JP2012168054A - Column packing material for liquid chromatography, method of manufacturing column packing material for liquid chromatography, and method of measuring hemoglobins - Google Patents

Abstract

The present invention provides a column filler having good performance reproducibility. Moreover, the manufacturing method of this column filler and the measuring method of hemoglobin using this column filler are provided.
A liquid chromatography column filler comprising hydrophilic crosslinked polymer particles, wherein the hydrophilic crosslinked polymer particles have a crosslinkable monomer and a hydrophilic monomer dispersed in an aqueous dispersion medium. A column for liquid chromatography, which is obtained by stirring the dispersion obtained above for at least 1 hour at a temperature not lower than the melting point of the crosslinkable monomer and at which the polymerization reaction does not proceed substantially. Agent.
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Description

The present invention relates to a column filler for liquid chromatography comprising hydrophilic crosslinked polymer particles. The present invention also relates to a method for producing the column filler for liquid chromatography, and a method for measuring hemoglobin using the column filler for liquid chromatography.

Liquid chromatography using a hydrophilic column filler is a very useful measurement method for measuring a hydrophilic substance, and is one of the currently widely used analytical methods. Examples of the measurement mode using a hydrophilic column filler include ion exchange chromatography, hydrophilic gel permeation chromatography, and normal phase chromatography.

A hydrophilic liquid chromatography column filler (hereinafter also simply referred to as a column filler) can be obtained by introducing a hydrophilic group into inorganic polymer particles or organic polymer particles. Examples of the material of the inorganic polymer particles include silica, ceramics, and glass. Examples of the material of the organic polymer particles include organic synthetic polymer materials such as polystyrene polymers and acrylic polymers, polyamino acids. And natural polymer materials such as polysaccharides.

Silica-based fillers widely used as inorganic column fillers have been reported to have disadvantages such as elution of silica itself under neutral or higher pH conditions and non-specific adsorption due to residual silanol groups. Among organic polymers, natural polymer fillers are highly hydrophilic and have little non-specific adsorption, but they are not mechanically strong and cannot be measured at high flow rates, greatly extending measurement time. There is a problem. On the other hand, the organic synthetic polymer column filler has an advantage that the inorganic column filler and the natural polymer column filler have few defects and can be produced by a simple method.

As an organic synthetic polymer column filler, for example, Patent Document 1 discloses that a monomer containing a carboxyl group that is a hydrophilic group is 5 to 90% by weight, a crosslinkable monomer is 10 to 95% by weight, and non-crosslinked A column packing material for ion exchange chromatography obtained by mixing and copolymerizing 0 to 85% by weight of a functional monomer is disclosed. Patent Document 2 discloses a column filler in which a hydrophilic monomer having a hydrophilic group is polymerized on the surface of a hydrophobic crosslinked polymer particle.

However, these organic synthetic polymer column fillers show variations in performance when applied to liquid chromatography. In order to obtain a column filler with good reproducibility, it was necessary to strictly control the conditions of the polymerization reaction for obtaining the organic synthetic polymer. However, its control is difficult and often results in a decrease in yield. In general, since column fillers and packed columns are expensive, a decrease in yield has been a serious detriment to cost reduction.

JP 57-178157 A Japanese Patent Laid-Open No. 03-073848

An object of the present invention is to provide a column packing material with good performance reproducibility. Another object of the present invention is to provide a method for producing the column filler and a method for measuring hemoglobin using the column filler.

The present invention 1 is a liquid chromatography column filler comprising hydrophilic cross-linked polymer particles, wherein the cross-linkable monomer and the hydrophilic monomer are dispersed in an aqueous dispersion medium. A column for liquid chromatography, which is obtained by stirring the dispersion obtained above for at least 1 hour at a temperature not lower than the melting point of the crosslinkable monomer and at which the polymerization reaction does not substantially proceed. It is a filler.
The present invention 2 is a liquid chromatography column filler comprising hydrophilic cross-linked polymer particles, wherein the hydrophilic cross-linked polymer particles comprise a dispersion obtained by dispersing a cross-linkable monomer in an aqueous dispersion medium. The hydrophilic monomer on the surface of the crosslinked polymer particles obtained by performing the polymerization reaction after stirring for 1 hour or more at a temperature not lower than the melting point of the crosslinkable monomer and at which the polymerization reaction does not substantially proceed. Is a column filler for liquid chromatography which is obtained by polymerizing.
The present invention is described in detail below.

The present inventor has found that the reason why the reproducibility of the performance of the conventional column packing material for liquid chromatography is poor is that the reproducibility of the particle size distribution is low and the amount of introduction of hydrophilic groups that greatly affects the separation performance. We thought that reproducibility was low. Therefore, the present inventor has found that the reproducibility of the performance of the column packing material is improved by prescribing the stirring conditions in the step prior to the polymerization reaction, which has not been conventionally omitted, rather than precise control of the polymerization reaction. The headline and the present invention have been completed.

The column filler according to the first aspect of the present invention includes a dispersion obtained by dispersing a crosslinkable monomer and a hydrophilic monomer in an aqueous dispersion medium, and having a polymerization reaction substantially equal to or higher than the melting point of the crosslinkable monomer. It consists of hydrophilic cross-linked polymer particles obtained by conducting a polymerization reaction after stirring for 1 hour or more at a temperature at which no progress.
The column filler according to the first aspect of the present invention includes a dispersion obtained by dispersing a crosslinkable monomer and a hydrophilic monomer in an aqueous dispersion medium, and having a polymerization reaction substantially equal to or higher than the melting point of the crosslinkable monomer. Can be produced by a method comprising a step 1 of stirring for 1 hour or more at a temperature that does not proceed and a step 2 of performing a polymerization reaction after performing the above step 1. Such a method for producing a column filler for liquid chromatography is also one aspect of the present invention.

The crosslinkable monomer is preferably a monomer having two or more vinyl groups in one molecule. The crosslinkable monomer is preferably a crosslinkable monomer that does not have an ion exchange group or that has a small amount even if it has an ion exchange group. Further, the crosslinkable monomer is more preferably an acrylic crosslinkable monomer.
In the present specification, the above “acrylic” means having an acrylic group or a methacrylic group. Further, “(meth) acrylate” means “acrylate or methacrylate”, and “(meth) acrylic acid” means “acrylic acid or methacrylic acid”.

Examples of the crosslinkable monomer include polyethylene glycol di (meth) acrylates, polypropylene glycol di (meth) acrylates, alkylene glycol di (meth) acrylates, hydroxyalkyl di (meth) acrylates, and at least in the molecule. Examples include alkylol alkane (meth) acrylates having two (meth) acryl groups.
Examples of polyethylene glycol di (meth) acrylates include ethylene glycol di (meth) acrylate, diethylene glycol di (meth) acrylate, triethylene glycol di (meth) acrylate, tetraethylene glycol di (meth) acrylate, and polyethylene glycol di ( And (meth) acrylate.
Examples of polypropylene glycol di (meth) acrylates include propylene glycol di (meth) acrylate, dipropylene glycol di (meth) acrylate, tripropylene glycol di (meth) acrylate, tetrapropylene glycol di (meth) acrylate, and polypropylene glycol. Examples include di (meth) acrylate.
Examples of the alkylene glycol di (meth) acrylates include 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 (meth) acrylate, And neopentyl glycol di (meth) acrylate.
Examples of hydroxyalkyl di (meth) acrylates include 2-hydroxy-1,3-di (meth) acryloxypropane, 2-hydroxy-1- (meth) acryloxy-3- (meth) acryloxypropane, 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-dioxy Undegan-2,6,10-triol, and the like.
Examples of alkylolalkane (meth) acrylates having at least two (meth) acrylic groups in the molecule include trimethylolpropane tri (meth) acrylate, tetramethylolpropane tri (meth) acrylate, and ditrimethylolpropanetetra ( Examples include (meth) acrylate, tetramethylolmethane tri (meth) acrylate, tetramethylolmethane tetra (meth) acrylate, trimethylolethane tri (meth) acrylate, and the like.
These crosslinkable monomers may be used independently and may use 2 or more types together.

The dispersion may contain a non-crosslinkable monomer as required in addition to the crosslinkable monomer.

