JP2006189427A - Filter for liquid chromatography, and column for liquid chromatography - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a filter for liquid chromatography that catches a foreign substance efficiently, prevents pressure increase due to foreign substance clogging, and can keep the performance for a long term. <P>SOLUTION: The filter for liquid chromatography has a three-layer structure where an outer layer, an inner layer, and another outer layer are stacked in this order. The hole diameter of the outer layers is larger than that of the inner layer, and the thickness ratio of outer layer : inner layer : outer layer is 10-40:20-80:10-40, when the whole thickness is set at 100. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a liquid chromatography filter capable of efficiently capturing foreign matter and free from pressure increase due to clogging of foreign matter, and a liquid chromatography column equipped with the liquid chromatography filter. .

  In the liquid chromatography reagent flow path, for example, filters such as a filter for filtering reagents and an in-line filter installed in the flow path from the sample injection device to the separation column section are installed. These filters capture solids, insoluble substances, poorly soluble substances (hereinafter also simply referred to as foreign substances), etc. in the mobile phase, so that before the interaction between the substance to be measured and the filler, It is often installed upstream from the separation column, and in some cases, in order to prevent these foreign substances from entering the detector when a part of the packing material flows out from the separation column. In addition, it may be installed on the flow path from the separation column unit to the detection unit, that is, on the downstream side of the separation column unit, and may be installed on both the upstream side and the downstream side of the separation column unit.

  Examples of the foreign matter captured by such a filter include those derived from reagents such as a mobile phase and a reaction reagent, those derived from movable parts such as a pump, and those derived from a sample. If it enters the separation column, it may be adsorbed on the surface of the column packing or on the detector cell, which may adversely affect the measurement of the sample. Various filters for liquid chromatography have been developed in order to reliably capture foreign substances.

  Conventionally, sintered filters made by sintering metal fibers, especially stainless steel fibers, have been widely used as filter materials, but metal filters are highly hydrophobic, so they analyze certain substances under certain conditions. In this case, the substance to be measured may be adsorbed nonspecifically on the filter surface. In order to prevent such nonspecific adsorption of the substance to be measured to the filter, for example, use of a filter made of an inert material such as made of Teflon (registered trademark) or ceramic has been studied. 1 discloses a technique using a filter made of polyetheretherketone (PEEK). Patent Document 2 also discloses a technique in which a blocking process is performed on a filter.

  On the other hand, under conditions where a large amount of specimens are continuously measured or specimens containing a large amount of foreign substances such as blood are measured, foreign substances cannot be sufficiently captured, or foreign substances are clogged with the filter and pressure increases. There is a problem that the sample cannot be measured accurately and quickly, and the performance of the filter becomes insufficient. Especially for measurements that require high measurement accuracy, such as clinical tests, the pressure rise should be as low as possible so that the effect on the measured value is minimized and the filter does not need to be replaced frequently. It was necessary to suppress.

  In order to prevent a pressure increase due to such clogging of foreign matters in the filter, Patent Document 3 discloses a method using a filter having a two-layer structure with different hole diameters. Patent Document 4 also discloses a method using a two-layer structure in which a filter paper and a filter are combined. Furthermore, a technique for improving the shape of the filter in order to reduce the gap in the packing for holding the filter is disclosed.

However, in the method using a two-layer filter, the effect of preventing pressure rise cannot be obtained unless the layer with the larger hole diameter is always placed upstream, and the filter is installed inadvertently. However, there was a problem that no effect was obtained.
JP 2000-32261 A JP 2000-131302 A Japanese Patent Laid-Open No. 2-262004 JP-A-5-203634

  An object of the present invention is to provide a liquid chromatography filter that can efficiently capture foreign substances and that is unlikely to cause a pressure increase due to clogging of foreign substances.

  Another object of the present invention is to provide a liquid chromatography column equipped with the liquid chromatography filter.

