JP2000146934A - Packing agent for liquid chromatography - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide packing agent for liquid chromatography whose column life is sufficient which can be used with a wide range of pH in which the fluctuation of performance can be reduced as packing agent for liquid chromatography. SOLUTION: This packing agent for liquid chromatography is made of polymer constituted of a monomer (A) containing at least one sulfonic group in one molecule (for example, 2-(meta)acrylamid-2-methylpropane sulfonic acid) and a bridge monomer (B) (for example, diethylene glycol dimethacrylate) as units. Also, this packing agent for liquid chromatography is made of polymer constituted of a monomer (A) containing at least one sulfonic group in one molecule (for example, allyl sulfonic acid), monomer (C) containing at least one carboxyl group in one molecule (for example, methacrylic acid, itaconic acid), and bridge monomer (B) (for example, divinylbenzene) as units.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a packing material for liquid chromatography, and more particularly to a packing material for cation exchange liquid chromatography.

[0002]

2. Description of the Related Art Liquid chromatography using a packing material having a carboxyl group or a sulfonic acid group as a cation exchange group (filling material containing a cation exchange group) is useful for the analysis of glycated hemoglobins and other biologically relevant substances. This is a very useful method for analysis of DNA.

The following two methods have been disclosed as conventional methods for producing a cation exchange group-containing filler composed of an organic synthetic polymer. (1) A method of reacting a cation exchange group-containing compound with crosslinkable particles. That is, the inorganic or organic particles having a reactive functional group are reacted with a compound having a group that reacts with the reactive functional group and a cation exchange group, so that the inorganic or organic particles have a cation exchange group. How to introduce. Specifically, for example, JP-A-1-262468 discloses a method in which a carboxyl group-containing compound or a sulfonic acid group-containing compound reacts with an epoxy group in a particle.

(2) A method of polymerizing a carboxyl group-containing monomer with a crosslinkable monomer. For example, Japanese Patent Publication No. 63-59463 discloses a carboxyl group-containing monomer of 5 to 90% by weight, a crosslinkable monomer of 10 to 90% by weight.
A method is disclosed in which 95% by weight and 0 to 85% by weight of a non-crosslinkable monomer are mixed and polymerized. In addition, Tokiko 8-71
No. 97 discloses a method of polymerizing a cation exchange group-containing monomer on the surface of a hydrophobic crosslinked polymer particle containing a polymerization initiator.

[0005]

The packing material for liquid chromatography obtained by the above methods (1) and (2) includes:
The following problems remain. It is generally known that it is difficult to quantitatively introduce a compound having a cation exchange group in the method of introducing a cation exchange group by post-treatment with the filler according to the method (1), It is inferior in the reproducibility of production as compared with the introduction of a cation exchange group derived from a monomer (Yoshimaki, Hosoya, Kizen, Tanaka: Chromatography, 16 (1) 7-12 (1995)). Therefore, there is a disadvantage that the separation performance as a packing material for liquid chromatography varies. Also, the post-processing method is extremely complicated and requires a long time.

Filler by the above-mentioned method (2): By controlling the amount of the carboxyl group-containing monomer added and the polymerization conditions, the filler can be quantitatively made to contain a carboxyl group. However, since the usable pH of a filler having a carboxyl group is limited to about 5.5 or more, there is a drawback that analysis with a low pH eluent cannot be performed. In addition, there is no disclosure of an example in which a sulfonic acid group-containing monomer is used to prepare a sulfonic acid group-containing packing material for liquid chromatography. Therefore, the conventional sulfonic acid group-containing filler is only the filler obtained by the above method (1), but the filler has a strong adsorption ability, and depending on the measurement conditions, causes nonspecific adsorption of interfering substances in the sample, As a result, there is a disadvantage that the life of the column is short.

