JP2007327776A - Carrier for liquid chromatography and its manufacturing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a carrier for liquid chromatography having high-pressure resistance, reduced in swelling/shrinkage and especially suitable for ion exchange liquid chromatography. <P>SOLUTION: In the carrier for liquid chromatography comprising a porous hydrophobic crosslinked polymer continuum, the porous hydrophobic crosslinked polymer continuum has 20 μeq/g or above of an ion exchange group only on its surface and has pores A with a diameter of below 100 nm and pores B with a diameter of 600 nm or above. The whole volume of the pores B is 10% or above of the whole pore volume of the porous hydrophobic crosslinked polymer continuum. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention relates to a liquid chromatography carrier having high pressure resistance and less swelling / shrinking, and particularly suitable for ion exchange liquid chromatography, and a method for producing a liquid chromatography carrier.

Among the liquid chromatography methods, in particular, the ion exchange liquid chromatography method is widely used for the separation of ionic substances, and in particular, it is an extremely effective method for the separation and analysis of various biological materials including glycated hemoglobin. Known as.

In the ion exchange liquid chromatography method, the measurement accuracy may decrease due to non-specific adsorption of the analyte or mixture, so it is necessary to suppress this non-specific adsorption. Because it is used, high measurement accuracy is required.

For example, Patent Document 1 discloses a packing material for liquid chromatography having an ion exchange group on the surface of a hydrophobic crosslinked polymer. This filler does not expand or contract even in an aqueous medium and is excellent in pressure resistance, so high measurement accuracy can be expected, but since it is particulate, when manufacturing a column, There is a problem that it is necessary to pack the packing material and adjustment is difficult, and there is a problem that the back pressure applied in the column and the flow path is large.

In order to solve such a problem, Non-Patent Document 1 discloses a method for producing a porous polymer continuous body having an ion exchange group. Since the porous polymer continuum produced by this method has no interparticle volume, it is tightly packed by being produced in the tube used for the column, and a very high transmittance can be obtained. The column efficiency is very high. In addition, since it is not necessary to pack particles and a column can be manufactured, adjustment is easy, and the porosity in the column is larger than the column obtained by packing particles. There is an advantage that the pressure is reduced.
However, the porous polymer continuum obtained by this production method is used for liquid chromatography, because the ion exchange group is present inside the polymer, so that it easily swells and shrinks in an aqueous medium and has poor pressure resistance. However, there is a problem that the peak becomes broad because the equilibration of the eluent is slow.
Japanese Patent Laid-Open No. 3-73848 Frechet et al. , Analytical Chemistry, 1997, volume 69, p. 3646-3649

The present invention has been made in view of the above situation, and aims to provide a liquid chromatography carrier having high pressure resistance, low swelling and shrinkage, and particularly suitable for ion exchange liquid chromatography, and a method for producing a liquid chromatography carrier. To do.

The present invention is a liquid chromatography carrier comprising a porous hydrophobic cross-linked polymer continuum, wherein the porous hydrophobic cross-linked polymer continuum has an ion exchange group of 20 μeq / g or more only on the surface, And a hole A having a diameter of less than 100 nm and a hole B having a diameter of 600 nm or more, and the total volume of the hole B is 10% or more of the total pore volume of the porous hydrophobic polymer continuous body This is a carrier for liquid chromatography.
The present invention is described in detail below.

As a result of intensive studies, the present inventors have determined that a carrier having an ion exchange group attached only to the surface of a porous hydrophobic crosslinked polymer continuous body having a small range of pores and a large range of pores. Has been found to be a carrier for liquid chromatography particularly suitable for ion exchange liquid chromatography because it does not swell and contract even in an aqueous medium and is excellent in pressure resistance, and has completed the present invention.

The carrier for liquid chromatography of the present invention comprises a porous hydrophobic crosslinked polymer continuous body, and has an ion exchange group only on the surface.
Thereby, the carrier for liquid chromatography of the present invention does not swell and shrink even in an aqueous medium, and has excellent pressure resistance.

The ion exchange group has a role of separating while preventing non-specific adsorption of hydrophilic substances such as proteins.
The ion exchange group is not particularly limited, and examples thereof include an N, N-diethylamino-2-hydroxypropyl group, a quaternary trimethylamino group, a sulfonic acid group, and a carboxymethyl group.

