Hydrophilic polymeric sorbent

 

(57) Abstract:

Usage: synthesis of hydrophilic organic polymeric sorbent for liquid chromatography for the separation of biopolymers. The inventive hydrophilic polymer sorbent is a product of the interaction of the copolymer epoxyethyl - (meth) acrylate of the General formula given in the text description, and a crosslinking monomer selected from the group consisting of di-, tri-, Tetra (meth) acrylates of pentaerythritol or mixtures thereof, with an organic hydroxyl-containing modifier is activated epichlorohydrin compound selected from the group of higher polyhydric alcohols, polyvinyl alcohols, or dextrans when the mass ratio of copolymer: modifier= (90,2 - 98,9):(1,1 - 9,8).

The invention relates to organic polymeric hydrophilic sorbents for liquid chromatography and can be used for the separation of biopolymers (proteins, enzymes, amino acids and their fragments, oligo - and polysaccharides, and so forth).

A known number of polymeric sorbents (carriers) for liquid chromatography based on (meth)acrylic monomers and cross-linking agents, which are easily modified, easy to prepare and efficiency is adopted, predstavljajushej a porous granular copolymer glycidylmethacrylate simple or complex esters with alkylanthraquinones ether-modified alkalophiles, polyethylene glycol or a triol in the disclosure epoxy ring [3]

The disadvantage of this medium is low hydrophilicity due to the absence of hydroxyl groups in the copolymer before modification and lack of them after modification, i.e. modifiers used are few hydroxyl groups, and the number of reactive epoxy groups in the copolymer is also small. Such media are ineffective for the separation of high molecular weight protein molecules.

Closest to the invention to the technical essence and the achieved result is hydrophilic separating carrier for liquid chromatography, the obtained water-suspension - tion homopolymerization pentaerythritol dimethacrylate or water-suspension copolymerization of pentaerythritol dimethacrylate and methacrylate monomer having a hydrophilic and/or hydrophilic transform group and modified hydrophilic epoxidation connection. Moreover, the copolymer containing an epoxy group, before the option will fetter the nonspecific adsorption of protein molecules due to insufficient hydrophilicity and difficult to obtain [4]

The task of the invention to provide hydrophilic polymeric macroporous sorbent with high mechanical and chemical stability, high resolution and efficiency in the separation of biopolymers.

This result is achieved by hydrophilic polymeric sorbent, which is a product of the interaction of the epoxy(meth)acrylate copolymer and an organic hydroxyl-containing modifier. Epoxy(meth)acrylate copolymer is a copolymer of amoxicil(meth)-acrylate of General formula

CH2= - -O-Q-CHH2where Q (CHR2)nCH2CH2OCHR2CH2CH2OCH2CH2OCHR2n 1-11, R1, R2H, CH3and a crosslinking monomer selected from the group consisting of di-, tri-, Tetra(meth)acrylates of pentaerythritol or mixtures thereof.

The amount of organic hydroxyl-containing modifier 1.1 to 9.8 percent by weight of the mixture of the modifier and copolymer.

Organic hydroxyl-containing modifier is a compound selected from the group of the epichlorohydrin-activated polyhydric alcohols or dextrans.

The maximum hydrophilic sorbents is achieved by modulating, is selected from the group petitou and hexitol (xylitol, D - and L-sorbitol, mannitol, dulcet and so on) polyvinyl alcohols or activated dextrans.

Sorbent receive water and the suspension copolymerization of amoxillin(meth)acrylate and a crosslinking monomer, taken in the ratio of from 10:90 to 90: 10, preferably from 40:60 to 60:40, in the presence of organic solvents proobraz - users that do not react with the epoxy ring. Suitable solvents are n-butanol, butyl acetate, ISO-amyl alcohol, cyclohexanol, octanol, dodecanol, monochlorbenzene and mixtures thereof.

As the radical polymerization initiator can be used dinitrosopentamethylene acid (DINIZ) or peroxide initiators.

As suspension stabilizers may be used polyvinyl alcohol, polyvinylpyrrolidone, carboxymethylcellulose and/or other organic and Neorganicheskie dispersers.

The polymerization was carried out under continuous stirring for 5-10 h at 70-80aboutC.

Activation of polyhydric alcohols or dextrans is performed as follows. In a reactor with a stirrer, thermometer and reflux condenser load 100 wt.h. hydroxysteroid is added 40 wt.h. of epichlorohydrin and stirring is continued for another 2 h at 50aboutWith the production of activated polyhydric alcohol or dextran.

P R I m e R 1. A solution containing 52 wt.h. glycidylmethacrylate, 48 wt.h technical mixture of di-, tri - and tetramethylsilane pentaerythritol, 3 wt.h. The Diniz, 65 wt.h. butyl acetate and 65 wt.h. isoamyl alcohol, suspension in 1000 ml of 1% aqueous solution of polyvinyl alcohol in water. Under continuous stirring at a speed of 400 Rev/min conducting the copolymerization at 70aboutWith over 6 hours of Pellet suspension was filtered and washed with hot water and alcohol, getting a spherical particle with the number of surface epoxy groups to 2.8 mEq/g dry. sorbent (determined by potentiometric titration of the excess Hydrobromic acid in acetone). The granules are subjected to modification activated mannitol mass ratio of 1:1 at 70aboutC for 4 h, After washing with water receive hydrophilic sorbent content associated mannitol to 5.7 wt. and nonspecific adsorption of protein standards is not more than 2%, the maximum exclusion (exclusion limit molecular weight) dextrans from 10000 to 10000000 daltons. Sorbents can withstand pressure up to 100 ATM.

