JPH02262054A - Filter for high-performance liquid chromatograph - Google Patents

Abstract

PURPOSE:To improve filtering accuracy, to prevent clogging for a long period and to improve arresting efficiency by changing the size of the diameters of the holes of filters which are provided in a liquid path in the flowing direction of liquid. CONSTITUTION:A main body 20 of a cylindrical column 20 is filled with a filler 21. A box-nut shaped coupling member 30 is screwed on the outside of the liquid input end part of the main body 20. Filters 10 are coupled into a cylindrical space part 31 of the member 30. The inner surface of the member 30 and the filters 10 are sealed with a sealing member 40 in a liquid tight manner. A sample is injected into an eluate. A pipe through which the eluate flows is provided and screwed into a coupling part 32 at the opposite end of the member 30 with respect to the side of the main body 20. The filters 10 comprise an outer filter layer 11 having the larger hole diameter and an inner filter layer 12 having the smaller hole diameter in the order from the coupling part 32. The hole diameter of the filter layer 12 is smaller than the hole diameter of the filler 21.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a filter for high performance liquid chromatography used as an analytical instrument in fields such as biochemistry and medicinal chemistry.

(Prior art) High-speed chromatographs, which are used as analytical instruments in the fields of biochemistry, medicinal chemistry, and other natural sciences, have recently been used not only for basic research but also for pharmaceutical analysis,
It is also used in clinical tests.

In high-speed chromatography, a sample is usually injected into the eluent while the eluent in the eluent container is sent to the analytical column via piping by a liquid pump, and the eluent containing the sample is transferred to the analytical column. pass it through and analyze it. In such high-speed chromatographs, in order to prevent clogging of piping and analytical columns, filters are installed in the eluent flow area.For example, some filters are installed in the eluent flow area. A solution filter is disposed between them to prevent foreign matter from entering the pipe when the eluent is fed into the pipe from the eluent container by the liquid pump. A line filter is installed in the piping connected to the analytical column in order to filter out foreign matter that has entered the eluent from the sample injected into the column, as well as worn parts of the seal parts of the liquid pump that feeds the eluent. Furthermore, column filters are provided at the liquid inflow end and the liquid outflow end of the column, respectively, to prevent the filler in the analytical column from flowing out of the column.

The column filter installed on the liquid inlet side of the column is
It also filters out foreign matter in the sample and wear of the liquid pump to prevent them from flowing into the column.

Among filters for high-speed chromatographs, line filters in particular filter out foreign substances such as solids in samples and worn parts of seal parts in liquid pumps, so they tend to get clogged and need to be replaced frequently. . The line filter usually uses short metal fibers of stainless steel, sintered bodies of fine metal powder, or a mixture of both, and the pore size varies depending on the type of sample. is approximately 1 μm to 10 μm.

(Problems to be Solved by the Invention) In high-speed chromatographs, samples containing solids are usually removed by filtration using a membrane filter or the like as a pretreatment, or by centrifugation. Separation takes place. However, depending on the measurement system, such preprocessing may not be performed because it reduces work efficiency, reduces sample recovery rate, or makes automation difficult.

For example, in clinical tests, whole blood or hemolyzed diluted red blood cells are used as samples for screening tests for diabetes and for measuring If-hemoglobin, which is an indicator of blood sugar control in diabetic patients. A dedicated automatic measuring device that applies the principles of high-speed chromatography is used to measure this glycated hemoglobin. The hemolyzed diluted sample contains solids such as red blood cell membranes,
This solid content is removed by filtration or by centrifugation. However, this removal work is difficult to automate, and even when it is done manually, the work is complicated and reduces measurement efficiency, so solid content removal work is rarely done. The reality is that this is not the case.

As described above, in a measurement system in which solid content removal work is not performed or in cases where this removal work is not performed properly, solid content contained in the sample is filtered by the line filter. For this reason, there is a problem in that the line filter is easily clogged. If the line filter becomes clogged during measurement, the pressure in the flow path increases, and in extreme cases, measurement becomes impossible. When this happens, it is necessary to immediately interrupt the measurement, stop feeding the eluent, and replace the line filter. Then, after replacing the line filter, feeding of the eluent is restarted, and the measurement is restarted after the measurement system becomes stable.

