JPH05203634A - Usage of filter for liquid chromatograph - Google Patents

Abstract

PURPOSE:To catch particles such as dust in sample, impurities in pump seal, dust in eluate, etc., by using a filter paper sheet for a filter, and prevent choking. CONSTITUTION:Eluate (a) is fed to an infection valve (d) by a feed pump (b). Sample is injected by the valve (d) to a column (c) to be separated, it is then sent to a detector (e) for detection. A filter paper sheet is disposed in a passage between the eluate (a) and the column (c), and it shall preferably be between the valve (d) and the column (c). The filter paper sheet may also be used as it is combined with a sintered body filer or a membrane filter. For the sort of the filter paper sheet, cellulose material, cellulose acetate material, polyester fiber material, glass fiber material, etc., may be used. The form of the filter paper sheet need not be specially specified. For example, one sort of the filter paper sheet may be used as one sheet, or plural sheets, or plural sorts of the sheets may be combined.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the use of filters in liquid chromatographs used as analytical means in fields such as biochemistry and medicinal chemistry.

[0002]

2. Description of the Related Art In the fields of biochemistry and medicinal chemistry, especially in the field of medicinal chemistry, a liquid chromatograph has recently been widely used as an analytical means. In a liquid chromatograph, by utilizing the fact that each component in a sample has different properties, the sample is subjected to an analytical column to separate and analyze each component. For this reason, if impurities in the sample, dust in the pump seal, dust in the eluent, etc. enter the analytical column, the accuracy of the analysis will be reduced, and sometimes the analysis will not be possible.

In a liquid chromatograph, a sample which may contain impurities is usually subjected to centrifugation or filtration using a membrane filter to thoroughly remove the impurities and then analyze the sample. .. For example, in clinical tests by liquid chromatography such as hemoglobin A1c, which is a test item for diabetes, and vanillyl mandelic acid and homovanillic acid, which are test items for screening pediatric neuroblastoma tumors, there are many measurement samples, so automation is essential. is there. In the measurement of hemoglobin A1c, blood is diluted with a solution containing a surfactant, and the diluted solution is used as a measurement sample. In the measurement of vanillyl mandelic acid and homovanillic acid, urine is used as a measurement sample as it is. In such an inspection item, the removal of impurities in the sample by the pretreatment is complicated in operation and difficult to automate, so the sample obtained without removing the impurities is directly used for the measurement.

[0004]

Pretreatment of a sample by centrifugation or filtration using a membrane filter is complicated in operation and is rarely performed when the number of samples to be measured is large. It is also possible to install a filter in front of the analytical column to capture impurities in the sample, but the filter is likely to be clogged, and if the pressure rises significantly, it becomes impossible to measure and the replacement is unavoidable. To be done. In order to replace the filter, it is necessary to interrupt the liquid supply of the pump, and the operation is complicated.

[0005]

DISCLOSURE OF THE INVENTION The inventors of the present invention have captured a particle such as an impurity in a sample, dust in a pump seal, dust in an eluent, etc., and are further less likely to be clogged with a filter for a liquid chromatograph. As a result of diligent study on −, it is possible to provide a filter for liquid chromatograph that is less likely to be clogged by using at least paper as a filter, and a sintered body filter or a membrane filter as a filter for liquid chromatograph. Even when using −, by disposing a paper filter having a capture particle size equal to or larger than those capture particle sizes,
The present invention has been completed by finding that it is possible to prevent clogging of a filter for a liquid chromatograph. That is, the present invention is a method of using a paper filter as a filter in a liquid chromatograph, and a method of using a paper filter and a sintered filter or a membrane filter as a filter in a liquid chromatograph. The present invention will be described in detail below.

The type of liquid chromatograph to which the present invention is applied is not limited, but when impurities in the sample have a great influence on the separation (analysis result), particularly when a separation column having a high separation ability is used. Is effective in. For example, in the measurement of glycated hemoglobin A1c,
A fully automatic measurement device has been developed and sold, and is widely used as a routine analysis in hospitals and clinics. Since such a device requires particularly simple operation and quick measurement, it is suitable for applying the method of the present invention.

In the present invention, the type of the sintered body filter and / or the membrane filter used in combination with the paper filter is not particularly limited. For example, the sintered body filter may be a ceramic type or a stainless type. Examples of the membrane filter include cellulose acetate type, nitrocellulose type, and tetrafluoroethylene resin type. Examples of the type of paper include cellulose type, cellulose acetate type, polyester fiber type, glass fiber type and the like. The pore size of these filters and filters depends on the column used,
Although it may be appropriately selected depending on the type of the measurement sample, a sample having a size of several microns to several tens of microns is usually used.

