DD237842A1 - METHOD FOR THE PRODUCTION OF ANION EXCHANGERS - Google Patents

Abstract

The invention relates to a process for the preparation of cellulose-based anion exchangers. The aim is to produce mechanically stable anion exchangers in an economically favorable way. The invention has for its object to achieve a homogeneous distribution of the bondability and a chemical and thermal Eluierbarkeit. The object is achieved by a method characterized in that molded or unformed macromolecular masses with chemically active fillers, which - from a macromolecular compound in a concentration of 5-80 vol .-%, - from a macromolecular compound with 4 , 6-Dihalo-1,3,5-triazine groupings in a concentration of 1-80 vol .-%, - of 2,4,6-trihalo-1,3,5-triazine in a concentration of 0.5 -50% by volume, of halides of metals in a concentration of up to 20% by volume and optionally buffering substances of up to 5% by volume, in one or more solvents containing one or more primary and / or secondary solvents linear, cyclic, heterocyclic, aromatic amines, diamines and / or polyamines or a macromolecular substance, each containing primary and / or secondary and optionally tertiary amino groups, reacted, if appropriate, with water and / or a salt solution and / or a dismutated Mi neralsaeure waescht, optionally quaternized with an alkylating agent, washed again and then dried. Field of application of the invention is molecular biology, biochemistry and genetic engineering.

Description

H ₉ MR »/

be implemented, where

R is an alkyl group having 2 to 24 C atoms, an aromatic group, an optionally substituted diphenyl group, an optionally

substituted naphthyl group, an optionally substituted bitolyl group, an optionally substituted diphenylmethane group or an optionally substituted heterocyclic group,

R ¹ and R ^{2 are} each independently hydrogen, an alkyl group of 2 to 18 carbon atoms, an optionally substituted phenyl group or a heterocyclic group.

3. The method according to item 1 or 2, characterized in that shaped or non-formed macromolecular masses are reacted with chemically active fillers in aqueous solution at a pH between 1 and 11 at room temperature with amines, substituted amines and / or polyamines.

4. The method according to item 1 to 3, characterized in that molded or non-formed macromolecular masses with chemically active fillers in mixtures of organic solvents and water having a pH adjusted in the range of 1 to 11 at room temperature with primary and / or secondary amines , Diamines and / or polyamines, with ω-hydroxy-alkylamines, with bis (a> -hydroxyalkyl) amines and / or N- (w-hydroxyalkyl) heterocyclic amines.

Process according to items 1 to 4, characterized in that the reaction is carried out within 1 to 60 minutes at a temperature of between 10 and 60 ^° C.

6. The method according to item 1 to 5, characterized in that are used as molded macromolecular masses mechanically stable cellulose papers, nonwovens, fabrics or coated with other macromolecular masses or mixed cellulose-containing products.

7. The method according to item 1 to 6, characterized in that as unformed macromolecular masses crystalline, spherical or fibrous cellulose, which may be partially or completely coated or mixed with other polymers, is used.

Field of application of the invention

The invention relates to a process for the preparation of cellulose-based anion exchangers which are used for analytical and preparative tasks in molecular biology, biochemistry and genetic engineering.

Characteristic of the known technical solutions

The preparation of cellulose-based ion exchangers is known, see the abstract of EAPeterson, Cellulosic Ion Exchangers in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol.2, ed. TS Work, E. Work, North Holland Publishing Company, Amsterdam-London 1970, p.225-394. Apart from a plurality of cellulose-based particulate ion exchangers, a cellulosic surface support having diethylaminoethyl groups (DEAE-cellulose) attached thereto is described. Although this ion exchanger has a relatively high binding capacity to nucleic acids, but has a low mechanical strength. A further planar ion exchanger, in which DEAE-cellulose is incorporated in a plastic membrane, has a higher mechanical stability but has only ¹ Ao to ¹ Aw of the binding capacity of the DEAE support.

Further, anion-active ion exchangers based on macromolecular compositions comprising dihalotriazine groups and containing 2,4,6-trihalo-1,3,5-triazines and metal halides as fillers are known. These are prepared by reacting these compounds with compounds of the general formula

XRN έΖ-Fii Υ "

wherein X may be an NH ₂ , OH or SH group, R is alkyl or aryl group, R ¹ , R ² , R ^{3 are} alkyl groups and 4 halides or OH. The reaction takes place within 12 hours at room temperature usually in acetonitrile as solvent.