  The present invention is a liquid chromatography filter having a three-layer structure in which an outer layer, an inner layer and an outer layer are laminated in this order, and the outer layer has a larger pore diameter than the inner layer and has an overall thickness of 100. The ratio of the thickness of the liquid chromatography filter is as follows: outer layer: inner layer: outer layer = 10-40: 20-80: 10-40.

  Hereinafter, the present invention will be described in detail.

  As a result of intensive studies, the present inventors have made a liquid chromatography filter a three-layer structure having an inner layer with a small pore size between outer layers with a large pore size, and the ratio of the thicknesses of the three layers within a certain range. By preparing, foreign matter can be captured efficiently, pressure rise due to clogging of foreign matter is unlikely to occur, and it is possible to prevent the filter from being mistakenly inadvertently inadvertently produced or used. As a result, the present invention has been completed.

  The liquid chromatography filter of the present invention has a three-layer structure in which an outer layer, an inner layer, and an outer layer are laminated in this order. In the present specification, the outer layer or the inner layer is a filter layer that can be regarded as having substantially the same pore diameter, and a plurality of filters having the same pore diameter or a plurality of pore diameters that are substantially equivalent. It is also assumed that such a layer is laminated. However, in the case of including a laminate of a plurality of layers having substantially the same pore diameter, it is preferable to increase the pore diameter toward the outer layer. Further, the size of the pore diameter may be continuously changed or discontinuous between the outer layer and the inner layer.

  FIG. 1 shows an example of the liquid chromatography filter of the present invention.

  As shown in FIG. 1, the liquid chromatography filter 1 includes an outer layer 2, an inner layer 3, and an outer layer 4, and an inner layer 3 having a pore diameter smaller than that of the outer layer 2 and the outer layer 4 is sandwiched between the outer layer 2 and the outer layer 4. It has a three-layer structure. Since the liquid chromatography filter 1 has such a structure, even when it is used as a liquid chromatography filter in either the liquid flow direction 1 or the liquid flow direction 2, foreign substances can be efficiently used. Can be trapped and the pressure rise due to clogging of foreign matter can be prevented. Therefore, the liquid chromatography filter 1 can be installed in the wrong direction due to carelessness during use.

  In the filter for liquid chromatography of the present invention, the outer layer has a larger pore size than the inner layer. Thereby, a large foreign matter can be captured by the outer layer, and an increase in pressure due to the foreign matter being clogged in the inner layer can be prevented. Further, the liquid chromatography filter of the present invention has a structure in which the inner layer is sandwiched between outer layers having the same pore diameter, so that the liquid chromatography filter is not installed in the wrong direction during use.

  The preferable lower limit of the pore diameter of the outer layer is 7 μm, and the preferable upper limit is 20 μm. If it is less than 7 μm, a pressure increase may occur due to clogging of foreign matter, and if it exceeds 20 μm, foreign matter may not be sufficiently captured. A more preferable lower limit is 8 μm, and a more preferable upper limit is 15 μm. In addition, in this specification, the said hole diameter means what is derived | led-out by the bubble point method prescribed | regulated to ISO4003.

  The outer layer is preferably made of metal, particularly stainless steel or titanium. Further, the outer layer may be made of a material different from that of the inner layer, or may be made of the same material. In the case where the outer layer and the inner layer are different materials, the outer layer is preferably produced using a material having higher strength than the inner layer. When the outer layer and the inner layer are made of the same material, it is preferable that the outer layer is made of fibers having a diameter larger than that of the inner layer.

  In the filter for liquid chromatography of the present invention, the inner layer has a pore diameter smaller than that of the outer layer. Thereby, when the foreign substance which passed the said outer layer is capture | acquired efficiently and it installs in the flow path of a liquid chromatography, the inflow of the foreign substance to a separation column part or the inflow of a foreign substance to a detection part can be prevented.

  The preferable lower limit of the pore diameter of the inner layer is 0.1 μm, and the preferable upper limit is 7 μm. If it is less than 0.1 μm, the pressure increase due to clogging of foreign matter may increase, and if it exceeds 7 μm, foreign matter may not be sufficiently captured. A more preferred lower limit is 1 μm, and a more preferred upper limit is 5 μm.

