JP2015114273A - Column for liquid chromatography having a convex filter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a filter that can be easily applied to a small-capacity analysis column in particular by the common method of fitting the filter and a column end to each other in a pressure-released state and can maintain a state of being densely filled under high pressure.SOLUTION: A filter for use at the column end of a column for liquid chromatography has a hole that does not allow passage of column filler to fill the column, a flat bottom and another face that is convex and at least partly invades into the column for liquid chromatography to occupy part of the internal space of the column.

Description

  The present invention relates to a liquid chromatography column having a filter having a convex shape.

  In the analysis by liquid chromatography, a small-capacity column (for example, inner diameter of 5 mm) packed with a finer column filler (hereinafter also simply referred to as fine particles) of 3 μm or less for high-speed and high-resolution analysis. , About 40 mm in length) is being used. The process of filling fine particles in such a small volume column is generally performed by dispensing a predetermined amount of a fine particle dispersion suspension into a reservoir attached to the column, and a tube having sufficient pressure resistance above the reservoir. Attach, pump the solvent using a high-pressure pump, pump the suspension in the reservoir into the column, fill the column with microparticles under high pressure, stop pumping and pump the reservoir to the column The step of attaching the column end equipped with a filter that does not allow the passage of fine particles with the pressure released and the pressure released, the more easily the fine particles are elastically deformed, the more densely packed under high pressure It is important to keep the pressure after the pressure is released. This is because the packed state of the fine particles slightly changes when the pressure is released, but the slight change can greatly affect the separation performance of the column. On the other hand, in a large column used for fractionation, there are devices disclosed in, for example, Patent Document 1 and Patent Document 2 as devices capable of maintaining a high-density packed state under high pressure when packed with a column filler. .

Japanese Patent Laid-Open No. 4-98156 Special table Hei 8-509551

  If a relatively large column is packed in a relatively large column, a general method of attaching a filter and a column end with the pressure released can obtain a sufficient effect. However, in the case where fine particles are packed into a small-capacity column by this method, it is difficult to maintain the state of the fine particles packed with high density under high pressure after the pressure is released. In the devices disclosed in Patent Document 1 and Patent Document 2, the structure of the column is complicated, and it is difficult to apply to a small-capacity column. It is inconvenient as an analytical column that is often used by replacing it every time, and when a movable part such as a movable stopper or a valve is clogged, the separation performance of the column is affected.

  Therefore, the present invention is easy to apply to a small-capacity analytical column by a general method of attaching a filter and a column end with the pressure released. The object is to provide a filter that can be retained.

The present invention made to achieve the above object is a filter used in a column for liquid chromatography, having a hole through which the column packing material to be packed does not pass, a bottom surface being a flat surface, and the other surface being a flat surface. It is a filter which is a convex surface and at least a part of which enters the liquid chromatography column and occupies a part of the internal space of the column. Hereinafter, the present invention will be described in detail.

  The filter of the present invention is used together with the column end of a liquid chromatography column. The column for liquid chromatography is usually made of metal or resin, and is usually cylindrical, and various types such as molecular exclusion liquid chromatography, ion exchange liquid chromatography, affinity liquid chromatography, reverse phase liquid chromatography, etc. The column for liquid chromatography. The column end is a member that closes both ends of the column, and may be made of a material different from that of the column or the same material. The mechanism for closing the column end by the column end can be exemplified by a screwing type to the column end, but there is no particular limitation.

  The column packing material is filled in the column with one end of the column previously closed by the first filter and the column end, and the other end is closed by the second filter and the column end after filling. Although the present invention is used as a second filter that closes a column after being filled with a column packing material, there is no limitation to use it as a first filter.

  The filter has pressure resistance and does not allow column fillers to pass through, but it is necessary to pass eluents used in liquid chromatography. Examples of the filter of the present invention include a porous sintered filter manufactured by sintering metal pieces, a porous filter manufactured using a resin such as PEEK, Teflon (registered trademark), and polyethylene. The pore size of the filter may be determined based on the particle size of the column packing material, and various types such as hydrophilicity or non-hydrophilicity may be used in order to suppress interference with an object to be analyzed by liquid chromatography. Processing may be performed. The shape of the filter may be any shape that can close the column end, and examples of the shape of the filter include a columnar shape and a polygonal columnar shape in view of a normal column being in a cylindrical state.

  The filter of the present invention is fixed to the end of a column for liquid chromatography by a column end, and serves as an inlet for a sample to be analyzed or an outlet for a sample separated by a column filler. Therefore, in order to prevent the diffusion from becoming non-uniform when the sample passes through the filter, the bottom surface is preferably a flat surface. The surface opposed to the bottom surface of the filter is a convex surface that partially enters the liquid chromatography column and occupies a part of the column internal space. Here, entering the column means that the capacity of the column is reduced when the column is fixed to the end of the column. In the present invention, a filter having a convex surface that reduces the column volume by 0.5% to 5% is preferable. Note that the convex surface can be considered as a protrusion formed on a plane from another viewpoint. For example, one or a plurality of protrusions having a rod shape, a cone shape, or a shape obtained by cutting a part of a sphere may be formed on one surface of a cylinder. It is particularly preferable that one protrusion having a shape that can uniformly apply pressure to the packed column packing material, for example, a shape obtained by cutting a part of a sphere (eg, a half-moon shape) on one plane of a cylinder. It can be exemplified that the column end is closed by the circular arc portion of the sphere.