Filter clogging depends on the filter's pore size and porosity (
This is related to the void volume ratio per unit volume of the filter.If the pore size is made smaller, smaller foreign substances can be filtered out and filtration accuracy improves, but as the porosity decreases, the filter The amount of foreign matter collected is small and it is easy to get clogged, resulting in poor collection efficiency.

Furthermore, solids that cannot be captured by filtration with a line filter must be filtered with a column filter on the liquid inflow side of the analytical column. If the solid content is not filtered by the column filter, the solid content will enter the packing material of the column, clogging the column and causing contamination, making the column unusable. If such column clogging or contamination occurs, the packing material must be removed from the column, washed, and then packed into the column. However, it is very difficult to fill a column with a filler, and in particular, it is almost impossible for an operator to fill a column for high-speed chromatography.

Recently, as fillers have become finer particles,
There is a need to reduce the pore size of column filters. If the pore size of the column filter becomes larger than the particle size of the filler, the filler will clog the column filter. As described above, when the pore size is made smaller, although the filtration accuracy is improved, there is a problem in that the collection efficiency is reduced.

The present invention solves the above-mentioned conventional problems, and its purpose is to provide a filter for high-speed chromatography that has excellent filtration accuracy, is free from clogging over a long period of time, and has excellent collection efficiency. It's about doing.

(Means for Solving Problem 11) The filter for high-speed chromatography of the present invention is a filter that is installed in a liquid flow area in a high-performance liquid chromatograph, and has pore sizes that are sequentially arranged in the liquid flow direction. It is characterized by the fact that it is changing, thereby achieving the above object.

(Example) The present invention will be described below with reference to Examples.

FIG. 1 is a sectional view of a main part of a column equipped with a column filter, which is an example of a filter for high-speed chromatography according to the present invention. The column has a cylindrical column body 20, and the column body 20 is filled with a packing material 21.

A connecting member 30 in the form of a cap nut is externally fitted and screwed onto the end of the column body 20 on the liquid inflow side, for example.

When the connecting member 30 is attached to the column main body 20, a continuous columnar space 31 is formed on the end face of the column main body 20, and a columnar space 31 of the present invention is provided in the space 31. A filter 10 is fitted therein. Connecting member 3
A cylindrical sealing member 40 made of, for example, fluororesin is interposed in the space 31 of No. 0 to liquid-tightly seal the filter 10 and the inner peripheral surface of the connecting member 30. At the end of the connecting member 30 opposite to the column body 20 side, a connecting part 32 is provided, into which a pipe through which an eluent in which a sample is injected flows is fitted and screwed. The connecting portion 32 communicates with the space 31 in which the column filter 10 is accommodated.

The column filter 10 accommodated in the space 31 of the connecting member 30 has an outer filter layer 11 with a large pore size arranged on the side of the connecting part 32 in the connecting member 3o, and an outer filter layer 11 with a large pore size arranged on the side of the column body 20. It has a small daylight side filter layer 12. The inner filter layer 12 is in contact with the end surface of the packing material 21 filled in the column body 20.

The pore size of the inner filter layer 12 in contact with the filler 21 is smaller than the particle size of the filler 21.

If the pore size of the inner filter layer 12 becomes larger than the particle size of the filler 21, the filler will clog the inner filter layer 12, increasing column pressure or creating gaps in the filler 21. In this way, the pore size of the inner filter layer 12C may be smaller than the particle size of the filler 21, but if the pore size is too small (too much), the pressure loss of the inner filter layer 12 will increase. If the flow rate of the liquid passing through the filter layer is the same, the flow rate increases as the pore diameter becomes smaller, and if the pore size remains the same, the flow rate increases as the thickness (length in the liquid passage direction) increases.For this reason, the inner filter layer The pore diameter of No. 12 is preferably 1 μm or more.

The pore size of the outer filter layer 11 is the same as that of the inner filter layer 12.
(but smaller than the foreign matter to be filtered. In normal high-speed chromatography, considering the size of the target foreign matter, it is preferably 2 μm or more and 20 μm or less, more preferably 2 μm. 10μm or more
The following shall apply.

The column filter 10 does not need to have two layers, the outer filter layer 11 and the inner filter layer 12, but has three or more layers, as long as the pore diameter changes sequentially such that the inner side is smaller and the outer side is larger. It may be a laminated structure or a structure in which the pore diameters are continuously different.

