WO1996031549A1

WO1996031549A1 - Dendrimeric graft polymers

Info

Publication number: WO1996031549A1
Application number: PCT/EP1995/001278
Authority: WO
Inventors: Egbert Müller; Margot Mack; Peter Poguntke; Dieter Lubda
Original assignee: Mueller Egbert; Margot Mack; Peter Poguntke; Dieter Lubda
Priority date: 1995-04-07
Filing date: 1995-04-07
Publication date: 1996-10-10

Abstract

The invention concerns dendrimeric graft polymers based on base carriers containing hydroxyl groups and on the surfaces of which polymers are covalently bound, the following conditions applying: a) the base carrier contains aliphatic hydroxyl groups; b) the covalently bound polymers are bound by an end-position monomer unit to the basic carrier; c) the polymers at the branching points of the dendrimeric structure contain monomer units of formula (II); and d) the dendrimeric polymers contain monomer units of formula (III); R?1, R2, R3¿ independently of one another stand for H or CH¿3, R?4 stands for H, C¿1?-C5 alkyl or C6-C12 aryl, n is an integer between 1 and 5, one group X is OH and the other group X is an end-position monomer unit of a further polymer chain, and Y stands for a group containing a separation effector. The invention also concerns the production of these dendrimeric graft polymers and their use as separating agents for liquid chromatography.

Description

Dendrimeric graft polymers

The invention relates to dendrimeric graft polymers and their use as separation materials for liquid core chromatography.

From DE 38 11 042 separation materials for chromatography are known which have linear graft polymers. These materials have clear advantages over separation materials that contain crosslinked polymers. The object of the present invention is. To provide release materials with improved properties.

DE 43 10 964 discloses oxirane-containing activated carrier materials in which monomers of the formula I are grafted onto a hydroxyl-containing base carrier,

woπn

R ¹ , R2 and R3 independently of one another

H or CH ₃ , R ₁ H, d-Cs-alkyl or C ₆ -C ₁₂ aryl and n is an integer between 1 and 5.

It has been found that these activated carrier materials can be converted into the graft polymers according to the invention with a dendrimeric structure. Linear polymers are in turn grafted onto the known graft polymer with a linear polymer chain (first generation polymer chain) (second generation polymer chain), this step being able to be repeated several times (higher generation protein chains).

ORIGINAL DOCUMENTS This gives rise to branched graft polymers which, however, have no crosslinking. In addition, separation effectors are introduced in accordance with the intended chromatographic separation processes. The resulting release materials have improved properties.

The separation effectors required for the various chromatographic separation processes are bound as a solid phase to a base support and interact with the analytes of the sample to different extents. Suitable separation effectors are known to the person skilled in the art for various chromatographic separation methods; for example: ionic or ion-forming (ionogenic) groups for ion exchange chromatography, affinity ligands for affinity chromatography, which also includes metal chelate chromatography, hydrophobic groups for hydrophobic interaction chromatography or reversed phase chromatography and predominantly reticulated porous hydrophilic groups for gel permeation chromatography. For the separation of low molecular weight analytes from protein-containing matrices, materials with hydrophilic and hydrophobic areas are known, which either by their pore structure (US 4,544,485; EP 0 173 233) or by hydrophilic shielding (US 5,277,813) avoid the undesired binding of the proteins to the hydrophobic areas .

The invention relates to dendrimeric graft polymers based on hydroxyl-containing base supports, on the surface of which polymers are covalently bonded, with a) the base support containing aliphatic hydroxyl groups, b) the covalently bonded polymers being bonded to the base support via a terminal monomer unit, c) the polymers contain monomer units of the formula II at the branching points of the dendrimeric structure

d) and the dendrimeric graft polymers contain monomer units of the formula III,

- (CR ¹ R ² -CR ³ ) - i IM

wherein

Ri, R2 and R3 independently

H or CH _3l R4 H. d-Cj-alkyl or C ₆ -C ₁₂ aryl, n represents an integer between 1 and 5, one radical X OH and the other radical X represents a terminal monomer unit of a further polymer chain, and

Y is a radical containing a separation effector.

