JP3352645B2 - Filler for liquid chromatography and measurement method using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a packing material for ion exchange liquid chromatography and a method for measuring a sample using the packing material.

[0002]

2. Description of the Related Art Ion exchange chromatography is widely used for separating ionic substances, and as a filler, particles composed of particles having a cation exchange group or an anion exchange group are used. This measurement method by ion exchange chromatography is a particularly effective measurement method for the analysis of glycated hemoglobins and various biological substances.

[0003] As a filler for ion exchange liquid chromatography, a filler obtained by reacting a compound having an ion exchange group with polymer particles or the like, or a monomer having an ion exchange group and a crosslinkable monomer can be used. Fillers and the like obtained by polymerizing are known.

[0004] Fillers obtained by reacting the above-mentioned polymer particles and the like with a compound having an ion-exchange group are disclosed in, for example, JP-A-1-262468 and JP-A-7-27754. First, inorganic or natural
It is obtained by preparing synthetic organic polymer particles, and then bonding a compound containing an ion exchange group to the polymer particles.

Here, an important physical property that determines the performance of the packing material for ion exchange chromatography is the ion exchange capacity. The ion exchange capacity of a conventional packing material for ion exchange chromatography is about several meq to several tens of meq / g.
It was about q / g. The ion exchange capacity depends on the particle size, specific surface area, pore diameter, pore volume, etc. of the filler, in addition to the amount of the ion exchange group-containing compound to be reacted and the reaction conditions. Therefore, in order to enable high-precision analysis, it is considered extremely important to optimize the physical property values for each of the ion exchange capacity, pore diameter, specific surface area, and pore volume.

Therefore, Japanese Patent Application Laid-Open No. 7-27754 discloses that the pore diameter is 20 to 2000 mm and the specific surface area is 0.1 to 10 mm.
A filler having an ion exchange capacity of 0.5 to 3.0 meq / g is disclosed by reacting an ion exchange group-containing compound with 0 m ² / g of porous particles.

However, since this filler is a filler obtained by reacting a compound having an ion exchange group with polymer particles or the like, the compound having an ion exchange group is quantitatively introduced into the polymer particles or the like. There was a problem that it was difficult and the reproducibility was poor (Yoshimiwa, Hosoya, Kizen, Tanaka: Chromatography, 16 (1) 7-12 (1995)). In addition, when the polymer particles are silica particles or the like, the pH range of the eluent is limited, the separation ability is reduced by non-specific adsorption, and the life of the column is shortened. There is a problem, and in the case of natural polymer particles, there is a problem that they are easily swelled and shrunk and have low pressure resistance.

On the other hand, a filler obtained by polymerizing a monomer having an ion exchange group and a crosslinkable monomer can be easily produced with good reproducibility, has a long column life, and is superior to the above-mentioned filler. It has excellent performance. However, with regard to this type of filler, there has been no study on a material having optimized properties such as pore diameter, specific surface area, pore volume, and ion exchange capacity.

[0009]

SUMMARY OF THE INVENTION In view of the above, an object of the present invention is to optimize the physical properties such as ion exchange capacity, pore diameter, specific surface area, and pore volume, especially for the measurement of biological samples. An object of the present invention is to provide a suitable packing material for ion exchange chromatography and a measuring method using the same.

[0010]

Means for Solving the Problems The invention according to claim 1 (hereinafter referred to as the present invention 1) comprises a polymer comprising a monomer having an ion-exchange group and a crosslinkable monomer, It has pores having a diameter of 10 to 100 °, a specific surface area of 0.05 to ⁵ m ² per 1 g of dry weight of the filler, and a pore volume of 0.1 to 10 g / g of dry weight of the filler.
It is a filler for ion exchange chromatography, which has a volume of 10 μl and an ion exchange capacity of 1 to 100 μeq per 1 g of a dry weight of the filler.

Hereinafter, the present invention 1 will be described. The present invention 1 comprises a polymer comprising a monomer having an ion exchange group and a crosslinkable monomer as constituent components. The monomer having an ion exchange group is a monomer having at least one ion exchange group and having at least one polymerizable functional group. Here, the ion-exchange group is a functional group that exhibits an ion-exchange ability at a certain pH, and is not particularly limited as long as it is a known ion-exchange group. For example, a carboxyl group, a sulfonic group, a phosphate group Tertiary amino group, 4
And a secondary amino group. The polymerizable functional group is not particularly limited as long as it is a known polymerizable functional group such as a vinyl group. In addition, if the monomer having an ion exchange group contains a benzene skeleton, it particularly non-specifically adsorbs hydrophobic substances such as proteins and reduces the life of the column. When preparing an agent, it is preferable to use one not containing a benzene skeleton.

