JPH01262468A - Carrier for chromatography - Google Patents

Abstract

PURPOSE:To facilitate the introduction of an ion exchange group to a hydroxyl group by subjecting the epoxy group on particle surfaces to ring opening modification by hydrolysis to the hydroxyl group and subjecting the epoxy group in the particles to self-crosslinking, then synthesizing the particles within a specific range of evarage particle sizes. CONSTITUTION:The crosslinked polymer particles having a glycidyl group are synthesized by subjecting glycidyl ester and polyvinyl ester as a crosslinking agent to an aq. suspension polymn. These particles are then suspended in an aq. soln. contg. a dispersant and mineral acid and the epoxy group based on the glycidyl group in the particle surface is subjected to the ring opening modification by the hydrolysis to the hydroxyl group. The epoxy group based on the glycidyl in the particles is then subjected to the self-crosslinking to intensify the mechanical strength of the particles. The crosslinked polymer particles having the hydroxyl group on the surface are synthesized in the 1-5mum average particle size. The carrier which can easily introduce the ion exchange group into the hydroxyl group is, therefore, obtd.

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention is useful for producing various chromatography packing materials, and provides a chromatography support and an ion exchange group introduced into the chromatography support. Regarding packing materials for ion-exchange chromatography, it is particularly useful for producing packing materials for ion-exchange chromatography that can be used for separation 1 analysis of biochemical substances such as proteins, oligopeptides, nucleic acids, and oligonucleotides. The present invention relates to a carrier for chromatography that can be used for chromatography, and a packing material for ion exchange chromatography in which an ion exchange group is introduced into the carrier.

[Conventional technology]

BACKGROUND ART A method of separating and analyzing biochemical substances such as proteins and nucleic acids using ion exchange chromatography has been conventionally used. However, the conventional method requires only one separation and two analyses.
It has the disadvantage that it requires a long time of 0 minutes or more. In recent years, there has been a strong demand in the industry to separate and analyze a small amount of sample in a short period of time. - In order to perform sample separation and analysis in a short time, it is necessary to fill a small column with extremely small particles. Conventionally, porous polymer glues, porous silica cells, and the like have been used as packing materials for ion exchange chromatography.

However, when the particle size of porous polymer glue is reduced, its mechanical strength is low, so there is a problem that it cannot be used at high pressure and high flow rate.

On the other hand, although porous silica gel has high mechanical strength, it has the disadvantage that it dissolves under weakly alkaline conditions, and this tendency becomes more pronounced as the particle size becomes smaller.

[Problem to be solved by the invention]

The purpose of the present invention is to overcome the drawbacks of the conventional packing material for ion exchange chromatography, to provide a packing material with excellent mechanical strength, withstand use under high pressure and high flow rate, and to be chemically stable and suitable for weakly alkaline conditions. A carrier for ion exchange chromatography that is sufficiently stable under the following conditions and has no volume change, and is useful for producing a packing material for ion exchange chromatography. The aim is to provide agents for

[Means to solve all problems]

The present invention provides a chromatography carrier useful for producing a chromatography filler capable of achieving the above object, and an ion exchange chromatography filler in which an ion exchange group is introduced into the chromatography carrier. be done.

That is, the present invention consists of crosslinked polymer particles having glycidyl groups, in which epoxy groups based on glycidyl groups on the surface of the particles are ring-opened and modified to hydroxyl groups by hydrolysis, and epoxy groups based on glycysol groups inside the particles. The present invention relates to a chromatography carrier having self-crosslinked groups and having an average particle size within the range of 1 to 5 μm.

The present invention also provides a cross-linked polymer particle having a glycidyl group, in which an epoxy group based on a glycidyl group on the surface of the particle is ring-opened and modified to a hydroxyl group by hydrolysis, and an epoxy group based on a glycidyl group inside the particle. A packing material for ion-exchange chromatography, characterized in that an ion-exchange group is introduced into the hydroxyl group of a chromatography carrier whose groups are self-crosslinked and whose average particle diameter falls within the range of 1 to 5 μm. .

Hereinafter, the chromatography carrier of the present invention and the ion exchange chromatography packing material in which an ion exchange group is introduced into the chromatography carrier will be explained.

The chromatographic carrier useful for producing the chromatographic packing material of the present invention can be synthesized through the steps shown below.

l) Synthesis of crosslinked polymer particles with glycidyl foundations.