The dendrimeric graft polymers according to the invention can be obtained by the following reaction steps: a) grafting of monomers of the formula I onto a hydroxyl group-containing base support in the presence of cerium IV ions,

wherein Ri, R2, R3, R and n have the meanings already mentioned; b) at least partially converting the oxirane groups into diol groups; c) grafting on further monomers, for example further monomers of the formula I, or else monomers which contain separation effectors in the presence of cerium IV ions; d) optional repetition of steps b) and c); e) introduction of residues with separation effectors, insofar as these have not already been introduced by the grafting reaction with monomers which contain separation effectors; f) optional ring opening of remaining oxirane groups.

The invention relates to the use of the dendrimeric graft polymers according to the invention in the separation of mixtures of at least two substances, in particular for the separation of biopolymers, by means of liquid chromatography, in particular by means of ion exchange and affinity chromatography. Another use according to the invention is the separation of low molecular weight analytes from protein-containing matrices.

The invention also relates to processes for the preparation of dimeric graft polymers with the following process steps: a) grafting of monomers of the formula I onto a hydroxyl group-containing base support in the presence of cerium IV ions,

wherein

Ri, R2 and R3 independently

H or CH ₃ , RH, C _r C ₅ alkyl or C ₆ -C ₁₂ aryl and n are an integer between 1 and 5, with activated carrier materials known from DE 43 10 964 being formed, b) at least partially converting the oxirane groups into diol groups; c) grafting of monomers of the formula I or of monomers of the formula V onto the diol groups formed in step b) in the presence of cerium (IV) ions,

CR ¹ R ² = CR ³

V

0 = C-W

10 where

W OH or NHRs and

R ⁸ C -C ₀ alkyl which with an amino, monoalkylamino,

Dialkylamino, trialkylammonium, carboxyl or AC sulfonic acid residue is substituted, where steps b) and c) can be repeated one or more times, • d) introduction of the residues which contain the separation effectors, insofar as Q has not already done so the grafting reaction with monomers of

Formula V has taken place and e) optional ring opening of remaining oxirane groups

The invention relates to processes for the separation of mixtures of at least two substances, in particular for the separation of biopolymers, by means of liquid chromatography, in particular by means of ion exchange, hydrophobic interaction or affinity chromatography, using the dendrimeric graft polymers according to the invention. Further methods according to the invention relate to the separation of low molecular weight analytes from protein-containing matrices with the aid of dendrimeric separation materials according to the invention.

5 Figure 1 shows the dependence of selectivity α (curve A) and binding capacity (curve B) on diethylamine-substituted (DEA) ion exchangers with different degrees of branching (sample number as abscissa). The experimental details can be found in application example A. It shows that the selectivity of branched material is significantly higher than that of unbranched material. The binding capacity increases compared to unbranched comparative material with little branching, but drops significantly with strong branching.

Figures 2 and 3 show the experimental setup for the separation of analytes from biological matrices, for example serum or plasma, using separation materials according to the invention. Figure 4 shows the results of a recovery test using a separating material produced according to Example 9. Further experimental details can be found in application example B.

The dendrimeric graft polymers according to the invention are distinguished by a tree-like branched structure, a first linear polymer being grafted onto a base support which contains aliphatic hydroxyl groups. Monomers of the formula I, where the radicals R ¹ , R2, R3 and R ⁴ and n have the meanings already mentioned, are grafted on in the presence of cerium IV ions, a linear graft polymer being formed. The basics of this reaction are described by G. Mino and S. Kaizerman (1958) J. Polymer Science __1, 242-243, and G. Mino et al. (1959) J. Polymer Science __, 393-401. The polymer initially contains oxirane residues, which are then completely or partially converted into diol groups. Treatment with dilute sulfuric acid is preferred.