The monomer having an ion exchange group and having a carboxyl group includes, for example,
(Meth) acrylic acid, 2- (meth) acryloyloxyethyl succinic acid, crotonic acid, itaconic acid, citraconic acid, mesaconic acid, maleic acid, fumaric acid and derivatives thereof.

The above-mentioned monomers having an ion exchange group and having a phosphate group include, for example, ((meth) acryloyloxyethyl) acid phosphate and (2- (meth) acryloyloxyethyl) acid phosphate , (3- (meth) acryloyloxypropyl) acid phosphate and derivatives thereof.

The monomer having an ion exchange group and having a sulfonic acid group includes, for example, (meth) allylsulfonic acid, 2- (meth) acrylamide-
Examples include 2-methylpropanesulfonic acid, (3-sulfopropyl) -itaconic acid, 3-sulfopropyl (meth) acrylic acid, and derivatives thereof.

Examples of the above-mentioned monomer having an ion exchange group and having a tertiary amino group include, for example, dimethylaminoethyl (meth) acrylate, diethylaminoethyl (meth) acrylate, 2-hydroxy-3-
Examples include dimethylaminopropyl (meth) acrylate and derivatives thereof.

The monomer having an ion exchange group and having a quaternary amino group includes, for example, 2-
(Meth) acryloyloxyethyltrimethylammonium chloride, 2- (meth) acryloyloxyethyltriethylammonium chloride, 2-hydroxy-3
-(Meth) acryloyloxypropyltrimethylammonium chloride and derivatives thereof.

The above-mentioned monomers having various ion exchange groups include:
In addition to the monomers described above, various derivatives thereof, salts such as sodium salts and potassium salts, and chlorides may be used.

In the present invention 1, as the monomer having an ion exchange group, a monomer having a functional group which can be converted into an ion exchange group by a chemical reaction is used. It may be obtained by converting the functional group into an ion exchange group. The chemical reaction includes a hydrolysis reaction and a transfer reaction. Examples of the functional group that can be converted into an ion exchange group by the chemical reaction include an ester group. As a specific example, using methyl methacrylate as a monomer, after polymerization, by heating under alkalinity, the ester bond is decomposed and converted into a carboxyl group, thereby obtaining a monomer having an ion exchange group. And a filler for ion exchange chromatography comprising a polymer comprising a crosslinkable monomer as a constituent component.

The amount of the monomer having an ion exchange group varies depending on the type of the monomer.
It is preferable to use 10 to 200 parts by weight with respect to 0 parts by weight. This is because if the amount is less than 10 parts by weight, the ion exchange capacity is too small, so that a sufficient ion exchange reaction is difficult to be performed, and the separation ability is reduced.
Hydrophilicity increases, pressure resistance and swelling resistance decrease, and
This is because, for example, when the eluent is switched, a long time is required for the equilibration, which causes a problem that the measurement time becomes long. Further, two or more of the above monomers may be used as a mixture, if necessary.

The crosslinking monomer in the present invention 1 includes:
For example, a monomer having two or more vinyl groups in one molecule, a derivative of a (meth) acrylic acid ester, an aliphatic diene compound and a derivative thereof as described later are exemplified.

Examples of the monomer having two or more vinyl groups in one molecule include styrene derivatives such as divinylbenzene, divinyltoluene, divinylxylene, divinylethylbenzene and divinylnaphthalene.

Examples of the (meth) acrylic acid ester derivatives include ethylene glycol di (meth) acrylate, polyethylene glycol di (meth) acrylate, 1,3-butylene glycol di (meth) acrylate, and 1,6-hexaene. Glycol di (meth) acrylate, neopentyl glycol di (meth) acrylate,
Polypropylene glycol di (meth) acrylate, trimethylolethanetri (meth) acrylate, trimethylolpropanetri (meth) acrylate, tetramethylolpropanetri (meth) acrylate, tetramethylolmethanetri (meth) acrylate, tetramethylolmethanetetra (meth) ) Acrylates and derivatives thereof.

