WO2019004440A1 - 液体クロマトグラフィー用充填剤及び液体クロマトグラフィー用カラム - Google Patents
液体クロマトグラフィー用充填剤及び液体クロマトグラフィー用カラム Download PDFInfo
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- 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
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
- B01J20/28011—Other properties, e.g. density, crush strength
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- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
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- 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
- B01J20/288—Polar phases
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- 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
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- 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
- B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
- B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
- B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
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- 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
- B01J20/3244—Non-macromolecular compounds
- B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
- B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
- B01J20/3251—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising at least two different types of heteroatoms selected from nitrogen, oxygen or sulphur
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- 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
- B01J20/3291—Characterised by the shape of the carrier, the coating or the obtained coated product
- B01J20/3293—Coatings on a core, the core being particle or fiber shaped, e.g. encapsulated particles, coated fibers
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- 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
- B01J39/08—Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
- B01J39/16—Organic material
- B01J39/18—Macromolecular compounds
- B01J39/20—Macromolecular compounds obtained by reactions only involving unsaturated carbon-to-carbon bonds
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- 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
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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
- B01J47/00—Ion-exchange processes in general; Apparatus therefor
- B01J47/014—Ion-exchange processes in general; Apparatus therefor in which the adsorbent properties of the ion-exchanger are involved, e.g. recovery of proteins or other high-molecular compounds
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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
- B01J47/00—Ion-exchange processes in general; Apparatus therefor
- B01J47/02—Column or bed processes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
- 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
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
- B01J2220/58—Use in a single column
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
Definitions
- the present invention relates to a packing for liquid chromatography having a sulfo group and a column for liquid chromatography packed with the packing.
- Liquid chromatography (hereinafter also referred to as "HPLC") is used in various fields as a wide range of sample analysis methods. Among them, cation exchange chromatography using a filler having a sulfo group is used for analysis of proteins, amino acids and the like.
- nonspecific adsorption of the proteins to the packing may occur. If nonspecific adsorption of protein occurs, not only accurate analysis results can not be obtained, but also a problem of accelerating deterioration of the column arises, so it is necessary to use a packing material which does not cause this nonspecific adsorption.
- Nonspecific adsorption of proteins is mainly due to the hydrophobic interaction between the packing agent and the protein. For this reason, as a means to control this, particles with high hydrophilicity are used for the filler.
- particles using a (meth) acrylate polymer as a filler are widely known.
- Patent Document 1 discloses a method of analyzing glucohemoglobin using an ion exchanger in which a sulfo group is introduced to particles made of a methacrylic acid ester-based polymer.
- Patent Document 2 discloses a filler obtained by forming a hydrophilic polymer layer such as methacrylic acid on the surface of a divinylbenzene hydrophobic particle.
- a layer of methacrylic acid ester is formed on the surface of the core of a polymer of divinylbenzene generated by a polymerization reaction in acetonitrile, particles are obtained, and epichlorohydrin is reacted with the particles.
- an example of analysis of protein using a filler having a sulfo group introduced therein is disclosed.
- an object of the present invention is to provide a packing material for liquid chromatography and a column for liquid chromatography that can be suitably used for analysis of proteins, amino acids and the like and can be efficiently produced.
- a filler for liquid chromatography comprising particles of a copolymer having a monomer unit derived from (meth) acrylic acid ester and a monomer unit derived from divinylbenzene, wherein the monomer unit derived from (meth) acrylic acid ester 70 to 90% by mass: 30 to 10% by mass of the monomer unit derived from the divinylbenzene, a structure in which a sulfo group is bonded to the particle surface, and a sulfo group is bonded, the formula (1) (Wherein, R represents a partial structure derived from the surface of the copolymer particle, X represents an oxygen atom or a sulfur atom, and n represents an integer of 2 to 4).
- a filler for liquid chromatography which comprises 40 ⁇ mol / g to 300 ⁇ mol / g of sulfo groups in particles.
- a method for producing the filler as described in 1 or 2 above which comprises copolymerizing a monomer mixture containing (meth) acrylate and divinylbenzene by suspension polymerization to obtain copolymer particles, And a step of reacting with a sulfonating agent in a solvent containing a secondary alcohol in the presence of a base and a method for producing a filler.
- the packing agent for liquid chromatography and the column for liquid chromatography in one embodiment of the present invention can be suitably used for analysis of proteins, amino acids and the like.
- the filler is based on an acrylic polymer
- the strength can be increased while the hydrophilicity is kept high, so that the particle size can be reduced, whereby the analysis ability is improved.
- the packing and the column can be produced efficiently.
- FIG. 7 is a chromatogram obtained by Analysis Example 1.
- 7 is a chromatogram obtained by Analysis Example 2.
- 7 is a chromatogram obtained by Analysis Example 3.
- 7 is a chromatogram obtained by Analysis Example 4.
- FIG. 6 is a SEM photograph (20,000 ⁇ magnification) of the filler particles obtained in Example 1.