The non-crosslinkable monomer is preferably an acrylic monomer, and specific examples include alkyl (meth) acrylates and non-crosslinkable acrylic monomers having a hydroxyl group.
Examples of alkyl (meth) acrylates include, for example, methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth) acrylate, isopropyl (meth) acrylate, butyl (meth) acrylate, (meth) Examples include 2-ethylhexyl acrylate.
Examples of the non-crosslinkable acrylic monomer having a hydroxyl group include polyethylene glycol mono (meth) acrylates, polypropylene glycol mono (meth) acrylates, alkylene glycol mono (meth) acrylates, and other hydroxyl groups (meta ) Acrylates and the like.
Examples of polyethylene glycol mono (meth) acrylates include ethylene glycol mono (meth) acrylate, diethylene glycol mono (meth) acrylate, triethylene glycol mono (meth) acrylate, tetraethylene glycol mono (meth) acrylate, polyethylene glycol mono ( Examples include meth) acrylate, methoxytri (poly) ethylene glycol mono (meth) acrylate, and methoxypolyethylene glycol mono (meth) acrylate.
Examples of polypropylene glycol mono (meth) acrylates include propylene glycol mono (meth) acrylate, dipropylene glycol mono (meth) acrylate, tripropylene glycol mono (meth) acrylate, tetrapropylene glycol mono (meth) acrylate, and polypropylene glycol. A mono (meth) acrylate etc. are mentioned.
Examples of the alkylene glycol mono (meth) acrylates include poly (ethylene glycol / propylene glycol) mono (meth) acrylate, poly (ethylene glycol / tetramethylene glycol) mono (meth) acrylate, and poly (propylene glycol / tetramethylene glycol). ) Mono (meth) acrylate, octoxy polyethylene glycol polypropylene glycol-mono (meth) acrylate, and the like.
Examples of other (meth) acrylates having a hydroxyl group include glycerin mono (meth) acrylate, 2-hydroxyethyl (meth) acrylate, 2-hydroxypropyl (meth) acrylate, and 2,3-dihydroxylethyl (meth). Examples thereof include acrylate and 2,3-dihydroxylpropyl (meth) acrylate.
These non-crosslinkable monomers may be used alone or in combination of two or more.

When using a non-crosslinkable monomer, it is preferable that content of a non-crosslinkable monomer is 100 weight part or less with respect to 100 weight part of a crosslinkable monomer. If the content of the non-crosslinkable monomer exceeds 100 parts by weight, the degree of crosslinking of the resulting crosslinked polymer particles will be lowered and the column filler will be inferior in pressure resistance, and swelling and shrinkage will occur, resulting in separation accuracy. It tends to decrease.

The dispersion contains a hydrophilic monomer having a hydrophilic functional group in addition to the crosslinkable monomer and the non-crosslinkable monomer. The hydrophilic functional group imparts hydrophilicity to the crosslinked polymer particles obtained after the polymerization, and plays a role of separation by interaction with each component in the sample at the time of measurement by liquid chromatography. Accordingly, the hydrophilic monomer can be appropriately selected depending on the measurement mode applied as the column filler.

For example, the hydrophilic monomer used when the column packing material of the present invention 1 is used for ion exchange chromatography is a hydrophilic monomer having one or more ion exchange groups in one molecule (hereinafter referred to as ion). (It is also referred to as an exchangeable monomer).
Examples of the ion exchange group include a cation exchange group such as a carboxyl group, a sulfonic acid group, and a phosphoric acid group, an anion exchange group such as a tertiary amino group, and a quaternary ammonium group.
In addition, in this specification, since the structure accompanying an ion exchange group does not ask | require in an ion exchange group, all the functional groups which have an ion exchange group in the terminal are included. For example, the “carboxyl group” includes all functional groups to which a carboxyl group is bonded, such as a carboxylethyl group and a carboxylpropyl group (hereinafter, when a functional group is described, it means the same structure).

Examples of the monomer having a carboxyl group include (meth) acrylic acid derivatives such as (meth) acrylic acid, 2- (meth) acryloyloxyethyl succinate, 2- (meth) acryloyloxyethylphthalic acid, Examples include crotonic acid, itaconic acid, citraconic acid, mesaconic acid, maleic acid, fumaric acid, and derivatives thereof.

Examples of the monomer having a sulfonic acid group include (meth) 2- (meth) acrylamido-2-methylpropanesulfonic acid, 2-sulfoethyl (meth) acrylate, 3-sulfopropyl (meth) acrylic acid, and the like. Examples include acrylic acid derivatives, styrene sulfonic acid, allyl sulfonic acid, (3-sulfopropyl) -itaconic acid, and derivatives thereof.

Examples of the monomer having a phosphoric acid group include ((meth) acryloyloxyethyl) acid phosphate, (2- (meth) acryloyloxyethyl) acid phosphate, (3- (meth) acryloyloxypropyl) acid phosphate, and These derivatives are mentioned.

Examples of the monomer having a tertiary amino group include dimethylaminoethyl (meth) acrylate, diethylaminoethyl (meth) acrylate, 2-hydroxy-3-dimethylaminopropyl (meth) acrylate, N, N-dimethyl (meta). ) Acrylamide and derivatives thereof.

Examples of the monomer having a quaternary ammonium group include 2- (meth) acryloyloxyethyltrimethylammonium chloride, 2- (meth) acryloyloxyethyltriethylammonium chloride, 2-hydroxy-3- (meth) acryloyloxy. Examples thereof include propyltrimethylammonium chloride and derivatives thereof.

As another method for using the column packing material of the present invention 1 for ion exchange chromatography, a compound having an ion exchange group (hereinafter also referred to as an ion exchange compound) instead of using an ion exchange monomer. A hydrophilic monomer (hereinafter also referred to as a reactive monomer) having a functional group capable of reacting with the polymer (hereinafter also referred to as a reactive group) is polymerized on the surface of the crosslinked polymer particles, and then the reaction is performed. The method of making an ion exchange compound react with a functional group is mentioned.

The reactive group is preferably a nonionic hydrophilic group.
Examples of the nonionic hydrophilic group include a hydroxyl group, a glycol group, an epoxy group, a glycidyl group, a primary amino group, a secondary amino group, a cyano group, and an aldehyde group. , A glycidyl group, a primary amino group, and a secondary amino group are preferable, and a hydroxyl group, an epoxy group, and a glycidyl group are more preferable.

The reactive monomer is preferably a (meth) acrylic acid ester, for example, polyethylene glycol mono (meth) acrylates, polypropylene glycol (meth) acrylates, alkylene glycol mono (meth) acrylates, epoxidation or hydroxylation (Meth) acrylates, aminated (meth) acrylates, aldehyded or cyanated (meth) acrylates and the like can be mentioned.
Examples of polyethylene glycol mono (meth) acrylates include ethylene glycol mono (meth) acrylate, diethylene glycol mono (meth) acrylate, triethylene glycol mono (meth) acrylate, tetraethylene glycol mono (meth) acrylate, polyethylene glycol mono ( Examples include meth) acrylate, methoxytri (poly) ethylene glycol mono (meth) acrylate, and methoxypolyethylene glycol mono (meth) acrylate.
Examples of the polypropylene glycol (meth) acrylates include propylene glycol mono (meth) acrylate, dipropylene glycol mono (meth) acrylate, tripropylene glycol mono (meth) acrylate, tetrapropylene glycol mono (meth) acrylate, and polypropylene glycol mono (Meth) acrylate etc. are mentioned.
Examples of the alkylene glycol mono (meth) acrylates include poly (ethylene glycol / propylene glycol) mono (meth) acrylate, poly (ethylene glycol / tetramethylene glycol) mono (meth) acrylate, and poly (propylene glycol / tetramethylene glycol). ) Mono (meth) acrylate, octoxy polyethylene glycol polypropylene glycol-mono (meth) acrylate, and the like.
Examples of epoxidized or hydroxylated (meth) acrylates include glycidyl (meth) acrylate, glycerin mono (meth) acrylate, 2-hydroxyethyl (meth) acrylate, 2-hydroxypropyl (meth) acrylate, 2,3- Examples include dihydroxylethyl (meth) acrylate and 2,3-dihydroxylpropyl (meth) acrylate.
Examples of aminated (meth) acrylates include 2-aminoethyl (meth) acrylate, 2,3-diaminoethyl (meth) acrylate, 2-aminopropyl (meth) acrylate, and 2,3-diaminopropyl (meth). Examples include acrylate and (meth) acrylamide.
Examples of aldehyde- or cyanated (meth) acrylates include (meth) acrolein, cyano (meth) acrylate, methyl cyanoacrylate, ethyl cyanoacrylate, butyl cyanoacrylate, (meth) acrylonitrile, and the like.
These reactive monomers may be used alone or in combination of two or more.

As a method of reacting an ion exchange compound with a reactive group, a known chemical reaction can be used. These known chemical reactions can be used without limitation as long as the conditions do not involve a reaction such as decomposition of the crosslinked polymer particles.

When the reactive group is a hydroxyl group, for example, a halogenated ethanesulfonic acid such as sodium bromoethanesulfonate or a halogenated acetic acid such as sodium chloroacetate is used as the ion-exchange compound. An ion exchange group can be introduced by reacting these ion exchange compounds with a crosslinked polymer particle having a hydroxyl group on the surface in an aqueous alkali hydroxide solution.
Moreover, an ion exchange group can be introduce | transduced by making the aldehyde compound which has an ion exchange group react with the crosslinked polymer particle which has a hydroxyl group on the surface by an acetalization reaction under an acid catalyst.
Furthermore, for example, a carboxyl group can be introduced on the surface of the crosslinked polymer particles by esterification by a dehydration reaction between a polyfunctional carboxylic compound such as tricarbanilic acid or butanetetracarboxylic acid and a hydroxyl group.
In addition, crosslinked polymer particles can be obtained by reacting 1,3-propane sultone, 1,4-butane sultone, or the like with a crosslinked polymer particle having a hydroxyl group on the surface thereof in an aqueous alkali hydroxide solution or an organic solvent solution. A sulfonic acid group can be introduced on the surface of the substrate.

