WO2006025556A1 - Surface-modified packing material for liquid chromatography and production method thereof - Google Patents

Surface-modified packing material for liquid chromatography and production method thereof Download PDF

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Publication number
WO2006025556A1
WO2006025556A1 PCT/JP2005/016174 JP2005016174W WO2006025556A1 WO 2006025556 A1 WO2006025556 A1 WO 2006025556A1 JP 2005016174 W JP2005016174 W JP 2005016174W WO 2006025556 A1 WO2006025556 A1 WO 2006025556A1
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WIPO (PCT)
Prior art keywords
water
packing material
liquid chromatography
soluble polymer
support
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PCT/JP2005/016174
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French (fr)
Inventor
Kuniaki Shimbo
Yoshiji Okada
Ryuji Takahashi
Original Assignee
Showa Denko K.K.
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Publication of WO2006025556A1 publication Critical patent/WO2006025556A1/en

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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
    • B01J20/328—Polymers on the carrier being further modified
    • 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
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • 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
    • B01J20/3231—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
    • B01J20/3272—Polymers obtained by reactions otherwise than 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
    • B01J20/30—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
    • B01J20/328—Polymers on the carrier being further modified
    • B01J20/3282—Crosslinked polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/34—Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation
    • 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

Definitions

  • the present invention relates to surface-modified packing material for liquid chromatography and production method therefor. More specifically, the invention relates to surface-modified packing material for liquid chromatography having huge pores, which is suitably used in separating or measuring water-soluble substance having a huge molecular weight such as sugar and lipid by gel filtration chromatography without adsorbing water-soluble substance having a hydrophobic site in its molecule such as protein which coexists in sample onto the surface of the packing material, production method thereof, and a column for analysis using the packing material.
  • viscosity-average molecular weight obtained by measuring viscosity of dilute polymer solution and weight-average molecular weight obtained by light scattering and ultracentrifugal method are employed.
  • gel filtration chromatography is widely employed for its quickness and simplicity.
  • Examples of packed column for gel filtration chromatography used in estimating molecular weight distribution of water-soluble polymer include TSK-GEL PW XL series (products of TOSOHCORPORATION) and Shodex OHpak SB-800 HQ series (products of SHOWA DENKO K.K.), which are columns packed with packing material obtained by crosslinking monomers of (raeth) acrylic acid ester.
  • the object of the invention is to provide a packing material which enables analysis of molecular weight and molecular weight distribution of water-soluble macromolecule sample such as hyaluronic acid having a viscosity-average molecular weight of 3,000,000 or more without reaching the exclusion limit of the column andwithout adsorbing the water-soluble macromolecule onto the surface of the packing material even when a water-soluble substance having a hydrophobic site in its molecule such as protein is present in the same sample.
  • the present inventors have found out that the above object can be achieved by coating the surface of support with a hydrophilic polymer to thereby crosslink the hydrophilic polymer surface by using a specific method, a packing material for liquid chromatography.
  • the present invention relates to the following surface-modified packing material for liquid chromatography, production method therefor, and a column using the packing material.
  • a surface-modified packing material for liquid chromatography which is obtained by first suspending a support containing a polar group in a water-soluble polymer solution, next removing water content therefrom to coat the support surface with the water-soluble polymer, and then suspending the resulting support having surface coated with the water-soluble polymer in a solvent which does not dissolve the water-soluble polymer to thereby crosslink the water-soluble polymer on the support surface.
  • a method of producing a surface-modified packing material for liquid chromatography which is obtained by first suspending a support containing a polar group in a water-soluble polymer solution, next removing water content therefrom to coat the support surface with the water-soluble polymer, and then suspending the resulting support having surface coated with the water-soluble polymer in a solvent which does not dissolve the water-soluble polymer to thereby crosslink the water-soluble polymer on the support surface.
  • the surface-modified packing material for liquid chromatography is a packing material whose surface is modified by coating the surface of support containing a polar group with water-soluble polymer.
  • the support is not particularly limited as far as the support contains a polar group.
  • examples thereof include synthetic polymer supports such as polyvinyl alcohol gel, poly(meth) acrylic acid gel, poly-N-vinylcarboxylic acid amide gel and polystyrene gel having an ion exchanger group such as sulfo group, quaternary ammonium group, tertiary amino group, secondary amino group or primary amino group introduced therein.
  • polystyrene gel having an ion exchange group introduced therein for its porosity and strength
  • sulfonated polystyrene having a sulfo group introduced therein for its availability.
  • the support be porous, that by using a column filled with the support particles and using tetrahydrofuran as eluent, polystyrene of molecular weight of 16,100,000 (product of TOSOH CORPORATION) as standard be eluted without reaching the exclusion limit, and that the molecular weight exclusion limit inferred by using polystyrene be approximately 200,000,000.
  • polystyrene of molecular weight of 16,100,000 (product of TOSOH CORPORATION) as standard be eluted without reaching the exclusion limit, and that the molecular weight exclusion limit inferred by using polystyrene be approximately 200,000,000.
  • the size of polystyrene having a molecular weight of 16, 100, 000 is about 1,100 nm
  • the size of polystyrene (diameter) having a molecular weight of about 200,000,000 is about 4,900 nm.
  • the surface-modified packing material used in the present invention be porous particles having a pore diameter of 100 to 5,000 niti.
  • the mass-average particle size of the support is preferably 10 to 100 ⁇ m.
