JP2008202992A - Packing for chromatographic column and the chromatographic column - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a packing for chromatographic column capable of rapidly controlling the properties of a fixed phase, and to provide a chromatographic column. <P>SOLUTION: In the chromatographic column 10, a column cylinder 12 comprising glass, or the like, is filled with the column packing 1 and a coil 11 is wound around the column cylinder 12. At least magnetic fine particles and the temperature responsive polymer layer formed to the peripheries of them are provided to the column packing 1. For example, the spherical magnetic fine particles are covered with the temperature-responsive polymer layer. Furthermore, at least magnetic fine particles are covered with a silica-based substance, and this silica-based substance may be covered with the temperature-responsive polymer layer. Furthermore, at least magnetic fine particles and at least silica-based particles may be covered with the temperature-responsive polymer layer. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a chromatography column filler suitable for temperature-responsive chromatography and a chromatography column.

  Chromatography is known as one method for separating and purifying substances. In chromatography, a substance called a mobile phase passes through a surface or inside of a substance called a stationary phase or a carrier and is separated. This separation occurs based on various factors such as the size, adsorption power, charge, mass, hydrophobicity of the substance. A solid is generally used for the stationary phase. The degree of interaction between the substance dissolved in the mobile phase and the surface of the stationary phase is adjusted by the type of liquid constituting the mobile phase, the mixing ratio of a plurality of liquids, and the like. In addition, by making the surface of the stationary phase hydrophobic, hydrophilic or ionic (anionic or cationic), or by introducing specific affinity groups, the properties of the surface can be changed and dissolved in the mobile phase. The difference in affinity with the substance to be separated may be used.

  One type of chromatography is one using a temperature-responsive polymer as a stationary phase (temperature-responsive chromatography). A polymer that recognizes and responds to environmental changes such as temperature and pH by itself is called a stimulus-responsive polymer, and by modifying it to a solid surface, a substance with a high-functional surface imparted with environmental responsiveness is obtained. It is possible. For example, certain polymer aqueous solutions such as poly (N-substituted acrylamide), poly (N-substituted methacrylamide), or methylcellulose become cloudy when heated to a temperature above the lower critical solution temperature (LCST). It is known that when cooled to the following temperature, it exhibits a reversible phase separation behavior of dissolving again and returning to transparency. Further, it has been reported that such a phase separation phenomenon is accompanied by a contraction phenomenon of a polymer chain called a coil-globule transition. That is, below LCST, the polymer chain dissolves and takes a random coil-like conformation due to the strong interaction between the amide group and water, but the hydrogen bond between the amide group and water becomes unstable due to an increase in water temperature. Therefore, dehydration occurs and the polymer chain contracts to become a globule. Furthermore, the macromolecules associate by hydrophobic interaction and phase separation occurs.

  Among such substances, PNIPAAm (N-isopropylacrylamide) is widely used as a temperature-responsive polymer because it undergoes a sharp and reversible change in response to an external temperature stimulus. Since PNIPAAm has a hydrogen bonding site in water, water molecules strongly adhere around the polymer chain and dissolve in water on the low temperature side. However, when the temperature is raised, it separates from water and becomes insoluble and precipitates. Therefore, PNIPAAm extends and hydrates at a temperature lower than the phase transition temperature of the polymer, but contracts and dehydrates at a high temperature. In this way, a sensitive and reversible change occurs in response to an external temperature stimulus. Specifically, when it is heated to a temperature of 32 ° C. or higher, it becomes cloudy, and when it is cooled to a temperature lower than that, it dissolves again and becomes transparent.