When the reactive group is an epoxy group or a glycidyl group, a method of reacting a crosslinked polymer particle having an epoxy group or a glycidyl group on the surface with an ion exchange compound such as sodium sulfate, taurine or glycolic acid, an epoxy group or After the crosslinked polymer particles having a glycidyl group and boron trifluoride etherate are bonded, an ion exchange group can be introduced by a method of heat treatment in an aqueous sodium sulfite solution.

When the reactive group is an amino group, an epoxy compound such as epichlorohydrin or triglycidyl ether is epoxidized by reacting in an aqueous solution of an alkali hydroxide or in an organic solvent solution, and then the above epoxy group or glycidyl group. An ion exchange group can be introduced by performing the same treatment.

The hydrophilic monomer used when the column filler of the present invention 1 is used in gel permeation chromatography is hydrophilic having one or more nonionic hydrophilic groups such as hydroxyl groups or epoxy groups in one molecule. A monomer is preferred. For example, monomers such as the above-mentioned polyethylene glycol mono (meth) acrylates, polypropylene glycol di (meth) acrylates, alkylene glycol mono (meth) acrylates, epoxidized or hydroxylated (meth) acrylates may be mentioned. .

The hydrophilic monomer used when the column packing material of the present invention 1 is used for normal phase chromatography has 1 nonionic hydrophilic group such as primary amino group, secondary amino group, cyano group and hydroxyl group. A hydrophilic monomer having one or more molecules in the molecule is preferred. For example, polyethylene glycol mono (meth) acrylates, polypropylene glycol di (meth) acrylates, alkylene glycol mono (meth) acrylates, aminated (meth) acrylates, aldehyded or cyanated (meth) acrylates In addition to monomers, monomers such as (meth) acrylamide, N-methyl (meth) acrylamide, N-ethyl (meth) acrylamide, N-isopropyl (meth) acrylamide, and other acrylamides and derivatives thereof Can be mentioned.

The hydrophilic monomer is preferably an acrylic monomer. The hydrophilic monomer may be a salt such as sodium salt or potassium salt, chloride or the like.
Two or more types of hydrophilic monomers may be mixed and used, and are not limited to the above-described measurement modes, but may be used in other measurement modes or mixed with the monomers listed in other measurement modes. Or may be used. Moreover, as a hydrophilic monomer, the monomer which has the same several functional group in 1 molecule may be used, and the monomer which has a different kind of functional group in 1 molecule may be used.

The content of the hydrophilic monomer in the dispersion may be a crosslinkable monomer or a mixture of a crosslinkable monomer and a non-crosslinkable monomer (hereinafter referred to as “monomer mixture”. The preferred lower limit is 10 parts by weight and the preferred upper limit is 200 parts by weight with respect to 100 parts by weight. If the content of the hydrophilic monomer is less than 10 parts by weight, the separation performance may be lowered when the obtained column filler is used. When the content of the hydrophilic monomer exceeds 200 parts by weight, the hydrophilicity increases, and the pressure resistance and swelling resistance may decrease. Further, when measurement is performed using a plurality of eluents, it takes a long time to equilibrate when switching the eluent, and the measurement time may be extended.

Examples of the aqueous dispersion medium include water, a water-soluble organic solvent, a mixture of water and a water-soluble organic solvent, and the like.
Examples of the water-soluble organic solvent include alcohols such as methanol, ethanol and isopropanol, acetone and acetonitrile.
When the water-soluble organic solvent is used as a mixture with water, the preferable upper limit of the concentration of the water-soluble organic solvent is 20% by weight.

The amount of the aqueous dispersion medium used is preferably 200 parts by weight with respect to 100 parts by weight of the monomer mixture and the hydrophilic monomer (hereinafter also referred to as monomers), and preferably 10,000 weights with respect to the upper limit. Part.

The dispersion may contain a substance having a surface-active effect (hereinafter also referred to as a dispersant) as necessary.
Examples of the dispersant include water-soluble polymer compounds such as polyvinyl alcohol, polyvinyl pyrrolidone, gelatin, starch, hydroxy cellulose, and polyvinyl ether, inorganic salts such as calcium phosphate, sodium lauryl sulfate, sodium lauryl benzene sulfonate, polyoxy Examples thereof include ionic surfactants such as sodium ethylene lauryl sulfate and sodium dialkylsulfosuccinate, and nonionic surfactants such as polyoxyethylene nonyl phenyl ether, polyethylene glycol monostearate and sorbitan monostearate.

The preferable lower limit of the content of the dispersant in the dispersion is 0.01% by weight, and the preferable upper limit is 20% by weight. When the content of the dispersant is less than 0.01% by weight, the added monomer is difficult to disperse and aggregates are likely to be generated. When the content of the dispersant exceeds 20% by weight, the particle size of the obtained hydrophilic crosslinked polymer particles becomes small, and aggregates are easily generated during the polymerization. The more preferable lower limit of the content of the dispersant is 0.1% by weight, and the more preferable upper limit is 10% by weight.

The dispersion may contain various other known additives.
Examples of additives include a pH regulator, an antifoaming agent, a porous agent for forming macropores in the resulting hydrophilic crosslinked polymer particles, various chain transfer agents for controlling the polymerization reaction, and monomers. Examples thereof include an organic compound or an inorganic compound for adjusting the solubility of the mixture.

“More than the melting point of the crosslinkable monomer” means that when a plurality of crosslinkable monomers are used, the melting point is higher than the melting point of all the crosslinkable monomers. When used in combination, it means that the melting point is higher than the melting point of all crosslinkable monomers and non-crosslinkable monomers.
“Temperature at which the polymerization reaction does not proceed substantially” means a temperature at which a polymer produced when stirring is performed at that temperature for at least 1 hour is 10% by weight or less of the total amount of monomers. Specifically, when the monomers exemplified above are used as the monomers and those exemplified below are used as the polymerization initiator, the temperature is preferably 5 to 40 ° C. If the temperature at which the dispersion is stirred is less than 5 ° C., some of the monomers are frozen and difficult to stir uniformly. If the temperature at which the dispersion is stirred exceeds 40 ° C., some of the monomers may be polymerized during stirring, and the reproducibility of the performance of the column filler obtained may be reduced. A more preferable lower limit of the temperature at which the dispersion is stirred is 10 ° C., and a more preferable upper limit is 30 ° C.

The lower limit of the stirring time when stirring the dispersion at a temperature equal to or higher than the melting point of the crosslinkable monomer and at which the polymerization reaction does not proceed substantially is 1 hour. When the stirring time is less than 1 hour, the monomers of the dispersion are not stable, and the reproducibility of the particle size of the resulting hydrophilic crosslinked polymer particles is deteriorated. A preferable lower limit of the stirring time is 3 hours, and a more preferable lower limit is 5 hours.
Even if the stirring time is long, only the operation time is extended, and the effect of improving reproducibility and separation performance cannot be obtained. Even if the reaction system is the above-mentioned “temperature at which the polymerization reaction does not substantially proceed”, since the polymerization may proceed, the preferable upper limit of the stirring time is substantially 48 hours. .

As the polymerization reaction, a known polymerization method can be used. For example, when performing a polymerization reaction by a radical reaction, the polymerization initiator is carried out by performing an initiation reaction for generating radicals in the presence of a polymerization initiator that is a radical generation source. The initiation reaction can be performed by a known technique such as an operation of adding a polymerization initiator, heating, or energy beam irradiation. A method of heating after adding the polymerization initiator is preferable, and a method of adding the monomer in which the polymerization initiator is dissolved to the aqueous dispersion medium and heating is more preferable.

As the polymerization initiator, a known water-soluble or oil-soluble radical polymerization initiator is preferably used. For example, persulfates such as potassium persulfate, sodium persulfate, and ammonium persulfate, cumene hydroperoxide, and benzoyl peroxide are used. Oxide, lauroyl peroxide, octanoyl peroxide, o-chlorobenzoyl peroxide, acetyl peroxide, t-butyl hydroperoxide, t-butyl peroxyacetate, t-butyl peroxyisobutyrate, 3, 5, 5 -Organic peroxides such as trimethylhexanoyl peroxide, t-butylperoxy-2-ethylhexanoate, di-t-butyl peroxide, 2,2'-azobisisobutyronitrile, 2,2 '-Azobis (2,4-dimethylvaleronitrile ), 4,4′-azobis (4-cyanopentanoic acid), 2,2′-azobis (2-methylbutyronitrile), azobiscyclohexanecarbonitrile and the like, and derivatives thereof. .