Monomers of the formula I can now be polymerized again in the presence of cerium IV ions onto the aliphatic hydroxyl groups of this polymer (first generation) thus formed. The resulting polymer (second generation) is linear in itself. However, the forest is branched. In order to obtain even more branched dendrimeric graft polymers, the above-mentioned steps can be repeated: conversion of the oxirane groups into diol groups and polymerization of monomers of the formula I in the presence of cerium IV ions.

In addition, the separation effectors required for the chromatographic separations are introduced, which are bound to a base support as a solid phase and which interact to different degrees with the analytes of the sample. Suitable separation effectors are known to the person skilled in the art for various chromatographic separation methods, for example: a) For ion exchange chromatography, ionic groups such as, for example, quaternary ammonium alkyl groups and the SO ₃ group, and also ionogenic groups which form ions under certain pH conditions are known. The last group includes, for example, the alkylated amino groups, as well as the carboxyl and phosphoric acid groups. b) A large number of affinity ligands are known to those skilled in the art for affinity chromatography, each of which forms a structurally defined bond with the analyte, and which as

Separation effectors are suitable, for example:

Table 1 :

Affinity ligand analyte (example) Protein A immunoglobulins

Concanavalin A glycoproteins

Biotin avidin / streptavidin

Avidin biotin

Streptavidin biotin 5'-adenosine monophosphate NAD-dependent oxidoreductases

2 ', 5'-Adenosine diphosphate NADP-dependent oxidoreductases

Aminoacridine RNA or DNA Table 1 (continued):

Affinity ligand analyte (example.

Boronic acid catecholamines

Boronic acid glycosylated hemoglobin iminodiacetic acid metalloproteins

"thiophile" ligands immunoglobulins

Cibachrome blue monoclonal antibodies

c) Uncharged hydrophobic separation effectors are customary for hydrophobic interaction chromatography, for example C ₁ -C ₂ o-alkyl,

C ₆ -C_ ₅ aryl, C ₇ -C ₂₅ alkylaryl or C ₇ -C ₂₅ arylalkyl, which can also be derivatized one or more times with nitrile or Ci-Cs-alkoxy, one or more non-adjacent CH ₂ Groups can be replaced by NH or O or one or more CH groups can be replaced by N, or polyoxyethylene or polyoxypropylene derivatives

[(CH ₂ ) π _. -O-] ₀ -R ⁹ , wherein m is 2 or 3, o is an integer between 1 and 200 and Rs is H or CrC ₅ alkyl. Residues with medium or low hydrophobicity are particularly preferred. These residues can be introduced as alkyl or aryl residues, as alkoxy or aroxy residues or as alkoyl or aroyl residues.

d) For gel permeation chromatography, hydrophilic compounds, which preferably form pores or networks, are used as the separation effector. These include (meth) acrylic acid derivatives such as acrylamide or methacrylamide, furthermore (2,3-dihydroxypropyl) methacrylate or N- (2-methoxyethyl) acrylamide or N- (2,3-dihydroxypropyl) acrylamide. They also include vinylated heterocycles such as 1-vinylimidazole, N-vinylpyrrolidone, 2-vinylpyridine, 4-vinylpyridine, 4-vinylpyrrolidone-N-oxide.

e) So-called shielded phases are used for the separation of low molecular weight substances from biological samples (eg urine or blood). These separating materials have both hydrophobic and hydrophilic areas. The hydrophobic areas, the structure of which corresponds to the hydrophobic separation materials mentioned above (see c)), interact with the low molecular weight analyte of the sample. The hydrophilic areas prevent the interaction of the high molecular weight portions of the sample (eg the proteins) with the hydrophobic areas. Dendrimeric graft polymers according to the present invention are outstandingly suitable as shielded phases for the

Separation of analytes from biological matrices.

Various reaction paths are possible for introducing the separation effectors: a) The separation effectors are reacted with the

Oxirane residues which are present after the polymerization with compounds of the formula I are built into the support; e.g .: a1) the reaction with sulphurous acid or its salts or with primary, secondary or tertiary amines, whereby ion exchangers are formed; a2) the reaction with iminodiacetic acid or the introduction of thiophilic ligands, or other affinity ligands such as protein A, producing carriers for affinity chromatography; a3) the reaction with alcohols, phenols or primary amines, whereby hydrophobic separation materials are formed.

