JPH11295288A - Column packing for ion exchange liquid chromatography and measuring method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To achieve satisfactory pressure tightness and swelling properties, to shorten the equilibration time of an eluant, and to enable highly accurate analysis in a shorter time and the long life of a column by bringing ion exchange capacity in a predetermined range per dry weight of a column packing. SOLUTION: A column packing for ion exchange liquid chromatography is manufactured by regulating the amount of ion exchange radicals present in column packing particles, so that the amount of ion exchange radicals may be 0.1-20 μeq per dry weight 1 g of the column packing. The manufacture of the column packing is performed by polymerizing, drying and classifying a mixture of, for example, tetraethyleneglycol dimethacrylate, nonaethyleneglycol dimethacrylate, and methacrylic acid in predetermined ratios in a polyvinyl alcohol aqueous solution with benzoyl peroxide as an initiator under predetermined conditions. The column packing obtained in this way does not comprise exchange radicals more than necessary, enables short-time and highly accurate measurement in the chromatography of, for example, hemoglobins, and exhibits stable results ion measurements in the partial different conditions.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a packing material for ion exchange liquid chromatography and a method for measuring a sample by liquid chromatography using the packing material.

[0002]

2. Description of the Related Art As a filler for liquid chromatography generally used for ion exchange chromatography, a filler obtained by introducing an ion exchange group into silica particles or polymer particles is widely used. This ion exchange capacity is generally several meters.
It is about eq, and about 0.2 meq at least (for example, JP-A-1-262468). This is because if the ion exchange capacity is insufficient, the separation may be insufficient due to the lack of interaction between the ion exchange group and the sample. Fillers were used.

In order to shorten the measurement time of a sample by liquid chromatography, the filler particles need to be excellent in pressure resistance and swelling resistance. was there. However, if the hydrophobicity is too strong, the protein components in the sample may cause non-specific adsorption to the filler particles, which may cause deterioration of the column performance. In order to reduce the deterioration of the column performance, a conventional packing material for ion exchange liquid chromatography tends to introduce an ion exchange group in an amount larger than a required amount for the purpose of improving hydrophilicity.

[0004]

The following problems arise when an ion-exchange group is introduced into the filler in an amount larger than a required amount. (1) Existence of an excessive amount of the hydrophilic group causes a decrease in pressure resistance and swelling resistance of the filler. If the pressure resistance of the packing material is low, high-pressure packing of the column cannot be performed, and the life of the column and the resolution will be reduced. In addition, since high flow velocity analysis cannot be performed, measurement time is long. If the swelling resistance is low, swelling and shrinkage occur when the eluent is switched, for example, and the pressure loss during the measurement fluctuates greatly, causing a variation in the measured value. Frequent repetition of swelling / shrinking causes a reduction in column life.

(2) In an analysis system using a plurality of eluents,
It takes a long time to equilibrate to the switched eluent. Therefore, the measurement time is extended.

The present invention solves the above-mentioned drawbacks of the packing material used in ion exchange liquid chromatography, and has an object to provide an excellent pressure resistance and swelling resistance and an equilibration time of an eluent. An object of the present invention is to provide a packing material that is short, can analyze a sample in a shorter time with high accuracy, and has a long column life, and a measuring method using the packing material.

[0007]

The packing for ion-exchange liquid chromatography of the present invention is characterized in that the ion-exchange capacity is 0.1 to 20 μeq per 1 g of dry weight of the packing.

Hereinafter, the filler according to the present invention will be described. In the present invention, a known titration method is used for measuring the ion exchange capacity. For example, in the case of a filler in which a cation exchange group is introduced, titration is performed using an aqueous solution of sodium hydroxide or potassium hydroxide, and in the case of a filler in which an anion exchange group is introduced, an aqueous solution of hydrochloric acid is used. This is a titration method.

