DD237841A1 - METHOD FOR PRODUCING ION EXCHANGE - Google Patents

Abstract

The aim is to produce an ion exchanger for molecular biological work, which has a high binding capacity with gentle elution. It is an object of the present invention to bind tertiary amines to polymers containing trihalo-s-triazine such that little or no hydrolysis products are formed. The object is achieved by reacting the polymeric material with one or more tertiary amines in the presence of substantially anhydrous organic solvents at temperatures between 20 and 100C within 1-180 min. The ion exchanger prepared by means of the invention is used for analytical and preparative work in microbiology, biochemistry and genetic engineering.

Description

be used, in the R ¹ , R ² and R ^{3 is} an optionally substituted by OH groups or halogen, sulfur, phosphorus and / or silicon-containing groups, unsaturated, saturated, straight-chain or branched aliphatic radical having 1-25 carbon atoms , a cycloaliphatic radical, an optionally substituted, aromatic radical or a heterocyclic radical.

3. The method according to item 1, characterized in that tertiary cyclic amines in which at least one nitrogen atom belongs to a ring are used.

4. The method according to claim 1 and 3, characterized in that bicyclic amines in which at least one nitrogen atom belongs to 2 rings are used.

5. The method according to item 1,3 and 4, characterized in that is used as the tertiary amine diazabicyclooctane.

6. The method according to item 1 or 2, characterized in that at least one nitrogen-substituted group is further substituted by a hydroxyl group.

7. Process according to items 1-6, characterized in that the polymers include cellulose, cellulose derivatives, modified cellulose, cellulose copolymers, grafted cellulose, acrylate polymers and copolymers, polyurethanes, polyamides, polyimides, polysiloxanes, polysulfones, polystyrene and copolymers , be used in the form of powders, granules, fibers, short fibers, fiber structures, tissues, papers, nonwovens, films, hollow fibers, membranes, greases and foams.

Field of application of the invention

The invention relates to a process for the preparation of ion exchangers from polymers and triazines, which are used for analytical and preparative processes in microbiology, biochemistry and gene technology.

Characteristic of the known technical solutions

Ion exchangers based on polymers of various types are known. Of these, a variety of technical products based on polystyrene and cellulose are produced. Other polymers and copolymers have also been described, but have not achieved the importance of these two compositions.

Cellulose-based ion exchangers are used e.g. prepared according to DE-OS 2908185 by reaction with iminodiacetic acid or Iminodipröpionsäure. In DE-OS 3130178 a process for the preparation of fibrous ion exchangers by agglomeration with a hydrophobic polymer is described. According to US Pat. No. 3,823,133, DEAE-cellulose is prepared by derivatization of cellulose with diethylaminoethyl chloride to obtain an anion exchanger. By this method one obtains cation or anion exchangers with very firm binding of the species to be bound and often very low mechanical stability.

DD-WP 148955 describes the reaction of cellulose (paper) with cyanuric chloride for the purpose of activation. No ion exchangers can be produced by this process, and the hydrolysis product also has no ion exchanger properties. An anion exchange paper prepared analogously to DEAE cellulose has a low mechanical stability and is therefore limited in scope in biochemistry.

The reaction of 2,4,6-trihalo-1,3,5-triazines with amines is known. When using tertiary amines are obtained mainly hydrolysis products. In the reaction of cyanuric chloride with pyridine in the presence of a little water is obtained according to SAU RE, Chem. Ber. 83,335 (1950) colorless crystals of dipyridyl dihydrochloride, in the presence of more water are obtained mainly the 2,4-dihydroxy-6- (4-pyridyl) -1,3,5-triazine, d. H. a product without ion exchange capabilities. According to FOYE and BUCKPITT (J.Amer.Pharmaceutical.Assoc., Vol. 41, No. 7, pp. 385, 1952), from 2-substituted-4,6-dihalogen, 1,3,5-triazines and from cyanuric chloride in Present tertiary amines only the Hydroylseprodukte obtained, d. H. Cyanuric acid in the case of cyanuric chloride cyanuric acid and in the 2-substituted compounds 2-substituted-4,6-dihydroxy-1,3,5-triazines, but in no case addition products of tertiary amines or anionic products.