2) Hydrolysis of epoxy groups based on glycidyl groups on the surface of crosslinked polymer particles having glycidyl groups with water.

3) Self-crosslinking of epoxy groups based on glycidyl groups inside crosslinked polymer particles having glycidyl groups.

To explain in more detail, the first step of producing a crosslinked polymer having a glycidyl group consists of (4) glycidyl ester or glycidyl monovinyl ether of unsaturated caldicic acid and (B) polyvinyl ester of polyhydric alcohol as a crosslinking agent. It can be produced by aqueous suspension polymerization according to any conventional method.

Glycinol acrylate, glycidyl methacrylate, etc. are used as the glycidyl ester of unsaturated caldenic acid as the prison component. In addition, as component (4) glycidyl monovinyl ether, allyl glycidyl ether,
Metaallyl glycidyl ether and the like are used.

On the other hand, as the polyvinyl ester of polyhydric alcohol as the component (B) as a crosslinking agent, ethylene glycol diacrylate, ethylene glycol dimethacrylate, propylene glycol diacrylate, propylene glycol dimethacrylate, glycerin diacrylate, glycerin diacrylate, glycerin triacrylate, etc. acrylate,
Examples include glycerin trimethacrylate, pentaerythritol diacrylate, pentaerythritol dimethacrylate, pentaerythritol trimethacrylate, nrubitol no methacrylate, and nrubitol trimethacrylate.

In the present invention, component (4) may be used as the main component, and all other monomers that can be copolymerized therewith may be used in combination as appropriate.

The 19-rivinyl ester of polyhydric alcohol as component (B) is used in an amount of 1 to 30 moles, preferably 5 to 20 moles, of the total polymerizable monomers.

If the proportion of component (B) increases, the amount of glycidyl groups on the surface of the crosslinked polymer particles decreases, which is preferable or not.

The initiator used in 1 polymerization may be a general radical polymerization initiator used in normal suspension polymerization, for example 2 polymerization initiators.
, 2'-azobisisobutyronitrile, 2,2'-azobis-(2,4-dimethylvaleronitrile) and other azo-based initiators, and peroxide-based initiators such as benzoyl peroxide and lauroyl peroxide. Agents can be used.

When carrying out suspension polymerization, it is preferable to add a dispersion stabilizer such as polyvinyl alcohol, carboxymethylcellulose, or gelatin to the aqueous phase, and also to add a carrier component and (
In order to prevent monomerizable monomers such as component B from dissolving into the water of the polymerization dispersion medium, salts such as sodium chloride, sodium sulfate, and calcium chloride may be dissolved in the water.

The amount of the aqueous layer used is from approximately the same volume or more to about 10 times the volume of the organic layer. The particle diameter of the crosslinked polymer particles can be controlled by the stirring speed when dispersing the monomer in the aqueous phase, and the monomer is preferably dispersed into oil droplets of 0.5 to 5 μm.

The polymerization reaction is usually carried out at 50 to 90°C for 3 to 16 hours. After completion of polymerization, the crosslinked polymer particles are filtered, washed with water, and further washed with an organic solvent. The crosslinked polymer particles having glycidyl groups obtained by pressing in this manner are substantially non-porous and are classified into particle sizes of 1 to 5 μm, preferably 2 to 4 μm. Classification can be carried out by conventional methods such as air classification or decantation several times in an organic solvent.

In the second step, the crosslinked polymer particles having glycidyl groups obtained in the first step are impregnated with an organic solvent that is poorly soluble in water, and then the particles are suspended in an aqueous solution containing a dispersant and a mineral acid. By heating at 50 to 90°C for 1 to 5 hours,
Epoxy groups based on glycidyl groups on the surface of crosslinked polymer particles are hydrolyzed to ring-open and modify into hydroxyl groups. Examples of the organic solvent with which the crosslinked polymer particles are impregnated include toluene, xylene, chlorobenzene, and non-roughin.

Further, examples of the mineral acid of the catalyst include hydrochloric acid and sulfuric acid. The concentration of mineral acid is preferably 0.05 to 0.5 normal.

Examples of the dispersant include polyvinyl alcohol and polyvinyl alcohol. The concentration of dispersant is 0.5-1
0% by weight is preferred.