Examples of the aliphatic diene compound include 1,3-butadiene, isoprene, 1,4-hexadiene and derivatives thereof.

The filler of the present invention 1 can be prepared from the above monomer by a known polymerization method such as suspension polymerization, emulsion polymerization and dispersion polymerization. Especially preferably,
As described in JP-A-7179, after preparing crosslinkable polymer particles using a crosslinkable monomer, the polymer particles are impregnated with a polymerization initiator, and a monomer having an ion exchange group is added. By polymerization. In this method, the polymerization temperature when the monomer having an ion exchange group is added is preferably 50 to 90 ° C., and the polymerization time is preferably 0.5 to 5 hours. This is because if the temperature is less than 50 ° C. or less than 0.5 hour, the polymerization of the monomer having an ion exchange group does not proceed sufficiently and the ion exchange capacity becomes insufficient. If it exceeds or exceeds 5 hours, aggregation occurs during polymerization.

In the present invention, the polymerization initiator used in the polymerization of the monomer is not particularly limited, and known water-soluble or oil-soluble radical polymerization initiators can be used. Examples thereof include potassium persulfate and persulfate. Persulfates such as sodium and ammonium persulfate; cumene hydroperoxide, benzoyl peroxide, lauroyl peroxide, octanoyl peroxide, o-chlorobenzoyl peroxide, acetyl peroxide, t-butyl hydroperoxide, t-butyl peroxide Oxyacetate, t-butyl peroxyisobutyrate, 3,
5,5-trimethylhexanoyl peroxide, t-
Butyl peroxy-2-ethylhexanoate, di-t
Organic peroxides such as -butyl peroxide; 2,2-azobisisobutyronitrile, 2,2-azobis (2,4
-Dimethylvaleronitrile), 4,4-azobis (4-
Azo compounds such as cyanopentanoic acid), 2,2-azobis (2-methylbutyronitrile), 2,2-azobis (2,4-dimethylvaleronitrile), and azobiscyclohexanecarbonitrile.

When the amount of the polymerization initiator is less than 0.05 part by weight based on 100 parts by weight of the crosslinking monomer,
The polymerization reaction may be insufficient or the polymerization may take a long time, and if it exceeds 1 part by weight, the reaction proceeds rapidly,
Since aggregates may be generated, it is preferable to use 0.05 to 1 part by weight. The polymerization initiator is preferably used by dissolving it in the crosslinking monomer.

As a component of the polymer, a non-crosslinkable monomer can be used in addition to the crosslinkable monomer and the like. Further, other additives may be added.

Examples of the non-crosslinkable monomer include styrene derivatives such as styrene, α-methylstyrene, p-methylstyrene and chloromethylstyrene; aliphatic monomers such as vinyl chloride; Vinyl esters such as vinyl propionate and vinyl stearate; methyl (meth) acrylate, ethyl (meth) acrylate,
Butyl (meth) acrylate, (meth) acrylic acid-2-
Examples thereof include acrylic acid derivatives such as ethylhexyl, stearyl (meth) acrylate, (meth) acrylamide, (meth) acrylonitrile, and glycidyl (meth) acrylate.

The amount of the non-crosslinkable monomer varies depending on the type of the monomer, but is preferably 0 to 100 parts by weight based on 100 parts by weight of the crosslinkable monomer. The non-crosslinkable monomers may be used as a mixture of two or more as necessary.

As the above-mentioned other additives, for example, an organic solvent, polymer particles and the like may be added to the monomer as required. Further, the present invention is not limited to these exemplified additives, and various known additives may be added.

The above organic solvent dissolves the monomer,
An organic solvent that does not dissolve the polymer, and the pores of the obtained polymer can be made larger. For example, aromatic hydrocarbons such as toluene, xylene, diethylbenzene and dodecylbenzene; hexane, heptane, octane,
Saturated hydrocarbons such as decane; alcohols such as isoamyl alcohol, hexyl alcohol, and octyl alcohol;

The amount of the organic solvent to be used is preferably from 0 to 100 parts by weight based on 100 parts by weight of the crosslinkable monomer.