- the filler for liquid chromatography in one embodiment of the present invention uses, as a base material, particles of a copolymer including a monomer unit derived from (meth) acrylic acid ester and a monomer unit derived from divinylbenzene.
- (meth) acrylic acid ester means acrylic acid ester or methacrylic acid ester.
- the copolymer in one embodiment of the present invention uses divinylbenzene as a crosslinkable monomer.
- divinylbenzene as the crosslinkable monomer
- the resulting copolymer has high mechanical strength. Therefore, even if the particle size of this copolymer is reduced, the strength is high, and even when this particle is used as a filler for a column, it withstands an increase in pressure during analysis associated with the reduction of the particle size. It is possible. This can enhance the separation performance of the column.
- Divinylbenzene as a monomer may be a mixture of meta and para isomers.
- (meth) acrylic acid ester which is a monomer of a copolymer
- methyl (meth) acrylate, ethyl (meth) acrylate, 2-hydroxyethyl (meth) acrylate, 4-hydroxybutyl (meth) acrylate, Glycerin di (meth) acrylate, glycidyl (meth) acrylate, etc. are mentioned, These can be used individually or in combination of 2 or more types.
- the (meth) acrylic acid ester has a hydroxyl group or a glycidyl group at least a certain amount. Although the amount can not be generally said, 70 mass% or more of the (meth) acrylic acid ester whole is preferable, for example.
- the (meth) acrylic acid ester having a hydroxyl group or glycidyl group include 2-hydroxyethyl (meth) acrylate, glycerin di (meth) acrylate, 4-hydroxybutyl (meth) acrylate, glycidyl (meth) acrylate and the like.
- glycidyl (meth) acrylate is more preferable because it can be suitably used for introducing a sulfo group structure and can be converted to a structure having a hydroxyl group by a ring opening reaction.
- Glycidyl methacrylate is most preferred because of easy availability.
- the (meth) acrylic acid ester highly hydrophobic ethylene glycol di (meth) acrylate, butyl (meth) acrylate and hexyl (meth) acrylate can be used in combination.
- the amount thereof used may be in a range that does not significantly affect the hydrophilicity of the copolymer, and specifically, it is 10% by mass or less with respect to the total amount of (meth) acrylic acid ester.
- the ratio of the (meth) acrylic acid ester-derived monomer unit to the divinylbenzene-derived monomer unit is (meth) acrylic acid ester-derived monomer units (when plural types are used) (Total amount of them): 70 to 90% by mass: 30 to 10% by mass of the monomer unit derived from the divinylbenzene, and more preferably 75 to 85% by mass: 25 to 15% by mass.
- the monomer unit derived from (meth) acrylic acid ester is less than 70% by mass, the hydrophobicity of the copolymer becomes high, and nonspecific adsorption of proteins is likely to occur. Moreover, when introduce
- the monomer unit derived from (meth) acrylate exceeds 90% by mass, the strength of the copolymer particles decreases, and when it is used as a column filler, the particles are deformed and the column is deteriorated. It is easy to invite.
- the polymer may contain other monomer units in addition to the monomer unit derived from (meth) acrylic acid ester and the monomer unit derived from divinylbenzene.
- the amount of other monomer units may be in a range that does not significantly affect the hydrophilicity of the copolymer, and is, for example, an amount of 10% by mass or less of the copolymer. The amount is preferably 5% by mass or less, more preferably 3% by mass or less.
- monomers of other monomer units include styrene, ethylstyrene, methylstyrene, propylstyrene, methoxystyrene, ⁇ -methylstyrene, butadiene, vinyl acetate and the like.
- the particle size of the copolymer is not particularly limited as long as it can be used as a column packing agent for liquid chromatography, but considering the packing and separation performance into the column, the average particle diameter is 1 ⁇ m to 10 ⁇ m. Is preferred. When the average particle size is 10 ⁇ m or less, the separation ability of the compound is high, and good analysis results can be obtained. On the other hand, if it is less than 1 ⁇ m, the pressure of the column becomes high, and the packing tends to be difficult.
- the average particle diameter of the particles is more preferably 2 ⁇ m to 7 ⁇ m, and most preferably 2 ⁇ m to 3 ⁇ m, in order to obtain a column with high separation capacity.
- the average particle size in the present specification is a volume average particle size and can be measured as follows. First, the particles are imaged so as to be 2000 or more by the particle size distribution measuring apparatus, and from the obtained two-dimensional particle image (still image is preferable), the equivalent circle diameter of each particle (the same area as the projected area of the particle image) Get the diameter of the circle with. It is a particle diameter obtained by calculating the volume of each particle from the equivalent circle diameter and averaging based on the calculated volume. At this time, each particle is regarded as a sphere having the same diameter as the equivalent circle diameter described above. As a particle size distribution measuring apparatus, FPIA-3000 (made by Sysmex Corporation) etc. can be used.
- the particles of the copolymer have few fine pores on the surface.