In the third step, the epoxy groups based on the glycidyl groups inside the crosslinked polymer particles are self-crosslinked to fully enhance the mechanical strength of the crosslinked polymer particles. Self-crosslinking is performed by adding the crosslinked polymer particles having hydroxyl groups on the surface produced in the second step in an organic solvent that does not react with epoxy groups based on glycidyl groups by a conventional method.
As a total catalyst of boron trifluoride accumulated etherate, 20℃~8
This is done by heating at 0°C for 1 to 5 hours. Examples of organic solvents used in this case include toluene, xylene, dioxane, and n-paraffin.

The crosslinked polymer particles obtained as described above have hydroxyl groups on the surface and self-crosslinked epoxy groups based on internal glycidyl groups have an average particle diameter within the range of 1 to 5 μm, and have hydroxyl groups. It is possible to easily introduce an ion-exchange fund into the ion-exchange chromatography packing material, and therefore it is extremely useful as a chromatography support for the production of all ion-exchange chromatography packing materials.

If the average particle diameter of the crosslinked polymer particles is less than 1 μm, the column pressure will be high when packed in a column, making it difficult for the eluent to flow. On the other hand, when the average particle diameter of the crosslinked polymer particles exceeds 5 μm, the surface area becomes small and the amount of ion exchange groups introduced onto the surface of the crosslinked polymer particles decreases.

Next, a method for introducing an ion exchange group into the hydroxyl group of the above-mentioned chromatography carrier will be explained. 1. The ion exchange group introduced into the hydroxyl group includes (a) a sulfone group, (b) a carboxyl group, and a CJ diethylamine ethyl group. groups, etc.) quaternary ammonium groups, etc.

The ion exchange capacity is preferably 50 to 500 M equivalent/9.

The introduction of sulfone groups is carried out by treating the crosslinked polymer particles having hydroxyl groups on the surface produced in the third step with glonosone sultone or 1,4
-The reaction temperature and reaction time are usually room temperature to 100°C for 2 to 2 hours.
It is 4 hours.

Introduction of carboxyl groups is carried out by adding 7-logonated acetic acid such as monochloroacetic acid or monobromoacetic acid, or halogenated acetate such as sodium monochloroacetate or potassium monochloroacetate to the crosslinked polymer particles having a hydroxyl group on the surface produced in the third step. It is carried out by reacting in the presence of sodium hydroxide or potassium hydroxide.

Introduction of diethylamine ethyl groups can be carried out by reacting the crosslinked polymer particles having hydroxyl groups on the surface produced in the third step with hydrochloric acid β-noethylaminoethyl chloride in an aqueous solution of alkali metal hydroxide. can.

The reaction temperature is preferably room temperature to 80°C, and the reaction time is preferably 1 to 8 hours. The concentration of an aqueous solution of alkali metal hydroxide is
5 to 40% by weight is preferred.

The quaternary ammonium group is introduced by reacting crosslinked polymer particles into which diethylaminoethyl groups have been introduced with methyl halide or ethyl halide in an organic solvent.

The thus obtained packing material for ion exchange chromatography consists of substantially non-porous crosslinked polymer particles with an average particle size in the range of 1 to 5 μm), and the surface of the crosslinked polymer particles has ion exchange groups. Once introduced, the interior of the crosslinked polymer particles consists of a densely crosslinked double structure.

In the prior art, porous carriers were used to increase the surface area of the carrier. In the present invention, the particle size of the carrier 'z
By reducing the size to 1 to 5 μm, the surface area of the carrier becomes louder and the mechanical strength of the carrier increases, so the crosslinking density is increased inside the carrier and the crosslinking density is lowered only on the surface of the carrier.
The hydroxyl group of the carrier is left behind, and an ion exchange group is introduced into the hydroxyl group to create an ion exchange effect.

The packing material for ion exchange chromatography according to the present invention has an average particle diameter within the range of 1 to 5 μm, and is a conventional packing material for ion exchange chromatography.

In other words, unlike porous carriers in which ion exchange foundations are introduced, the carrier is substantially non-porous and has a highly cross-linked interior, so it has strong mechanical strength and has a small particle size of 1 to 5 lzm. There are also many features that can be used.

〔Example〕

Dan, below, example? The present invention will now be described in further detail.