The above-mentioned polymer particles have a uniform particle size distribution, and are added to the above-mentioned crosslinkable monomer or a mixture of a monomer having an ion exchange group and a crosslinkable monomer before polymerization. By doing so, a filler having a uniform particle size distribution can be obtained. Examples of the polymer particles include the non-crosslinkable monomers such as a styrene polymer, a styrene-divinylbenzene copolymer, a methyl (meth) acrylate polymer, and an ethyl (meth) acrylate polymer. Alternatively, non-crosslinkable polymer particles which are copolymers can be used. Further, as the polymer particles, crosslinked copolymer particles composed of the non-crosslinkable monomer, the crosslinkable monomer, and the like may be used.
In this case, it is preferable to use low crosslinkable polymer particles having a crosslinkable polymer ratio of 10% or less.

As the polymer particles, those prepared by a known polymerization method can be used, and can be polymerized by, for example, emulsion polymerization, soap-free polymerization, dispersion polymerization, suspension polymerization and the like.

The average particle size of the polymer particles is 0.1 to 1
0 μm, and variation in particle size is CV (%) = 15% or less (C
V (%) = (standard deviation / average particle size) × 100) is preferable. The amount of the polymer particles used is 10
0.5 to 100 parts by weight with respect to 0 parts by weight is preferred.

The filler of the present invention 1 has an average diameter of 10 to 1
It has pores that are 00 °. This is because pores having an average diameter of less than 10 ° are difficult to control in production and difficult to prepare reproducible ones. Further, if the average diameter exceeds 100 °, swelling and shrinkage are liable to occur, and there is a possibility that the pressure loss may fluctuate during the measurement. This causes a problem that it takes time to reach equilibrium in the column.

The specific surface area of the filler of the present invention 1 is 0.05 to ⁵ m ² per 1 g of the dry weight of the filler. this is,
0.05m is less than ² / g, since the grain size is increased, resolution is lowered, if it exceeds 5 m ² / g, particle size becomes very small, a problem that causes an increase in pressure loss Because there is.

The pore volume of the filler of the present invention 1 is 0.1 to 10 μl per 1 g of the dry weight of the filler. this is,
If the amount is less than 0.1 μl / g, it is difficult to control the production and it is difficult to prepare a filler having reproducible performance.
If the amount exceeds μl / g, swelling and shrinkage are liable to occur, and the pressure loss may fluctuate during the measurement. This is because it takes time to reach the equilibrium.

The ion exchange capacity of the filler of the present invention 1 is 1 to 100 μeq / g of the dry weight of the filler. This is because if it is less than 1 μeq / g, an ion exchange reaction with the measurement sample hardly occurs, and the separation performance decreases.
If it exceeds 00 μeq / g, it takes a long time for equilibration, and there is a problem that the measurement time becomes longer and the pressure resistance decreases.

In the present invention 1, the physical properties of the average pore diameter, the specific surface area, the pore volume, and the ion exchange capacity must be within the ranges specified above, respectively, and these are indispensable in the present invention. It is a configuration requirement. Therefore, when at least one of the physical property values is not within the above range, the effects unique to the present invention cannot be exhibited.

The filler of the present invention 1 has an average particle size of 1 to 20.
μm is preferred, and more preferably 5 to 10 μm, and the variation in particle size is preferably CV (%) = 15% or less. If necessary, classification may be performed by a known dry or wet classification method so as to fall within the above range.

The filler of the present invention 1 is usually used by packing it in a column made of stainless steel or the like. The method of filling is not particularly limited, but it is particularly preferable to use a wet method (slurry method). In this wet method, packing can be performed by dispersing a filler in a solvent or the like used as an eluent and press-fitting it into a column via a packer or the like.

The liquid chromatography using the packing material of the present invention 1 is not particularly limited, and for example, a known one composed of a liquid feed pump, a sample introduction device, a column, a detection device and the like can be used. Further, other accessories such as a thermostat and an eluent deaerator may be appropriately attached.

The substance that can be measured using the filler of the present invention 1 is not particularly limited, but biologically-related substances such as catecholamine derivatives, nucleotides, peptides, and proteins are particularly suitable.

For the measurement by liquid chromatography using the packing material of the present invention 1, a known eluent can be used, and there is no particular limitation. For example, phosphoric acid, nitric acid, hydrochloric acid,
Inorganic acids such as perchloric acid and salts thereof, acetic acid, malic acid,
Various buffers containing organic acids such as tartaric acid, succinic acid, citric acid and salts or halides thereof, basic substances such as sodium hydroxide and potassium hydroxide, or
Acetone, acetonitrile, dioxane, methanol,
A mixture of an organic solvent such as ethanol and the above-mentioned buffer or water can be used.

