WO1992009889A1 - Procede de preparation d'un porteur pour la chromatographie liquide a echange de cations, et procede de determination quantitative de l'hemoglobine saccharifiee a l'aide dudit porteur - Google Patents

Procede de preparation d'un porteur pour la chromatographie liquide a echange de cations, et procede de determination quantitative de l'hemoglobine saccharifiee a l'aide dudit porteur Download PDF

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Publication number
WO1992009889A1
WO1992009889A1 PCT/JP1990/001522 JP9001522W WO9209889A1 WO 1992009889 A1 WO1992009889 A1 WO 1992009889A1 JP 9001522 W JP9001522 W JP 9001522W WO 9209889 A1 WO9209889 A1 WO 9209889A1
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WIPO (PCT)
Prior art keywords
monomer
hydrophobic
carrier
group
polymer particles
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PCT/JP1990/001522
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English (en)
Japanese (ja)
Inventor
Kazuyuki Oishi
Kazutoshi Yamazaki
Toshiki Kawabe
Masahiro Takechi
Makoto Takahara
Original Assignee
Sekisui Kagaku Kogyo Kabushiki Kaisha
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Application filed by Sekisui Kagaku Kogyo Kabushiki Kaisha filed Critical Sekisui Kagaku Kogyo Kabushiki Kaisha
Priority to DE69022250T priority Critical patent/DE69022250D1/de
Priority to CA002074177A priority patent/CA2074177C/fr
Priority to AT91900342T priority patent/ATE127586T1/de
Priority to US07/915,685 priority patent/US5292818A/en
Priority to PCT/JP1990/001522 priority patent/WO1992009889A1/fr
Priority to KR1019920701714A priority patent/KR920704135A/ko
Priority to EP91900342A priority patent/EP0596106B1/fr
Priority claimed from CA002074177A external-priority patent/CA2074177C/fr
Publication of WO1992009889A1 publication Critical patent/WO1992009889A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/327—Polymers obtained by reactions involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/321—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J39/00—Cation exchange; Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
    • B01J39/26—Cation exchangers for chromatographic processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/54—Sorbents specially adapted for analytical or investigative chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood

Definitions

  • the present invention relates to a method for producing a carrier suitable for cation exchange liquid chromatography and a method for quantifying glycated hemoglobin using the carrier obtained by the production method.
  • Liquid chromatography is used for the separation or detection of various substances, and ion exchange chromatography is used for separation or detection of hydrophilic substances, such as the separation of proteins from biological samples.
  • the ion-exchange chromatography method is a method in which a carrier having an ion-exchange group is used and separation is performed based on the difference in the ion-bonding strength of the component to be separated, i-one, to the carrier.
  • carboxyl S are useful for weak cation exchange chromatography and for the analysis of proteins and peptides.
  • glycated hemoglobin in blood is measured by ion exchange chromatography using such a carrier.
  • Glycated hemoglobin is formed by non-enzymatic reaction of hemoglobin in red blood cells with glucose in blood. Since the average concentration of dalcose in blood can be determined by measuring glycated hemoglobin, the measurement of glycated hemoglobin is widely used for diagnosis of diabetes.
  • glycated hemoglobin is mainly used for high-performance liquid chromatography (HPLC) using the above carriers. ) Is determined. According to the HPLC method, it is possible to perform a quicker measurement than the conventional force ram chromatography method, electrophoresis method, colorimetric method and the like.
  • an organic polymer-based carrier or an inorganic carrier is used as a carrier for weak cation exchange chromatography.
  • One of the most commonly used organic polymer-based gels is a carrier obtained by introducing a sulfoxyl group into the surface of crosslinked styrene divinylbenzene particles.
  • Such a carrier is obtained by introducing a carboxyl group into a styrenedivinylbenzene crosslinked copolymer particle by a chemical reaction.
  • an alkyl halide is introduced into the benzene ring of styrene in the copolymer by treating the above copolymer particles with methyl ether or the like, and the carboxyl group is obtained by hydrolyzing and oxidizing the alkyl halide. be introduced.
  • JP-A-58-221164 discloses a copolymer of an ester of acrylic acid or methacrylic acid such as tetramethylol methacrylate and acrylic acid or methacrylic acid. Different entities are disclosed.
  • a carrier is generally referred to in
  • the above-mentioned crosslinked copolymer particles are prepared by adding a polymerization initiator to a crosslinkable monomer and a monomer having a carboxyl group and subjecting them to suspension polymerization by the method disclosed in No. 58-221164. Is done. Or styrene, divinylbenzene and After copolymerizing with a monomer having a functional group capable of forming a carboxylic acid group by a hydrolysis reaction (hereinafter referred to as “hydrolyzable group”), the functional group is converted into a carboxyl group by a hydrolysis reaction. Can also be used. In order to improve the pressure resistance of such a carrier, it is necessary to increase the degree of cross-linking.
  • the cross-linking part is water-phobic, increasing the cross-linking degree increases the hydrophobicity of the gel and increases the nonspecificity of Adsorption occurs. For this reason, the amount of the crosslinking agent is limited, and it is difficult to obtain sufficient pressure resistance. Further, the carrier obtained by the above method has swelling and shrinking in an aqueous solvent because the carboxylic acid group is dispersed throughout the polymer particles, and for this reason, the pressure resistance is insufficient. is there.
  • quantification of glycated hemoglobin is performed by a step or continuous Daragen method using two types of eluents, a solution with a low solution output (hereinafter referred to as solution 1) and a solution with a strong solution output (hereinafter solution 2). It is implemented by.
  • the first liquid increases free carboxyl groups in the carrier particles.
  • hemoglobin other than glycated hemoglobin in the sample is not retained by the carrier, and glycated hemoglobin is separated and eluted.
  • the second solution has a high ion strength, the free carboxyl group becomes a salt. Therefore, the retained glycated hemoglobin other than hemoglobin elutes quickly.
