JP2010236907A - COLUMN PACKING FOR SEPARATING HEMOGLOBINS, METHOD FOR MEASURING HEMOGLOBIN A1c AND ABNORMAL HEMOGLOBINS, AND METHOD FOR MANUFACTURING COLUMN FILLER FOR SEPARATING HEMOGLOBINS - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a column filler for separating hemoglobins, capable of reducing the non-specific adsorption of hemoglobins and capable of enhancing measuring precision and the life of a column, a method for measuring hemoglobin A1c and abnormal hemoglobins by liquid chromatography using the column packing for separating hemoglobins, and a method for manufacturing the column filler for separating hemoglobins. <P>SOLUTION: The column filler for separating hemoglobins is composed of crosslinked polymer particles which are obtained by polymerizing a monomer mixture, containing a crosslinkable acrylic monomer and consisting of only an acrylic monomer, and the acrylic polymer layer polymerized on the surfaces of the crosslinked polymer particles, wherein the acrylic polymer layer has a cation-exchange group and an anion-exchange group. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention relates to a column packing material for separating hemoglobins that can reduce non-specific adsorption of hemoglobins and improve measurement accuracy and column life. The present invention also relates to a method for measuring hemoglobin A1c and abnormal hemoglobin by liquid chromatography using the column filler for separating hemoglobins, and a method for producing the column filler for separating hemoglobins.

In the field of clinical examination, hemoglobin A1c is generally measured as an index for diagnosis of diabetes. Hemoglobin A1c is measured by liquid chromatography (hereinafter also referred to as HPLC), immunization method, enzyme method, and the like. Among them, the HPLC method is particularly accurate and can be measured in a short time. Used for value management. In general, in order to be used for management of hemoglobin A1c value of diabetic patients, it is necessary that the CV value of the simultaneous reproducibility test is about 1.0% or less.

The requirement for column packing with high column durability (column life) that can measure hemoglobin A1c in a short time and with high accuracy and that can be measured at low cost is the non-specific adsorption of components in the sample to be measured. It is to suppress. The non-specific adsorption of the components in the sample to the column filler reduces the measurement accuracy of the hemoglobin A1c value by deforming the chromatogram and shortens the column durability.

As a column filler for measuring hemoglobin A1c, for example, Patent Document 1 discloses crosslinked polymer particles into which an ion exchange group is introduced. In Patent Document 1, hemoglobin A1c is obtained by a column filler obtained by a general method, that is, a method of epoxidizing a hydroxyl group of a crosslinked polymer particle and then introducing a sulfonic acid group with sodium sulfate. It is disclosed that it can be measured.
However, the column packing obtained by such an ion exchange group introduction method retains the hydrophobicity of the surface portion where no ion exchange group exists, and nonspecific adsorption to this portion cannot be suppressed.

In order to suppress such non-specific adsorption, a method of preparing filler particles with a hydrophilic material is conceivable. However, if the crosslinked polymer particles themselves are made more hydrophilic, pressure resistance and swelling resistance are lowered, and rapid analysis cannot be performed.
As a means for making the column filler hydrophilic without reducing the strength such as pressure resistance of the column filler, it is known to use a two-layered column filler whose surface is covered with a hydrophilic polymer. For example, Patent Document 2 discloses a filler in which an ion exchange group is introduced by coating the surface of the filler with polyvinyl alcohol, which is a hydrophilic polymer. However, since such a coating layer preparation method is based on physical adsorption depending on the hydrophobicity of the polymer chain, when a large number of samples are measured, the coating layer peels off, and the measurement accuracy decreases. The potential is great.

On the other hand, as a method of more strongly coating the surface of the crosslinked polymer particles with a hydrophilic polymer to form a two-layer structure, there is a method in which a hydrophilic monomer is polymerized and coated on the surface of the crosslinked polymer particles. Patent Document 3 discloses a method of forming a hydrophilic polymer layer on the surface of crosslinked polymer particles. In Patent Document 3, hemoglobin A1c can be separated with high accuracy by using cation exchange polymer particles as hydrophilic polymer particles. However, at the present time when higher speed and higher accuracy of measurement are desired, the column packing material of Patent Document 3 has insufficient separation performance. In order to improve the separation performance using the technique of Patent Document 3, it is possible to change the type of the cation exchange polymer on the surface of the crosslinked polymer particles, or to increase or decrease the amount of the cation exchange group. There was a limit to the improvement of separation performance with optimization technology.

The measurement of hemoglobin A1c has a strong demand for high-accuracy measurement within a short time in the field of clinical examination, and it has been necessary to realize higher separation performance after solving the above-mentioned problem of non-specific adsorption.

Japanese Patent Laid-Open No. 03-255360 Japanese Patent Laid-Open No. 11-183460 Japanese Patent Laid-Open No. 03-73848

An object of the present invention is to provide a column packing material for separating hemoglobins that can reduce nonspecific adsorption of hemoglobins and improve measurement accuracy and column life. Another object of the present invention is to provide a method for measuring hemoglobin A1c and abnormal hemoglobin by liquid chromatography using the column filler for separating hemoglobins, and a method for producing the column filler for separating hemoglobins. And

The present invention includes a crosslinked polymer particle obtained by polymerizing a monomer mixture containing a crosslinkable acrylic monomer and consisting only of an acrylic monomer, and polymerization on the surface of the crosslinked polymer particle. The acrylic polymer layer is a column filler for separating hemoglobins having a cation exchange group and an anion exchange group.
The present invention is described in detail below.

The present inventor has improved separation performance by introducing anion exchange groups into column packings for cation exchange using carboxyl groups or sulfonic acid groups, which have been used for the separation of hemoglobins by the conventional HPLC method. I found. That is, a crosslinked polymer particle containing a crosslinkable acrylic monomer and obtained by polymerizing a monomer mixture composed only of an acrylic monomer, and a cation exchange polymerized on the surface thereof By using a column filler composed of an acrylic polymer layer having a group and an anion exchange group, it was found that high separation performance was obtained and that the separation performance was not easily deteriorated even during long-term use.

The column filler for separating hemoglobins of the present invention is based on cross-linked polymer particles obtained by polymerizing a monomer mixture containing a cross-linkable acrylic monomer and consisting only of an acrylic monomer. And
In the present specification, “acrylic” means having an acryl group or a methacryl group. In the present specification, “(meth) acrylic acid” means “acrylic acid or methacrylic acid”.