The invention according to claim 2 (hereinafter referred to as invention 2) is a method for measuring a sample by liquid chromatography, characterized by using the filler according to claim 1. As a sample to be measured by the measurement method of the second aspect of the present invention, a biological substance is suitable. The present invention 2 is particularly suitable for simultaneously measuring glycated hemoglobins and abnormal hemoglobins.

The case where glycated hemoglobins and abnormal hemoglobins are measured simultaneously by the measurement method of the present invention 2 will be described. In this case, human hemolyzed blood is used as a sample, and hemolysis can be performed using a buffer containing a hemolytic reagent such as a known surfactant.

The glycated hemoglobin (hereinafter, hemoglobin is also referred to as “Hb”) is mainly composed of stable HbA.
1c. Also, as abnormal hemoglobin,
There are many species such as HbS and HbC, and HbF and HbA
Thalassemia and the like such as 2 are also included.

[0049]

EXAMPLES Example 1 2.0 g of benzoyl peroxide was mixed and dissolved in a mixture of 400 g of triethylene glycol dimethacrylate and 20 g of methyl methacrylate (manufactured by Wako Pure Chemical), and 2.5 L of 4 wt% polyvinyl It was dispersed in an aqueous alcohol solution. This was heated with stirring under a nitrogen atmosphere, and polymerized at 80 ° C. for 1.2 hours. After the reaction system was cooled to 35 ° C., 200 g of methacrylic acid (manufactured by Wako Pure Chemical Industries, Ltd.) was added thereto, and the mixture was stirred for 1 hour.
Polymerization was carried out at 1.2 ° C for 1.2 hours. After the polymerization, the polymer was washed and classified to obtain a filler having an average particle size of 6.5 μm.

Example 2 2.0 g of benzoyl peroxide was mixed and dissolved in 450 g of tetraethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.), and 2.5 l
In a 4% by weight aqueous solution of polyvinyl alcohol.
This was heated with stirring under a nitrogen atmosphere, and polymerized at 80 ° C. for 1.5 hours. After cooling the reaction system to 35 ° C,
200 g of 2-acrylamide-2-methylpropanesulfonic acid (manufactured by Tokyo Chemical Industry) was added, stirred for 1 hour, and polymerized again at 80 ° C. for 1.3 hours. After the polymerization, the polymer was washed and classified to obtain a filler having an average particle size of 6.5 μm.

Example 3 400 g of diethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical) and 100 g of tetramethylol methane triacrylate (manufactured by Shin-Nakamura Chemical)
In this mixture, 2.0 g of benzoyl peroxide was dissolved and dispersed in 2.5 l of a 4% by weight aqueous solution of polyvinyl alcohol. This was heated in a nitrogen atmosphere while stirring, and polymerized at 80 ° C. for 1.2 hours. Reaction system at 35 ° C
After cooling, 150 g of 2-acrylamido-2-methylpropanesulfonic acid (manufactured by Tokyo Kasei) and 150 g of methacrylic acid were added, and the mixture was stirred for 1 hour and again at 80 ° C for 1.5 hours.
Polymerized for hours. After the polymerization, the polymer was washed and classified to obtain a filler having an average particle size of 6.5 μm.

Example 4 20 g of isoamyl alcohol (manufactured by Wako Pure Chemical Industries, Ltd.) and 2.0 g of benzoyl peroxide were mixed and dissolved in 400 g of triethylene glycol dimethacrylate and dispersed in 2.5 l of a 4% by weight aqueous solution of polyvinyl alcohol. I let it. This was heated with stirring under a nitrogen atmosphere, and polymerized at 80 ° C. for 1.5 hours. Further, 150 g of 2-acryloyloxyethyltrimethylammonium chloride (Kyoeisha Chemical) was added to the reaction system, and the mixture was polymerized at 80 ° C. for 1.2 hours. After the polymerization, the polymer was washed and classified to obtain a filler having an average particle size of 6.5 μm.

