CS209268B1 - Polymere ion exchanger for liquid chromatography and method of its preparation - Google Patents

Description

The invention describes the properties, the process and the use of a novel ion exchange type polymer based on the kepelymer of ethyl methacrylate and ethylene glycol dimethyl methacrylate (manufactured by both Separon H or Spheron).

The polymer anion exchanger is prepared by a simple reaction and an intermediate, which is the subject of a separate application, the reaction proceeds with a high conversion and without extraordinary demands on chemicals and equipment and provides a material with suitable parameters for '; high efficient liquid ehromateography,

The use is documented by the high separating ability of mixtures of natural substances and a group of nucleic acid components and promises practical application in the field of biochemistry and clinical analyzes,

The invention relates to a polymer exchange resin for liquid chromatography and to a process for its preparation.

Polymeric ion exchange materials are used for analytical and preparative separation of mixtures of inorganic and organic compounds. Of particular importance have recently been the ion exchangers for so-called high performance liquid chromatography (HPLC), which, in conjunction with suitable sensitive detectors, enables rapid and efficient analysis of mixtures. The polymeric material used must fulfill other conditions for this purpose, eg homogeneous changes, low compressibility at high operating pressures, low volume change when changing ionic strength, polarity and pH of the eluent, and finally must be chemically stable under extreme conditions. These parameters depend, on the one hand, on the properties of the basic polymer matrix, and on the properties and method of bonding the groups bound to the matrix. λ

A suitable polymer for preparing such an ion exchange resin is a macroporous glycol methacrylate gel. i.e., a copolymer of 2-hydroxyethyl methacrylate and ethylene glycol dimethacrylate (Separon H or Spheron), see hereinafter Separon H, whose matrix contains chemically stable units of the 2-hydroxyethyl carboxylic acid type 2-esters (cf. pat. certificate 150 819).

Recent work has reported the preparation of some Separone H-based ion exchangers (carboxymethylsulfonyl phosphoryl dimethyl dimethylaminoethyl derivative) (O. Mikeš, J. Zbrozek, J. Coupek: J. Chromatogr. 119, 339 (1976): O. Mikes, P. Strop, J. Column: J. Chromatogr., 153, 23 (1978)). However, in order to achieve a sufficiently high degree of modification of the base polymer, the reaction had to be carried out repeatedly using less readily available or expensive reagents. These disadvantages are overcome by the present invention of the novel polymer exchange resin, which achieves maximum substitution of the polymeric material in a single reaction and under mild conditions.

SUMMARY OF THE INVENTION The present invention is based on a new liquid ion exchange polymer polymer whose structural unit has the general formula I (I)

From ^CH 2

CH, -C--COOCH CH.OCH.CHCH, ^O ^ y ^CH 2 ^{N / CH 3/2}

CH 2 I 2

OH

The essence of the preparation of this ion exchanger is that 1- (4-chloromethylphenyloxy) -2-hydroxy-3-propyl ether of a copolymer of 2-hydroxyethyl methacrylate and ethyleneglycol dimethacrylate, whose structural unit has the general formula II (Π) /

AND

AND

CH,

CH ₃ - C ~ COOCH ₂ CH ₂ OCH ₂ - CHCH ₂ O - ^ "^ - CH ₂ Cl? ^H 2

The OH is reacted with solvent-free dimethylamine or in an environment of dioxane, methanol, ethanol, chloroform, dimethylformamide or water at temperatures from 20 ° C to 100 ° C, whereupon the resulting product is filtered off and washed, optionally dried.

The starting polymer compound of formula II is readily available from Separon H sequentially by reaction with phenylglyddyl ether and chloromethyl ether (cf. No. 209267) and contains practically all substitutable functional groups etherified with a chloromethyl ligand that is sufficiently stable in the absence of strong nucleophilic agents, but sufficiently reactive to ammonia and amines. Therefore, the reaction of the compound of formula II with dimethylamine takes place under relatively mild conditions. The dimethylamine can be used for the reaction in a commercially available concentrated aqueous solution, optionally directly as a readily recoverable solvent, or it can finally be diluted with other inert solvents (methanol, ethanol, chloroform, dioxane, dimethylformamide and others). The reaction is preferably carried out at a slightly elevated temperature, which shortens the reaction time. Due to the volatility of the dimethylamine, it is also suitable to carry out the reaction in a closed vessel or in a pressure vessel.

