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WO1999038594A1 - Procede de separation de melanges de substances au moyen de polysaccharides - Google Patents

Procede de separation de melanges de substances au moyen de polysaccharides Download PDF

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Publication number
WO1999038594A1
WO1999038594A1 PCT/EP1999/000473 EP9900473W WO9938594A1 WO 1999038594 A1 WO1999038594 A1 WO 1999038594A1 EP 9900473 W EP9900473 W EP 9900473W WO 9938594 A1 WO9938594 A1 WO 9938594A1
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WO
WIPO (PCT)
Prior art keywords
separation
polysaccharides
chromatography
microparticles
poly
Prior art date
Application number
PCT/EP1999/000473
Other languages
German (de)
English (en)
Inventor
Holger Bengs
Arnold Schneller
Gitte Böhm
Doris Andert
Original Assignee
Aventis Research & Technologies Gmbh & Co. Kg
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 Aventis Research & Technologies Gmbh & Co. Kg filed Critical Aventis Research & Technologies Gmbh & Co. Kg
Priority to JP2000529314A priority Critical patent/JP2002501811A/ja
Priority to EP99907417A priority patent/EP1071499A1/fr
Priority to AU27189/99A priority patent/AU2718999A/en
Priority to CA002319386A priority patent/CA2319386A1/fr
Publication of WO1999038594A1 publication Critical patent/WO1999038594A1/fr

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Classifications

    • 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
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
    • B01D15/3833—Chiral chromatography
    • 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
    • B01J20/262—Synthetic macromolecular compounds obtained otherwise than by reactions only involving carbon to carbon unsaturated bonds, e.g. obtained by polycondensation
    • 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
    • B01J20/282—Porous sorbents
    • B01J20/285—Porous sorbents based on 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
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/29—Chiral phases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B57/00—Separation of optically-active compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B63/00—Purification; Separation; Stabilisation; Use of additives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C29/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring
    • C07C29/74—Separation; Purification; Use of additives, e.g. for stabilisation
    • C07C29/76—Separation; Purification; Use of additives, e.g. for stabilisation by physical treatment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
    • C07C37/68—Purification; separation; Use of additives, e.g. for stabilisation
    • C07C37/70—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment
    • C07C37/82—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment by solid-liquid treatment; by chemisorption
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2602/00—Systems containing two condensed rings
    • C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
    • C07C2602/14—All rings being cycloaliphatic
    • C07C2602/26—All rings being cycloaliphatic the ring system containing ten carbon atoms
    • C07C2602/28—Hydrogenated naphthalenes