Example 1 (1) Synthesis of crosslinked polymer particles having glycidyl groups Glycidyl methacrylate 36 g of 324.1-ethylene glycol dimethacrylate and azobisisobutyronitrile 14,9' (H mixed), 96 g of polyvinyl alcohol The mixture was stirred at high speed to adjust the particle size of the oil droplets to 0.5 to 5 μm, and then heated to 60°C and kept for 10 hours.Next, the reaction The product was cooled to room temperature and diluted with water. The resulting crosslinked polymer particles were centrifuged and then suspended in water and centrifuged. After repeating this operation three times, the crosslinked polymer particles were diluted with acetone. The crosslinked polymer particles were suspended in water, filtered through a glass filter, and washed with acetone from above the glass filter.The crosslinked polymer particles were air-dried, then vacuum-dried at 10°C for 8 hours, and then air-classified using a conventional method. , crosslinked polymer particles 110I having a particle diameter of 2 to 4 μm were obtained.

(2) Hydrolysis of epoxy groups based on glycidyl groups on the surface of crosslinked polymer particles with water. 30 g of xylene was added to the log of crosslinked polymer particles obtained in (1) above to completely impregnate the crosslinked polymer particles with xylene. Next, the interior of the crosslinked polymer particles completely impregnated with xylene was placed in an aqueous solution containing 4 g of polyvinyl alcohol and 1.479 g of concentrated sulfuric acid dissolved in 100 g of water, and the mixture was vigorously stirred. This suspended wave sound was heated to 80°C and maintained for 1 hour. The reaction mixture was diluted with gold water and centrifuged. The precipitated crosslinked polymer particles were transferred onto a glass filter and washed with water and then with acetone. The crosslinked polymer particles were air-dried at room temperature and further vacuum-dried at 40° C. for 8 hours. The amount of hydroxyl groups on the surface of the obtained crosslinked polymer particles was 0.6 mm/! 9
Met.

(3) Self-crosslinking of epoxy groups based on glycidyl groups inside crosslinked polymer particles having hydroxyl groups on the surface (2) above
Crosslinked polymer particles 109 with hydroxyl group on the surface obtained in
Suspend in 50 g of total sooxane.

0.5 mi of boron trifluoride etherate (55%) was added and heated at 60°C for 8 hours. Next, it was filtered through a reaction exchange glass filter, washed with acetone, and then air-dried at room temperature.

(4) Introduction of sulfone group Into 2g'tlOmZ of the crosslinked polymer particles obtained in (3) above, which have hydroxyl groups on the surface and are self-crosslinked by the epoxy groups based on internal glycidyl groups, Suspend this with pro-P sultone 0.59 and 1
It was added to 1.2.9' of 0% sodium hydroxide and kept at 30° C. for 6 hours with stirring. Next, the obtained reaction product was washed with water.

A portion of the reactant was taken and the sulfone group was determined by neutralization titration. As a result, 0.15 mmol//g (dry g
el ) sulfone group was observed.

(5) Introduction of carboxyl groups 2 g of the crosslinked polymer particles obtained in (3) above, which have hydroxyl groups on the surface and are self-crosslinked with epoxy groups based on internal glycidyl groups, are suspended in water (9). Sodium monochloroacetate 3.5g, potassium iodide 2.9.50%
Sodium hydroxide 69', 1 added.

The mixture was kept at 60° C. for 3 hours while stirring. Next, the reaction product was washed with water. Neutralization titration was performed using 1 part of the reactant and 9.0.1N aqueous sodium hydroxide solution rL gold, and the carboxyl group was completely determined. As a result, 0.2 mmol/9 (dry
get ) carboxyl group was observed.

(6) Introduction of diethylamine ethyl group Is there a hydroxyl group on the surface obtained in (3) above? Crosslinked polymer particles having self-crosslinked epoxy groups based on internal glycidyl groups2.
9 Suspended in total 20% sodium hydroxide 12y, β-hydrochloric acid
-1 g of soethylaminoethyl chloride was added and kept at 60°C for 3 hours with stirring. The reactants were then washed with water.

One part of the reactant was taken and subjected to neutralization titration using all 0.1N aqueous hydrochloric acid solution, and the result was 0.25 mmol/'! j(dr
ygel) diethylaminoethyl group was observed.

