WO1989009643A1 - Milieux nouveaux de chromatographie en cellulose reticulee - Google Patents

Milieux nouveaux de chromatographie en cellulose reticulee Download PDF

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Publication number
WO1989009643A1
WO1989009643A1 PCT/US1989/001488 US8901488W WO8909643A1 WO 1989009643 A1 WO1989009643 A1 WO 1989009643A1 US 8901488 W US8901488 W US 8901488W WO 8909643 A1 WO8909643 A1 WO 8909643A1
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WO
WIPO (PCT)
Prior art keywords
cellulose
crosslinked
regain
water
naoh
Prior art date
Application number
PCT/US1989/001488
Other languages
English (en)
Inventor
Peter Grandics
Susan Szathmary
Original Assignee
Peter Grandics
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Peter Grandics filed Critical Peter Grandics
Publication of WO1989009643A1 publication Critical patent/WO1989009643A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J39/00—Cation exchange; Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
    • B01J39/08—Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
    • B01J39/16—Organic material
    • B01J39/18—Macromolecular compounds
    • B01J39/22—Cellulose or wood; Derivatives thereof
    • 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
    • B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
    • 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
    • B01J41/00—Anion exchange; Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
    • B01J41/08—Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
    • B01J41/12—Macromolecular compounds
    • B01J41/16—Cellulose or wood; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B15/00—Preparation of other cellulose derivatives or modified cellulose, e.g. complexes
    • C08B15/005—Crosslinking of cellulose derivatives
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B15/00—Preparation of other cellulose derivatives or modified cellulose, e.g. complexes
    • C08B15/10—Crosslinking of cellulose

