DE19803415A1 - Separation of mixtures using polysaccharides - Google Patents

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

The invention relates to a method for separating mixtures of substances, especially enantiomers, using polysaccharides as Release material.

Chromatography is an effective method for complete separation of components of a mixture, in particular for the separation of Mixtures of similar compounds, e.g. B. of stereoisomers. Because of the increasing interest in pure enantiomers for pharmaceutical and biochemically active substances and pesticides is the chromatographic separation of enantiomeric compounds of particular Interest. For the purification of these compounds are constantly developed improved processes and searched for better separation materials, especially based on the polysaccharides cellulose and starch, two easily accessible and inexpensive polymers with chiral atoms.

The article describes Chromatographie 65, LaborPraxis, 730-738 (1990) the use of microcrystalline tribenzoyl cellulose with grain sizes of 10 to 20 µm compared to triacetyl cellulose as a sorbent for the Enantiomer separation in chromatography. Both substances can be used for analytical and preparative separations can be used. you are through Derivatization of cellulose accessible. Unmodified polymers are coming  not used. The introduction of smaller grain sizes down to 5 µm to Dead volumes and a compression of the Avoiding the pillar is described as desirable.

WO 95/05 879 also deals with the separation of enantiomers Liquid chromatography. Particular attention is paid here to dedicated to the mobile phase to improve separation performance. As a stationary Phase are carbamate derivatives of cellulose and amylose and ester Derivatives of cellulose used.

DE-A 43 17 139 describes a special method for Enantiomer separation of inhalation anesthetics using preparative Gas chromatography. As a stationary phase with ester groups derivatized cyclodextrins in polysiloxane solution on porous support material (Chromosorb) used. In order to obtain suitable separating material, the cyclodextrins chemically modified, dissolved in polysiloxane and on a suitable carrier material can be fixed.

Polymers containing cyclodextrin derivatives are also described in US Pat. No. 5,403,898 Siloxanes as chiral stationary phase in the analytical and preparative Gas chromatography (GC), chromatography with supercritical Liquids (SFC) and liquid chromatography (LC) are used. This Peralkyl cyclodextrins are chemically bound to the polysiloxanes. The The material used is therefore modified by chemical modification Cyclodextrins and subsequent chemical fixation to polysiloxanes receive.

In US 5,302,633 to insolubility of the chiral stationary phase achieve a vinyl derivative of a polysaccharide (e.g. cellulose) on the Surface of a porous support (e.g. silica gel) first adsorbed, then polymerized. A second variant is the copolymerization of one Porous carrier containing vinyl groups (e.g. modified silica gel)  a vinyl derivative of a polysaccharide. Here are chemical too Modifications necessary to obtain suitable release material.

EP-B-0 157 365 describes stationary phases for enantiomer and Isomer separation and for gel permeation chromatography based of polysaccharide carbamate derivatives. The polysaccharides are cellulose, Amylose, chitosan, xylan, dextran and inulin are suitable. Here it can manufactured powder with a particle diameter of 1 µm to 300 µm directly or after physical or chemical fixation on a porous Carrier can be used. So it's a chemical modification necessary to obtain suitable separating material.

In DE-C 26 55 292 and DE-C 26 55 361 porous gels are made Dextran derivatives as separation media in electrophoretic Separation materials used. To achieve insolubility, you must Dextran derivatives containing vinyl groups by radical polymerization be networked.

So there are already numerous polysaccharides known in chromatographic separation processes are used. However, these must always to ensure good separation performance, grain size distribution, Achieve solvent stability and resistance, chemically and / or be physically modified. This is always with an increase in Associated costs.

Are under chemical and / or physical modifications especially derivatization through the introduction of special groups, covalent fixations on a carrier material as well as subsequent understand chemical and / or physical networking.

The object of the invention is therefore to simplify and reduce the cost Chromatography bypassing chemical and physical modifications of the chromatography material used.

This task is accomplished using a chromatographic separation technique of mixtures of substances using spherical microparticles chemically and physically unmodified, water-insoluble, linear Polysaccharides dissolved as a separating material.