Under the reaction conditions used, about 10% of the triazine groups present are substituted. As a result, although a higher binding capacity than in the above-mentioned plastic membrane, but achieved a much lower than DEAE cellulose. Furthermore, the heterogeneous reaction in two steps leads to an inhomogeneous carrier material. Overall, the reaction in heterogeneous phase in two steps is relatively complicated and expensive.

Object of the invention

The invention has the aim of producing economically stable pathways mechanically stable anion exchangers, which are particularly useful in molecular biology, biochemistry and genetic engineering.

Explanation of the essence of the invention

The object of the invention is to produce anion exchangers which have a homogeneous distribution of the bondability as well as chemical and thermal eluability.

The object is achieved by a process for the preparation of anion exchangers, which is characterized in that molded or non-formed macromolecular masses with chemically active fillers, the

From a macromolecular compound in a concentration of 5-80VoI .-%,

From a macromolecular compound with 4,6-dihalo-1,3,5-triazine groupings in a concentration of 1-80VoI .-%,

From 2,4,6-trihalo-1,3,5-triazine in a concentration of 0.5-50% by volume,

From halides in a concentration of up to 20% by volume and

- optionally from buffering substances up to 5VoI .-%

consist in one or more solvents with one or more primary and / or secondary, linear, cyclic, heterocyclic, aromatic amines, diamines and / or polyamines or a macromolecular substance, each containing primary and / or secondary and optionally tertiary amino groups , optionally with water and / or a salt solution and / or a dilute mineral acid and then dried.

In the process according to the invention, preference is given to diamines having a primary or secondary and a primary, secondary or tertiary amino group of the general formula

i _n

R <£

wherein R is an alkyl group having 2 to 24 carbon atoms, a phenylene group, a diphenyl group, diphenylmethane group, naphthyl group, bitolyl group, a heterocyclic group which may optionally be further substituted, R ^{1 is} hydrogen, an alkyl group of 2-18 C atoms, a Phenyl group or a heterocyclic group and R ^{2 is} hydrogen, an alkyl group of 2-18 C atoms, an optionally substituted phenyl group or a heterocyclic group.

In the reaction of 2,4,6-triahalogen-1,3,5-triazines or monosubstituted 2,4-dihalo-1,3,5-triazines with amino groups in certain solvents or solvent mixtures, hydrogen halide is split off, which with the second amino group reacts under quaternization, so as to obtain at least one quaternary ammonium group. The reaction is preferably carried out in water-immiscible solvents, in water-miscible solvents or mixtures thereof at room temperature within 1 to 60 minutes. Lower or higher temperatures are also possible, but require more effort. Suitable, water-immiscible solvents are, for. Toluene, xylene, benzene, chloroform, methylene chloride, dichloroethane, carbon tetrachloride, trichlorethylene, etc. Suitable water-miscible solvents are e.g. For example, acetone, dioxane, methyl ethyl ketone, alkyl acetate, cyclohexanone, etc. Depending on the reaction conditions and the duration of the reaction in the direction of mono- or disubstitution proceed, monosubstituted products arise at a shorter reaction time and in the event that no after-washing is carried out, carrier materials which still have the ability to covalently bond.

Another embodiment of the method according to the invention consists in carrying out the entire reaction in aqueous solution. In this embodiment, the pH of the amine solution is adjusted by the addition of dilute mineral acid to values between 1 and 9, but preferably between 6 and 8 and the reaction then optionally carried out with the addition of water-miscible solvents at room temperature. As a result, support materials are obtained which are unable to form covalent bonds, but have a high binding capacity. Instead of the diamines, polyamines can be used. These polyamines contain z. B. primary and secondary amino groups. Examples of this class of compounds are diethylenetriamine, triethylenetetramine, dipropylenetriamine, tripropylenetetramine, tetrapropylenepentamine, etc. Furthermore, polyamines can be used, as they are, for. B. in the polymerization of ethyleneimine, and then containing both primary, secondary and tertiary amino groups (by branching reactions) in the molecule.