The crosslinkable acrylic monomer is not particularly limited. For example, polyethylene glycol di (meth) acrylates, polypropylene glycol di (meth) acrylates, alkylene glycol di (meth) acrylates, and at least two in the molecule. Examples include alkylol alkane (meth) acrylates having a (meth) acryl group.
The polyethylene glycol di (meth) acrylates are not particularly limited. For example, ethylene glycol di (meth) acrylate, diethylene glycol di (meth) acrylate, triethylene glycol di (meth) acrylate, tetraethylene glycol di (meth) acrylate, The polypropylene glycol di (meth) acrylates such as polyethylene glycol di (meth) acrylate are not particularly limited. For example, propylene glycol di (meth) acrylate, dipropylene glycol di (meth) acrylate, tripropylene glycol di ( Examples include meth) acrylate, tetrapropylene glycol di (meth) acrylate, and polypropylene glycol di (meth) acrylate.
The alkylene glycol di (meth) acrylates are not particularly limited. For example, polytetramethylene glycol di (meth) acrylate, poly (propylene glycol-tetramethylene glycol) -di (meth) acrylate, polyethylene glycol polypropylene glycol polyethylene glycol- Di (meth) acrylate, 1,3-butylene glycol di (meth) acrylate, 1,4-butylene glycol di (meth) acrylate, 1,6-hexaglycol di (meth) acrylate, 1,9-nonanediol di ( And (meth) acrylate, neopentyl glycol di (meth) acrylate, and the like.
The hydroxyalkyl di (meth) acrylates are not particularly limited, and examples thereof include 2-hydroxy-1,3-di (meth) acryloxypropane, 2-hydroxy-1- (meth) acryloxy-3- (meth) acrylic. Roxypropane, 2-hydroxy-3- (meth) acryloyloxypropyl (meth) acrylate, glycerol di (meth) acrylate, glycerol acrylate methacrylate, urethane (meth) diacrylate, isocyanuric acid di (meth) acrylate, isocyanuric acid tri ( (Meth) acrylate, 1,10-di (meth) acryloxy-4,7-dioxadecane-2,9-diol, 1,10-di (meth) acryloxy-5-methyl-4,7-dioxadecane-2,9- Diol, 1,11-di (meth) acryloxy-4 8-di-oxa-undecane cancer-2,6,10-triol, and the like.
The alkylolalkane (meth) acrylate having at least two (meth) acrylic groups in the molecule is not particularly limited, and examples thereof include trimethylolpropane tri (meth) acrylate, tetramethylolpropane tri (meth) acrylate, and ditril. Examples include methylolpropane tetra (meth) acrylate, tetramethylolmethane tri (meth) acrylate, tetramethylolmethane tetra (meth) acrylate, and trimethylolethane tri (meth) acrylate.
These crosslinkable acrylic monomers may be used alone or in combination of two or more.
Moreover, it is preferable that the said crosslinkable acrylic monomer is a structure which does not have an ion exchange group.

The monomer mixture may contain other non-crosslinkable acrylic monomers as necessary in addition to the crosslinkable acrylic monomers. The non-crosslinkable acrylic monomer is not particularly limited. For example, alkyl (meth) acrylates, polyethylene glycol mono (meth) acrylates, polypropylene glycol mono (meth) acrylates, alkylene glycol mono (meth) acrylate And (meth) acrylates having a hydroxyl group or a glycidyl group.
The alkyl (meth) acrylates are not particularly limited. For example, methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth) acrylate, butyl (meth) acrylate, (meth) acrylic acid 2 -Ethylhexyl etc. are mentioned.
The polyethylene glycol mono (meth) acrylates are not particularly limited. For example, ethylene glycol mono (meth) acrylate, diethylene glycol mono (meth) acrylate, triethylene glycol mono (meth) acrylate, tetraethylene glycol mono (meth) acrylate, Examples include polyethylene glycol mono (meth) acrylate, methoxytri (poly) ethylene glycol mono (meth) acrylate, and methoxypolyethylene glycol mono (meth) acrylate.
The polypropylene glycol mono (meth) acrylates are not particularly limited. For example, propylene glycol mono (meth) acrylate, dipropylene glycol mono (meth) acrylate, tripropylene glycol mono (meth) acrylate, tetrapropylene glycol mono (meth) Examples thereof include acrylate and polypropylene glycol mono (meth) acrylate.
The alkylene glycol mono (meth) acrylates are not particularly limited. For example, poly (ethylene glycol / propylene glycol) mono (meth) acrylate, poly (ethylene glycol / tetramethylene glycol) mono (meth) acrylate, poly (propylene glycol) -Tetramethylene glycol) mono (meth) acrylate, octoxy polyethylene glycol polypropylene glycol-mono (meth) acrylate, and the like.
The (meth) acrylates having a hydroxyl group or a glycidyl group are not particularly limited. For example, glycidyl (meth) acrylate, glycerin mono (meth) acrylate, 2-hydroxyethyl (meth) acrylate, 2-hydroxypropyl (meth) acrylate 2,3-dihydroxylethyl (meth) acrylate, 2,3-dihydroxylpropyl (meth) acrylate, and the like.
These non-crosslinkable acrylic monomers may be used alone or in combination of two or more.
Moreover, it is preferable that the said non-crosslinkable acrylic monomer is a structure which does not have an ion exchange group.

A preferable upper limit of the blending amount of the non-crosslinkable acrylic monomer when the monomer mixture is 100% by weight is 20% by weight. When the blending amount of the non-crosslinkable acrylic monomer exceeds 20% by weight, the degree of cross-linking decreases, resulting in a decrease in pressure resistance and swelling resistance. May decrease. A more preferable upper limit of the blending amount of the non-crosslinkable acrylic monomer is 15% by weight.

The shape of the crosslinked polymer particles is not particularly limited, but is preferably spherical and more preferably true spherical.
The particle diameter of the crosslinked polymer particles is not particularly limited, but a preferable lower limit is 0.1 μm and a preferable upper limit is 50 μm. If the particle diameter of the crosslinked polymer particles is less than 0.1 μm, the column pressure increases, and special parts for imparting pressure resistance to the HPLC are required, which is not preferable. When the particle diameter of the crosslinked polymer particles exceeds 50 μm, the porosity in the column increases, the sample tends to diffuse, and the measurement accuracy may decrease due to broadening of the peak. The more preferable lower limit of the particle diameter of the crosslinked polymer particles is 0.5 μm, and the more preferable upper limit is 30 μm.

