JPH0518843B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides water-soluble substances with a molecular weight of 1000 or less, such as
This invention relates to a method for separating oligosaccharides, oligopeptides, and glycoprotein sugars by gel permeation chromatography using a hydrophilic resin.

Conventionally, as packing materials for gel permeation chromatography, crosslinked dextran, which is obtained by crosslinking dextran with epichlorohydrin, agarose, or crosslinked polyacrylamide, which is a compound of acrylamide and methylene bisacrylamide, etc., have been used.

However, since these fillers have a low degree of crosslinking and a wide gel mesh structure, the particles are soft and have low mechanical strength. Therefore, chromatography operations under pressure have disadvantages such as deformation of particles and generation of pressure loss. on the other hand,
To improve this problem, a gel is created by ring-opening the epoxy group of a copolymer of unsaturated carboxylic acid glycidyl ester crosslinked with ethylene glycol dimethacrylate, etc. with water or a modifier such as lower alkylene glycol, polyalkyne glycol, or polyol. Permeation chromatography packing materials are known. (For example, see JP-A-53-1087)
However, although this product has improved strength, in order to separate water-soluble substances with a molecular weight of 1000 or less, the retention capacity, that is, the elution capacity of the upper limit molecular weight substance and the elution capacity of the lower limit molecular weight substance in the fractionation range of the substance. There is not enough difference. Therefore, when the fractionation range of a substance is constant, the larger the retention capacity, the gentler the slope of the calibration curve in gel permeation chromatography, which plots the molecular weight of the substance on the vertical axis and the elution volume of the substance on the horizontal axis. Considering that the distance between adjacent peaks becomes longer and the separation property is improved, this filler is still not sufficient.

As a result of intensive studies to solve these drawbacks, the present inventors have developed a method for separating water-soluble substances with a molecular weight of 1000 or less in descending order of molecular weight by gel permeation chromatography using a hydrophilic resin. The present invention was achieved by discovering a hydrophilic resin that has good separability and high mechanical strength.

That is, the present invention provides a method for separating water-soluble substances having a molecular weight of 1000 or less by gel permeation chromatography using a hydrophilic resin, and a glycidyl ester of an unsaturated carboxylic acid as the hydrophilic resin and polyvinyl as a crosslinking agent. A crosslinked copolymer with a compound is a _C3 to _C5 lower fatty acid ketone, a _C2 to _C4
A fat produced by ring-opening the glycidyl group of the crosslinked copolymer with polyethylene glycol or glycerin in the presence of a solvent that swells the copolymer selected from lower aliphatic ethers or cyclic ethers is used. A method for separating a low molecular weight water-soluble substance is provided.

The present invention will be explained in more detail below.

The crosslinked copolymer used in the present invention can be prepared by mixing a glycidyl ester of an unsaturated carboxylic acid with a polyvinyl compound as a crosslinking agent in a conventional manner, for example, in
Manufactured by copolymerization as described in No. 1087.

As the glycidyl ester of unsaturated carboxylic acid, glycidyl acrylate, glycidyl methacrylate, glycidyl crotonate, diglycidyl itagonate, diglycidyl fumarate, etc. are used. On the other hand, examples of the polyvinyl compound as a crosslinking agent include alkylene glycols having 2 to 3 carbon atoms, or esters of these polyalkylene glycols with acrylic acid or methacrylic acid;
For example, ethylene glycol diacrylate, ethylene glycol dimethacrylate, propylene glycol diacrylate, propylene glycol dimethacrylate, polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, polypropylene glycol diacrylate, polypropylene glycol dimethacrylate, etc. are used. Further, esters of acrylic acid or methacrylic acid of polyols such as propanetriol lyacrylate and propanetriol trimethacrylate, aromatic compounds such as divinylbenzene, divinyltoluene, divinylxylene, divinylethylbenzene, and divinylnaphthalene, and the like are used.

The polyvinyl compound contains 5 of the total polymerizable monomers.
(weight)% or more, preferably 15 (weight)% or more.

As a diluent during polymerization, a diluent that is a solvent for the polymerizable monomer and is inert to the polymerization reaction is used. Further, since polymerization is usually carried out by suspension polymerization, the diluent must be insoluble or sparingly soluble in water, which is a polymerization dispersion medium.