In the packing for ion exchange liquid chromatography of the present invention, the ion exchange capacity is limited to 0.1 to 20 μeq per 1 g of dry weight of the packing. When the ion exchange capacity is less than 0.1 μeq, effective ion exchange interaction with the sample to be measured hardly occurs,
If it exceeds 20 μeq, it takes time to equilibrate to changes in the external environment due to changes in the composition of the eluent, so that the measurement time tends to be extended, and swelling / shrinkage tends to occur, which tends to shorten the column life. .

The filler of the present invention is not particularly limited except that the ion exchange capacity is limited. The particle size of the filler is not particularly limited, but is preferably 0.5 to 20 μm. The particle size distribution of the filler is represented by a coefficient of variation (CV value).
20% as (standard deviation of particle size 粒径 average particle size × 100)
Or less, more preferably 15% or less.

[0011] The method for producing the filler of the present invention is characterized in that the ion exchange capacity is 0.1 to 20 µg / g of the dry weight of the filler.
The method is not particularly limited as long as the method can achieve eq. For example, (a) a method of introducing an ion-exchange group into polymer particles, and (b) a method of polymerizing a monomer having an ion-exchange group to form a polymer particle And (c) mixing a polymerizable ion-exchange group-containing ester (eg, methyl (meth) acrylate, ethyl (meth) acrylate) with a crosslinking monomer and polymerizing in the presence of a polymerization initiator. And a method of subjecting the obtained particles to a hydrolysis treatment to convert an ester into an ion-exchange group.

The method (a) for introducing an ion-exchange group into polymer particles will be described below. Examples of the polymer particles include inorganic particles such as silica and zirconia; natural polymer particles such as cellulose, polyamino acid, and chitosan; and synthetic polymer particles such as polystyrene and polyacrylate.

As the above-mentioned polymer particles, the constituent components other than the ion-exchange groups to be introduced are preferably more hydrophilic, and those having a high degree of crosslinking are preferable from the viewpoint of pressure resistance and swelling resistance.

When the polymer particles are silica particles, the silica particles can be obtained, for example, by adding an alkoxylane derivative to a mixed solvent of alcohol / water and polymerizing in the presence of a catalyst. Further, if necessary, an appropriate functional group can be introduced by reacting with a functional group-containing silane coupling agent.

When the polymer particles are cellulose particles, the cellulose particles are obtained by dissolving cellulose triacetate in an organic solvent, adding an aqueous gelatin solution to remove the organic solvent, and hydrolyzing under alkaline conditions. Obtainable.

When the polymer particles are polyamino acid particles, the polyamino acid particles can be obtained, for example, by polymerizing N-carboxylic anhydride.

When the polymer particles are synthetic polymer particles, the synthetic polymer particles may be, for example, styrene, divirbenzene,
Divinyltoluene; alkyl (meth) acrylates such as methyl (meth) acrylate and ethyl (meth) acrylate; monomers such as polyethylene glycol di (meth) acrylate and polypropylene glycol di (meth) acrylate in the presence of a polymerization initiator It can be obtained by polymerization.

The method of introducing an ion exchange group into a polymer particle is such that the polymer particle has a reactive functional group (for example, a hydroxyl group,
Amino group, carboxyl group, epoxy group, etc. (Hereinafter, referred to as a reactive group), a compound having both a functional group having a reactivity with the reactive group and an ion exchange group may be chemically bonded to the polymer particles. To prepare the polymer particles having the reactive group, for example,
Hydroxyl group-containing monomers (such as 2-hydroxyethyl (meth) acrylate, 2-hydroxymethyl (meth) acrylate, hydroxypropyl (meth) acrylate, (poly) ethylene glycol mono (meth) acrylate); amino group-containing monomers (2-aminoethyl (meth)
Acrylate, aminopropyl (meth) acrylate,
Dimethylaminoethyl (meth) acrylate, diethylaminoethyl (meth) acrylate, allylamine, etc.); carboxyl group-containing monomer ((meth) acrylic acid, itaconic acid, etc.); or epoxy group-containing monomer (glycidyl (meth) acrylate) ) May be mixed with a crosslinkable monomer or the like and polymerized in the presence of a polymerization initiator.