Hydrophobic separation effectors can also be introduced, for example, by ester bonds to hydroxyl groups, such as those formed by hydrolysis of the oxirane residues.

In the reaction sequence according to a1), for example, compounds are formed in which the radical Y from formula III has the meaning of formula IV,

0 = C-0- (CH ₂ ) _π -CH-CHR ⁴ IV

II currently woπn

R ^{4 is} H, d-Cj-alkyl or C ₆ -C ₁₂ aryl, n is an integer between 1 and 5, one radical Z OH and the other radical Z is a radical selected from the group NR5R6, N + R5R6R ?, po ₄ H ₂ and S0 ₃ H, and Rs, R6 and R ⁷ independently

C 1 -C ₄ -alkyl, where one or both radicals R5 or Re can also be H. b) In the last graft polymerization, the monomers known from DE 38 11 042 can be used instead of the monomers of the formula I, the groups known from this publication being introduced as separation effectors. These include, for example, monomers of the formula V

CR ¹ R ² = CR ³

O = C-W

wherein

W OH or NHR ^β and

R ⁸ CrCio-alkyl, which is substituted with an amino, monoalkylamino, dialkylamino, trialkylammonium, carboxyl or sulfonic acid residue.

In the support materials produced by process variant b), the radical Y from formula III denotes a radical according to formula VI,

VI

0 = CW wherein

W OH or NHR ^β and

Rs d-C-io-alkyl, which is substituted with an amino, monoalkylamino, dialkylamino, trialkylammonium, carboxyl or sulfonic acid residue.

Preferred monomers of the formula V are those in which W has one of the following meanings: OH, NH (CH ₂ ) ₂ N (CH ₃ ) ₂ , NH (CH ₂ ) ₂ N (C ₂ H ₅ ) ₂ , NH (CH ₂ ) ₂ N ⁺ (CH ₃ ) ₃ , NHC (CH ₃ ) ₂ CH ₂ S0 ₃ H or NH (CH ₂ ) ₂ S0 ₃ H.

After the reactions described have been carried out, any remaining oxirane residues can be hydrolyzed, for example by a final treatment with dilute sulfuric acid.

Even without further explanations, it is assumed that a person skilled in the art can use the above description to the greatest extent. The preferred embodiments are therefore only to be understood as a descriptive disclosure, in no way as a limitation in any way.

The complete disclosure of all of the applications, patents and publications mentioned above and below, in particular German application P 43 34 351, filed on October 8, 1993, is incorporated by reference into this application.

The following examples are intended to explain the subject in more detail; these examples do not represent a restriction of the subject matter of the invention. Examples

Manufacturing examples

In the following production examples, room temperature (RT) means

15-30 ° C. The polymerization is carried out in a suitably sized three-necked flask equipped with a stirrer, dropping funnel and thermometer. It is washed by suction on a glass frit (G2).

Example 1: Production of an oxirane-activated carrier starting from Fractogel®-TSK HW 65 (S)

A suspension of 100 ml of sedimented Fractogel®-TSK HW 65 (S) and 66 ml of water is mixed with 3 g of ammonium cerium (IV) nitrate (dissolved in a

Mixture of 180 ml water and 3 g HN0 ₃ (65%)) mixed at room temperature with vigorous stirring. After 1 minute, a solution of 6 g (2,3-epoxypropyl) methacrylate in 44 ml of dioxane is added. It is stirred for an hour. The reaction product is then washed twice with 200 ml of water, three times with 100 ml of acetone and three times with 200 ml of water.