[Comparative Example 1] 350 g of glycidyl methacrylate (manufactured by NOF Corporation), 50 g of ethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical) and 10 g of azobisisobutyronitrile (manufactured by Wako Pure Chemical Industries) were mixed, and 2.5 l of the mixture was mixed. It was dispersed in a 4% by weight aqueous solution of polyvinyl alcohol, and the temperature was increased while stirring under a nitrogen atmosphere, and polymerization was performed at 60 ° C. for 10 hours. After washing the obtained polymer, it was classified to obtain a polymer having an average particle size of 3.1 μm. 300 g of xylene was added to 100 g of the polymer. The polymer impregnated in xylene was added to 1 liter of a 4% by weight aqueous solution of polyvinyl alcohol containing 15 g of concentrated sulfuric acid, and heated at 80 ° C. for 1 hour with stirring. Then, it was washed with water and acetone and dried.
100 g of the obtained polymer was dispersed in 500 ml of dioxane, and 5 ml of boron trifluoride etherate (55%) was dispersed.
And heated at 50 ° C. for 8 hours. This was washed with acetone and dried. Further, 20 g of the polymer was added to 100 g
Disperse in 0 ml of water and add sodium monochloroacetate 35
g, 20 g of potassium iodide and 60 g of 50% by weight sodium hydroxide were added and reacted at 60 ° C. for 3 hours with stirring. The obtained polymer was washed and dried to obtain a filler.

Comparative Example 2 400 g of 2-hydroxyethyl methacrylate (manufactured by Shin-Nakamura Chemical), 50 g of diethylene glycol dimethacrylate, and methyl methacrylate 5
0 g and benzoyl peroxide 1.5 g,
It was dispersed in 2.5 l of a 4% by weight aqueous solution of polyvinyl alcohol. This was heated with stirring under a nitrogen atmosphere, and
Polymerization was carried out at 0 ° C. for 8 hours. After washing the obtained polymer, it was classified to obtain a polymer having an average particle size of 2.9 μm. A 20% by weight aqueous sodium hydroxide solution 1 was added to 100 g of the polymer.
Dispersed in 00 ml. And epichlorohydrin 4
0 g was added and reacted for 5 hours. After 100 g of the obtained epoxy group-containing polymer was dispersed in 100 ml of a 20% by weight aqueous sodium sulfate solution, the mixture was reacted at 80 ° C. for 15 hours. The obtained polymer was washed and dried to obtain a filler.

Comparative Example 3 125 g of ethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical) and 2.5 g of benzoyl peroxide were mixed and dissolved. This is 2.
It was dispersed in 5 l of a 4.0% by weight aqueous solution of polyvinyl alcohol, and heated at 80 ° C. with stirring under a nitrogen atmosphere.
After 1 hour, 125 g of 2-acrylamido-2-methylpropanesulfonic acid was added to the reaction system,
For 24 hours. After the polymerization, the product was washed and classified to obtain a filler having an average particle size of 6.5 μm.

Comparative Example 4 150 g of isoamyl alcohol (manufactured by Wako Pure Chemical Industries, Ltd.) and 1.5 g of benzoyl peroxide were added to 400 g of triethylene glycol dimethacrylate.
g was dissolved and dispersed in 2.5 l of a 5% by weight aqueous solution of polyvinyl alcohol. The temperature was raised with stirring, and polymerization was carried out at 80 ° C. for 8 hours. The obtained product was washed with water and acetone to obtain crosslinked polymer particles. 300g of these particles
Was added to 1 liter of an acetone solution containing 1.0 g of benzoyl peroxide, and the particles were impregnated with a polymerization initiator. Next, acetone was distilled off under reduced pressure at 20 ° C. The obtained polymerization initiator-containing particles are dispersed in 2.5 l of a 1% aqueous solution of polyvinyl alcohol, and methacrylic acid 2 is dispersed with stirring.
After adding 00 g and purging with nitrogen, polymerization was carried out at 80 ° C. for 1 hour. After the polymerization, the polymer was washed and classified to obtain a filler having an average particle size of 6.5 μm.

[Evaluation] (1) Evaluation of Physical Properties The following physical properties were evaluated for the dried filler.
For the pore distribution, specific surface area and pore volume, a high-speed specific surface area / pore distribution measuring device (NOVA manufactured by Yuasa Ionics)
-1200) by the gas adsorption method. Also,
The ion exchange capacity is determined using a potentiometric titrator (Kyoto Electronics)
AT-310). Table 1 shows the measurement results.

[0058]

[Table 1]

(2) Packing into a column 0.7 g of each of the above packing materials was added to a 50 mM phosphate buffer (pH
6.0) After dispersing in 30 ml and sonicating for 5 minutes,
Stir well. The whole amount was filled with a stainless steel empty column (4.6
(φ × 35 mm) was injected into a packer (manufactured by Umeya Seiki). A liquid feed pump (manufactured by Sanuki Kogyo KK) was connected to the packer, and filling was performed at a constant pressure of 200 kg / cm ² .