The lower limit of the amount of the ion exchange group is 20 μeq / g. If it is less than 20 μeq / g, sufficient separation ability as a carrier for liquid chromatography cannot be exhibited. A preferred lower limit is 30 μeq / g, and a preferred upper limit is 200 μeq / g.

As the porous hydrophobic crosslinked polymer continuum, for example, a hydrophobic crosslinked monomer composition containing a crosslinkable monomer composed of a (meth) acrylic acid derivative or a styrene derivative having a water solubility of 5% by weight or less is polymerized. Can be used.
By using a polymer obtained by polymerizing such a hydrophobic crosslinking monomer composition, the resulting porous hydrophobic crosslinked polymer continuum has almost no ion exchange group (hydrophilic group) inside, It does not swell or shrink even in an aqueous medium.
Here, the water solubility means that 20 mL of a crosslinkable monomer is added to 100 mL of water, stirred at room temperature for 10 minutes × 3 times, left overnight in a 20 ° C. incubator, and then the amount of the crosslinkable monomer dissolved in water is determined by hydrogen It is a value calculated by measurement by a double bond (PSDB) method by flame gas chromatography.

The (meth) acrylic acid derivative having a water solubility of 5% by weight or less is not particularly limited. For example, ethylene glycol di (meth) acrylate, polyethylene glycol di (meth) acrylate, 1,3-butylene glycol di (meth) Acrylate, 1,6-hexaglycol di (meth) acrylate, neopentyl glycol di (meth) acrylate, propylene glycol di (meth) acrylate, polypropylene glycol di (meth) acrylate, trimethylolethane tri (meth) acrylate, trimethylol Propane tri (meth) acrylate, triethylene glycol dimethacrylate, tetramethylol methane triacrylate, tetramethylol methane tetraacrylate, tetramethylol propane trimethacrylate Like the inside of the molecule a polymerizable unsaturated bond having 2 or more.

The styrene derivative having a water solubility of 5% by weight or less is not particularly limited. For example, divinylbenzene, divinyltoluene, divinylxylene, divinylethylbenzene, divinylnaphthalene and the like having two or more polymerizable unsaturated bonds in the molecule Is mentioned.

The hydrophobic crosslinking monomer composition may further contain a non-crosslinking monomer. The non-crosslinkable monomer preferably has a water solubility of 5% by weight or less. When the amount exceeding 5% by weight is used, an ion exchange group (hydrophilic group) may be generated inside the resulting porous hydrophobic crosslinked polymer continuous body, and may swell and shrink in an aqueous medium.
The non-crosslinkable monomer is not particularly limited as long as it is a hydrophobic polymerizable monomer that can be polymerized with the crosslinkable monomer. For example, vinyl esters such as vinyl chloride, vinyl acetate, vinyl propionate, and vinyl stearate Unsaturated nitriles such as (meth) acrylonitrile; acid amides such as (meth) acrylamide, N-methyl (meth) acrylamide, N-ethyl (meth) acrylamide, methyl (meth) acrylate, ethyl (meth) acrylate, Propyl (meth) acrylate, isopropyl (meth) acrylate, butyl (meth) acrylate, t-butyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, stearyl (meth) acrylate, cyclohexyl (meth) acrylate, acrylonitrile, glycerin (Meth) acrylic acid ester derivatives such as Gilles methacrylate; styrene, alpha-methyl styrene, p- methyl styrene, include those having one polymerizable unsaturated bond in the molecule, such as styrene derivatives, such as chloromethylstyrene.

In the hydrophobic crosslinking monomer composition, the preferred lower limit of the content of the crosslinking monomer is 25% by weight. If it is less than 25% by weight, the obtained liquid chromatography carrier may swell and shrink in an aqueous medium. A more preferred lower limit is 50% by weight.

The porous hydrophobic crosslinked polymer continuum constituting the liquid chromatography carrier of the present invention has pores A having a diameter of less than 100 nm and pores B having a diameter of 600 nm or more.

The hole A plays a role of sufficiently increasing the surface area of the liquid chromatography carrier of the present invention and exhibiting sufficient separation ability as a liquid chromatography carrier. If it exceeds 100 nm, the substantial surface area becomes small, and sufficient resolution cannot be obtained. Moreover, since it does not contribute to ion exchange, a minimum is 0.1 nm. A preferable range of the diameter is 0.8 to 50 nm, and a more preferable range is 1 to 25 nm.
The diameter of the hole A can be measured using an automatic specific surface area measuring device or the like.