[0007] On the other hand, in the case of a short-time analysis including a peak whose elution position is close to each other, especially in a case where separation cannot be performed only by changing elution conditions, selection of a filler is important. However, the conventional carboxyl group-containing filler and the sulfonic acid group-containing filler have too different cation exchange capacities, making it difficult to fine-tune the filler.

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and has as its object to reduce the variation in performance as a liquid chromatography filler, to be able to be used in a wide range of pH, and to provide a liquid chromatography having a sufficient column life. The purpose is to provide a filler.

[0009]

The invention according to claim 1 (hereinafter referred to as the present invention 1) comprises a monomer (A) containing at least one sulfonic acid group in one molecule and a crosslinkable monomer (A). B)
A filler for liquid chromatography, comprising a polymer having the following structural unit.

The invention according to claim 2 (hereinafter referred to as the present invention 2) is a monomer (A) having at least one sulfonic acid group in one molecule, and a monomer having at least one carboxyl group in one molecule. A filler for liquid chromatography, comprising a polymer comprising a monomer (C) and a crosslinkable monomer (B) as constituent units.

Hereinafter, the present invention will be described in detail. (Monomer (A) Containing at least One Sulfonate Group in One Molecule) In the present inventions 1 and 2, the monomer (A) containing at least one sulfonic acid group in one molecule includes:
For example, a monomer having at least one sulfonic acid group and at least one vinyl group in one molecule is exemplified.
Examples of such a monomer include styrenesulfonic acid, (meth) allylsulfonic acid, 2- (meth) acrylamido-2-methylpropanesulfonic acid, (3-sulfopropyl) -itaconic acid, and 3-sulfopropyl (Meta)
Acrylic acid and derivatives and salts thereof are exemplified. As the monomer (A), a monomer having no benzene skeleton in the molecule is particularly preferable. The benzene skeleton is not preferable in the analysis of biological substances, in particular, because it induces nonspecific adsorption of substances in the sample such as proteins.

When the amount of the monomer (A) is too small, the cation exchange capacity of the obtained polymer is too small, so that a sufficient cation exchange reaction with the substance to be measured becomes difficult.
When the amount increases, the hydrophilicity of the obtained polymer becomes too large, the pressure resistance decreases, and aggregation easily occurs during polymerization. Therefore, 10 to 100 parts by weight with respect to 100 parts by weight of the crosslinkable monomer (B). Parts are preferred.

(Crosslinkable Monomer (B)) In the present inventions 1 and 2, the crosslinkable monomer (B) is a monomer having two or more vinyl groups in one molecule, and is an ion exchange group. It is preferable to use a monomer which has no or a small amount of a monomer, and which is more hydrophobic than the monomers (A) and (C)). Examples of such a crosslinkable monomer (B) include styrene derivatives such as divinylbenzene, divinyltoluene, divinylxylene, divinylethylbenzene, and divinylnaphthalene; ethylene glycol di (meth)
Acrylate, polyethylene glycol di (meth) acrylate, 1,3-butylene glycol di (meth) acrylate, 1,6-hexane glycol di (meth) acrylate, neopentyl glycol di (meth) acrylate, polypropylene glycol di (meth) acrylate , Trimethylolethanetri (meth) acrylate,
Derivatives of (meth) acrylates such as trimethylolpropane tri (meth) acrylate, tetramethylolpropane tri (meth) acrylate, tetramethylolmethane tri (meth) acrylate, tetramethylolmethanetetra (meth) acrylate; 1,3- Aliphatic diene compounds such as butadiene, isoprene and 1,4-hexanediene; and derivatives of the above monomers. These may be used as a mixture of two or more.

(Monomer (C) containing at least one carboxyl group in one molecule)
Examples of the monomer (C) containing at least one carboxyl group in one molecule include, for example, at least one monomer in one molecule.
And a monomer having one carboxyl group and at least one vinyl group. Examples of such a monomer include (meth) acrylic acid, crotonic acid, β- (meth)
Acryloyloxyethyl hydrogen succinate, itaconic acid, citraconic acid, mesaconic acid, maleic acid, fumaric acid, and derivatives and salts thereof. As the monomer (C), a monomer having no benzene skeleton in the molecule is particularly preferable. The benzene skeleton is not preferable in the analysis of biological substances, in particular, because it induces nonspecific adsorption of substances in the sample such as proteins.

