JPH07171389A - Manufacture of separating agent - Google Patents

Abstract

PURPOSE:To provide a separating agent especially suitable for liquid-phase chromatography by adding methacryloyl isocyanate, etc., to a carrier having an active hydrogen group on the surface and then copolymerizing the carrier with a polymerizable vinyl monomer. CONSTITUTION:A carrier made of silica gel, polystyrene, etc., having an active hydrogen group e.g. hydroxyl group, carboxyl group, etc., on the surface is used as a carrier for liquid-phase chromatography. Addition reaction of the carrier with a compound, e.g. methacryloyl isocyanate having the formula I or the formula II (R1 is hydrogen or methyl group; n is 1-2 integer) is carried out. Further, the resulting addition reaction product is copolymerized with a copolymerizable vinyl monomer e.g. acrylic acid, acrylamide, etc. Consequently, a polymer part can be introduced into the surface of the carrier and thus a separating agent by which pressure loss is kept from changing and which is suitable for liquid-phase chromatography is provided.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a separating agent, and more particularly to a method for producing a separating agent which is preferably used as a packing material for liquid chromatography.

[0002]

2. Description of the Related Art Carriers used as packing materials for liquid chromatography include inorganic carriers such as silica gel and hydroxyapatite, natural polymer carriers such as dextran, cellulose and chitosan, polystyrene and poly (meth) acrylic acid. Synthetic polymer carriers such as esters are known. Although these carriers may be used in some separation modes such as hydrophobic chromatography and reverse phase chromatography even if they are used as they are, they can be used in various other separation modes with the progress of technology in recent years. In order to make it possible, it is important to provide a technique for imparting various functional groups to these carriers.

Among the above carriers, for example, in the case of silica gel, it is possible to react a silanol group of silica gel with a silane coupling agent having various functional groups and, if necessary, further introduce a functional group. Further, if it is an organic carrier, it is possible to introduce a functional group by various chemical reactions, and if it is a synthetic polymer carrier, it is also possible to introduce a functional group by using a compound having a functional group as a raw material during synthesis. .

However, in these methods, when functionalization is insufficient, phenomena such as deterioration of separability and nonspecific adsorption of an object to be separated on a carrier may occur. Further, particularly in the organic carrier, there is a problem that the functional strength is lowered when it is functionalized. In order to solve these problems, various methods for introducing a high density of functional groups only on the surface of the carrier have been studied. For example, JP-A 1-217
No. 035 discloses that a glycidyl group of a glycidyl monovinyl ester or a copolymer of glycidyl monovinyl ether and a polyvinyl ester of a polyhydric alcohol, or a glycidyl monovinyl ester or a copolymer of a glycidyl monovinyl ether and a polyvinyl ether of a polyhydric alcohol is hydrolyzed. After the reaction, an alkyl halide having an oxirane ring is reacted, and a compound having an oxirane ring is graft-polymerized thereto.
However, this method can achieve hydrophilicity with a graft polymer of the oxirane ring, but it is difficult to introduce further various functional groups.

Further, JP-A-1-310744 discloses a separating agent having a support having a hydroxyl group as a base material, the surface of which is coated with a covalently bonded polymer having a degree of polymerization of 2 to 100. There is. Since this polymer is bound in a so-called "tentacle-type", the spread of the polymer is significantly changed by the environmental change around the separating agent. This is not preferable because it causes a change in pressure loss in liquid chromatography.

[0006]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a separating agent which solves the above problems and is particularly suitable for liquid chromatography.

[0007]

In the present invention, a carrier (A) having an active hydrogen group on its surface is subjected to an addition reaction with a compound (B) represented by the following general formula (I) or general formula (II). Then, a separating agent obtained by introducing a polymer portion onto the surface of the carrier (A) by copolymerizing the addition reaction product and the polymerizable vinyl monomer (C) is used to solve the above problems. Therefore, the present invention has been reached, and the present invention has been reached.

[0008]

[Chemical 2]

(In the formula, R ₁ represents hydrogen or a methyl group, and n represents an integer of 1 to 2) The carrier (A) used in the present invention has an active hydrogen group on its surface. The composition and the presence or absence of porosity are not particularly limited. When the separating agent obtained by the method of the present invention is used as a packing material for liquid chromatography, silica gel, an inorganic carrier such as hydroxyapatite, a natural polymer carrier such as dextran, cellulose, chitosan and polystyrene, poly ( A synthetic polymer carrier in which an active hydrogen group is introduced on the surface of a crosslinked synthetic polymer such as (meth) acrylic acid ester is preferable.

