparatus without pretreatment or dilution. The measurement was carried out until 50 to 100 specimens were measured per batch and the pressure increase was confirmed remarkably. This measurement was performed on three columns made in the same way. The results of measurement injection frequency and column pressure {measured with a pressure sensor (113)} are shown in FIG. In all the columns, the system pressure reached 10 MPa by measuring 450 samples or more.

(Comparative Example 1)
The measurement was performed in the same manner as in Example 1 except for the outlet side column filter of “TSKgel Lipopropak-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 deformed powder made of stainless steel was used. This deformed powder sintered filter was provided by Sakai Manufacturing Co., Ltd. This measurement was performed on three columns made in the same way. The results of measurement injection frequency and column pressure {measured with a pressure sensor (113)} are shown in FIG. In any column, the system pressure reached 10 MPa by measuring 300 samples or less.

  In Example 1, the pressure increase was moderate in all the columns as compared with the column in Comparative Example 1. This is because the spherical powder sintered filter formed by sintering the spherical powder made of stainless steel was adopted as the outlet side column filter in Example 1, and it was derived from blood deposited or aggregated inside the column. It was suggested that the substance was the result of accelerated outflow without remaining in the outlet side column filter.

(Comparative Example 2)
Spherical powder sintered filter (φ3.1 mm × thickness 2.0 mm, filtration accuracy, formed by sintering stainless steel spherical powder with a particle size classified in the range of 50 to 100 μm in diameter. 2 μm) was provided by Sakai Manufacturing Co., Ltd.

  This spherical powder sintered filter is applied to the column filter on the outlet side of “TSKgel Lipopropak-AEXII” (manufactured by Tosoh Corporation), a dedicated column for automatic lipoprotein analyzer (trade name “HLC-729LPII”, manufactured by Tosoh Corporation). did. When the column was packed with a packing material having a particle size of 2.5 μm having a DEAE group as an anion exchange group and having a center value, gel leakage occurred in this example. This is the main role of the spherical powder sintered filter formed by sintering stainless steel spherical powder having a particle diameter of 50 μm or more so that the column filler does not flow out of the column. The filter function was not fulfilled.

(001) Column filter sleeve (002) Inflow side (inlet side) deformed powder sintered column filter (003) Column filler (004) Outlet 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) Autosampler (108) Column (109) Column filter (110) Cholesterol reaction liquid (111) Pump (112) for feeding cholesterol reaction liquid 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 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 sintered particles are characterized in that the particles are maintained as spherical particles. A column filter for liquid chromatography having a thickness of 1.5 mm or more.   A column for liquid chromatography, wherein the column filter on the outlet side in the liquid feeding direction is the column filter according to claim 1.

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