Definitions

  • the invention relates to a method for separating mixtures of substances, in particular enantiomers, using spherical microparticles made of chemically and physically unmodified, water-insoluble, linear polysaccharides as the separating material.
  • Chromatography is an effective method for the complete separation of components of a mixture, in particular for the separation of mixtures of similar compounds, e.g. of stereoisomers. Due to the increasing interest in pure enantiomers for pharmaceutically and biochemically active substances and crop protection agents, the chromatographic separation of enantiomeric compounds is of particular interest. For the purification of these compounds, continuously improved processes are being developed and better separating materials are sought, in particular based on the polysaccharides cellulose and starch, two easily accessible and inexpensive polymers with chiral atoms.
  • the article Chromatographie 65, LaborPraxis, 730-738 (1990) describes the use of microcrystalline tribenzoyl cellulose with grain sizes of 10 to 20 ⁇ m in comparison to triacetyl cellulose as a sorbent for the enantiomer separation in chromatography. Both substances can be used for analytical and preparative separations. They are accessible through the derivatization of cellulose. Unmodified polymers are not used. The introduction of smaller grain sizes down to 5 ⁇ m in order to avoid dead volumes and a compression of the column that occurs during operation is described as desirable.
  • WO 95/05879 also deals with the separation of enantiomers by liquid chromatography. Particular attention is paid here to the mobile phase to improve the separation performance. As a stationary phase 2
  • Carbamate derivatives of cellulose and amylose and ester derivatives of cellulose are used.
  • DE-A-43 17 139 describes a special process for the enantiomer separation of inhalation anesthetics by means of preparative gas chromatography.
  • Cyclodextrins in polysiloxane solution on a porous carrier material (Chromosorb) derivatized with ester groups are used as the stationary phase.
  • the cyclodextrins have to be chemically modified, dissolved in polysiloxane and fixed on a suitable carrier material.
  • polymeric siloxanes containing cyclodextrin derivatives are used as chiral stationary phase in analytical and preparative gas chromatography (GC), chromatoraphy (LC). These peralkyl cyclodextrins are chemically bound to the polysiloxanes. The material used is thus obtained by chemical modification of the cyclodextrins and subsequent chemical fixation to polysiloxanes.
  • a vinyl derivative of a polysaccharide e.g. cellulose
  • a porous support e.g. silica gel
  • a second variant is the copolymerization of a porous carrier (e.g. modified silica gel) containing vinyl groups with a vinyl derivative of a polysaccharide.
  • a porous carrier e.g. modified silica gel
  • chemical modifications are necessary in order to obtain suitable separating material.
  • EP-B-0 157 365 describes stationary phases for enantiomer and isomer separation and for gel permeation chromatography based on polysaccharide carbamate derivatives.
  • Cellulose, amylose, chitosan, xylan, dextran and inulin are suitable as polysaccharides.
  • the powder produced can have a particle diameter of 1 ⁇ m to 300 ⁇ m directly or according to physical 3 or chemical fixation can be used on a porous support. A chemical modification is therefore necessary in order to obtain suitable separating material.
  • DE-C 26 55 292 and DE-C 25 55 361 use porous gels from dextran derivatives as separation media in electrophoretic separation materials.
  • dextran derivatives containing vinyl groups In order to achieve insolubility, dextran derivatives containing vinyl groups must be crosslinked by radical polymerization.
  • Chemical and / or physical modifications are to be understood in particular as derivatizations through the introduction of special groups, covalent fixations on a carrier material and subsequent chemical and / or physical crosslinking.
  • the object of the invention is therefore to simplify and reduce the cost of the chromatography process by circumventing chemical and physical modifications to the separation material used.
  • separation material spherical microparticles made of chemically and physically unmodified, water-insoluble, linear polysaccharides (hereinafter called separation material according to the invention) as separation material.
  • Spherical microparticles are to be understood as meaning microparticles which are approximately spherical in shape.
  • the 4 Define the radius of the sphere in all spatial directions, for the spherical microparticles the axis lengths can deviate from 1% to 40%.