The amount of the polymerization initiator used is preferably 0.05 parts by weight and preferably 5 parts by weight with respect to 100 parts by weight of monomers. If the amount of the polymerization initiator used is less than 0.05 parts by weight, the polymerization reaction may not proceed sufficiently, and unreacted monomers may remain in the reaction system. Moreover, it takes a long time to complete the polymerization reaction, and the working efficiency is lowered. When the amount of the polymerization initiator used exceeds 5 parts by weight, an agglomerate is likely to be generated due to rapid progress of the reaction.

When performing a polymerization reaction by heating, the preferable minimum of reaction temperature is 40 degreeC, and a preferable upper limit is 100 degreeC. If the reaction temperature is less than 40 ° C., it is difficult to control the temperature, and the reaction may take a long time. When the reaction temperature exceeds 100 ° C., aggregates are likely to be generated.
Moreover, the minimum with the preferable reaction time in a polymerization reaction is 0.3 hour, and a preferable upper limit is 48 hours. When the reaction time is less than 0.3 hours, the polymerization of the monomers becomes insufficient, and the reproducibility of the performance of the obtained column filler may be lowered. When the reaction time exceeds 48 hours, aggregates are likely to be generated.

After carrying out the polymerization reaction, the obtained polymer is taken out of the reaction system and washed with water, an organic solvent, a mixture of water and an organic solvent or the like as necessary to make the surface of the crosslinked polymer particles hydrophilic. Hydrophilic crosslinked polymer particles having a functional polymer can be obtained. Moreover, you may perform the hydrophilization process by a well-known technique as needed to the obtained hydrophilic crosslinked polymer particle.
The hydrophilization treatment is, for example, a method of adsorbing a compound having a hydrophilic group such as a protein such as hemoglobin, albumin or globulin, a saccharide, or a nonionic surfactant, as disclosed in JP-A-2001-91505. A method of performing ozone treatment by the method described in Japanese Unexamined Patent Publication No. 2004-295368 is preferable.

The column filler of the present invention 2 is the same as that of the column filler of the present invention 1 before the stirring step of stirring for 1 hour or more at a temperature not lower than the melting point of the crosslinkable monomer and substantially not allowing the polymerization reaction to proceed. The hydrophilic monomer is not added to the aqueous dispersion medium together with the monomer mixture, and after the polymerization reaction of the monomer mixture proceeds, the hydrophilic monomer is polymerized on the surface of the obtained crosslinked polymer particles. Except for the above, it consists of hydrophilic crosslinked polymer particles obtained under the same conditions as in the present invention 1.
In the column packing material of the present invention 2, since the hydrophilic functional group that interacts with the measurement sample exists only near the surface of the crosslinked polymer, higher separation performance can be obtained.

The monomer mixture constituting the crosslinked polymer particles in the column filler of the present invention 2 is preferably the same monomer mixture as that of the column filler of the present invention 1, and the aqueous dispersion medium used and the polymerization start are used. It is preferable to use the same agent as in the case of the column filler of the present invention 1. As in the case of the column filler of the present invention 1, the hydrophilic monomer in the column filler of the present invention 2 can be selected depending on the measurement mode to which the column filler is applied. Examples thereof include the same hydrophilic monomers as in the case of the agent.

In the column packing material of the present invention 2, “the temperature at which the polymerization reaction does not proceed substantially” means that the polymer produced when stirring at that temperature for at least 1 hour is 10% by weight or less of the total amount of the monomer mixture Means the temperature at which Specifically, when the above-exemplified examples are used as the monomer mixture and the above-described examples are used as the polymerization initiator, the temperature is preferably 5 to 40 ° C. If the temperature at which the dispersion is stirred is less than 5 ° C., a part of the monomer mixture is frozen and it becomes difficult to stir uniformly. When the temperature at the time of stirring the dispersion exceeds 40 ° C., a part of the monomer mixture may be polymerized during stirring, and the reproducibility of the performance of the obtained column filler may be lowered. A more preferable lower limit of the temperature at which the dispersion is stirred is 10 ° C., and a more preferable upper limit is 30 ° C.

Hereinafter, a method for producing the column filler for liquid chromatography of the second aspect of the present invention will be shown.
First, a dispersion obtained by dispersing the monomer mixture was stirred in the same manner as in the column filler of the present invention 1, and then the monomer mixture was polymerized until the polymerization reaction was completed. A hydrophilic monomer is added to a dispersion obtained by dispersing a crosslinked polymer in an aqueous dispersion medium. Thereafter, the hydrophilic monomer is polymerized on the surface of the crosslinked polymer particles by performing a polymerization reaction in the presence of a polymerization initiator using a dispersion containing the crosslinked polymer particles and the hydrophilic monomer. be able to.

In order to efficiently polymerize the hydrophilic monomer on the surface of the crosslinked polymer particles, it is preferable to previously contain a polymerization initiator in the crosslinked polymer particles. Examples of the method of incorporating the polymerization initiator in the crosslinked polymer particles include, for example, dissolving the polymerization initiator in an organic solvent that can swell the crosslinked polymer particles and dissolve the polymerization initiator, and further crosslink the polymer particles. And the like.

When the hydrophilic monomer polymerization reaction is carried out by heating, the preferred lower limit of the reaction temperature is 40 ° C and the preferred upper limit is 100 ° C. If the reaction temperature of the polymerization reaction of the hydrophilic monomer is less than 40 ° C., the reaction may take a long time. When the reaction temperature of the polymerization reaction of the hydrophilic monomer exceeds 100 ° C., aggregates are likely to be generated.
The preferable lower limit of the reaction time in the polymerization reaction of the hydrophilic monomer is 0.3 hours, and the preferable upper limit is 12 hours. If the reaction time in the polymerization reaction of the hydrophilic monomer is less than 0.3 hours, the polymerization of the hydrophilic monomer becomes insufficient, and the reproducibility of the performance of the obtained column filler may be lowered. If the reaction time in the polymerization reaction of the hydrophilic monomer exceeds 12 hours, aggregates are likely to be generated. The more preferable lower limit of the reaction time in the polymerization reaction of the hydrophilic monomer is 1 hour, and the more preferable upper limit is 6 hours.

After carrying out the polymerization reaction of the hydrophilic monomer, the obtained polymer is taken out from the reaction system and washed with water, an organic solvent, a mixture of water and an organic solvent, etc. Hydrophilic crosslinked polymer particles having a hydrophilic polymer on the surface of the coalesced particles can be obtained. Further, as in the case of the column filler of the first aspect of the present invention, the obtained hydrophilic crosslinked polymer particles may be subjected to a hydrophilic treatment by a known technique, if necessary.

Another method for producing the column filler of the present invention 2 is that before the polymerization reaction in preparing the crosslinked polymer particles by the polymerization reaction of the monomer mixture is completed, that is, the polymerization initiator is completely consumed. A method of polymerizing the hydrophilic monomer on the surface of the crosslinked polymer particles by adding a hydrophilic monomer, and using this method, the polymerization initiator added first is used. The hydrophilic monomer can be efficiently polymerized on the surface of the crosslinked polymer particles.

A method for producing the column filler of the present invention 2, wherein a dispersion obtained by dispersing a crosslinkable monomer in an aqueous dispersion medium is a polymerization reaction substantially equal to or higher than the melting point of the crosslinkable monomer. Step 1 of stirring at a temperature that does not proceed for 1 hour or more, Step 2 of performing the first polymerization reaction after performing Step 1 above, and obtaining crosslinked polymer particles, and before the completion of the first polymerization reaction A method for producing a column filler comprising the step 3 of adding a hydrophilic monomer to carry out a second polymerization reaction and polymerizing the hydrophilic monomer on the surface of the crosslinked polymer particles is also disclosed in 1 of the present invention. One.

When the first polymerization reaction is carried out by heating, the reaction temperature of the first polymerization reaction varies depending on the type of monomer used and the type of polymerization initiator, but the preferred lower limit is 40 ° C. and the preferred upper limit is 100 ° C. When the reaction temperature of the first polymerization reaction is less than 40 ° C., the reaction may take a long time. When the reaction temperature of the first polymerization reaction exceeds 100 ° C., aggregates are likely to be generated.
The preferable lower limit of the reaction time in the first polymerization reaction is 0.3 hours, and the preferable upper limit is 12 hours. When the reaction time in the first polymerization reaction is less than 0.3 hour, the polymerization of the monomer mixture becomes insufficient, and the reproducibility of the performance of the obtained column filler may be lowered. When the reaction time in the first polymerization reaction exceeds 12 hours, the polymerization initiator is consumed, and the reaction rate of the second polymerization reaction may decrease. The more preferable lower limit of the reaction time in the first polymerization reaction is 1 hour, and the more preferable upper limit is 6 hours.