The column filler for separating hemoglobins of the present invention includes a crosslinked polymer particle prepared from the monomer mixture, and an acrylic polymer having a cation exchange group and an anion exchange group, which is polymerized on the surface of the crosslinked polymer particle. It is comprised by the type | system | group polymer (henceforth only ion-exchange polymer) layer.
The “cation exchange group” of the ion exchange polymer refers to a functional group having a known cation exchange property, and examples thereof include a cation exchange group such as a carboxyl group, a phosphate group, and a sulfonate group. The “anion exchange group” of the ion exchange polymer refers to a known functional group having anion exchange properties, and examples thereof include anion exchange groups such as a tertiary amino group and a quaternary ammonium group. The cation exchange group in the ionic polymer is particularly preferably a sulfonic acid group, and the anion exchange group is preferably a tertiary amino group.
The cation exchange group or anion exchange group in the present invention (hereinafter also referred to simply as “ion exchange group”) may have any structure associated with these ion exchange groups. All functional groups possessed by For example, the “carboxyl group” in the present invention includes all functional groups to which a carboxyl group is bonded, such as a carboxylethyl group and a carboxylpropyl group.
Moreover, each ion exchange group may have multiple types of ion exchange groups, for example, a carboxyl group and a sulfonic acid group as a cation exchange group, and a tertiary amino group and a quaternary ammonium group as an anion exchange group.

The ion exchange polymer may be (1) a polymer obtained by polymerizing an acrylic monomer having an ion exchange group, or (2) an acrylic single molecule having a functional group convertible to an ion exchange group. It is a polymer obtained by polymerizing a monomer and then converting the functional group to an ion exchange group.

The “acrylic monomer having an ion exchange group” constituting the ion exchange polymer of the above (1) is an ion exchange group, that is, the cation exchange group such as a carboxyl group, a phosphate group, or a sulfonate group. Alternatively, acrylic monomers having any one of the anion exchange groups such as a tertiary amino group and a quaternary ammonium group, or both the cation exchange group and the anion exchange group may be mentioned.

The monomer having the carboxyl group is not particularly limited. For example, (meth) acrylic acid such as (meth) acrylic acid, 2- (meth) acryloyloxyethyl succinate, 2- (meth) acryloyloxyethylphthalic acid, and the like. Derivatives are mentioned.

The monomer having a phosphoric acid group is not particularly limited. For example, ((meth) acryloyloxyethyl) acid phosphate, (2- (meth) acryloyloxyethyl) acid phosphate, (3- (meth) acryloyloxypropyl) ) (Meth) acrylic acid derivatives such as acid phosphate.

The monomer having a sulfonic acid group is not particularly limited, and examples thereof include 2- (meth) acrylamido-2-methylpropanesulfonic acid, 2-sulfoethyl (meth) acrylate, and 3-sulfopropyl (meth) acrylic acid. And (meth) acrylic acid derivatives.

The acrylic monomer having an anion exchange group is not particularly limited, and examples thereof include (meth) acrylates having a tertiary amino group and (meth) acrylic acid derivatives having a quaternary ammonium group.
The (meth) acrylates having the tertiary amino group are not particularly limited. For example, dimethylaminoethyl (meth) acrylate, diethylaminoethyl (meth) acrylate, diethylaminopropyl (meth) acrylate, 2-hydroxy-3-dimethylamino Examples thereof include propyl (meth) acrylate and N, N-dimethyl (meth) acrylamide.
The (meth) acrylic acid derivatives having a quaternary ammonium group are not particularly limited. For example, dimethylaminoethyl (meth) acrylate quaternized product, 2- (meth) acryloyloxyethyltrimethylammonium chloride, 2- (meth) Examples include acryloyloxyethyl triethylammonium chloride and 2-hydroxy-3- (meth) acryloyloxypropyltrimethylammonium chloride.

On the other hand, the “acrylic monomer having a functional group convertible to an ion exchange group” constituting the ion exchange polymer of the above (2) is a functional group that can be converted to an ion exchange group by a chemical reaction or the like. Or an acrylic monomer having a functional group (hereinafter also referred to as a reactive functional group) capable of reacting with a compound having an ion exchange group described later.
The reactive functional group here is not particularly limited as long as it is a non-ion-exchangeable functional group capable of reacting with a compound having an ion-exchange group, which will be described later. For example, a hydroxyl group, a glycol group, Examples include an epoxy group, a glycidyl group, a primary amino group, a secondary amino group, a cyano group, and an aldehyde group, preferably a hydroxyl group, an epoxy group, a glycidyl group, a primary amino group, and a secondary amino group, and more preferably Are a hydroxyl group, an epoxy group, and a glycidyl group.

As the acrylic monomer having a reactive functional group, (meth) acrylic acid esters are preferable. For example, epoxidized (meth) acrylates, hydroxylated (meth) acrylates, aminated (meth) acrylates , Aldehyded (meth) acrylates, cyanated (meth) acrylates, and the like.
The epoxidized (meth) acrylates are not particularly limited, and examples thereof include glycidyl (meth) acrylate.
The hydroxylated (meth) acrylates are not particularly limited. For example, glycerin mono (meth) acrylate, 2-hydroxyethyl (meth) acrylate, 2-hydroxypropyl (meth) acrylate, 2,3-dihydroxylethyl (meta ) Acrylate, 2,3-dihydroxylpropyl (meth) acrylate, and the like.
The aminated (meth) acrylates are not particularly limited, and examples thereof include 2-aminoethyl (meth) acrylate, 2,3-diaminoethyl (meth) acrylate, 2-aminopropyl (meth) acrylate, and 2,3-diamino. Examples thereof include propyl (meth) acrylate and (meth) acrylamide.
The said aldehyde-ized (meth) acrylate is not specifically limited, For example, (meth) acrolein etc. are mentioned.
The cyanated (meth) acrylates are not particularly limited, and examples thereof include cyano (meth) acrylate and ethyl-2-cyanoacrylate.
These acrylic monomers having a reactive functional group may be used alone or in combination of two or more.

The conversion to an ion exchange group for preparing the ion exchange polymer of (2) above is carried out by polymerization of an acrylic monomer having the reactive functional group, which is a polymer having a reactive functional group. The reaction is performed by reacting the reactive functional group of the coalescence with a compound having an ion exchange group.

As a method for introducing an ion exchange group into the polymer having a reactive functional group, a technique based on a known chemical reaction can be used. The methods exemplified below are all known chemical reactions, and any known reaction can be used without limitation as long as it does not involve a reaction such as decomposition of the crosslinked polymer.