Spherical microparticles are to be understood as meaning microparticles which are approximately spherical in shape. If a sphere is described by axes of the same length, starting from a common origin, which point into the space and define the radius of the sphere in all spatial directions, the spherical microparticles may deviate from the ideal length of the sphere by 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" should not exceed 20% of the average diameter of the spherical microparticles. Figs. 1a to d show scanning electron micrographs (SEM) (Camscan S-4) of the spherical microparticles used.
1a: scanning electron microscope image of the spherical used
Microparticles in 5000x magnification
1b: Scanning electron microscope image of the spherical microparticles used in 10000x magnification
1c: scanning electron microscope image of the spherical microparticles used in 5000x magnification
1d: Scanning electron microscope image of the spherical microparticles used in 20000x magnification
By avoiding chemical and physical modifications of the linear polysaccharides, additional cost-intensive process steps can be avoided and the economic efficiency of the chromatography process can thereby be increased. Another advantage is the very good reproducibility of the chromatography process.

Physical modifications are not the usual ones Standard laboratory procedures such as stirring, centrifuging, To understand slurries, washing, filtering, drying, etc.

Linear polymers according to the present invention can be polyglucans or other linear polysaccharides such as pullulans, pectins, mannans or Be polyfructans. Poly (1,4-α-D-glucan) is particularly preferred.

According to the invention, the material can be used for the chromatographic separation of Mixtures of substances, particularly preferably stereoisomers, very particularly preferably enantiomers are used. Therefore, the use is special as chromatographic material in preparative and analytical Chromatography such as gas chromatography, preparative and analytical Thin layer chromatography and particularly preferably in the Column chromatography, especially high performance liquid Chromatography (HPLC), of interest. Furthermore, the use of the Material in gel permeation chromatography (GPC) during separation of polymers of different molecular weights of interest. A Application that also falls within the scope of the invention is Separation of substances from solutions, emulsions or suspensions through specific or non-specific interactions with low molecular weight or polymeric compounds. But also ionic Compounds, especially metal cations, are of interest to the Application. Special effects are particularly due to the structural Similarity to polysaccharides in the separation of monosaccharides, Disaccharides and oligosaccharides can be achieved. Furthermore, the Treatment of water, especially wastewater, treatment and Analysis of blood, or the separation or separation of genetic material  or related compounds (e.g. oligonucleotides, peptides, proteins) from Interest.

Chromatography means physical in the sense of the invention Separation processes in which the separation of substances by distribution between a stationary and a mobile phase happens. A division enables the Combination of the phase states solid, liquid and gaseous for the mobile and stationary phase.

Under HPLC (High Performance (or) High Pressure Liquid Chromatography) is the high performance (or) high pressure liquid understand chromatography. One works with the HPLC with very fine Material (3-10 µm), because the separation performance of a column decreases Grain size of the stationary phase increases. The fineness of the Separating materials require high pressures (up to 400 bar).

In gel permeation chromatography (GPC), also exclusion chromatography, which can also be operated as HPLC, exists stationary phase from beads with a heteroporous swollen network, whose pore size distribution varies over several orders of magnitude, so that fractionation by molecular size is done, which is the rapid determination the molecular size distribution of polymers allowed.

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.

Linear polysaccharides are polysaccharides made from monosaccharides, Disaccharides or other monomeric building blocks (monomer) such are built up that the monosaccharides, disaccharides or others monomeric units are always linked together in the same way. Each basic unit or module defined in this way has exactly two links, one each to another "monomer" built into the polymer (Repeating unit in the polymer). The two are basic units except which form the beginning and the end of the polysaccharide. This Basic units have only one link to another "monomer". With three links (covalent bonds) one speaks of one Branch. Branches do not occur or only to a minor extent on, so that they are the conventional with very small branch proportions analytical methods are not accessible.

Nature-identical polysaccharides are understood to mean polymers that either not occurring in nature or only in a mixture with other compounds, which can also be other polymers. The Such nature-identical polymers can be produced by processes which under the broadest definition of the term bio- and genetic engineering processes fall. On the one hand, there are biotechnological ones Understand procedures or processes as defined below but also those which, through the use and modification of the Example bacteria, fungi or algae to corresponding compounds to lead. On the other hand, the term also includes, for example Polysaccharides that can be obtained by biotechnological or genetic engineering Procedures can be applied to higher plants so that separation can be done from the plant. Such plants include, in particular Potatoes, corn, cereals, cassava, rice and peas. But others too Plants that produce polysaccharides are among those of the present invention Classify aspect in this category.  

Linear, water-insoluble are preferred in the context of the invention Polysaccharides used in a biotechnical, in particular a biocatalytic, also biotransformatory, or a fermentative process.