  Hereinafter, one embodiment of the present invention described in the drawings will be described. FIG. 1 is a view showing a filter and a column end of the present invention. The filter 4 is incorporated into the column end via the seal member 3 and is fixed to the column end by screwing the column end into one end of the cylindrical column main body 1. The bottom surface (column end side) of the filter 4 is a flat surface (flat), but the opposite surface (column filler side) 4a formed a protrusion obtained by cutting off a part of a sphere on the surface of a cylindrical body. Convex shape. The convex surface 4a has a high density before pressure release by pressurizing the packing in the column whose pressure has been released again by pressing the convex part into the column when the column fitting is attached to the column end. Reproduce the state filled in.

  In the example of FIG. 1, the column has an inner diameter of 5 mm, a length of 20 mm, and a volume of 392 μl. The filter convex portion 4 a that penetrates into the column of the filter 4 and occupies a part of its inner space is It is designed to occupy 3% of the volume (column internal space).

  By using the filter of the present invention, for example, when packing fine particles of 3 μm or less into a small-capacity column having an inner diameter of 5 mm and a length of about 40 mm, the pressure in the column once released from the pressure is re-pressurized. It is possible to reproduce the state of being filled with high density before opening. As a result, it is possible to suppress fluctuations in column separation performance due to changes in the packing state of the fine particles, and it is possible to manufacture a high-performance and homogeneous column in large quantities. Further, as shown in the examples, it is possible to provide a column that is not easily deteriorated (a highly durable column). Moreover, the present invention can be applied to conventional columns. Therefore, there is no waste of cost burden and production of a new column in the implementation.

FIG. 1 is a diagram for explaining the present invention, and is a cross-sectional view showing how a column, a filter, and a column end are attached. FIG. 2 is a diagram illustrating the relationship between the number of samples and the number of theoretical plates in Example 1. FIG. 3 is a diagram illustrating the relationship between the number of samples and the number of theoretical plates in Comparative Example 1.

1 Column for liquid chromatography 2 Column end 3 Shield member 4 Filter 4a Convex part of filter

    Examples will be shown to describe the present invention in more detail, but the present invention is not limited to the examples.

Example 1
A non-porous cation packing material (1 ml) having a particle size of 3 μm was packed into a SUS column having an inner diameter of 5 mm and a length of 20 mm at a packing pressure of 50 MPa. The column end has the shape shown in FIG. 1, and the SUS filter (the flat portion is a circle with a diameter of 5 mm and a porosity of 1 μm) is incorporated into the column end made of SUS via a seal member (material Teflon (registered trademark)). It is fixed by crimping to the column end by screwing the column end into the column body. The bottom surface (column end side) of the filter is a flat surface (flat), but the opposite surface (column filler side) is a projection (height 0. 0) cut out of a part of a sphere on the surface of the cylindrical body. 8 mm). This convex surface occupies 3% of the internal space of the column.

  The above column was attached to a commercially available glycohemoglobin analyzer (trade name HLC-723G9, manufactured by Tosoh Corporation), and commercial products for the apparatus (G9HSi eluent 1 liquid, G9HSi eluent 2 liquid, and G9HSi eluent 3) were used as the eluent. Using liquids, both trade names, manufactured by Tosoh Corporation), stable glycated hemoglobin (hereinafter referred to as HbA1c) in human blood was measured. The column pressure at this time was about 11 MPa.

  Column durability was evaluated using the number of theoretical plates of the HbA1c peak, which is a measure for separating HbA1c. The results are shown in FIG. Comparing the number of theoretical plates at the time of 0 sample measurement and the number of theoretical plates at the time of 2000 sample measurement, the decrease in the number of theoretical plates is about 20%, and it can be seen that sufficient durability is obtained for the separation of hemoglobins.

Comparative Example 1
A non-porous cation filler having a particle diameter of 3 μm was packed in the same column as in Example 1 by performing the same operation. However, unlike Example 1, as the filter, a cylindrical filter having no convex portion was used.

  The results of evaluating column durability under the same conditions as in Example 1 are shown in FIG. The column pressure was about 11 MPa, which was not apparently different from Example 1. However, when the number of theoretical plates at the time of 0 sample measurement and the number of theoretical plates at the time of 850 sample measurement were compared, the column pressure was significantly reduced to 70% or more. It can be seen that sufficient durability is not obtained as compared with the column.

Claims (3)

A filter used for a column end of a column for liquid chromatography, having a hole through which a column filler to be packed does not pass, a bottom surface being a flat surface, and the other surface being a convex surface and at least a part thereof Is a filter that penetrates into the liquid chromatography column and occupies a part of the internal space of the column. A method for producing a column for liquid chromatography, comprising a hole through which a column packing material to be packed does not pass, a bottom surface being a flat surface, and the other surface being a convex surface, at least a part of which is liquid chromatography A method for producing a column for liquid chromatography, characterized in that a filter that penetrates into the interior of the column and occupies a part of the internal space of the column is crimped and fixed to the end of the column. 3. The liquid chromatography according to claim 2, wherein the convex portion of the filter that penetrates into the column and occupies a part of the internal space of the column occupies 0.5% to 5% of the internal space of the column. Column manufacturing method.

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