DD296005A5 - METHOD FOR PRODUCING PERLFOERMERIC CELLULOSE PRODUCTS FOR USE AS SEPARATOR AND TRAITER MATERIALS - Google Patents

Abstract

The invention relates to bead-shaped cellulose products and a process for manufacturing bead-shaped cellulose products for separating and carrier materials. Macroporous cellulose beads moistened with water are used as the starting material and dried under specified conditions. The cellulose matrix is then crosslinked and at the same time hydroxyalkyl groups are introduced. The products so obtained are characterized by a porosity which can be adjusted to any desired value within wide limits, good mechanical and chemical stability and the absence of non-specific interactions. The products are used preferably as chromatographic separating materials for analytical and preparatory purposes.

Description

Field of application of the invention. ,

The invention relates to a process for the preparation of bead-shaped cellulose products with adjustable porosity, which can be used as separation and support materials in chemistry, biotechnology, medicine and pharmacy for analytical and preparative purposes.

Characteristic of the known state of the art

Particulate cellulosic materials have become increasingly important in recent years, especially as stationary phases in chromatographic separation and purification processes, but also as selective specific adsorbents. For producing such more or less porous cellulose particles, a variety of methods have been proposed. An essential objective of these methods is the production of spherical porous particles in order to optimally adapt the resulting products to the respective rather special separation tasks, for example the separation of proteins by gel chromatography.

The preparation of porous spherical cellulose particles occurs in most cases by preparation of a cellulose or cellulose derivative solution, deformation of the solution into droplets, coagulation of the droplets and regeneration of the cellulose. Differences exist mainly in the manner of droplet formation, e.g. by dropping the cellulose solution into a suitable precipitation bath or by dispersing the cellulose solution in a suitable solvent and the manner of coagulation or regeneration of the cellulose (see US Pat , DE-OS 2138905, DD-PS 118887, JP-PS 80-44312)

 The problem with all these methods is the setting of a defined porosity of the cellulose matrix.

For this reason, various methods have been proposed, such as z. B. on the cellulose concentration (DE-OS 1792 230) or by additions to the cellulose derivative solution as a pore former (US-PS 4312980), the pore size may vary. Good results have been obtained when the pore structure is fixed by the additional use of a bifunctional crosslinking agent of the general formula X-R-Z, where X and Z are halogen or epoxy groups and R is a C3 to C10 alkylene radical (US Pat. No. 3,598,245). Although the products obtained in this case are in principle suitable for gel chromatographic separation of dissolved high molecular weight substances, unmodified cellulose particles have the general disadvantage that they interact with various proteins in an unspecific interaction, which limits their universal applicability.

Cheaper properties have hydroxyethyl or hydroxypropylgruppenhaltige products which can be prepared according to US-PS 3598245 by simultaneous reaction of dried Regeneratcelluloseteilchen with a crosslinking agent. However, this solution has several disadvantages. Thus, for example, the running behavior in chromatographic columns is unfavorably influenced by the use of fibrous regenerated cellulose particles. Another disadvantage is the inadequate mechanical stability of the gels, which can lead to reduced flow rates or even clogging of the columns, in particular given a high content of hydroxypropyl groups and a low degree of crosslinking. It is also disadvantageous that these highly swollen gels already partially dissolve in water (JS Ayers, MJ Petersen, BE Sheerin, GS Bethell: J. Chromatography 294 [1984], pp. 195-205) and thus can not be used for chromatographic purposes , The porosity range which can be exploited for gel chromatographic separations is relatively narrow and is less than 5 × 10 ⁴ daltons.

Object of the invention

The aim of the invention is a process for producing bead-shaped porous cellulose products having improved properties, in particular a hydrophilic reversibly swellable cellulose matrix with a pore structure which can be specifically adjusted within wide limits while simultaneously suppressing non-specific interactions with biomacromolecules.

Explanation of the essence of the invention

The invention has for its object to come by a suitable pretreatment of Regeneratcelluloseteilchen associated with a chemical modification to bead-shaped cellulose products having advantageous properties, in particular a hydrophilic, reversibly swellable cellulose matrix with defined porosity and good mechanical stability and possible no unspecific interactions with proteins respectively.