  • the mass-average molecular weight can be measured with a Coulter Counter (Registered Trademark of a product manufactured by Beckman Coulter, Inc.) .
  • the sulfo group may be modified with a surface modifying agent for the purpose of enhancing its hydrophilicity.
  • the modifying agent used here is not particularly limited as far as the agent can react with the sulfo group to enhance hydrophilicity. Examples thereof include sorbitol polyglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether and glycidol. Further, glycidol is preferred for its handleability and availability.
  • the amount of the modifying agent used for the purpose of enhancing hydrophilicity is not particularly limited as far as the amount does not affect the pore size of the packing material.
  • the modifying agent is used in an amount 0.5 to 2 times by mass the amount of the support, preferably 1 to 1.5 times by mass the amount of the support.
  • catalyst may be appropriately used in order to accelerate the reaction.
  • the catalyst examples include inorganic acids such as hydrochloric acid and sulfuric acid, alkalis such as sodium hydroxide and potassium hydroxide, quaternary ammonium salts such as triethylbenzyl ammonium chloride and tetramethyl ammonium chloride, tertiary amines such as benzyldimethyl amine and tributyl amine, imidazole compounds such as 2-methyl-4-ethylimidazole and 2-methyl imidazole and Lewis acids such boron trifluoride, zinc fluoroborate and tin chloride.
  • Lewis acid is preferred, and particularly preferred is zinc fluoroborate in view of reaction control.
  • the water-soluble polymer used in modifying the support surface is not particularly limited as far as the polymer has a property to be dissolved in water.
  • Examples thereof include synthetic water soluble polymers such as polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylamide, polyacrylic acidandpolyethylene oxide, animal water-soluble polymers such as gelatin and casein,, and polysaccharides such as dexstran pullulan and cellulose and derivatives thereof .
  • synthetic water soluble polymers such as polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylamide, polyacrylic acidandpolyethylene oxide, animal water-soluble polymers such as gelatin and casein, and polysaccharides such as dexstran pullulan and cellulose and derivatives thereof .
  • those having a hydroxyl group are preferred in terms of strong hydrophilicity, and particularly preferred are cellulose derivatives consisting of polysaccharides since the variety of the derivatives enables selectionwith awide scope .
  • the method for coating the support surface with water-soluble polymer for the purpose of surface modification is not particularly limited.
  • water content is removed by filtration or the like.
  • the pressure be 30 to 200 hpa, more preferably 50 to 100 hpa in consideration for reduction in removal time and boiling by reducing the pressure.
  • the temperature in order to remove the water quickly under reduced pressure, however, it is not preferred to raise the temperature to an extremely high temperature under reduced pressure, since the support spatters out of the solution, resultingindecrease in yield. It is preferable that the temperature be raised to 70 0 C or less, and more preferable that the heating be conducted within a range of 30 to 50 0 C.
  • the amount of the water-soluble polymer coating the support surface in the present invention is not particularly limited, and is determined to such an amount that can modify the surface without affecting porosity of the support. It is preferable that the amount of the water-soluble polymer be 0.0005 to 0.5 times by mass the amount of the support to be surface-modified, more preferably 0.002 to 0.2 times by mass.
  • the solvent in which the water-soluble polymer coating the support is not dissolved but in which the water-soluble polymer is dispersed to be used for crosslinking reaction is not particularly limited as far as the solvent does not dissolve the water-soluble polymer. Since solubility varies depending on the polymerization degree and molecular weight distribution of the water-soluble polymer used, a solvent which does not substantially dissolve the water-soluble polymer is selected.
  • a solvent does not substantially dissolve the water-soluble polymer
  • the mass decrease rate of the water soluble polymer is 50 % or less based on the mass before the immersion, preferably25% or less, morepreferably 5 % or less.
  • the solvent include aromatic compounds such as toluene, xylene and ethylbenzene, and polar organic solvents such as dioxane anddimethyl formamide.
  • aromatic compounds such as toluene, xylene and ethylbenzene
  • polar organic solvents such as dioxane anddimethyl formamide.
  • polarorganic solvent is preferable for its handleability, and more preferred is dioxane for its performance in reaction.
  • the crosslinking agent used for crosslinking the water-soluble polymer coating the support is not particularly limited as far as the agent is a polyfunctional compound which reacts with a functional group of the water-soluble polymer.
  • the agent may be a radical generator such as organic peroxides.
  • a polyfunctional compoundreactivewiththese functional groups may be used.
  • Examples thereof include dialdehydes such as glutaric aldehyde and terephthalic aldehyde, diglycidyls such as ethylene glycol diglycidyl ether, diethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, glycerol diglycidyl ether and 1,4-butane diglycidyl ether and epichlorohydrin.
  • dialdehydes such as glutaric aldehyde and terephthalic aldehyde
  • diglycidyls such as ethylene glycol diglycidyl ether, diethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, glycerol diglycidyl ether and 1,4-butane diglycidyl ether and epichlorohydrin.
  • diglycidyl is preferred in view of reaction control, and particularly preferred is ethylene glycol diglycidyl ether in consideration for its availability.
  • catalyst When crosslinking reaction is conducted by using the above diglycidyl compound, catalyst may be appropriately used for the purpose of accelerating the reaction.