  • the above carriers in particular, hydrophobic and hydrophilic monomers (carboxyl groups or monomers capable of generating carboxyl groups)
  • ion-exchange groups mainly derived from the hydrophilic monomer are distributed throughout the carrier particles.
  • the support swells and the pressure in the ram increases.
  • the operation of flowing the first solution and freezing the carboxyl groups is required to measure the next sample.
  • Glycated hemoglobin can be measured at a relatively high speed by HPLC using a polymer-based carrier, but if the measurement speed is to be further increased, as described above, the ion-exchange groups in the nursery school will not be sufficiently exchanged. Alternatively, the separation performance is reduced because the carrier swells. For high-precision separation, it is necessary to reduce the melting rate.
  • JP-A-63-75558 discloses a porous silica gel in which a carboxyl group is chemically bonded to the surface of a porous silica gel.
  • a carrier is disclosed.
  • This suspension is excellent in pressure resistance, capable of relatively high-speed processing, and excellent in separation.
  • this gel has the property of adsorbing substances having basic groups such as proteins due to the residual hairs of silanol groups on the surface.
  • silica gel is dissolved with acid and alcohol, so the pH of the solution is limited to 3-8.
  • JP-A-56-151712, JP-A-59-18705, JP-A-62-63856 and JP-A-63-79064 disclose a so-called seed polymerization method.
  • a crosslinked polymer particle is impregnated with a polymerization initiator and a monomer, and is further subjected to suspension polymerization to obtain particles having a two-layer structure.
  • a carrier for weak exchange chromatography can be obtained.
  • the same weak cation exchange chromatography carrier can be obtained by impregnating a monomer having a functional group capable of generating a hydrolyzable group, polymerizing the resultant, and then performing hydrolysis.
  • a carboxyl group is present inside the obtained particles, the particles are easily swelled or shrunk in an aqueous solvent for the same reason as described above, and thus have insufficient pressure resistance.
  • the separation performance of the liquid chromatography is poor, or the separation time is long.
  • the carrier for cathon exchange liquid chromatography of the present invention is a carrier for cathon exchange liquid chromatography of the present invention.
  • the first manufacturing method solves the various problems described in the section of the background art above,
  • a polymerizable monomer having a carboxyl group is added to the aqueous dispersion, and the monomer is polymerized on the surface of the hydrophobic crosslinked polymer particles. Obtaining coated polymer particles on which a polymer layer having a carbonyl group is formed.
  • the hydrophobic cross-linked polymer is obtained by homopolymerizing or co-polymerizing at least one hydrophobic crosslinkable monomer, or at least one hydrophobic crosslinkable monomer. It can be obtained by copolymerizing a monomer with at least one hydrophobic non-crosslinkable monomer.
  • the hydrophobic crosslinking monomer comprises di (meth) acrylate, poly (meth) acrylate of a polyhydric alcohol, and at least two poly (meth) acrylates. At least one selected from the group consisting of aromatic compounds having a vinyl group
  • the hydrophobic non-crosslinkable monomer is at least one selected from the group consisting of (meth) acrylate, vinyl dioxylate, and styrene-based monomer. Is a seed.
  • the polymerizable monomer having a carboxyl group is at least one selected from the group consisting of acrylic acid, methacrylic acid, maleic acid, fumaric acid, and ⁇ -tonic acid. And one kind.
  • the coated polymer particles are porous particles having substantially no pores or having an average radius of 10,000 A or less.
  • the aqueous dispersion in which the hydrophobic crosslinking polymer to which the polymerization initiator is attached is dispersed is formed by polymerizing a monomer containing the hydrophobic crosslinking monomer. It is obtained by reacting with an initiator.
  • the second method for producing a carrier for cation exchange liquid chromatography of the present invention is an aqueous dispersion in which hydrophobic palpitated polymer particles having no hydrolyzable group to which a polymerization initiator is attached are dispersed.
  • the step of obtaining the particles is carried out.
  • the hydrophobically crosslinked polymer is obtained by homopolymerizing or copolymerizing at least one hydrophobic crosslinkable monomer.
  • the hydrophobic rack ⁇ monomer one hydrophobic non-rack of It can be obtained by copolymerizing a monomer.
  • the water-hydrophobic crosslinking monomer is an aromatic compound having at least two vinyl groups.
  • the water-hydrophobic non-crosslinkable monomer is a styrene monomer.
  • the monomer having a functional group capable of forming a carboxyl group by a chemical decomposition reaction is (meth) alkyl acrylate, (meta) It is selected from the group consisting of acrylamide and (meta) acrylonitrile.
  • the coated polymer in the above-mentioned second production method is a porous particle having substantially no pores or having pores having an average radius of ⁇ , ⁇ or less.
  • the aqueous dispersion in which the hydrophobic crosslinked polymer to which the polymerization initiator is attached is dispersed is a hydrophobic crosslinkable monomer having no hydrolyzable group. It is obtained by reacting a monomer containing with a polymerization initiator.
  • the carrier used for the liquid chromatography is obtained by the first or second production method.
  • Cationic exchange suitable for the separation of hydrophilic substances such as proteins Provided is a method for producing a carrier for liquid chromatography, which has high pressure resistance, low swelling / shrinkage, and low non-specific adsorption such as protein;
  • Fig. 1, Fig. 3, Fig. 5, Fig. 8, Fig. 10 and Fig. 12 are Example 1, Example 2 and Comparative Example 1, Example 5, Example 6, and Comparative Example 5, respectively.
  • Fig. 3 shows a chromatogram obtained when an analysis of glycated hemoglobin in blood was performed using a force ram filled with the carrier obtained in the above.
  • Figures 2, 4, 6, and 7 show the analysis of protein using the columns packed with the carriers obtained in Example 1, Example 2, Comparative Example 1 and Comparative Example 3, respectively. The chromatogram obtained when the test was performed is shown.
  • FIGS. 9, 11 and 13 to 15 show the separation of various proteins using the carrier-filled rams obtained in Example 5, Example 6 and Comparative Examples 5 to 7, respectively. The mouth matogram obtained when the test was performed is shown.