  The ratio of the thicknesses of the outer layer and the inner layer when the total thickness of the filter for liquid chromatography of the present invention is 100 is outer layer: inner layer: outer layer = 10-40: 20-80: 10-40. If the thickness ratio of the outer layer becomes smaller than the above range, or conversely, the thickness ratio of the inner layer becomes smaller than the above range, foreign matters cannot be efficiently captured, resulting in a liquid chromatography filter. The life of the is shortened. Therefore, by setting the ratio of the thickness of the outer layer and the inner layer within the above range, a liquid chromatography filter that exhibits particularly excellent effects when analyzing a biological sample containing foreign substances of various sizes, can do. The biological sample is a sample measured at the time of clinical examination, and mainly includes blood, serum, plasma, urine and the like.

  The preferable lower limit of the total thickness of the liquid chromatography filter of the present invention is 0.1 mm, and the preferable upper limit is 10 mm. If the thickness is less than 0.1 mm, the absolute amount of foreign matter that can be captured is small, which may shorten the life of the filter. If it exceeds 10 mm, the diffusion of the liquid becomes large, which may cause a decrease in separation performance. A more preferable lower limit is 0.2 mm, and a more preferable upper limit is 3 mm.

  The liquid chromatography filter of the present invention is preferably subjected to a surface treatment on all or part thereof. In this specification, the surface treatment means modifying the properties of the filter surface for liquid chromatography by subjecting it to chemical treatment and / or physical treatment. This surface treatment effectively avoids this problem even under analytical conditions where problems such as non-specific adsorption may occur when using a liquid chromatography filter that has not been surface-treated. can do.

  A method for modifying a part or all of the liquid chromatography filter by a chemical reaction is not particularly limited, and examples thereof include a method of modifying by heating or an oxidation reaction with an acid or the like. By performing the oxidation treatment, a part of the liquid chromatography filter surface is modified, and non-specific adsorption or the like of the hydrophobic substance can be suppressed.

  The surface treatment includes modifying the surface properties by coating with other substances. The method for coating the other substances is not particularly limited. For example, a so-called blocking treatment in which a liquid chromatography filter surface is coated with a substance having a desired characteristic such as a hydrophilic substance or a hydrophobic substance. It is done.

  Although it does not specifically limit as said other substance, For example, proteins, such as bovine serum albumin, globulin, lactoferrin, skim milk, silicone, a fluororesin, etc. are mentioned. By coating the above substance on the surface of a liquid chromatography filter, nonspecific adsorption of the hydrophobic substance can be suppressed.

  The shape of the liquid chromatography filter of the present invention is not particularly limited, but is preferably a disc shape or a cylindrical shape. Further, the diameter when the liquid chromatography filter of the present invention is cylindrical is not particularly limited, but the preferred lower limit is 1 mm and the preferred upper limit is 20 mm. If it is less than 1 mm, the absolute amount of foreign matter that can be captured is small, which may shorten the life of the filter. If it exceeds 20 mm, the diffusion of the liquid becomes large, which may cause a decrease in separation performance. A more preferable lower limit is 2 mm, and a more preferable upper limit is 10 mm.

  The method for producing the filter for liquid chromatography of the present invention is not particularly limited, and the outer layer and the inner layer may be separately produced and then superposed to form a three-layer structure, or for liquid chromatography having a three-layer structure at a time. A filter may be manufactured. Moreover, the hole diameter of an outer layer and an inner layer can be adjusted with a well-known method. For example, in the case of a fiber sintered filter, it can be adjusted to various hole diameters by changing the diameter of the metal fiber used or changing the pressure during processing.

  When the liquid chromatography filter of the present invention is installed in a flow channel of liquid chromatography, it is preferable to use it in a packing or a holder. The packing and holder are not particularly limited as long as they can be installed in a liquid chromatography channel and the mobile phase does not leak.