[0008] The usage form of the paper used is not particularly limited. For example, one kind of filter paper may be used by stacking one or several sheets, or several kinds of filter paper may be stacked. In particular, when several kinds of paper are stacked and used, it is preferable to arrange the one having a large capture particle size on the side far from the column or the gel packed in the column. When a paper filter and a sintered filter or a membrane filter are used in combination, it is preferable to arrange the paper filter on the side far from the column or the gel filled in the column.

When the sintered body filter or the membrane filter is used alone, clogging gradually occurs as the number of particles to be captured increases, and the flow velocity decreases accordingly. On the other hand, in the paper filter used in the present invention, due to its structure, when the number of particles to be captured is large, they cannot be completely captured, but clogging is less likely to occur, and a decrease in flow velocity is less likely to occur. Have Therefore, when the filter paper and the sintered filter and / or the membrane filter are used in combination, it is preferable to dispose the filter paper on the side far from the column or the gel or the like packed in the column. In addition, since the sample to be subjected to the liquid chromatograph usually contains only particles (dust) that can be captured by the filter paper, according to the present invention, particles (dust) in the sample are substantially removed. Possible,
In addition, it is possible to realize a liquid chromatograph that is unlikely to cause a decrease in flow velocity due to clogging.

In the present invention, the position where the paper filter is placed is, for its purpose, a flow path for the sample or the eluent, and is limited as long as the solution does not come into contact with the gel or the like packed in the analytical column. There is no. More specifically, it is possible to exemplify the flow path from the liquid feed pump for the sample or the eluent to the analysis column, but it should be placed immediately before the gel or the like filled in it, for example, by embedding paper in the analysis column. Can also be illustrated.
The position where the paper is placed will be described below with reference to the drawings.

In a liquid chromatograph, as shown in FIG. 4, an eluent a is usually sent by a feed pump b to an injection valve d provided in front of a column c. The sample injected into the column by the injection valve is separated and then sent to the detector e for detection. In such a liquid chromatograph, for example, the eluent a
Although it is possible to exemplify that a paper filter is arranged in the flow path between the column c and the column c, it is preferable that the filter paper is arranged between the injection valve d and the column c. The position of f immediately before is particularly preferable.

For example, as shown in FIG. 5, the column side has a housing structure in which a paper filter is sandwiched between a nut portion g and a bolt portion h so that the particularly preferable paper filter arrangement can be achieved. Can be realized. Further, for example, if the liquid feed pump side of the column is modified to have a structure in which a paper filter, a membrane filter or the like can be sandwiched and the column and the paper filter are integrated, the housing and the like can be omitted.

FIG. 6 shows an example of the structure on the liquid feed pump side of the column for realizing the method of the present invention using a paper filter and other filters. For example, the paper m and the sintered body filter-1 can be sandwiched by pressing the fitting portion k into the main body j at the end of the column on the liquid feed pump side and fitting it.

[0014]

EXAMPLES Examples will be described below to show the present invention in more detail, but the present invention is not limited to these examples.

Example 1 Fresh blood collected by adding ethylenediaminetetraacetic acid sodium salt, which is an anticoagulant, was used as 0.1% Triton X-
20 mM sodium phosphate buffer pH 6.
A 20-fold diluted solution of 0 was used as a sample.

An injection valve equipped with a 20 μl sample loop was provided in the flow path after the liquid delivery pump,
In the flow path behind the injection valve, a filter-a membrane filter housing with a flow path diameter of 8 mm is used, and a filter paper with a retention particle diameter of 0.8 μm and a thickness of 1.0 mm is used as a filter. Sodium buffer solution pH 7.0 flow rate 1.5m by pump
The sample was injected every 3 minutes by an injection valve, and clogging of the filter was detected as pressure. The obtained pressure change is shown by a black circle (●) in Fig. 1.
Indicate.

Example 2 Instead of the filter paper having a retained particle size of 0.8 micron and a thickness of 1.0 mm used in Example 1, a filter paper having a retained particle size of 0.8 micron and a thickness of 0.5 mm was used. And perform the same operation,
A pressure change was detected. The obtained pressure change is indicated by a white triangle (Δ) in FIG.

Example 3 A similar operation was carried out by using a paper filter having a particle size of 4 μm and a thickness of 1 mm instead of the filter paper having a particle size of 0.8 μm and a thickness of 1.0 mm used in Example 1. Then, the pressure change was detected. The obtained pressure change is shown by a black triangle (▲) in FIG.

Example 4 The same as in Example 1 except that the retained paper having a particle diameter of 0.8 μm and a thickness of 1.0 mm was replaced by a paper filter having a retained particle diameter of 1 μm and a thickness of 0.72 mm. Then, the pressure change was detected. The obtained pressure change is indicated by a white square (□) in FIG. 1.