  • the catalyst include inorganic acids such as hydrochloric acid and sulfuric acid, alkalis such as sodium hydroxide and potassium hydroxide, quaternary ammonium salts such as triethylbenzyl ammonium chloride and tetramethyl ammonium chloride, tertiary amines such as benzyldimethyl amine and tributyl amine, imidazole compounds such as 2-methyl-4-ethylimidazole and 2-methyl imidazole and Lewis acids such boron trifluoride, zinc fluoroborate and tin chloride.
  • Lewis acid is preferred, and particularly preferred is zinc fluoroborate in view of reaction control.
  • the crosslinking agent is usually used in an excessive amount which is larger than the amount actually required for crosslinking the water-soluble polymer coating the support surface.
  • the use amount is 0.1 to 2 times by mass the amount of the support, preferably 0.25 to 1.5 times by mass.
  • the surface-modified packing material according to the present invention is a packing material for liquid chromatography which has a polar group in the molecule and whose surface is modified by coating with water-soluble polymer .
  • Example 1 5 g of particles (Shodex KS-807 GEL, registered trademark of a product manufactured by SHOWA DENKO K.K.) where counter ions had been changed to hydrogen-type ions by using sulfonated polystyrene gel was dispersed in 25 g of dioxane. Next, 5 g of glycidol was added thereto at room temperature, and the resultant mixture was stirred for 30 minutes. Subsequently, 1 g of zinc fluoroborate was added thereto, the temperature was increased to 80 °C, and reaction was conducted for 4 hours. After cooling, the particles surface-modified with glycidol were separated by filtration with filter paper No.101.
  • the particles were spread on a stainless steel tray and dried in air. 5 gof the sulfonatedparticles having surface treated with glycidol was placed in a 200 mL eggplant-shaped flask and further 25 mL of 0.05 mass % aqueous solution of hydroxyethyl cellulose was added thereto to disperse the particles. Water was removed by using a rotary evaporator to thereby form hydroxyethyl cellulose film on the particle surface.
  • This column was connected with high-performance liquid chromatography apparatus, 0.2M sodium chloride solution as eluent was flowed at a rate of 0.5 mL/min, and 100 ⁇ l of a sample prepared by dissolving hyaluronic acid having a viscosity-average molecular weight of 3,370,000 (product of SHISEIDO Co., Ltd.) in an eluent to be a concentration of 0.03 mass % was injected, and the molecular weight was measured.
  • Shodex a differential refractometer detector (registered trademark : RI-71, product of SHOWA DENKO K.K.) was used as a detector.
  • the recovery rate of bovine serum albumin defined by dividing the area value of the chromatogram obtained by using the column packed with the surface-modified packing material by the area value of the chromatogram obtained by using the reference column (polytetrafluoroethylene tube) was 78 %.
  • Comparative Example 1 A stainless steel column for liquid chromatography having an inner diameter of 8 mm and a length of 300 mm was charged with the same polystyrene gel particles having a sulfo group introduced therein as used in Example 1, by using a packer and a high-pressure pump by equilibrium slurrymethod in the same manner as in Example 1.
  • This column was connected with high-performance liquid chromatography apparatus, 0.2M sodium chloride solution as eluent was flowed at a rate of 0.5 mL/min, and a sample of hyaluronic acid having a viscosity-average molecular weight of 3, 370, 000 (product of SHISEIDO Co., Ltd.) was analyzed in the same manner as in Example 1. From the obtained chromatogram, it was confirmed that analysis of the molecular weight distribution had been conducted, including the maximum molecular weight of the hyaluronic acid, without reaching the exclusion limit.
  • a stainless steel column for liquid chromatography having an inner diameter of 8 mm and a length of 300 mm was charged with sulfonated polystyrene particles after the process of surface treatment with glycidol as in Example 1 / by using a packer and a high-pressure pump.by equilibrium slurry method in the same manner as in Example 1.
  • This column was connected with high-performance liquid chromatography apparatus, 0.2M sodium chloride solution as eluent was flowed at a rate of 0.5 mL/min, and a sample of hyaluronic acid having a viscosity-average molecular weight of 3, 370, 000 (product of SHISEIDO Co ., Ltd.) was analyzed in the same manner as in Example 1.
  • a surface-modified packing material for liquid chromatography can be produced by a relatively simple method, and by using this packing material for liquid chromatography, samples of macromolecules such as hyaluronic acid having a viscosity-average molecular weight of 3, 000, 000 or more can be analyzed without reaching the exclusion limit and without adsorbingproteins suchas albuminwhichcoexist inthe sample.

Abstract

The invention relates to a surface-modified packing material for liquid chromatography, which is obtained by first suspending a support containing a polar group in a water-soluble polymer solution, next removing water content therefrom to coat the support surface with the water-soluble polymer, and then suspending the resulting support having surface coated with the water-soluble polymer in a solvent which does not dissolve the water-soluble polymer to thereby crosslink the water-soluble polymer on the support surface, a production method thereof and a column for liquid chromatography using the packing material. The packing material for liquid chromatography of the invention, which has a surface modified without affecting 100-5000nm giant pores of a porous support and which does not adsorb water-soluble substances such as proteins having a hydrophobic site, is suitable for gel filtration chromatography for water- soluble macromolecules such as sugar and fat.

Description

DESCRIPTION
SURFACE-MODIFIED PACKING MATERIAL FOR LIQUID CHROMATOGRAPHY AND PRODUCTION METHOD THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
This is an application filed pursuant to 35 U.S.C.
Section 111 (a) with claiming the benefit of U.S. provisional application Serial No. 60/607,634 filed September 8, 2004 under the provision of 35 U.S.C. lll(b), pursuant to 35 U.S.C.