  The filter for liquid chromatography of the present invention is a pre-filter installed in a flow path leading to a separation column section or a column end filter installed in a flow path leading from a separation column section to a detection section when analyzing a biological sample. Can be used as When used for such biological sample analysis, foreign substances can be efficiently captured even when a large amount of specimens are continuously measured or specimens that contain a lot of foreign substances such as blood, and pressure rise is prevented. it can.

  In addition, the liquid chromatography filter may be separated from the liquid chromatography column having the separation column part, and may be appropriately installed in the flow path of the liquid chromatography, or may be integrated with the separation column part. It may be configured to be installed in a simple housing and incorporated in a liquid chromatography column.

  The filter for liquid chromatography according to the present invention efficiently captures foreign matters when used in liquid chromatography, and prevents the inflow of foreign matters to the downstream separation column when used as a prefilter. In addition, when used as a column end filter, it is possible to prevent the inflow of foreign matter into the detector, and the pressure rise due to clogging with foreign matter is small, so the filter must be replaced. Many specimens can be measured. Moreover, these performances can be maintained over a long period of time. Furthermore, the filter is not installed in the wrong direction due to carelessness during use.

  Moreover, since the liquid chromatography column of the present invention is equipped with the liquid chromatography filter, excellent separation performance can be maintained over a long period of time.

In order to describe the present invention in more detail, examples will be given below, but the present invention is not limited to these examples.
Example 1
Stainless steel liquid chromatography having a three-layer structure in which a filter layer 1 (outer layer) having a pore size of 12 μm, a filter layer 2 (inner layer) having a pore size of 3 μm, and a filter layer 3 (outer layer) having a pore size of 12 μm are laminated in this order. A filter was prepared. In addition, the thickness of the filter layers 1 and 3 of the obtained filter was 0.25 mm respectively, the thickness of the filter layer 2 was 0.50 mm, and the whole thickness was 1 mm. The outer diameter of the filter was 5 mm.
(Example 2)
A filter for liquid chromatography was produced in the same manner as in Example 1 except that the thickness of each of the filter layers 1 and 3 (outer layer) was 0.15 mm and the thickness of the filter layer 2 (inner layer) was 0.70 mm.
(Example 3)
A filter for liquid chromatography was produced in the same manner as in Example 1 except that the thickness of each of the filter layers 1 and 3 (outer layer) was 0.35 mm and the thickness of the filter layer 2 (inner layer) was 0.30 mm.
Example 4
A liquid chromatography filter was produced in the same manner as in Example 1, except that the thickness of each of the filter layers 1 and 3 (outer layer) was 0.10 mm, and the thickness of the filter layer 2 (inner layer) was 0.80 mm.
(Example 5)
A liquid chromatography filter was produced in the same manner as in Example 1, except that the thickness of each of the filter layers 1 and 3 (outer layer) was 0.40 mm, and the thickness of the filter layer 2 (inner layer) was 0.20 mm.
(Comparative Example 1)
A stainless steel liquid chromatography filter having a two-layer structure in which a filter layer 1 having a pore diameter of 12 μm and a filter layer 2 having a pore diameter of 3 μm were laminated was produced. In addition, the thickness of the filter layer 1 of the obtained filter was 0.50 mm, the thickness of the filter layer 2 was 0.50 mm, and the whole thickness was 1 mm. The outer diameter of the filter was 5 mm.
(Comparative Example 2)
A stainless steel liquid chromatography filter comprising one layer of the filter layer 1 having a pore diameter of 3 μm was prepared. The total thickness of the obtained filter was 1 mm. The outer diameter of the filter was 5 mm.
(Comparative Example 3)
A liquid chromatography filter was produced in the same manner as in Comparative Example 2 except that the pore size of the filter layer 1 was 12 μm.
(Comparative Example 4)
A filter for liquid chromatography was produced in the same manner as in Example 1 except that the thickness of the filter layers 1 and 3 (outer layer) was 0.05 mm and the thickness of the filter layer 2 (inner layer) was 0.90 mm.
(Comparative Example 5)
A liquid chromatography filter was produced in the same manner as in Example 1, except that the thickness of each of the filter layers 1 and 3 (outer layer) was 0.45 mm, and the thickness of the filter layer 2 (inner layer) was 0.10 mm.
(Evaluation)
The following evaluation was performed about the filter for liquid chromatography obtained in Examples 1-5 and Comparative Examples 1-5.
(1) Evaluation of pressure increase of liquid chromatography filter and membrane filter The obtained liquid chromatography filter was fitted into a PEEK packing and attached to a PEEK holder provided with a threaded portion that can be connected to a flow path. The liquid chromatography shown in FIG.