  • the surface of the spherical microparticles can be compared macroscopically with that of a raspberry, the depth of the “indentations” or “incisions” being a maximum of 20% of the average diameter of the spherical microparticles.
  • Figures 1 to 4 show scanning electron microscope (SEM) images (Camscan S-4) of the spherical microparticles used.
  • Linear polysaccharides according to the present invention can be polyglucans or other linear polysaccharides such as pullulans, pectins, mannans or polyfructans.
  • poly- (1,4- ⁇ -D-glucan) is particularly preferred.
  • the separating material according to the invention can be used for the chromatographic separation of substance mixtures, particularly preferably stereoisomers, very particularly preferably enantiomers. Therefore, the use of the separating material according to the invention in preparative and analytical chromatography such as gas chromatography, preparative and analytical thin layer chromatography and in particular high performance liquid chromatography (HPLC) is of particular interest. Furthermore, the use of the separating material according to the invention in gel permeation chromatography (GPC) 5 of interest in the separation of polymers of different molecular weights.
  • GPC gel permeation chromatography
  • One application that also falls within the scope of the invention is the separation of substances from solutions, emulsions or suspensions through specific or non-specific interactions with low molecular weight or polymeric compounds. However, ionic compounds, in particular metal cations, are also of interest for use. Special effects can be achieved in particular due to the structural similarity to polysaccharides in the separation of monosaccharides, disaccharides and oligos
  • the invention also relates to the treatment of water, in particular waste water, the analysis of biological material, in particular blood and sera, or for example the separation or separation of genetic material (nucleic acids) or other biogenic compounds (e.g. oligonucleotides, peptides, proteins).
  • genetic material nucleic acids
  • biogenic compounds e.g. oligonucleotides, peptides, proteins
  • Chromatography in the sense of the invention is understood to mean physical separation processes in which the separation of substances takes place by distribution and / or adsorption between a stationary and a mobile phase.
  • the separating material according to the invention preferably poly (1,4-alpha-D-glucan) is used as the stationary phase and combined with a liquid and / or gaseous mobile phase.
  • HPLC high-LC, GPC, GC, DC and further or any specifications of chromatographic methods or methods similar to chromatography. In principle, any chromatographic method of the invention is accessible.
  • HPLC High Performance (or) High Pressure Liquid Chromatography
  • HPLC works with very fine material (3-10 ⁇ m), since the separation performance of a column increases with decreasing grain size of the stationary phase. The fine particle size of the separating materials requires high pressures (up to 400 bar).
  • GPC Gel permeation chromatography
  • Exclusion chromatography which can also be operated as HPLC, the stationary phase consists of beads with a heteroporous swollen network, the pore size distribution of which varies over several orders of magnitude, so that fractionation takes place according to molecular size, which allows the molecular size distribution of polymers to be determined quickly.
  • Gas chromatography is used to separate mixtures of substances that are in gaseous form or that can be vaporized without decomposition, with a gas serving as the mobile phase.
  • Gas chromatographic analysis begins with the application of a gas, an evaporable liquid or an evaporable solid to the thermostatted separation column. With the help of the carrier gas (He, N 2 or H 2 ) the substances are transported through the column, where the chromatographic separation takes place.
  • He, N 2 or H 2 carrier gas
  • Thin layer chromatography is a chromatographic process with a multi-stage distribution process, the stationary phase (the separating material is called sorbent here) being a thin layer on a suitable support, for example glass, polyester or aluminum. This layer separates by elution with the eluent (mobile phase).
  • the invention therefore relates to the separating material according to the invention as such, containing spherical microparticles of chemically and physically unmodified, water-insoluble, linear polysaccharides and, if appropriate, further auxiliaries, additives and carriers.
  • Linear polysaccharides are polysaccharides which are built up from monosaccharides, disaccharides or other monomeric units in such a way that the monosaccharides, disaccharides or other monomeric units are always linked to one another in the same way.