According to the invention, this object is achieved in that water-wet beaded macroporous Regeneratcellu loose beads with a water content of 85 to 95g / 100g total mass initially dried to a defined water content between 10 and 95 g / 100g total mass and then in alkaline medium with a bifunctional C ₂ -C ₄ -halogen and / or epoxy compound as a crosslinking agent and a monofunctional halogen or epoxy compound are reacted as Hydroxyalkylierungmittel in a closed system at 313 to 373K for 10 to 360 min. The workup or purification of the resulting porous cellulose derivative particles is carried out in a known manner by neutralization of excess alkali hydroxide, thorough washing of the by-products, classification and optionally drying. In the case of drying, a previous at least partial displacement of the water by methanol, ethanol or acetone or a freeze-drying proves to be advantageous.

According to the invention, water-wet macroporous bead-shaped particles of regenerated cellulose having an average degree of polymerisation (DP) of from 150 to 500, the exclusion limit for which is between 10 ⁶ and 3 × 10 ⁷ daltons, are used as cellulosic starting material. It is advantageous for later use, for example in chromatographic columns, to start from spherical cellulose particles whose particle size can range from 1 to 2,000 μm, depending on the intended use, but is preferably from 50 to 250 μm.

Essential for the selective adjustment of a defined porosity of the cellulose matrix is the partial drying of the cellulose particles to a residual water content of 10 to 95 g / 100 g total mass, wherein the drying in usually z. B. by evaporation of the water at elevated temperature, optionally also by displacement with a water-miscible solvent such. As C ₂ -C ₃ alcohols, acetone, dioxane, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide can be done. The cellulose material pretreated in this way is then reacted in a closed vessel under pressure with the bifunctional crosslinking agent and the monofunctional halogen or epoxy compound as hydroxyalkylating agent with addition of alkali metal hydroxide. As bifunctional crosslinking agents, for example, epichlorohydrin, dichlorohydrin, dibromopropanol or diepoxybutane can be used, while preferred monofunctional hydroxyalkylating agents are ethylene oxide, propylene oxide, 2-chloroethanol and 2-chloropropanol. In addition to the extent of pre-drying or water removal, the product properties are determined by the amounts of crosslinking or hydroxyalkylating agents used.

According to the invention, from 0.01 to 0.5 mol of crosslinking agent and from 0 to 2.5 mol, preferably from 0.01 to 2.5 mol, of hydroxyalkylating agent per mole of monomer unit of the cellulose are used to achieve the desired porosity properties. The alkaline medium used is an aqueous solution of an alkali metal hydroxide, preferably sodium hydroxide, containing 5 to 45% by mass, the amount to be used being at least equal to the stoichiometric consumption by the reactants, but in particular 0.1 to 10 mol, but preferably 0.5 to 5 Corresponds to moles per mole of monomer unit of cellulose.

The water content in the reaction mixture is kept as low as possible in order to achieve high reagent yields. Inert or substantially inert organic solvents, such as. As toluene, benzene, chlorobenzene, ethanol, propanol or acetone, for better mixing and for better distribution of the reactants are added. The amount of solvent to be added is preferably 2 to 50 times, based on the cellulose mass used.

In a special embodiment of the method according to the invention, further etherifying agents are added to introduce additional functional groups. The addition can be carried out simultaneously with the crosslinking agent, the hydroxyalkylating agent and the sodium hydroxide solution or even before drying the water-moist macroporous cellulose. Suitable etherifying agents for introducing ionic groups are haloalkylcarboxylic acids such as 2 B monochloroacetic acid, haloalkylsulfonic acids, such as Chl B chloromethylphosphonic acid, haloalkylsulfonic acids, ζ Β 2-chloroethylsulfonic acid, or salts thereof, and haloalkylamines such as ζ B 2-chloroethyldiethylamine The amounts of etherifying agent required to achieve customary exchange capacities are from 0.2 to 1.0 mol per mole of monomer unit the cellulose

Above all, the advantages of the process are that the pore system of the cellulose can be adjusted in a defined manner through the use of the macroporous cellulose matrix as the starting material and its targeted partial drying. The perlformed cellulose materials obtained in this way are particularly suitable for gel chromatography due to their good mechanical properties Purpose up to molecular weights of 10 ⁶ Daltons suitable The simultaneous introduction of highly hydrophilic hydroxyalkyl groups suppresses the unspecific interaction with biomacromolecules In addition, the products are due to their highly hydrophilic nature, in contrast to unmodified regenerated cellulose, after suitable drying almost completely reversibly swellable Not only the handling is improved, but also the possible uses are extended