  FIG. 2 is a schematic diagram showing a part of liquid chromatography used in this evaluation. As shown in FIG. 2, the moving bed 6 is fed to the membrane filter 10 via the liquid feed pump 7, the sample injection device 8, and the liquid chromatography filter 9. The mobile phase 6 was 50 mmol / l phosphate buffer having a pH of 6, and the solution was fed at a flow rate of 1.5 mL / min. The membrane filter 10 is a stack of four membrane filters having a pore diameter of 0.65 μm, and is used to capture what has passed through the filter 9 for liquid chromatography, and is a separation column in an actual analysis system. To act as a department.

  As a sample, a sample obtained by diluting human healthy human blood 200 times with a phosphate buffer solution of pH 7 containing a surfactant was used to measure 1000 samples of human healthy human blood continuously. The difference between the pressure value before measurement of the membrane filter (0 MPa) and the pressure value after measurement of 1000 specimens was determined. The results are shown in Table 1. In addition, as for the liquid flow direction of Table 1, inflow of a moving layer is the order of filter layers 1, 2, and 3 (Examples 1-5, comparative example 4, comparative example 5) and the order of filter layers 1 and 2 (comparison). In the case of Example 1), the liquid passing direction was 1, and in the order of the filter layers 3, 2, 1 (Example 1) and in the order of the filter layers 2, 1 (Comparative Example 1), the liquid passing direction was 2.

表１に示すように、実施例１、２及び３の液体クロマトグラフィー用フィルタは、液体クロマトグラフィー用フィルタ圧力値、メンブランフィルタ圧力値ともに上昇がなかった。また、実施例１の液体クロマトグラフィー用フィルタは、通液方向１及び２の両方向からの通液に対して、圧力値の上昇がなかった。これは、どちらの向きに設置しても異物を充分に捕捉でき、かつ、多量検体測定時においても詰まり等の問題の発生が少ないことを示す。

As shown in Table 1, in the liquid chromatography filters of Examples 1, 2, and 3, neither the liquid chromatography filter pressure value nor the membrane filter pressure value increased. In addition, the liquid chromatography filter of Example 1 did not increase in pressure value with respect to liquid flow from both the liquid flow directions 1 and 2. This indicates that foreign matter can be sufficiently captured regardless of the orientation, and problems such as clogging are less likely to occur when measuring a large amount of samples.

  The liquid chromatography filters of Examples 4 and 5 were capable of measuring 1000 samples, although there was a slight increase in the pressure value for liquid chromatography.

  On the other hand, in the liquid chromatography filter of Comparative Example 1, the filter pressure value did not increase in the liquid passing direction 1, but the filter pressure value increased in the liquid passing direction 2. The membrane filter pressure value did not increase in either direction. This means that the liquid chromatography filter of Comparative Example 1 does not damage the separation column part, but in the case of the liquid flow direction 2, the usable period is shorter than that of the liquid flow direction 1. That is, if the direction in which the liquid chromatography filter is installed in the wrong direction during use, it means that the life of the liquid chromatography filter is shortened.

In the liquid chromatography filters of Comparative Examples 2, 4, and 5, the liquid chromatography filter pressure value increased, but the membrane filter pressure value did not increase. Further, the liquid chromatography filter of Comparative Example 3 did not increase the liquid chromatography filter pressure value, but the membrane filter pressure value increased.
(2) Evaluation of inhibitory effect of nonspecific adsorption of Hb Surface treatment was performed by immersing the liquid chromatography filter of Example 1 in a phosphate buffer solution of 0.01% bovine serum albumin at 40 ° C. for 1 hour. It was. Next, using the liquid chromatography filter with the above surface treatment and the liquid chromatography filter without the above surface treatment, Hb is recovered under the following conditions by a liquid chromatography system (Shimadzu LC-6A system). A rate test was performed.