P R I m e R 100 wt.h. n-butanol and 40 wt.h. butyl acetate, suspended in 1000 ml of 2% aqueous solution of polyvinylpyrrolidone in water. The copolymerization is conducted analogously to example 1. Get spherical particles containing 2,3 mg-EQ./g dry. sorbent epoxy groups. Polymer granules modify activated xylitol in a mass ratio of 1: 0.5 to obtaining sorbent containing 1.1% linked xylitol. The sorbent has a nonspecific sorption of protein standards to 5% and the limit of exclusion from 100-10000 daltons. The sorbent can withstand pressure up to 30 ATM.

P R I m e R 3. A solution containing 50 wt.h. glycidylmethacrylate, 50 wt.h. technical mixtures of di-, tri - and tetramethylsilane pentaerythritol, 2 wt. CH Diniz, 60 wt.h. butyl acetate, 60 wt.h. ISO-amyl alcohol and 20 wt. h n-octanol, suspended in 1000 ml of 1% aqueous solution of polyvinyl alcohol in water. The copolymerization is conducted analogously to example 1. Get spherical particles containing a 2.8 mEq/g dry. sorbent epoxy groups, which modify the activated D-sorbitol in a mass ratio of 1:2 with obtaining sorbent containing 7.2% of bound D-sorbitol. The sorbent has a nonspecific sorption of less than 3% and the limit of exclusion from 10000 to 2000000 D. Sorbent withstands pressure up to 100 ATM.

P R I m e R 5. The solution containing 45 wt.h. 10,11-epoxiderivatives, 55 wt. including pentaerythritol dimethacrylate, 2 wt.h. The Diniz, 60 wt.h. butyl acetate, 60 wt.h. cyclohexanol and 20 wt.h. dodecanol, suspended in 100 ml of 2% aqueous solution of polyvinyl alcohol in water. The copolymerization is conducted analogously to example 1. The obtained spherical particles containing 1.7 mg-equiv/g of dry sorbent epoxy groups modified activated polyvinyl alcohol (mol.m. 10000) in a mass ratio of 1:1 to receive sorbent, containing 1.7% of the linked PVA. The sorbent is non-specific adsorption of protein molecules to the 5% limit exclusion from 1000-100000 daltons. The sorbent can withstand pressures up to 50 ATM.

P R I m e R 6. All operations carried out analogously to example 1, except that as abaxially(meth)acrylate monomer used glycidylmethacrylate and as a modifier activated dextran. Before modification the sorbent contains related dextran 5.2 wt. and nonspecific adsorption of protein standards is not more than 2% Limit exclusion 1000-1000000 daltons. The sorbent can withstand pressure up to 100 ATM.

The conditions determining the exclusion limit molecular weight is the same for all carriers: column chromatographic 0,9x12 cm, eluent the distillated water, the rate of elution of 0.3-0.4 cm3/min, the concentration of dextrans in the sample is 0.1% of the volume of the sample 5010-3cm, detector flow Refractometer, temperature 20aboutWith the diameter of spherical particles of the medium 40-120 microns.

Conditions determine the nonspecific adsorption of protein molecules: column 0,9x12 cm, eluent 0.1 N. phosphate buffer pH 7, the rate of elution of 0.6 cm3/min, detector spectrophotometer 280 nm and 410 nm, the temperature of the 20aboutWith the diameter of the carrier 10-40 microns: protein standards bovine serum albumin, cytochrome C, ferritin, egg albumin.

Determination of surface epoxy groups conduct potentiometric on the device And-120.

Conditions of separation: column h,5 cm, eluent 0.02 M Hcl buffer pH 6.8, 0,6 ml/min, temperature 20aboutC, detector UV 280 nm.

The use of the inventive sorbent allows to achieve high efficiency in the separation process biopolymer epoxy(meth)acrylate of General formula

< / BR>
where Q -(CHR2-)n, -CH2CH2OCHR2-, -CH2CH2OCH2CH2OCHR2-;

n 1 11;

R1and R2-H, -CH3,

and a crosslinking monomer compounds selected from the group consisting of di-, tri-, Tetra(meth)acrylates of pentaerythritol or mixtures thereof with modifier - activated epichlorohydrin polyhydric alcohol selected from the group consisting of pentity, exity, polyvinyl alcohols or dextrans, with a ratio of modifier copolymer of 1,1 9,8 98,9 90,2 wt. when this porous product has an exclusion limit 10000 10000000.

 

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30 cl, 5 ex, 2 tbl

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19 cl, 6 tbl, 16 ex

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22 cl, 3 dwg, 1 ex

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EFFECT: improved preparing method.

22 cl, 3 dwg, 1 ex

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