The hole B plays a role of allowing a sample and an eluent to permeate at a relatively low back pressure when used as a liquid chromatography carrier. If it is less than 600 nm, the back pressure applied to the flow path becomes too high. In addition, the upper limit is 10,000 nm because the resolution is lowered. A preferable range of the diameter is 800 to 4000 nm, and a more preferable range is 1000 to 4000 nm.
The diameter of the hole B can be measured using a mercury porosimeter or the like.

The lower limit of the total volume of the pores B is 10% with respect to the total pore volume of the porous hydrophobic polymer continuous body. When it is less than 10%, when the column is produced using the liquid chromatography carrier of the present invention, the back pressure applied in the column and the flow path becomes too high. A preferred lower limit is 20%, a preferred upper limit is 90%, a more preferred lower limit is 50%, and a more preferred upper limit is 99%.

The method for producing the carrier for liquid chromatography of the present invention is not particularly limited. For example, the hydrophobic crosslinking monomer composition and the porogen are in a volume ratio of 10:90 to 60:40, and the polymerization initiator is the above hydrophobic. Step 1 for preparing a porous hydrophobic crosslinked polymer continuous body by polymerizing a mixed composition containing 0.2 to 5 parts by weight with respect to 100 parts by weight of the crosslinked monomer composition, and the porous hydrophobic crosslinked polymer The continuum is washed with a washing solvent to remove the unreacted hydrophobic crosslinked monomer composition and the porogen, step 2, the porous hydrophobic crosslinked polymer continuum, and the porous By reacting with a substance that reacts with a functional group present on the surface of the hydrophobic crosslinked polymer continuum to generate an ion exchange group, the surface of the porous hydrophobic crosslinked polymer continuum is reacted with. And a method comprising a step 3 for imparting ion-exchange group.
Such a method for producing a carrier for liquid chromatography is also one aspect of the present invention.
Hereinafter, a method for producing the liquid chromatography carrier will be described in detail.

In the method for producing a carrier for liquid chromatography of the present invention, the volume ratio of the hydrophobic crosslinking monomer composition and the porogen is 10:90 to 60:40, and the polymerization initiator is 100 parts by weight of the hydrophobic crosslinking monomer composition. Step 1 of polymerizing a mixed composition containing 0.2 to 5 parts by weight to prepare a porous hydrophobic crosslinked polymer continuous body.

The hydrophobic crosslinking monomer composition is not particularly limited, and as described above, it preferably contains a crosslinking monomer composed of a (meth) acrylic acid derivative or a styrene derivative having a water solubility of 5% by weight or less.

The porogen is used for the purpose of generating pores in the copolymer of the mixed composition.
The porogen is not particularly limited, and examples thereof include water, aliphatic hydrocarbons, aromatic hydrocarbons, esters, alcohols, ketones, ethers, soluble polymer solutions, and mixtures thereof. It is done.

In the mixed composition, the hydrophobic crosslinking monomer composition and the porogen are preferably 10:90 to 60:40 in volume ratio. When the hydrophobic crosslinking monomer composition is less than 10 in volume ratio, the number of pores is too large and sufficient strength may not be obtained. When the hydrophobic crosslinking monomer composition exceeds 60 in volume ratio A sufficient number of holes may not be obtained. More preferably, the hydrophobic crosslinking monomer composition and the porogen are 20:80 to 50:50 by volume ratio.

The polymerization initiator is not particularly limited, and examples thereof include persulfates such as sodium persulfate, potassium persulfate, and ammonium persulfate, o-benzoyl peroxide, lauroyl peroxide, octanoyl peroxide, and o-chlorobenzoyl peroxide. Acetyl peroxide, t-butyl hydroperoxide, t-butyl peroxyacetate, t-butyl peroxyisobutyrate, 3,5,5-trimethylhexanoyl peroxide, t-butylperoxy-2-ethylhexa Organic peroxides such as noate and di-t-butyl peroxide; 2,2-azobisisobutyronitrile, 2,2-azobis (2,4-dimethylvaleronitrile), 4,4-azobis (4 -Cyanopentanoic acid), 2,2-azobis (2-methylbutyro) Tolyl), 2,2-azobis (2,4-dimethylvaleronitrile), include those which generate free radicals azo compounds such as azo-bis-cyclohexane carbonitrile.