The filtration efficiency improves as the overall thickness (length in the liquid flow direction) of the column filter IO becomes thicker, provided that pressure loss, liquid diffusion, etc. do not affect measurement. Usually, it is 1 mm or more and 5 mm or less, preferably 1 mm or more and 3 m+a or less. The thickness of each filter layer 11 and 12 is
They don't have to be equal.

The material of the column filter 10 may be any material as long as it is inert to the eluent and the sample, and for example, metals such as stainless steel or ceramics are used.

When the column filter 10 has a laminated structure of the outer filter layer 11 and the inner filter layer 12 as in the above embodiment, for example, metal powder and scrap metal fibers as raw materials for each layer are heated at high temperature and under high pressure. There are two methods: sintering at the bottom to form single layers and stacking them, and sintering multiple layers at the same time.

Such a column filter 10 may be disposed only at the end of the column on the liquid inflow side, and a conventional filter may be disposed at the end of the column on the liquid outflow side. If the column body has a structure in which the liquid inflow side and liquid outflow side are not defined and liquid can flow in from either end, a column filter similar to the above example is installed at each end with a pore diameter The filter layer with a large pore size may be placed on the outside, and the filter layer with a small pore size may be placed on the inside (on the column body side).

FIG. 2 is a sectional view of a line filter mounting structure which is another embodiment of the high speed chromatography filter of the present invention. The line filter 50 of the present invention is interposed in the flow path of the eluent into which the sample is injected. The line filter 50 is disposed within a holder 60 in which each half body 61 is integrated by screw connection. Each half 61 of the holder 60 is arranged in parallel in the flow direction of the eluent into which the sample is injected. Each half body 61 has cylindrical spaces 61a and 61a formed on the respective negative sides so that openings thereof face each other, and a cylindrical seal member extends between the rain spaces 61a. 70 is fitted. A cylindrical line filter 50 is supported within the cylindrical seal member 70.

The sealing member 70 is made of, for example, fluororesin, and seals the line filter 50 and the peripheral surface of the space 61a of each half 61 of the holder 60 in a liquid-tight state.

Each half 61 in the holder 60 has a respective space 61a on the side opposite to the side where each space 61a is provided.
A connecting recess 61b communicating with each other is provided respectively.

Each connecting recess 61b is screwed with each pipe through which an eluent in which a sample is injected flows.

The line filter 50 is supported by the upstream portion of the seal member 70 in the eluent flow direction, and a space is formed inside the downstream portion of the seal member 70 in the eluent flow direction.

The line filter 50 also has an inflow side filter layer 51 with a large pore diameter, similar to the column filter IO of the above embodiment.
The inlet filter layer 51 has a large pore size on the upstream side in the eluent flow direction, and the outlet filter layer 52 has a small pore size on the upstream side in the eluent flow direction. It is located on the downstream side in the flow direction.

Outflow side filter layer 5 in the line filter 50
The pore diameter of 2 is 1. The pore diameter is the same or slightly smaller than the column filter installed in the column connected via piping. If the pore size of the outflow side filter layer 52 becomes larger than the pore size of the column filter, foreign matter that is not captured by the outflow side filter 52 will be captured by the column filter or the packing material in the column, resulting in clogging of the column. The pore size of the column filter varies depending on the particle size of the filler and the inner diameter of the column, but is usually 5 μm or less, so the pore size of the outflow filter layer 52 is 5 μm or less.

If the pore size becomes too small, the outflow side filter layer 52
pressure loss increases. Pressure loss increases as the pore diameter becomes smaller if the flow rate of liquid passing through the filter layer is the same, and increases as the thickness (length in the liquid passage direction) increases if the pore diameter remains the same. For this reason,
The pore diameter of the outflow side filter layer 52 is preferably 1 μm or more.

The pore diameter of the inflow side filter layer 51 only needs to be larger than the pore diameter of the outflow side filter layer 52 and smaller than the foreign matter to be filtered. Since the size of foreign particles differs depending on the substance to be measured, the pore diameter of the inflow side filter layer 51 varies depending on the substance to be measured, but in a normal high-speed chromatograph, considering the size of the target foreign substance, it is 2 μm or more and 20 μm. Hereinafter, the thickness is preferably 2 μm or more and 10 μm or less.