  The temperature (LCST) causing such dissolution-precipitation change can be adjusted by the copolymer composition of the polymer, and can be controlled in a narrow temperature range. Since LCST strongly depends on the molecular structure of the polymer chain, the hydrophobic monomer and NIPAAm are copolymerized and shifted to the low temperature side by forming a hydrophobic copolymer, and the hydrophilic monomer and NIPAAm are copolymerized. And shift to a high temperature side by using a hydrophilic polymer. That is, the properties of the copolymer to be synthesized can be controlled by adjusting the balance of the NIPAAm monomer, hydrophobic substance and hydrophilic substance introduced in the copolymerization. For example, when butyl methacrylate (BMA), which is a hydrophobic substance of about 5 mol%, and NIPAAm are copolymerized, the LCST of the resulting copolymer shifts to the low temperature side to around 21 ° C. And when such a functional polymer is modified to a solid surface, the surface property is reversibly changed to a hydrophilic property at a temperature lower than the phase transition temperature of the polymer and a hydrophobic property at a higher temperature by a temperature stimulus. Surface material can be obtained.

  In temperature-responsive chromatography, a solid whose surface exhibits a change between hydrophilicity and hydrophobicity is used as a packing material for high performance liquid chromatography (HPLC). According to this temperature-responsive chromatography, it is possible to control the separation by changing the interaction with the sample (separation phase) by an external stimulus. Therefore, since the separation selectivity can be controlled by temperature even in a mobile phase containing only water, various separations can be performed. That is, it was necessary to use an organic solvent or the like in the previous distribution chromatography, but according to the temperature-responsive chromatography, it is not necessary to use such a substance.

  However, in the conventional temperature-responsive chromatography, it is necessary to control the temperature of the mobile phase itself. For this reason, there exists a problem that temperature control will take time.

Japanese Patent Laid-Open No. 2005-60244 JP 2005-82538 A JP 2000-212144 A

  An object of the present invention is to provide a chromatography column packing material and a chromatography column capable of quickly controlling the properties of the stationary phase.

  As a result of intensive studies to solve the above problems, the present inventor has come up with various aspects of the invention described below.

  The chromatography column filler according to the present invention is characterized by having magnetic fine particles and a temperature-responsive polymer layer surrounding the magnetic fine particles.

  The chromatography column according to the present invention includes a column cylinder and a column filler packed in the column cylinder, and the column filler includes magnetic fine particles and a temperature-responsive polymer layer surrounding the magnetic fine particles. It is characterized by having.

  According to the present invention, since the surface characteristics of the temperature-responsive polymer layer change depending on whether or not a magnetic field is applied to the magnetic fine particles, the properties of the stationary phase can be controlled only by controlling the magnetic field.

  Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. FIG. 1 is a schematic diagram showing a chromatography column according to an embodiment of the present invention.

  In the chromatography column 10 according to this embodiment, a column packing 1 is packed in a column cylinder 12 made of glass or the like. The particle diameter of the column filler 1 is about several μm to several tens of μm. A coil 11 is wound around the column cylinder 12. The column filler 1 is provided with at least magnetic fine particles and a temperature-responsive polymer layer formed around the magnetic fine particles. For example, as shown in FIG. 2A, a column filler 1 a configured by covering spherical magnetic fine particles 2 with a temperature-responsive polymer layer 3 is used as the column filler 1. Further, as shown in FIG. 2B, a column filler 1 b configured such that one or more magnetic fine particles 2 are covered with a silica-based material 4 and the silica-based material 4 is covered with a temperature-responsive polymer layer 3. May be used as column filler 1. Furthermore, as shown in FIG. 2C, a column filler 1c constituted by covering one or more magnetic fine particles 2 and one or two or more silica-based particles 5 with a temperature-responsive polymer layer 3 is a column filler. 1 may be used.

  In the example shown in FIG. 2A, a temperature-responsive polymer (hydrated graft structure) layer 3 made of an acrylamide derivative or the like that expresses a sensitive temperature-responsiveness is fixed to the magnetic fine particles 2 made of iron oxide or the like by covalent bonds. Has been.

  Here, a method for producing the column filler 1a as described above will be described.

  As the magnetic fine particles 2, for example, fine particles of iron oxide compounds such as hematite, maghemite or magnetite can be used, and compounds in which iron atoms in these iron oxide compounds are replaced with atoms such as manganese and Co are also available. Can be used.