Object of the invention

The invention has the aim of producing ion exchangers in a simple manner, which are particularly suitable for molecular biological work, d. H. which have a high binding capacity with gentle elution.

Explanation of the essence of the invention

It is an object of the present invention to bind tertiary amines to trihalo-s-triazine-containing polymers which covalently bond the triazine and / or contain it as a filler, resulting in little or no hydrolysis products.

The solution of Aufgabhe carried out in that the polymeric material with one or more tertiary amines in the presence of substantially anhydrous organic solvents at temperatures between -20 ⁰ C and 100 ° C within 1-180 min reacted and then dried or optionally with Solvents, water, saline solutions and / or dilute acids and dried.

Surprisingly, it has been found that in the reaction of tertiary amino groups with 2,4,6-trihalo-1,3,5-triazine bound to polymeric materials, substitution of the triazine ring to form quaternary ammonium groups is achieved when the reaction is carried out in solvents Practically no water or this only in small amounts commonly used in laboratory practice. The course of the reaction can then be formally described as follows:

In a parallel reaction, free and bound 2,4,6-trichloro-1,3,5-triazine react with the tertiary amine. Because of its highest reactivity, first the first chlorine atom of the free trichlorotriazine will react, at the same time and then the second chlorine atoms of both the substituted on the polymeric carrier and the free trichlorotriazine. Under these reaction conditions, a substitution of the third chlorine atom is possible, so that in the final product in addition to the going in solution and remaining there substituted triazine both by the tertiary amine mono- and disubstituted, fixed to the carrier triazine residues are prepared.

Suitable solvents for the reaction are benzene, toluene, xylene, ethylbenzene, mesitylene, dioxane, chloroform, acetone, methyl ethyl ketone, decalin, tetralin, cyclohexanone, cyclohexane, trichlorethylene, etc.

Suitable tertiary amines are compounds of the general formula

R-

(3)

in which R ¹ , R ² and R ^{3 are} each independently an aliphatic radical having 1 to 25 C atoms, a cycloaliphatic radical, an optionally substituted aromatic radical, a heterocyclic or alkenyl radical. Of importance are also cyclic amines, z. B. substituted pyrimidines, piperidines, piperazines, also diazabicyclooctane, diazabicycloundecane, imidazole, N-substituted morpholines, etc. In addition, the amines may in turn be substituted, for example, ω-hydroxyalkyl groups, substituted by tertiary amines phenols, substituted by carboxylic acid groups, sulfonic acid groups or phosphoric acid group tertiary Phenylamines are used. The possibility of direct reaction of, for example, tertiary amines with acid groups and the 2,4,6-trihalo-1,3,5-triazines bound to polymeric materials makes it possible to rapidly obtain cation exchangers.

Of particular interest in certain biochemical work are the reaction products of diazabicyclooctane, in which two tertiary nitrogen atoms are present in a strained ring system with exposed lone pairs on the nitrogen atoms:

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This reaction is advantageously carried out in acetone or dioxane. After the relatively rapid reaction (i.e., within 1 to 10 minutes), the second nitrogen atom can also be quaternized by washing with dilute mineral acids.