(3) System Configuration The following was used as the system configuration. Liquid pump: LC-9A (manufactured by Shimadzu Corporation) Autosampler: ASU-420 (manufactured by Sekisui Chemical Co., Ltd.) Detector: SPD-6AV (manufactured by Shimadzu Corporation)

(4) Measurement of Protein Mixture Using the above system, a mixture of protein standard substances was measured. (Measurement conditions) Eluent: Eluent A: 100 mM phosphate buffer (pH 7.0) Eluent B: Eluent A with NaCl added to 500 mM (pH 7.0) Elution method: Eluent A 100% Gradient eluent to eluent B 100% Flow rate: 1.5 ml / min Detection wavelength: 254 nm Sample injection volume: 10 μl (measurement sample) myoglobin, α-chymotrypsinogen, ribonuclease A, lysozyme (all Sig
ma). (Measurement Results) The results of measurement using the fillers of Example 1, Comparative Examples 1 and 4 are shown in FIGS. 1, 2 and 3, respectively. It can be seen that the filler of Example 1 has a higher resolution of each peak than the fillers of Comparative Examples 1 and 4, despite the shorter measurement time.

(5) Measurement of Peptide Mixture Using the above system, a mixture of peptide standards was measured. (Measurement conditions) Eluent: Eluent C: 30 mM acetic acid (pH 2.8) Eluent D: 30 mM acetic acid to which Na ₂ SO ₄ was added to 300 mM Elution method: From eluent C 100% to eluent D 100% Linear gradient method Flow rate: 1.5 ml / min Detection wavelength: 215 nm Sample injection amount: 10 μl (measurement sample) γ-endorphin, calcitonin, substance P, insulin, β-endorphin (Si
gma) was used. (Measurement Results) The results of measurement using the fillers of Example 2 and Comparative Examples 2 and 3 are shown in FIGS. 4, 5 and 6, respectively. It can be seen that the filler of Example 2 has higher resolution of each peak than the fillers of Comparative Examples 2 and 3, despite the shorter measurement time.

(6) Measurement of glycated Hb in human blood Using the above system, glycated Hb in human blood was measured. (Measurement conditions) Eluent: Eluent E: 15 to 100 mM phosphate buffer containing perchlorate (pH 5.0 to 6.0) Eluent F: 300 mM phosphate buffer containing perchlorate (pH 7.0) 8.5) The salt concentration and pH of the eluent A were adjusted within the above ranges so that the retention time of the stabilized HbA1c was optimized, and the step gradient elution was performed with the buffers A and B. Flow rate: 1.5 ml / min Detection wavelength: 415 nm Sample injection amount: 10 μl (Measurement sample) Normal human blood was collected by sodium fluoride, and collected.
An aqueous glucose solution was added to a concentration of 0 mg / dl,
After heating at 7 ° C. for 5 hours, a hemolysis diluent (0.1% polyethylene glycol mono-4-octylphenyl ether (Triton X-100) (Tokyo Kasei) phosphate buffer (pH 7.0))
And diluted 150-fold to obtain a measurement sample. (Measurement Results) The results of measurement using the fillers of Example 2 and Comparative Examples 2 and 3 are shown in FIGS. 7, 8 and 9, respectively. Here, in order to maintain good quantitativeness of HbF and stable HbA1c which is an indicator of diabetes, it is necessary to elute HbF, unstable HbA1c, stable HbA1c, and HbA0 in this order. This is because the HbF peak is usually smaller than the peaks of HbA1c and HbA0, and therefore, if eluted between stable HbA1c and HbA0, for example, the quantitativeness of HbF is extremely reduced. In the case of the filler of Example 2, each peak appears in the above-described order, and it can be seen that the separation is excellent even though the measurement time is short. On the other hand, in Comparative Example 3, the elution order was reversed, causing a problem in the quantitativeness of HbF and stable HbA1c. In both Comparative Examples 2 and 3, the resolution was low despite the long measurement time.