In the present invention 2, the amount of the monomer (C) is 10 to 1 based on 100 parts by weight of the crosslinkable monomer (B).
00 parts by weight is preferred.

The polymer used for the packing material for liquid chromatography of the present inventions 1 and 2 may contain, if necessary, a non-crosslinkable monomer other than the monomers (A) and (C). (D) may be a part of a structural unit. Examples of the non-crosslinkable monomer (D) include styrene derivatives such as styrene, α-methylstyrene, p-methylstyrene and chloromethylstyrene; vinyl halides such as vinyl chloride; vinyl acetate and propionic acid Vinyl esters such as vinyl and vinyl stearate; (methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, and stearyl (meth) acrylate (Meth) acrylic esters; (meth) acrylamide, (meth)
(Meth) acrylic acid derivatives such as acrylonitrile.

When the amount of the non-crosslinkable monomer (D) with respect to the crosslinkable monomer (B) increases, the voids in the polymer particles become large, and swelling and shrinkage tend to occur. When a plurality of eluents are used, it takes a long time to equilibrate with the eluents, so that the amount is preferably 0 to 100 parts by weight based on 100 parts by weight of the crosslinkable monomer (B).

As the polymer used for the packing material for liquid chromatography of the present inventions 1 and 2, polymer particles (E) having a uniform particle size distribution are used at the time of production thereof, and the polymer particles (E) and The particle size may be made more uniform by manufacturing by absorbing the monomer (C). The polymer particles (E) refer to polymer particles having a uniform particle size distribution, for example, a non-crosslinked polymer composed of a homopolymer or a copolymer such as the non-crosslinkable monomer (D). Examples of the particles include a styrene polymer, a methyl (meth) acrylate polymer, and an ethyl (meth) acrylate polymer. Further, as the polymer particles (E), crosslinked copolymer particles (for example, styrene-divinylbenzene) which are copolymers of the non-crosslinkable monomer (D) and the crosslinkable monomer (B) are used. In this case, the proportion of the crosslinkable monomer (B) may be 10
Low-crosslinked polymer particles obtained by copolymerization at a weight percent or less are preferred.

The method for producing the polymer particles (E) may be a known polymerization method, for example, emulsion polymerization, soap-free polymerization, dispersion polymerization, suspension polymerization and the like.

The average particle size of the polymer particles (E) is 0.1.
The variation in particle size is preferably 15 as a variation coefficient (CV) (= standard deviation ÷ average particle size × 100).
% Or less is preferable.

The amount of the polymer particles (E) is preferably 0.5 to 100 parts by weight based on 100 parts by weight of the crosslinkable monomer (B).

When the above-mentioned polymer particles (E) are used, the crosslinkable monomer (B) (including the non-crosslinkable monomer (D) as required) and the polymerization initiator are used. It is preferable that the polymerization be promoted by absorption into the coalesced particles (E).

The polymer used for the packing material for liquid chromatography of the present inventions 1 and 2 can be produced by a known polymerization method, for example, suspension polymerization, emulsion polymerization, dispersion polymerization and the like. For example, a method of mixing the above-mentioned monomer (A) (and the above-mentioned monomer (C)) and the above-mentioned monomer (B), adding a predetermined amount of a polymerization initiator thereto, and raising the temperature to carry out polymerization. No. Further, after preparing crosslinkable polymer particles using the crosslinkable monomer (B), the polymer particles are impregnated with a polymerization initiator, and then the monomer (A) (and the above monomer ( C)) may be added to the polymerization system for polymerization.