- the total pore volume is 0.05 cm 3 / g or less with respect to the mass of the particles measured by a gas adsorption method, and more preferably less 0.02 cm 3 / g. Particles with few fine pores have improved strength.
- HPLC analysis is performed using a filler based on the particles, compounds such as proteins contained in the sample do not diffuse in the pores, which enables rapid analysis.
- the total pore volume can be measured using Cantachrome Autosorb® iQ as an apparatus.
- the filler for liquid chromatography in one embodiment of the present invention is obtained by obtaining particles of a copolymer having a monomer unit derived from (meth) acrylic acid ester and a monomer unit derived from divinylbenzene, and then forming a sulfo group on the particle surface. It can be manufactured by introducing it.
- a well-known oil-soluble radical polymerization initiator can be used for the said copolymerization.
- the polymerization initiator include 2,2′-azobisisobutyronitrile (hereinafter also referred to as AIBN), 2,2′-azobis (2,4-dimethylvaleronitrile) (hereinafter also referred to as V-65), 2 And 2,2'-azobisisobutyrate dimethyl, 2,2'-azobis (2-methylbutyronitrile), benzoyl peroxide, lauroyl peroxide and the like.
- the use amount of the polymerization initiator may be appropriately determined depending on the kind and amount of the monomer, but is usually 0.1 to 5 parts by mass with respect to 100 parts by mass of the total amount of the monomers.
- a suspension polymerization method for example, a suspension polymerization method, an emulsion polymerization method, a precipitation polymerization method and the like can be used.
- a suitable organic solvent in which the monomer is soluble but not the polymer particles of uniform particle size can be obtained.
- aggregation of particles may occur during polymerization, and it may be difficult to synthesize monodisperse particles with high yield.
- the suspension polymerization method or the emulsion polymerization method is preferable, and the suspension polymerization method is more preferable in that aggregation of particles is less likely to occur.
- SPG membrane suspension polymerization method using a Shirasu porous glass (SPG) membrane with controlled pore size and an internal pressure micro kit MN-20 manufactured by SGP Techno Co., Ltd.
- microchannel suspension A microchannel suspension polymerization method using an apparatus manufactured by EPTEC Co., Ltd. is more preferable because particles having uniform particle diameter can be obtained.
- the polymerization reaction can be carried out, for example, by heating at 60 to 80 ° C. for 6 to 20 hours.
- classification is carried out using a sieve or classifier (for example, a semi-free-vortex classifier, Aerofine Classifier AC, manufactured by Nisshin Engineering Co., Ltd.) to obtain an average particle size, uniformity, and fine particle content.
- a sieve or classifier for example, a semi-free-vortex classifier, Aerofine Classifier AC, manufactured by Nisshin Engineering Co., Ltd.
- the amount etc can be adjusted.
- a sulfonating agent can be used.
- a sulfonating agent sodium 2-hydroxyethane sulfonate, sodium 3-hydroxypropane sulfonate, sodium 2-mercaptoethane sulfonate, sodium 3-mercaptopropane sulfonate, sodium 2-bromopropane sulfonate, 3-bromopropane Sulfonates such as sodium sulfonate, 1,3-propane sultone, sultone compounds such as 1,3-butane sultone, 1,4-butane sultone, 2,4-butane sultone and the like can be mentioned.
- sodium 3-mercaptopropanone sulfonate and 1,3-propane sultone are preferable in view of easiness of reaction, and 1,3-propane sultone
- sulfonating agents are bonded as sulfo groups utilizing a reaction with reactive functional groups present on the surface of the copolymer particles.
- a reactive functional group a hydroxyl group or an epoxy group is preferable, and an epoxy group is more preferable.
- the sulfonation agent directly reacts with the reactive functional group derived from the copolymer present on the particle surface, and also causes the reactive functional group to react with a spacer, or the epoxy is used when the reactive functional group is an epoxy group. It is also possible to react with a sulfonating agent after ring-opening reaction of the group. Preferably, the reaction is carried out directly, and the separation performance of the column using the packing obtained thereby is improved, and the production efficiency is also excellent.
- direct sulfonation of the epoxy group is more effective for separation of the desired protein than sulfonation after the ring opening reaction of the epoxy group present on the surface of the copolymer particle. Will be better.
- the reaction of the copolymer particles with the sulfonating agent can be carried out in a suitable manner depending on the type of sulfonating agent.
- a base when introducing a sulfo group into a monomer unit derived from glycidyl methacrylate using a sultone compound as a sulfo agent, a base can be used.
- inorganic bases such as sodium hydroxide, potassium hydroxide and lithium hydroxide, amines such as triethylamine and N, N-diisopropylethylamine, sodium methoxide, potassium methoxide, potassium t-butoxide, 1,8 And organic bases such as -diazabicyclo [5.4.0] -7-undecene.
- sodium hydroxide, potassium hydroxide and potassium t-butoxide are preferable, and sodium hydroxide and potassium hydroxide are more preferable.