  As the temperature-responsive polymer constituting the temperature-responsive polymer layer 3, for example, a polymer or copolymer containing an acrylamide derivative represented by the following general formula (I) (Chemical Formula 1) can be used. Such a temperature-responsive polymer is disclosed in Patent Document 3, for example.

However, in general formula (I),
R ¹ represents a hydrogen atom or a linear or branched alkyl group having 1 to 4 carbon atoms,
R ² represents a linear or branched alkyl group having 1 to 4 carbon atoms,
R ³ represents a methylene group having 1 to 6 carbon atoms,
X is a hydrogen atom, an amino group, a hydroxyl group, a halogen atom, a carboxyl group, or —COOR ⁴ (R ⁴ is a linear or branched alkyl group having 1 to 6 carbon atoms, a phenyl group, a substituted phenyl group, a benzyl group, or a substituted group. Represents a benzyl group),
Y represents an amino group, a hydroxyl group, a halogen atom, a carboxyl group, or —COOR ⁴ (R ⁴ represents a linear or branched alkyl group having 1 to 6 carbon atoms, a phenyl group, a substituted phenyl group, a benzyl group, or a substituted benzyl group. Represents.) However, R ² and R ³ may be combined to form a ring.

Examples of the linear or branched alkyl group having 1 to 4 carbon atoms of R ¹ and R ² constituting the general formula (I) include a methyl group, an ethyl group, an n-propyl group, an i-propyl group, An n-butyl group, an i-butyl group, an s-butyl group, and a t-butyl group can be exemplified. Besides these, an n-pentyl group and an n-hexyl group can also be exemplified.

Examples of the methylene group having 1 to 6 carbon atoms of R ³ include a monomethylene group, a dimethylene group, a trimethylene group, and a tetramethylene group, and a monomethylene group is preferably used.

  Examples of the halogen atom of X constituting the general formula include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.

Examples of the substituted benzyl group for R ⁴ include a diphenyl group and a triphenylmethyl group.

In the general formula (I), when R ² and R ³ are combined to form a ring, preferred examples of the ring include cyclopropane, cyclobutane, cyclopentane, cyclohexane and the like.

  The degree of polymerization of the polymer or copolymer having a repeating unit represented by the general formula (I) may be 2 or more, and preferably 10 to 500.

  And when immobilizing the above-mentioned temperature-responsive polymer layer 3 on the magnetic fine particles 2, it is preferable to first perform the following pretreatment on the magnetic fine particles 2. That is, by reacting the magnetic fine particles 2 with a compound represented by the following general formula (II) (Chemical Formula 2), a functional group is introduced using a silane coupling reagent, and then the above-described general formula (I) is used. It is preferable to make it react with the polymer or copolymer containing the acrylamide derivative to be made. When such pretreatment is performed, the column filler 1b or 1c is obtained as shown in FIG. 2B.

However, in general formula (II),
R ⁵ represents a linear or branched alkyl group having 1 to 6 carbon atoms,
X ′ is an amino group, an alkyl-substituted amino group, a carboxyl group, —COOR ⁶ (R ⁶ is a linear or branched alkyl group having 1 to 6 carbon atoms, a phenyl group, a substituted phenyl group, a benzyl group, or a substituted benzyl group. Represents a hydroxyl group, an epoxy group or a halogen atom,
Y ′ represents a halogen atom or an alkoxy group.

  In order to increase the reactivity between the magnetic fine particles 2 and the compound represented by the general formula (II), it is effective to subject the magnetic fine particles 2 to vitrification. Such vitrification treatment may be performed by, for example, reacting sodium silicate with hydrochloric acid, sulfuric acid, hydrogen peroxide, or the like in water and then drying.

  Examples of the alkyl-substituted amino group of X ′ constituting the general formula (II) include a dimethylamino group, a diethylamino group, a methylethylamino group, a trimethylamino group, a dimethylethylamino group, and a triethylamino group. Can do.