All biocatalytic processes (= biotransformer processes, that is, processes involving enzymes or in combining with organisms that have intra- or extracellular proteins form who can take on this task, can be carried out) or fermentative processes that occur with or in nature recombinant organisms can be carried out.

Linear polysaccharides produced by biocatalysis (also: Biotransformation) in the context of this invention means that the linear Polysaccharide through catalytic reaction of monomeric building blocks such as oligomeric saccharides, e.g. B. of mono- and / or disaccharides, is produced by a so-called biocatalyst, usually a Enzyme, under suitable conditions for use in the implementation is being used.

Linear polysaccharides from fermentations are used in the Invention of linear polysaccharides by fermentative processes under the Use of organisms occurring in nature, such as Fungi, algae or bacteria or not using in nature occurring organisms, but with the help of genetic engineering Methods of general definition modified natural organisms, such as for example, fungi, algae or bacteria can be obtained from or under Activation and with the help of fermentative processes can.  

In addition, linear polysaccharides can be used to achieve that in the effects described in the present invention can also be obtained by that non-linear polysaccharides containing branches, so with one or more enzymes are treated that the branches are split off from the backbone of the polysaccharide, so that after their Separation linear polysaccharides are present. It is with these enzymes are for example amylases, iso-amylases, pullulanases or Gluconohydrolases.

Production of uniform spherical microparticles

The preferred poly- (1,4-α-D-glucan) can be used different ways are made. It will be a very beneficial method described in WO 95/31 553. The revelation of Scripture will explicitly related here. In it the poly (1,4-α-D-glucan) is used a biocatalytic (biotransformatory) process with the help of Amylosucrase manufactured. Furthermore, the poly- (1,4-α-D-glucan) under Use of polysaccharide synthases, starch synthases, glycol transferases, α-1,4-glucan transferases, glycogen synthases or Phosphorylases are produced using a biocatalytic process.

The earlier priority unpublished application DE-A 197 37 481 describes the production of spherical microparticles from linear water-insoluble polysaccharides, and their molecular weights and Diameter. Reference is made here to the above-mentioned application.

The spherical microparticles are produced by dissolving the water-insoluble, linear polysaccharide, especially of the poly- (1,4-α-D- glucans), in a solvent, particularly preferably in DMSO the solution in a precipitant, preferably water, cooling the  resulting mixture to preferably 10 ° C to -10 ° C and separating the formed microparticles.

The properties can be determined by using suitable additives of the particles like size, structure of the surface, porosity etc. as well as on the Litigation influence. Suitable additives are, for example surfactants such as sodium dodecyl sulfate or N- Methyl gluconamide, sugar, e.g. B. 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 by means of gel permeation chromatography in comparison to a calibration using pullulin standards.

The particles can have average 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 one Uniformity.

The microparticles made of poly (1,4-α-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 different solvents show that a wide variety of solvents for use in the Chromatography methods according to the invention are suitable. As Solvents, as a function of the eluent, are e.g. B. n-hexane, Dichloromethane, tetrahydrofuran, 1,4-dioxane, ethanol, acetone, acetonitrile, Methanol, n-heptane and water are used.

Chromatography procedure

According to the invention, microparticles are formed in the chromatography process linear polysaccharides, particularly preferably poly (1,4-α-D-glucan), especially in a solvent, e.g. B. n-heptane, slurried, by means of whirled up in an Ultraturrax and degassed in an ultrasonic bath. The suspension is filled into a column and by constantly knocking on the column, constant Shake in combination with brief treatment in an ultrasonic bath evenly and tightly packed, so that dead volume packs are created. The separations are in a state of the art Chromatography system for the HPLC performed. The is Sample concentration 2.0 to 0.025 mg / l solvent, particularly preferred 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 vary. The prints used are strong on the polarity of the Solvent dependent. You lie i. a. in a range between 30 bar and 200 bar. For example, the following pressures were measured: 138 up 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. It can be an inner Standard, e.g. B. 1,3,5-tri-tert-butylbenzene can be used.

The following examples explain the invention in more detail.

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) Amylosucrase.

10 l of a 20% sucrose solution are placed in a sterilized (steam sterilization) 15 l container. The enzyme extract, containing amylosucrase, is added in one portion. The enzyme activity in this experiment is 20 units (1 unit = 1 µmol sucrose × min ^-1 × 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 (Vacuubrand GmbH & Co, CVC 2). The mass is 651 g (yield 65%).