- the inorganic base is used in aqueous solution of appropriate concentration.
- a sulfo group using a sulfonate as a sulfo agent it can carry out using a base similarly.
- a sulfonate having a halogen group a sulfo group is introduced by a halogen substitution reaction with a hydroxyl group generated on the copolymer particles during the reaction, and when a sulfonate having a hydroxyl group or a mercapto group is used, copolymer particles
- the sulfo group is introduced by a nucleophilic reaction involving the ring opening of the above epoxy group.
- Examples of the solvent used for the sulfonation reaction include water, dimethylsulfoxide, dimethylacetamide, dimethylformamide, N-methylpyrrolidone, alcohol and the like.
- alcohols are preferred, and secondary alcohols are more preferred from the viewpoint of reaction efficiency and reactivity.
- 2-propanol, 2-butanol, 3-methyl-2-butanol, 2-pentanol, 3-pentanol and the like can be mentioned, and from the economical viewpoint, 2-propanol is preferable.
- secondary alcohols are particularly preferable from the viewpoint of reaction efficiency.
- the reactivity is not sufficient.
- a Lewis acid when used, the hydrophilicity of the obtained filler is low and it does not disperse in an aqueous solution, so that it can not be packed in a column and does not exhibit the function as a filler for protein separation.
- the amount of sulfo group bound to the copolymer particles is preferably 20 ⁇ mol / g to 300 ⁇ mol / g, more preferably 40 ⁇ mol / g to 300 ⁇ mol / g. If the amount of sulfo group to be bound is less than 40 ⁇ mol / g, the retention time in liquid chromatography analysis such as HPLC becomes short, and good separation can not be performed.
- the water dispersibility of a filler is favorable in it being 40 micromol / g or more, and it can obtain a favorable column for analysis of protein.
- the amount of sulfo group to be bound exceeds 300 ⁇ mol / g, the retention of the sample becomes too strong, and the analysis time tends to be long, which is not preferable.
- the amount of sulfo group can be measured by the following method. That is, 10 mL of 0.5 M hydrochloric acid is added to 1 g of particles (sample) dried under vacuum, dispersed, filtered, and washed with water. As a result, the sulfo group is in an acid form to obtain particles in a washed state. Next, 10 mL of 0.5 M sodium hydroxide is added to the particles, which is filtered with a container for collecting the filtrate, and then the particles are washed with 10 mL water. The filtrate and washings mixture is titrated with 0.1 M hydrochloric acid. The number of moles of hydrochloric acid required at this time is determined, and the value obtained by subtracting the number of moles of sodium hydroxide (5 mM in the above case) is taken as the amount of sulfo group.
- This structure is copolymerized using, for example, glycidyl (meth) acrylate as at least one of (meth) acrylic acid esters, and then the epoxy group is opened by a sulfo agent and bound as a sulfo group, or an epoxy group is It is formed by ring opening to a diol or monool monothiol followed by coupling of a sulfo group with a sulfo agent.
- copolymerization may be performed using a diol structure-containing ester or a monool monothiol-containing ester as at least one of (meth) acrylic acid esters, and then forming a sulfo group by bonding with a sulfo group.
- the surface of the particle means a portion where solid copolymer particles can contact with the reagent in the reaction solvent, and usually does not include the solid interior.
- a sulfo group may be bound inside the individual.
- the packing material for liquid chromatography in one embodiment of the present invention is one obtained by bonding a specific structure having a sulfo group to the surface of the above-mentioned copolymer particle.
- the liquid chromatography column in one embodiment of the present invention comprises the above-mentioned packing material.
- the column is obtained by packing the above-mentioned filler into a column by a known packing method such as a slurry method.
- the packing column can be made of PEEK or SUS as the housing material, but if it is made of SUS, there is a possibility that proteins will be adsorbed, so if analyzing a sample containing protein, PEEK is preferable .
- a frit is provided in the column for preventing the flow of the filler particles, it is preferable to use a frit made of PEEK to suppress adsorption.
- a buffer such as phosphoric acid can be preferably used as the eluent.
- the average particle size measured in Examples and Comparative Examples is a volume average particle size, and was measured by the method described in the section of the embodiment of the present invention.
- glycidyl methacrylate made by NOF Corporation
- divinylbenzene made by Nippon Steel Sumikin Chemical Co., Ltd., purity 99%
- the obtained mixture was made into oil droplets by a microchannel suspension apparatus (manufactured by EPTEC Co., Ltd.) using 500 g of a 1% aqueous solution of sodium alkyldiphenylether sulfonate as an aqueous phase, and polymerization reaction was further performed to obtain copolymer particles. .
- the average particle size of the obtained particles was 2.6 ⁇ m.
- 1.0 g of the filler particle 1 dried in vacuum was dispersed by adding 10 mL of 0.5 M hydrochloric acid, and after filtration, the dispersion was washed with water to obtain particles in which the sulfo group was in an acid form.