Definitions

  • Crystalline alpha-cellulose has been widely used as a support medium in a variety of chromatographic applications because of its hydrophilic nature and enormous surface area.
  • Cellulose is inexpensive and readily available on an industrial scale. It can be derivatized with all the chemistries having significance in chromatography, thereby attaching affinity ligands, ionizable, hydrophobic, or reactive groups to the support for binding proteins or other bioactive substances.
  • Crosslinking did increase flow rates but swelling of cellulose ion exhange resins in alkali and the dimensional changes of ion exchange resins in high salt have not been eliminated by the crosslinking process. This remains a significant obstacle for fast-flow applications as changes in the hydrodynamic resistance of the resin bed may lead to channel formation or plugging up the column.
  • a crosslinked cellulose has been developed which retains the highly rigid structure of cellulose fibers upon chemical derivatization in aqueous solutions and at high pH and ionic strenght.
  • the method of this invention involves a crosslinking process which is carried out in organic solvents.
  • the organic solvent crosslinking medium prevents the hydrogen-bonded structure of cellulose fibers from being dissociated resulting in fibers which not only retain their original, compact and rigid structure of native cellulose but are superior to their native counterpart in dimensional stability and swelling characteristics.
  • the crosslinked product can be readily derivatized by ionic, hydrophobic, or reactive groups and is suitable for the immobilization of proteins or other biomolecules of interest.
  • a method for a novel type of crosslinking of cellulose chromatography media which retains the native structure and excellent physicochemical characteristics of cellulose fibers.
  • native fibrous (microcrystalline) cellulose regenerated cellulose in the form of spherical beads or as a microcrystalline powder can be crosslinked by the method.
  • the improved chromatography support can be derivatized with a variety of chemistries, including but not limited to affinity ligands, ion exchange-, hydrophobic-, or reactive groups having the capacity of binding proteins or other biomolecules .
  • the process for achieving the stabilization of the native structure of cellulose fibers is carried out in a nonaqueous medium.
  • a nonaqueous medium promotes hydrogen bonding between the individual cellulose polymeric strands and thus permits the stabilization of the native crystalline structure.
  • the reaction medium may, for example, contain methanol, ethanol, propanol, acetone, dioxane, N,N'-dimethylformamide or dimethylsulfoxide.
  • polar organic solvents capable of dissolving the crosslinking reagent may be employed.
  • crosslinking reagents may be employed in stabilizing the structure of cellulose fibers.
  • the crosslinking agents are chemicals that have at least two reactive groups, both of which are reactive with cellulose.
  • the crosslinking reagent will have the structure R1-A-R2, wherein Ri and R 2 are moieties capable of covalently binding with hydroxyl moieties on cellulose, and A is any group capable of linking Ri and R ⁇ .
  • Ri and R- moieties include vinylsulfone, epoxy, and aliphatic or aromatic halogenides.
  • Crosslinking agents suitable for use with cellulose are well-known, and any of the conventional agents that are soluble in organic solvents may be used.
  • Such conventional crosslinking agents include divinylsulfone, 1 , 3-dibromo-2-propanol, 1 , 4-butanedioldiglycidyl ether, epichlorohydrine or cyanuric chloride.
  • the concentration of the crosslinking agent is between 1 to 20 percent based on volume with 5 to- 10 percent being most preferred.
  • the crosslinking reaction is catalyzed either by acid or base catalysts. Reagents like ZnCl ⁇ , BF3 , KOH or NaOH may be employed to act as catalyst in the crosslinking reaction.
  • the rection is suitably conducted at temperatures ranging from 0 C to 100 C preferably 20 C to 60 C with room temperature being most preferred.
  • the reaction is carried out with a concentration of solids that is conveniently handled.
  • the reaction mixture contains 1 to 50 percent and preferably 10 to 30 percent of cellulose material.
  • the completion of the reaction takes several hours with 16 to 24 h being preferred.
  • the crosslinked product is suitably washed to remove reactants and tested for regain and dimensional stability.
  • Regain is an empirical parameter which relates to the ability of cellulose to swell and is defined as the gram weight of water retained by 1 gram of dried cellulose.
  • the dimensional stability of crosslinked celluloses have been tested in an HPLC instrument at various flow-rates while the pressure in the system has been recorded. This test provides information on the stability of cellulose particles and on the linear flow-rates a cellulose-based chromatography column filling can withstand.
  • Method A Microcrystalline cellulose (50 g) obtained from Whatman Paper Ltd., Maidstone, Kent, UK, is extensively washed with dry dioxane on a sintered glass filter and transferred into a 1 L three-neck round bottom flask equipped with a teflon impeller stirrer, a thermometer and a 100 ml dropper funnel. Then, 200 ml of dry dioxane and 5 g of epichlorohydrine-BF3 complex mixed in 50 ml of dry dioxane is added over a period of 30 min under continuous stirring. The mixture is allowed to stand overnight at room temperature and then poured on a sintered disk filter funnel and washed with dry dioxane, dioxane/water (1:), and distilled water, respectively.
  • Method B Microcrystalline cellulose (50 g) obtained from Whatman Paper Ltd., Maidstone, Kent, UK, is extensively washed with dry methanol on a sintered glass filter and transferred into a 1 L three-neck round bottom flask equipped with a teflon impeller stirrer, a thermometer and a 100 ml dropper funnel. Then, 250 ml of 0.5 M methanolic KOH is added followed by 5 g of epichlorohydrine mixed with 50 ml of dry methanol is added over a period of 30 min under continuous stirring. The temperature is kept around 25-30 C during the addition of reagents and then the mixture is allowed to stand overnight at room temperature. The mixture is poured on a sintered disk filter funnel and washed with dry methanol, methanol/water (1:), and distilled water, respectively.
  • EXAMPLE 2 EXAMPLE 2
  • the anion exchange derivative is prepared by mixing 25 g of crosslinked cellulose, prepared in accordance with Example 1, with
  • the cation exchange derivative is prepared by mixing 25 g of crosslinked cellulose, prepared in accordance with Example 1, with 70 ml of distilled water and 40 ' ml of 47% NaOH for 10 min. The matrix was collected by filtration and 50 ml isopropylalcohol and 2 g sodium monochloroacetate is added and the mixture is heated to 80 C with stirring for 30 min. The product was filtered and washed thoroughly with distilled water. The resulting material comprised an effective anion exchange resin.
  • the reactive group is introduced into the support by stirring 25 g of crosslinked support, prepared in accordance with Example 1, with 20 ml of 1 M NaOH, 4 ml epichlorohydrine and 100 mg NaBH 4 at room temperature for 4 h.
  • the activated resin is recovered by filtration, washed with distilled water to neutrality, with 50 ml of acetone and then with distilled water to remove acetone.
  • the hydrophobic group is introduced into the support from Example 4 by stirring 25 g of activated support with 20 ml of 0.1 M NaHCO3/acetone (1:1), pH 8.5 , containing 0.5 ml of n-hexylamine for 4 h.
  • the resin is recovered by filtration, washed with distilled water/acetone (1:1) and then with water to remove acetone.
  • the resulting material comprises a-cellulose substrate with hydrophobic n-hexylamine moieties covalently bound thereto.
  • Immunoglobulin (mouse IgG) is coupled to the activated support by stirring 25 g of activated resin with 20 ml of 0.1 M NaHCO 3 , pH 8.0, containing 0.2 g of antibody at 4 C for 16 h .
  • the coupled resin is recovered by filtration, washed with 0.1 M NaHCO3 and the residual reactive groups are inactivated by incubation in a 0.1 M ethanolamine, pH 8.5, for 4 h.
  • the coupled resin is stored in phosphate-buffered saline at 4 C.
  • a cellulose sample is placed in 10 times of the resin volume of either distilled water or 0.5 M NaOH and allowed to stand for 10 rain. The material is filtered and the alkali-treated sample is washed until the pH of the supernatant falls to 8. The samples are then transferred into Gooch crucibles, moistened with water and placed in centrifuge cups. The centrifuge is spun at approximately 3000xg for 60 min and the water content of the samples is measured after drying overnight at 105 C.
  • the subject invention is aimed at rectifying these shortcomings and introduces a crosslinking method which prevents swelling and even improves upon the initial rigidity of microcrystalline alpha-cellulose.
  • the method is based upon crosslinking cellulose in a nonaqueous medium with the omission of extreme pH conditions.
  • the resulting product swells in NaOH to a lower degree than the inital cellulose and exhibits much lower swelling than its conventionally-crosslinked counterpart which is reflected by the regain values (Table 1).
  • g/g 0.5 M NaOH 0.84 3.5 0.52 evidences the extreme stability of the cellulose chromatography matrix of subject invention.
  • Native microcrystalline cellulose, conventionally crosslinked cellulose and a crosslinked cellulose prepared by our method has been compared in an HPLC apparatus. Columns of equal dimensions have been packed and run at various flow-rates while the pressure in the system has been recorded (Table 2) .