Section 119 (e) (1) .
TECHNICAL FIELD
The present invention relates to surface-modified packing material for liquid chromatography and production method therefor. More specifically, the invention relates to surface-modified packing material for liquid chromatography having huge pores, which is suitably used in separating or measuring water-soluble substance having a huge molecular weight such as sugar and lipid by gel filtration chromatography without adsorbing water-soluble substance having a hydrophobic site in its molecule such as protein which coexists in sample onto the surface of the packing material, production method thereof, and a column for analysis using the packing material.
BACKGROUND ART
Conventionally, for measuring an average molecular weight of a water-soluble polymer, viscosity-average molecular weight obtained by measuring viscosity of dilute polymer solution and weight-average molecular weight obtained by light scattering and ultracentrifugal method are employed. On the other hand, in measurement of molecular weight distribution, gel filtration chromatography is widely employed for its quickness and simplicity.
Examples of packed column for gel filtration chromatography used in estimating molecular weight distribution of water-soluble polymer include TSK-GEL PWXL series (products of TOSOHCORPORATION) and Shodex OHpak SB-800 HQ series (products of SHOWA DENKO K.K.), which are columns packed with packing material obtained by crosslinking monomers of (raeth) acrylic acid ester. However, for example, in measurement of molecular weight distribution of a water-soluble huge polymer such as hyaluronic acid having a viscosity-average molecular weight of 3,000,000 or more, when TSK-GEL G6000PWXL whose molecular weight exclusion limit is the largest among the TSK-GEL PWXL series products, is used, it is assumed that the molecular weight of the polymer exceeds the exclusion limit (Akinori Ono, et al . , "Measurement of SEC of polymer hyaluronic acid" (by SHISEIDO Co., Ltd. Kakegawa Factory) in the summary of the lectures in the 49th annual meeting of The Japan Society for Analytical Chemistry, page 15, 2A11. On the other hand, even when Shodex OHpak SB-806HQwhosemolecularweight exclusion limit is the largest among the Shodex OHpak SB-800 HQ series products, is used, hyaluronic acid having a huge viscosity-average molecular weight of 3,000,000 or more goes beyond the exclusion limit (Masahide NAKAMURA, et al . , "SEC-MALS analysis using a column for water-soluble ultra-high molecular weight polymer", pages 107-108 III-10 in summary report of the 6th discussion meeting on polymer analysis, published by Shodex Service Center of SHOKO Co., Ltd.) . Thus, among columns packed with packing material obtained by crosslinking monomers of (meth) acrylic acid ester, there is no column which can analyze a sample having a molecular weight as huge as a viscosity-average molecular weight of 3, 000, 000 or more (in the molecular weight distribution, the maximum molecular weight reaches a level of 4,000,000 to 5,000,000) without reaching the exclusion limit and therefore, it is difficult to precisely estimate the molecular weight distribution of such a polymer.
With the above problem in the background, a packing material which can enable measurement of molecular weight distribution of huge polymer material such as hyaluronic acidhaving a viscosity-average molecular weight of 3, 000, 000 or more quickly and easily by gel filtration chromatography has been demanded. As column packed with water-soluble polymers other than (meth) acrylic acid ester polymers which is used for gel filtration chromatography, there is a column packed with packing material obtained by making polystyrene hydrophilic through sulfonation, for example, Shodex SUGAR KS series (products of SHOWA DENKO K.K.) . It was reported that among the Shodex SUGAR KS series, Shodex SUGAR KS-807, which has the largest molecular weight exclusion limit, enables analysis without reaching the molecular weight exclusion limit (Kei Oide, et al . "Analysis of hyaluronic acid in biological liquid by column switching" in Summaries of the 123rd Annual Meeting of Pharmaceutical Society of Japan in 2003, in page 37, 28 (Pl) 1-276 , (DENKI KAGAKU KOGYO KABUSHIKI KAISHA, SHOKO Co., Ltd. SHOWA DENKO K.K.) ) .
However, such a column packed with packing material obtained by making polystyrene hydrophilic through sulfonation is defective in that, water-soluble substance having a hydrophobic site in its molecule such as protein, which coexists in sample, is adsorbed onto the surface of the packing material.
DISCLOSURE OF INVENTION
Therefore, the object of the invention is to provide a packing material which enables analysis of molecular weight and molecular weight distribution of water-soluble macromolecule sample such as hyaluronic acid having a viscosity-average molecular weight of 3,000,000 or more without reaching the exclusion limit of the column andwithout adsorbing the water-soluble macromolecule onto the surface of the packing material even when a water-soluble substance having a hydrophobic site in its molecule such as protein is present in the same sample.
As a result of intensive studies, the present inventors have found out that the above object can be achieved by coating the surface of support with a hydrophilic polymer to thereby crosslink the hydrophilic polymer surface by using a specific method, a packing material for liquid chromatography.
That is, the present invention relates to the following surface-modified packing material for liquid chromatography, production method therefor, and a column using the packing material.
1. A surface-modified packing material for liquid chromatography, which is obtained by first suspending a support containing a polar group in a water-soluble polymer solution, next removing water content therefrom to coat the support surface with the water-soluble polymer, and then suspending the resulting support having surface coated with the water-soluble polymer in a solvent which does not dissolve the water-soluble polymer to thereby crosslink the water-soluble polymer on the support surface.