  • Each basic unit or building block defined in this way has exactly two links, one each to a different monomer. The two basic units, which form the beginning and the end of the polysaccharide, are excluded from this. These basic units have only one link to another monomer. With three links (covalent bonds) one speaks of a branch. Branches do not occur or only to a minor extent, so that they are not accessible to the conventional analytical methods with very small branch fractions.
  • Examples of preferred water-insoluble linear polysaccharides are linear poly-D-glucans, the type of linkage being immaterial as long as there is linearity in the sense of the invention.
  • Examples are poly (1,4-alpha-D-glucan) and poly (1,3-beta-D-glucan), with poly (1,4-alpha-D-glucan) being particularly preferred.
  • branching If the basic unit has three or more links, this is referred to as branching.
  • the linear water-insoluble polysaccharides have a degree of branching of less than 8%, i.e. they have less than 8 branches per 100 basic units.
  • the degree of branching is preferably less than 4% and in particular a maximum of 1.5%.
  • the degree of branching in the 6-position is less than 4%, preferably a maximum of 2% and in particular a maximum of 0.5% and the degree of branching in the 8 other positions not involved in the linear linkage, for example the 2- or 3-position in the case of the preferred poly (1,4-alpha-D-glucan), is preferably in each case a maximum of 2% and in particular a maximum of 1%.
  • Polysaccharides in particular poly-alpha-D-glucans, are particularly preferred which have no branches or whose degree of branching is so minimal that it can no longer be detected using conventional methods.
  • Nature-identical polysaccharides are understood to mean polymers which either do not occur in nature or only in a mixture with other compounds, which can also be other polymers.
  • Such nature-identical polymers can be produced by processes which fall under the broadest definition of the term biotechnological and genetic engineering processes. On the one hand, this includes biotechnical processes or processes, as will be defined below, but also those that lead to corresponding connections through the use and modification of, for example, bacteria, fungi or algae.
  • the term also includes, for example, polysaccharides which can be obtained by applying bio- or genetic engineering processes to higher plants, so that separation from the plant can take place. Such plants include, in particular, the potato, corn, cereals, cassava, rice and peas. However, other plants which produce polysaccharides can also be classified in this category from the aspect of the invention.
  • Linear, water-insoluble polysaccharides which are used in a biotechnical, in particular a biocatalyzed 9 tables can also be produced using a biotransformer or a fermentative process.
  • Linear polysaccharides produced by biocatalysis in the context of this invention means that the linear polysaccharide by catalytic reaction of monomeric building blocks such as oligomeric saccharides, e.g. of mono- and / or disaccharides is produced by using a so-called biocatalyst, usually an enzyme, under suitable conditions for use in the reaction.
  • biocatalyst usually an enzyme
  • Linear polysaccharides from fermentations are, in the parlance of the invention, linear polysaccharides which are obtained by fermentative processes using organisms found in nature, such as, for example, fungi, algae or bacteria, or using organisms not occurring in nature, but with the aid of genetic engineering Methods of general definition modified natural organisms, such as fungi, algae or bacteria can be obtained.
  • linear polysaccharides can also be obtained by treating nonlinear polysaccharides containing branches with one or more enzymes such that the branches are cleaved from the backbone of the polysaccharide so that after it Separation linear polysaccharides are present.
  • enzymes are, for example, amylases, iso-amylases, pullulanases or also gluconohydrolases. 10
  • sparingly soluble to practically insoluble compounds in particular very sparingly soluble to practically insoluble compounds, are preferred.
  • the preferred poly- (1,4- ⁇ -D-glucan) can be produced in various ways. A very advantageous method is described in WO 95/31553. The revelation of Scripture is explicitly mentioned here 11 related.
  • the poly- (1,4- ⁇ -D-glucan) is produced by means of a biocatalytic (biotransformatory) process using amylosucrase.
  • the poly- (1,4- ⁇ -D-glucan) can be produced using polysaccharide synthases, starch synthases, glycol transferases, 1,4- ⁇ -D-glucan transferases, glycogen synthases or phosphorylases by means of a biocatalytic process.