When introducing an ion exchange group through a hydroxyl group of the polymer having the reactive functional group, for example, halogenated ethanesulfonic acid such as sodium bromoethanesulfonate, and halogenated acetic acid such as sodium chloroacetate, It can introduce | transduce into the said polymer by using as a compound which has an ion exchange group, and making these compounds react in alkaline hydroxide aqueous solution.
Moreover, an ion exchange group can be similarly introduced by reacting an aldehyde compound having an ion exchange group with a hydroxyl group by an acetal reaction under an acid catalyst.
Furthermore, for example, a carboxyl group can be introduced by esterification by dehydration reaction of a polyfunctional carboxylic compound such as tricarbanilic acid or butanetetracarboxylic acid with a hydroxyl group.
In addition, the sulfonic acid group can also be introduced by reacting 1,3-propane sultone, 1,4-butane sultone or the like in an alkali hydroxide aqueous solution or an alkali hydroxide organic solvent solution.

In addition, when an ion exchange group is introduced via an epoxy group, a glycidyl group, or an amino group, for example, an epoxy compound such as epichlorohydrin or triglycidyl ether is added to a polymer having a reactive functional group in an aqueous alkali hydroxide solution. After reacting in an organic solvent solution of alkali or alkali hydroxide and epoxidizing, reacting with a compound containing a cation exchange group such as sodium sulfate, taurine, glycolic acid, etc., after binding boron trifluoride etherate, sodium sulfite The method etc. which heat-process in aqueous solution are mentioned.
Similarly, a tertiary amino group can be introduced by adding an aqueous 2-chlorotriethylamine hydrochloride solution to an aqueous alkali hydroxide solution or an organic solvent solution of an alkali hydroxide.
When the reactive functional group is an epoxy group or a glycidyl group, it may be subjected to the above treatment as it is.

The acrylic monomer having an anion exchange group or the anion exchange group when the acrylic monomer having a total ion exchange group (including an acrylic monomer having a reactive functional group) is 100% by weight The preferable lower limit of the amount of the acrylic monomer having a functional group that can be converted to is 0.5% by weight, and the preferable upper limit is 20% by weight. When the amount of the acrylic monomer having an anion exchange group or the acrylic monomer having a functional group convertible to the anion exchange group is less than 0.5% by weight, the effect of introducing the anion exchange group May not appear. Cation exchange reaction for separating hemoglobins when the amount of the acrylic monomer having an anion exchange group or the acrylic monomer having a functional group convertible to the anion exchange group exceeds 20% by weight May be hindered and the separation performance may deteriorate. The more preferable lower limit of the amount of the acrylic monomer having an anion exchange group or the acrylic monomer having a functional group convertible to the anion exchange group is 1% by weight, and a more preferable upper limit is 15% by weight. .

Moreover, the preferable minimum of the amount of anion exchange groups which the said ion exchange polymer has per 1g of dry weight of a column packing material is 1 μeq, and the preferable upper limit is 100 μeq. When the amount of anion exchange groups contained in the ion exchange polymer is less than 1 μeq / g, the effect of introducing anion exchange groups may not appear. When the amount of anion exchange groups possessed by the ion exchange polymer exceeds 100 μeq / g, the cation exchange reaction for separating hemoglobins may be inhibited, resulting in poor separation performance.

A column filler for separating hemoglobins of the present invention is a crosslinked polymer particle obtained by polymerizing a monomer mixture containing the above-mentioned crosslinkable acrylic monomer and consisting only of an acrylic monomer, And it can obtain by polymerizing the acrylic monomer which has the said ion exchange group in presence of a polymerization initiator, and forming the ion exchange polymer layer on the surface of the said crosslinked polymer particle. Such a manufacturing method is also one aspect of the present invention.

In the production method of the present invention, first, crosslinked polymer particles are prepared. The crosslinked polymer particles comprise a monomer mixture containing the crosslinkable acrylic monomer and, if necessary, the non-crosslinkable acrylic monomer, and consisting only of the acrylic monomer. And prepared by conducting a polymerization reaction in the presence of a polymerization initiator. The polymerization reaction method is not particularly limited, and for example, a known polymerization reaction such as an emulsion polymerization method, a soap-free polymerization method, a dispersion polymerization method, a suspension polymerization method, or a seed polymerization method can be applied. Among these, it is preferable to use a dispersion polymerization method, a suspension polymerization method, a seed polymerization method, or the like.

Specifically, for example, in the case of suspension polymerization, a polymerization initiator is dissolved in the monomer mixture and dispersed in a suitable dispersion medium, and then, if necessary, in an inert gas atmosphere such as nitrogen gas. By heating with stirring, true spherical polymer particles suitable as a column filler can be obtained.

The said polymerization initiator is not specifically limited, A water-soluble or oil-soluble well-known radical polymerization initiator can be used, For example, a persulfate, an organic peroxide, an azo compound etc. are mentioned.
The persulfate is not particularly limited, and examples thereof include potassium persulfate, sodium persulfate, and ammonium persulfate.
The organic peroxide is not particularly limited. For example, cumene hydroperoxide, benzoyl peroxide, lauroyl peroxide, octanoyl peroxide, o-chlorobenzoyl peroxide, acetyl peroxide, t-butyl hydroperoxide, t -Butyl peroxyacetate, t-butylperoxyisobutyrate, 3,5,5-trimethylhexanoyl peroxide, t-butylperoxy-2-ethylhexanoate, di-t-butyl peroxide, etc. It is done.
The azo compound is not particularly limited. For example, 2,2-azobisisobutyronitrile, 2,2-azobis (2,4-dimethylvaleronitrile), 4,4-azobis (4-cyanopentanoic acid), Examples include 2,2-azobis (2-methylbutyronitrile), 2,2-azobis (2,4-dimethylvaleronitrile), azobiscyclohexanecarbonitrile, and the like.

The preferable lower limit of the blending amount of the polymerization initiator with respect to 100 parts by weight of the monomer mixture is 0.05 parts by weight, and the preferable upper limit is 5 parts by weight. If the amount of the polymerization initiator is less than 0.05 parts by weight, the polymerization reaction may be insufficient or the polymerization reaction may take a long time. When the amount of the polymerization initiator exceeds 5 parts by weight, an agglomerate may be generated due to a rapid reaction.
The polymerization initiator is preferably used after being dissolved in the monomer mixture.

In the polymerization, various additives used in known polymerization methods may be added.
The additive is not particularly limited. For example, a porous agent for forming macropores in the polymer, various chain transfer agents for controlling the polymerization reaction, a dispersant for stabilizing suspended particles, and the like. Is mentioned.

Specifically, for example, an organic solvent that dissolves the monomer mixture but does not dissolve the polymer as a porosifying agent is added to the monomer mixture to perform a polymerization reaction, and the porous agent is added after the polymerization reaction. By removing, macropores can be formed in the resulting polymer. Examples of such organic solvents include aromatic hydrocarbons, saturated hydrocarbons, alcohols and the like.
The aromatic hydrocarbons are not particularly limited, and examples thereof include toluene, xylene, diethylbenzene, dodecylbenzene and the like.
The saturated hydrocarbons are not particularly limited, and examples thereof include hexane, heptane, octane, and decane.
The alcohols are not particularly limited, and examples thereof include alcohols such as isoamyl alcohol and octyl alcohol.
A preferable upper limit of the amount of the porous agent to be added relative to 100 parts by weight of the monomer mixture is 100 parts by weight.