- To the particles was added 10 mL of 0.5 M sodium hydroxide and filtered with the vessel in place to collect the filtrate, then the particles were washed with 10 mL water.
- the mixed solution of the filtrate and the washing solution collected in a container installed was titrated with 0.1 M hydrochloric acid.
- the amount of sulfo group was determined from the difference between the number of moles of sodium hydroxide (5 mM) used initially and the number of moles of hydrochloric acid required for the titration. As a result, the amount of sulfo groups based on the mass of the particles was 200 ⁇ mol / g.
- the filler particles 1 were observed by SEM. No pores were identified. The observation results are shown in FIG. As a result of pore measurement by gas adsorption (Autosorb (registered trademark) iQ manufactured by Cantachrome Co., Ltd. was used as an apparatus used), the pore volume was a very small value of 0.01 cm 3 / g.
- the obtained filler particles 1 were packed in a PEEK column with an inner diameter of 4.6 mm and a length of 10 mm by a slurry method to obtain a column 1.
- Example 1 The column 1 manufactured in Example 1 was attached to a liquid chromatograph (Agilent (registered trademark) 1260 Infinity, manufactured by Agilent Technologies, Inc.), and a glycated hemoglobin control sample (manufactured by ARKRAY, Inc.) under the following analysis conditions Analysis was carried out.
- Eluent phosphate buffer (pH 5.3)
- Flow rate 1.7mL / min
- Detector UV-Vis detector (wavelength: 415 nm)
- Temperature 35 ° C
- the obtained chromatogram is shown in FIG.
- the theoretical plate height of the stable glycated hemoglobin peak at this time was 33 ⁇ m.
- the obtained mixture was added to a 5% aqueous solution of polyvinyl alcohol (Kuraray Poval PVA 224, manufactured by Kuraray Co., Ltd.), and polymerized at 80 ° C. for 24 hours by a suspension polymerization method.
- the obtained particles were classified to obtain filler particles 2 having an average particle diameter of 6.5 ⁇ m.
- the obtained filler particles 2 were packed into a SUS column of inner diameter 4.6 mm ⁇ length 20 mm by a slurry method to obtain a column 2.
- Analysis example 3 The analysis was performed using the column 2 obtained in Comparative Example 1 under the same conditions as in Analysis Example 1. The obtained chromatogram is shown in FIG. The theoretical plate height of the glycated hemoglobin peak at this time was 81 ⁇ m. Since the average particle size can not be reduced, the theoretical plate height is larger than that of Analysis Example 1. Furthermore, since sufficient separation was not obtained, the column length was required, and the time required for analysis was about 1.3 times that for Analysis Example 1.
- Comparative example 3 To 3 g of the copolymer particles obtained in Example 1, 24 g of tetrahydrofuran was added and dispersed. To this, 3 g of 1,3-propane sultone and 0.1 g of boron trifluoride diethyl etherate were added, and allowed to react at 50 ° C. for 6 hours. The reaction solution was filtered, and particles were obtained by washing sequentially with tetrahydrofuran, water, 0.5 M hydrochloric acid, water, 0.5 M aqueous sodium hydroxide solution, and water.
- the amount of sulfo groups based on the mass of particles was 38.2 mmol / g.
- the particles obtained had the property of not being dispersed in water.
- the reason for not dispersing in water is presumed to be because the amount of sulfo group introduced by the reaction of propane sultone is small in the acidic condition. As it did not disperse in water, no column suitable for protein analysis was obtained.
- Example 2 3 g of the copolymer particles obtained in Example 1 was dispersed in 8 mL of water, 8 mL of 0.5 M sulfuric acid was added, and the mixture was reacted at 70 ° C. for 6 hours to open the epoxy ring. It was confirmed by FT-IR that the peak derived from the epoxy group was burned off that the epoxy ring was opened by the reaction with sulfuric acid.
- the obtained particles were subjected to a modification step with 1,3-propane sultone by the same operation as in Example 1 to obtain 3 g of filler particles 4.
- the amount of sulfo groups based on the mass of the particles was 180 ⁇ mol / g.
- the obtained filler particles 4 were packed in a PEEK column with an inner diameter of 4.6 mm and a length of 10 mm by a slurry method to obtain a column 4.