  • the spherical microparticles are produced by dissolving the water-insoluble, linear polysaccharide, in particular the poly- (1,4-D-glucan), in a solvent, particularly preferably in DMSO, introducing the solution into a precipitant, preferably water, cooling the resulting mixture to preferably 10 ° C to -10 ° C and separating the microparticles formed.
  • a precipitant preferably water
  • suitable additives are, for example, surfactants such as sodium dodecyl sulfate or N-methylglucanoamide, sugar, e.g. Fructose, sucrose, glucose.
  • the molecular weights M w of the linear polysaccharides used according to the invention can vary within a wide range from 10 3 g / mol to 10 7 g / mol.
  • the molecular weight M w describes the weight average of the molecular weight and is determined using gel per 12 meation chromatography compared to a calibration with pullulin standards.
  • the particles can have mean diameters (number average) such as 1 nm to 100 ⁇ m, preferably 100 nm to 10 ⁇ m, particularly preferably 1 ⁇ m to 5 ⁇ m.
  • the distribution of the diameters is characterized by a high level of uniformity.
  • microparticles made of poly (1,4-D-glucan) are distinguished by a specific surface area of 1 m 2 / g to 100 m 2 / g, particularly preferably 3 m 2 / g to 10 m 2 / g.
  • n-Hexane e.g. n-Hexane, dichloromethane, tetrahydrofuran, 1,4-dioxane, ethanol, acetone, acetonitrile, methanol, n-heptane and water are used.
  • eluent e.g. n-Hexane, dichloromethane, tetrahydrofuran, 1,4-dioxane, ethanol, acetone, acetonitrile, methanol, n-heptane and water are used.
  • microparticles of linear polysaccharides are slurried, whirled up by means of an Ultraturrax and degassed in an ultrasonic bath.
  • a solvent for example n-heptane
  • the supension is filled into a column and packed evenly and densely by constant tapping on the column, constant shaking in combination with brief treatment in an ultrasonic bath, so that low-volume packs are created.
  • the separations are carried out in a state-of-the-art chromatography system for HPLC.
  • the sample concentration is 2.0 to 0.025 mg / l solvent, particularly preferably 0.25 to 0.05 mg / l solvent.
  • the elution rate is 2.0 to 0.25 ml / min, preferably 1.0 to 0.4 ml / min, particularly preferably 0.5 ml / min.
  • This information may vary depending on the solvent used.
  • the prints used are strongly dependent on the polarity of the solvent. 13 pending. They are generally in a range between 30 bar and 200 bar. For example, the following pressures were measured: 138 to 159 bar for acetonitrile, about 100 bar for ethyl acetate, about 62 bar for t-butyl methyl ether and 54 to 70 bar for n-heptane.
  • An internal standard for example 1,3,5-tri-tert-butylbenzene, can be used.
  • Fig. 1 Scanning electron microscope image (see Example 4) of the spherical microparticles used in 5000x magnification.
  • Fig. 2 Scanning electron microscope image (see Example 4) of the spherical microparticles used in 10000x magnification.
  • Fig. 3 Scanning electron microscope image (see Example 4) of the spherical microparticles used in 15000x magnification.
  • Fig. 4 Scanning electron microscope image (see Example 4) of the spherical microparticles used in 20000x magnification.
  • 5 Agarose gel (detection with ethidium bromide 0.5 pg / ml at 256 nm) according to
  • Example 12 see legend. 6: agarose gel (detection with ethidium bromide 0.5 pg / ml at 256 nm) according to
  • a) 400 g of poly (1,4- ⁇ -D-glucan) are dissolved in 2 l of dimethyl sulfoxide (DMSO, p.a. from Riedel-de-Haen) at 60 ° C within 1.5 hours. Then it is stirred for one hour at room temperature. The solution is added in 20 l of double-distilled water with stirring through a dropping funnel over a period of 2 h. The mixture is stored at 4 ° C. for 44 h. A fine suspension is formed. The particles are separated by first decanting the supernatant. The sediment is slurried and centrifuged in small portions (RC5C ultracentrifuge: 5 minutes each at 5000 revolutions per minute).
  • DMSO dimethyl sulfoxide
  • the measurements of the specific surface are made with a Sorptomatic 1990 (from Fisons Instruments). The ' default method sorptomatic ' method is used for evaluation. For the examination, the samples are dried at 80 ° C. in a vacuum (membrane pump from Vacuubrand GmbH & Co., CVC 2) overnight. The potato starch and the poly- (1,4- ⁇ -D-glucan) obtained directly from the biotransformation are ground beforehand so that the mass of the 18th
  • Particle is in the range of less than 200 microns.
  • a commercially available mill is used for this (Waring®).
