WO1999038594A1

WO1999038594A1 - Separation of substance mixtures using polysaccharides

Info

Publication number: WO1999038594A1
Application number: PCT/EP1999/000473
Authority: WO
Inventors: Holger Bengs; Arnold Schneller; Gitte Böhm; Doris Andert
Original assignee: Aventis Research & Technologies Gmbh & Co. Kg
Priority date: 1998-01-29
Filing date: 1999-01-26
Publication date: 1999-08-05
Also published as: AU2718999A; EP1071499A1; CA2319386A1; JP2002501811A; CN1289263A; DE19803415A1

Abstract

The invention relates to a method for separating substance mixtures by chromatography using spherical microparticles consisting of chemically and physically unmodified, water-insoluble, linear polysaccharides.

Description

1

description

Separation of mixtures using polysaccharides

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.

For example, the article Chromatographie 65, LaborPraxis, 730-738 (1990) describes the use of microcrystalline tribenzoyl cellulose with grain sizes of 10 to 20 μm 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 μm 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. In order to obtain suitable separating material, the cyclodextrins have to be chemically modified, dissolved in polysiloxane and fixed on a suitable carrier material.

No. 5,403,898, 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.

In US 5,302,633, in order to achieve insolubility of the chiral stationary phase, a vinyl derivative of a polysaccharide (e.g. cellulose) is first adsorbed on the surface of a porous support (e.g. silica gel), then polymerized. 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. Here too, 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 μm to 300 μm 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. In order to achieve insolubility, dextran derivatives containing vinyl groups must be crosslinked by radical polymerization.

Numerous polysaccharides are therefore already known which are used in chromatographic separation processes. However, these must always be chemically and / or physically modified in order to achieve good separation performance, particle size distribution, solvent stability and resistance. This is always associated with an increase in costs.

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.

This object is achieved by a method for the chromatographic separation of mixtures of substances using 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. When describing a sphere by axes of the same length, starting from a common origin, directed into space, 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%. Spherical microparticles with deviations of up to 25%, particularly preferably up to 15%, are preferably obtained. 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.

By bypassing chemical and physical modifications of the linear polysaccharides, additional cost-intensive process steps can be avoided, thereby increasing the economy of the chromatography process. Another advantage is the very good reproducibility of the chromatography process.

Physical modifications are not to be understood as the usual work-up steps such as stirring, centrifuging, slurrying, etc.

Linear polysaccharides according to the present invention can be polyglucans or other linear polysaccharides such as pullulans, pectins, mannans or polyfructans. However, poly- (1,4-α-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. 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 oligosaccharides.

Also for reasons of biocompatibility of the separation material according to the invention, 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).

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. In the context of this invention, 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. The following are therefore to be seen as special embodiments: HPLC, 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) is to be understood as high performance (or) high pressure liquid chromatography. HPLC works with very fine material (3-10 μm), 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). Gel permeation chromatography (GPC), too

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 (GC) 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.

Thin layer chromatography (DC) 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.

Another object of the invention is the use of the separating material according to the invention and, if appropriate, further auxiliaries and additives for the separation or retention of substances in the sense of a filter medium or adsorbent (cf. Example 12). 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.

If the basic unit has three or more links, this is referred to as branching. The number of hydroxyl groups per 100 basic units, which are not involved in the construction of the linear polymer backbone and form the branches, results in the so-called degree of branching.

According to the invention, 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%.

If the water-insoluble linear polysaccharide is a polyglucan, for example poly- (1,4-alpha-D-glucan), 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.

According to the invention, the prefixes "alpha", "beta" or "D" relate solely to the linkages which form the polymer backbone and not to the branches.

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. On the other hand, 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. Biotechnical processes include all biocatalytic (= biotransformatory processes, i.e. processes that can only be carried out with enzymes or in combination with organisms that form intra- or extracellular proteins that take on this task) or fermentative processes that are carried out with in naturally occurring or recombinant organisms can be carried out.

Linear polysaccharides produced by biocatalysis (also: biotransformation) 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.

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.