  • the material of the hydrophobic crosslinked polymer particles used in the first and second production methods of the present invention includes a hydrophobic polymer obtained by homopolymerizing or copolymerizing at least one type of hydrophobic crosslinkable monomer.
  • a crosslinked polymer or a copolymer of at least one type of hydrophobic crosslinkable monomer and at least one type of hydrophobic non-crosslinkable monomer is used.
  • hydrophobic crosslinking monomer used in the first production method examples include ethylene glycol di (meth) acrylate, polyethylene glycol di (meta) acrylate, and propylene.
  • Di (meth) acrylic acid esters such as glycol alcohol (meta) acrylate and polypropylene glycol (meta) acrylate; tetramethylol methacrylate (meta)
  • Poly (meth) acrylic acid esters of polyhydric alcohols such as relate, tetramethylol methane tetra (meth) acrylate; divinylpentene, divinyltoluene, divinylxylene, divinylnaphthalene
  • an aromatic compound having two or more vinyl groups such as an aromatic compound is used as the water-based non-crosslinkable monomer used in the first production method described above.
  • any non-crosslinkable polymerizable monomer can be used, for example, methyl (meth) acrylate, ethyl (meta) acrylate, (Meta) acrylate, isopropyl (meta) acrylate, butyl (meta) acrylate, t-butyl (meta) acrylate And styrene-based monomers such as styrene, methylstyrene, etc.
  • crosslinkability is used.
  • the monomer is used in an amount of 10 parts by weight or more, preferably 20 parts by weight or more, based on 100 parts by weight of the whole monomer.
  • the monomer used for forming the polymer layer having a carboxyl group formed on the surface of the hydrophobic crosslinked polymer particles has a carboxyl group.
  • Monomers are used. This includes, for example, acrylic acid, methacrylic acid, maleic acid, fumaric acid, crotonic acid or a mixture thereof.
  • Other polymerizable monomers having a carboxyl group are used.
  • the polymerizable monomer having a carboxyl group may be used as a mixture of two or more as necessary. The use amount of such a monomer varies depending on the kind of the monomer, but a ratio of 5 to 80 parts by weight to 100 parts by weight of the hydrophobic cross-linked polymer is appropriate.
  • a hydrophobic cross-linkable monomer is (co) A (co) polymer obtained by polymerization or a copolymer of Sounaga & Jai-Ji-Single-Chain-Hydrophod tt ⁇ - ⁇ - ⁇ - ⁇ - ⁇ -H ⁇ - ⁇ is used.
  • the above-mentioned hydrophobic cross-linkable monomer and the hydrophobic non-insertable monomer can be used alone or in combination of two or more.
  • the hydrophobic crosslinking monomer for example, an aromatic compound having two or more butyl groups, such as divinylbenzene, divinyltoluene, divinylxylene, and divinylnaphthalene, is used.
  • the hydrophobic non-crosslinkable monomer for example, a styrene monomer such as styrene or methylstyrene is used.
  • the crosslinkable and non-crosslinkable monomers are mixed and used, the crosslinkable monomer is at least 10 parts by weight, preferably at least 20 parts by weight, based on 100 parts by weight of the total monomers. Used to be
  • a monomer used to form a polymer covering the hydrophobic cross-linked polymer particles is a monomer having a hydrolyzable group.
  • ⁇ click 1 J Rusanme chill such as main Tak Lil Sanme chill, Ata acrylic acid Or alkyl esters of methacrylic acid (hereinafter referred to as (meth) acrylic acid); (meta) acrylyl amide, (meta) acrylonitrile and the like.
  • the above-mentioned monomers having a hydrolyzable group can be used as a mixture of two or more as necessary.
  • the amount of the monomer having a hydrolyzable group varies depending on the type of the monomer, but is preferably 5 to 80 parts by weight based on 00 parts by weight of the hydrophobic crosslinking polymer.
  • hydrophobic crosslinked polymer particles are prepared by the following method.
  • the hydrophobic crosslinked polymer particles can be prepared by any known aqueous suspension polymerization method.
  • the above-mentioned hydrophobic cross-linking monomer and, if necessary, the hydrophobic non-cross-linking monomer and a polymerization initiator are mixed. These may be dissolved in the diluent if necessary.
  • the organic solvent as the diluent is dispersed in the obtained polymer particles.By removing the organic solvent after the polymerization, the porous spherical particles are formed.
  • hydrophobic crosslinked polymer particles need not be porous, it is not necessary to add a diluent.
  • a diluent it is usually used in a proportion of 200 parts by weight or less based on 100 parts by weight of the monomer mixture.
  • a diluent of this monomer, or a combination of a monomer and a polymerization initiator with polyvinyl alcohol Suspension polymerization is carried out by adding to an aqueous solution in which a suspending agent such as acid lucidum is dissolved, replacing with nitrogen and heating to 40 to 100 with stirring.
  • the polymerization initiator used here and the polymerization initiator to be attached to the obtained hydrophobic crosslinked polymer particles are catalysts that generate radicals, and are not particularly limited as long as they are hydrophobic.
  • organic peroxides such as benzoyl peroxide, acetyl baroxide and cumene peroxide
  • known radical generation such as azo compounds such as azobisisobutyronitrile and azobisisobutyramide Any of the catalysts can be used.
  • any organic solvent that dissolves the above-mentioned monomer and does not dissolve the polymer can be used.
  • aromatic hydrocarbons such as toluene, xylene, getylpentene, and dodecylbenzene
  • saturated hydrocarbons such as hexane, heptane, octane and decane
  • isoamyl alcohol hexyl alcohol
  • Alcohols such as octyl alcohol.
  • a polymerization initiator is attached to the obtained hydrophobic crosslinked polymer particles.
  • “to attach the polymerization initiator to the hydrophobic cross-linked polymer particles” means that the polymerization initiator is attached to the surface of the aqueous crosslinked polymer particles, or the polymerization initiator is attached to the surface of the particles. And infiltrating the polymerization initiator near the surface of the particles.