Exemplary embodiments

example 1

91 g of moist perlformige Regeneratcellulose with a DP of 250 (= 10g = 62mmol cellulose) with a particle size of 80 to 200 μηι, a water content of 89g / 100g total mass and an exclusion limit of 10 ⁷ daltons are 15ml 30% by weight containing in% sodium hydroxide ( 150 mmol), 5 ml of propylene oxide (72 mmol) and 0.4 ml of epichlorohydrin (5 mmol) are thoroughly mixed and transferred to a pressure-resistant steel cup with a capacity of 300 ml. After the cup has been closed, the reaction mixture is stirred in a water bath for 1.5 h while the beaker is being stirred heated to 333K Then the cup is cooled to about 303 K, opened and the reaction mixture in a beaker with 500 ml of water. The product is filtered off with suction on a frit, residual alkali with dilute (1 N) hydrochloric acid and washed until free from chloride with water obtained a white perlformiges cellulose derivative (10.4g dry matter) with a Sedi mentation volume of 13.4 ml / g and a water holding capacity of 485% The distribution coefficients (K _av ) determined by means of dextranes of different molecular weights (MG) and of proteins as model substances are shown in Table 1 (sample 1)

Example 2

As in Example 1, water-moist perlformige regenerated cellulose is denatured, wherein instead of 0.4 ml of epichlorohydrin 1.6 ml of epichlorohydrin (20 mmol) are used

A white perlformiges cellulose derivative (sample 2) is obtained with a sedimentation volume of 14.9 ml / g and a water holding capacity of 462%.

Surprising result is that the swelling of the products, which is a measure of the pore volume, virtually no difference between Examples 1 and 2, although the amount of crosslinking agent has been widely varied. Also the K _av values are of the same order of magnitude

Example 3

Water-wet perlformige regenerated cellulose having a particle size of 80-200 μηη and a water content of 89 g / 100 g total mass is first dried in a rotary evaporator to a water content of 49g / 100g total mass 20g of the predried material as in Example 1 with 15 ml 30 parts by weight in % containing sodium hydroxide solution, 5 ml of propylene oxide and 0.4 ml of epichlorohydrin reacted and worked up

A white pearl-shaped cellulose product is obtained with a sedimentation volume of 16 ml / g and a water retention capacity of 550%. The K _av values are shown in Table 3 (sample 3). They are markedly lower compared to sample 1.

Example 4

Moisture macroporous cellulose with a particle size of 80-200 μνη and a water content of 89g / 100g total mass is first dried in a rotary evaporator at 333 K to a residual water content of about 10g / 100 g total mass 11,0g of the pre-dried Perlcellulosematerials as in Example 1 with 15 ml of 30 parts by mass in% sodium hydroxide solution, 5 ml of propylene oxide and 0.4 ml of epichlorohydrin reacted and worked up a nearly translucent swollen cellulose derivative obtained (11.0 g dry matter) with a sedimentation volume of 21.9 ml / g and a water holding capacity of 655% Die K _av values are also shown in Table 1. They are significantly lower than in Samples 1 and 3

Example 5

Perlcellulose is dried and denatured analogously to Example 4 using, instead of 0.4 ml of epichlorohydrin, 1.6 ml of epichlorohydrin (20 mmol) (sample 5) or 0.2 ml of epichlorohydrin (2.5 mmol) (sample 6). The sedimentation volumes of the resulting products are 7.9 and 47.4 ml / g and the values for water retention 294 and 894%, respectively. In contrast to the moist macroporous pearl celluloses, the swelling values of the predried pearl celluloses increase considerably as the amount of crosslinker decreases. However, the K _av values are higher despite increased swelling values significantly lower dls for the predried cellulose beads with a water content of 10 and 50 g / 100 g total mass (Table 1)

Example 6

91 g of water-wet macroporous cellulose Perlcellulose is mixed with a solution of 3g 2-chloroethyl-diethylamine hydrochloride in 30 ml of water for 30 min in a rotary evaporator and then dried to a residual water content of 10g / 100g total mass. The impregnated cellulose beads are transferred into a stainless steel beaker, mixed with 15 ml of sodium hydroxide containing 15 parts by mass in%, 5 ml of propylene oxide and 1.6 ml of epichlorohydrin and heated to 353 K for 1.5 h after closing the beaker. The work-up of the product is carried out as in Example 1. The diethylaminoethyl-perl cellulose obtained has an exchange capacity of 0.8 meq / g and a sedimentation volume of 5.2 ml / g.

Example 7

Perl cellulose is modified analogously to Example 6, but 6 g of 2-chloroethyl-diethylamine hydrochloride, 0.4 ml of epichlorohydrin, but no propylene oxide is used. The resulting diethylaminoethyl perl cellulose has an exchange capacity of 1.3 meq / g and a sedimentation volume of 7.2 ml / g.