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Abstract
Description
特許文献2ではジビニルベンゼン系疎水性粒子の表面にメタクリル酸などの親水性重合体層を形成し得られた充填剤が開示されている。
非特許文献1にはアセトニトリル中の重合反応により、発生させたジビニルベンゼンの重合体のコアの表面にメタクリル酸エステルの層を形成し粒子を取得し、さらに、該粒子にエピクロロヒドリンを反応させたうえ、スルホ基を導入した充填剤を用いたタンパク質の分析例が開示されている。
また、特許文献2のように、疎水性粒子の表面に親水性重合体層を形成する方法では、親水性層を形成する必要性から作業工程が増え、生産性が低下するという問題がある。
[1](メタ)アクリル酸エステル由来のモノマー単位とジビニルベンゼン由来のモノマー単位を有する共重合体の粒子を含む液体クロマトグラフィー用充填剤であって、前記(メタ)アクリル酸エステル由来のモノマー単位:前記ジビニルベンゼン由来のモノマー単位が70~90質量%:30~10質量%であり、前記粒子表面にスルホ基が結合してなり、スルホ基が結合した構造として式(1)
[2]前記(メタ)アクリル酸エステルがグリシジルメタクリレートを含む前記1に記載の液体クロマトグラフィー用充填剤。
[3]前記1または2に記載の充填剤の製造方法であって、(メタ)アクリル酸エステルとジビニルベンゼンとを含むモノマー混合物を懸濁重合により共重合し、共重合体粒子を得る工程、および塩基の存在下、第二級アルコールを含む溶媒中でスルホ化剤と反応させる工程を有する充填剤の製造方法。
[4]前記の共重合体粒子を得る工程の後、得られた粒子を別の化学反応に供することなく、前記のスルホ化剤と反応させる工程を実施する前記3に記載の充填剤の製造方法。
[5](メタ)アクリル酸エステルがグリシジルメタクリレートであり、スルホ化剤がプロパンスルトンであり、第二級アルコールが2-プロパノールである前記3または4に記載の充填剤の製造方法。
[6]前記1または2に記載の液体クロマトグラフィー用充填剤を充填してなる糖化ヘモグロビン分析用の液体クロマトグラフィー用カラム。
[7]前記1または2に記載の液体クロマトグラフィー用充填剤を充填してなるカラムを用いた糖化ヘモグロビンの分析方法。
また、前記充填剤及びカラムは効率的に生産することができる。
単量体としてのジビニルベンゼンは、メタ及びパラ位の異性体混合物であっても良い。
全細孔容積は、装置としてカンタクローム社製Autosorb(登録商標) iQを用いて測定することができる。
重合開始剤の使用量はモノマーの種類や量によって適切に定めれば良いが、通常、モノマーの合計量100質量部に対して0.1~5質量部である。
スルホ化剤は、粒子表面に存在する共重合体由来の反応性官能基に直接反応させるほか、前記反応性官能基にスペーサーを反応させたり、前記反応性官能基がエポキシ基である場合にエポキシ基を開環反応させた後に、スルホ化剤と反応させることもできる。好ましくは、直接反応させるものであり、それにより得られる充填剤を用いたカラムの分離性能が向上するほか、製造効率にも優れる。特に、糖化ヘモグロビンの分析においては、共重合体粒子の表面に存在するエポキシ基の開環反応を行った後にスルホ化するよりも、エポキシ基を直接スルホ化する方が、目的とするたんぱく質の分離が良好になる。
このように、得られた共重合体粒子を、エポキシ基の開環反応も含め、他の反応に供することなく、スルホ基化剤との反応に用いることが好ましい。
スルホ剤としてスルホン酸塩を用いてスルホ基を導入する場合にも、同様に塩基を用いて行うことができる。ハロゲン基を有するスルホン酸塩を用いる場合、反応中に共重合体粒子上に生じる水酸基によるハロゲン置換反応によりスルホ基が導入され、水酸基あるいはメルカプト基を有するスルホン酸塩を用いる場合、共重合体粒子上のエポキシ基の開環を伴う求核反応によりスルホ基を導入される。
共重合体粒子とスルホ化剤の反応に酸性触媒を用いた場合には反応性が十分ではない。特に、ルイス酸を用いた場合には、得られた充填剤の親水性が低く、水溶液に分散しないため、カラムに充填できないなど、タンパク質分離用充填剤としての機能を発揮しない。
で示される構造を有することが望ましい。
本発明の一実施態様における液体クロマトグラフィー用カラムは、上記充填剤を有してなる。カラムは上記充填剤をスラリー法等の公知の充填法によってカラムに充填することによって得られる。充填用のカラムは、筐体の材質としてPEEK製またはSUS製のものが使用できるが、SUS製の場合はタンパク質が吸着するおそれもあるため、タンパク質を含む試料を分析する場合はPEEK製が好ましい。カラム内には充填剤粒子が流出しないためのフリットが設置されるが、そのフリットも吸着を抑制するためにPEEK製のものを使用することが好ましい。
実施例及び比較例にて測定した平均粒子径は、体積平均粒子径であり、発明を実施する形態の欄に記載した方法により測定した。