The next step in the production method of the present invention is a step of polymerizing the acrylic monomer having the ion exchange group on the surface of the obtained crosslinked polymer particles. In this step, the acrylic monomer having an ion exchange group is polymerized on the surface of the crosslinked polymer particles by performing a polymerization reaction in the presence of the crosslinked polymer particles and the polymerization initiator. Reaction takes place. The reaction carried out at a portion other than the surface of the crosslinked polymer particles, that is, the polymer consisting only of the acrylic monomer having the ion exchange group is removed by washing after the polymerization reaction.

In order to efficiently polymerize the acrylic monomer having the ion exchange group on the surface of the crosslinked polymer particle, the ion exchange group is added in a state where the polymerization initiator is contained in the crosslinked polymer particle. It is preferable to use a method of polymerizing the acrylic monomer. Examples of the method for causing the crosslinked polymer particles to contain the polymerization initiator include dissolving the polymerization initiator in an organic solvent capable of swelling the crosslinked polymer particles and dissolving the polymerization initiator. Furthermore, a method of impregnating the crosslinked polymer particles with the organic solvent may be mentioned. By this method, the polymerization initiator can be contained in the crosslinked polymer particles. The crosslinked polymer particles containing the polymerization initiator are dispersed in the appropriate dispersion medium described above, and an acrylic monomer having the ion exchange group is added to perform a polymerization reaction.

Further, in the polymerization reaction when preparing the crosslinked polymer particles, before the polymerization reaction is completed, that is, before the polymerization initiator is completely consumed, the acrylic monomer having the ion exchange group is added. By adding to the reaction system, the acrylic monomer having an ion exchange group can be efficiently polymerized on the surface of the crosslinked polymer particles by using the polymerization initiator added first.

Moreover, the column filler for separating hemoglobins of the present invention is a crosslinked polymer obtained by polymerizing a monomer mixture containing the above-mentioned crosslinkable acrylic monomer and consisting only of the acrylic monomer. In the presence of the particles and the polymerization initiator, the acrylic monomer having the reactive functional group is polymerized to form a polymer layer having the reactive functional group on the surface of the crosslinked polymer particle. Furthermore, it can be obtained by reacting the compound having the ion exchange group with the reactive functional group to convert the polymer having the reactive functional group into an ion exchangeable polymer. Such a manufacturing method is also one aspect of the present invention.

The production method of the present invention when an acrylic monomer having a reactive functional group is used can be performed in the same manner as when the above-described acrylic monomer having an ion exchange group is used. That is, after the polymer layer having the reactive functional group is formed on the surface of the crosslinked polymer particle, the polymer layer is converted into an ion-exchangeable polymer by the method described above.

Moreover, when preparing the said ion exchange polymer, the acrylic monomer which has the said ion exchange group, and the acrylic monomer which has the said reactive functional group can also be used together. Specifically, for example, an acrylic monomer having a cation exchange group and an acrylic monomer having a functional group that can be converted into an anion exchange group are polymerized on the surface of the crosslinked polymer particle, and then an anion is obtained. Exchange groups can also be introduced by the methods described above.

Hemoglobins can be measured by liquid chromatography (HPLC) using the column filler for separating hemoglobins of the present invention. Such a measuring method is also one aspect of the present invention.
When measuring hemoglobins such as hemoglobin A1c by HPLC using the column packing material for separating hemoglobins of the present invention, a known HPLC system equipped with a pump, sampler, detector and the like for feeding an eluent is used. The column filled with the column filler for separating hemoglobins of the present invention can be connected to measure hemoglobins in a blood sample.

As the eluent used in the HPLC method using the column filler for separating hemoglobins of the present invention, it is preferable to use buffers and organic solvents containing known salt compounds. , Inorganic acids, salts thereof, amino acids, Good's buffer, and the like.
The organic acid is not particularly limited, and examples thereof include citric acid, succinic acid, tartaric acid, malic acid and the like.
The inorganic acid is not particularly limited, and examples thereof include hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, boric acid, and acetic acid.
The amino acids are not particularly limited, and examples thereof include glycine, taurine, arginine and the like.
In addition, other commonly added substances such as surfactants, various polymers, hydrophilic low molecular weight compounds and the like may be appropriately added to the buffer solution.
The preferable lower limit of the salt concentration of the buffer solution when measuring hemoglobin A1c is 10 mmol / L, and the preferable upper limit is 1000 mmol / L. If the salt concentration of the buffer solution is less than 10 mmol / L, the ion exchange reaction may not be performed, and hemoglobin may not be separated. When the salt concentration of the buffer solution exceeds 1000 mmol / L, the salt may precipitate and adversely affect the system.

In addition to the hemoglobin A1c, abnormal hemoglobin can be measured by HPLC using the column filler for separating hemoglobin of the present invention.
Such a measuring method is also one aspect of the present invention.
Examples of abnormal hemoglobins that can be measured by the measurement method of the present invention include hemoglobin S and hemoglobin C. Moreover, hemoglobin F (fetal hemoglobin) and hemoglobin A2 can also be measured.

The column filler for separating hemoglobins of the present invention is composed of crosslinkable polymer particles and an ion exchange polymer layer having a cation exchange group and an anion exchange group, which are polymerized on the surface thereof. Since the ion exchange polymer has a cation exchange group and an anion exchange group, a higher separation performance can be obtained in a shorter time than the measurement using a conventional column filler, and as a result, a highly accurate measurement can be obtained. In addition, the durability of the column can be improved. Particularly in the measurement of hemoglobin A1c, high-precision measurement and improved column durability are achieved.
That is, according to the present invention, it is possible to provide a column filler for separating hemoglobins that can reduce nonspecific adsorption of hemoglobins and improve measurement accuracy and column life. The present invention also provides a method for measuring hemoglobin A1c and abnormal hemoglobin by liquid chromatography using the column filler for separating hemoglobins, and a method for producing the column filler for separating hemoglobins. Can do.

It is the chromatogram obtained when measuring hemoglobin A1c using the column packing material of Example 1. It is the chromatogram obtained when measuring hemoglobin A1c using the column filler of the comparative example 1. FIG. 2 is a chromatogram obtained when abnormal hemoglobins were measured using the column packing material of Example 1. It is the chromatogram obtained when measuring hemoglobin A2 using the column packing material of Example 1. It is a result of durability evaluation of the column fillers of Examples 1 and 2 and Comparative Examples 1 and 3.