Example 5 A filter-a housing for a membrane filter having a flow passage diameter of 8 mm, and a retained particle diameter of 0.8 μm and a thickness of 0.5 mm.
The filter paper was used as a filter, and a sintered body filter housing having a diameter of 3.5 mm and a thickness of 3.5 mm was provided immediately after the flow path, and a ceramic sintered body having a pore diameter of 1 micron was provided.

As described above, the same operation as in Example 1 was performed using the two types of filters to detect the pressure change. The obtained pressure change is shown by a black circle (●) in FIG.

Example 6 The same operation was carried out by using a paper filter having a retention particle size of 4 μm and a thickness of 1 mm instead of the filter paper having a retention particle size of 0.8 μm and a thickness of 0.5 mm used in Example 5. Then, the pressure change was detected. The obtained pressure change is shown by a white triangle (Δ) in FIG.

Example 7 The same procedure was carried out by using a paper filter having a retention particle size of 1 micron and a thickness of 0.72 mm instead of the retention paper particle having a retention particle size of 0.8 micron and a thickness of 0.5 mm used in Example 5. Then, the pressure change was detected. The obtained pressure change is shown by a black triangle (▲) in FIG. Example 8 A filter-a membrane filter housing having a channel diameter of 8 mm-has a retained particle diameter of 0.8 microns and a thickness of 0.5 mm.
Paper filter and 0.45 micron pore size membrane filter
Were stacked so that the paper filter was positioned in front of the flow path (on the injection valve side), and the same operation as in Example 1 was performed to detect the pressure change. The obtained pressure change is shown by a white triangle (Δ) in FIG.

Example 9 In a filter-housing for a membrane filter having a channel diameter of 8 mm, a filter paper having a retained particle diameter of 4 microns and a thickness of 1 mm and a membrane filter having a pore diameter of 1.0 micron were placed at the rear of the channel. Then, the same operation as in Example 1 was performed and the pressure change was detected. The obtained pressure change is shown by a black triangle (▲) in FIG.

Example 10 In a housing for a filter-a membrane filter having a channel diameter of 8 mm, a filter paper having a retained particle diameter of 0.8 micron and a thickness of 1 mm and a membrane filter having a pore diameter of 1.0 micron-are placed behind the flow channel. Then, the pressure change was detected by carrying out the same operation as in Example 1 by superimposing so as to be located at. The obtained pressure change is shown by a white square (□) in FIG.

Comparative Example 1 Used in Example 5, the diameter is 3.5 mm and the thickness is 3.5 m.
The operation was performed in the same manner as in Example 1 except that only the ceramic sintered body having a pore diameter of 1 micron was used as the filter in the sintered body filter housing of m. The obtained pressure changes are shown by white circles (◯) in FIGS. 1 and 2.

Comparative Example 2 A pressure change was detected by performing the same operation as in Example 8 except that only a membrane filter having a pore diameter of 0.45 μm was used. The obtained pressure change is shown by a white circle (◯) in FIG.

Comparative Example 3 A pressure change was detected in the same manner as in Example 8 except that only a membrane filter having a pore size of 1.0 micron was used. The obtained pressure change is shown by a black circle (●) in FIG.

[0029]

As is apparent from the above description, according to the present invention, when the sample to be measured contains impurities, for example, a biological sample such as blood, a filter which is less likely to be clogged. An effective liquid chromatograph using paper can be carried out.

[Brief description of drawings]

FIG. 1 shows pressure changes obtained in Examples 1 to 4 and Comparative Example 1.

FIG. 2 shows pressure changes obtained in Examples 5 to 7 and Comparative Example 1.

FIG. 3 is a diagram showing pressure changes obtained in Examples 8 to 10 and Comparative Examples 2 and 3.

FIG. 4 shows an example of a normal configuration of a liquid chromatograph and a place for arranging a paper filter as a filter.

FIG. 5 shows an example of a housing structure for use in placement of paper filters.

FIG. 6 shows an example of the structure of the end of the column on the liquid feed pump side for use in arranging paper filters.

[Explanation of symbols]

 a: Eluent b: Liquid feed pump c: Column d: Injection valve e: Detection part f: Filter- (paper filter) g: Nut part h: Bolt part i: Filter- (paper filter) j: Liquid transfer of column Pump side end body k: Inset part l: Filter- (sintered body filter-) m: Filter- (paper filter and membrane filter-)

Claims (2)

[Claims] 1. A filter in a liquid chromatograph
How to use paper as a paper. 2. A filter in a liquid chromatograph
A method of using a paper filter and a sintered body filter or a membrane filter as the above.