In the case of the polymer particles having no reactive group, the reactive group may be introduced into the polymer particles using various known chemical reactions.

The ion exchange group introduced into the polymer particles is
It may be a known one and there is no particular limitation. For example, a cation exchange group such as a carboxyl group, a sulfonic group, and a phosphate group;
Anion exchange groups such as a tertiary amino group and a quaternary ammonium group are exemplified.

The method (b) of polymerizing a monomer having an ion exchange group into polymer particles will be described below. When the ion exchange group is a carboxyl group, examples of the monomer having an ion exchange group include (meth) acrylic acid, itaconic acid, fumaric acid, and maleic acid. Then, for example,
Styrenesulfonic acid, 3-sulfopropyl (meth) acrylate, 3-sulfopropylitacolate, N- (3
-Sulfopropyl) -N- (meth) acryloyloxyethyl-N, N-dimethylammonium betaine, 2-
Acrylamide-2-methylpropanesulfonic acid and the like. In the case of a phosphoric acid group, for example, ((meth) acryloyloxymethyl) acid phosphate, (2- (meth) acryloyloxyethyl) acid phosphate, (3 -(Meth) acryloyloxypropyl) acid phosphate and the like. For example, in the case of a tertiary amino group or a quaternary ammonium group, dimethylaminoethyl (meth) acrylate, diethylaminoethyl (meth) acrylate, ( (Meth) acrylate hydroxypropyltrimethylammonium chloride, (meth) acrylatedimethylaminoethyltrimethylammonium chloride and the like.

The above polymerization method includes, for example, a method of mixing the above monomer and a crosslinkable monomer and polymerizing in the presence of a polymerization initiator. Further, as disclosed in JP-B-8-7197, after the above-mentioned crosslinkable monomer is polymerized, a monomer having an ion-exchange group may be added for polymerization.

In producing the filler of the present invention, the ion exchange capacity can be controlled by controlling the amount of ion exchange groups present in the filler particles. Therefore, in the case of the ion exchange group introduction method (a), the conditions of chemical treatment (type and amount of polymer particles; type and amount of ion exchange group-containing compound; reaction time and temperature; Depending on the method (b), the monomer having an ion-exchange group (such as type and amount) and the preparation conditions of the polymer particles (reaction temperature and time; type and amount of catalyst) Can be controlled by

The conditions for these controls are completely different depending on the case, but can be controlled by, for example, the amount of the added monomer in the following examples. When using (poly) ethylene glycol diacrylate, acrylic acid as a monomer and benzoyl peroxide as a polymerization initiator, and mixing these to carry out suspension polymerization, the amount of acrylic acid to be added should be (poly) ethylene glycol For 100 parts by weight of diacrylate,
The ion exchange capacity is preferably 0.1 to 20 μe per 1 g of the dry weight of the filler by adding so that the content is preferably in the range of 1 to 15 parts by weight, more preferably 2 to 10 parts by weight.
Particles that are q can be prepared.

However, in the polymerization method disclosed in Japanese Patent Publication No. 8-7197, not all the added monomers are polymerized, so that the amount is controlled by the amount of the monomers as in the above example. The method is not applicable.

Next, the measuring method of the present invention will be described. The measurement method of the present invention is a method for measuring a sample by liquid chromatography, characterized by using the filler of the present invention.

In carrying out the measurement method of the present invention, the packing material is packed in a column and used for liquid chromatography measurement. The column is not particularly limited, such as a well-known stainless steel, glass, or resin column. The column size is preferably 0.5 to 10 mm in inner diameter and 5 to 300 mm in length. As a method for filling the column with the filler, any known method can be used, but a slurry filling method is more preferable. Specifically, for example, it is performed by pressing a slurry in which filler particles are dispersed in a buffer such as an eluent into a column by a liquid sending pump or the like.