The polymerization initiator is not particularly limited, and a known water-soluble or oil-soluble radical polymerization initiator is used. Specific examples of the polymerization initiator include persulfates such as potassium persulfate, sodium persulfate, and ammonium persulfate; cumene hydroperoxide, benzoyl peroxide, lauroyl peroxide, octanoyl peroxide, o-chlorobenzoyl. Peroxide, acetyl peroxide, t-butyl hydroperoxide, t-butyl peroxyacetate,
organic peroxides such as t-butylperoxyisobutyrate, 3,5,5-trimethylhexanoyl peroxide, t-butylperoxy-2-ethylhexanoate, di-t-butyl peroxide; 2'-azobisisobutyronitrile, 2,2'-azobis (2,4-dimethylvaleronitrile), 4,4'-azobis (4-cyanopentanoic acid), 2,2'-azobis (2-methyl Azo compounds such as butyronitrile) and azobiscyclohexanecarbonitrile.

The amount of the polymerization initiator used is preferably 0.05 to 5 parts by weight based on 100 parts by weight of the crosslinkable monomer (B). If the amount of the polymerization initiator is less than 0.05 part by weight, the polymerization reaction may be insufficient or the polymerization may take a long time. Things may occur. This polymerization initiator may be used by dissolving it in the crosslinkable monomer (B).

The polymer used for the packing material for liquid chromatography of the present inventions 1 and 2 may be made porous by being manufactured by using a porogen at the time of its manufacture.

When producing a porous polymer,
An organic solvent that dissolves the monomer as a porosifying agent but does not dissolve the polymer may be added to the polymerization reaction system for polymerization.
As such a porosifying agent, known ones may be used, for example, aromatic hydrocarbons such as toluene, xylene, diethylbenzene, dodecylbenzene; hexane, heptane,
Saturated hydrocarbons such as octane and decane; and alcohols such as isoamyl alcohol, hexyl alcohol and octyl alcohol.

When the amount of the above-mentioned porosifying agent increases,
Since the pressure resistance of the obtained polymer is reduced, swelling and shrinkage are easy to occur, and aggregation is liable to occur during polymerization. Therefore, 0 to 100 parts by weight of the crosslinkable monomer (B) is used. Parts by weight are preferred.

(Particle Size of Packing Material for Liquid Chromatography of the Present Invention) The packing material for liquid chromatography of the present inventions 1 and 2 preferably has an average particle size of 0.5 to 100 μm. CV) (= standard deviation ÷ average particle size × 100) is preferably 15% or less. Such a packing material for liquid chromatography of the present invention can be obtained by classifying polymer particles obtained by polymerization as necessary. For classification, a known method such as a dry method or a wet method can be used.

(Use of the packing material for liquid chromatography of the present invention) The packing material for liquid chromatography of the present inventions 1 and 2 is packed in a column made of stainless steel or the like and used for cation exchange liquid chromatography or the like. An appropriate method can be used for filling the column, but a wet method (slurry method) in which a predetermined amount of a filler is dispersed in a dispersion medium such as a solvent used as an eluent, and the mixture is pressed into the column via a packer or the like. Method) is particularly preferred.

The substances to be measured in the separation and measurement using the packing material for liquid chromatography of the present invention 1 or 2 are all substances conventionally separated by cation exchange liquid chromatography or ion chromatography. In particular, biologically-related substances such as catecholamine derivatives, nucleotides, peptides, and proteins are suitable.

The liquid chromatograph to which the packing material of the present invention 1 or 2 can be applied may be a known one, and includes, for example, a liquid sending pump, a sample introduction device, a column, a detector and the like. In addition, other accessories (such as a thermostat and an eluent deaerator) may be appropriately attached to these.