  • the procedure is as follows: 100 mg of the particles from Example 3 are overlaid with 3 ml of the solvent in a crimp-top vial. The observation time is 24 hours. The particles are examined every hour for changes. For this purpose, the reaction vessel is swiveled.
  • Example 3a 5 g of the particles from Example 3a are slurried in about 60 ml of heptane.
  • the suspension is briefly whirled up using an Ultraturrax (Ika) and then degassed in an ultrasonic bath (Sonorex Super RK510H).
  • the suspension is then filled under maximum flow into a column measuring 125 mm in length and 4 mm in diameter (Hibar® finished column from Merck AG). Constant tapping on the column, or constant shaking of the column in combination with short-term, approximately one-minute treatment in the ultrasonic bath ensures a tight and even packing of the separating material, so that packs with low dead volume result.
  • the separations are carried out in a Lichograph® chromatography system from Merck.
  • the individual components are: a gradient pump L 6200 A, a UV detector L-400 (measurement at 254 nm), a chromato-integrator D-2500 and a separation column Hibar® RT-125-4.
  • the sample concentration is ideally 0.05 to 0.1 mg / ml solvent. 1,3,5-tritert.-butylbenzene (from Fluka) can be used as the internal standard.
  • the chromatography column is prepared with n-heptane (Aldrich for HPLC). The plant is operated with n-heptane. The elution rate is 0.5 ml / min. The pressure is 54 bar. The substances to be separated are: indole and 2-methylindole. 21
  • Example 10 The test is carried out analogously to Example 10. In this example the pressure is around 70 bar. The concentration of the samples is 0.25 mg / ml solvent. 22
  • Disposable centrifuge filters (Schleicher & Schull, eg Centrix, catalog number 467012 (April 1996): "Filter 1" in FIGS. 5 and 6) are used with 580 mg of the microparticles produced as in Example 3 as filter material or adsorbent with 3 ml of a buffer 1 (see below) Equilibrated solution (possibly centrifuge (2000 rpm)) Then 2 ml of an aqueous DNA plasmid solution (plasmid used: pBluescript II SK) with a concentration of 50 ⁇ g / ml are added to the filter (centrifuge if necessary) (5min at 2000 rpm)) There is a reference run with (“Filter 2" in Fig. 5 and 6) Qiagen® Midi drapep (Hilden, Germany) and another reference run with Qiagen® Midi ebookp - cartridges without Qiagen® filter medium, which were equipped with the separating material.
  • Each 5 ⁇ l eluate is provided with about 1/10 of the concentration with staining reagent (marker) and applied to an agarose gel plate (60% sucrose, 20 mM EDTA, 0.025% bromophenol blue) with subsequent gel electrophoresis (company Biorad, Power Supply, model 100 / 200).
  • staining reagent labeled with staining reagent
  • an agarose gel plate (60% sucrose, 20 mM EDTA, 0.025% bromophenol blue) with subsequent gel electrophoresis (company Biorad, Power Supply, model 100 / 200).
  • a marker MG 5,000
  • the DNA plasmid solution used are applied as a reference.
  • Fig. 5 shows that on 23 separating material according to the invention (here filter means) the sample DNA was retained.
  • buffer 2 a second buffer (buffer 2) are added to the column (filter) for elution and processed quickly by centrifugation (5 min at 2000 rpm).
  • the eluates (references as mentioned above) are applied to an agarose gel plate (60% sucrose, 20 mM EDTA, 0.025% bromophenol blue) and then gel electrophoresis (company Biorad, Power Supply, model 100/200) is carried out (see FIG. 6).
  • a marker MW 5,000
  • the DNA plasmid solution lane 7
  • FIG. 6 shows that when the separating material according to the invention is used, the plasmid DNA used is again eluted from the column.
  • the comparison with commercially available filter media shows the quality of the filter process, which has at least the same quality.
  • Buffer 1 750 mM NaCl
  • Lane t marker (Boehringer Mannheim DNA Molecular Weight Marker X) lane 2 filter 2 without separating material (blind reference) lane 3 filter 2 filled with separating material according to the invention lane 4 filter 1: Qiagen® cartridge with separating material according to the invention lane 5 filter 1: Qiagen® Midi drapep (Hilden , Germany) Reference lane 6 Filter 2 with separation material lane 7 pure DNA plasmid solution (commercially available plasmid pBluescript II SK)
  • Lane marker (Boehringer Mannheim DNA Molecular Weight Marker X) lane 2 filter 2 without separating material (blind reference)
  • Track 3 filter 2 filled with separating material according to the invention.
  • Track 4 filter 1 Qiagen® cartridge with separating material according to the invention.
  • Track 5 filter 1 Qiagen® Midi drapep (Hilden, Germany).
  • Track 7 pure DNA plasmid solution (commercial plasmid pBluescript II SK)