  Moreover, as a halogen atom of Y ', a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom can be mentioned. Examples of the alkoxy group for Y include a methoxy group and an ethoxy group.

  In addition, the solvent used for the reaction of the compound represented by the general formula (II) and the magnetic fine particles 2 is not particularly limited as long as it does not participate in the reaction. For example, water, methanol, ethanol, n-propanol, i-propanol, acetic acid, acetone, 2-butanone, tetrahydrofuran, dioxane, acetonitrile, benzene, toluene, xylene, chlorobenzene, chloroform, methylene chloride, carbon tetrachloride, dimethylformamide, dimethylacetamide, dimethylsulfoxide, and the like can be used. Moreover, as long as it is a solvent miscible with each other, a mixture of two or more selected from these may be used. The reaction is desirably performed at 0 ° C. or higher and 100 ° C. or lower.

  In the reaction of fixing the polymer or copolymer represented by the general formula (I) to the magnetic fine particles 2 by covalent bonding, the reaction is performed in advance on the X or Y in the general formula (I) and the magnetic fine particles 2 in advance. What is necessary is just to make it react with X 'in general formula (II) to make.

  For example, in order to facilitate the reaction, when X or Y in the general formula (I) is a carboxyl group, X ′ in the general formula (II) is preferably a hydroxyl group or an amino group. Moreover, when X or Y in general formula (I) is a hydroxyl group or an amino group, it is preferable that X 'in general formula (II) is a carboxyl group. In this case, in particular, when X or Y in the general formula (I) or X in the general formula (II) is a carboxyl group, the reaction is extremely difficult when the succinimide group, the p-nitrophenyl ester group or the like is changed. Proceed easily. Even when such a change does not occur, the reaction can be promoted by using a condensing agent. Examples of the condensing agent include dicyclohexylcarbodiimide, diisopropylcarbodiimide, N-ethyl-N′-3-dimethylaminopropylcarbodiimide, benzotriazol-1-yl-tris (dimethylamino) phosphonium hexafluorophosphide, and diphenylphosphoryl azide. be able to. These condensing agents can be used alone or in combination with N-hydroxysuccinimide or 1-hydroxybenzotriazole.

  In addition, the reaction for immobilizing the polymer or copolymer containing the acrylamide derivative represented by the general formula (I) to the magnetic fine particles 2 by a covalent bond is preferably performed in a solvent that does not participate in the reaction. Examples of such a solvent include water, methanol, ethanol, n-propanol, i-propanol, acetic acid, acetone, 2-butanone, tetrahydrofuran, dioxane, acetonitrile, benzene, toluene, xylene, chlorobenzene, chloroform, methylene chloride, Examples thereof include carbon tetrachloride, dimethylformamide, dimethylacetamide, dimethyl sulfoxide and the like. Moreover, as long as it is a solvent miscible with each other, a mixture of two or more selected from these may be used. The reaction is desirably performed at 0 ° C. or higher and 100 ° C. or lower.

  In order to smoothly proceed the reaction of immobilizing the polymer or copolymer containing the acrylamide derivative represented by the general formula (I) to the magnetic fine particles 2 by covalent bonding, it is preferably performed under basic conditions. In order to create such basic conditions, it is preferable to use potassium hydroxide, sodium hydroxide, potassium carbonate, sodium carbonate, pyridine, N, N-dimethylpyridine, trimethylamine, triethylamine, triethanolamine and the like.

  The particle size of the column filler 1 obtained by such a method is, for example, about several μm to several tens of μm.

  The reaction between the compound represented by the general formula (II) and the magnetic fine particles 2 can be confirmed using an X-ray photoelectron spectrometer and an infrared spectrometer. The reaction between the magnetic fine particles 2 reacted with the general formula (II) and the polymer or copolymer containing the acrylamide derivative represented by the general formula (I) can be confirmed using a transmission electron microscope. .