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  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Materials Engineering (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Analytical Chemistry (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

Une cellulose réticulée retient la structure très rigide de la cellulose lorsqu'elle est soumise à une dérivation chimique dans une solution aqueuse à un pH et une résistance ionique élevés. Un procédé comprend la réticulation dans des solvants organiques. Le solvant organique de réticulation empêche la structure liée par l'hydrogène des fibres de cellulose de se dissocier, ce qui permet d'obtenir des fibres qui non seulement conservent la structure origninelle rigide et compacte de la cellulose naturelle mais présentent des caractéristiques de gonflage et de stabilité dimensionnelle d'une qualité supérieure à celles de la cellulose naturelle. Le produit réticulé se laisse aisément dériver lorsqu'il est exposé à des groupes réactifs, hydrophobes ou ioniques et convient pour mobiliser des protéines ou d'autres biomolécules.
PCT/US1989/001488 1988-04-12 1989-04-10 Milieux nouveaux de chromatographie en cellulose reticulee WO1989009643A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US180,537 1980-08-22
US18053788A 1988-04-12 1988-04-12

Publications (1)

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WO1989009643A1 true WO1989009643A1 (fr) 1989-10-19

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991009878A1 (fr) * 1989-12-29 1991-07-11 Institut Für Polymerenchemie 'erich Correns' Produits cellulosiques en perles destines a des substances de separation et de support, ainsi que leur fabrication
US5447859A (en) * 1993-07-16 1995-09-05 Viagene Method for the purification or removal of retroviruses using sulfated cellulose
WO1995023885A1 (fr) * 1994-03-01 1995-09-08 Lenzing Aktiengesellschaft Procede de purification de solutions aqueuses d'amino-oxydes tertiaires
US5527902A (en) * 1989-12-29 1996-06-18 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V. Bead-shaped cellulose products for separating and carrier materials and their manufacture
WO1999024459A1 (fr) * 1997-11-10 1999-05-20 Peter Grandics RETICULATION ET FONCTIONNALISATION INDUITE PAR Mn(IV) DE SUPPORTS CHROMATOGRAPHIQUES
EP1260632A1 (fr) * 2001-05-23 2002-11-27 Linificio e Canapificio Nazionale S.p.A. Finissage antifroisse de fibres et fils de lin
EP1260522A1 (fr) * 2001-05-23 2002-11-27 Linificio e Canapificio Nazionale S.p.A. Ethers cellulosiques O-alkyles et procédé de préparation