Before the first polymerization reaction is completed, the method for adding the hydrophilic monomer when adding the hydrophilic monomer to the reaction system depends on the type and amount of the hydrophilic monomer to be used. The most appropriate method can be selected. For example, a method of adding the whole amount of the hydrophilic monomer at once, a method of adding a part thereof, a method of adding it over several minutes to several hours, and the like can be mentioned. Moreover, you may change the temperature of a reaction system at the time of addition of a hydrophilic monomer.

When the second polymerization reaction is carried out by heating, the reaction temperature of the second polymerization reaction varies depending on the type of monomer used and the type of polymerization initiator, but the preferred lower limit is 40 ° C. and the preferred upper limit is 100 ° C. If the reaction temperature of the second polymerization reaction is less than 40 ° C., it is difficult to control the temperature, and the reaction may take a long time. When the reaction temperature of the second polymerization reaction exceeds 100 ° C., aggregates are likely to be generated.
The preferable lower limit of the reaction time in the second polymerization reaction is 0.3 hours, and the preferable upper limit is 12 hours. If the reaction time in the second polymerization reaction is less than 0.3 hours, the polymerization of the hydrophilic monomer may be insufficient, and the reproducibility of the performance of the obtained column filler may be lowered. When the reaction time in the second polymerization reaction exceeds 12 hours, an aggregate mainly due to a homopolymer of the hydrophilic monomer is likely to be generated. The more preferable lower limit of the reaction time in the second polymerization reaction is 1 hour, and the more preferable upper limit is 6 hours.

After carrying out the second polymerization reaction, the obtained polymer is taken out of the reaction system and, if necessary, washed with water, an organic solvent, a mixture of water and an organic solvent, etc. Hydrophilic crosslinked polymer particles having a hydrophilic polymer on the surface can be obtained. Further, as in the case of the column filler of the first aspect of the present invention, the obtained hydrophilic crosslinked polymer particles may be subjected to a hydrophilic treatment by a known technique, if necessary.

The column filler of the present invention 1 and the column filler of the present invention 2 (hereinafter also referred to as the column filler of the present invention) can be applied to liquid chromatography by filling the column. In the liquid chromatography using the column filler of the present invention, a liquid chromatograph having a known configuration and an eluent having a known composition can be used.

The column filler of the present invention is particularly effective in measuring hemoglobins.
The method for measuring hemoglobin by liquid chromatography using the column filler of the present invention 1 or the column filler of the present invention 2 is also one aspect of the present invention.

The method for measuring hemoglobin of the present invention can measure hemoglobins contained in healthy human blood such as hemoglobin A0, hemoglobin A1c, hemoglobin F (fetal hemoglobin) and hemoglobin A2 in a short time with high accuracy. Furthermore, abnormal hemoglobins such as hemoglobin S, hemoglobin C, hemoglobin D, hemoglobin E and the like can be measured.

ADVANTAGE OF THE INVENTION According to this invention, the column filler with favorable reproducibility of performance can be provided. Moreover, according to this invention, the manufacturing method of this column filler and the measuring method of hemoglobin using this column filler can be provided.

It is the chromatogram obtained when measuring proteins using the column packing material obtained in Example 1. It is the chromatogram obtained when measuring the hemoglobin of healthy human blood using the column packing material obtained in Example 1. It is the chromatogram obtained when measuring proteins using the column packing material obtained in Example 2. It is the chromatogram obtained when measuring proteins using the column filler obtained in Example 3. It is the chromatogram obtained when measuring the saccharides using the column filler obtained in Example 4. It is the chromatogram obtained when measuring proteins using the column packing material obtained in Example 5. It is the chromatogram obtained when the hemoglobin of normal human blood was measured using the column filler obtained in Example 5. It is the chromatogram obtained when measuring proteins using the column packing material obtained in Example 6. It is the chromatogram obtained when measuring proteins using the column packing material obtained in Example 7. It is the chromatogram obtained when measuring the saccharide | sugar using the column filler obtained in Example 8. FIG. It is the chromatogram obtained when measuring abnormal hemoglobin using the column packing material obtained in Example 5. It is the chromatogram obtained when measuring abnormal hemoglobin using the column packing material obtained in the comparative example 5. It is the chromatogram obtained when measuring hemoglobin containing hemoglobin A2 using the column packing material obtained in Example 5. It is a chromatogram obtained when measuring hemoglobin containing hemoglobin A2 using the column packing material obtained in Comparative Example 5.

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
In Example 1, the column filler of the present invention 1 was prepared as a column filler for cation exchange chromatography, and the performance was confirmed.

(1) Preparation of column filler As a crosslinkable monomer, 150 g of tetraethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.), 150 g of diethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.), and hydrophilic single monomer 100 g of 2-acrylamido-2-methylpropanesulfonic acid (manufactured by Toagosei Co., Ltd.) as a monomer and 1.0 g of benzoyl peroxide (manufactured by Kishida Chemical Co., Ltd.) as a polymerization initiator are mixed and dissolved, and 5 as a dispersant. It was dispersed in 2000 mL of an aqueous solution of wt% polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Co., Ltd., “GOHSENOL GH-20”) The obtained dispersion was stirred at 25 ° C. for 8 hours (stirring step).
Next, the reaction system was heated to 80 ° C. under a nitrogen atmosphere to carry out a polymerization reaction for 24 hours. The obtained polymer was washed to obtain a column filler.
As a result of measuring the particle size distribution of the obtained column filler with a particle size distribution measuring device (“Accurizer 780” manufactured by Nikonp Co., Ltd.), the average particle size was 8.2 μm.

(2) Packing into a column 0.8 g of the column packing obtained in “(1) Preparation of column packing” is dispersed in 30 mL of 50 mmol / L phosphate buffer (pH 6.0), and stirred. Turned into. After sonication for 5 minutes, the entire amount of the slurry was injected into a packer (manufactured by ASONE) connected to a stainless steel empty column (4.6φ × 35 mm). A liquid feed pump (GL Science, “PU-614”) was connected to the packer, and the liquid was fed at a pressure of 20 MPa and filled at a constant pressure.

(3) Performance evaluation by liquid chromatography 1

A standard substance mixture of proteins was measured using the column obtained in “(2) Packing in a column”. The measurement conditions are shown below.
System: LC-10A system (manufactured by Shimadzu Corporation)
Detection wavelength: 280 nm
Eluent A: 100 mmol / L phosphate buffer (pH 6.9)
Eluent B: Eluent A + 500 mmol / L NaCl (pH 6.9)
Elution conditions: Linear gradient from eluent A 100% to eluent B 100% Flow rate: 1.5 mL / min Sample to be measured: Mixture of trypsinogen, α-chymotrypsinogen, ribonuclease A and lysozyme (all of which are manufactured by Sigma-Aldrich)

The obtained chromatogram is shown in FIG. In FIG. 1, peak 1 represents trypsinogen, peak 2 represents α-chymotrypsinogen, peak 3 represents ribonuclease A, and peak 4 represents lysozyme. From FIG. 1, it was confirmed that each peak can be satisfactorily separated in a short time. Table 1 shows the reproducibility of the retention time of lysozyme when the same measurement is repeated 10 times. The CV value of the retention time of lysozyme when measured 10 times continuously was 3.2%, and the reproducibility was good.

(4) Performance evaluation 2 by liquid chromatography
Using the column obtained in “(2) Packing the column”, hemoglobins including hemoglobin A1c in the blood of healthy persons were measured. The measurement conditions are shown below.
System: LC-10A system (manufactured by Shimadzu Corporation)
Detection wavelength: 415 nm
Eluent C: 30 mmol / L phosphate buffer (pH 5.8)
Eluent D: 220 mmol / L phosphate buffer (pH 8.1)
Elution conditions: Step gradient from eluent C 100% to eluent D 100% Flow rate: 1.5 mL / min Measurement sample: 0.05% Triton X-100 (manufactured by Sigma-Aldrich) ) Diluted with phosphate buffer (pH 6.7)

The obtained chromatogram is shown in FIG. In FIG. 2, peak 11 represents hemoglobin F, peak 12 represents hemoglobin A1c, and peak 13 represents hemoglobin A0. From FIG. 2, it was confirmed that each peak can be separated well in a short time. Table 1 shows the reproducibility of the retention time of hemoglobin A1c when the same measurement is repeated 10 times. The CV value of the retention time of hemoglobin A1c when measured 10 times continuously was 2.4%, and the reproducibility was good.

(5) Evaluation of polymerization reproducibility Column fillers were prepared 20 times (20 lots) under the same conditions as in “(1) Preparation of column filler”. Each lot was subjected to “(4) Performance evaluation 2 by liquid chromatography” to confirm the reproducibility of the retention time of hemoglobin A1c. In addition, the average value when the measurement of each lot was performed three times was used as the measurement value of the corresponding lot. The obtained results are shown in Table 1. The CV value of the retention time of hemoglobin A1c when 20 lots were measured was 3.8%, and the reproducibility was good.