  Next, the chromatography using the column 10 for chromatography provided with the column filler 1 comprised as mentioned above is demonstrated. In this chromatography, a mobile phase is passed through the chromatography column 10 while supplying an alternating current to the coil 11.

  When an alternating current is supplied to the coil 11, an alternating magnetic field is generated in the column 10. As a result, the magnetic fine particles 2 generate heat. In addition, the temperature-responsive polymer constituting the temperature-responsive polymer layer 3 is dissolved in water at a temperature lower than a specific temperature and insolubilized at a high temperature. That is, it becomes hydrophilic on the low temperature side and hydrophobic on the high temperature side. Therefore, the properties of the column filler 1 change depending on the presence or absence of an alternating magnetic field. For example, when a mobile phase containing two kinds of substances is allowed to flow through the column 10 without supplying an alternating current, the temperature-responsive polymer shows hydrophilicity, and the two kinds of substances are almost simultaneously shown in FIG. 3A. Is eluted. On the other hand, when a mobile phase containing two kinds of substances is allowed to flow through the column 10 while supplying an alternating current, the temperature-responsive polymer exhibits hydrophobicity, and as shown in FIG. Is eluted. In the case where the temperature-responsive polymer is hydrophilic, the alternating current may not be supplied when the discharge timing of the two types of substances is more likely to shift.

  As described above, according to the present embodiment, it is possible to change the characteristics of the temperature-responsive polymer layer 3 only by switching on / off of an alternating current (alternating magnetic field) to separate substances in the mobile phase. . That is, the substances can be separated without adjusting the temperature of the entire column as in the prior art.

  Furthermore, it is possible to shorten the time required for elution by switching on / off of an alternating current (alternating magnetic field) during the separation of substances. As shown in FIG. 4 (a), when an alternating magnetic field is not applied, an unnecessary object and a target object are eluted almost simultaneously. On the other hand, when an alternating magnetic field is applied, as shown in FIG. 4 (b), the target object is eluted with a delay from the unnecessary material, so that they are separated. However, it may take a long time from the end of elution of unnecessary substances to the start of elution of target substances. In such a case, as shown in FIG. 4 (c), the application of the AC magnetic field is continued until the elution of the unnecessary material is completed, and the application of the AC magnetic field is completed after the elution of the unnecessary material is completed. do it. By controlling such a magnetic field, the target is eluted early, and the time required for separation is shortened. That is, according to the present embodiment, the surface characteristics of the stationary phase can be changed in a short time, so that the target object can be collected quickly. In conventional temperature-responsive chromatography, it is necessary to change the temperature of the entire column from the outside when controlling the temperature.For example, the temperature of the entire mobile phase needs to be changed. It is very difficult to change the surface characteristics of the surface.

  The silica-based material 4 or the silica-based particles 5 function as a binder that chemically bonds the magnetic fine particles 2 and the temperature-responsive polymer layer 3, but other materials or particles may be used. For example, non-silica ceramics or hydroxyapatite may be used. Moreover, you may use the metal compound which does not show magnetism. Furthermore, organic substances can be used.

  Hereinafter, the contents and results of tests actually conducted by the present inventors will be described in detail.

(First test)
In the first test, the relationship between the presence or absence of magnetization of the magnetic fine particles 2 and the change in characteristics was investigated. Specifically, 3.0 g of column filler 1 and 10 ml of water that were not magnetized were placed in a test tube, and the column filler 1 was dispersed in water by stirring. Next, an alternating magnetic field of 1000 W and 300 kHz was applied to the column filler 1, and the water temperature was measured over time. The initial water temperature was 15.0 ° C. The same measurement was performed on the column filler 1 magnetized using a fixed magnetic field type magnetizer. These results are shown in FIG.

  As shown in FIG. 5, the water temperature rose immediately after application of the alternating magnetic field regardless of the presence or absence of magnetization, and rose to about 35.0 ° C. after 20 minutes. From the results of this test, it can be said that a sufficient effect can be obtained regardless of whether the magnetic fine particles 2 are magnetized or not magnetized.