Examples of such diluents include aliphatic halogenated hydrocarbons such as dichloromethane, dichloroethane, and trichloroethane, and aliphatic or aromatic esters such as ethyl acetate, butyl acetate, dimethyl phthalate, and diethyl phthalate. .

The diluent accounts for at least 30% by volume or more, preferably 40% to 40% by volume, based on the total amount of the polymerizable monomer and diluent.
Use so that the capacity is 80%.

The polymerization initiator is usually a peroxide such as benzoyl peroxide, t-butyl hydroperoxide, lauroyl peroxide, cumene hydroperoxide, or methyl ethyl ketone peroxide, or azobisisobutyronitrile,
Azo polymerization initiators such as azobis α,α-dimethylvaleronitrile, azobis α-methylbutyronitrile, and azobisisobutyramide are used.
The amount used is 0.1 to 10 (weight)% of the polymerizable monomer.
It is.

During polymerization, salts such as sodium chloride and calcium chloride may be dissolved in water to prevent the polymerizable monomer from dissolving in water, which is a polymerization dispersion medium. Also, in order to stabilize the dispersion,
Dispersion stabilizers such as gelatin, polyvinyl alcohol, carboxymethylcellulose, etc. can also be added to the water.

There is no particular restriction on the volume of the water tank, which is approximately the same volume or more than the volume of the organic layer, but it is used up to about 10 times the volume.

The polymerization reaction is usually carried out at 50 to 80°C for 3 to 8 hours. After completion of the polymerization, the copolymer having a glycidyl group is filtered and washed with water, and if necessary, subjected to a treatment such as extraction and drying, and then separated by an appropriate method.

When the crosslinked copolymer obtained as described above is made hydrophilic by ring-opening the glycidyl group with polyethylene glycol or glycerin,
By reacting the copolymer selected from C ₃ to _{C 5} lower aliphatic ketones, C ₂ to _{C 4} lower aliphatic ethers, or cyclic ethers in the presence of a swelling solvent, a water-soluble polymer with a molecular weight of 1000 or less It is possible to use a hydrophilic resin that exhibits good separation properties in gel permeation chromatography, which separates substances in descending order of molecular weight, and also has high mechanical strength.

The polyethylene glycol used when ring-opening the glycidyl group of a crosslinked copolymer having a glycidyl group usually has a molecular weight of 200 or more. Further, as the glycerin, a usual commercially available product may be used.

The solvent that swells the copolymer used in the ring-opening reaction is usually a solvent that has the property of dissolving polyethylene glycol and glycerin, and the property of dissolving the linear glycidyl polymer. Specifically, C ₃ such as acetone and acetonylacetone
~ _C5 lower aliphatic ketone, diethyl ether, 1,
A _C2 to _{C4 lower aliphatic ether or cyclic ether such as 4} -dioxane is used. In the ring-opening reaction of the glycidyl group of a crosslinked copolymer containing glycidyl groups, using the above-mentioned solvents will result in a faster ring-opening reaction than when the solvent is simply hydrolyzed with water or when the ring-opening reaction is performed with polyalkylene glycol or polyol. This is preferable because the degree of swelling of the copolymer becomes large.

For example, when using a glycerin solution of 1,4-dioxane, the swelling degree of a 25% crosslinked copolymer is 1.1 at a concentration of 25% by volume and 1.3 at a concentration of 50% by volume, assuming that the volume basis of glycerin is 1.0. , 1.5 at 75 volume % concentration, 100 volume % concentration (quasi-1,4-dioxane)
It becomes 1.7 times larger, and swelling starts at 25% volume or more.

The amount of solvent used varies depending on the type of solvent, but is generally 5 to 20 ml per 1 g of copolymer.
It is preferable to use 25% by volume or more, preferably 40 to 80% by volume, based on the total amount of polyethylene glycol or glycerin and the solvent. The solvent may be reacted with the copolymer as a mixed solution with polyethylene glycol or glycerin, or the copolymer may be swollen in advance with a solvent and then reacted with polyethylene glycol or glycerin.

The ring-opening reaction of the glycidyl group is carried out in the presence of the above solvent.
The reaction is carried out using an acid catalyst in a catalytic amount. For example, if sulfuric acid is used in an amount of 5 mg to 50 mg per 1 g of copolymer and the reaction is carried out at 50°C to 100%°C for 3 to 10 hours, the ester bonds in the crosslinked copolymer will not be substantially cut, and the hydrophilic The ring-opening reaction for the conversion can be carried out selectively.