After completion of the reaction, the polymeric material is washed to remove solvents and salts with dilute hydrochloric acid, water, and water-removing solvents. The Separone H polymer of the formula I obtained is in the chloride cycle. The nitrogen content, which determines the presence of dimethylamino, corresponds to the complete conversion of the chloromethyl groups in the compound of formula II into dimethylaminomethyl groups.

The nature of the polymer exchange resin of formula I thus prepared is determined by the presence of covalently (ether) bonded groups bearing a tertiary amine group of the benzyldimethylamine type: the compound of formula I is a moderately basic anion exchanger having a pK value of 7.7 (titration curve is shown in FIG. 1). Therefore, it can be advantageously used for the separation of alkaline, neutral and moderately acidic substances. Because this material further comprises aromatic groups fenyletherové may _be: used as partitioning ionic interactions with components of the split mixture. Finally, due to the markoporous nature of the carrier, the ion exchanger capacity of formula I can also be utilized to separate substances on the basis of their molecular weights. F

The reaction of the compound of formula II with dimethylamine does not significantly change the physical parameters for use in chromatography and in particular HPCL (particle size, relative flow rate, etc.) as compared to ^! The basic (unsubstituted) Separone H. Ionex is stable in aqueous buffer solutions, dilute inorganic and organic acids and base solutions, as well as organic solvents (eg ethanol, methanol, acetone, 2-propanol, dioxane, dimethylformamide, ether). With the change in pressure, ionic strength, pH of the solution, there are no significant volume changes on the columns of the support I. It can be regenerated with 2N hydrochloric acid and 2N sodium or potassium hydroxide. '

The usefulness of the polymer exchanger of formula I for separation of mixtures of organic substances is documented by analyzes of mixtures of nucleic acid components (heterocyclic bases, ribonucleosides, 2'-deoxyribonucleosides and their nucleotide derivatives), as well as some of their derivatives (see Figure 2). This example analysis is of importance in the fields of nucleic acid chemistry and biochemistry, molecular genetics and clinical biochemistry. The elution time values shown (Table 1) and the separation diagrams of some mixtures (Fig. 2) demonstrate the high separation capability of the prepared ion exchanger of formula (I), especially when using a borate elution solution, where acid complexes with cis-diol-containing substances grouping. These data illustrate the suitability of the ion exchanger of formula (I) for both microanalysis of natural material mixtures and for enzyme kinetics.

In the following, the invention is illustrated by the following non-limiting examples. and

Example 1

A mixture of 20 g of a compound of formula II (prepared from Separon H-40) (10-15 μ), 100 ml of dioxane and 40 g of anhydrous dimethylamine is heated in a pressure vessel at 70 ° C for 6 hours, then allowed to stand overnight at The suspension was filtered off with suction and the product on the filter was washed successively (2 x 100 ml) with dioxane, water / dioxane (1: 3), water and left to stand in 2 N hydrochloric acid for 2 hours. It is then filtered off with suction, washed with water until the filtrate is neutral and left to stand with 2 N sodium hydroxide for 1 hour. After washing with water until neutral, the procedure with 2 N hydrochloric acid is repeated, finally washed with water until neutral, acetone, ether and dried over phosphorus pentoxide in vacuo. 20 g of compound I are obtained in the chloride cycle. Dimethylamino content 1.9 meq / g dry weight.

Example 2

A mixture of 2 g of a compound of the formula II (prepared from Separon H-1000) (25-63 μ) and 20 ml of a 2M solution of dimethylamine in methanol is treated as described in Example 1. 2.0 g of the compound of the formula I are obtained. chloride content, dimethylamino content 1.95 meq / g dry weight.

Example 3

A mixture of 2 g of a compound of formula II (prepared from Separon H-1000) (25-63 μ) and 20 ml of anhydrous dimethylamine is allowed to stand in a closed vessel for 48 hours at room temperature. The dimethylamine is then distilled off and the residue is worked up as outlined in Example 1. 2 g of the compound of formula I in the chloride cycle are obtained, having a content of 1.87 meq. dimethylamino groups / g dry weight.

Example 4

A mixture of 2 g of the compound of formula II (prepared with Separon H-40) (10-15 μm), 20 ml of chloroform and 5 g of dimethylamine is heated in a pressure vessel at 50 ° C for 8 hours and worked up as described in Example 1. 2 g of the compound of formula I are obtained, containing 1.92 meq. dimethylamino groups / g dry weight.