(Second test)
In the second test, the dissolution behavior when three types of steroids (hydrocortisone, dexamethasone and testosterone) were included in the mobile phase was investigated. Comparing the hydrophobicity of hydrocortisone, dexamethasone and testosterone, testosterone has the highest hydrophobicity and hydrocortisone has the lowest hydrophobicity. FIG. 6A shows the results when hydrocortisone is used, FIG. 6B shows the results when dexamethasone is used, and FIG. 6C shows the results when testosterone is used. In addition, the continuous line in FIG. 6A-FIG. 6C has shown the change of the elution amount when an alternating magnetic field is not applied, and the broken line has shown the change of the elution amount when an alternating magnetic field is applied.

  As shown in FIG. 6A to FIG. 6C, in any steroid, elution was delayed when an alternating magnetic field was applied than when it was not applied. This tendency became more prominent as the degree of hydrophobicity increased. That is, the elution delay was most prominent in testosterone. From the result of this test, it can be said that the surface of the column packing 1 is surely hydrophobic by application of an alternating current.

(Third test)
The third test relates to conventional temperature-responsive chromatography. Specifically, the elution behavior in a mobile phase containing three types of steroids (hydrocortisone, dexamethasone and testosterone) was investigated using a column packed with a conventional packing material. As the conventional filler, a filler composed of a temperature-responsive polymer layer bonded to the surface of silica-based fine particles was used. That is, a material not containing magnetic fine particles was used. And the change of the elution amount when the temperature of a mobile phase was 20 degreeC and the change of the elution amount when it was 50 degreeC were measured. FIG. 7A shows the result at 20 ° C., and FIG. 7B shows the result at 50 ° C. 7A and 7B, A represents the elution peak of hydrocortisone, B represents the elution peak of dexamethasone, and C represents the elution peak of testosterone.

  As shown in FIGS. 7A and 7B, the timing at which the elution peak of each steroid appears at 50 ° C. is later than at 20 ° C. This indicates that the surface of the conventional column filler has become hydrophobic due to the increase in temperature.

(Fourth test)
The fourth test relates to fine particle preparation. Specifically, first, 12.0 g of iron (II) chloride tetrahydrate and 24.0 g of iron (III) chloride were dissolved in 50 ml of distilled water and kept at 75 ° C. Next, after adding silica gel under the conditions shown in Table 1, 100 ml of an aqueous ammonium chloride solution (concentration: 28% by mass) was slowly added with stirring.

  And it stirred at 75 degreeC for further 30 minutes. Subsequently, after cooling to room temperature, filtration and drying under reduced pressure were performed. Further, it was dried in an oven kept at 80 ° C. As a result, silica magnetic composite particles having the mass shown in Table 1 were collected. In any of the reference examples, it can be said that the composite fine particles were prepared with high yield.

  Next, 7.5 g of silica magnetic composite fine particles prepared under the conditions of Reference Example 1, 2 or 3 was added to 75 ml of acetic acid aqueous solution (concentration: 1% by mass), and further 2.5 ml of 3-aminopropyltrimethoxy. Silane was added. Subsequently, after making it react at room temperature for 12 hours, it filtered and dried under reduced pressure. As a result, fine particles into which amino groups having masses shown in Table 2 were introduced were collected.

  It can be said that amino groups were introduced with high yield when any silica magnetic composite fine particles were used.

  Further, 7.5 g of silica magnetic composite fine particles prepared under the conditions of Reference Example 3 and 2.5 ml of 3 epoxypropylmethoxysilane were added to 75 ml of toluene, reacted at room temperature for 12 hours, filtered and dried under reduced pressure. Went. As a result, fine particles into which 6.8 g of epoxy groups had been introduced were recovered. That is, it can be said that even if the type of the silane coupling agent is changed, it can be recovered with a high yield.