Although it does not specifically limit as content of the said polymerization initiator in the said mixed composition, A preferable minimum is 0.2 weight part with respect to 100 weight part of hydrophobic crosslinking monomer compositions, and a preferable upper limit is 5 weight part. If it is less than 0.2 parts by weight, polymerization hardly occurs, and even if it exceeds 5 parts by weight, no further effect can be obtained.

The mixed composition is preferably polymerized after being degassed by a method of bubbling an inert gas such as nitrogen or argon into the mixed composition.

The polymerization method is not particularly limited, and after adding the above mixed composition in a reaction vessel, the reaction is appropriately performed in an inert atmosphere such as nitrogen or argon for 2 to 24 hours depending on the type of initiator and monomer used. What is necessary is just to heat in a 50-90 degreeC hot water bath, an oil bath etc .. The mixed composition may be added to the reaction vessel all at once, or may be added in portions. In the latter case, polymerization may be performed after each addition until the next addition is performed.

The material of the reaction vessel is not particularly limited as long as the volume does not change during the polymerization of the mixed composition, and examples thereof include metals, glass, and hard polymers.

When a tube is used as the reaction container, it is possible to omit the work of filling the column after producing the liquid chromatography carrier, and it is preferable because this tube can be used in the column as it is. In this case, each end of the tube may be closed with a fitting suitable for connecting to liquid chromatography.
In this case, the porous hydrophobic crosslinked polymer continuous body obtained by polymerizing the mixed composition is in a state of extending completely across the inner cross-sectional area of the tube, and the length is about 5 to 200 mm. Become.
When it is desired to increase the length of the porous hydrophobic crosslinked polymer continuous body, several kinds of porous hydrophobic crosslinked polymer continuous bodies may be produced in the same tube.

The method for producing a carrier for liquid chromatography according to the present invention removes the unreacted hydrophobic crosslinked monomer composition and the porogen by washing the porous hydrophobic crosslinked polymer continuum using a washing solvent. Step 2 is performed.

It does not specifically limit as said washing | cleaning solvent, For example, methanol, ethanol, benzene, toluene, acetone, tetrahydrofuran, a dioxane etc. are mentioned.
When washing with the washing solvent, washing with a washing solvent, followed by washing with water, further washing with a washing solvent, or continuous washing using a washing solvent may be performed.
In particular, when a tube is used as the reaction vessel, the cleaning solvent can be pumped through the tube clogged with a porous hydrophobic crosslinked polymer obtained by polymerization. Good.

In the method for producing a carrier for liquid chromatography according to the present invention, the porous hydrophobic crosslinked polymer continuum reacts with a functional group present on the surface of the porous hydrophobic crosslinked polymer continuum to form an ion exchange group. It has the process 3 which provides an ion exchange group only on the surface of the said porous hydrophobic crosslinked polymer continuous body by making it react with the substance to produce | generate.
In the method for producing a carrier for liquid chromatography of the present invention, a hydrophobic crosslinked polymer continuum is prepared in Step 1 and Step 2, and further, Step 3 is performed to form a surface of the hydrophobic crosslinked polymer continuum. Since only an ion exchange group can be imparted, the obtained liquid chromatography carrier does not swell or shrink even in an aqueous medium, and has excellent pressure resistance.

The substance that reacts with the functional group present on the surface of the porous hydrophobic crosslinked polymer continuum to generate an ion exchange group is not particularly limited. For example, diethylamine, triethylamine hydrochloride, sodium sulfite, chloroacetic acid, fuming sulfuric acid Examples include -1,4-dioxane complex.
For example, when the porous hydrophobic cross-linked polymer continuum contains a polymer having an epoxy group, an N, N-diethylamino-2-hydroxypropyl group is imparted by reaction with diethylamine, resulting in a reaction with triethylamine hydrochloride. A quaternary trimethylamino group is imparted by reaction, a sulfonic acid group is imparted by reaction with sodium sulfite, and after the epoxy group is hydrolyzed, a carboxymethyl group is imparted by reaction with chloroacetic acid, Sulfonic acid groups are imparted by reaction with fuming sulfuric acid-1,4-dioxane complex.