Example 2: Production of an oxirane-activated carrier starting from LiChrospher® diol

The preparation is carried out according to Example 1, LiChrospher®-DIOL (particle size 15-25 μm, pore size 80 nm) is used as the base support instead of Fractogel®-TSK HW 65 (S). Example 3: Grafting a polymer onto a diol-containing polymer (producing the dendrimeric structure)

Stage 1: Transfer of the oxirane into diol groups 100 ml of extracted oxirane-activated carrier material produced according to

Example 1 are hydrolyzed with 200 ml of 0.5 M sulfuric acid (1 hour, 50 ° C.) and the oxirane groups are thus converted into diol groups. It is then washed three times with 200 ml of water each time.

Step 2: grafting on another polymer chain

To a suspension of 100 ml of sedimented material from stage 1 and 66 ml of water are mixed with 3 g of ammonium cerium (IV) nitrate (dissolved in a mixture of 180 ml of water and 3 g of HNO ₃ (65%)) at room temperature with vigorous stirring. After 1 minute, a solution of 6 g (2,3-epoxypropyl) methacrylate in 44 ml of dioxane is added. It is stirred for an hour. The reaction product is then washed twice with 200 ml of water, three times with 100 ml of acetone and three times with 200 ml of water.

The result is a single-branched dendrimary material with oxirane residues.

Example 4: Grafting a polymer onto a diol-containing polymer (generation of multi-branched denim structures)

The material from Example 3 is again subjected to the reaction sequence described in Example 3. A doubly branched denim material with oxirane residues is formed.

This material can in turn be subjected to the reaction sequence described in Example 3. This creates dendrimary materials with oxirane residues and with higher degree of branching. - 14 -

Example 5: Synthesis of a dendrimeric separation material substituted with diethylamine

100 ml of filtered gel prepared according to Example 3 (single branch) are suspended in 100 ml of water and 100 ml of diethylamine are added.

Then stirring is continued for 20 hours at room temperature. The reaction product is then washed twice with 100 ml of water.

The washed reaction product is suspended in 100 ml of a 0.5 M sulfuric acid solution and slowly stirred at 40 ° C. for two hours. Then it is washed with 0.25 M phosphate buffer (pH 7) to the neutral point, then with water. The gel is stored in aqueous suspension with the addition of 0.02% sodium azide.

Example 6: Synthesis of a double-branched dendrimeric separation material substituted with diethylamine

100 ml of filtered gel prepared according to Example 4 (branched twice) are reacted with diethylamine as described in Example 5.

Correspondingly, even more branched dendrimeric separation materials substituted with diethylamine are accessible.

Example 7: Preparation of an affinity support for metal chelate chromatography

A solution of 15 g NaOH and 25 g iminodiacetic acid in 100 ml water is adjusted to pH 11 with concentrated HCl and decolorized with 1 g activated carbon. 50 ml of suctioned-off oxirane-activated carrier material prepared according to Example 4 (branched twice) are added to this solution. The solution is stirred at 45 ° C for 20 hours. The reaction product is filtered off, washed with 250 ml of 0.5 M NaOH and washed with water. The unreacted oxirane groups were hydrolyzed by treatment with 100 ml of 0.5 M sulfuric acid (2 hours, 45 ° C.). The affinity carrier material is then washed once with 100 ml of 0.5 M sulfuric acid, twice with 100 ml of water, once with 100 ml of 0.5 M phosphate buffer pH 7 and once with 100 ml of 1 M NaCl solution. The gel is stored in 0.02 M phosphate buffer pH 7 with the addition of 1 M NaCl and 0.02% NaN ₃ .

Example 8: Preparation of an acidic ion exchanger

Step 1 :

100 ml of gel prepared according to Example 3 are suspended in 160 ml of 0.5 M sulfuric acid and stirred at 45 ° C. for one hour. It is then washed three times with 500 ml of water each.

Level 2:

10 g of NaOH are dissolved in 200 ml of water and 2-acrylamido-2-methylpropanesulfonic acid is added until the pH is 4 (approx. 45 g; monomer solution). As a starter solution for the polymerization, 8.2 g of ammonium cerium (IV) nitrate are dissolved in 50 ml of 0.5 M nitric acid.