Likewise, cyclic amines can be reacted by the process according to the invention, provided they still have a nitrogen atom with a hydrogen atom, ζ. As morpholine, pyrimidine, piperazine, N-methylpiperazine, N- (2-hydroxyethyl-lpiperazine, N- (2-sulfoethyl-piperazine, etc.) Substituted cyclic amines and substituted primary and secondary amines can be implemented by the novel process, since Thus, for example, ethanolamine or propanolamine can be reacted in aqueous solution or in water-acetone mixtures to give supports with quaternary nitrogen bases in which the charge is directly linked to the triazine ring which remain sufficiently hydrophilic for the binding reaction with nucleic acids but are thermally labile, thereby allowing for cleavage by a thermal reaction Under the reaction conditions used, only the amino group reacts with the halogen of the triazine ring and not the hydroxyl group (or sulfo group) ,

The process according to the invention consequently represents an economical way of obtaining anion exchangers of granular or planar structure with very different properties. These properties can be used in a variety of ways in genetic engineering, microbiology and biochemistry. The different strength of the binding allows the use of gentle elution methods with mostly quantitative recovery of the bound nucleic acids. In addition, the high binding capacity of the support materials allows working with high concentrations and high yields of the individual steps of the operations. Thus, they are new-grade support materials that offer significant advantages when working with nucleic acids.

embodiments

In the exemplary embodiments, in each case a cone carrier obtained according to DD-WP Note C08 L / 262 265/7 by reaction of cellulose with 2,4,6-trichloro-1,3,5-triazine and on the fiber surface 0, 8 wt .-% 2,4,6-trichloro, 3,5-triazine and 0.65Gew .-% sodium chloride absorbs described. This material is referred to as base carrier A.

example 1

A strip of 50 cm ^{2 of} the base carrier A is shaken in a closable glass dish for 30 minutes at room temperature with a solution of 1.0 g of diethylamine in 19 g of acetone. After this time, it is squeezed onto filter paper, washed 4x with 40 ml of water and dried in air. The following tests are carried out with the carrier:

Binding Assay:

To 0.5 cm ^{2 of} the carrier, add 100 μL of a solution containing 250 mg of calf thymus DNA and allow to stand for 12 hours at room temperature. The supernatant is then pipetted off and washed 3 times with 1 ml of water each time. After the last wash, the carrier is dried between filter paper. Subsequently, the absorbed amount of DNA (as ³² P derivative) is measured. The binding capacity is calculated from the exit value and the measured value. Binding capacity of the carrier: 236 / xg / cm ²

Stability of binding:

0.5 cm ^{2 of} the support is added with about 20% of its capacity with an oligonucleotide and allowed to dry. After drying, treated in each case 1 ml of water at different temperatures and the remaining amount of oligonucleotide measured and reported in%

 Stability after 1 h at 22 ° C 96%

after 1 h at 40 ^° C. 70%

after 1 h at 60 ^° C. 34%

after 10 min at 90 ° C 12%

after 1 h with 10% piperidine at 90 ° C 4%

Example 2

50 cm ^{2 of} base support A are shaken as in Example 1 with 19 g of acetone and 1 g of diethanolamine for 30 minutes at room temperature and treated further as in Example 1. After the tests, the following values are obtained: Binding capacity: 192 / ig / cm ²

Stability after 1 h at 22 ^° C. 88%

after 1h at 40 ° C 58%

after 1 h at 60 ^° C. 22%

after 3 hours at 60 ^° C. 12%

after 30 min at 90 ° C 7%

Example 3

50 cm ^{2 of} base support A are shaken as in Example 1 with a solution of 1 g of diethanolamine adjusted to pH 8.0 by 1.0 N in 19 ml of water for 30 minutes at room temperature and treated further as in Example 1. After the tests, the following values are obtained:

Binding capacity: 230 / xg / cm ²

Stability after 1 h at 22 ⁰ C 89%

after 1 h at 40 ° C 44%

after 1 h at 60 ^° C. 16%

after 10 min at 90 ° C 6%

Example 4

50 cm ^{2 of} base support A are shaken as in Example 1 with a solution of 1.1 g of dimethylaminopropylamine in 19 g of toluene for 10 minutes at room temperature and treated further as in Example 1. After the tests the following values are obtained: Binding capacity: 285μg / cm ²

Stability according to IStd.bei 22 ⁰ C 100%

according to IStd.bei 40 ⁰ C 100%

after IStd.bei 6O ⁰ C 100%

after 30 minutes at 90 ° C 60%

after 15 min at 00 ° C 55%

after 1 h with 10% piperidine at 9O ⁰ C 5%

Example 5

50 cm ² base support A are shaken as in Example 4 with 1.0 g dimethylaminopropylamine in 19 g acetone for 10 min at room temperature and then treated as there. After the tests the following values are obtained: Binding capacity: 220 ^ g / cm ²