  • the polymerization initiator is converted into a solvent having a low boiling point and a good affinity for the hydrophobic cross-linked polymer. After dissolving, the hydrophobic crosslinked polymer particles are impregnated. This allows the polymerization initiator to penetrate into the particles.
  • the polymerization initiator-adhered particles are dispersed in an aqueous dispersion medium in which the polymerizable monomer having a propyloxyl group is dissolved, or the particles are dispersed.
  • the above-mentioned monomer is added to and dissolved in the aqueous medium.
  • the polymerization reaction is carried out by replacing with nitrogen and heating with stirring.
  • a dispersion stabilizer such as hydroxypropylcellulose or polyvinyl alcohol may be added to the aqueous dispersion medium.
  • the polymerization temperature and time vary depending on the type of the above-mentioned monomer to be reacted and the type of the polymerization initiator, but are 0.5 to 40 hours at 40 to 100 I :.
  • the preparation of the hydrophobic crosslinking polymer particles may be followed by the preparation of the polymerizable monomer having a carboxyl group.
  • the polymer particles having the two-layer structure can also be prepared by a continuous method of reacting a monomer. In this method, first, a polymerization reaction for preparing the cross-linked polymer particles is started. When the polymerization proceeds to some extent and the unreacted polymerization initiator remains, the above monomer is added to the reaction system.
  • the polymerization initiator is present in the oil phase in the system and the inside of the generated hydrophobic cross-linked polymer particles. Then, a polymer layer having a carboxyl group is formed so as to cover the surface portion of the hydrophobic crosslinked polymer particles.
  • the polymer particles obtained by each of the above methods are thoroughly washed with hot water, an organic solvent, or the like, and the suspension stabilizers, solvents, residual monomers, etc., which are contained or adhered to the particles, Is removed. If necessary, the particles are classified to obtain a carrier for cation exchange liquid chromatography.
  • the polymerization initiator-attached particles are dispersed in a dispersion medium in which the above-mentioned monomer having a hydrolyzable group is dissolved, or in a dispersion medium in which the particles are dispersed. Then, a monomer having a hydrolyzable group is added to the mixture, and the mixture is dissolved. By this polymerization, a monomer having a hydrolyzable group is polymerized on the surface of the hydrophobic crosslinked polymer particle, and coats the particle.
  • the dispersion medium water or an organic solvent capable of dissolving a monomer having a hydrolyzable group, or a mixture of both is used.
  • a dispersion stabilizer such as carboxymethyl cellulose or polyvinyl alcohol may be used.
  • the polymerization temperature and time vary depending on the type of the monomer having a hydrolyzable group to be reacted and the type of the polymerization initiator, but it is about 0.5 to 40 hours at 40 to 100 hours. is there.
  • the polymer particles having the two-layer structure are prepared.
  • Hydrophobic polymer particles to which the above-mentioned polymerization initiator is adhered are hydrolyzed.
  • a continuous method of reacting a monomer having a hydrolyzable group subsequent to the preparation of the hydrophobically crosslinked polymer particles is also used.
  • Bilayer polymer particles can be prepared. In this method, first, a polymerization reaction for producing the hydrophobic crosslinked polymer particles is started. When the polymerization has progressed to some extent and unreacted monomers remain, the above-mentioned monomer having a hydrolyzable group is added to the reaction system.
  • the polymer particles obtained by each of the above methods are sufficiently washed with hot water, an organic solvent, or the like to remove a suspension stabilizer, a solvent, a residual monomer, or the like contained in or attached to the particles.
  • the obtained polymer particles are treated with an acid catalyst or an alkaline catalyst with, for example, 40 to 100: for 0.5 to 50 hours to perform hydrolysis, whereby hydrolysis existing in the coating layer on the particle surface is performed.
  • the acidic functional group is hydrolyzed to a carboxyl group.
  • the polymer particles are dissolved in a 15 to 25% by weight solution of sodium hydroxide in methanol. By reacting at a temperature of 60 to 80 for 4 to 20 hours, the —C00CH 3 group on the particle surface becomes a lipoxyl group.
  • the polymer particles are collected by filtration, washed with water and dried, If necessary, the particles are classified to obtain a carrier for weak cation exchange chromatography.
  • the average particle size of the polymer obtained by the method of the present invention varies depending on the stirring speed at the time of preparing the hydrophobic crosslinked polymer, the type and amount of the dispersing solvent, the type of the hydrophobic monomer, and the like. um, preferably in the range of 2 to 100 im.
  • the carrier of the present invention does not need to be porous, but if it is porous, its average pore radius is 10,000 A or less, preferably 5,000 A or less.
  • the carrier obtained by the production method is a polymer particle having a two-layer structure in which a hydrophobic crosslinked polymer is used as a skeleton and a surface portion of the hydrophobic crosslinked polymer is coated with a polymer having a carboxyl group.
  • the average thickness of the S-layer to be formed of a polymer having a lipoxyl group is preferably from 10 to 300 A.
  • the thickness of the coating layer is measured in accordance with the “method for measuring the average thickness of the coating layer” described in Examples described later. If the average thickness is less than 10 mm, the coating is incomplete, and the surface of the hydrophobic cross-linked polymer particles is likely to be exposed. If there is such an exposed portion, the substance to be separated (eg, protein) may be non-specifically adsorbed to the carrier. If the average thickness exceeds 300 A, the swelling and shrinkage of the coating layer itself will increase, and during the analysis, the separation ability of the carrier will decrease and the pressure will increase. In addition, the time required for equilibration with the solution becomes longer, and the analysis time becomes longer.