Example 2 Determination of the molecular weight of the synthesized with amylosucrase water-insoluble poly (1,4-α-D-glucans) from Example 1

2 mg of the poly- (1,4-α-D-glucan) from Example 1 are dissolved in dimethyl sulfoxide (DMSO, pa von 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 using an RI detector and evaluated against pullulan 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.

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, pa from Riedel-de-Haen) at 60 ° C. within 1.5 h. Then it is stirred for one hour at room temperature. The solution is added in 20 l of bidistilled 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 images (SEM) (Camscan S-4) are used to characterize the particles. Figs. 1a to d show images of the particles, which make it clear that it is spherical, highly uniform in terms of particle shape, size and surface roughness.

Example 5 Investigations of the size distributions of the particles from Example 3

To characterize the size distributions of the particles from Examples 3, investigations were carried out using a Mastersizer (from Malvern Instruments). 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%.

Table 1

Characterization of the particle diameter from Examples 3

Example 6 Determination of the specific surface area of poly- (1,4-α-D-glucan) (Comparative example) and microparticles made of poly (1,4-α-D-glucan) and Potato starch (comparative example)

The measurements of the specific surface are carried out with a Sorptomatic 1990 (Fisons Instraments). The "default method sorptomatic "method. For the investigation, the Samples at 80 ° C in a vacuum (membrane pump from Vacuubrand GmbH & Co, CVC 2) dried overnight. The potato starch and that straight out  the biotransformation obtained poly- (1,4-α-D-glucan) beforehand ground so that the mass of the particles in the range less than 200 microns lies. A commercially available mill is used for this (Waring®).

Table 2

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

Example 7 Experiments on the stability of the microparticles in different solvents for determining the solvents that can be used

To test the suitability of various solvents in the chromatographic application with microparticles made of poly (1,4-α-D- The procedure is as follows: 100 mg of the particles from Example 3 are Covered in a crimp-top vial with 3 ml of the solvent. The observation time is 24 hours. At hourly intervals the particles are examined after changes. For this purpose the reaction vessel is pivoted.  

The results show that a wide variety of solvents for the here described chromatographic use can be used. Exclude only toluene, ethyl acetate and dimethyl sulfoxide. I'm not Listed individual case, the solvent must first on its Usability to be checked. The results of the tests are in Table 3 recorded. The dielectric constants, as well as the Dipole moment and / or the Snyder polarity index given by a To give an indication of the polarity of the solvent.

Table 3

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

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 The suspension is then transferred to a column with dimensions 125 under maximum flow mm length and 4 mm diameter (Hibar® finished column from Merck AG) filled. Constant tapping on the column, or constant shaking of the column in Combination with short-term, approximately one-minute treatment in Ultrasonic bath ensures a tight and even packing of the Separating material, so that low-volume packs are created.

Example 9 Separation of indole and 2-methylindole in heptane

The separations are carried out in a Lichograph® chromatography system Carried out by the Merck company. The individual components are: one Gradient pump L 6200 A, a UV detector L-4000 (measurement at 254 mn), a Chromato integrator D-2500 and a separation column Hibar® finished column RT- 125-4. The sample concentration is ideally 0.05 to 0.1 mg / ml Solvent. As an internal standard, 1,3,5-tri-tert-butylbenzene (Fa. Fluka) can be used.  

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.

Table 4

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

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

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 lies the pressure at approx. 70 bar. The concentration of the samples is 0.25 mg / ml Solvent.  

Table 6

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

Claims (10)

1. Procedure for the chromatographic separation of mixtures under Use of spherical microparticles from chemically and physically unmodified, water-insoluble, linear polysaccharides. 2. The method according to claim 1, characterized in that the separations be carried out by column chromatography. 3. The method according to claim 2, characterized in that the separations with Using HPLC (high performance (or) high pressure liquid chromatography). 4. The method according to any one of claims 1 to 3, characterized in that the Mixtures of substances are stereoisomers. 5. The method according to claim 4, characterized in that the substance mixtures Are enantiomers. 6. The method according to any one of claims 1 to 5, characterized in that the Microparticles have a diameter of 10 nm to 100 microns. 7. The method according to claim 6, characterized in that the microparticles have a diameter of 1 µm to 5 µm. 8. The method according to any one of claims 1 to 7, characterized in that as Polysaccharide poly- (1,4-α-D-glucane) can be used. 9. The method according to any one of claims 1 to 8, characterized in that Polysaccharides are used by a biotechnical process are producible. 10. Use of spherical microparticles made of linear polysaccharides for the chromatographic separation of substance mixtures.