(7) Measurement of Abnormal Hbs Control blood containing HbA2, HbS and HbC as abnormal Hbs (Helena, AFSC control)
Was measured under the same conditions as in the case of saccharified Hb. The results of measurement using the fillers of Example 2 and Comparative Example 2 were
These are shown in FIGS. In the filler of Example 2,
It can be seen that, compared to the filler of Comparative Example 2, the resolution of each peak is higher despite the shorter measurement time.

(8) Column durability test In the above Hb measurement, a sample of a healthy person was repeatedly measured.
The fluctuation of the stable HbA1c value was observed. The stable HbA1c value was determined by the following formula: stable HbA1c value (%) = (area of stable HbA1c peak) ÷ (total peak area) × 100. The results are shown in FIG. The filler of Example 2 is the same as Comparative Example 2
As compared with the filler of No. 3, the measured value was stable for a long time.

(9) Production Reproducibility Test The fillers of Example 2 and Comparative Examples 2 and 3 were each prepared 30 times (30 lots) under the same conditions, and the saccharified Hb was measured. The eluent was adjusted so that the retention time of the stable HbA1c was about 5.0 minutes. As shown in Table 2, when the variation in the retention time of the stable HbA1c and the variation in the stable HbA1c value in each lot were compared, the filler of Example 2 was superior in the production reproducibility as compared with Comparative Examples 2.3. I was In Comparative Example 3, aggregates were generated during polymerization in 7 out of 30 lots, and the column could not be packed.

[0067]

[Table 2]

[0068]

EFFECT OF THE INVENTION The packing material for liquid chromatography of the present invention is extremely excellent in durability and reproducibility of production, and has a very high separation ability, particularly for the measurement of biological samples such as proteins. Peaks that could not be separated with the ion-exchange chromatography packing material can be separated in a short time.

[Brief description of the drawings]

FIG. 1 is a diagram showing a chromatogram obtained by measuring a protein mixture using the filler obtained in Example 1.

FIG. 2 is a diagram showing a chromatogram obtained by measuring a protein mixture using the filler obtained in Comparative Example 1.

FIG. 3 is a diagram showing a chromatogram obtained by measuring a protein mixture using the filler obtained in Comparative Example 4.

FIG. 4 is a diagram showing a chromatogram obtained by measuring a peptide mixture using the filler obtained in Example 2.

FIG. 5 is a view showing a chromatogram obtained by measuring a peptide mixture using the filler obtained in Comparative Example 2.

FIG. 6 is a diagram showing a chromatogram obtained by measuring a peptide mixture using the filler obtained in Comparative Example 3.

FIG. 7 is a diagram showing a chromatogram obtained by measuring glycated hemoglobins using the filler obtained in Example 2.

FIG. 8 is a diagram showing a chromatogram obtained by measuring glycated hemoglobins using the filler obtained in Comparative Example 2.

FIG. 9 is a diagram showing a chromatogram obtained by measuring glycated hemoglobins using the filler obtained in Comparative Example 3.

FIG. 10: Using the filler obtained in Example 2,
The figure which shows the chromatogram obtained by measuring abnormal hemoglobins.

FIG. 11: Using the filler obtained in Comparative Example 2,
The figure which shows the chromatogram obtained by measuring abnormal hemoglobins.

FIG. 12 is a view showing the results of a column durability test performed using the fillers obtained in Example 2 and Comparative Examples 2.3.

[Explanation of symbols]

 Reference Signs List 1 myoglobin 2 α-chymotrypsinogen 3 ribonuclease A 4 lysozyme 11 γ-endorphin 12 calcitonin 13 substance P14 insulin 15 β-endorphin 21 HbA1a and HbA1b 22 HbF23 and HbA1b 24 HbA2Ab H2Ab H2Ab H2Ab H2A1b

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI G01N 30/88 G01N 30/88 Q // C08J 5/20 CER C08J 5/20 CER C08L 101: 02 C08L 101: 02 (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 30/48 G01N 30/88

Claims (2)

(57) [Claims] 1. A polymer comprising a monomer having an ion exchange group and a crosslinkable monomer as constituents, and having an average diameter of 1
0 to 100 °, having a specific surface area of 0.05 to ⁵ m ² per 1 g of dry weight of the filler, and a pore volume of 0.1 to 10 μl per 1 g of dry weight of the filler. , The ion exchange capacity is 1 / g of dry weight of the filler
A filler for ion exchange chromatography, which has a particle size of from 100 μeq to 100 μeq. 2. A method for measuring a sample by liquid chromatography, comprising using the filler according to claim 1.