The sample to be measured by the method of the present invention can be any sample that can be measured by conventional ion exchange chromatography, and is not particularly limited. For example, biological samples such as proteins and nucleic acids; drugs; saccharides; surfactants;

The liquid chromatograph used for the above measurement may be a known one, and includes, for example, a liquid sending pump, a sample injection device (sampler), a column, a detector and the like. Further, other accessory devices (such as a column thermostat and an eluent deaerator) may be appropriately attached.

The eluent used for the measurement is selected from known eluents used for ion exchange chromatography. Specifically, an inorganic buffer containing phosphoric acid, boric acid and a salt thereof; an organic acid buffer containing various amino acids, citric acid, succinic acid, phthalic acid and a salt thereof; B
is-Tris (bis (2-hydroxyethyl) iminotris- (hydroxymethyl) methane), HEPES
Good's buffers such as (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) and MES (2- (N-morpholino) ethanesulfonic acid). Also, if necessary, various salts such as sodium chloride, potassium chloride, sodium sulfate and sodium hydrogen carbonate; inorganic acids such as hydrochloric acid; organic acids such as acetic acid; bases such as sodium hydroxide and potassium hydroxide; Or a water-soluble organic solvent such as acetonitrile may be added or used alone.

The concentration of the eluent varies depending on other measurement conditions, but is preferably 1 to 1000 mM, more preferably 5 mM.
８００800 mM. Further, a plurality of the above eluents may be mixed and used. Further, the eluent may be switched during the measurement, or gradient elution may be performed.

(Action) Since the packing for ion exchange liquid chromatography of the present invention has an ion exchange capacity of 0.1 to 20 μeq per 1 g of the dry weight of the packing, there is no unnecessary ion exchange group. Excellent pressure resistance and swelling resistance, and short equilibration time to eluent. Therefore, the column life is long and high resolution is obtained. Since the method for measuring a sample by liquid chromatography of the present invention uses the above-mentioned filler, proteins such as hemoglobins can be measured in a short time and with high accuracy.

[0033]

The present invention will be further clarified by the following non-limiting examples.

Example 1 300 g of tetraethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical), nonaethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical) 1
1.5 g of benzoyl peroxide (manufactured by Wako Pure Chemical Industries) was dissolved in 00 g and 40 g of methacrylic acid (manufactured by Wako Pure Chemical Industries). This was dispersed in 2500 ml of a 4% by weight aqueous solution of polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Co., Ltd.), and the temperature was raised to 70 ° C. under a nitrogen atmosphere with stirring, and polymerization was carried out for 4 hours. After polymerization,
After washing, drying and classification, particles having an average particle diameter of 6 μm were obtained. When the ion exchange capacity was measured by an automatic potentiometric titrator (AT-310, manufactured by Kyoto Denshi), it was 14.6 μeq per gram of dry particles.

(Example 2) Styrene (manufactured by Wako Pure Chemical Industries) 1
00 g, divinylbenzene (manufactured by Kishida Chemical) 100
g, methyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.) 100 g,
1.5 g of benzoyl peroxide (manufactured by Wako Pure Chemical Industries) was dissolved in 40 g of toluene and 65 g of acrylic acid. This was mixed with a 4% by weight aqueous solution of polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Company) 2
Dispersed in 500 ml, and stirred under a nitrogen atmosphere for 6 minutes.
The temperature was raised to 5 ° C., and polymerization was performed for 5 hours. After polymerization, the polymer was washed, dried, and classified to obtain particles having an average particle size of 6 μm. The ion exchange capacity was measured using an automatic potentiometric titrator (AT-310, manufactured by Kyoto Denshi).
μeq.

Example 3 350 g of triethylene glycol dimethacrylate (Shin-Nakamura Chemical Co., Ltd.) and 50 g of trimethylolmethane triacrylate (Shin-Nakamura Chemical Co., Ltd.)
In addition, 1.5 g of benzoyl peroxide (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 40 g of methacrylate hydroxypropyltrimethylammonium chloride (manufactured by NOF Corporation). This is 4
The resultant was dispersed in 2500 ml of an aqueous solution of polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Co., Ltd.) by weight, heated to 65 ° C. under a nitrogen atmosphere with stirring, and polymerized for 5 hours. After polymerization, the polymer was washed, dried, and classified to obtain particles having an average particle size of 6 μm. The ion exchange capacity was measured by an automatic potentiometric titrator (AT-310, manufactured by Kyoto Denshi).
It was 4.2 μeq.