  • the carrier particles obtained by the production method of the present invention are The use of a high-grade polymer results in extremely high mechanical strength and excellent pressure resistance. Furthermore, since there is no hydrophilic group in the skeleton portion of this carrier, the degree of swelling and shrinking is extremely low. Since the surface is coated with a hydrophilic polymer having a carboxyl group, there is no non-specific adsorption of proteins and the like. By selecting a monomer having an appropriate carboxyl group, the degree of hydrophilicity of the particle surface and the ion exchange capacity can be adjusted. Depending on the desired force, a carrier for carbon exchange can be obtained. This carrier can be used in a wide pH range. Furthermore, as described above, the pressure resistance is large and the degree of swelling and shrinking is extremely low, so that the particle size can be reduced, and as a result, separation with high precision is possible. Analysis can be done quickly because it can be used under pressure conditions.
  • the measurement of the mog ⁇ bin in blood glycation is performed.
  • blood of a sample is hemolyzed as necessary. This is applied to a column filled with the above-mentioned carrier, and quantification of saccharified hemoglobin is carried out by a conventional technique of liquid chromatography. By selecting an appropriate buffer, saccharified hemoglobin in the sample and then other hemoglobins are separated and eluted sequentially.
  • the carrier used in the method of the present invention is composed of polymer particles having a two-layer structure in which a hydrophobic cross-linked polymer has a skeleton and a surface portion of the hydrophobic cross-linked polymer is coated with a polymer having a carboxyl group.
  • Bone By using a polymer having a high degree of cross-linking as compared with the upper part, a carrier for liquid chromatography having extremely high mechanical strength and excellent pressure resistance can be obtained. Since there is no hydrophilic group in the skeleton of the carrier, the degree of swelling and shrinking is extremely low. Therefore, in the quantification of glycated hemoglobin, the pressure rise during the passage of the second liquid is extremely small.
  • the separation performance of glycated hemoglobin is excellent, and measurement can be performed in a short time. Furthermore, no non-specific adsorption of proteins is observed.
  • a section having a thickness of about 900 A is obtained using a microtome ULTRACUTE manufactured by Reichert-Jung. This section was labeled using a silver nitrate solution (for volumetric analysis, manufactured by Wako Pure Chemical Industries, Ltd.), and observed and photographed with a transmission electron microscope JEM100S manufactured by HONDA ELECTRONICS CO., LTD. Was evaluated, and the average thickness of the coating layer was measured.
  • the obtained carrier was filled in a stainless steel ram having an inner diameter of 6 mm and a length of 75 mm, and pressure resistance and swelling property in water were examined. Pressure resistance was measured by flowing purified water through a column, changing the flow rate, and measuring the relationship between the flow rate and pressure loss. The swellability was determined from the change in the force-ram pressure when liquids having different ionic strengths were passed.
  • the carboxyl group on the gel surface was quantified using an automatic potentiometric titrator AT-310 manufactured by Kyoto Electronics Industry Co., Ltd.
  • solution A is equivalent to the first solution (solution with weak dissolution power) described in the section of Background Art!
  • the best separation conditions were determined by using a melting zone obtained by further diluting the above solution A in the range of ⁇ to J0. Using these separation conditions, the separation ability was evaluated. Separately, using a liquid chromatography ⁇ -matography system SSLC-20 manufactured by Sekisui Chemical Co., Ltd., proteins of samples containing several kinds of proteins were separated.
  • SSLC-20 liquid chromatography ⁇ -matography system manufactured by Sekisui Chemical Co., Ltd.
  • the pressure resistance and the swelling property were evaluated by the above methods.
  • the pressure loss was proportional to the flow velocity up to 150 kgZcnf.
  • the swellability test no change in column pressure was observed when the eluent was changed from 40 mM phosphate buffer to 200 mM phosphate buffer.
  • the ion exchange capacity of the above carrier was determined by titration to be 0.8 meq / g.
  • the carrier was treated with a silver nitrate solution, and the thickness of the coating layer was measured according to the method described above.
  • the specific surface area was 50 mVg, and the average pore diameter was 40A.
  • Fig. 1 shows the resulting chromatogram.
  • Fig. 1 and Fig. 3 Fig. 5, Fig. 8, Fig. 10, and Fig. 12 described later, 1 is HbA, a and A lb , 2 is fetal Hb (P), 3 is unstable HbA , C , 4 are stable HbA lc , and 5 is HbA. Due to Beak.
  • HbA lc total of stable type and unstable type is calculated by the following equation.
  • peak 6 is myoglobin (derived from pharmacoskeleton)
  • 7 is ⁇ -chymotrypsinogen (derived from citrus kidney)
  • 8 is ribonuclease ⁇ ( It is a beak caused by 9-beam zozyme (derived from nitted egg white).
  • a hydrophobic frame was prepared according to Example 1.
  • Bridge polymer particles were prepared. Further, 190 g of fine polymer particles were obtained by the same operation method as in Example 1 using 50 g of methacrylic acid as a monomer having a carboxyl group. This was classified to obtain a carrier having a particle size of 8 to 10 / m.
  • the pressure loss was proportional to the flow rate up to 150 kg / cm 2 in the pressure resistance.
  • the swelling test when the eluent was changed from a 40 mM phosphate buffer to a 200 mM phosphate buffer, no increase in force ram pressure was observed.
  • the ion-exchange capacity of the suspended work as measured by titration was 0.7 meq / g.
  • the thickness of the coating layer measured by treating the carrier particles with a silver nitrate solution was about 100A.
  • the specific surface area was 30 m 2 , and the average pore radius was 20A.
  • human blood was analyzed using Hi-AUTO A, c manufactured by Kyoto Daiichi Kagaku.
  • Figure 3 shows the resulting chromatogram. There were no changes in the chromatograms and measured values after 3,000 repetitions of the measurement using the same samples.
  • the protein was further analyzed using a liquid chromatograph SSLC-20 manufactured by Sekisui Chemical Co., Ltd.
  • Figure 4 shows the resulting chromatogram.
  • Example 1 The reaction mixture was heated again to 80 t and polymerized for 1 hour. The product was washed successively with hot water and acetone and dried to obtain 450 g of fine polymer particles. Classification was performed in the same manner as in Example 1, and particles having a particle size of 6 to 9 ⁇ m were collected to obtain a carrier. This was evaluated in the same manner as in Example 1.