  Further, an isopropylacrylamide-2-carboxyisopropylacrylamide copolymer (containing 10 mol% carboxyl group), which is a temperature-responsive copolymer, is weighed in the amount shown in Table 3, and dissolved in 10 ml of distilled water. It was. This was then placed in a 50 cc sample tube. Further, the silica magnetic composite particles into which amino groups were introduced in Reference Example 8 (aminated silica magnetic composite fine particles) were weighed by the amounts shown in Table 3 and placed in the 50 cc sample tube. Further, N-ethyl-N′-3-dimethylaminopropylcarbodiimide, which is a condensing agent, is weighed 0.5 times equivalent to the carboxyl group of the polymer, and this is dissolved in 3.5 ml of distilled water at 4 ° C., The resulting liquid was added to the sample tube.

  And it was made to react for 12 hours, keeping the solution in said sample tube at 4 degreeC, and also it was made to react at room temperature for 12 hours. Subsequently, fine particles obtained by filtration were sufficiently washed with cold water. As a result, the silica magnetic composite fine particles having the mass-responsive polymer having the mass shown in Table 3 immobilized thereon were recovered. In any case, it can be said that the silica magnetic composite fine particles having the temperature-responsive polymer immobilized thereon in a high yield were recovered.

  Further, 500 g of the above-mentioned silica magnetic composite particles (epoxidized silica magnetic composite fine particles) having 500 g of epoxy group introduced therein were weighed, and 100 mg of isopropylacrylamide-2-aminoisopropylacrylamide copolymer (Table 4) The temperature-responsive copolymer) was added to the amount of distilled water listed in Table 4 and stirred for 24 hours.

  The fine particles obtained by filtration were sufficiently washed with cold water and dried under reduced pressure. As a result, silica magnetic composite particles having the mass-responsive polymer having the mass shown in Table 4 immobilized thereon were recovered. In any case, it can be said that the silica magnetic composite fine particles having the temperature-responsive polymer immobilized thereon in a high yield were recovered.

  Patent Documents 1 and 2 describe a protein separation method using magnetic fine particles, but the particle size of the magnetic fine particles is too small for use in chromatography. In addition, Patent Documents 1 and 2 do not have a description regarding chromatography.

It is a schematic diagram which shows the column for chromatography which concerns on embodiment of this invention. It is sectional drawing which shows the example of a column filler. It is sectional drawing which shows the other example of a column filler. It is sectional drawing which shows the other example of the column filler. It is a graph which shows the elution behavior when not applying an alternating current (alternating magnetic field). It is a graph which shows the elution behavior in the case of applying an alternating current (alternating magnetic field). It is a figure which shows the example of the chromatography using the column for chromatography which concerns on embodiment. It is a graph which shows the result of a 1st test. It is a graph which shows the elution behavior of hydrocortisone in the second test. It is a graph which shows the elution behavior of dexamethasone in a 2nd test. It is a graph which shows the elution behavior of testosterone in the second test. It is a graph which shows the elution behavior in 20 degreeC in a 3rd test. It is a graph which shows the elution behavior in 50 degreeC in a 3rd test.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1, 1a, 1b, 1c: Column packing material 2: Magnetic fine particle 3: Temperature-responsive polymer layer 4: Silica type material 5: Silica type particle 10: Column 11: Coil 12: Column cylinder

Claims (4)

Magnetic fine particles,
A temperature-responsive polymer layer surrounding the magnetic fine particles;
A chromatography column filler characterized by comprising:   The chromatography column filler according to claim 1, further comprising a binder that chemically bonds the magnetic fine particles and the temperature-responsive polymer layer. A column tube,
A column filler packed in the column cylinder;
Have
The column filler is
Magnetic fine particles,
A temperature-responsive polymer layer surrounding the magnetic fine particles;
A chromatography column characterized by comprising:   The chromatography column according to claim 3, wherein the column filler includes a binder that chemically bonds the magnetic fine particles and the temperature-responsive polymer layer.

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