Example 8

Perl cellulose is modified analogously to Example 6, but instead of 2-chloroethyl-diethylamine hydrochloride, 3 g of sodium monochloroacetate are used as etherifying agent. The obtained carboxymethyl perl cellulose has an exchange capacity of 1.0 meq / g and a sedimentation volume of 10.1 ml / g.

Example 9

Perlcellulose is modified analogously to Example 6, using 6 g of sodium monochloroacetate as the etherifying agent, 0.4 ml of epichlorohydrin, but no propylene oxide instead of 2-chloroethyldiethylamine hydrochloride. The obtained carboxymethyl perl cellulose has an exchange capacity of 1.5 meq / g and a sedimentation volume of 8.2 ml / g.

Table 1: Distribution coefficients (K _av ) for crosslinked hydroxypropyl perl cellulose (bed volume: 20 ml)

dextran Sample 1 Sample 2 K : _av values Sample 5 Sample 6 Dalton 0,298 0.256 Sample 3 Sample 4 0.0 0.0 2 000000 0,520 0,460 0.0 0.0 0.0 0,007 500,000 0,695 0,655 0.046 0.0 0.0 0,050 100000 0,216 0,020 0,020 0,160 35000 0.348 0,080 0.034 0,429 8,000 0.535 0,310 0.143 0.545 4000 0.623 0,425 0.463 0,878 180 * 0.743 0,730

glucose

Claims (7)

1. A process for the preparation of bead-shaped cellulose products for use as release and support materials by crosslinking and simultaneous hydroxyalkylation of Regeneratcelluloseteilchen in alkaline medium, characterized in that water-wet macroporous 1 to 2000 μιη large Regeneratcelluloseperlen with a water content of 85 to 95g per 100g total mass and an exclusion limit from 10 ^{5 to} 3 × 10 ⁷ daltons to a defined water content between 10 and 90 g per 100 g total mass dried, then in an aqueous alkaline medium with 0.1 to 10 moles of alkali metal hydroxide as aqueous solution in a concentration of 5 to 45 parts by weight alkali hydroxide in%, per Mol monomer unit of the cellulose with 0.01 to 0.5 mol of a bifunctional compound of the formula X-R'-Y, where X, Y is a halogen and / or epoxy group and R 'is a C ₂ - to C ₄ -alkylene radical, CH (CH ₂ OH) -CH ₂ - or -CH ₂ -CH (OH) -CH ₂ - to C ₄ -alkylene, -CH (CH ₂ OH) -CH ₂ -or-CH ₂ -CH (OH ) -CH ₂ -, per mole of monomer unit of cellulose and 0 to 2.5 moles of a monofunctional compound of formula ZR, where Z is either an epoxide group and R is H or CH ₃ , or Z is a halogen and R is C ₂ H ₄ -OH, CH ₂ -CH (OH) -CH ₂ - or C ₃ H ^ -O HG group is, per mole of monomer unit of the cellulose, optionally with the addition of substantially inert organic solvents and / or from 0.2 to 1, 0 mol of further etherifying agent per mole of monomer unit of the cellulose in a closed system, reacted at temperatures of 213 to 373 K and in a time of 10 to 360 minutes, washed in a known manner, classified and optionally dried. 2. The method according to claim 1, characterized in that the macroporous bead-shaped cellulose particles used as starting material have a particle size of 50 to 250 μιη. Process according to claims 1 and 2, characterized in that the bifunctional compound used is epichlorohydrin in an amount of from 0.1 to 0.4 mole per mole of monomer unit of the cellulose. 4. The method according to claim 1 and 2, characterized in that is used as the monofunctional epoxy compound propylene oxide in an amount of 0.01 to 2.5 moles per cellulose. 5. The method according to claim 1, characterized in that are used as further etherifying haloalkylcarboxylic acids, -phosphonic acids, -sulfonic acids and / or their salts or haloalkylamines. 6. The method according to claim 1 and 5, characterized in that 2-chloroethyl diethylamine hydrochloride or monochloroacetic acid and / or sodium monochloroacetate is used as a further etherifying agent. 7. The method according to claim 1, characterized in that as substantially inert solvent C _{1 to} C ₃ -AIkOhOIe, acetone, dimethylformamide and / or dimethyl sulfoxide in amounts of from 10 to 80g, based on 100 g of liquid phase used.