(Example 2)
In Example 2, the column filler of the present invention 1 was prepared as a column filler for anion exchange chromatography, and the performance was confirmed.

(1) Preparation of column filler 300 g of triethylene glycol dimethacrylate (made by Shin-Nakamura Chemical Co., Ltd.) as a crosslinkable monomer, 100 g of diethylaminoethyl methacrylate (made by Wako Pure Chemical Industries, Ltd.) as a hydrophilic monomer, and As a polymerization initiator, 1.5 g of benzoyl peroxide was mixed and dissolved, and dispersed in 2000 mL of a 5 wt% aqueous polyvinyl alcohol solution. The obtained dispersion was stirred at 20 ° C. for 20 hours (stirring step).
Next, the reaction system was heated to 80 ° C. under a nitrogen atmosphere to carry out a polymerization reaction for 24 hours. The obtained polymer was washed to obtain a column filler.
As a result of measuring the particle size distribution of the obtained column filler in the same manner as in Example 1, the average particle size was 6.7 μm.

(2) Packing into a column In the same manner as in Example 1, 1.4 g of the obtained column filler was packed into a stainless steel empty column (4.6 φ × 50 mm).

(3) Performance Evaluation by Liquid Chromatography A protein standard substance mixture was measured using the column obtained in “(2) Packing into a column”. The measurement conditions are shown below.
System: LC-10A system (manufactured by Shimadzu Corporation)
Detection wavelength: 280 nm
Eluent E: 50 mmol / L Tris-HCl buffer (pH 8.2)
Eluent F: Eluent E + 500 mmol / L NaCl (pH 8.2)
Elution conditions: Linear gradient from eluent E 100% to eluent F 100% Flow rate: 1.5 mL / min Sample to be measured: Conalbumin, ovalbumin, trypsin inhibitor mixture

The obtained chromatogram is shown in FIG. In FIG. 3, peak 21 shows conalbumin, peak 22 shows ovalbumin, and peak 23 shows trypsin inhibitor. From FIG. 3, it was confirmed that each peak can be satisfactorily separated in a short time. Table 1 shows the reproducibility of the retention time of trypsin inhibitor when the same measurement is repeated 10 times in succession. The CV value of the retention time of the trypsin inhibitor when measured 10 times continuously was 3.4%, and the reproducibility was good.

(4) Evaluation of polymerization reproducibility Column fillers were prepared 20 times (20 lots) under the same conditions as in “(1) Preparation of column filler”. Each lot was subjected to “(3) performance evaluation by liquid chromatography” to confirm the reproducibility of the retention time of trypsin inhibitor. The obtained results are shown in Table 1. The CV value of the retention time of trypsin inhibitor when measuring 20 lots was 4.7%, and the reproducibility was good.

(Example 3)
In Example 3, the column filler of the present invention 1 was prepared as a column filler for aqueous gel permeation chromatography, and the performance was confirmed.

(1) Preparation of Column Filler 300 g of tetraethylene glycol dimethacrylate (made by Shin-Nakamura Chemical Co., Ltd.) as a crosslinkable monomer, 100 g of 2-hydroxyethyl methacrylate (made by Wako Pure Chemical Industries, Ltd.) as a hydrophilic monomer, 100 g of polyethylene glycol monomethacrylate (manufactured by NOF Corporation) as a non-crosslinkable monomer, 400 g of toluene, and 1.5 g of benzoyl peroxide as a polymerization initiator are mixed and dissolved to obtain 2500 mL of 6 wt% polyvinyl alcohol. Dispersed in an aqueous solution. The obtained dispersion was stirred at 15 ° C. for 10 hours (stirring step).
Next, the reaction system was heated to 80 ° C. under a nitrogen atmosphere to carry out a polymerization reaction for 24 hours. The obtained polymer was washed to obtain a column filler.
As a result of measuring the particle size distribution of the obtained column filler in the same manner as in Example 1, the average particle size was 10.5 μm.

(2) Packing into column In the same manner as in Example 1, 3.2 g of the obtained column filler was packed into a stainless steel empty column (6.0 φ × 100 mm).

(3) Performance Evaluation by Liquid Chromatography A mixture of molecular weight standard substances was measured using the column obtained in “(2) Packing into a column”. The measurement conditions are shown below.
System: LC-10A system (manufactured by Shimadzu Corporation)
Detector: Differential refractometer SE-51 (Showa Denko)
Eluent G: 80 mmol / L phosphate buffer (pH 6.5) containing 60 mmol / L sodium sulfate
Flow rate: 1.2 mL / min Measurement sample: thyroglobulin (molecular weight 640,000), γ-globulin (molecular weight 155000), ovalbumin (molecular weight 47000), ribonuclease (molecular weight 13700) (all are manufactured by Wako Pure Chemical Industries, Ltd.) Mixture of

The obtained chromatogram is shown in FIG. In FIG. 4, peak 31 shows thyroglobulin, peak 32 shows γ-globulin, peak 33 shows ovalbumin, and peak 34 shows ribonuclease. From FIG. 4, it was confirmed that each peak can be satisfactorily separated in a short time. Table 1 shows the reproducibility of the retention time of ribonuclease when the same measurement is repeated 10 times in succession. The CV value of the retention time of ribonuclease when measured 10 times continuously was 4.3%, and the reproducibility was good.

(4) Evaluation of polymerization reproducibility Column fillers were prepared 20 times (20 lots) under the same conditions as in “(1) Preparation of column filler”. Each lot was subjected to “(3) Performance Evaluation by Liquid Chromatography” to confirm reproducibility of ribonuclease retention time. The obtained results are shown in Table 1. The ribonuclease retention time CV value when measuring 20 lots was 4.9%, and the reproducibility was good.

Example 4
In Example 4, the column filler of the present invention 1 was prepared as a column filler for normal phase chromatography, and the performance was confirmed.

(1) Preparation of column filler 1,10-dimethacryloxy-4,7-dioxadecane-2,9-diol (manufactured by Kyoeisha Chemical Co., Ltd.) 100 g as a crosslinkable monomer, and tetraethylene glycol dimethacrylate (Shin Nakamura Chemical) 100 g of 2-aminoethyl methacrylate (manufactured by NOF Corporation) as a hydrophilic monomer and 1.5 g of benzoyl peroxide as a polymerization initiator are mixed and dissolved, and 2000 mL of 4% by weight polyvinyl Dispersed in an aqueous alcohol solution. The obtained dispersion was stirred at 30 ° C. for 5 hours (stirring step).
Next, the reaction system was heated to 80 ° C. under a nitrogen atmosphere to carry out a polymerization reaction for 24 hours. The obtained polymer was washed to obtain a column filler.
As a result of measuring the particle size distribution of the obtained column filler in the same manner as in Example 1, the average particle size was 5.5 μm.

(2) Packing into a column In the same manner as in Example 1, 1.4 g of the obtained column filler was packed into a stainless steel empty column (4.6 φ × 50 mm).

(3) Performance Evaluation by Liquid Chromatography A saccharide standard substance mixture was measured using the column obtained in “(2) Packing into a column”. The measurement conditions are shown below.
System: LC-10A system (manufactured by Shimadzu Corporation)
Detection wavelength: 280 nm
Eluent H: acetonitrile: water = 75: 25 mixed aqueous solution Flow rate: 1.0 mL / min Sample to be measured: mixture of fructose, glucose, sucrose, maltose (all of which are manufactured by Wako Pure Chemical Industries, Ltd.)

The obtained chromatogram is shown in FIG. In FIG. 5, peak 41 indicates fructose, peak 42 indicates glucose, peak 43 indicates sucrose, and peak 44 indicates maltose. From FIG. 5, it was confirmed that each peak can be satisfactorily separated in a short time. Table 1 shows the reproducibility of the maltose retention time when the same measurement was repeated 10 times. The CV value of the retention time of maltose when measured 10 times continuously was 3.2%, and the reproducibility was good.

(4) Evaluation of polymerization reproducibility Column fillers were prepared 20 times (20 lots) under the same conditions as in “(1) Preparation of column filler”. Each lot was subjected to “(3) Performance Evaluation by Liquid Chromatography” to confirm the reproducibility of maltose retention time. The obtained results are shown in Table 1. The CV value of the maltose retention time when 20 lots were measured was 3.9%, and the reproducibility was good.

(Example 5)
In Example 5, the column filler of the present invention 2 was prepared as a column filler for cation exchange chromatography, and the performance was confirmed.