The filtration efficiency improves as the overall thickness (length in the liquid flow direction) of the line filter 50 becomes thicker, provided that pressure loss, liquid diffusion, etc. do not affect measurement. Therefore, it is preferably 1 mm or more and 5 mm or less, more preferably 1 mm or more and 3 rm or less. The thickness of each filter layer 51 and 52 does not need to be equal.

The line filter 50 does not need to have a two-layer structure like the column filter 10 of the above embodiment, but may have a laminated structure of three or more layers, or a structure with continuously different pore sizes. The material used is also the same as that of the column filter.

Next, the performance of the high-speed chromatography filter of the present invention was tested and will be described below.

(Experiment Example 1) The thickness of each filter layer is 1.5 μm, and the pore diameter of each filter layer is 21 μm and 5 μm.
One structure filter (a), each filter layer has a thickness of 1.5r11m, and the two-layer structure filter (b) has a pore size of 10 μm and 2 μm, each filter layer has a thickness of 1.5×11 m. The pore size of each filter layer is 5μm and 2μm, respectively.
A filter with a two-layer structure (C), each filter layer having a thickness of 1. on+, the filter (d) is made of 3 mm with the pore sizes of each filter layer being 10 μm, 5 μm, and 2 μm, and the thickness of one filter layer is 2.0 μm.
The pore size was 10 μm, and the thickness of the other filter layer was 1. oaox and a two-layer filter (e) having a pore size of 5 μm were manufactured.

The material of each filter was a metal sintered body made of a mixture of metal single fibers made of stainless steel and metal fine powder, and each filter was cylindrical and had a diameter of 5 m+n. The pore size of each filter is JIS standard B asoil! It was determined by measuring the bubble point pressure in the excessive particle size test. and,
Each filter was stamped into a cylindrical sealing member made of fluororesin and mounted in a holder shown in FIG. 2. At this time, the filtration area of each filter is 3 m in diameter.
It was a circular shape.

Each holder 60 with each filter mounted in this manner was set in the test apparatus shown in FIG. 3. The test apparatus is configured to cause distilled water in a container 81 to flow through a pipe line 83 using a constant flow pump 82. The pipe line 83
A standard particle liquid container 84 containing a standard particle liquid and a holder 85 are interposed in this order. The holder 8
5 is equipped with the above-described multilayered filter such that the filter layer with the larger pore size is located on the upstream side.

The standard particle liquid container contains standard particles (manufactured by Sekisui Chemical Co., Ltd., trade name: rMI) having a particle size distribution as shown in Figure 4.
CRONEX AlC-H5nJ) with distilled water
．． A standard particle solution (hereinafter referred to as sample stock solution) dispersed at a concentration of 0.02 g/L is stored. Distilled water is flowed into the pipe line 83 by a constant flow pump 82 at a rate of 5 ml/m.
The standard particle liquid in the standard particle liquid container 84 was fed to the holder 85 and filtered by the filter in the holder 85. The filtered filtrate is stored in a container 86.
Collected at.

The collected filtrate 2el was diluted 100 times with an electrolytic solution (manufactured by Coulter Co., trade name: rl5OTQN-INJ), and the particle size distribution of the diluted solution was measured using a Coulter Counter (manufactured by Coulter Co., Ltd.). did.

Such a test was conducted by mounting each of the above-mentioned filters a'=e on the holder 85. The results are shown in FIG. In FIG. 5, the horizontal axis shows the particle diameter of the standard particles, and the vertical axis shows the collection efficiency (I) for the sample stock solution. The collection efficiency (H) was determined by the following formula.

H= ((n − k) /n) XIQO (%) n: Number of particles in the sample stock solution: i! ! The number of particles in the liquid and the particle diameter when the collection efficiency is 95% are defined as filtration accuracy, and the results are shown in Table 1.

For comparison, a single-layer filter (Comparative Example 1) with a thickness of 3.0 mm and a pore diameter of 10 μm was used.

A similar test was conducted using a single-layer structure filter with a pore size of 5 μm (Comparative Example 2) and a single-layer structure filter with a thickness of 3.0 wm and a pore size of 2 μm. The results are also shown in FIG. 5 and Table 1.

As is clear from Table 1, filters b, c and d are:
Filters a and e have substantially the same filtration precision as the filter of Comparative Example 3, and filters a and e have substantially the same filtration precision as Comparative Example 2. In other words, the filtration accuracy of the filter of the present invention is
Determined by the pore size of the small pore filter layer.