The hydrophilic resin of the present invention is obtained by washing the reaction mixture with pure water after cooling.

By using a hydrophilic resin obtained by a specific method, the present invention has the characteristics that the resin has a large retention capacity and high separability.
Water-soluble substances with low molecular weight (1000 or less) can be separated better than gel permeation chromatography.

Next, the present invention will be explained in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to the following Examples unless it exceeds the gist thereof.

Example 1 (a) Production of crosslinked copolymer particles having glycidyl ester groups 75 g of ethylene glycol dimethacrylate,
225 g of glycidyl methacrylate, 432 g of 1,2-dichloroethane and 2,2'-azobis-
A mixture of 15 g of 2,4-dimethylvaleronitrile was added to a solution of 21 g of polyvinyl alcohol and 84 g of sodium chloride dissolved in 2100 ml of demineralized water, and suspension polymerization was carried out at 65° C. for 8 hours while stirring at high speed.

After cooling the reaction product, the produced copolymer particles were taken and washed with water. Next, this copolymer was placed in a mixture of 1125 ml of toluene and 375 ml of water, stirred at room temperature for 3 hours, and then vortexed. Further, this copolymer was poured into 1.5 methanol and stirred twice, and then dried at 80°C for 8 hours.

The particles that have gone through the above operations are sieved and the particle size is 8~
100 g of 12 μm copolymer particles were obtained (degree of crosslinking: 25
%).

(b) Ring-opening reaction in polyethylene glycol solution 20 g of the copolymer particles obtained in (a) are placed in 100 ml of 1,4-dioxane, mixed well, and then swollen at room temperature for about 20 hours. Next, add 100 ml of polyethylene glycol (average molecular weight 600) and mix well. To this, add 0.5 ml of 97% sulfuric acid as a catalyst.
Add.

Heat at 80°C for 8 hours with stirring. After cooling, the obtained hydrophilic resin is washed with pure water and filtered to obtain a hydrophilic resin to which polyethylene glycol is chemically bonded.

(c) Performance test of resin The above hydrophilic resin was classified, and particles having a particle diameter of 8 to 10 μm were packed into a stainless steel column with an inner diameter of 7.5 mm and a length of 600 mm. Using this column, polyethylene glycol of known molecule and a 0.5% aqueous solution of ethylene glycol were tested at a temperature of 25%.
The measurement was carried out at a flow rate of 0.5 ml/mm at a temperature of 0.5 ml/mm, and a calibration curve was prepared with the logarithm of the molecular weight on the vertical axis and the elution volume on the horizontal axis, as shown in Figure 1. In polyethylene glycol of known molecular weight, molecular weight 200 and 600.
The value of the elution volume was taken as the average value of the elution curve. From the calibration curve, the retention capacity between ethylene glycol (molecular weight 62) and polyethylene glycol having a molecular weight of 1×10 ³ was 5.7 ml. When polyethylene glycol with an average molecular weight of 200 was analyzed using this packed column, it was well separated as shown in Figure 2.
At this time, the effective retention capacity (capacity from the beginning to the end of the chromatogram) is 3.9ml.
It was hot. Note that peak detection was performed using refractive index.

Example 2 Ring-opening reaction in glycerin solution Using copolymer particles obtained in the same manner as in Example 1-(a), glycerin was used instead of polyethylene glycol in Example 1-(b). A hydrophilic resin to which glycerin was chemically bonded was obtained by carrying out the same procedure except for that. This hydrophilic resin was classified in the same manner as in Example 1-(c), and particles with a particle diameter of 8 to 10 μm were classified into particles with an inner diameter of 7.5 μm.
packed in a stainless steel column with a length of 600 mm.
When this column was used to analyze polyethylene gelicol of known molecular weight and a 0.5% aqueous solution of ethylene glycol to create a calibration curve, the result was as shown in Figure 3. According to the calibration curve, the retention capacity for the molecular weight range of 62 to 1×10 ³ was 59 ml. When polyethylene glycol having an average molecular weight of 200 was analyzed using this packed column, it was well separated as in Example 1.