Example 4 Benzoyl peroxide was added to 300 g of tetraethylene glycol dimethacrylate and 100 g of glycerol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.).
5 g was dissolved and dispersed in 2500 ml of a 4% by weight polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Industry) aqueous solution. The temperature was raised under a nitrogen atmosphere with stirring, and polymerization was carried out at 80 ° C. for 1 hour.
One hour later, 100 g of 2-acrylamido-2-methylpropanesulfonic acid was added, and the mixture was further polymerized at 65 ° C for 3 hours. After polymerization, washing, drying, and classification, the average particle size is 6
μm particles were obtained. Automatic potentiometric titrator (Kyoto Electronics A
When the ion exchange capacity was measured by T-310),
It was 11.3 μeq / g of dry particles.

Comparative Example 1 An ion exchange filler was prepared by the method described in JP-B-63-59463. 500 g of diethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) and 100 g of methacrylic acid were mixed, and 1.5 g of benzoyl peroxide was dissolved. This was mixed with a 4% by weight aqueous solution of polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Company) 2500
The mixture was dispersed in the same, and the temperature was raised to 80 ° C. under a nitrogen atmosphere with stirring, and the polymerization was carried out for 8 hours. After polymerization, washing and drying,
After classification, particles having an average particle size of 6 μm were obtained. When the ion exchange capacity was measured using an automatic potentiometric titrator (AT-310, manufactured by Kyoto Electronics Co., Ltd.), 36.9 μeq per 1 g of dry particles was measured.
Met.

Comparative Example 2 An ion-exchange filler was prepared by the method described in JP-A-1-262468. Glycidyl methacrylate (manufactured by NOF Corporation) 300 g, ethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.)
Dissolve 1.5 g of benzoyl peroxide in 40 g and add 4% by weight
It was dispersed in 2500 ml of an aqueous polyvinyl alcohol solution.
The temperature was raised under a nitrogen atmosphere with stirring, and polymerization was carried out at 80 ° C. for 8 hours. After polymerization, the polymer was washed, dried, and classified to obtain particles having an average particle size of 6 μm. 300 ml of xylene was added to 100 g of the obtained particles, and the mixture was further added to 1000 ml of a 4% by weight aqueous solution of polyvinyl alcohol containing 15 g of sulfuric acid, and reacted at 80 ° C. for 3 hours. After the reaction, the particles were washed and dried. This is added to 500 ml of dioxane,
Add 5 ml of boron trifluoride etherate, and add
The reaction was performed for 0 hours. After the reaction, the particles were washed and dried. 20 g of the obtained particles were dispersed in 100 ml of water, and 35 g of sodium monochloroacetate, 20 g of potassium iodide, and 50 g of potassium iodide were added.
60 ml of an aqueous solution of sodium hydroxide by weight
The reaction was carried out at a temperature of 3 ° C. for 3 hours. After the reaction, the particles were washed and dried. When the ion exchange capacity was measured by an automatic potentiometric titrator (AT-310, manufactured by Kyoto Denshi), it was 172.3 μeq per gram of dry particles.

Comparative Example 3 Benzoyl peroxide (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 350 g of tetraethylene glycol dimethacrylate and 150 g of methacrylate hydroxypropyltrimethylammonium chloride (manufactured by NOF Corporation).
5 g were dissolved. This was dispersed in 2500 ml of a 4% by weight aqueous solution of polyvinyl alcohol, and the temperature was raised to 70 ° C. in a nitrogen atmosphere with stirring, and polymerization was carried out for 6 hours. After polymerization, the polymer was washed, dried, and classified to obtain particles having an average particle size of 6 μm.
When the ion exchange capacity was measured by an automatic potentiometric titrator (AT-310, manufactured by Kyoto Electronics Co., Ltd.), it was 75.2 μeq per gram of dry particles.