For the liquid chromatography analysis using the packing material of the present invention 1 or 2, a known eluent is used.
For example, various buffers containing the following substances and the like can be mentioned. Inorganic acids such as phosphoric acid, nitric acid, hydrochloric acid and perchloric acid and salts thereof; organic acids such as acetic acid, malic acid, tartaric acid, succinic acid and citric acid and salts or halides thereof; sodium hydroxide and potassium hydroxide Basic substances. Further, for example, an organic solvent such as acetone, acetonitrile, dioxane, methanol, and ethanol can be used, and water or a mixture of the above-mentioned buffer and an organic solvent can also be used.

[0034]

The filler of the present invention 1 is a filler for liquid chromatography comprising polymer fine particles containing a sulfonic acid group, and the sulfonic acid group is converted into a monomer containing at least one sulfonic acid group. It is derived from. Therefore, unlike the filler obtained by the method of introducing a sulfonic acid group by the post-treatment according to the conventional method, the filler has less variation in performance as a filler for liquid chromatography. Further, since the packing material of the present invention 1 has less nonspecific adsorption of proteins and the like, the column life is longer than that of a column packed with a conventional packing material.

The packing material of the present invention 2 is a packing material for liquid chromatography comprising polymer fine particles containing both a carboxyl group and a sulfonic acid group, and the carboxyl group is a simple packing containing at least one carboxyl group. The sulfonic acid group is derived from a monomer containing at least one sulfonic acid group. By adding and polymerizing these monomers at a certain ratio, it becomes possible to fine-tune the ion exchange capacity of the filler, and to remove substances that have conventionally been difficult to separate, or substances that have conventionally taken a long time to separate, Separation can be performed with high precision under simple and easy elution conditions.

[0036]

Examples of the present invention will be described below. Example 1 200 g of 2- (meth) acrylamide-2-methylpropanesulfonic acid (monomer (A): manufactured by Tokyo Chemical Industry Co., Ltd.), diethylene glycol dimethacrylate (crosslinkable monomer (B): Shin-Nakamura Chemical 400 g) and 1.5 g of benzoyl peroxide (polymerization initiator: manufactured by Wako Pure Chemical Industries, Ltd.) were mixed and dispersed in 2.5 liters of a 4% by weight aqueous solution of polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Co., Ltd.). The temperature was increased with stirring under a nitrogen atmosphere,
Polymerized for hours. After polymerization, wash, classify and average particle size 5 μm
Was obtained.

Example 2 100 g of methacrylic acid (monomer (C): manufactured by Wako Pure Chemical Industries, Ltd.), 50 g of itaconic acid (monomer (C): manufactured by Kishida Chemical Co., Ltd.), allylsulfonic acid (monomer) (A): 50 g of Kishida Chemical Co., Ltd., 20 g of styrene (non-crosslinkable monomer (D)), 400 g of divinylbenzene (crosslinkable monomer (B): Kishida Chemical Co.), toluene (porous agent) 100 g) and 1.5 g of benzoyl peroxide were mixed and dispersed in 2.5 liters of a 6% by weight aqueous solution of polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Co., Ltd.). The temperature was raised while stirring under a nitrogen atmosphere, and polymerization was carried out at 80 ° C. for 8 hours. After polymerization, wash, classify and average particle size 5μ
m of filler were obtained.

Example 3 Triethylene glycol dimethacrylate (Crosslinkable monomer (B): manufactured by Shin-Nakamura Chemical Co., Ltd.)
Benzoyl peroxide (polymerization initiator) in 400g
1.5g mixed and dissolved, 2.5 liters 4% by weight
It was dispersed in an aqueous polyvinyl alcohol solution. The temperature was raised while stirring under a nitrogen atmosphere, and polymerization was carried out at 80 ° C. for 1 hour. After the polymerization, the reaction system was cooled to 35 ° C, and acrylamide-
After adding 200 g of 2-methylpropanesulfonic acid (monomer (A): manufactured by Tokyo Chemical Industry Co., Ltd.) and stirring at 35 ° C. for 1 hour, 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 5 μm.