Since the carrier for liquid chromatography of the present invention uses a polymer having a high degree of cross-linking as the hydrophobic cross-linked polymer continuous body, it has extremely high mechanical strength and excellent pressure resistance.
Furthermore, since there is no ion exchange group inside the hydrophobic crosslinked polymer continuous body, the degree of swelling and shrinkage is extremely low.
Since the surface of the porous hydrophobic crosslinked polymer continuous body is covered with ion exchange groups, there is no non-specific adsorption of proteins and the like. In addition, when an ion exchange group is added to the surface of the porous hydrophobic crosslinked polymer continuous body, by changing the amount / concentration of the substance that reacts with the functional group present on the surface, the reaction time, the reaction temperature, and the reaction pH, Since the amount of ion-exchange groups imparted to the surface of the porous hydrophobic crosslinked polymer continuum can be adjusted, it becomes a desired carrier for ion-exchange chromatography according to the type of hydrophilic substance for analysis or separation, It can be used in a wide pH range.
Since the carrier for liquid chromatography of the present invention is a porous polymer continuum, the interparticle volume does not exist, so the column can be closely packed, and a very high transmittance is obtained. Column efficiency is very high. Further, since the back pressure applied to the column and the flow path is small, rapid analysis / separation is possible, and liquid leakage from the piping caused by the high back pressure hardly occurs.

According to the present invention, it is possible to provide a liquid chromatography carrier and a liquid chromatography carrier production method that have high pressure resistance and little swelling / shrinkage and are particularly suitable for ion exchange liquid chromatography.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

Example 1
(1) Preparation of column One end of a stainless steel tube (inner diameter 4.6 mm, length 35 mm) provided with a fitting suitable for attachment to liquid chromatography at both ends is closed with a steel nut stopper, After the tube was purged with nitrogen, the other end was closed with a silicone rubber septum.
2.0 g glycidyl methacrylate as non-crosslinkable monomer, 1.3 g trimethylene glycol dimethacrylate as crosslinkable monomer, 0.5 g 2-hydroxy-1,3-dimethacryloxypropane, 0.15 g tetramethylolmethane Triacrylate, 0.05 g tetramethylol methane tetraacrylate, 0.6 g water as porogen, 2.4 g methanol, 3.0 g ethylene glycol, and 0.04 g azobisisobutyronitrile as polymerization initiator A mixed composition was prepared by mixing. The oxygen present was removed by bubbling nitrogen through the mixture composition for 10 minutes. Polymerization was initiated by pouring 0.7 mL of this mixed composition through the septum into the tube and heating in an oil bath at 60 ° C. with a thermocouple. After 5 hours, the tube was removed from the oil bath and allowed to cool to room temperature, after which the stoppers at both ends of the tube were removed and replaced with standard chromatography column fittings. At this point, the column contained a porous hydrophobic cross-linked polymer continuum having a length of 35 mm. Unreacted monomer and porogen were removed by feeding methanol to this column and washing.
20 mL of 1M sodium sulfite aqueous solution (pH 7.0) was fed to this column over 15 hours under the condition of 75 ° C. The epoxy group of the polymerized glycidyl methacrylate unit reacted with sulfite ion to generate a sulfonic acid group. After the reaction was completed, the column was washed with water to obtain a column in which a porous hydrophobic crosslinked polymer continuous body was formed.

(2) Analysis using a column In order to evaluate the protein separation ability as a cation exchange column, a mixture of several kinds of proteins was analyzed. Elution was performed using a 50 mM phosphate buffer (pH 7.0, hereinafter referred to as A solution); and a mixture of A solution and 500 mM NaCl (pH 7.0) (hereinafter referred to as B solution), and 100% of A solution. To B solution 100% by a linear gradient method. The obtained chromatogram is shown in FIG.
Furthermore, human blood was analyzed using ARKRAY Hi-AUTO A1c (hereinafter also referred to as this apparatus), and the separation ability and the like were compared. The measuring method is as follows.
As a human blood sample, a sample obtained by adding heparin immediately after collecting blood of the same person (healthy person) was used. The blood sample is automatically diluted and hemolyzed 290 times by the dedicated hemolyzed blood 21L (phosphate buffer containing a nonionic surfactant) attached to the apparatus. Eluents are C reagent (pH 5.4 phosphate buffer solution), D solution (pH 8.1 phosphate buffer solution) and E solution (pH 5.4 phosphate buffer solution), which are dedicated reagents attached to this device. used. The obtained chromatogram is shown in FIG.