(Evaluation) The fillers of Examples 1 to 4 and Comparative Examples 1 to 3 were packed in columns, and the following evaluations were made. (1) Packing method of column: 0.7 g of particles obtained in each example
Was dispersed in 30 ml of 50 mM phosphate buffer (pH 6.0), sonicated for 5 minutes, and then stirred well. The whole amount was injected into a packer (manufactured by Umeya Seiki Co., Ltd.) to which a stainless steel empty column (4.6 φ × 35 mm) was connected. Connect the liquid feed pump (manufactured by Sanuki Kogyo) to the packer,
It was filled at a constant pressure of g / cm ² .

(2) Swelling resistance test: Solvents having different pH and ionic strength were passed through a column filled with the filler obtained in Example 1 and Comparative Example 1 in the following manner, and pressure loss was caused. The swelling resistance was confirmed by examining the variation in the swelling resistance. A liquid sending pump (LC-9A: manufactured by Shimadzu Corporation) was connected to the obtained column, and a 50 mM phosphate buffer solution (pH 5.
0: liquid a) at a flow rate of 1.0 ml / min for 30 minutes. Thereafter, the passing solution is changed to a 300 mM phosphate buffer (p
H8.0: b solution). Fluctuation of the pressure indicated on the liquid sending pump during liquid sending was monitored. Further, the time until the pressure was stabilized after switching to the liquid b was measured.
Table 1 shows the results.

[0043]

[Table 1]

As is clear from Table 1, it was found that Example 1 had a lower degree of swelling and faster equilibration to the external environment than Comparative Example 1.

(3) Measurement of hemoglobins (Hb) Hemoglobins were separated from the columns packed with the packing materials of Examples 1 and 4 and Comparative Examples 1 and 2. Measurement condition system: Liquid pump: LC-9A (manufactured by Shimadzu Corporation) Autosampler: ASU-420 (manufactured by Sekisui Chemical) Detector: SPD-6AV (manufactured by Shimadzu Corporation) Eluent: Eluent A: 10 mM 100 mM saline solution (pH 5.7) containing 2- (N-morpholino) ethanesulfonic acid (manufactured by Wako Pure Chemical Industries) Eluent B: 300 mM phosphate buffer (pH 7.2) With respect to the packing material of Example 1, the eluent A flows for 0 to 1 minute from the start of the measurement, the eluent B flows for 1 to 1.1 minutes, and the eluent for 1.1 to 2 minutes. A was flushed. With respect to the packing materials of Example 4 and Comparative Examples 1 and 2, the flow time of the eluents A and B was changed so as to achieve as good a separation as possible within a practical measurement time. Flow rate: 1.5 ml / min Detection wavelength: 415 nm Sample injection volume: 10 μl

Measurement Sample Blood of a healthy human was collected from sodium fluoride, and 500 mg / dl was collected.
Aqueous glucose solution at 37 ° C. for 3 hours to obtain unstable hemoglobin A1c (unstable H
bA1c) was increased. This was diluted with a hemolysis diluent (0.1 wt% Triton X-100 in a phosphate buffer solution (pH 7.
0)), hemolyzed and diluted 150-fold to obtain a sample.

Measurement Results The chromatograms obtained by measuring the sample under the above measurement conditions are shown in FIGS. 1 is a chromatogram of the filler obtained in Example 1, FIG. 2 is a chromatogram of the filler obtained in Example 4, and FIG. 3 is a chromatogram of the filler obtained in Comparative Example 1. The chromatogram of the filler obtained in Comparative Example 2 was the same as that in FIG. In each peak, the peak 1 was hemoglobin A1a (HbA
1a) and hemoglobin A1b (HbA1b), peak 2 is hemoglobin F (HbF), peak 3 is unstable hemoglobin A1c (unstable HbA1c), peak 4 is stable hemoglobin A1c (stable HbA1c), and peak 5 is hemoglobin A0 ( HbA0). 1 and 2, the peaks 3 and 4 are well separated in a short time. On the other hand, in FIG. 3, the separation performance is inferior despite the measurement time being extended as compared with FIGS. 1 and 2.