100 ml of gel from stage 1 are suspended in the monomer solution and poured into a three-necked flask, the starter solution is poured into the dropping funnel connected to it. The apparatus is evacuated three times and gassed with argon. The starter solution is then run in with stirring (150 rpm) and then stirred at 40 ° C. for a further four hours. The product is then washed with 200 ml of 1 M sodium sulfite in 1 M sulfuric acid, with 1 I of water, with 3 I of 0.1 M NaOH, with 500 ml of 1 M sodium acetate buffer pH 7 and with 500 ml of water. The product is stored in 20 mM phosphate buffer pH 7 with the addition of 0.02% NaN ₃ . Example 9: Preparation of a support material for the determination of low molecular weight substances in biological matrices

Step 1: Preparation of a Glycidyloxypropyl Carrier

10 g of a LiChrospher® Si 60, particle size 25 μm, with a specific surface area of 380 m2 / g and an average pore diameter of 9 nm (E. Merck, Darmstadt) are suspended in 50 ml of toluene and after adding 3.8 ml ( 4 mmol / m ² ) Gycidyloxypropyl-methyl-dimethoxysilane boiled under reflux for 5 hours with stirring. After the material has been suctioned off, it is washed with toluene and methanol and dried.

Stage 2: Ring opening of the glycidyloxypropyl carrier to the diol phase. The product obtained from stage 1 is suspended in 50 ml of a 5% sulfuric acid solution and refluxed for 3 hours with slow stirring to open the epoxy ring. After the reaction suspension has been suctioned off, it is washed free of sulfate with water and, after washing again, is dried with methanol. A diol carrier (carbon content 7.6%; corresponding to 2.81 mmol / m∑ diol groups) is obtained.

Stage 3: First graft polymerization

To a suspension of 40 ml sedimented LiChrospher® diol

Stage 2 and 60 ml of water are mixed with 0.8 g of ammonium cerium (IV) nitrate (dissolved in a mixture of 40 ml of water and 1.2 g of HNO ₃ (65%)) at room temperature with vigorous stirring. After 1 minute, a solution of 1.2 g (2,3-epoxypropyl) methacrylate in 15 ml of dioxane is added. It is stirred for an hour. The reaction product is then washed twice with 200 ml of water, three times with 100 ml of acetone and three times with 200 ml of water. Stage 4: sulfuric acid hydrolysis

40 ml of gel from stage 3 are suspended in 160 ml of 0.5 M sulfuric acid and stirred at 45 ° C. for one hour. It is then washed three times with 500 ml of water each.

Step 5: Second graft polymerization

0.8 g of ammonium cerium (IV) nitrate are dissolved in a mixture of 40 ml of water and 1.2 g of HNO ₃ (65%). This solution is added to a suspension of 40 ml of sedimented grafted LiChrospher® diol from stage 4 and 60 ml of water at room temperature with vigorous stirring. To

A solution of 1.2 g (2,3-epoxypropyl) methacrylate in 15 ml of dioxane is added in 1 minute. It is stirred for an hour. The reaction product is then washed twice with 200 ml of water, three times with 100 ml of acetone and three times with 200 ml of water.

Step 6: Reaction of the dendrimeric graft polymer with hydrophobic

Ligands 5 g of the dried carrier material from stage 5 are suspended at 0 ° C. in dry chloroform. 60 ml of dried triethylamine are added to this solution. A solution of 2 g of stearoyl chloride in 25 ml of chloroform is then added over the course of 3 hours, while cooling to 4 ° C.

After the acid chloride has been added, the mixture is stirred at room temperature for 48 hours and the gel is then washed with 50 ml of chloroform, methanol, water and methanol and dried.

Stage 7: sulfuric acid hydrolysis

40 ml of gel from stage 6 are suspended in 160 ml of 0.5 M sulfuric acid and stirred at 45 ° C. for one hour. It is then washed three times with 500 ml of water each.

In the manner described above, a dendrimeric shielded phase separation material is provided, the hydrophobic residues of which are shielded by hydrophilic diol groups. The following application example shows the influence of the degree of branching on the binding capacity and separating capacity of the separating material.