Example 1
A mixture of 400 g of triethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) and 100 g of tetramethylolmethane triacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) as a crosslinkable acrylic monomer, and benzoyl peroxide (Nacalai) as a polymerization initiator 1.0 g of Tesque) was dissolved. The obtained monomer mixture was dispersed in 5 L of a 4 wt% aqueous solution of polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Co., Ltd., “GOHSENOL GH-20”), heated to 80 ° C. in a nitrogen atmosphere with stirring, A time polymerization reaction was carried out. After cooling the temperature to 30 ° C., 195 g of 2-methacrylamide-2-methylpropanesulfonic acid (manufactured by Toagosei Co., Ltd.) as an acrylic monomer having a cation exchange group, and an acrylic monomer having an anion exchange group As a body, 5 g of diethylaminoethyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the reaction system, and the mixture was heated again to 80 ° C. to conduct a polymerization reaction for 1 hour. The obtained crosslinked polymer particles were washed with ion-exchanged water and acetone to obtain crosslinked polymer particles (column filler for separating hemoglobins) into which a sulfonic acid group and a tertiary amino group were introduced.
The obtained column filler for separating hemoglobins was packed into a stainless steel column having an inner diameter of 3 mm and a length of 30 mm to prepare a hemoglobin separation column.

The hemoglobin separation column was connected to an HPLC system (manufactured by Shimadzu Corporation, “LC-10A”). As a measurement sample, healthy human blood collected from sodium fluoride was diluted 150 times with a phosphate buffer (pH 6.8) containing 0.05% Triton X-100 (manufactured by Sigma-Aldrich). Using. Two eluents, 200 mmol / L phosphate buffer (pH 5.3) as eluent A and 400 mmol / L phosphate buffer (pH 7.5) as eluent B, were used at a flow rate of 1.0 mL. And the eluent A was separated from the eluent B by the stepwise gradient method, and the absorbance at 415 nm was measured. As a result, the chromatogram of FIG. 1 was obtained. In FIG. 1, peak 1 is hemoglobin A1c, and peak 2 is hemoglobin A0. Hemoglobin A1c and other hemoglobin fractions were well separated within a short time.

(Example 2)
A mixture of 400 g of tetraethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) as a crosslinkable acrylic monomer and 100 g of 2-hydroxyethyl methacrylate (manufactured by Shin Nakamura Chemical Co., Ltd.) as a non-crosslinkable acrylic monomer. Then, 1.0 g of benzoyl peroxide (manufactured by Nacalai Tesque) was dissolved as a polymerization initiator. The obtained monomer mixture was dispersed in 5 L of a 4 wt% aqueous solution of polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Co., Ltd., “GOHSENOL GH-20”), heated to 80 ° C. in a nitrogen atmosphere with stirring, A time polymerization reaction was carried out. After cooling the temperature to 30 ° C., 170 g of 3-sulfopropylmethacrylic acid (manufactured by Wako Pure Chemical Industries, Ltd.) as an acrylic monomer having a cation exchange group, and 2 as an acrylic monomer having an anion exchange group Then, 30 g of 3-diaminopropyl methacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) was added to the reaction system, and the mixture was heated again to 80 ° C. to conduct a polymerization reaction for 1 hour. The obtained crosslinked polymer particles were washed with ion-exchanged water and acetone to obtain crosslinked polymer particles (column filler for separating hemoglobins) into which a sulfonic acid group and a tertiary amino group were introduced.
The obtained column filler for separating hemoglobins was packed into a stainless steel column having an inner diameter of 3 mm and a length of 30 mm to prepare a hemoglobin separation column.
When measurement was performed using blood from a healthy human subject by the same method as in Example 1, a chromatogram similar to that in FIG. 1 was obtained.

Example 3
A mixture of 450 g of triethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) and 50 g of tetramethylol methane triacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) as a crosslinkable acrylic monomer and benzoyl peroxide (Nacalai) as a polymerization initiator 1.0 g of Tesque) was dissolved. The obtained monomer mixture was dispersed in 5 L of a 4 wt% aqueous solution of polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Co., Ltd., “GOHSENOL GH-20”), heated to 80 ° C. in a nitrogen atmosphere with stirring, A time polymerization reaction was carried out. After cooling the temperature to 30 ° C., 30 g of diethylaminoethyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.) as an acrylic monomer having an anion exchange group, and an acrylic monomer having a functional group that can be converted into a cation exchange group 100 g of glycerin methacrylate (manufactured by NOF Corporation) was added to the reaction system as a body, and the mixture was heated again to 80 ° C. to conduct a polymerization reaction for 1 hour. The obtained crosslinked polymer particles were washed with ion exchange water and acetone to obtain crosslinked polymer particles into which a hydroxyl group that can be converted into a cation exchange group and an anion exchange group were introduced.
50 g of the obtained crosslinked polymer particles were dispersed in 100 mL of a 10% by weight aqueous sodium hydroxide solution, and 40 g of epichlorohydrin was added at room temperature and reacted for 5 hours. 40 g of the obtained epoxidized polymer particles were dispersed in a 20 wt% aqueous sodium sulfate solution and reacted at 80 ° C. for 20 hours. The obtained reaction product was washed with ion-exchanged water to obtain crosslinked polymer particles (hemoglobin separation column filler) into which sulfonic acid groups were introduced in addition to anion exchange groups.
The obtained column filler for separating hemoglobins was packed into a stainless steel column having an inner diameter of 3 mm and a length of 30 mm to prepare a hemoglobin separation column.
When measurement was performed using blood from a healthy human subject by the same method as in Example 1, a chromatogram similar to that in FIG. 1 was obtained.