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3208994A (en) * 1961-10-25 1965-09-28 Pharmacia Ab Process for preparing hydrophilic copolymerization and product obtained thereby
US3959080A (en) * 1972-09-26 1976-05-25 Merck Patent Gesellschaft Mit Beschrankter Haftung Carrier matrix for the fixation of biochemically effective substances and process for the preparation thereof
US4158703A (en) * 1976-05-03 1979-06-19 Beckman Instruments, Inc. T3 uptake test employing covalently bound bovine serum albumin

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3208994A (en) * 1961-10-25 1965-09-28 Pharmacia Ab Process for preparing hydrophilic copolymerization and product obtained thereby
US3959080A (en) * 1972-09-26 1976-05-25 Merck Patent Gesellschaft Mit Beschrankter Haftung Carrier matrix for the fixation of biochemically effective substances and process for the preparation thereof
US4158703A (en) * 1976-05-03 1979-06-19 Beckman Instruments, Inc. T3 uptake test employing covalently bound bovine serum albumin

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF APPLIED POLYMER SCIENCE, Vol. 7, pp 1371-1389, 1983 "The action of epichlohydrin in the presence of alkalies and various salts on the crease recovery of cotton". (MCKELVEY) see the reaction scheme at page 1371. *
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, pp. 1965-1969, 1932 "Esterification of polyhydric alcohols", (FAIRBORNE). See the entire document. *
TEXTILE RESEARCH JOURNAL, February 1957: "Epoxy resins in the crease proofing of cotton", (SCHROEDER), see pages 136, 138 and 139. *
TEXTILE RESEARCH JOURNAL, Vol. 33, No. 8, pp 583-599, August 1963, "The base-catalyzed condensation of cellulosic fabrics with volatile epoxides", (FRANCIS). See the abstract and pages 583 and 584. *
TEXTILE RESEARCH JOURNAL, Vol. 35, No. 8, pp. 446-452, May 1965, "A vapor-phase epichlorhydrin process for wet and dry wrinkle recovery", (FERRANTE). See the entire document. *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991009878A1 (fr) * 1989-12-29 1991-07-11 Institut Für Polymerenchemie 'erich Correns' Produits cellulosiques en perles destines a des substances de separation et de support, ainsi que leur fabrication
US5527902A (en) * 1989-12-29 1996-06-18 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V. Bead-shaped cellulose products for separating and carrier materials and their manufacture
US5447859A (en) * 1993-07-16 1995-09-05 Viagene Method for the purification or removal of retroviruses using sulfated cellulose
WO1995023885A1 (fr) * 1994-03-01 1995-09-08 Lenzing Aktiengesellschaft Procede de purification de solutions aqueuses d'amino-oxydes tertiaires
GB2291833A (en) * 1994-03-01 1996-02-07 Chemiefaser Lenzing Ag Process for purifying aqueous solutions of tertiary amino oxides
GB2291833B (en) * 1994-03-01 1997-11-26 Chemiefaser Lenzing Ag Process for purifying aqueous solutions of tertiary amino oxides
WO1999024459A1 (fr) * 1997-11-10 1999-05-20 Peter Grandics RETICULATION ET FONCTIONNALISATION INDUITE PAR Mn(IV) DE SUPPORTS CHROMATOGRAPHIQUES
EP1260632A1 (fr) * 2001-05-23 2002-11-27 Linificio e Canapificio Nazionale S.p.A. Finissage antifroisse de fibres et fils de lin
EP1260522A1 (fr) * 2001-05-23 2002-11-27 Linificio e Canapificio Nazionale S.p.A. Ethers cellulosiques O-alkyles et procédé de préparation

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Publication number Publication date
AU3539989A (en) 1989-11-03

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