Application example A: Influence of the degree of branching

Single to seven-fold branched DEA-derived separation materials (samples 2-7) are produced in accordance with examples 5 and 6, unbranched comparison material (sample 1) is produced in analogy to example 5, the linear polymer from example 1 being used instead of the dendrimeric oxirane-containing polymer becomes.

These separating materials are each filled into a SuperFormance ^ glass column (50 x 10 mm) and equilibrated with the application buffer (50 mM TRIS buffer, pH 8.3). A solution of bovine serum albumin (10 mg / ml) in this buffer is applied continuously (flow: 0.5 ml / min) and the elution diagram is measured by photometry at 280 nm. The capacity is determined from the breakthrough curve. The separation behavior for bovine serum albumin is also determined and expressed as the selectivity factor α. The results are summarized in Figure 1.

It can be seen that the selectivity α (curve A) of branched material is significantly higher than that of unbranched material. The binding capacity (curve B) increases compared to unbranched comparison material with little branching, but drops significantly with strong branching.

Example of use B: Recovery of carbamazepine in plasma

a) Apparatus

2 shows the apparatus structure, in which mean:

1. Guard column buffer 2. Analysis buffer

3.HPLC pump (L-6000) 4.HPLC pump (L-6200)

5.Automatic sampler (AS-4000) 6.Automatic switch valve (ELV-7000)

7th guard column 8th analytical column 9. Detector 10. Integrator (D-2500)

11. Waste container (devices from E. Merck, Darmstadt, Germany)

In Fig. 3, the line connections between the modules in

Dependence on the position of the changeover valve (6) shown: Fig. 3a: Position "L" (LOAD) Fig. 3b: Position "I" (INJECT)

b) Chromatographic conditions:

Guard column ((8); 25 x 4 mm): Separating material, manufactured according to Example 9;

Guard column buffer (1): 0.05 M Na phosphate (pH 5.0); analytical column ((8);

LiChrospher® 60 RP-select B, 5 μm, 125 x 4 mm); Analysis buffer (2):

0.05 M Na phosphate (pH 4.0) / acetonitrile (80:20; V: V); Detection UV 210 nm.

c) Analysis cycle

After the plasma sample (100 μl) has been injected by the automatic sample dispenser (5) into position "L" of the changeover valve (6), the sample reaches the pre-column buffer ((1); pumped by the HPLC pump (3); flow rate 0 , 5 ml / min) on the guard column (7). The analyte (carbamazepine) is selectively retained by the separating material of the guard column, while matrix components, in particular proteins, are eluted into the waste container (11) within 12 minutes.

After switching the valve (6) to position "I", the analyte is completely removed from the guard column within 5 minutes with the help of the analysis buffer ((2); flow rate 0.8 ml / min), which is pumped by the HPLC pump (4) (7) eluted and transferred to the downstream analytical column (8).

After switching the valve (6) to the "L" position, the analytical separation takes place under isocratic conditions (flow rate 0.8 ml / min). The eluted connections are measured in the detector (9) and evaluated in the integrator (10). At the same time, the guard column is conditioned for a new analysis cycle using the HPLC pump (3). d) result

In Fig. 4, the elution diagrams for A are: a calibrator containing 0.5 µg carbamazepine, and

B: a plasma sample (human plasma) which also contains 0.5 μg carbamazepine.

As can be seen, the analyte is completely found again in the plasma sample.

Claims

- 21 -

Expectations

1. Dendrimeric graft polymers based on hydroxyl-containing base supports, on the surfaces of which polymers are covalently bound, characterized in that a) the base support contains aliphatic hydroxyl groups, b) the covalently bound polymers are bound to the base support via a terminal monomer unit, c) the polymers contain monomer units of the formula II at the branching points of the dendrimeric structure

d) the dendrimeric polymers contain monomer units of the formula III,

- (CR ¹ R ² -CR ³ ) - ...