  • the pressure loss was proportional to the flow velocity up to 600 kg / cm 2 .
  • the ion exchange capacity of the carrier was determined by titration to be 0.05 meq / g.
  • the thickness of the coating layer measured by treating the carrier with a silver nitrate solution was about 90 persons.
  • the specific surface area was 0.5 m 2 / g, and no pores were present.
  • Comparative Example 1 Dissolve 100 g of styrene, 200 g of divinylpentene, 150 g of acrylic acid, and penzyl peroxyside lg in 270 g of toluene, add to 4% aqueous solution of polyvinyl alcohol 2.5 and stir to homogenize the oil phase. After that, the mixture was heated at 80 to carry out suspension polymerization.
  • Example 2 After polymerizing for 8 hours, the product was subjected to the same operation as in Example 1 to obtain 420 g of fine polymer particles. This was classified to obtain a carrier having a particle size of 6 to 9 / .m.
  • Example 2 when 300 g of acrylic acid was used as a monomer having a carboxylic acid group, polymerization was carried out in the same manner as in Example 1. Aggregation occurred during the reaction, and 180 g of commercially available fine polymer particles were obtained. When the polymer particles were classified as Example 1 and di-distilled, and it was attempted to raise the power to 7 ⁇ m, the particles could not be filled due to the poor quality of the particles.
  • the ion exchange capacity of the carrier particles was determined by titration to be 1.2 meq / g.
  • the specific surface area was 0.6 m 2 / g, and no pores were present. When the thickness of the coating layer of the carrier particles was measured by treating with a silver nitrate solution, it was about 400 A.
  • a hydrophobic apple polymer was prepared in the same manner as in Example 1. Further, 10 g of acrylic acid was used as a monomer having a sulfoxyl group, By operating in the same manner as in Example 1, 180 g of fine polymer particles were obtained. This was separated to obtain a carrier having a particle size of 6 to 9 ⁇ . Next, a review of the body was performed.
  • a liquid chromatograph SSLC-20 manufactured by Jishui Chemical Industry Co., Ltd. proteins containing several types of proteins were separated.
  • Fig. 7 shows the resulting mouthmatogram. The elution order is different from that in Fig. 2 because of the hydrophobic interaction between the uncoated layer and the protein.
  • Example 1 The impregnated hydrophobic cross-linked polymer was suspended in 2 g of a 20% sodium chloride aqueous solution containing 5 g of polyvinyl alcohol, and the polymerization reaction was carried out at 80 at 80 for 5 hours after purging with nitrogen. The product was washed successively with hot water and acetone and dried to obtain 460 g of fine polymer particles. Classification was performed in the same manner as in Example 1 to collect particles having a particle size of 6 to 9 xm, to obtain a carrier. This was evaluated in the same manner as in Example 1.
  • the pressure loss was proportional to the flow velocity up to 400 kg / m 2 .
  • the force ram pressure increased by 15 kg / cm 2 .
  • the ion exchange capacity measured by titration was 0.2 meq / g.
  • the specific surface area is ⁇ . ⁇ 2 / ⁇ , there are no pores and ⁇ ⁇ ⁇
  • Example 1 a hydrophobic crosslinked polymer was prepared. 200 g of the hydrophobic crosslinked polymer particles are immersed in 1 lb of acetone in which 0.5 g of acetyl peroxide (polymerization initiator) is dissolved, and the polymerization initiator is attached. Was. Next, the acetate was distilled off under reduced pressure at 20 :. Disperse the above-mentioned hydrophobic crosslinked polymer in 2 £ of 50% methanol aqueous solution, add 50 g of methyl acrylate (monomer having a hydrolyzable group) with stirring, and add nitrogen. After the replacement, the polymerization reaction was performed at 70 t for 5 hours.
  • acetyl peroxide polymerization initiator
  • the product was very washed with hot water and acetone and dried to obtain 180 g of fine polymer particles.
  • 150 g of the obtained polymer particles of small size are added to 500 ml of a 20% by weight methanol solution of sodium hydroxide, and 5 By heating for a period of time, the ester portion of the resin caused by polymethyl acrylate was hydrolyzed. After cooling the reaction mixture to room temperature, the polymer particles were collected, washed several times and dried.
  • the obtained polymer particles are classified by an air classifier Turbo Classifier TC-15N manufactured by Nisshin Engineering Co., Ltd. to collect particles having a particle size of 8 to 10 ⁇ m.
  • a 75 mm stainless steel production ram was filled. Charging was performed by charging 2 g of carrier particles into purified water 35; ⁇ , stirring for 5 minutes, and then charging at a constant flow rate of 2.OmZ.
  • This carrier was evaluated by the method of Example 1.
  • the pressure loss was proportional to the flow velocity up to 150 kg / cm 2 .
  • the ion exchange capacity of the carrier was determined by titration to be 0.7 meq / g.
  • the carrier was treated with the silver nitrate solution according to the method described above, and the average thickness of the coating layer was measured.
  • the specific surface area was 50 m 2 / g, and the average pore radius was 30 A. ⁇ Human blood was analyzed using Hi-AUTO A IC manufactured by Kyoto Daiichi Kagaku. The resulting chromatogram is shown in FIG.
  • a hydrophobic crosslinked polymer was prepared. 200 g of the hydrophobic cross-linked polymer particles were immersed in 1 lb of acetone in which 0.5 g of acetyl chloride (polymerization initiator) was dissolved, and the polymerization initiator was attached. Next, the acetate was distilled off under reduced pressure at 20 :. The immersion-treated aqueous polymer was suspended in 2% of a 50% aqueous methanol solution, and 50 g of atalonitrile (monomer having a hydrolyzable group) was added with stirring. After the replacement, the polymerization reaction was performed at 70 t: for 10 hours.
  • Example 5 The product was washed successively with hot water and acetone, and dried to obtain 180 g of fine polymer particles.