(Example 4) Acrylamido-2-methylpropanesulfonic acid in Example 3 (monomer (A))
Instead of 200 g, methacrylic acid (monomer (C)) 4
Example 3 except that a mixture of 0 g, itaconic acid (monomer (C)) 50 g and acrylamido-2-methylpropanesulfonic acid (monomer (A)) 50 g was used.
A filler was prepared in the same manner as described above.

Comparative Example 1 Example of only a monomer containing one carboxyl group: methacrylic acid (monomer (C)) 200
g, 400 g of tetraethylene glycol dimethacrylate (crosslinkable monomer (B): manufactured by Shin-Nakamura Chemical Co., Ltd.) and 1.5 g of benzoyl peroxide (polymerization initiator) were mixed,
It was dispersed in 2.5 liters of a 4% by weight aqueous solution of polyvinyl alcohol. The temperature was increased while stirring under a nitrogen atmosphere, and 8
Polymerization was carried out at 0 ° C. for 8 hours. After the polymerization, the polymer was washed and classified to obtain a filler having an average particle size of 5 μm.

(Comparative Example 2) Examples of fillers obtained by introducing a sulfonic acid group by post-treatment: 200 g of 2-hydroxyethyl methacrylate, 40 g of ethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.), methyl methacrylate (sum A mixed solution of 15 g of Kojun Pharmaceutical Co., Ltd. and 5 g of tert-butyl perpivalate (polymerization initiator: Wako Pure Chemical Co., Ltd.) is dispersed in 2.5 L of a 4% by weight aqueous solution of polyvinyl alcohol in which 20 g of sodium sulfate is dissolved. Was. The temperature was raised while stirring under a nitrogen atmosphere, and polymerization was carried out at 60 ° C. for 12 hours. After polymerization, the particles were washed and classified to obtain particles having an average particle size of 5 μm. 100 g of the particles are dispersed in 100 ml of a 20% by weight aqueous sodium hydroxide solution, and 40 g of epichlorohydrin is dispersed.
Was added and reacted at room temperature for 4 hours. Further, 100 g of the obtained particles was dispersed in 100 ml of a 20% by weight aqueous sodium sulfate solution, and reacted at 80 ° C. for 20 hours. The obtained particles were washed to obtain a filler.

(Evaluation of Performance) The performance of the fillers obtained in Examples and Comparative Examples was evaluated as follows. (1) Production of a column for liquid chromatography: 0.7 g of a filler was collected, and a 50 mM phosphate buffer (pH 6.
0) Dispersed in 30 ml, sonicated for 5 minutes, and then stirred well. The whole amount is made of stainless steel empty column (4.6φ × 3
(5 mm) was injected into a connected packer (Umeya Seiki Co., Ltd.). A liquid pump (manufactured by Sanuki Kogyo KK) was connected to the packer, and the solution was packed at a constant pressure of 200 kg / cm ² to produce a column for liquid chromatography.

(2) Measurement of protein mixture: A mixture of protein standard substances was measured using columns filled with the fillers obtained in Examples 1 and 2 and Comparative Examples 1 and 2. (Measurement conditions) System: Liquid pump: LC-9A (manufactured by Shimadzu Corporation) Autosampler: ASU-420 (manufactured by Sekisui Chemical) Detector: SPD-6AV (manufactured by Shimadzu Corporation) Eluent: The following eluent A100 % To 100% eluent B by a linear gradient method. Eluent A: 100 mM phosphate buffer (pH 5.7) Eluent B: Eluent A + 500 mM NaCl (pH 5.5)
7) Flow rate: 1.5 ml / min Detection wavelength: 254 nm Sample injection volume: 10 μl

(Measurement sample) Mixture of myoglobin, α-chymotrypsinogen, ribonuclease A, and lysozyme (all manufactured by Sigma)

(Measurement Results) FIG. 1 shows a chromatogram obtained when the filler obtained in Example 1 was used, and FIG. 2 shows a chromatogram obtained when the filler obtained in Comparative Example 1 was used. The chromatogram obtained when the filler obtained in Example 2 was used was the same as that shown in FIG. 1, and the chromatogram obtained when the filler obtained in Comparative Example 2 was used was the same as that shown in FIG. 2.) In the figure,
Peak 1 indicates myoglobin, peak 2 indicates α-chymotrypsinogen, peak 3 indicates ribonuclease A, and peak 4 indicates lysozyme. FIG. 1 shows that the separation of each peak is better than that of FIG.