2. The surface-modified packing material for liquid chromatography as described in 1, wherein the water-soluble polymer has a hydroxyl group in its molecule.
3. The surface-modified packing material for liquid chromatography as described in 1 or 2, wherein the water-soluble polymer is a polysaccharide. 4. The surface-modified packing material for liquid chromatography as described in 3, wherein the polysaccharide as water-soluble polymer is hydroxyethyl cellulose.
5. The surface-modified packing material for liquid chromatography as described in 1, wherein the water-soluble polymer coating the support surface is crosslinked by using a crosslinking agent.
6. The surface-modified packing material for liquid chromatography as described in 5, wherein the crosslinking agent is ethylene glycol diglycidyl ether. 7. The surface-modified packing material for liquid chromatography as described in 1, wherein the support is a synthetic polymer which has a polar group in its molecule. 8. The surface-modified packing material for liquid chromatography as described in 7, wherein the support is a synthetic polymer which has a sulfo group as polar group in its molecule. 9. The surface-modified packing material for liquid chromatography as described in any one of 1, 7 and 8, wherein the support is sulfonated polystyrene.
10. The surface-modified packing material for liquid chromatography as described in any one of 1 and 7 to 9, wherein the support has fine pores having a diameter of 100 to 5,000 nm.
11. The surface-modified packing material for liquid chromatography as described in any one of 1 and 7 to 10, wherein the mass-average particle size of the support is from 10 to 100 μm.
12. A method of producing a surface-modified packing material for liquid chromatography, which is obtained by first suspending a support containing a polar group in a water-soluble polymer solution, next removing water content therefrom to coat the support surface with the water-soluble polymer, and then suspending the resulting support having surface coated with the water-soluble polymer in a solvent which does not dissolve the water-soluble polymer to thereby crosslink the water-soluble polymer on the support surface.
13. The method of producing a surface-modified packing material for liquid chromatographyas described in 12, wherein the water-soluble polymer used for the surface modification has a hydroxyl group in its molecule. 14. The method of producing a surface-modified packing material for liquid chromatography as described in 12 or 13, wherein the water-soluble polymer used for the surface modification is a polysaccharide. 15. The method of producing a surface-modified packing material for liquidchromatography as described in 14, wherein the polysaccharide as water-soluble polymer used for the surface modification is hydroxyethyl cellulose.
16. The method of producing a surface-modified packing material for liquidchromatographyas described in 12, wherein the water-soluble polymer used for the surface modification is crosslinked by using a crosslinking agent.
17. The method of producing a surface-modified packing material for liquid chromatography as described in 16, wherein the crosslinking agent is ethylene glycol diglycidyl ether.
18. The method of producing a surface-modified packing material for liquidchromatographyas described in 12, wherein the support whose surface is to be modified is a synthetic polymer which has a polar group in its molecule.
19. The method of producing a surface-modified packing material for liquidchromatographyas described in 18, wherein the polar group in the molecule of the synthetic polymer support is a sulfo group.
20. The method of producing a surface-modified packing material for liquidchromatography as described in 19, wherein the sulfo group in the molecule of the synthetic polymer support is modified with a modifying agent.
21. The method of producing a surface-modified packing material for liquid chromatographyas describedin 20, wherein the modifying agent to modify the sulfo group is glycidol .
22. A column which is packed with the packing material for liquid chromatography as described in any one of 1 to
11.
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, mode for carrying out the invention is described in detail, however, the present invention is by no means limited thereby. The surface-modified packing material for liquid chromatography according to the present invention is a packing material whose surface is modified by coating the surface of support containing a polar group with water-soluble polymer.
The support is not particularly limited as far as the support contains a polar group. Examples thereof include synthetic polymer supports such as polyvinyl alcohol gel, poly(meth) acrylic acid gel, poly-N-vinylcarboxylic acid amide gel and polystyrene gel having an ion exchanger group such as sulfo group, quaternary ammonium group, tertiary amino group, secondary amino group or primary amino group introduced therein. Among these, preferred is polystyrene gel having an ion exchange group introduced therein for its porosity and strength, and still more preferred is sulfonated polystyrene having a sulfo group introduced therein for its availability.
Also, it is preferable that the support be porous, that by using a column filled with the support particles and using tetrahydrofuran as eluent, polystyrene of molecular weight of 16,100,000 (product of TOSOH CORPORATION) as standard be eluted without reaching the exclusion limit, and that the molecular weight exclusion limit inferred by using polystyrene be approximately 200,000,000. According to the equation defined by Istvan Hal asz and Korn el Martin in "Pore Sizes of Solids", Angew. Chem. Int. Ed. Engl., 17. 901-908 (1978), the size of polystyrene having a molecular weight of 16, 100, 000 is about 1,100 nm, and the size of polystyrene (diameter) having a molecular weight of about 200,000,000 is about 4,900 nm. Accordingly, it is more preferable that the surface-modified packing material used in the present invention be porous particles having a pore diameter of 100 to 5,000 niti.
The mass-average particle size of the support is preferably 10 to 100 μm. The mass-average molecular weight can be measured with a Coulter Counter (Registered Trademark of a product manufactured by Beckman Coulter, Inc.) .
In the sulfonated polystyrene gel support having a sulfo group introduced therein, which is preferably used in the present invention, the sulfo group may be modified with a surface modifying agent for the purpose of enhancing its hydrophilicity. The modifying agent used here is not particularly limited as far as the agent can react with the sulfo group to enhance hydrophilicity. Examples thereof include sorbitol polyglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether and glycidol. Further, glycidol is preferred for its handleability and availability.