  • the polymer particles were hydrolyzed in the same manner as in Example 5.
  • the obtained polymer particles were classified to obtain a carrier having a particle size of 8 to 10 ⁇ m. This carrier was evaluated in the same manner as in Example 1.
  • the pressure loss was proportional to the flow velocity up to 150 kg / cm 2 for pressure resistance.
  • the ion exchange capacity of the carrier measured by titration was 0.8 meq / g.
  • the carrier was treated with a silver nitrate solution and the average thickness of the coating layer was measured to be about 70 A.
  • the specific surface area was 50 m 2 / g, and the average pore radius was 30 A.
  • Human blood was divided in the same manner as in Example 1. As a result The resulting chromatogram is shown in FIG. There were no changes in the chromatograms and measured values after 3,000 repetitions of the measurement. Next, a sample containing several kinds of proteins was separated in the same manner as in Example 5. The obtained chromatogram is shown in FIG.
  • styrene 100 g of styrene, 200 g of divinylbenzene and benzoyl peroxide were used in a continuous method, in which a monomer having a hydrolyzable group was reacted following the preparation of the aqueous crosslinked polymer particles.
  • lg was dissolved in 200 g of toluene. This was added to a 4% aqueous polyvinyl alcohol solution 2.5, and the mixture was stirred to disperse the oil phase uniformly. Then, the mixture was heated to 80 t under a nitrogen atmosphere to carry out suspension polymerization.
  • acrylamide (a monomer having a hydrolyzable group) was added to the above system, and polymerization was further performed at 80: 2 for 2 hours.
  • the product was washed successively with hot water and acetone, and dried to obtain 260 g of fine polymer particles.
  • 200 g of the polymer particles were added to 500 ml of a 20-ring: sodium hydroxide solution of sodium hydroxide and heated at 75 for 10 hours. After the reaction mixture was cooled to room temperature, it was washed several times with water and dried.
  • the obtained polymer particles were classified in the same manner as in Example 1 to obtain a carrier having a particle size of 8 to 10 ⁇ m. This was evaluated in the same manner as in Example 1.
  • the obtained polymer particles were subjected to a hydrolysis treatment in the same manner as in Example 5 and classified to obtain a carrier of 8 to 10 / m. This was evaluated in the same manner as in Example 1. As a result, with respect to pressure resistance, pressure loss and flow velocity were proportional to 150 kg / cm 2 . In the swelling test, no increase in pressure was observed even when the solution was changed from a 40 ⁇ phosphate buffer to a 200 mM phosphate buffer.
  • the ion exchange capacity of the carrier was determined by titration to be 0.6 (n qe / g.
  • the specific surface area was 60 m 2 / g-the average pore radius was 40 A.
  • the carrier was treated with a silver nitrate solution.
  • the average thickness of the coating layer measured by the above method was about 100 A.
  • the analysis of human blood was performed in the same manner as in Example 1, and various proteins were analyzed in the same manner as in Example 5. Separation of the sample containing the sample was performed, and the results obtained were similar to those shown in Fig. 8 and Fig. 9. After the human blood analysis was repeated 3,000 times, the cuticle was obtained. There were no changes in tomograms and measurements.
  • Example 1 100 g of styrene, 200 g of divinylbenzene, 70 g of methyl acrylate (single amount having a hydrolyzable group) and 200 g of acetylpropyl peroxide were dissolved in 200 g of toluene. This was added to 2.5% of a 4% aqueous polyvinyl alcohol solution and stirred to uniformly disperse the oil phase. The mixture was heated to 70 t and subjected to suspension polymerization. After polymerization for 8 hours, 340 g of fine polymer particles were obtained. The product was subjected to a hydrolysis treatment in the same manner as in Example 5. Classification was performed in the same manner as in Example 1 to obtain carrier particles having a particle size of 6 to 9 m. This was evaluated in the same manner as in Example 1.
  • the pressure loss was proportional to the flow rate up to 80 kg / cm 2 .
  • the eluent should be 40 mM phosphate buffer.
  • the force ram pressure increased by 20 kg / cm 2 .
  • the ion exchange capacity of the carrier particles measured by titration was 1.0 meq / g.
  • the specific surface area was 60 m 2 / g, and the average pore radius was 30.
  • Fig. 12 shows the results. Since this carrier has weak retention, the solution was diluted and the separation conditions were tested. As a result, the optimal pattern was obtained when the solution A was diluted 1.43 times and used. It can be seen that this carrier is clearly inferior in separation ability when Fig. 12 is compared with Fig. 8. The chromatogram after 3000 repeated human blood analyzes showed that the separation ability was further improved. It was found that the measured values of HbA and c were also reduced to 60% of that of the initial planting.Next, the samples containing several types of proteins were separated in the same manner as in Example 5. The result is shown in Fig. 13. Fig. 13 shows the result.
  • hydrophobic crosslinked polymer particles were prepared. Using 300 g of methyl acrylate as a monomer having a hydrolyzable group, polymerization was carried out in the same manner as in Example 5, and the hydrophobic polymer particles were coated to obtain 180 g of fine polymer particles. Obtained. The product It was subjected to a hydrolysis treatment in the same manner as in Example 5. Separation was performed in the same manner as in Example 1 to obtain carrier particles having a particle size of 6 to 9 m. This was evaluated in the same manner as in Example 1.
  • the pressure drop was proportional to the pressure loss up to 150 kg / cm 2 .
  • the column pressure was increased by 25 kg / cm 2 when the eluent was changed from 40 mM phosphate buffer to 200 mM phosphate buffer. This is considered to be due to the poor swelling resistance of the carrier particles of the wood comparative example because the coating layer having a carboxyl group is thick.
  • the ion exchange capacity of the carrier was measured by titration and was found to be 1.2 meq / g.
  • the carrier particles were treated with a silver nitrate solution, and the average thickness of the covered layer was about 400 A.
  • the specific surface area was 1.5ni 2 / g.
  • the coating layer was thick and had few pores.