Examples of the active hydrogen group existing on the surface of the carrier (A) include a hydroxyl group, a carboxyl group, a mercapto group, an amino group and a silanol group.
The amount of the functional group is 1 × 10 ⁻⁵ to 1 × 10 ⁻² per 1 g of the carrier.
Molar is preferred. When the carrier (A) does not have these active hydrogen groups, it may be introduced by a chemical reaction.
As an example of introducing an active hydrogen group into an inorganic carrier, an example of introducing a hydroxyl group by hydrolyzing an epoxy group such as silica gel containing an epoxy group (for example, Waters Protein Pack Affinity: Millipore product) under weakly acidic conditions Is mentioned. In addition, examples of introduction into a synthetic polymer carrier include an example in which a nitrile group of a styrene-divinylbenzene-acrylonitrile copolymer is hydrolyzed under alkaline conditions to introduce a carboxyl group, and glycidyl methacrylate-ethylene glycol dimethacrylate. Examples thereof include introducing a hydroxyl group by hydrolyzing the glycidyl group of the copolymer under acidic conditions.

Examples of the compound (B) represented by the general formula (I) or the general formula (II) include acryloyl isocyanate, methacryloyl isocyanate, isocyanate methyl (meth) acrylate and isocyanate ethyl (meth) acrylate. From the viewpoint of reactivity in the addition reaction to the carrier (A), acryloyl isocyanate or methacryloyl isocyanate is preferable.

In order to carry out the addition reaction of the compound (B) to the carrier (A), they are generally reacted in a solvent. As the solvent, acetic acid esters such as ethyl acetate and butyl acetate,
Examples thereof include aromatic hydrocarbons such as toluene and aliphatic hydrocarbons such as hexane and cyclohexane. Also,
Examples of the catalyst used in the addition reaction include organic tin compounds such as dibutyltin dilaurate and tertiary amines such as triethylamine. However, when acryloyl isocyanate or methacryloyl isocyanate is used as the compound (B), the reaction can be performed without a catalyst, which is preferable to the case where a catalyst is used.

The reaction temperature is preferably 0 to 70 ° C., and the reaction time is preferably 1 minute to 12 hours.
The amount of the compound (B) to be added to the carrier (A) is 0.05 to 40 with respect to the carrier (A) in the amount used.
%, Preferably 0.1 to 30% by weight. If this amount is too small, only a small amount of (C) can be copolymerized when the addition reaction product and the polymerizable vinyl monomer (C) are copolymerized. On the other hand, if the amount is too large, the balance with the copolymerization amount of (C) may be unfavorable.

As the method for copolymerizing the polymerizable vinyl monomer (C) with the addition reaction product of the carrier (A) and the compound (B), there are radical polymerization method, ionic polymerization method, thermal polymerization method and ultraviolet irradiation. Although the method etc. are used, the radical polymerization method is preferable. As the polymerization initiator used in this case,
2,2'-azobisisobutyronitrile, 2,2'-azobis (2,4-dimethylvaleronitrile), 4,4-
Azo-based polymerization initiators such as azobis (4-cyanopentanoic acid), t-butyl hydroperoxide, di-t-butyl peroxide, benzoyl peroxide, di-isopropyl peroxydicarbonate, t-butyl peroxyisobutyrate and hydrogen peroxide. Examples thereof include peroxide type polymerization initiators such as potassium persulfate and ammonium persulfate, and systems in which a reducing agent such as amine and sodium bisulfite is added to these.

As the polymerizable vinyl monomer (C), unsaturated carboxylic acids such as (meth) acrylic acid, itaconic acid and maleic acid; methyl (meth) acrylate, ethyl (meth) acrylate, (meth) Butyl acrylate, alkyl (meth) acrylates such as stearyl (meth) acrylate, hydroxyethyl (meth) acrylate, hydroxypropyl (meth) acrylate, phenyl (meth) acrylate, (meth) acrylic acid 3- Chloro-2-hydroxypropyl, 2-chloroethyl (meth) acrylate, glycidyl (meth) acrylate, dimethylaminoethyl (meth) acrylate, diethylaminoethyl (meth) acrylate, tetrahydrofurfuryl (meth) acrylate, etc. (Meth) acrylic acid esters;