(Test of Column Life) In the above measurement of hemoglobins, a stable hemoglobin A1c (stable hemoglobin A1c) was obtained by repeatedly performing the measurement using the fillers obtained in Examples 1, 3 and Comparative Example 1. HbA1c) measured value (ratio of stable HbA1c peak area value to total hemoglobin peak area value) was examined. FIG. 4 shows the degree of change of the measured value after the repetition when the initial value in the repetitive measurement is set to 100. As a result, the filler of Example 3 maintained good measurement accuracy even after 2,000 repetitions, but the filler of Comparative Example 1 showed a marked decrease in measured values.

(4) Measurement of Protein Mixture Using the fillers obtained in Example 3 and Comparative Example 3, a mixture of protein standard products was measured. Measurement condition system: Liquid pump: LC-9A (manufactured by Shimadzu Corporation) Autosampler: ASU-420 (manufactured by Sekisui Chemical Co., Ltd.) Detector: SPD-6AV (manufactured by Shimadzu Corporation) Eluent: Eluent A: 20 mM Tris -Phosphate buffer (pH 8.0) Eluent B: Equivalent mixture of eluent A and 500 mM saline solution A linear gradient method was used from eluent A 100% to eluent B 100%. Flow rate: 2.0 ml / min Detection wavelength: 254 nm Sample injection volume: 20 μl

Measurement Sample A solution prepared by dissolving a mixture of protein albumins of conalbumin, transferrin, ovalbumin and trypsin inhibitor (all manufactured by Sigma) in eluent A was used as a sample.

Measurement Results Chromatograms obtained by measuring the sample under the above measurement conditions are shown in FIG. 5 (Example 3) and FIG. 6 (Comparative Example 3). In each figure, peak 6 is conalbumin, peak 7
Indicates transferrin, peak 8 indicates ovalbumin, and peak 9 indicates trypsin inhibitor. In FIG. 5, the separation is good in a short time, but in FIG. 6, a long time is required and the separation performance is poor.

[0052]

The packing material for ion exchange liquid chromatography according to the present invention has an ion exchange capacity of 0.1 to 20 μeq per 1 g of dry weight of the packing material. Excellent swelling resistance,
In addition, the equilibration time of the eluent is short, the sample can be analyzed in a shorter time with high accuracy, and the packing material has a long column life.

Since the measuring method according to the present invention uses the filler of the present invention, when used for measurement of samples such as proteins such as hemoglobins, high-precision analysis can be performed in a shorter time. . In addition, the life of the column can be extended.

[Brief description of the drawings]

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

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

FIG. 3 is a diagram showing a chromatogram obtained by measuring hemoglobins using the filler obtained in Comparative Example 1.

FIG. 4 shows that when the measurement of hemoglobins is repeatedly performed using the fillers obtained in Example 1, Example 3 and Comparative Example 1, the change in the stable HbA1c value was 10
FIG.

FIG. 5 is a diagram showing a chromatogram obtained by measuring a protein standard mixture using the filler obtained in Example 3.

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

[Explanation of symbols]

 1 Peak of HbA1a and HbA1b 2 Peak of HbF 3 Peak of unstable HbA1c 4 Peak of stable HbA1c 5 Peak of HbA0 6 Peak of conalbumin 7 Peak of transferrin 8 Peak of ovalbumin 9 Peak of trypsin inhibitor

Claims (2)

[Claims] (1) The ion exchange capacity is 1% by the dry weight of the filler.
A packing material for ion-exchange liquid chromatography, wherein the packing material is 0.1 to 20 μeq per g. 2. A method for measuring a sample by liquid chromatography, comprising using the filler according to claim 1.