  • Example 5 As in Example 5, a sample containing several types of proteins was separated. The resulting chromatogram is shown in FIG. From Fig. 14, it can be seen that the carrier of this comparative example has a weaker holding force than the carriers of Examples 5 and 6 (Fig. 9 and Fig. 11). This is considered to be due to the fact that the surface area was small and the surface area was small.
  • hydrophobic crosslinked polymer particles were prepared. Using 10 g of methyl acrylate as a monomer having a hydrolyzable group, polymerization was carried out in the same manner as in Example 5, and the hydrophobic polymer particles were coated to obtain 180 g of fine polymer particles. Obtained. The product It was subjected to a hydrolysis treatment in the same manner as in Example 5. Classification was performed in the same manner as in Example 1 to obtain carrier particles having a particle size of 6 to 9 jam. This was evaluated in the same manner as in Example 1.
  • the ion exchange capacity of the carrier particles was measured by titration and was found to be 0.1 lmeq / g.
  • the specific surface area was 70 m 2 / g, and the average pore radius was 60 A.
  • the carrier particles were treated with a silver nitrate solution, and the average thickness of the coating layer was measured to be about 8 A. An uncoated portion was present on a part of the carrier particle surface.
  • Example 5 As in Example 5, a sample containing several types of proteins was separated. The resulting chromatogram is shown in FIG. The elution order in the chromatogram in Fig. 15 is different from that in Figs. 9 and 11, but this is due to insufficient interaction of the hydrophobic cross-linked polymer particles and hydrophobic interaction. it is conceivable that.
  • a carrier for cation exchange liquid chromatography which has excellent pressure resistance, has little swelling and shrinkage, and has no nonspecific adsorption of proteins can be obtained.
  • Such a carrier can be widely used for singulation or analysis of various hydrophilic substances.
  • Such carriers are particularly useful for determining glycated hemoglobin. It is effective for the amount of saccharified hemoglobin with high accuracy and in a short time. Diagnosis of diabetes can be made quickly and accurately by measuring hemoglobin to glycation in blood 0

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)

Abstract

Procédé de préparation d'un porteur adapté à la chromatographie liquide à échange de cations. Le procédé consiste à prendre une particule hydrophobe à laquelle adhère un initiateur de polymérisation, à ajouter à une dispersion aqueuse de la particule polymère réticulée et hydrophobe un monomère polymérisable possédant un groupe carboxyle, et à polymériser le monomère à la surface de la particule polymère. Ainsi, on obtient une particule polymère enrobée comportant une particule polymère réticulée et hydrophobe et, formée à la surface de celle-ci, une couche d'un polymère possédant un groupe carboxyle.
PCT/JP1990/001522 1982-07-20 1990-11-21 Procede de preparation d'un porteur pour la chromatographie liquide a echange de cations, et procede de determination quantitative de l'hemoglobine saccharifiee a l'aide dudit porteur WO1992009889A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
DE69022250T DE69022250D1 (de) 1990-11-21 1990-11-21 Verfahren zur herstellung von trägern für die kationenaustausch-flüssigchromatographie sowie verfahren zur quantitativen bestimmung von glucose-hämoglobin unter benutzung dieses trägers.
CA002074177A CA2074177C (fr) 1990-11-21 1990-11-21 Methode de production d'un support pour la chromatographie liquide par echange de cations et methode de determination de l'hemoglobine glycosylee a l'aide de ce support
AT91900342T ATE127586T1 (de) 1990-11-21 1990-11-21 Verfahren zur herstellung von trägern für die kationenaustausch-flüssigchromatographie sowie verfahren zur quantitativen bestimmung von glucose-hämoglobin unter benutzung dieses trägers.
US07/915,685 US5292818A (en) 1982-07-20 1990-11-21 Method for producing a carrier for cation exchange liquid chromatography and a method for determining glycosylated hemoglobins using the carrier
PCT/JP1990/001522 WO1992009889A1 (fr) 1990-11-21 1990-11-21 Procede de preparation d'un porteur pour la chromatographie liquide a echange de cations, et procede de determination quantitative de l'hemoglobine saccharifiee a l'aide dudit porteur
KR1019920701714A KR920704135A (ko) 1990-11-21 1990-11-21 카치온 교환액체 크로마토그라피용 담체의 제조방법 및 그 담체를 사용한 당화 헤모글로빈의 정량방법
EP91900342A EP0596106B1 (fr) 1990-11-21 1990-11-21 Procede de preparation d'un porteur pour la chromatographie liquide a echange de cations, et procede de determination quantitative de l'hemoglobine glucosique a l'aide dudit porteur

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CA002074177A CA2074177C (fr) 1990-11-21 1990-11-21 Methode de production d'un support pour la chromatographie liquide par echange de cations et methode de determination de l'hemoglobine glycosylee a l'aide de ce support
PCT/JP1990/001522 WO1992009889A1 (fr) 1990-11-21 1990-11-21 Procede de preparation d'un porteur pour la chromatographie liquide a echange de cations, et procede de determination quantitative de l'hemoglobine saccharifiee a l'aide dudit porteur

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01165955A (ja) * 1987-12-22 1989-06-29 Sekisui Chem Co Ltd 糖化ヘモグロビン測定用溶離液
JPH01314962A (ja) * 1988-06-15 1989-12-20 Kao Corp 液体クロマトグラフィー用充填剤
JPH0285758A (ja) * 1988-06-07 1990-03-27 Rohm & Haas Co イオンクロマトグラフィー用のイオン交換性複合組成物

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01165955A (ja) * 1987-12-22 1989-06-29 Sekisui Chem Co Ltd 糖化ヘモグロビン測定用溶離液
JPH0285758A (ja) * 1988-06-07 1990-03-27 Rohm & Haas Co イオンクロマトグラフィー用のイオン交換性複合組成物
JPH01314962A (ja) * 1988-06-15 1989-12-20 Kao Corp 液体クロマトグラフィー用充填剤

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