The amount of the modifying agent used for the purpose of enhancing hydrophilicity is not particularly limited as far as the amount does not affect the pore size of the packing material. In the present invention, the modifying agent is used in an amount 0.5 to 2 times by mass the amount of the support, preferably 1 to 1.5 times by mass the amount of the support. When the sulfo group is modified with a surface modifying agent, catalyst may be appropriately used in order to accelerate the reaction. Examples of the catalyst include inorganic acids such as hydrochloric acid and sulfuric acid, alkalis such as sodium hydroxide and potassium hydroxide, quaternary ammonium salts such as triethylbenzyl ammonium chloride and tetramethyl ammonium chloride, tertiary amines such as benzyldimethyl amine and tributyl amine, imidazole compounds such as 2-methyl-4-ethylimidazole and 2-methyl imidazole and Lewis acids such boron trifluoride, zinc fluoroborate and tin chloride. In terms of reaction efficiency, Lewis acid is preferred, and particularly preferred is zinc fluoroborate in view of reaction control.
The water-soluble polymer used in modifying the support surface is not particularly limited as far as the polymer has a property to be dissolved in water. Examples thereof include synthetic water soluble polymers such as polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylamide, polyacrylic acidandpolyethylene oxide, animal water-soluble polymers such as gelatin and casein,, and polysaccharides such as dexstran pullulan and cellulose and derivatives thereof . As the water-soluble polymer, those having a hydroxyl group are preferred in terms of strong hydrophilicity, and particularly preferred are cellulose derivatives consisting of polysaccharides since the variety of the derivatives enables selectionwith awide scope . Among them, hydroxyethyl cellulose is particularly preferred for its solubility and availability.
The method for coating the support surface with water-soluble polymer for the purpose of surface modification is not particularly limited. For example, after support is added into a solution having water-soluble polymer dissolved therein so that the water-soluble polymer is adsorbed onto the support surface, water content is removed by filtration or the like. However, such a process is cumbersome, resulting in deterioration of reproducibility. On the other hand, a process where support is suspended in a water-soluble polymer solution and then water content is removed under reduced pressure by using an apparatus such as evaporator, which is simple and highly reproducible, is preferred. In order to remove the water quickly, it is preferable that the pressure be 30 to 200 hpa, more preferably 50 to 100 hpa in consideration for reduction in removal time and boiling by reducing the pressure.
Further, it is preferable to increase the temperature in order to remove the water quickly under reduced pressure, however, it is not preferred to raise the temperature to an extremely high temperature under reduced pressure, since the support spatters out of the solution, resultingindecrease in yield. It is preferable that the temperature be raised to 70 0C or less, and more preferable that the heating be conducted within a range of 30 to 50 0C. The amount of the water-soluble polymer coating the support surface in the present invention is not particularly limited, and is determined to such an amount that can modify the surface without affecting porosity of the support. It is preferable that the amount of the water-soluble polymer be 0.0005 to 0.5 times by mass the amount of the support to be surface-modified, more preferably 0.002 to 0.2 times by mass.
In producing the packing material of the present invention, the solvent in which the water-soluble polymer coating the support is not dissolved but in which the water-soluble polymer is dispersed to be used for crosslinking reaction is not particularly limited as far as the solvent does not dissolve the water-soluble polymer. Since solubility varies depending on the polymerization degree and molecular weight distribution of the water-soluble polymer used, a solvent which does not substantially dissolve the water-soluble polymer is selected. Here, the term "a solvent does not substantially dissolve the water-soluble polymer"means thatwhen solubilityof awater-solublepolymer in an organic solvent is measured by the change in mass after support coated with the water-soluble polymer is immersed in the solvent, the mass decrease rate of the water soluble polymer is 50 % or less based on the mass before the immersion, preferably25% or less, morepreferably 5 % or less. Examples of the solvent include aromatic compounds such as toluene, xylene and ethylbenzene, and polar organic solvents such as dioxane anddimethyl formamide. Amongthese, polarorganic solvent is preferable for its handleability, and more preferred is dioxane for its performance in reaction.
The crosslinking agent used for crosslinking the water-soluble polymer coating the support is not particularly limited as far as the agent is a polyfunctional compound which reacts with a functional group of the water-soluble polymer. Moreover, the agent may be a radical generator such as organic peroxides. In a case where the water-soluble polymer has a hydroxyl group and/or sulfo group, a polyfunctional compoundreactivewiththese functional groups may be used. Examples thereof include dialdehydes such as glutaric aldehyde and terephthalic aldehyde, diglycidyls such as ethylene glycol diglycidyl ether, diethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, glycerol diglycidyl ether and 1,4-butane diglycidyl ether and epichlorohydrin. Among these, diglycidyl is preferred in view of reaction control, and particularly preferred is ethylene glycol diglycidyl ether in consideration for its availability.
When crosslinking reaction is conducted by using the above diglycidyl compound, catalyst may be appropriately used for the purpose of accelerating the reaction. Examples of the catalyst include inorganic acids such as hydrochloric acid and sulfuric acid, alkalis such as sodium hydroxide and potassium hydroxide, quaternary ammonium salts such as triethylbenzyl ammonium chloride and tetramethyl ammonium chloride, tertiary amines such as benzyldimethyl amine and tributyl amine, imidazole compounds such as 2-methyl-4-ethylimidazole and 2-methyl imidazole and Lewis acids such boron trifluoride, zinc fluoroborate and tin chloride. In terms of reaction efficiency, Lewis acid is preferred, and particularly preferred is zinc fluoroborate in view of reaction control.