(3) Measurement of hemoglobins in human blood: Using the columns filled with the fillers obtained in Examples 3 and 4 and Comparative Examples 1 and 2, each of which serves as an indicator for diagnosing diabetes. Containing glycated hemoglobin (Hb) in water
The measurement of class b was performed. (Measurement conditions) System: Same as the system used in the above (2) Measurement of protein mixture. Eluent: Elution was performed by a step gradient method using two kinds of buffers containing phosphate and perchlorate. The salt concentration and pH of the eluent C were appropriately adjusted so that the retention time of the stable hemoglobin A1c (stable HbA1c) was appropriate. Eluent C: salt concentration 15-100 mM, pH 5.6-
Adjusted between 6.0. Eluent D: salt concentration 300 mM, pH 7.2. Flow rate: 1.5 ml / min Detection wavelength: 415 nm Sample injection volume: 10 μl

(Measurement sample) Blood of a healthy human was collected with sodium fluoride, glucose was added thereto at a concentration of 500 mg / dl, reacted at 37 ° C. for 5 hours, and then diluted with a hemolytic diluent (0.1% by weight polyethylene). Hemolysis was performed with a phosphate buffer solution (pH 7.0) of glycol mono-4-octylphenyl ether (Triton X-100) (manufactured by Tokyo Chemical Industry Co., Ltd.), and the sample was diluted 150-fold to obtain a measurement sample.

(Measurement results) FIG. 3 shows the chromatogram obtained when the filler obtained in Example 3 was used (the chromatogram obtained when the filler obtained in Example 4 was used was the same as FIG. 3). FIG. 4 shows a chromatogram obtained when the filler obtained in Comparative Example 1 was used, and FIG. 5 shows a chromatogram obtained when the filler obtained in Comparative Example 2 was used. In the figure, peak 5
Is hemoglobin A1a and b (HbA1a and b); peak 6 is hemoglobin F (HbF); peak 7 is unstable HbA1c; peak 8 is stable HbA1c; peak 9
Indicates hemoglobin A0 (HbA0). In order to ensure good quantification of HbF (peak 6) and stable HbA1c peak (peak 8) as an indicator of diabetes diagnosis, HbF, unstable HbA1c, stable HbA1c,
Although it is necessary to elute in the order of HbA0, FIG. 3 shows that each peak elutes in this order, and that the separation of each peak is good despite the short-time measurement. FIG.
And in FIG. 5, the separation of each peak was poor despite the long measurement time, and some peaks were not detected.

(4) Evaluation of the difference between lots of the filler: the filler having a sulfonic acid group of the present invention (Example 3) and the conventional method of introducing a sulfonic acid group by post-treatment (Comparative Example 2) Was checked as follows. Polymerization was carried out 30 times each, and the dispersion in performance among the obtained 30 lots of the filler was observed. The measurement of hemoglobins in human blood in the above evaluation (3) was performed for each 30 lots of each filler, and the retention time of stable HbA1c at peak 8 was examined. The eluent C was prepared for each filler so that the retention time of the peak 8 was about 2 minutes.

(Results of Measurement) The average retention time of the filler having a sulfonic acid group (Example 3) was 2.2 minutes, the standard deviation was 0.15 minutes, and the coefficient of variation (CV (%) = (Standard deviation ÷ average retention time × 100) was 6.82%. On the other hand, the average retention time of the conventional filler (Comparative Example 2) was 2.2 minutes, the standard deviation was 0.91 minutes, and the coefficient of variation (CV (%) = standard deviation ÷ average retention time ×
100) was 41.4%. From the above, it was found that the filler of the present invention was excellent in production reproducibility.