Example 4
A mixture of 450 g of triethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) and 50 g of tetramethylol methane triacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) as a crosslinkable acrylic monomer and benzoyl peroxide (Nacalai) as a polymerization initiator 1.0 g of Tesque) was dissolved. The obtained monomer mixture was dispersed in 5 L of a 4 wt% aqueous solution of polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Co., Ltd., “GOHSENOL GH-20”), heated to 80 ° C. in a nitrogen atmosphere with stirring, A time polymerization reaction was carried out. After cooling the temperature to 30 ° C., 170 g of 2-methacrylamide-2-methylpropanesulfonic acid (manufactured by Toagosei Co., Ltd.) as an acrylic monomer having a cation exchange group, and a functional group that can be converted into an anion exchange group 30 g of glycidyl methacrylate (manufactured by NOF Corp.) was added to the reaction system as an acrylic monomer having a temperature of 80 ° C., and the polymerization reaction was carried out for 1 hour. The obtained crosslinked polymer particles were washed with ion exchange water and acetone to obtain crosslinked polymer particles into which glycidyl groups that can be converted into anion exchange groups and cation exchange groups were introduced.
50 g of the obtained crosslinked polymer particles were dispersed in 100 mL of a 10% by weight sodium hydroxide aqueous solution, 50 mL of a 5% by weight 2-chlorotrimethylamine hydrochloride aqueous solution was added, and reacted at 80 ° C. for 20 hours. The obtained reaction product was washed with ion-exchanged water to obtain crosslinked polymer particles (column filler for separating hemoglobins) in which tertiary amino groups were introduced in addition to cation exchange groups.
The obtained column filler for separating hemoglobins was packed into a stainless steel column having an inner diameter of 3 mm and a length of 30 mm to prepare a hemoglobin separation column.
When measurement was performed using blood from a healthy human subject by the same method as in Example 1, a chromatogram similar to that in FIG. 1 was obtained.

(Comparative Example 1)
In this comparative example, an example is shown in which a column filler (a filler having only a cation exchange group) is prepared without using the acrylic monomer having an anion exchange group in Example 1.
A crosslinked polymer in which only a sulfonic acid group was introduced by carrying out a polymerization reaction in the same manner as in Example 1 except that the acrylic monomer (diethylaminoethyl methacrylate) having an anion exchange group in Example 1 was not used. Particles (column filler for separating hemoglobins) and a hemoglobin separation column were obtained.
When the measurement was performed using the blood of a healthy human subject by the same method as in Example 1, the chromatogram of FIG. 2 was obtained. In FIG. 2, peak 1 is hemoglobin A1c and peak 2 is hemoglobin A0. Although the measurement time was extended from the chromatogram obtained with the column packing material of Example 1 (FIG. 1), the separation of hemoglobin A1c was poor.

(Comparative Example 2)
This comparative example shows an example in which a column filler (filler having only an anion exchange group) was prepared without using the acrylic monomer having a cation exchange group in Example 2.
A polymerization reaction was carried out in the same manner as in Example 2 except that the acrylic monomer having a cation exchange group in Example 2 (3-sulfopropylmethacrylic acid) was not used, and only a tertiary amino group was introduced. Crosslinked polymer particles (column filler for separating hemoglobins) and a hemoglobin separation column were obtained.
When measured using blood from healthy human subjects by the same method as in Example 1, hemoglobins were not separated at all and all components were eluted as one peak. This is presumably because the positively charged anion exchange groups on the surface of the column packing material and the positively charged hemoglobins repel each other and the ion exchange reaction was not performed.

(Comparative Example 3)
In this comparative example, an example is shown in which the crosslinked polymer particles in Example 1 contain a component derived from a monomer other than acrylic.
As a polymerization initiator, a mixture of 450 g of triethylene glycol dimethacrylate (made by Shin-Nakamura Chemical Co., Ltd.) as a crosslinkable acrylic monomer and 50 g of divinylbenzene (made by Kishida Chemical Co., Ltd.) as another crosslinkable monomer 1.0 g of benzoyl peroxide (manufactured by Nacalai Tesque) was dissolved. The obtained monomer mixture was dispersed in 5 L of a 4 wt% aqueous solution of polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Co., Ltd., “GOHSENOL GH-20”), heated to 80 ° C. in a nitrogen atmosphere with stirring, A time polymerization reaction was carried out. After cooling the temperature to 30 ° C., 195 g of 2-methacrylamide-2-methylpropanesulfonic acid (manufactured by Toagosei Co., Ltd.) as an acrylic monomer having a cation exchange group, and an acrylic monomer having an anion exchange group As a body, 5 g of diethylaminoethyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the reaction system, and the mixture was heated again to 80 ° C. to conduct a polymerization reaction for 1 hour. The obtained crosslinked polymer particles were washed with ion-exchanged water and acetone to obtain crosslinked polymer particles (column filler for separating hemoglobins) into which a sulfonic acid group and a tertiary amino group were introduced.
The obtained column filler for separating hemoglobins was packed into a stainless steel column having an inner diameter of 3 mm and a length of 30 mm to prepare a hemoglobin separation column.
When measured using the blood of a healthy human subject by the same method as in Example 1, the same chromatogram as in FIG. 2 was obtained, and the separation of hemoglobin A1c was poor.

Furthermore, the following evaluation was performed using the hemoglobin separation column obtained in Examples and Comparative Examples.

(1) Recovery test The recovery test was performed using the hemoglobin separation column prepared in the examples and comparative examples. The recovery rate was obtained by attaching a PEEK pipe having an inner diameter of 0.25 mm and a length of 1000 mm to the HPLC instead of the column, and preparing the hemoglobin separation column prepared for the peak total area when the samples used in the examples and comparative examples were measured. It was determined by the ratio (%) of the total peak area when mounted and measured. The results are shown in Table 1.

In the hemoglobin separation column prepared in the example, a recovery rate of almost 100% was obtained.
On the other hand, the recovery rate of the hemoglobin separation column prepared in the comparative example was as low as 80% or less. This is presumably because the hemoglobin as the measurement target did not elute due to nonspecific adsorption to the column packing material.

(2) Separation performance evaluation of modified hemoglobins Instead of human healthy human blood used in the examples, samples containing modified hemoglobins were artificially prepared and measured, and the separation performance from the hemoglobin A1c peak was evaluated. .
Three types of samples containing modified hemoglobins were prepared by a known method: a sample containing Rayval hemoglobin A1c (sample L), a sample containing acetylated hemoglobin (sample A), and a sample containing carbamylated hemoglobin (sample C). .
That is, the sample L was prepared by adding glucose to 2500 mg / dL of human healthy human blood and heating at 37 ° C. for 3 hours. Sample A was prepared by adding acetaldehyde to healthy human blood so as to be 60 mg / dL and heating at 37 ° C. for 2 hours. Sample C was prepared by adding sodium cyanate to 60 mg / dL of human healthy human blood and heating at 37 ° C. for 2 hours.
Samples containing the obtained modified hemoglobins (Sample L, Sample A, Sample C) and human healthy human blood (unmodified products) used for the preparation of the samples containing the modified hemoglobins are shown in Examples and Comparative Examples. It measured using the prepared hemoglobin separation column, and the measured value of hemoglobin A1c was compared. The separation performance was evaluated by calculating and comparing a value (Δ value) obtained by subtracting the hemoglobin A1c value of the unmodified product from the hemoglobin A1c value of the sample containing the modified hemoglobin. The results are shown in Table 2.