(Example 2)
1.4 g glycidyl methacrylate as non-crosslinkable monomer, 1.7 g trimethylene glycol dimethacrylate as crosslinkable monomer, 0.7 g 2-hydroxy 1,3-dimethacryloxypropane, 0.2 g tetramethylol methane tri A column in which a porous hydrophobic crosslinked polymer continuous body was formed was obtained in the same manner as in Example 1 except that 0.07 g of tetramethylolmethane tetraacrylate was used.
In order to evaluate the protein separation ability as a cation exchange column, human blood was analyzed in the same manner as in Example 1. The obtained chromatogram is shown in FIG.

(Example 3)
3.6 g glycidyl methacrylate as non-crosslinkable monomer, 0.3 g trimethylene glycol dimethacrylate as crosslinkable monomer, 0.1 g 2-hydroxy-1,3-dimethacryloxypropane, 0.03 g tetramethylolmethane A column on which a porous hydrophobic crosslinked polymer continuous body was formed was obtained in the same manner as in Example 1 except that triacrylate and 0.01 g of tetramethylolmethane tetraacrylate were used.
In order to evaluate the protein separation ability as a cation exchange column, human blood was analyzed in the same manner as in Example 1. The obtained chromatogram is shown in FIG.

Example 4
In the same manner as in Example 1, a column in which a porous hydrophobic crosslinked polymer continuous body from which unreacted monomers and porogen were removed was obtained. To this column, 20 mL of diethylamine was fed over 15 hours under the condition of 56 ° C. The epoxy group of the polymerized glycidyl methacrylate unit reacted with diethylamine to produce an N, N-diethylamino-2-hydroxypropyl group. After this reaction was completed, the column was washed with methanol and water to obtain a column in which a porous hydrophobic crosslinked polymer continuous body was formed.
In order to evaluate the protein separation ability as an anion exchange column, a mixture of several proteins was analyzed. Elution was performed using a 20 mM piperazine-hydrochloric acid buffer (pH 6.0, hereinafter referred to as F solution); and an equivalent mixture of F solution and 500 mM NaCl (pH 6.0) (hereinafter referred to as G solution). % To G solution 100% was performed by a linear gradient method. The obtained chromatogram is shown in FIG.

(Comparative Example 1)
A column in which a porous hydrophobic crosslinked polymer continuous body was formed was obtained in the same manner as in Example 1 except that 0.6 g of water and 5.4 g of methanol were used as the porogen. The column was connected to a pump and water was fed at a constant pressure of 60 MPa, but no water flow through the column was observed.

(Comparative Example 2)
In the same manner as in Example 1, a porous hydrophobic crosslinked polymer continuous body from which unreacted monomers and porogen were removed was obtained. To this column, 2.7 mL of 1M aqueous sodium sulfite solution (pH 7.0) was fed over 2 hours under the condition of 75 ° C. After the reaction was completed, the column was washed with water to obtain a column in which a porous hydrophobic crosslinked polymer continuous body was formed.
In order to evaluate the protein separation ability as a cation exchange column, human blood was analyzed in the same manner as in Example 1. The obtained chromatogram is shown in FIG. When the result of FIG. 6 was compared with FIG. 2, it turned out that a peak becomes broad clearly and the resolution is inferior.

<Evaluation>
The following evaluation was performed about the support | carrier and column for liquid chromatography obtained in Examples 1-4 and Comparative Examples 1-2. The results are shown in Table 1.

(1) Measurement of the amount of ion-exchange groups in the carrier For those having a cation-exchange group, the amount of ion-exchange groups is obtained by feeding a 1 mM aqueous copper sulfate solution and measuring the amount of copper ions adsorbed on the ion-exchange groups. It was.
For those having an anion exchange group, first, a 500 mM phosphate buffer solution (pH 7.1) is sent to the column, and the solution is sent to the 20 mM phosphate buffer solution (pH 7.1). The amount of ion exchange groups was determined by measuring the pH displacement of the eluate.