[重合工程]
グリシジルメタクリレート(日油株式会社製)及びジビニルベンゼン(新日鉄住金化学株式会社製,純度99%)(グリシジルメタクリレート:ジビニルベンゼン=82:17(質量比))からなる単量体混合物50gに、重合開始剤としての過酸化ラウロイル(ナカライテスク株式会社製)0.5g(前記単量体混合物の1質量%に相当する量)を添加し、混合物を得た。得られた混合物を、1%アルキルジフェニルエーテルスルホン酸ナトリウム水溶液500gを水相として、マイクロチャネル懸濁装置(株式会社イーピーテック製)により油滴とし、さらに重合反応を行い、共重合体粒子を得た。
得られた粒子の平均粒子径は2.6μmであった。
上記共重合体粒子3gに2-プロパノール24g及び1,3-プロパンスルトン(東京化成工業株式会社製)3gを加え、50℃に加熱した。これに8M水酸化カリウム水溶液1.2gを加え、6時間撹拌した後に濾過し、0.5M塩酸、水、0.5M水酸化ナトリウム水溶液、水で順次洗浄することで充填剤粒子1を得た。
真空乾燥させた充填剤粒子1の1.0gを、0.5M塩酸10mLを加えて分散させ、濾過した後に水で洗浄し、スルホ基が酸型になった粒子を得た。前記粒子に0.5M水酸化ナトリウム10mLを加え、濾液を集めるための容器を設置した状態でそれを濾過し、次いで粒子を水10mLで洗浄した。設置した容器に採取された濾液と洗浄液の混合液を0.1M塩酸で滴定した。初めに使用した水酸化ナトリウムのモル数(5mM)と、滴定に要した塩酸のモル数の差から、スルホ基の量を求めた。その結果、粒子の質量に対するスルホ基量は200μmol/gであった。
充填剤粒子1をSEM観察した。細孔は確認されなかった。観察結果を図5に示す。ガス吸着による細孔測定(使用装置としてカンタクローム社製Autosorb(登録商標)iQを用いた。)の結果、細孔容積は0.01cm3/gと非常に小さい値であった。
得られた充填剤粒子1を、内径4.6mm×長さ10mmのPEEK製カラムにスラリー法で充填し、カラム1を得た。
実施例1にて製造したカラム1を液体クロマトグラフ(Agilent(登録商標) 1260 Infinity,アジレント・テクノロジー株式会社製)に装着し、下記の分析条件にて糖化ヘモグロビンコントロールサンプル(アークレイ株式会社製)の分析を行った。
溶離液:リン酸緩衝液(pH5.3)
流速:1.7mL/min
検出器:UV-Vis検出器(波長:415nm)
温度:35℃
得られたクロマトグラムを図1に示す。この際の安定型糖化ヘモグロビンピークの理論段高さは33μmであった。
実施例1にて製造したカラム1を液体クロマトグラフ(Agilent(登録商標) 1260 Infinity,アジレント・テクノロジー株式会社製)に装着し、下記の分析条件にてタンパク質の分析を行った。
溶離液:(A)20mM 2-モルホリノエタンスルホン酸緩衝液(pH5.6)
(B)(A)+0.5M Na2SO4
リニアグラジエント 0minから2min、(A)から(B)
流速:1.7mL/min
検出器:UV-Vis検出器(波長:280nm)
温度:室温
サンプル:1.オボアルブミン、2.トリプシノーゲン、3.リボヌクレアーゼA、4.シトクロムC、5.リゾチーム(シグマアルドリッチ社製)
得られたクロマトグラムを図2に示す。1.5分という短時間で5種類のタンパク質が良好に分離されていることがわかる。
ジエチレングリコールジメタクリレート(日油株式会社製)30g、2-アクリルアミド-2-メチルプロパンスルホン酸(東京化成工業株式会社製)10g(ジエチレングリコールジメタクリレート:2-アクリルアミド-2-メチルプロパンスルホン酸=75:25(質量比))からなる単量体混合物に、重合開始剤としての過酸化ベンゾイル0.1g(前記単量体混合物の0.25質量%に相当する量)を溶解させ混合物を得た。得られた混合物を、5%ポリビニルアルコール(クラレポバールPVA224,株式会社クラレ製)水溶液に加え、懸濁重合法により80℃にて24時間重合させた。得られた粒子を分級し、平均粒子径6.5μmの充填剤粒子2を得た。
得られた充填剤粒子2をスラリー法にて内径4.6mm×長さ20mmのSUS製カラムに充填し、カラム2を得た。
比較例1にて得られたカラム2を用いて、分析例1と同じ条件で分析を行った。得られたクロマトグラムを図3に示す。この際の糖化ヘモグロビンピークの理論段高さは81μmであった。
平均粒子径を小さくできないので、分析例1と比べ、理論段高さが大きい。さらに、十分な分離が得られないためカラム長さが必要になり、分析例1に比べ、分析に要する時間が約1.3倍となった。
[重合工程]
グリシジルメタクリレート56gとエチレングリコールジメタクリレート24g(グリシジルメタクリレート:エチレングリコールジメタクリレート=70:30(質量比))からなる単量体混合物に、重合開始剤としてのV-65 1.5g(前記単量体混合物の1.88質量%に相当する量)を溶解させ混合物を得た。水400gにポリビニルアルコール4gと塩化ナトリウム4gを溶解させた水溶液に、得られた混合物を加え、懸濁重合法により60℃で10時間重合させた。得られた粒子を分級し、平均粒子径2.5μmの共重合体粒子を得た。