(5) Evaluation of Column Life: Using the columns filled with the packing materials of Examples 3, 4 and Comparative Example 2, the measurement of hemoglobin in human blood in the above evaluation (3) was repeated. Stable HbA1 of peak 8 represented by the following formula:
The change in the area value of c was examined. Area value of peak 8 (%) = (area of peak 8) / (area of all peaks) × 100

(Measurement Results) The measurement results are shown in FIG. In FIG. 6, the horizontal axis represents the number of repeated measurements, and the vertical axis represents the area value of each stable HbA1c and the initial measurement value of 10 times.
It is a value (%) indicated by a relative percentage as 0. From FIG. 6, the fillers of Examples 3 and 4 show good measured values even after 3000 times of measurement, while the fillers of Comparative Example 2 have decreased measured values as the number of measurements increases, It turns out that accurate measurement cannot be performed.

[0053]

The filler of the present invention 1 differs from the filler obtained by the method of introducing a sulfonic acid group by the post-treatment according to the conventional method, in that there is little variation in the performance as a filler for liquid chromatography. Further, since non-specific adsorption of proteins and the like is small, the column life is longer than that of a column packed with a conventional packing material.

The filler of the present invention 2 has all the effects of the above-mentioned present invention 1 and has a fine adjustment of the cation exchange capacity of 2
The composition can be easily changed only by changing the mixing ratio of the monomers (monomer having a carboxyl group and monomer having a sulfonic acid group), so that the composition most suitable for the substance to be measured can be easily selected. This makes it possible to separate a substance that has conventionally been difficult to separate or a substance that has conventionally taken a long time with high precision under a simple and easy elution condition. As described above, the fillers of the present inventions 1 and 2 are particularly suitable for analyzing proteins.

[Brief description of the drawings]

FIG. 1 is a diagram showing a chromatogram obtained when a protein mixture was measured using the filler obtained in Example 1.

FIG. 2 is a view showing a chromatogram obtained when a protein mixture is measured using the filler obtained in Comparative Example 1.

FIG. 3 shows that Hb was obtained using the filler obtained in Example 3.
The figure which shows the chromatogram obtained when performing the measurement of the class.

FIG. 4 shows that Hb was obtained using the filler obtained in Comparative Example 1.
The figure which shows the chromatogram obtained when performing the measurement of the class.

FIG. 5 shows that Hb was obtained using the filler obtained in Comparative Example 2.
The figure which shows the chromatogram obtained when performing the measurement of the class.

FIG. 6 is a graph showing the results of column life evaluation;
The horizontal axis represents the number of repeated measurements, and the vertical axis represents the value (%) of the area value of the stable HbA1c in each time expressed as a relative percentage with the initial measurement value as 100.

[Explanation of symbols]

 1 myoglobin 2 α-chymotrypsinogen 3 ribonuclease A 4 lysozyme 5 HbA1a and b 6 HbF 7 unstable HbA1c 8 stable HbA1c 9 HbA0

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) C08F 236/04 C08F 236/04 F term (Reference) 4J100 AB07Q AB16P AJ01R AJ02R AJ08R AJ09R AL08R AL62P AL63P AL66P AP01Q AS02P AS03P AS11P BA08P BA15R BA16Q BA16R BA35Q BA56Q CA04 CA05 CA23 JA16 JA17

Claims (2)

[Claims] (1) at least one sulfonic acid group per molecule;
A filler for liquid chromatography, comprising a polymer having a monomer (A) and a crosslinkable monomer (B) each as a constituent unit. 2. A compound having at least one sulfonic acid group in one molecule.
Liquid comprising a monomer (A) containing at least one carboxyl group in one molecule (C) and a polymer having a crosslinkable monomer (B) as a constituent unit. Packing material for chromatography.