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

Abstract

L'invention concerne un procédé de séparation, par chromatographie, de mélanges de substances au moyen de microparticules sphériques constituées de polysaccharides linéaires, insolubles dans l'eau, non modifiés chimiquement et physiquement.
PCT/EP1999/000473 1998-01-29 1999-01-26 Procede de separation de melanges de substances au moyen de polysaccharides WO1999038594A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP2000529314A JP2002501811A (ja) 1998-01-29 1999-01-26 多糖類を使用する物質混合物の分離
EP99907417A EP1071499A1 (fr) 1998-01-29 1999-01-26 Procede de separation de melanges de substances au moyen de polysaccharides
AU27189/99A AU2718999A (en) 1998-01-29 1999-01-26 Separation of substance mixtures using polysaccharides
CA002319386A CA2319386A1 (fr) 1998-01-29 1999-01-26 Procede de separation de melanges de substances au moyen de polysaccharides

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19803415.6 1998-01-29
DE19803415A DE19803415A1 (de) 1998-01-29 1998-01-29 Trennung von Stoffgemischen unter Einsatz von Polysacchariden

Publications (1)

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WO1999038594A1 true WO1999038594A1 (fr) 1999-08-05

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DE19860371A1 (de) 1998-12-28 2000-06-29 Aventis Res & Tech Gmbh & Co Kosmetische oder medizinische Zubereitung für die topische Anwendung
JPWO2006082968A1 (ja) * 2005-02-07 2008-06-26 江崎グリコ株式会社 α−１，４−グルカンを含有する吸着剤およびその製造方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0377046A1 (fr) * 1988-04-05 1990-07-11 Kanebo Ltd. Particules fines de cellulose poreuse echangeuse d'ions, procede pour leur production, et porteur d'affinite
WO1995005879A1 (fr) * 1993-08-27 1995-03-02 The Dow Chemical Company Procede pour la separation d'enantiomeres
WO1997038018A1 (fr) * 1996-04-11 1997-10-16 Amersham Pharmacia Biotech Ab Procede de production d'un gel polysaccharidique reticule poreux et son utilisation comme support pour filtration sur gel et en chromatographie

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EP0321597B1 (fr) * 1986-09-09 1992-11-11 Fraunhofer-Gesellschaft Zur Förderung Der Angewandten Forschung E.V. Adsorbant sélectif pour lier les lipoprotéines de faible densité
JPS6419024A (en) * 1987-07-15 1989-01-23 Tosoh Corp Separation of immunoglobulin g subclass

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0377046A1 (fr) * 1988-04-05 1990-07-11 Kanebo Ltd. Particules fines de cellulose poreuse echangeuse d'ions, procede pour leur production, et porteur d'affinite
WO1995005879A1 (fr) * 1993-08-27 1995-03-02 The Dow Chemical Company Procede pour la separation d'enantiomeres
WO1997038018A1 (fr) * 1996-04-11 1997-10-16 Amersham Pharmacia Biotech Ab Procede de production d'un gel polysaccharidique reticule poreux et son utilisation comme support pour filtration sur gel et en chromatographie

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EP1071499A1 (fr) 2001-01-31
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CN1289263A (zh) 2001-03-28
DE19803415A1 (de) 1999-08-05
CA2319386A1 (fr) 1999-08-05

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