Application examples The cation exchange resins obtained in Examples 1 to (4) were packed into a stainless steel column (inner diameter 4.6 m x length 35 mm), and 20 mM phosphate buffer Cp + 47.0 was added as an eluent.
) all used, 20 mM as eluent (B)! Using J acid buffer + 0.5M sodium chloride (7.0%), transfer the eluent (B) from the eluent to the eluent (B) at a flow rate of L5-/min.
0 minutes of linear graphene) was performed, and ■ myoglobin,
When a mixture of (1) ribonuclease A, (2) cytochrome C1, and (3) lysozyme was analyzed, four proteins could be separated within 5 minutes as shown in Figure 1.

Application Example 2 The anion exchange resin obtained in Example 1-(6) was packed into a stainless steel column (inner diameter 4.6 x length 35 columns), and 20mM lanone (pH 6.0) was added as the eluent (4). ) and 20 mM Bi-Zinone+05 as elution i (B).
Using M sodium chloride (d(6,0), flow rate 1.5 m
A 10-minute linear gradient was performed from eluent (A) to eluent (B) at +>min, and a mixture of ■ conalbumin, ■ transferrin, ■ ovalbumin, and ■ trizone inhibitor was analyzed. As shown in Figure 1, one protein could be separated within 6 minutes.

Application example 3 The column used in application example 2 was used as an eluent at 25 mM.
Phosphate buffer + 0.2 M sodium chloride (,I(6,
0)-i, 25mM phosphoric acid as eluent (B)
Using J solution + IM sodium chloride (PH6,0), perform a 30-minute linear gradient from the eluent to the eluent at a flow rate of 15 g/min to separate oligoazonylic acid. As shown in Figure 3, the oligomers of oligoazonylic acid could be separated.

〔Effect of the invention〕

The chromatography carrier of the present invention has excellent mechanical strength, is chemically stable, is sufficiently stable under weakly alkaline conditions, and has no volume change, and thus can be used to produce a packing material for ion exchange chromatography. It is extremely useful as a carrier for

Moreover, the packing material for ion exchange chromatography obtained according to the present invention has all the following advantages as compared to the conventional packing material for ion exchange chromatography.

1) Since it has high mechanical strength and a small particle size of 1 to 5 μm, it can perform high-performance separation when used as a packing material for high performance liquid chromatography.

2) Since the degree of crosslinking inside the crosslinked polymer particles is high, the degree of swelling is low and it can be used at high salt concentrations.

3) Since it is substantially non-porous, separation can be performed only on the surface of the packing material for ion exchange chromatography, reducing the spread of sample peaks due to diffusion.

4) Stable over a wide range of conditions 1. For example, it can be stably used under alkaline conditions, which is not applicable to fillers using silica gel as a carrier.

[Brief explanation of the drawings] Figure 1 is a chromatogram obtained when a protein mixture was subjected to ion exchange chromatography using the cation exchange resin obtained in Example 1-(4). It is. In the figure, ■ is myoglobin, ■ is bonuclease A, and ■ is
indicates each peak of cytochrome C1 and harizozyme. FIG. 2 is a chromatogram obtained when a protein mixture was subjected to ion exchange chromatography using the anion exchange resin obtained in Example 1-(6). In the figure, ■ indicates the peaks of conalbumin, ■ indicates the peaks of transferrin, ■ indicates the peaks of odialbumin, and ■ indicates the peaks of the bean trigsin inhibitor. FIG. 3 shows a chromatogram in which oligomers of oligoazonylic acid were separated using the anion exchange resin obtained in Example 1-(6).

Claims (2)

[Claims] (1) Consisting of crosslinked polymer particles having glycidyl groups,
An epoxy group based on a glycidyl group on the surface of the particle is ring-opened and modified to a hydroxyl group by hydrolysis, and an epoxy group based on a glycidyl group inside the particle is self-crosslinked.
A carrier for chromatography having an average particle diameter within the range of 1 to 5 μm. (2) consisting of crosslinked polymer particles having glycidyl groups,
An epoxy group based on a glycidyl group on the surface of the particle is ring-opened and modified to a hydroxyl group by hydrolysis, and an epoxy group based on a glycidyl group inside the particle is self-crosslinked.
A packing material for ion exchange chromatography, characterized in that an ion exchange group is introduced into the hydroxyl group of the chromatography carrier having an average particle diameter within the range of 1 to 5 μm.