In addition, to achieve the effects described in the present invention, 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. These enzymes are, for example, amylases, iso-amylases, pullulanases or also gluconohydrolases. 10

For the present invention, the term “water-insoluble polysaccharides” is understood to mean compounds which, according to the definition of the German Medicinal Products Book (DAB = German Medicinal Products Book, Scientific Publishing House mbH, Stuttgart, Govi-Verlag, Frankfurt, edition, 1987) correspond to classes 4 to 7 fall under the categories "sparingly soluble", "sparingly soluble", "very sparingly soluble" or "practically insoluble".

In the case of the polysaccharides used according to the invention, this means that at least 98% of the amount used, in particular at least 99.5%, under normal conditions (T = 25 ° C. +/- 20%, p = 101325 Pascal +/- 20%) in water is insoluble (according to classes 4 and 5).

For the present invention, sparingly soluble to practically insoluble compounds, in particular very sparingly soluble to practically insoluble compounds, are preferred.

"Very poorly soluble" according to class 6 can be illustrated by the following test description: One gram of the polyglucan / saccharide to be examined is heated in 1 l of deionized water to 130 ° C. under a pressure of 1 bar. The resulting solution only remains stable for a short time over a few minutes. When cooling under normal conditions, the substance precipitates again. After cooling to room temperature and separation by centrifugation, taking into account the experimental losses, at least 66% of the amount used can be recovered.

Production of uniform spherical microparticles

The preferred poly- (1,4-α-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. In it, the poly- (1,4-α-D-glucan) is produced by means of a biocatalytic (biotransformatory) process using amylosucrase. Furthermore, the poly- (1,4-α-D-glucan) can be produced using polysaccharide synthases, starch synthases, glycol transferases, 1,4-α-D-glucan transferases, glycogen synthases or phosphorylases by means of a biocatalytic process.

The patent application DE-A-197 37481 describes the production of spherical microparticles from linear water-insoluble polysaccharides, as well as their molecular weights and diameters. Reference is made here to the above-mentioned application.

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. By using suitable additives, the properties of the particles, such as size, structure of the surface, porosity, etc., as well as the process control can be influenced. Suitable additives are, for example, surfactants such as sodium dodecyl sulfate or N-methylglucanoamide, sugar, e.g. Fructose, sucrose, glucose.

Properties of the spherical microparticles

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. Molecular weights M _w in the range from 10 ⁴ g / mol to 10 ⁵ g / mol, in particular 2 × 10 ⁴ g / mol to 5 ×, are preferred for the preferably used linear polysaccharide poly- (1,4-α-D-glucan) 10 ⁴ g / mol used. 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 μm, preferably 100 nm to 10 μm, particularly preferably 1 μm to 5 μm. The distribution of the diameters is characterized by a high level of uniformity.

The 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.

Experiments on the stability of the microparticles in various solvents show that a wide variety of solvents are suitable for use in the chromatography process according to the invention. As a solvent, in function of the eluent, e.g. n-Hexane, dichloromethane, tetrahydrofuran, 1,4-dioxane, ethanol, acetone, acetonitrile, methanol, n-heptane and water are used.

Chromatography procedure

In the chromatography process, microparticles of linear polysaccharides, particularly preferably poly (1,4-α-D-glucan), in particular in a solvent, for example n-heptane, are slurried, whirled up by means of an Ultraturrax and degassed in an ultrasonic bath. 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.

Description of the figures:

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

Example 12, see legend.

The following examples explain the invention in more detail without restricting the invention to these examples.

14

Examples: Example 1

In vitro production of a linear water-insoluble polysaccharide in a biocatalytic process using the example of poly- (1,4-α-D-glucan) with amylosucrase

10 I of a 20% sucrose solution are placed in a sterilized (steam sterilization) 15 I vessel. The enzyme extract, containing amylosucrase, is added in one portion. The enzyme activity in this experiment is 20 units (1 unit = 1 μmol sucrose x min ¹ x mg enzyme). The apparatus is equipped with a KPG stirrer, also sterilized. The vessel is closed, stored at 37 ° C and stirred. A white precipitate forms after only a few hours. The reaction is stopped after 96 hours. The precipitate is filtered off and washed several times with water to separate off low-molecular sugar. The residue remaining in the filter is dried at 40 ° C. in a drying cabinet by applying a vacuum with the aid of a membrane pump (company Vacuubrand GmbH & Co., CVC 2). The mass is 651 g (yield 65%).