(1) Preparation of column filler As a crosslinkable monomer, 120 g of tetraethylene glycol dimethacrylate (made by Shin-Nakamura Chemical Co., Ltd.), 200 g of triethylene glycol dimethacrylate (made by Shin-Nakamura Chemical Co., Ltd.), and 2-hydroxy A monomer mixture obtained by mixing 60 g of -1,3-dimethacryloxypropane (manufactured by Shin-Nakamura Chemical Co., Ltd.) was dissolved by mixing 1.0 g of benzoyl peroxide as a polymerization initiator and dissolving 5 wt. It was dispersed in 2000 mL of an aqueous polyvinyl alcohol solution. The obtained dispersion was stirred at 25 ° C. for 15 hours (stirring step).
Next, the reaction system was heated to 80 ° C. under a nitrogen atmosphere to carry out a polymerization reaction for 1.2 hours.
Next, 200 g of 2-acrylamido-2-methylpropanesulfonic acid (manufactured by Toagosei Co., Ltd.) and 50 g of polyethylene glycol monomethacrylate (manufactured by NOF Corporation) as a hydrophilic monomer, 100 g of methanol, and ion-exchanged water A mixture obtained by dissolving in 200 mL of a mixed solution was added, and a polymerization reaction was further performed at 80 ° C. for 2 hours in a nitrogen atmosphere while stirring. The obtained polymer was washed to obtain a column filler.
As a result of measuring the particle size distribution of the obtained column filler in the same manner as in Example 1, the average particle size was 7.5 μm.

(2) Packing into a column In the same manner as in Example 1, 0.7 g of the obtained column filler was packed into a stainless steel empty column (4.6 φ × 25 mm).

(3) Performance evaluation by liquid chromatography 1

A standard substance mixture of proteins was measured using the column obtained in “(2) Packing in a column”. The measurement conditions are shown below.
System: LC-10A system (manufactured by Shimadzu Corporation)
Detection wavelength: 280 nm
Eluent A: 100 mmol / L phosphate buffer (pH 5.7)
Eluent B: Eluent A + 500 mmol / L NaCl (pH 5.7)
Elution conditions: Linear gradient from eluent A 100% to eluent B 100% Flow rate: 1.5 mL / min Sample to be measured: Mixture of trypsinogen, α-chymotrypsinogen, ribonuclease A and lysozyme (all of which are manufactured by Sigma-Aldrich)

The obtained chromatogram is shown in FIG. From FIG. 6, it was confirmed that each peak can be satisfactorily separated in a short time. Table 1 shows the reproducibility of the retention time of lysozyme when the same measurement is repeated 10 times. The CV value of the retention time of lysozyme when measured 10 times continuously was 0.9%, and the reproducibility was good.

(4) Performance evaluation 2 by liquid chromatography
Using the column obtained in “(2) Packing the column”, hemoglobins including hemoglobin A1c in the blood of healthy persons were measured. The measurement conditions are shown below.
System: LC-10A system (manufactured by Shimadzu Corporation)
Detection wavelength: 415 nm
Eluent I: 100 mmol / L phosphate buffer (pH 5.3)
Eluent J: 320 mmol / L phosphate buffer (pH 8.2)
Elution conditions: Step gradient from eluent I 100% to eluent J 100% Flow rate: 1.2 mL / min Measurement sample: 0.05% Triton X-100 (manufactured by Sigma-Aldrich) ) Diluted with phosphate buffer (pH 6.7)

The obtained chromatogram is shown in FIG. From FIG. 7, it was confirmed that each peak can be satisfactorily separated in a short time. Table 1 shows the reproducibility of the retention time of hemoglobin A1c when the same measurement is repeated 10 times. The CV value of the retention time of hemoglobin A1c when measured 10 times continuously was 0.8%, and the reproducibility was good.

(5) Evaluation of polymerization reproducibility Column fillers were prepared 20 times (20 lots) under the same conditions as in “(1) Preparation of column filler”. Each lot was subjected to “(4) Performance evaluation 2 by liquid chromatography” to confirm the reproducibility of the retention time of hemoglobin A1c. The obtained results are shown in Table 1. The CV value of the retention time of hemoglobin A1c when measuring 20 lots was 2.1%, and the reproducibility was good.

(Example 6)
In Example 6, the column filler of the present invention 2 was prepared as a column filler for anion exchange chromatography, and the performance was confirmed.

(1) Preparation of column filler 100 g of tetramethylolpropane triacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) and 300 g of triethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) as a crosslinkable monomer, and polymerization start As an agent, 1.5 g of benzoyl peroxide was mixed and dissolved, and dispersed in 2000 mL of a 5 wt% polyvinyl alcohol aqueous solution. The obtained dispersion was stirred at 15 ° C. for 10 hours (stirring step).
Next, the reaction system was heated to 80 ° C. under a nitrogen atmosphere to conduct a polymerization reaction for 2 hours.
Next, 300 mL of a 50 wt% methanol aqueous solution in which 200 g of diethylaminoethyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved as a hydrophilic monomer was added, and the mixture was further stirred at 80 ° C. for 1.5% under stirring in a nitrogen atmosphere. A time polymerization reaction was carried out. The obtained polymer was washed to obtain a column filler.
As a result of measuring the particle size distribution of the obtained column filler in the same manner as in Example 1, the average particle size was 7.9 μm.

(2) Packing into a column In the same manner as in Example 1, 1.4 g of the obtained column filler was packed into a stainless steel empty column (4.6 φ × 50 mm).

(3) Performance Evaluation by Liquid Chromatography A protein standard substance mixture was measured under the same conditions as in Example 2 using the column obtained in “(2) Packing into a column”. The obtained chromatogram is shown in FIG. From FIG. 8, it was confirmed that each peak can be satisfactorily separated in a short time. Table 1 shows the reproducibility of the retention time of trypsin inhibitor when the same measurement is repeated 10 times in succession. The CV value of the retention time of trypsin inhibitor when measured 10 times continuously was 1.0%, and the reproducibility was good.

(4) Evaluation of polymerization reproducibility Under the same conditions as in Example 2, column fillers were prepared 20 times (20 lots). Each lot was subjected to “(3) performance evaluation by liquid chromatography” to confirm the reproducibility of the retention time of trypsin inhibitor. The obtained results are shown in Table 1. The CV value of the retention time of trypsin inhibitor when 20 lots were measured was 1.5%, and the reproducibility was good.

(Example 7)
In Example 7, the column filler of the present invention 2 was prepared as a column filler for aqueous gel permeation chromatography, and the performance was confirmed.

(1) Preparation of column fillers Tetraethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) 300 g as a crosslinkable monomer, 100 g of polyethylene glycol monomethacrylate (manufactured by NOF Corporation) as a non-crosslinkable monomer, and 300 g of toluene and 1.5 g of benzoyl peroxide were mixed and dissolved, and dispersed in 2500 mL of a 6% by weight polyvinyl alcohol aqueous solution. The obtained dispersion was stirred at 10 ° C. for 18 hours (stirring step).
Next, the reaction system was heated to 80 ° C. under a nitrogen atmosphere, and a polymerization reaction was performed for 0.8 hours.
Next, 300 mL of a 40 wt% aqueous methanol solution in which 200 g of 2,3-dihydroxyethyl methacrylate (manufactured by NOF Corporation) was dissolved as a hydrophilic monomer was added, and the mixture was further stirred at 80 ° C. in a nitrogen atmosphere while stirring. The polymerization reaction was performed for 2.2 hours. The obtained polymer was washed to obtain a column filler.
As a result of measuring the particle size distribution of the obtained column filler in the same manner as in Example 1, the average particle size was 10.3 μm.

(2) Packing into column In the same manner as in Example 1, 3.2 g of the obtained column filler was packed into a stainless steel empty column (6.0 φ × 100 mm).

(3) Performance Evaluation by Liquid Chromatography Proteins were measured under the same conditions as in Example 3 using the column obtained in “(2) Packing into column”. The obtained chromatogram is shown in FIG. From FIG. 9, it was confirmed that each peak can be satisfactorily separated in a short time. Table 1 shows the reproducibility of the retention time of ribonuclease when the same measurement is repeated 10 times in succession. The CV value of the retention time of ribonuclease when measured 10 times continuously was 1.9%, and the reproducibility was good.

(4) Evaluation of polymerization reproducibility Under the same conditions as in Example 3, column fillers were prepared 20 times (20 lots). Each lot was subjected to “(3) Performance Evaluation by Liquid Chromatography” to confirm reproducibility of ribonuclease retention time. The obtained results are shown in Table 1. The ribonuclease retention time CV value when measuring 20 lots was 2.3%, and the reproducibility was good.

(Example 8)
In Example 8, the column filler of the present invention 2 was prepared as a column filler for normal phase chromatography, and the performance was confirmed.

(1) Preparation of column filler 100 g of tetramethylolpropane triacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) and 300 g of tetraethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) as a crosslinkable monomer, and polymerization initiation As an agent, 1.5 g of benzoyl peroxide was mixed and dissolved, and dispersed in 2000 mL of a 4 wt% aqueous polyvinyl alcohol solution. The obtained dispersion was stirred at 20 ° C. for 15 hours (stirring step).
Next, the reaction system was heated to 80 ° C. under a nitrogen atmosphere to carry out a polymerization reaction for 24 hours.
Next, 300 mL of a 40 wt% aqueous methanol solution in which 220 g of 2-aminoethyl methacrylate (manufactured by NOF Corporation) was dissolved as a hydrophilic monomer was added, and the mixture was further stirred at 80 ° C. under a nitrogen atmosphere while stirring. A time polymerization reaction was carried out. The obtained polymer was washed to obtain a column filler.
As a result of measuring the particle size distribution of the column filler obtained in the same manner as in Example 1, the average particle size was 6.3 μm.