(Meth) acrylamide, N, N-dimethyl (meth) acrylamide, N, N-dimethylaminopropyl (meth) acrylamide, 2-acrylamide-
(Meth) acrylamide derivatives such as 2-methylpropanesulfonic acid; styrenes such as styrene, methylstyrene, α-methylstyrene, chlorostyrene and chloromethylstyrene and alkyl or halogen substituted products thereof; vinyl such as vinyl acetate and vinyl propionate Esters; Vinyl ethers such as methyl vinyl ether, ethyl vinyl ether, butyl vinyl ether and octadecyl vinyl ether;

Allyl alcohol and its esters or ethers; allylamine derivatives such as allylamine and diallylamine; vinylamine derivatives such as N-vinylformamide and N-vinylacetamide; (meth) acrylonitrile, acrolein, sodium vinylsulfonate, sodium allylsulfonate, p-sodium styrenesulfonate, vinylpyridine, vinylpyrrolidone and other vinyl compounds;

And the like. These polymerizable vinyl monomers can be used alone or in a mixture of two or more kinds. When these monomers are used, depending on the characteristics of the functional group of the monomer itself, after the copolymerization, it can be used as a separating agent as it is, and after the copolymerization, the functional group of the monomer is different from other functional groups. It may be used as a separating agent by a method such as substitution or conversion to another functional group by hydrolysis or the like.

Particularly in the case of a packing material for liquid chromatography, for example, by using a polymerizable vinyl monomer having an ionic dissociative group, the packing material for ion exchange chromatography can be prepared as C ₄ , C ₈ , C _18, etc. The use of a polymerizable vinyl monomer having a C ₄ or higher alkyl group as a filler for reverse phase chromatography, and the use of a polymerizable vinyl monomer having a C ₄ or lower low molecular weight alkyl group or a phenyl group By using a packing material for hydrophobic chromatography, a packing material for optical resolution chromatography by using a polymerizable vinyl monomer having an optically active group, and a polymerizable vinyl monomer having a hydrophilic functional group such as a hydroxyl group. Each of them can be used to produce a packing material for water-based size exclusion chromatography.

The addition reaction product of the carrier (A) and the compound (B), more specifically, the copolymerization reaction of the unsaturated double bond derived from (B) and the polymerizable vinyl monomer (C), The reaction is carried out in a solvent containing the polymerizable vinyl monomer (C) at a reaction temperature of 0 to 100 ° C. and a reaction time of 30 minutes to 24 hours.

The amount of the polymerizable vinyl monomer (C) copolymerized with the addition reaction product of the carrier (A) and the compound (B) is 1 to 50% by weight based on the addition reaction product, preferably Is 2
-40% by weight. If this amount is too small, the polymer portion derived from (C) introduced on the surface of the resulting separating agent cannot sufficiently cover the surface, and if it is too large, a surface coating layer is required. As the thickness increases, problems such as deterioration of separation performance occur.

[0022]

EXAMPLES The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

Reference Example 1 (Synthesis of Carrier Having Active Hydrogen Group on Surface) In a reactor equipped with a stirrer, a condenser and a thermometer, the weight ratio of glycidyl methacrylate to ethylene glycol dimethacrylate was 70.4: 29. 6 spherical spherical copolymer particles (average particle size 4.52 μm, specific surface area 44.2 m ² / g, most frequent radius 29.8 nm) produced by copolymerization as 6 were added, and 10% by weight sulfuric acid aqueous solution was added. By adding 50 parts by weight and heating at 50 ° C. for 5 hours, the epoxy group derived from glycidyl methacrylate was hydrolyzed and converted into a hydroxyl group. After completion of the reaction, the particles were thoroughly washed with demineralized water and dried.

Example 1 Particles 5 obtained in Reference Example 1 above were placed in a reactor equipped with a stirrer, condenser, thermometer and nitrogen gas inlet tube.
Part by weight was added, and 45 parts by weight of ethyl acetate was added to disperse the particles. After replacing the reactor with nitrogen gas, 1 part by weight of methacryloyl isocyanate was added, and the addition reaction was carried out at 25 ° C. for 1 hour while stirring under a nitrogen atmosphere.

After completion of the reaction, the particles obtained by the above addition reaction were thoroughly washed with methanol and acetone. Then, the particles were put into a reactor equipped with a stirrer, a condenser, a thermometer and a nitrogen gas introducing tube, and N, N
Dimethylaminopropyl methacrylamide 2 parts by weight, 1
11.8 parts by weight of N-hydrochloric acid and 35.2 parts by weight of demineralized water were added. After replacing the reactor with nitrogen gas, 1 part by weight of a 3.2% by weight aqueous solution of potassium persulfate was added, and the mixture was heated to 70 ° C. in a nitrogen atmosphere and polymerized for 6 hours to obtain a separating agent.