The following is an example of the use of the polymeric ion exchange resins of the invention for separating substances on a column packed with a compound of formula I. The 0.8 x 10.5 cm column was packed with the suspension method of a 15% carrier suspension prepared according to Example 1 in 1 M boric acid buffer - sodium hydroxide pH 8.60 and elution carried out with the same buffer (concentration shown in Table 1 and Figure 2) (flow rate 124 ml / h). Working pressure 1 MPa (proportional program micro pump PPM 68005, CSAV Developing Workshops), temperature 20 ° C ± 0.1 ° C, spraying 10 to 20 μΐ. Detection was performed on a UV III Monitor 1203 (LDC, USA) at 257 nm; sensitivity of 0.256 and 0.512 absorption units per scale range, using a TZ 21 S recorder (12 cm / hr). The elution time data are shown in Table 1, diagrams of some of the split mixtures in Figure 2.

Figure 2 (Figure 2) contains ABGD plots showing the separation of a mixture of substances on a column of a polymer exchanger of formula I.

Figure A, eluent 0.0025 M buffer; Figure B, eluent 0.01 M buffer, linear gradient of 0.01 to 0.1 M buffer over 30 min in 8.5 min. Graph C, eluent 0.001 M buffer, 10 min linear gradient 0.01 to 0.05 M buffer over 30 min, GrafD, eluent 0.15 M buffer.

DHPA means 9- (2,3-dihydroxypropyl) adenine. The other abbreviations in the Charts are explained in Table 1.

Table 1

Elution times of some compounds. (Time t in minutes.) For conditions see usage example.

Substance Concentrate of eluent (x 10 ³ M) 1 2.5 5 10 50 100 ALIGN! 150 200 Cytosine (Cyt) 2.25 2.25 2.25 Uráčil (Ura) 6.0 5.50 4.0 Thymine 5.25 4.0 Adenin 11.50 10.50 10.50 9.5 7.25 Guanin 8.0 7.0 6.0 Hypoxanthin (Hx) 17.50 13.50 11.75 9.5 6.75 Xanthin 23.75 15.0 9.50 Cytidine (Cyd) 5.50 4.50 Uridin 13.75 9.50 3.75 3-Methyluridine 10.0 Adenosine (Ado) 8.0 9.0 8.50 7.25 4.00 Guanosine (Guo) Xanthosin 18.00 Inosin 7.5 2'-Deoxycytidine 2,0 2'-Deoxyuridine 5.25 2'-Deoxythymidine 4,5 2'-Deoxyadenosine 4,5 2'-Deoxyguanosine 8.0 1- (α-L-Lyxofuranosyl) uracil 4.25 J - (β-D-Lyxofuranosyl) uracil 3.50 J - (β-D-Ribopyranosyl) uracil 3.25 1- (β-D-Xylofuranosyl) uracil 3.75 1- (β-D-Arabinofuranosyl) uracil 3.25 5-Bromo-2'-deoxyuridine 9.0 Cytidine-5'-phosphate (CMP) 15.50 7.50 6.50 Adenosine 5'-phosphate (AMP) 25.0 10.5 8.50 Guanosine-5'-phosphate (GMP) 21.50 17.0 Uridine-5'-phosphate (UMP) 33.0 16.25 13.50 Uridine-2 ', 3'-cyclophosphate 7.0 Urea-3'-phosphate methyl ester 5.5 -

SUBJECT OF THE INVENTION

Claims (2)

1. Liquid chromatography ion exchange polymer whose structure has the formula I '(I) From ^CH 2 CH-C-COOCH "CH" OCH "CHCH CH-N 3 '2 CHI ² OH 2. A process according to claim 1, wherein the 1- (4-chloromethylphenyloxy) -2-hydroxy-3-propyl ether copolymer of 2-hydroxyethyl methacrylate and ethylene glycol dimethacrylate, whose structural unit has the general formula II _{/ C} h ₂ ch ₃ -c —Cooch ₂ ch ₂ and ₂ ch ₂ o ( ^H 2) - ^CH 2 ^Cl The OH (II) is reacted with non-solvent-free dimethylamine at temperatures from 20 ° C to 100 ° C, then the resulting 1a or in dioxane, methanol, ethanol is filtered off and washed or optionally dried. chloroform, dimethylformamide or water;