The Δ values of the examples shown in Table 2 are 0.2% or less, and it can be seen that hemoglobin A1c can be measured accurately even in samples containing modified hemoglobins. On the other hand, in the case where the hemoglobin separation column of Comparative Example 1 was used, if modified hemoglobin was present, the hemoglobin A1c value fluctuated greatly, and the hemoglobin A1c value could not be measured accurately. Moreover, the hemoglobin separation column of Comparative Examples 2 and 3 could not separate the modified hemoglobin at all.

(3) Separation performance evaluation of abnormal hemoglobins Samples containing hemoglobin S and hemoglobin C as abnormal hemoglobin using the hemoglobin separation column prepared in the examples and comparative examples ("AFSC hemocontrol" manufactured by Helena Laboratories) Was measured.
The chromatogram obtained as a result of measurement using the hemoglobin separation column of Example 1 is shown in FIG. In FIG. 3, peak 1 represents hemoglobin A1c, peak 2 represents hemoglobin A0, peak 3 represents hemoglobin F (fetal Hb), peak 4 represents hemoglobin S, and peak 5 represents hemoglobin C. In the hemoglobin separation column of Example 1, hemoglobin S and hemoglobin C, which are abnormal hemoglobins, were successfully separated. Even when the column fillers of other examples were used, almost the same separation performance was exhibited.
On the other hand, when measured using the hemoglobin separation column of the comparative example, hemoglobin S and hemoglobin C, which are abnormal hemoglobins, could not be separated.

(4) Separation performance evaluation of hemoglobin A2 Using the hemoglobin separation column prepared in the examples and comparative examples, A2 control (level 2) (manufactured by Bio-Rad) was measured as a sample containing hemoglobin A2.
A chromatogram obtained by measurement using the column filler of Example 1 is shown in FIG. In FIG. 4, peak 1 represents hemoglobin A1c, peak 2 represents hemoglobin A0, peak 3 represents hemoglobin F (fetal Hb), and peak 6 represents hemoglobin A2. In the hemoglobin separation column of Example 1, hemoglobin A2 was successfully separated. Even when the column fillers of other examples were used, almost the same separation performance was exhibited.
On the other hand, when measured using the column filler of the comparative example, hemoglobin A2 could not be separated.

(5) Evaluation of column durability Using the hemoglobin separation columns prepared in Examples 1 and 2 and Comparative Examples 1 and 3, the same human healthy blood sample was continuously measured, and the transition of hemoglobin A1c value was confirmed. . The results are shown in FIG.
In the hemoglobin separation column prepared in Examples 1 and 2, the measurement value did not change even after 3000 times measurement, but when the hemoglobin separation column prepared in Comparative Examples 1 and 3 was used, the measurement value decreased. It was confirmed that the column life was short.

ADVANTAGE OF THE INVENTION According to this invention, the column packing material for hemoglobin separation which can reduce the nonspecific adsorption | suction of hemoglobin and can improve a measurement precision and column lifetime can be provided. The present invention also provides a method for measuring hemoglobin A1c and abnormal hemoglobin by liquid chromatography using the column filler for separating hemoglobins, and a method for producing the column filler for separating hemoglobins. Can do.

1 Hemoglobin A1c
2 Hemoglobin A0
3 Hemoglobin F (Fetal Hb)
4 Hemoglobin S
5 Hemoglobin C
6 Hemoglobin A2

Claims (9)

A cross-linked polymer particle obtained by polymerizing a monomer mixture containing a cross-linkable acrylic monomer and comprising only an acrylic monomer, and an acrylic polymer polymerized on the surface of the cross-linked polymer particle A polymer layer,
The acrylic polymer layer has a cation exchange group and an anion exchange group, and is a column filler for separating hemoglobins. The acrylic polymer layer is obtained by polymerizing an acrylic monomer having a cation exchange group and / or an anion exchange group on the surface of the crosslinked polymer particle. Column filler for separation of hemoglobins. The acrylic polymer layer is formed by polymerizing an acrylic monomer having a functional group that can be converted into a cation exchange group and / or an anion exchange group on the surface of the crosslinked polymer particles, and then the cation exchange group and / or The column packing material for separating hemoglobins according to claim 1, wherein the column packing material is obtained by converting a functional group that can be converted into an anion exchange group into a cation exchange group and / or an anion exchange group. The column filler for separating hemoglobins according to claim 1, 2 or 3, wherein the amount of anion exchange groups per gram of dry weight of the column filler is 0.01 to 100 µeq. A method for measuring hemoglobin A1c by liquid chromatography, wherein the column packing material for separating hemoglobins according to claim 1, 2, 3, or 4 is used. A method for measuring hemoglobin A1c and abnormal hemoglobin by liquid chromatography, wherein the column filler for separating hemoglobins according to claim 1, 2, 3, or 4 is used. A method for producing a column filler for separating hemoglobins according to claim 1, 2, 3 or 4,
A step of polymerizing a monomer mixture containing a crosslinkable acrylic monomer and comprising only an acrylic monomer to form a crosslinked polymer particle; and
A polymerization initiator comprising the crosslinked polymer particles and an acrylic monomer having a cation exchange group and an acrylic monomer having an anion exchange group, or a monomer having both a cation exchange group and an anion exchange group A method for producing a column filler for separating hemoglobins, comprising a step of polymerizing in the presence of a polymer and forming a polymer layer having a cation exchange group and an anion exchange group on the surface of the crosslinked polymer particles. A method for producing a column filler for separating hemoglobins according to claim 1, 2, 3 or 4,
A step of polymerizing a monomer mixture containing a crosslinkable acrylic monomer and comprising only an acrylic monomer to form a crosslinked polymer particle; and
The crosslinked polymer particles are polymerized with an acrylic monomer having a functional group that can be converted into a cation exchange group and / or an anion exchange group in the presence of a polymerization initiator, and a cation is formed on the surface of the crosslinked polymer particles. A polymer having a functional group convertible to an exchange group and / or anion exchange group is formed, and the functional group convertible to the cation exchange group and / or anion exchange group is converted to a cation exchange group and / or an anion exchange group A method for producing a column filler for separating hemoglobins, comprising the step of: An acrylic monomer having an anion exchange group-containing acrylic monomer or an acrylic monomer having a functional group convertible to an anion exchange group of 0.5 to 20% by weight and having a cation exchange group The column for separating hemoglobins according to claim 7 or 8, wherein the amount of the acrylic monomer having a functional group that can be converted into a monomer or a cation exchange group is 80 to 99.5% by weight. A method for producing a filler.

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