上記重合体粒子を実施例1と同様に修飾し、充填剤粒子3を得た。この充填剤粒子3を実施例1と同様にカラムに充填したところ、充填途中で充填剤粒子の変形により圧力が上昇し、送液が停止してしまいカラムに充填できなかった。
実施例1で得られた共重合体粒子3gにテトラヒドロフラン24gを加え分散させた。これに1,3-プロパンスルトン3gと三フッ化ホウ素ジエチルエーテル錯体0.1gを加え50℃で6時間反応させた。反応液を濾過し、テトラヒドロフラン、水、0.5M塩酸、水、0.5M水酸化ナトリウム水溶液、水の順に洗浄することで粒子を得た。
得られた粒子1.0gに、2-プロパノール1mlと0.5M塩酸10mLを加えて分散させ、濾過した後に水で洗浄し、スルホ基が酸型になった粒子を得た。前記粒子に0.5M水酸化ナトリウム10mLを加え、濾液を集めるための容器を設置した状態でそれを濾過し、次いで粒子を水10mLで洗浄した。設置した容器に採取された濾液と洗浄液の混合液に純水70mLを加え、0.1M塩酸で滴定した。計算の結果、粒子の質量に対するスルホ基量は38.2mmol/gであった。
得られた粒子は、水に分散しない性質を有していた。
水に分散しない理由としては、酸性条件では、プロパンスルトンの反応による導入されるスルホ基量が少ないからではないかと推測される。水に分散しないため、タンパク質の分析に適したカラムは得られなかった。
実施例1で得た共重合体粒子3gを8mLの水に分散させ、0.5M硫酸8mLを加えて70℃で6時間反応させて、エポキシ環を開環させた。エポキシ環が硫酸との反応により開環したことは、FT-IRでエポキシ基由来のピークが焼失したことにより確認した。得られた粒子に対し、実施例1と同様な操作によって1,3-プロパンスルトンとの修飾工程を行い、充填剤粒子4を3g得た。粒子の質量に対するスルホ基量は180μmol/gであった。
得られた充填剤粒子4を、内径4.6mm×長さ10mmのPEEK製カラムにスラリー法で充填し、カラム4を得た。
カラム4を用いて、溶離液条件として0minから0.5minは(A)リン酸緩衝液(pH5.3)、0.5minから1.0minは(B)リン酸緩衝液(pH8.5)であるステップグラジエントとした以外は分析例1と同様に測定を行った。得られたクロマトグラムを図4に示す。図1と同様に、糖化ヘモグロビンが短時間で良好に分離した。比較した場合、糖化ヘモグロビンと不安定型糖化ヘモグロビンの分離に関しては、図1のクロマトグラムが、やや勝っていた。
Claims (7)
- (メタ)アクリル酸エステル由来のモノマー単位とジビニルベンゼン由来のモノマー単位を有する共重合体の粒子を含む液体クロマトグラフィー用充填剤であって、前記(メタ)アクリル酸エステル由来のモノマー単位:前記ジビニルベンゼン由来のモノマー単位が70~90質量%:30~10質量%であり、前記粒子表面にスルホ基が結合してなり、スルホ基が結合した構造として式(1)
- 前記(メタ)アクリル酸エステルがグリシジルメタクリレートを含む請求項1に記載の液体クロマトグラフィー用充填剤。
- 請求項1または2に記載の充填剤の製造方法であって、(メタ)アクリル酸エステルとジビニルベンゼンとを含むモノマー混合物を懸濁重合により共重合し、共重合体粒子を得る工程、および塩基の存在下、第二級アルコールを含む溶媒中でスルホ化剤と反応させる工程を有する充填剤の製造方法。
- 前記の共重合体粒子を得る工程の後、得られた粒子を別の化学反応に供することなく、前記のスルホ化剤と反応させる工程を実施する請求項3に記載の充填剤の製造方法。
- (メタ)アクリル酸エステルがグリシジルメタクリレートであり、スルホ化剤がプロパンスルトンであり、第二級アルコールが2-プロパノールである請求項3または4に記載の充填剤の製造方法。
- 請求項1または2に記載の液体クロマトグラフィー用充填剤を充填してなる糖化ヘモグロビン分析用の液体クロマトグラフィー用カラム。
- 請求項1または2に記載の液体クロマトグラフィー用充填剤を充填してなるカラムを用いた糖化ヘモグロビンの分析方法。
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CN112742359A (zh) * | 2019-10-31 | 2021-05-04 | 昭和电工株式会社 | 糖化血红蛋白分析用柱填料的制造方法 |
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CN113786823A (zh) * | 2021-09-18 | 2021-12-14 | 浙江中医药大学 | 超小粒径水热碳球乳胶附聚型高效离子色谱填料的制备方法 |
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