Stability after 1h at 22 ° C 100%

after 1h at 40 ° C 100%

after 1h at 60 ° C 100%

after 30 minutes at 90 ° C 66%

after 1 h with 10% piperidine at 9O ⁰ C 4%

Example 6

50 cm ² base support A are shaken as in Example 1 with a solution of 1.2 g of hexamethylenediamine in 22.5 g of toluene for 30 min at room temperature and then treated further as well. After the last wash, however, it is washed once with 10 ml of 0.75% HCl for 5 min. It is then dried and the following values are obtained after the tests: Binding capacity: 430 / xg / cm ²

Stability after 1 h at 40 ° C 96%

according to IStd. at 60 ⁰ C 89%

according to IStd. at 9O ⁰ C 56%

after 1 h with 10% piperidine at 90 ° C 3%

Example 7

50 cm ² base carrier A are shaken as in Example 6 with a solution of 0.95 g of hexamethylenediamine in 19 g of acetone for 10 min at room temperature and treated as there. After the tests the following values are obtained: Binding capacity: 445 ^ g / cm ²

Stability according to IStd.bei 40 ° C 100%

according to IStd. at 60 ° C 100%

after 30 min at 90 ^° C. 50%

after 10 minutes at 100 ^° C. 27%

after 1 h with 10% piperidine at 9O ⁰ C 7%

Example 8

50 cm ^{2 of} base carrier A are shaken with a solution of 1.1 g of benzidine in 10 ml of acetone and 10 ml of toluene for 20 min at room temperature and treated as in Example 6. After the tests the following values are obtained: Binding capacity: 270 //, g / cm ²

Stability after 1 h at 23 ^° C. 100%

after 1 hr. At 40 ⁰ C. 90%

according to IStd.bei 6O ⁰ C '90%

after 1 h with 10% piperidine at 90 ° C 4%

Example 9

50 cm ^{2 of} base support A are shaken with a solution of 1 g of m-phenylenediamine in 15 ml of acetone and 2.5 ml of toluene for 40 min at room temperature and treated further as in Example 6. After the tests the following values are obtained: Binding capacity: 255μg / cm ²

Stability according to IStd.bei 23 ⁰ C 100%

after 1 h at 40 ^° C. 100%

after IStd.bei 6O ⁰ C 100%

after 1 hour with 10% piperidine at 90 ° C 4%

Example 10

50 cm ^{2 of} base carrier A are treated with a solution of 1.2 g of N- (2-hydroxyethyl) -piperazine in 19 ml of water which has been adjusted to a pH of 7.6 by 1 H ₂ SO ₄ in an open glass dish 1 Std. Shaken at room temperature. Thereafter, it is first washed once with 20 ml of 0.01 N HCl, then with water until the pH of the wash water has reached 6.2. Then it is dried in air. The tests are carried out as in Example 1 and the following values are obtained: Binding capacity: 245 / μg / cm ²

Stability after 1h at 23 ° C 98%

after 1 h at 40 ° C 95%

after 1h at 60 ° C 81%

after 30 min at 90 ° C 50%

n:: _i: _ ι: nno

Example 11

50 cm ^{2 of} base carrier A are shaken as in Example 1 with a solution of 1.3 g of diethylenetriamine in 10 ml of acetone and 10 ml of chloroform for 30 min at room temperature and treated as there. The following values are obtained after the tests: Binding capacity: 410 / xg / cm ²

Stability after 1 h at 23 ° C 98%

after 1 h at 40 ^° C. 98%

after 1 h at 60 ° C 96%

after 30 min at 90 ^° C. 88%

after 30 min in 10% piperidine at 90 ^° C. 6%

Claims (1)

Invention claim: Process for the preparation of anion exchangers based on macromolecular masses with chemically active fillers, characterized in that shaped or non-formed macromolecular masses with chemically active fillers, the From a macromolecular compound in a concentration of 5-80VoI .-%, From a macromolecular compound with 4,6-dihalo-1,3,5-triazine groupings in a concentration of 1-80 vol.%, From 2,4,6-trihalo-1,3,5-triazine in a concentration of 0.5-50% by volume, From halides of metals in concentrations up to 20% by volume and - optionally buffering substances up to 5% by volume consist in one or more solvents with one or more primary and / or secondary linear, cyclic, heterocyclic, aromatic amines, diamines and / or polyamines or a macromolecular substance, each containing primary and / or secondary and optionally tertiary amino groups, reacting, if appropriate, washing with water and / or a salt solution and / or a dilute mineral acid, optionally quaternized with an alkylating agent, washed again and then dried. A method according to item 1, characterized in that the shaped or unformed macromolecular masses with chemically active fillers in one or more solvents with diamines of the general formula