(2) Packing into a column In the same manner as in Example 1, 1.4 g of the obtained column filler was packed into a stainless steel empty column (4.6 φ × 50 mm).

(3) Performance Evaluation by Liquid Chromatography Saccharides were measured under the same conditions as in Example 3 using the column obtained in “(2) Packing into the column”. The obtained chromatogram is shown in FIG. From FIG. 10, it was confirmed that each peak can be satisfactorily separated in a short time. Table 1 shows the reproducibility of the maltose retention time when the same measurement was repeated 10 times. The CV value of the retention time of maltose when measured 10 times continuously was 1.4%, and the reproducibility was good.

(4) Evaluation of polymerization reproducibility Under the same conditions as in Example 4, column fillers were prepared 20 times (20 lots). Each lot was subjected to “(3) Performance Evaluation by Liquid Chromatography” to confirm the reproducibility of maltose retention time. The obtained results are shown in Table 1. The CV value of the retention time of maltose when measuring 20 lots was 2.5%, and the reproducibility was good.

(Comparative Examples 1-8)
In the comparative example, a column filler that does not satisfy the conditions of the stirring step in the present invention was prepared. Table 1 shows the conditions of the stirring step in Comparative Examples 1-8.
The conditions other than the stirring step were as follows: Comparative Example 1 was Example 1, Comparative Example 2 was Example 2, Comparative Example 3 was Example 3, Comparative Example 4 was Example 4, Comparative Example 5 was Example 5, and Comparative Example 6 Were prepared in the same manner as in Example 6, Comparative Example 7 as in Example 7, and Comparative Example 8 as in Example 8. In Comparative Example 4 and Comparative Example 6, in the stirring step, some of the monomers and monomer mixture in the dispersion were frozen and could not be stirred uniformly.
Table 1 shows the evaluation results of the reproducibility of polymerization (n = 20) according to the retention time in the comparative example. The CV value was larger than that of the example.

(Performance evaluation of separation performance)
In order to confirm the influence on the separation performance of the column packing material by defining the conditions of the stirring step, various hemoglobins were measured using the column packing materials for cation exchange chromatography of Examples and Comparative Examples, Separation performance was evaluated.

(1) Measurement of modified hemoglobins The artificially prepared modified hemoglobins were measured using the column fillers for cation exchange chromatography of Example 1, Example 5, Comparative Example 1 and Comparative Example 5. It was. As samples containing modified hemoglobins, three types of samples including unstable hemoglobin A1c (sample L), acetylated hemoglobin-containing sample (sample A), and carbamylated hemoglobin-containing sample (sample C) were prepared by a known method.
Sample L was prepared by adding glucose to healthy human blood at 2000 mg / dL and heating at 37 ° C. for 3 hours. Sample A was prepared by adding acetaldehyde at 50 mg / dL to healthy human blood and heating at 37 ° C. for 2 hours. Sample C was prepared by adding sodium cyanate at 50 mg / dL to healthy human blood and heating at 37 ° C. for 2 hours.
For the separation performance, a value (Δ value) obtained by subtracting the hemoglobin A1c value of healthy human blood (unmodified product) used for the preparation of the sample containing the modified hemoglobin from the hemoglobin A1c value of the sample containing the modified hemoglobin is calculated. The two were compared and evaluated. The results are shown in Table 2.
From Table 2, the Δ value when the column fillers of Example 1 and Example 5 were used was as small as 0.2% or less, and hemoglobin A1c could be accurately measured even in samples containing modified hemoglobins. On the other hand, when the column fillers of Comparative Example 1 and Comparative Example 5 were used, the Δ value was large, and the hemoglobin A1c value was affected by the modified hemoglobins.

(2) Measurement of abnormal hemoglobin Samples containing hemoglobin S and hemoglobin C, which are abnormal hemoglobins (“AFSC hemoglobin” manufactured by Helena Laboratories, Inc.) using the column packing material for cation exchange chromatography of Example 5 and Comparative Example 5. Control ") was measured.
The chromatogram obtained as a result of measurement using the column packing material of Example 5 is shown in FIG. In FIG. 11, peak 14 indicates hemoglobin S, and peak 15 indicates hemoglobin C. From FIG. 11, when the column filler obtained in Example 5 was used, abnormal hemoglobins could be separated satisfactorily. When the column packing obtained in Comparative Example 5 was used, the separation performance of abnormal hemoglobins was poor as shown in FIG.

(3) Measurement of hemoglobin A2 Using the column packing material for cation exchange chromatography of Example 5 and Comparative Example 5, a sample containing hemoglobin A2 (manufactured by Bio-Rad, “A2 control (level 2)”) was measured. .
A chromatogram obtained by measurement using the filler of Example 5 is shown in FIG. In FIG. 13, a peak 16 indicates hemoglobin A2. From FIG. 13, when the column filler obtained in Example 5 was used, hemoglobin A2 could be satisfactorily separated. When the column filler obtained in Comparative Example 5 was used, hemoglobin A2 could not be separated as shown in FIG.
From the measurement performance evaluation of the above hemoglobins, by defining the conditions of the stirring step, not only the reproducibility of polymerization of the column filler but also the separation performance was improved.

ADVANTAGE OF THE INVENTION According to this invention, the column filler with favorable reproducibility of performance can be provided. Moreover, according to this invention, the manufacturing method of this column filler and the measuring method of hemoglobin using this column filler can be provided.

1 Trypsinogen 2 α-Chymotrypsinogen 3 Ribonuclease A
4 Lysozyme 11 Hemoglobin F
12 Hemoglobin A1c
13 Hemoglobin A0
14 Hemoglobin S
15 Hemoglobin C
16 Hemoglobin A2
21 Conalbumin 22 Ovalbumin 23 Trypsin inhibitor 31 Thyroglobulin 32 γ-globulin 33 Ovalbumin 34 Ribonuclease 41 Fructose 42 Glucose 43 Sucrose 44 Maltose

Claims (7)

A liquid chromatography column filler comprising hydrophilic crosslinked polymer particles,
The hydrophilic cross-linked polymer particles are prepared by dispersing a cross-linkable monomer and a dispersion liquid obtained by dispersing the hydrophilic monomer in an aqueous dispersion medium at a temperature equal to or higher than the melting point of the cross-linkable monomer. A column filler for liquid chromatography, which is obtained by performing a polymerization reaction after stirring for 1 hour or more at a temperature at which does not proceed. A method for producing the liquid chromatography column filler according to claim 1, comprising:
A dispersion obtained by dispersing a crosslinkable monomer and a hydrophilic monomer in an aqueous dispersion medium is stirred for 1 hour or more at a temperature not lower than the melting point of the crosslinkable monomer and a temperature at which the polymerization reaction does not substantially proceed. Step 1 to perform,
The manufacturing method of the column filler for liquid chromatography which has the process 2 which performs a polymerization reaction after performing the said process 1. FIG. The method for producing a column filler for liquid chromatography according to claim 2, wherein the temperature which is not lower than the melting point of the crosslinkable monomer and at which the polymerization does not proceed substantially is 5 to 40 ° C. A liquid chromatography column filler comprising hydrophilic crosslinked polymer particles,
The hydrophilic crosslinked polymer particles are prepared by dispersing a dispersion obtained by dispersing a crosslinkable monomer in an aqueous dispersion medium at a temperature equal to or higher than the melting point of the crosslinkable monomer and at which the polymerization reaction does not proceed substantially. A column filler for liquid chromatography, which is obtained by polymerizing a hydrophilic monomer on the surface of a crosslinked polymer particle obtained by carrying out a polymerization reaction after stirring for more than an hour. A method for producing a liquid chromatography column filler according to claim 4,
A step 1 of stirring a dispersion obtained by dispersing a crosslinkable monomer in an aqueous dispersion medium at a temperature equal to or higher than the melting point of the crosslinkable monomer and at a temperature at which the polymerization reaction does not substantially proceed;
A step 2 of performing a first polymerization reaction after performing the step 1 to obtain crosslinked polymer particles;
A liquid chromatograph having a step 3 of polymerizing a hydrophilic monomer on the surface of the crosslinked polymer particle by adding a hydrophilic monomer before the completion of the first polymerization reaction and performing a second polymerization reaction. A method for producing a column filler for lithography. 6. The method for producing a column filler for liquid chromatography according to claim 5, wherein a temperature not lower than the melting point of the crosslinkable monomer and the temperature at which the polymerization does not proceed substantially is 5 to 40 ° C. A method for measuring hemoglobin by liquid chromatography using the column filler for liquid chromatography according to claim 1 or 4.

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