Example 2

Determination of the molecular weight of the water-insoluble poly- (1,4-α-D-glucan) synthesized with amylosucrase from Example 1

2 mg of the poly (1,4-α-D-glucan) from Example 1 are dissolved in dimethyl sulfoxide (DMSO, pa from Riedel-de-Haen) at room temperature and filtered (2 μm filter). Part of the solution is injected into a gel permeation chromatography column. DMSO is used as the eluent. The signal intensity is measured by means of an RI detector and evaluated against pullulin standards (company Polymer Standard Systems). The flow rate is 1.0 ml per minute. The measurement gives a number average molecular weight (M _n ) of 7,000 g / mol and a weight average molecular weight (M _w ) of 34,000 g / mol. 15

Example 3

Production of microparticles from poly (1,4-α-D-glucan)

a) 400 g of poly (1,4-α-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). The solid residue is slurried three times with bidistilled water and centrifuged again. The solids are collected and the suspension of approx. 1000 ml freeze-dried (Christ Delta 1-24 KD). 283 g of white solid are isolated (yield 71%).

b) The collected supernatants are kept at a temperature of 18 ° C overnight. The processing takes place as described. A further 55 g of the white solid are isolated (yield 14%).

The overall yield of this process is 85%.

Example 4

Investigations of the microparticles from Example 3 using electron microscopy

Scanning electron microscope (SEM) (Camscan S-4) images are used to characterize the particles. Figures 1 to 4 show photographs of the particles, which clarify that they are spherical, very uniform particles in terms of shape, size and surface roughness. 16

Example 5

Investigations of the size distributions of the particles from Example 3

To characterize the size distribution of the particles from Examples 3, tests were carried out using a Mastersizer (Malvern Insturments). The investigation was carried out in Fraunhofer mode (evaluation: multimodal, number) assuming a density of 1.080 g / cm ³ and a volume concentration in the range from 0.012% to 0.014%.

17

Table 1 :

Characterization of the particle diameter from Examples 3

Examples diameter particle distribution

1

No. dn ^{* 2} Dw ^{* 2} dw / dn ^{* 3} d (10%) ^{* 4} D (50%) ^{* 5} d (90%) ^{* 6}

(μm) (μm) (μm) (μm) (μm)

3a 1, 664 4.184 2.541 0.873 1, 504 2.624

3b 0.945 2.345 2.481 0.587 0.871 1.399

^{* 1} dn: average number of the diameter ^{* 2} dw: average weight of the diameter

* ₃ dw / dn: ispersity of the particle diameter d (10%): 10% of all particles have a smaller diameter than the specified value d (50%): 50% of all particles have a smaller diameter than the specified value

* 6 d (90%): 90% of all particles have a smaller diameter than the specified value

Example 6

Determination of the specific surface area of poly (1,4-α-D-glucan) (comparative example) and microparticles of poly (1,4-α-D-glucan) and potato starch (comparative example)

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-α-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®).

Table 2:

Characterization of the specific surface area of the microparticles made of poly- (1,4-α-D-glucan) (see Example 3)

Example No. Substance Specific surface area m ² / g

1 poly (1,4-α-D-glucan) 2,243

3 microparticles

Poly- (1,4-α-D-glucan) 4,527

- potato starch

(Toffena, South Starch) 1, 280

Example 7

Experiments on the stability of the microparticles in various solvents

Determination of the solvents that can be used

To test the suitability of various solvents in the chromatographic application with microparticles of poly- (1,4-α-D-glucan), 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.

The results show that a wide variety of solvents can be used for the chromatographic use described here. Exclude only toluene, ethyl acetate and dimethyl sulfoxide. In the individual case not listed, the solvent must first be checked for its usability. The results of the tests are in table 19

3 recorded. The dielectric constants, the dipole moment and / or the polarity index according to Snyder were given in order to give an indication of the polarity of the solvent.