Importance has to adjust the hydrophilicity and cleavability in an aqueous medium, the use of substituted amines and their feasibility under mild reaction conditions. The inventive method can be in a rapid step anionic ion exchangers with terminal hydrophilic groups z. As hydroxyl groups, produce, wherein the hydroxyl groups practically do not react under these conditions. Suitable amines for this purpose are, for. B. alkanolamines different degrees of substitution, eg. As Dimethylethano'lamin, diethylethanolamine, methyldiethanolamine, dimethylpropanolamine, 4- (dimethylaminomethyl) phenol, 2,4,6-tris (dimethylaminomethyl) phenol, etc. Depending on the number of hydroxyl groups used and the length of the alkyl chains or the number The aromatics results in a different hydrophilicity of the carrier. Furthermore, suitable for this reaction N- (a> -hydroxyalkyl) heterocycles, z. N- (2-hydroxyethyl) piperidine, N-bis (2-hydroxyethyl) piperazine, N- (2-hydroxyethyl) N'-ethylpiperanzine, etc. For this reaction, water-miscible and water-immiscible Solvents are used, provided that the water content is kept low.

As polymeric materials, it is possible to use virtually all of the polymers which covalently bond 2,4,6-trihalo-1,3,5-triazine and / or contain it as a filler. Such polymers include cellulose, cellulose derivatives, modified cellulose, acrylate polymers and copolymers, polyurethanes, polyamides, polyimides, cellulose copolymers, polysiloxanes, polysulfones, etc. These polymers may be in the form of powders, granules, fibers, short fibers, fiber structures, fabrics, nonwovens , Papers, foils, hollow fibers, membranes, hoses and foams.

The process according to the invention provides ion exchange materials in various geometric forms from customary raw materials by a simple reaction which has a high binding capacity for biological and medical materials under normal conditions and can be recovered by simple operations of these biological substances. They are therefore not only suitable for analytical but also for preparative separation processes and for reactions with biological materials while these are fixed. In addition, by using specially substituted amines, support materials can be prepared for specific reactions as well as quantitation-based test strips.

embodiments

In the embodiments support materials are used, namely base support A: a cellulosic sheet carrier, which according to DD-WP-Anm. C 08 C / 262 265/7 with 2,4,6-trichloro-1,3,5-triazine

was reacted and on the surface still free 2,4,6-trichloro-1,3,5-triazine and sodium chloride base support B: a flat carrier of a copolymer of microcrystalline cellulose, polyamide short fibers, polyacrylate and carboxymethyl cellulose, the DD-WP -Anm. C 08 C / 262 265/7 with 2,4,6-trichloro-1,3,5-triazine

reacted was

 Base carrier C: a granular carrier of a copolymer of microcrystalline cellulose and polystyrene, which after

DD-WP-n. C 08 L / 262 265/7 was reacted with 2,4,6-trichloro-1,3,5-triazine. Base support D: a foam-like support based on polyurethane (from glucose polyether alcohol, amine polyether alcohol, water and diphenylmethane diisocyanate) with 3 wt .-% of microcrystalline cellulose as a filler, which after

DD-WP-n. C 08 L / 262 265/7 with 2,4,6-trichloro-1,3,5-triazine. Binding capacity:

0.5 cm ^{2 of} the activated carrier is mixed with 100 μΙΙ of a solution containing 250 / xg DNA from calf thymus and then allowed to stand for 12 hours at room temperature. Thereafter, the supernatant solution is pipetted off and washed 3 times with 1 ml of water. After the last wash, the carrier is dried between filter paper. Subsequently, the bound amount of DNA (as ³² P-labeled) is measured. From the initial value and the measured value, the binding capacity is calculated according to:

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Stability of binding:

0.5 cm ^{2 of} the activated support is added with about ³² % of its capacity to a ³² P-labeled oligonucleotide and allowed to dry. After drying, the predetermined temperature is treated in each case in 1 ml of water and the still bound amount of oligonucleotide is measured and stated in% of the starting amount.

example 1

A sample of 50 cm ^{2 of} the base support A is shaken with 19 g of xylene and 1 g of triethylamine in a glass dish for 10 minutes at room temperature. Thereafter, washed 4x each 30 ml of water and dried at room temperature in the air.