Example 3 240 g of glycidyl methacrylate, 60 g of ethylene glycol dimethacrylate, 432 g of 1,2-dichlorothane and 2,2'-azobis-2,4-
A crosslinked copolymer was produced in the same manner as in Example 1-(a), except that a homogeneous mixed solution containing 5 g of dimethylvaleronitrile was used as the polymerization solution, and 100 g of copolymer particles with a particle size of 8 to 12 μm were obtained. . (degree of crosslinking 20%).

Using the obtained copolymer particles, the same procedure as in Example 1-(b) was performed except that glycerin was used instead of polyethylene glycol to obtain a hydrophilic resin to which glycerin was chemically bonded. . Example 1
- Classify this hydrophilic resin in the same way as (c), and
Particles with a particle diameter of
% aqueous solution and created a calibration curve.
It looked like Figure 4. 62 to 1×10 ³ from the calibration curve
The retention capacity for the molecular weight range of is 7.1 ml, and Example 1
Similarly, when polyethylene glycol with an average molecular weight of 200 was analyzed, the effective holding capacity was 4.0 ml.
Separability was also good.

Comparative Example 1 20g of copolymer particles produced in Example 1-(a) were
Pour into 40 ml of 0.1N sulfuric acid aqueous solution containing 1,4-dioxane and mix well. Do this on a water bath
The mixture was heated to 50°C and reacted for 3 hours with stirring to open the glycidyl group. After cooling, the obtained hydrophilic resin was washed with pure water. This hydrophilic resin was classified and
Particles with a particle size of ~10 μm, inner diameter 7.5 mm, length 600 mm
was packed into a stainless steel column. Using this column, polyethylene glycol of known molecular weight and a 0.5% aqueous solution of ethylene glycol were analyzed to create a calibration curve, as shown in Figure 5. According to the calibration curve, the retention capacity for the molecular weight range of 62 to 1×10 ³ was 5.0 ml. Using this column,
When polyethylene glycol with an average molecular weight of 200 was analyzed, the results shown in Figure 6 were obtained, indicating that it was not suitable for separating water-soluble substances with a molecular weight of 1000 or less.

Although the holding capacity is 5.0 ml, which is slightly smaller than that of the hydrophilic resin of the present invention, it is thought that the degree of hydrophilicity is lower than that of the hydrophilic resin of the present invention, resulting in poor separation performance.

[Brief explanation of drawings]

FIG. 1 is a calibration curve obtained for a column packed with the hydrophilic resin obtained in Example 1 using polyethylene glycol of known molecular weight and an aqueous solution of ethylene glycol. FIG. 2 is a chromatogram obtained by analyzing polyethylene glycol having an average molecular weight of 200 using the packed column of Example 1 as a sample. FIG. 3 is a calibration curve obtained for the column packed with the hydrophilic resin obtained in Example 2 using polyethylene glycol of known molecular weight and an aqueous solution of ethylene glycol. FIG. 4 is a calibration curve obtained for the column packed with the hydrophilic resin obtained in Example 3 using polyethylene glycol of known molecular weight and an aqueous solution of ethylene glycol. FIG. 5 is a calibration curve obtained for the column packed with the hydrophilic resin obtained in Comparative Example 1 using polyethylene glycol of known molecular weight and an aqueous solution of ethylene glycol. FIG. 6 is a chromatogram obtained by analyzing polyethylene glycol with an average molecular weight of 200 as a sample using the packed column of Comparative Example 1. In Figures 1 to 6, the vertical axis represents the molecular weight,
The horizontal axis represents elution volume (ml).

Claims (1)

[Claims] 1. In a method of separating an aqueous substance with a molecular weight of 1000 or less by gel permeation chromatography using a hydrophilic resin, a crosslinking method using a glycidyl ester of an unsaturated carboxylic acid as the hydrophilic resin and a polyvinyl compound as a crosslinking agent is used. polymer,
The crosslinked copolymer is treated with polyethylene glycol or glycerin in the presence of a solvent that swells the copolymer selected from _{C3 to C5} lower aliphatic ketones, C2 _{to C4} lower aliphatic ethers, or cyclic ethers. A method for separating a low molecular weight water-soluble substance, characterized by using a resin produced by ring-opening reaction of glycidyl groups.