Furthermore, the filtration life of each filter was measured using the apparatus shown in FIG. In this apparatus, a standard particle liquid similar to the standard particle liquid used in the apparatus shown in FIG.
A constant flow pump 92 supplies the standard particle liquid to the pipe 9 from the standard particle liquid container 91 containing the standard particle liquid dispersed at a concentration of g/L.
3. A pressure gauge 94 and a holder 95 similar to the holder 85 used in the apparatus shown in FIG. 3 are successively installed in the conduit 93. The liquid that has passed through the holder 95 is collected into a container 96.

The standard particle liquid in the standard particle liquid container 91 is stirred by a magnetic cooler (not shown) while being stirred by a constant flow pump 92.
The liquid is fed to the conduit 93 at a flow rate of 1 ml/sin, and passes through a filter installed in the holder 95. At this time, the relationship between the amount of liquid fed and the pressure was measured using a pressure gauge 94. FIG. 7 shows the relationship between the amount of liquid fed to the filter a and the pressure (
pressure characteristic curve).

In FIG. 7, the horizontal axis shows the amount of standard particle liquid sent, and the vertical axis shows the pressure. In the pressure characteristic curve, the amount of liquid fed at the intersection point V between the tangent at point A where the liquid fed amount was 0 and the extension of the linear portion of the pressure characteristic curve was defined as the filtration life. The filtration life of each filter a to e is also listed in Table 1.

Furthermore, the filtration life of each of the filters of Comparative Examples 1 to 3 was determined in the same manner. The results are also listed in Table 1.

As is clear from Table 1, the filtration life of each filter of the present invention is approximately determined by the pore size and thickness of the filter layer with a large pore size, and Filter a is approximately 1.5 times longer than the filter of Comparative Example 2.
．． 7 times, filter e is approximately 1.8 times that of Comparative Example 2, filter b is approximately 2.1 times that of Comparative Example 3, and each filter of the present invention has a similar filter layer with a small pore size. It has a better filtration life than single layer filters with pore size.

(Experimental Example 2) Instead of the standard particle liquid of Experimental Example 1, human hemolysate, which was obtained by diluting fresh human whole blood 50 times with distilled water and causing hemolysis, was used. Then, the filtration life of each filter was determined in the same manner as in Experimental Example 1. The results are also listed in Table 1.

Human hemolysate contains membrane components of red blood cells, and filters are likely to become clogged by these membrane components. Therefore, the greater the amount of liquid fed that indicates the filtration life, the better the collection efficiency will be. In this experimental example, the filtration life of filters a and e is approximately 1.8 times that of the filter of Comparative Example 2, and the filtration life of filter b is approximately 2.2 times that of the filter of Comparative Example 3. The filter has a filter layer with a small pore size and a better filtration life than a single layer filter with a similar pore size.

(Margin below) Table 1 (Effects of the invention) As described above, the high performance liquid chromatography filter of the present invention has pore diameters that change sequentially in the liquid flow direction.
It has high filtration accuracy and excellent collection efficiency. As a result, it has excellent filtration life, suppresses column deterioration due to foreign matter clogging, and enables highly accurate measurements. B Fig. 1 is a cross-sectional view of a main part of a column equipped with a column filter which is an example of a filter for high-performance liquid chromatograph of the present invention, and Fig. 2 is a line sectional view of a column which is another example of the filter for high-performance liquid chromatograph of the present invention. A cross-sectional view of the holder with a filter attached, Figure 3 is a schematic diagram of the equipment used for filter performance experiments, Figure 4 is a particle size distribution diagram of the standard particle liquid used in the equipment, and Figure 5 is a diagram of the equipment used in the equipment. A graph showing the experimental results, FIG. 6 is a schematic diagram of the apparatus used for other performance tests of the filter, and FIG. 'M7 is a graph for explaining the filtration life.

10, 50...filter, 11.12.51.52
...Filter layer, 20...Column body, 21...
- Filler, 3o... Connecting member, 31... Space, 4
0. ．． 70... Seal member, 60... Holder first
Figure 2 Figure 4 Grain size for ib (1 m) Figure 5 #P, quasiparticle 1h Katsura Kabuto m)

Claims (1)

[Claims] 1. A filter for high-performance liquid chromatograph, which is installed in a liquid flow area in a high-performance liquid chromatograph, and is characterized in that the pore size changes sequentially in the liquid flow direction.