After drying, the tests are carried out and the following values are obtained:

Binding capacity: 200μg / cm ²

Stability: after 30 min / 22 ° C 71%

after 30 min / 40 ⁰ C 69%

after 60 minutes / 60 ^° C. 62%

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Example 2

50 cm ^{2 of} base carrier A are shaken with 1 g of dimethylethanolamine and 19 g of toluene for 10 minutes at room temperature.

Thereafter, 4 χ is washed with 30 ml of water and dried (sample a). The experiment is repeated, but moreover

washed with 30 ml of 0.25% HCl (b).

Binding capacity: (a) 216 / xg / cm ² (b) 230 g / cm ²

Stability: 'after 60 min / 22 ⁰ C 100% 100%

after 60 min / 40 ° C 90% 95%

after 16h / 60 ° C 74% 88%.

Example 3

50 cm ^{2 of} base support A are shaken with a solution of 1.25 g of 2,4,6-tris (dimethylaminomethyl) phenol in 19 g of xylene for 20 min at room temperature in a glass dish. Thereafter, 5 times with 30 ml of water once with a 1% NaCl solution g'ewaschen.

Binding capacity: 186 / * m / cm ²

Stability: after 1h7 / 22 ° C ~ 99%

after 1h / 40 ° C 98%

after 3h / 60 ° C 92%

after 10 minutes / 90 ^° C. 45%

Example 4

50 cm ^{2 of} base carrier A are shaken with a solution of 1.5 g of diazabicyclooctane in 18.5 g of acetone for 30 min at room temperature in a glass dish. It is then washed 4 times with 30 ml of water each time and dried in air. Binding capacity: 288 / xg / cm ² ,

Stability: after 1h / 22h 99%

after 1h / 40 ° C 60%

after 1h / 60 ° C 45%

after 30 minutes / 90 ^° C. 15%

Example 5

50 cm ^{2 of} base carrier A are shaken with a solution of 1 g of methyldiethanolamine in 19 g of acetone for 30 minutes at room temperature in a glass dish. Then it is washed 4x with 40 ml of water and dried in air. Binding capacity: T60 / xg / cm ²

Stability: after 45 min / 22 ⁰ C 89%

after 60 min / 40 ⁰ C 80%

after 60 min / 60 ⁰ C 50%

after 30 minutes / 90 ^° C. 20%

Example 6

50 cm ^{2 of} base support B are shaken with a solution of 1 g of dimethylethanolamine in 19 g of toluene for 15 min at room temperature in a glass dish. Then it is washed with 30 ml of 1: 1 acetone-water mixture, 3x with 30 ml of water and dried.

Binding capacity: 268 / jg / cm ²

Stability: after 1h / 22 ° C 96%

after 1h / 40 ° C 95%

after 3h / 60 ° C 91%

Example 7

5 g of base carrier C are suspended in 50 ml of acetone and 10 ml of a 10% solution of dimethylethanolamine in toluene are added. The mixture is stirred for 1 hour at room temperature, filtered off, squeezed, washed with 2 x 20 ml of water and dried on filter paper

 Binding capacity: 4mg / g

Example 8

A glass column of 20 cm in length and 40 mm in diameter is filled with base support D. It is then pre-swollen with toluene and then slowly eluted with a 1% solution of dimethylethanolamine in toluene until the pH of the eluate is constant. It is then washed first with acetone and then to neutral with water.

Claims (2)

Invention claim: 1. A process for the preparation of ion exchangers based on natural and / or synthetic polymers, characterized in that polymers with covalently bound 2,4,6-trihalo-s-triazines and / or with 2,4,6-trihalogen-s- Triazines reacted as a filler with one or more tertiary amines in the presence of substantially anhydrous solvents at temperatures between -20 ⁰ C and 100 ⁰ C within 1 to 180 minutes and then dried or optionally with solvents, water, salt solutions and / or diluted Acids are washed and dried. 2. The method according to item!, Characterized in that tertiary amines of the general formula