Table 3:

Results of the studies on the stability of microparticles made from poly- (1,4-α-D-glucan) (see Example 3) in various solvents

Solvent dielectric dipole polarity observation

Constant (ε) ^"1 moment D ^{* 2} index ^{* 3} (after 24 h) n-hexane 1, 8865 - 0.0 no change n-heptane 1, 9209 - 0.0 no change

Toluene 2,379 0,375 2,3 Transparent, slightly swollen material, cloudy overhang

Ethyl acetate 6.0814 1, 78 - particles stick together, cloudy supernatant

Dichloromethane 8.93 1, 60 3.4 no change

Tetrahydrofuran 7.2 1, 75 4.2 no change

1,4-dioxane 2.2189 - 4.8 no change

Ethanol 25.3 1, 69 5.2 no change

Acetone 21, 01 2.88 5.4 no change

Acetonitrile 36.64 3.924 6.2 no change

Methanol 33.0 1, 7 6.6 no change

Dimethylformamide 38.25 3.82 - no change

Dimethyl sulfoxide 47.24 3.96 - dissolved

Water 80,100 1, 854 9.0 no change

^{* 1} dielectric constant according to “Handbook of Chemistry and Physics, 77 ^th

Edition " ^{* 2} Dipole moment according to" Handbook of Chemistry and Physics, 77 Edition "

^* 3 Snyder polarity index from: Merck, ChromBook 20th

Example 8

Filling a chromatography column with microparticles from 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.

Example 9

Separation of indole and 2-methylindole in heptane

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

Table 4

Results of the separation of indole and 2-methylindole in heptane

Substance response time

(min)

1,3,5-tri-tert-butylbenzene 1,86 (standard)

Indole 9.12

Indole / 2-methylindole 8.98 / 7.37

2-methylindole 7.20

Example 10

Separation of indole and 2-naphthol in heptane

The test is carried out as described in Example 10.

Table 5:

Results of the separation of 2-naphthol and indole in heptane

Substance retention time (min)

2-naphthol 2.24

2-naphthol / indole 1, 92 / 9.16

Indole 9.12

Example 11

Separation of a mixture of enantiomers: racemic 1- (2-naphthyl) ethanol

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

Table 6:

Results of the separation of racemic 1- (2-naphthyl) ethanol in heptane

Substance retention time (min)

1,3,5-tri-tert-butylbenzene 1,89

(Default)

S - (-) - 1 - (2-Naphthyl) ethanol 32.03

1,3,5-tri-tert-butylbenzene 1,87

(Default)

R - (+) - 1 - (2-Napthyl) ethanol 36.30

Example 12

Separation or retention of substances, use of the separating material according to the invention as a filter medium

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 μg / 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® Midipräp (Hilden, Germany) and another reference run with Qiagen® Midipräp - cartridges without Qiagen® filter medium, which were equipped with the separating material.

Each 5 μl 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). As a reference for the estimation and classification of detected plasmids, a marker (MG 5,000) in lane 1 and 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.

Then 5 ml of 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). As a comparison, a marker (MW 5,000) (lane 1) and the DNA plasmid solution (lane 7) were applied.

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 description:

Buffer 1: 750 mM NaCl

50 mM MOPS (3- [N-morpholino] propanesulfonic acid, pK _a 7.2) 15% isopropanol 0.15% ^Triton® X-100

Buffer 2: 1, 25 M NaCl

50 mM Tris, Tris ^* Clm, Ph 8.5 15% isopropanol 24

Fig 5:

Left to right:

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® Midipräp (Hilden , Germany) Reference lane 6 Filter 2 with separation material lane 7 pure DNA plasmid solution (commercially available plasmid pBluescript II SK)

Fig. 6

Left to right:

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® Midipräp (Hilden, Germany). Reference track 6 filter 2 with separating material. Track 7 pure DNA plasmid solution (commercial plasmid pBluescript II SK)

Claims

25 claims

1. Process for the chromatographic separation of substance mixtures using spherical microparticles made of chemically and physically unmodified, water-insoluble, linear polysaccharides.

2. The method according to claim 1, characterized in that the separations are carried out by column chromatography.

3. The method according to claim 2, characterized in that the separations are carried out with the aid of HPLC (high performance (or) high pressure liquid chromatography).

4. The method according to claim 1, characterized in that the separations are carried out by thin layer chromatography.

5. The method according to claim 1, characterized in that the separation can be carried out with the aid of gel permeation chromatography.

6. The method according to any one of claims 1 to 5, characterized in that the substance mixtures are stereoisomers.

7. The method according to claim 6, characterized in that the substance mixtures are enantiomers.

8. The method according to any one of claims 1 to 7, characterized in that the microparticles have a diameter of 10 nm to 100 microns.

9. The method according to claim 8, characterized in that the microparticles have a diameter of 1 micron to 5 microns. 1.3

Jι * ^Q

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1 μm