wherein

Ri, R and R3 independently

H or CH ₃ , R ⁴ H, C Cs-alkyl or C ₆ -C ₁₂ aryl, n is an integer between 1 and 5, one radical X OH and the other radical X represents a terminal monomer unit of a further polymer chain, and

Y is a radical containing a separation effector. 2. Dendrimeric graft polymers obtainable by the following reaction steps: a) grafting of monomers of the formula I onto a hydroxyl group-containing base support in the presence of cerium IV ions,

CR ¹ R ² = CR ³

wherein

R 1, R ² and R, independently of one another, denote H or CH ₃ , 5 R ⁴ H, CC ₅ alkyl or C ₆ -C ₁₂ aryl and n is an integer between 1 and 5, b) at least partially converting the oxirane groups into o diol groups; c) grafting on further monomers, for example of further monomers of the formula I, or else monomers which contain separation effectors, in the presence of cerium IV ions; d) optional repetition of steps b) and c); 5 e) introduction of residues with separation effectors, insofar as these have not already been introduced by the grafting reaction with monomers which contain separation effectors; f) optional ring opening of remaining oxirane groups.

3. Dendrimeric graft polymers according to claim 1 or 2, characterized in that the separation effector contains an ionic or ionogenic radical.

5 Dendrimeric graft polymers according to claim 3, characterized in that a radical Y according to formula IV is present,

0 = C-0- (CH ₂ ) _π -CH-CHR ⁴ IV

I I z z

wherein

R ⁴ H, CC ₅ alkyl or C ₆ -C ₁₂ aryl, n is an integer between 1 and 5, one radical Z OH and the other radical Z is a radical selected from

Group NRsRe, N-RsR ^β R?, P0 ₄ H ₂ and S0 ₃ H, and

Rs, R6 and R ⁷ independently of one another are dd-alkyl, where one or both radicals Rs or R ~ can also be H.

5. dendrimeric graft polymers according to claim 3, characterized gekenn¬ characterized in that a radical Y is contained according to formula VI,

VI

0 = C-W

wonn

W OH or NHR ^β and

R ^β Cι-C ₁₀ alkyl with an amino, monoalkylamino,

Dialkylamino, trialkylammonium, carboxyl or

Substituted sulfonic acid mean.

6. Dendrimeric graft polymers according to claim 1 or 2, characterized in that the radical Y contains an affinity ligand. 7. Dendrimeric graft polymers according to claim 1 or 2, characterized in that the radical Y contains a hydrophobic radical.

8. Use of dendrimeric graft polymers with the features of claim 1 or 2 in the separation of mixtures of at least two substances, in particular for the separation of biopolymers, by means of liquid chromatography, in particular by means of ion exchange, hydrophobic interaction or affinity chromatography.

9. Use according to claim 8, wherein analytes are separated from biological matrices.

10. Process for the preparation of dendrimeric graft polymers, characterized by the following process steps: a) grafting of monomers of the formula I onto a hydroxyl group-containing base support in the presence of cerium IV ions,

wherein

Ri, R2 and R3 independently

H or CH ₃ , R ⁴ H, CC ₅ alkyl or C ₆ -C ₁₂ aryl and n are an integer between 1 and 5, b) at least partially converting the oxirane groups into diol groups; c) grafting of monomers of the formula I or of monomers of the formula V onto the diol groups formed in step b) in the presence of cerium (IV) ions,

wherein

W OH or NHR ^β and

Rß CC ₁₀ alkyl, which with an amino, monoalkylamino,

Dialkylamino, trialkylammonium, carboxyl or

Sulfonic acid residue is substituted mean

wherein steps b) and c) can be repeated once or several times, and d) introduction of the radicals which contain the separation effectors, provided that this has not already been done by the grafting reaction with monomers of the formula V.

11. Process for the separation of mixtures of at least two substances, in particular for the separation of biopolymers, by means of liquid chromatography, in particular by means of ion exchange, hydrophobic interaction or affinity chromatography, using dendrimeric graft polymers with the features of claim 1 or 2 .

12. The method according to claim 11, wherein analytes are separated from biological matrices.