The anion exchange capacity of the obtained separating agent derived from N, N-dimethylaminopropylmethacrylamide was measured by a titration method and found to be 0.20 meq / g. The separating agent thus obtained had an inner diameter of 8.0 mm,
It was packed in a glass column having a length of 100 mm and the protein adsorption / desorption capacity was measured.

[0027]

[Table 1] Measurement conditions: Measurement device: LC-6A system (manufactured by Shimadzu Corporation) Adsorption liquid: 14 mM trishydroxymethylaminomethane / hydrochloric acid buffer solution (pH 8.0) Desorption liquid: 14 mM trishydroxymethylaminomethane / hydrochloric acid buffer solution (PH 8.0) +0.5 M sodium chloride Flow rate: 1.0 ml / min Detection: UV280 nm Temperature: 25 ° C. Sample: Bovine serum albumin (manufactured by Sigma)

Under the above conditions, the adsorbent was passed through, and the sample (bovine serum albumin) dissolved in the adsorbent was injected into the column for adsorption. After saturated adsorption, the amount of bovine serum albumin adsorbed on the column was determined by quantifying the bovine serum albumin that was not adsorbed and was eluted by the absorbance at UV 280 nm. Met. Next, the desorption solution was passed through to elute the bovine serum albumin adsorbed on the column. When the amount of bovine serum albumin recovered was determined from the absorbance at UV280 nm, the desorption capacity per ml of the separating agent was 47.3 mg, and the recovery rate for the adsorption capacity was 9%.
It was 9.8%.

Comparative Example 1 5 parts by weight of the particles obtained in Reference Example 1 were placed in a reactor equipped with a stirrer, a condenser and a thermometer.
24 parts by weight of N-NaOH aqueous solution was added, and the mixture was stirred at room temperature for 30 minutes under ultrasonic dispersion. Then, 29.4 parts by weight of 51% β-diethylaminoethyl chloride hydrochloride aqueous solution was added, and the mixture was reacted at 50 ° C. for 6 hours to introduce a diethylaminoethyl group to obtain a separating agent (Comparative Example 1). The anion exchange capacity derived from the diethylaminoethyl group of the obtained separating agent was measured by a titration method to be 0.56.
It was meq / g.

The adsorption / desorption capacity of bovine serum albumin was measured for this separating agent in the same manner as in Example 1. The adsorbing capacity of bovine serum albumin per ml of the separating agent was 70 m.
Although the amount was g, the desorption capacity per ml was 36 mg, and the recovery rate was 51.4%, indicating that the degree of nonspecific adsorption of bovine serum albumin to particles was high.

[0031]

INDUSTRIAL APPLICABILITY The separating agent obtained by the present invention has a polymer moiety derived from the polymerizable vinyl monomer (C) introduced into the surface of the polymerizable vinyl monomer (C) at a high density by a covalent bond. C) -derived functional group or polymerizable vinyl monomer (C)
The functional group introduced into the derived polymer portion provides suitable separation performance in various separation modes. Also,
The polymeric portion derived from the polymerizable vinyl monomer (C) is covalently bonded to a plurality of polymerizable unsaturated double bonds derived from the compound (B) added to the surface of the carrier to coat the surface of the carrier in a layered manner. This is different from the separating agent described in JP-A No. 1-310744. This structure solves problems such as deterioration of separability due to the properties of the carrier itself and non-specific adsorption of the separation target on the carrier, and in particular, in organic carriers, the functionalization of the entire carrier causes a decrease in mechanical strength. There is no. Furthermore, when it is used as a packing material for liquid chromatography, it is possible to minimize changes in pressure loss due to changes in the composition of the eluent, and it is possible to use it particularly preferably in high performance liquid chromatography.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C08F 292/00 MPZ 7308-4J // C08F 20/58 MNG

Claims (2)

[Claims] 1. A carrier (A) having an active hydrogen group on its surface.
Is subjected to an addition reaction with a compound (B) represented by the following general formula (I) or general formula (II), and then the addition reaction product and a polymerizable vinyl monomer (C) are copolymerized,
A method for producing a separating agent, which comprises introducing a polymer portion into the surface of the carrier (A). [Chemical 1] (In the formula, R ₁ represents hydrogen or a methyl group, and n is 1 to 2
Represents an integer) 2. The method for producing a separating agent according to claim 1, wherein the compound (B) is methacryloyl isocyanate.