In the support surface modification, the crosslinking agent is usually used in an excessive amount which is larger than the amount actually required for crosslinking the water-soluble polymer coating the support surface. The use amount is 0.1 to 2 times by mass the amount of the support, preferably 0.25 to 1.5 times by mass. The surface-modified packing material according to the present invention is a packing material for liquid chromatography which has a polar group in the molecule and whose surface is modified by coating with water-soluble polymer .
EXAMPLES
Hereinafter, the present invention is described in more detail, however, the present invention is not limited by the following Examples.
Example 1 : 5 g of particles (Shodex KS-807 GEL, registered trademark of a product manufactured by SHOWA DENKO K.K.) where counter ions had been changed to hydrogen-type ions by using sulfonated polystyrene gel was dispersed in 25 g of dioxane. Next, 5 g of glycidol was added thereto at room temperature, and the resultant mixture was stirred for 30 minutes. Subsequently, 1 g of zinc fluoroborate was added thereto, the temperature was increased to 80 °C, and reaction was conducted for 4 hours. After cooling, the particles surface-modified with glycidol were separated by filtration with filter paper No.101. Then after washed with 100 mL of deionized water and further washed with 100 mL of acetone, the particles were spread on a stainless steel tray and dried in air. 5 gof the sulfonatedparticles having surface treated with glycidol was placed in a 200 mL eggplant-shaped flask and further 25 mL of 0.05 mass % aqueous solution of hydroxyethyl cellulose was added thereto to disperse the particles. Water was removed by using a rotary evaporator to thereby form hydroxyethyl cellulose film on the particle surface.
Into the eggplant-shaped flask containing the particles having hydroxyethyl cellulose film on the surface, 50 g of dioxane was added and the particles were sufficiently dispersed. Subsequently, 2.5 gof ethylene glycol diglycidyl ether was added thereto and stirring was continued for 10 minutes. Then, 2.5 g of zinc fluoroborate was added thereto to conduct reaction at 80 °C for 3 hours to thereby crosslink the hydroxyethyl cellulose film on the surface. A stainless steel column for liquid chromatography having an inner diameter of 8 mm and a length of 300 mm was charged with the thus obtained particles having surface modified with hydroxyethyl cellulose by using a packer and a high pressure pump by equilibrium slurry method under high pressure.
This column was connected with high-performance liquid chromatography apparatus, 0.2M sodium chloride solution as eluent was flowed at a rate of 0.5 mL/min, and 100 μl of a sample prepared by dissolving hyaluronic acid having a viscosity-average molecular weight of 3,370,000 (product of SHISEIDO Co., Ltd.) in an eluent to be a concentration of 0.03 mass % was injected, and the molecular weight was measured. Shodex, a differential refractometer detector (registered trademark : RI-71, product of SHOWA DENKO K.K.) was used as a detector. From the obtained chromatogram, it was confirmed that analysis of the molecular weight distribution had been conducted, including the maximum molecular weight of the hyaluronic acid, without reaching the exclusion limit. Further, the column was connected with another high- performance chromatography apparatus, a 100 mM phosphoric acid buffer solution (pH7.0) as eluent was flowed in at 1.0 mL/min, and a sample of 10 μL of 0.03 mass % solution of bovine serum albumin (product of SIGMA-ALDRICH) was injected. By using a UV detector, Shodex (registered trademark: UV-41, product of SHOWA DENKO K.K.) , a chromatogram was obtained at detection wavelength of 280 nm and the area value was calculated. Subsequently, the column was taken off the high-performance chromatography apparatus and instead, a polytetrafluoroethylene tube having an inner diameter of 0.5 mm and a length of 10 m as a reference column which does not adsorb bovine serum albumin was connected with the high-performance chromatography apparatus . In the same way as in the above, a sample of 10 μL of 0.03 mass % solution of bovine serum albumin was injected to thereby obtain a chromatogram, and the area value was calculated.
The recovery rate of bovine serum albumin defined by dividing the area value of the chromatogram obtained by using the column packed with the surface-modified packing material by the area value of the chromatogram obtained by using the reference column (polytetrafluoroethylene tube) was 78 %.
Comparative Example 1 : A stainless steel column for liquid chromatography having an inner diameter of 8 mm and a length of 300 mm was charged with the same polystyrene gel particles having a sulfo group introduced therein as used in Example 1, by using a packer and a high-pressure pump by equilibrium slurrymethod in the same manner as in Example 1.
This column was connected with high-performance liquid chromatography apparatus, 0.2M sodium chloride solution as eluent was flowed at a rate of 0.5 mL/min, and a sample of hyaluronic acid having a viscosity-average molecular weight of 3, 370, 000 (product of SHISEIDO Co., Ltd.) was analyzed in the same manner as in Example 1. From the obtained chromatogram, it was confirmed that analysis of the molecular weight distribution had been conducted, including the maximum molecular weight of the hyaluronic acid, without reaching the exclusion limit.
Further, into the column, a sample of 10 μl of 0.03 mass % solution of bovine serum albumin was injected under the same conditions as in Example 1, however, since most of the bovine serum albumin was adsorbed by the packing material and little amount of the bovine serum albumin flowed out of the column, a precise chromatogram of the bovine serum albumin could not be obtained.