  The Hb recovery rate was derived from the average value of the peak area ratios of the latter three samples when the same sample was measured 10 times. The peak area ratio is the liquid chromatography filter with the above surface treatment or the liquid chromatography without the above surface treatment when the peak area obtained without setting the liquid chromatography filter is 100%. It is a ratio of peak areas obtained when a filter is set. Therefore, the closer the peak area ratio is to 100%, the more non-specific adsorption of Hb on the liquid chromatography filter surface is suppressed.

Mobile phase: Solution A = 50 mM phosphate buffer (pH 5)
Solution B = 500 mM phosphate buffer (pH 5)
Flow rate: 1.5 ml / min Detection wavelength: 415 nm
Sample to be measured: Healthy human blood (diluted 200 times with phosphate buffer (pH 7) containing surfactant)

表２に示すように、上記表面処理をした液体クロマトグラフィー用フィルタの場合は、Ｈｂ回収率が１００％に近い値を示したのに対して、上記表面処理をしていない液体クロマトグラフィー用フィルタの場合は、非常に低い値を示した。これは、上記表面処理をしていない液体クロマトグラフィー用フィルタの場合には、液体クロマトグラフィー用フィルタ表面にＨｂが非特異吸着したためであると考えられる。即ち、非特異吸着が問題とならない検体を測定する場合には特に必要ではないが、非特異吸着が生じるような検体を測定する場合には、表面処理を施すことが非常に効果的であることがわかる。

As shown in Table 2, in the case of the liquid chromatography filter subjected to the above surface treatment, the Hb recovery rate was close to 100%, whereas the liquid chromatography filter not subjected to the above surface treatment. In the case of, it showed a very low value. This is considered to be due to non-specific adsorption of Hb on the surface of the liquid chromatography filter in the case of the liquid chromatography filter not subjected to the surface treatment. In other words, it is not particularly necessary when measuring specimens where nonspecific adsorption is not a problem, but it is very effective to perform surface treatment when measuring specimens that cause nonspecific adsorption. I understand.

  In view of the above-mentioned present situation, the present invention is a liquid chromatography filter capable of efficiently capturing foreign matter and free from pressure increase due to clogging of foreign matter, and a liquid equipped with the liquid chromatography filter A chromatographic column can be provided.

It is an example of the filter for liquid chromatography of this invention. It is a part of liquid chromatography containing the filter for liquid chromatography of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Filter 2 for liquid chromatography, 4 Outer layer 3 Inner layer 5 Liquid chromatography 6 Moving layer 7 Feed pump 8 Sample injection device 9 Filter 10 for liquid chromatography Membrane filter

Claims (6)

  The outer layer, the inner layer, and the outer layer are liquid chromatography filters having a three-layer structure in which the layers are laminated in this order, and the outer layer has a larger pore diameter than the inner layer and the total thickness is 100. The ratio of thickness is outer layer: inner layer: outer layer = 10-40: 20-80: 10-40, The filter for liquid chromatography characterized by the above-mentioned.   The filter for liquid chromatography according to claim 1, wherein the pore diameter of the outer layer is 7 to 20 µm, and the pore diameter of the inner layer is 0.1 to 7 µm.   3. The liquid chromatography filter according to claim 1, wherein the total thickness is 0.1 to 10 mm.   4. The liquid chromatography filter according to claim 1, wherein the surface treatment is performed.   The liquid chromatography filter according to claim 4, wherein the surface treatment suppresses nonspecific adsorption of the hydrophobic substance.   A liquid chromatography column having a separation column part, wherein the liquid chromatography filter according to claim 1, 2, 3, 4 or 5 is mounted on the upstream side or downstream side of the flow path of the separation column part. A column for liquid chromatography characterized by comprising:

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