(2) The polymerized carrier was taken out from the column for measuring the pore diameter and pore volume of the carrier, dried with a vacuum dryer, and pulverized into small pieces.
The pore diameter and pore volume of pores of 100 nm or less were measured using a mercury porosimeter using about 1 cm square dry pieces.
Further, the pore diameter and pore volume of pores of 600 nm or more were measured using an automatic specific surface area measuring apparatus using a dried piece of about 0.5 cm square.

(3) Pressure resistance evaluation column of support The column was connected to a pump, and water was fed at a constant pressure of 60 MPa. Thereafter, the column was opened, the carrier was taken out, observed with an optical microscope to confirm the state, and evaluated according to the following criteria.
○: The same shape as that before the constant pressure liquid feeding was maintained.
Δ: A part of the carrier was damaged.
X: The carrier was almost broken.

(4) Back pressure measurement The back pressure was measured using a pressure gauge of a liquid chromatography liquid pump.

According to the present invention, it is possible to provide a liquid chromatography carrier and a liquid chromatography carrier production method that have high pressure resistance and little swelling / shrinkage and are particularly suitable for ion exchange liquid chromatography.

2 is a chromatogram obtained as a result of analyzing a protein mixture using the column obtained in Example 1. FIG. 2 is a chromatogram obtained as a result of analyzing a mixture of human blood using the column obtained in Example 1. FIG. It is the chromatogram obtained as a result of analyzing the mixture of human blood using the column obtained in Example 2. It is the chromatogram obtained as a result of analyzing the mixture of human blood using the column obtained in Example 3. It is the chromatogram obtained as a result of analyzing the mixture of proteins using the column obtained in Example 4. It is the chromatogram obtained as a result of analyzing the mixture of human blood using the column obtained in Comparative Example 2.

Explanation of symbols

1 Peak attributed to myoglobin (derived from horse skeleton) 2 Peak attributed to α-chymotrypsinogen (derived from bovine pancreas) 3 Peak attributed to ribonuclease A (derived from bovine pancreas) 4 Peak attributed to lysozyme (derived from chicken egg white) 5 HbA1a And peak attributed to A1b 6 peak attributed to fetal Hb (F) 7 peak attributed to unstable HbA1c 8 peak attributed to stable HbA1c 9 peak attributed to HbA0 10 peak attributed to conalbumin 11 attributed to transferrin Peak 12 Peak due to ovalbumin

Claims (4)

A liquid chromatography carrier comprising a porous hydrophobic crosslinked polymer continuum,
The porous hydrophobic cross-linked polymer continuum has an ion exchange group of 20 μeq / g or more only on the surface, and a hole A having a diameter of less than 100 nm and a hole B having a diameter of 600 nm or more. The carrier for liquid chromatography, wherein the total volume of the pores B is 10% or more of the total pore volume of the porous hydrophobic polymer continuous body. The porous hydrophobic crosslinked polymer continuum is obtained by polymerizing a hydrophobic crosslinked monomer composition containing a crosslinking monomer composed of a (meth) acrylic acid derivative or a styrene derivative having a water solubility of 5% by weight or less. The liquid chromatography carrier according to claim 1, wherein the content of the crosslinkable monomer in the hydrophobic crosslinkable monomer composition is 25% by weight or more. A method for producing a liquid chromatography carrier according to claim 1,
Mixing which contains hydrophobic crosslinking monomer composition and porogen 10: 90-60: 40 by volume ratio, and 0.2-5 weight part of polymerization initiator with respect to 100 weight part of said hydrophobic crosslinking monomer composition. Step 1 for polymerizing the composition to prepare a porous hydrophobic crosslinked polymer continuum;
Removing the unreacted hydrophobic crosslinking monomer composition and the porogen by washing the porous hydrophobic crosslinked polymer continuum with a washing solvent; and
By reacting the porous hydrophobic cross-linked polymer continuum with a substance that reacts with a functional group present on the surface of the porous hydrophobic cross-linked polymer continuum to generate an ion exchange group. And a step 3 of imparting an ion exchange group only to the surface of the hydrophobic crosslinked polymer continuous body. 4. The production of a carrier for liquid chromatography according to claim 3, wherein the hydrophobic crosslinking monomer composition contains a crosslinking monomer comprising a (meth) acrylic acid derivative or a styrene derivative having a water solubility of 5% by weight or less. Method.

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