Comparative Example 2:
A stainless steel column for liquid chromatography having an inner diameter of 8 mm and a length of 300 mm was charged with sulfonated polystyrene particles after the process of surface treatment with glycidol as in Example 1/ by using a packer and a high-pressure pump.by equilibrium slurry method in the same manner as in Example 1. This column was connected with high-performance liquid chromatography apparatus, 0.2M sodium chloride solution as eluent was flowed at a rate of 0.5 mL/min, and a sample of hyaluronic acid having a viscosity-average molecular weight of 3, 370, 000 (product of SHISEIDO Co ., Ltd.) was analyzed in the same manner as in Example 1. From the obtained chromatogram, it was confirmed that analysis of the molecular weight distribution had been conducted, including the maximum molecular weight of the hyaluronic acid, without reaching the exclusion limit. Further, with respect to the column, the recovery rate of bovine serum albumin, which was calculated in the same manner as in Example 1, was 42 %.
INDUSTRIAL APPLICABILITY According to the present invention, a surface-modified packing material for liquid chromatography can be produced by a relatively simple method, and by using this packing material for liquid chromatography, samples of macromolecules such as hyaluronic acid having a viscosity-average molecular weight of 3, 000, 000 or more can be analyzed without reaching the exclusion limit and without adsorbingproteins suchas albuminwhichcoexist inthe sample.

Claims

1. A surface-modified packing material for liquid chromatography, which is obtained by first suspending a support containing a polar group in a water-soluble polymer solution, next removing water content therefrom to coat the support surface with the water-soluble polymer, and then suspending the resulting support having surface coated with the water-soluble polymer in a solvent which does not dissolve the water-soluble polymer to thereby crosslink the water-soluble polymer on the support surface.
2. The surface-modified packing material for liquid chromatography as claimed in claim 1, wherein the water-soluble polymer has a hydroxyl group in its molecule.
3. The surface-modified packing material for liquid chromatography as claimed in claim 2, wherein the water-soluble polymer is a polysaccharide.
4. The surface-modified packing material for liquid chromatography as claimed in claim 3, wherein the polysaccharide as water-soluble polymer is hydroxyethyl cellulose.
5. The surface-modified packing material for liquid chromatography as claimed in claim 1, wherein the water-soluble polymer coating the support surface is crosslinked by using a crosslinking agent.
6. The surface-modified packing material for liquid chromatography as claimed in claim 5, wherein the crosslinking agent is ethylene glycol diglycidyl ether.
7. The surface-modified packing material for liquid chromatography as claimed in claim 1, wherein the support is a synthetic polymer which has a polar group in its molecule.
8. The surface-modified packing material for liquid chromatography as claimed in claim 7, wherein the support is a synthetic polymer which has a sulfo group as polar group in its molecule.
9. The surface-modified packing material for liquid chromatography as claimed in claim 8, wherein the support is sulfonated polystyrene.
10. The surface-modified packing material for liquid chromatography as claimed in claim 1, wherein the support has fine pores having a diameter of 100 to 5,000 nm.
11. The surface-modified packing material for liquid chromatographyas claimed in claim 1, wherein themass-average particle size of the support is from 10 to 100 μm.
12. A method of producing a surface-modified packing material for liquid chromatography, which is obtained by first suspending a support containing a polar group in a water-soluble polymer solution, next removing water content therefrom to coat the support surface with the water-soluble polymer, and then suspending the resulting support having surface coated with the water-soluble polymer in a solvent which does not dissolve the water-soluble polymer to thereby crosslink the water-soluble polymer on the support surface.
13. The method of producing a surface-modified packing material for liquid chromatography as claimed in claim 12, wherein the water-soluble polymer used for the surface modification has a hydroxyl group in its molecule.
14. The method of producing a surface-modified packing material for liquid chromatography as claimed in claim 13, wherein the water-soluble polymer used for the surface modification is a polysaccharide.
15. The method of producing a surface-modified packing material for liquid chromatography as claimed in claim 14, wherein the polysaccharide as water-soluble polymer used for the surface modification is hydroxyethyl cellulose.
16. The method of producing a surface-modified packing material for liquid chromatography as claimed in claim 12, wherein the water-soluble polymer used for the surface modification is crosslinked by using a crosslinking agent.
17. The method of producing a surface-modified packing material for liquid chromatography as claimed in claim 16, wherein the crosslinking agent is ethylene glycol diglycidyl ether.
18. The method of producing a surface-modified packing material for liquid chromatography as claimed in claim 12, wherein the support whose surface is to be modified is a synthetic polymer which has a polar group in its molecule.
19. The method of producing a surface-modified packing material for liquid chromatography as claimed in claim 18, wherein the polar group in the molecule of the synthetic polymer support is a sulfo group.
20. The method of producing a surface-modified packing material for liquid chromatography as claimed in claim 19, wherein the sulfo group in the molecule of the synthetic polymer support is modified with a modifying agent.
21. The method of producing a surface-modified packing material for liquid chromatography as claimed in claim 20, wherein the modifying agent to modify the sulfo group is glycidol.
22. A column which is packed with the packing material for liquid chromatography as claimed in any one of claims 1 to 11.
PCT/JP2005/016174 2004-08-31 2005-08-30 Surface-modified packing material for liquid chromatography and production method thereof WO2006025556A1 (en)

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