JPH02227144A - Anion-exchanger and liquid chromatography column filled with it for sugar separation - Google Patents

Abstract

PURPOSE:To heighten separation efficiency between monosaccharides and disaccharides by preparing an anion-exchanger from a copolymer of a vinyl alcohol unit having specified hydroxy group and anion-exchangeable group each and a crosslinkable monomer. CONSTITUTION:A part of ester groups of a copolymer prepared by copolymerization of vinyl carboxylate and a crosslinkable monomer is saponified or ester interchanged. Then, after a part of hydroxy groups is reacted with halogenocarboxylic acid halide, epichlorohydrin, etc., the resultant copolymer is reacted with ammonia or various amines to give an anion-exchanger. The anion-exchanger has 0.5-4.0meq/g of hydroxy derived from vinyl alcohol unit, 0.02-5.0meq/g anion-exchange capacity, 0.5-4.0g/g of water retaining capacity, and crosslinking monomer ratio X; 0.45<=X<=0.8.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention provides an anion exchanger having a hydroxyl group and an anion exchange group and having excellent mechanical strength and chemical stability, and an anion exchanger having a hydroxyl group and an anion exchange group and having excellent mechanical strength and chemical stability. Place it in a packed liquid chromatography column for separating sugars.

[Conventional technology]

Saccharides are widely distributed in the natural world and in living organisms, and their state of existence is also diverse. *I not only naturally exists in a free state as a single monosaccharide (from the oligo I! class, which is formed by bonding several monosaccharides together, to starch and cellulose, in which a large number of several types of monosaccharides are bonded together). There are various molecular forms, including polysaccharides.
There are ketoses, which have a hydroxyl group and a carboxyl group, and aldoses, which have a hydroxyl group and an aldehyde group, and each group has many stereoisomers that differ in the coordination of the hydroxyl group.

Liquid chromatography is suitable for separating and analyzing sugars.
It is commonly used. Liquid chromatography is used to separate and analyze relatively low molecular weight sugars such as monosaccharides and trisaccharides formed by bonding two molecules of monosaccharides, using polystyrene gels with cation exchange groups such as sulfonic acid groups. There is a method in which a gel into which metal ions are introduced as counterions is used as a packing material, and water or a mixture of water and a water-soluble organic solvent is used as an eluent for separation. Collection of materials, supervised by Kenji Soda, 200 pages, NTS, 1982-
(See Publication No. 162953).

Although this method is suitable for the separation and analysis of Class Ii, it has the disadvantage that the metal ions bound to the cation exchange resin are easily desorbed and its durability is relatively poor. Also, increase the column temperature to 80°C.
It has the disadvantage of being difficult to handle because it needs to be kept at a relatively high temperature.

Another method is to use a crosslinked polystyrene resin having an anion exchange group such as a quaternary ammonium base as a packing material, use a borate buffer as an eluent, and adjust the concentration of the buffer in stages or A method of separation using a gradient method that changes continuously can be mentioned (“
Comprehensive collection of technical materials for the commercialization of liquid chromatography in the field of biotechnology, supervised by Kenji Soda, p. 197, see NTS).

Although this method has excellent separation ability for monosaccharides and trisaccharides,
Since it uses a gradient, the operation is complicated and separation takes a long time.

Still another method is to use a filler in which an amino group such as an aminopropyl group is introduced into silica gel, and use a mixture of water and a water-soluble organic solvent such as a7tonitrile as the eluent. [Koizumi et al., Starch Science, 34.
308 (87)].

This method uses an eluent with a constant composition, so it is easy to operate, and the column temperature is about 40°C, so it is relatively easy to handle. However, silica gel fillers having amino groups have the disadvantage of poor durability.

In addition, as an anion exchanger containing an amino group, JP-A-6
There is an anion exchanger described in Japanese Patent No. 0-150839. The anion exchanger described in this publication is mainly intended for the separation and analysis of proteins and peptides, and has a low molecular weight of 1! It is not suitable for the separation and analysis of species.

[Problem to be solved by the invention]

As mentioned above, conventional separation analysis methods are complicated and time-consuming, and the packing materials used for separation have poor durability.
It has the disadvantage that it deteriorates during use, and therefore it is difficult to obtain results with good reproducibility over a long period of time.

Therefore, the present inventors set out to overcome the drawbacks of such prior art (
As a result of extensive research, we have developed a packing material with excellent chemical stability, high strength, and excellent ability to separate low-molecular-weight sugars such as monoclass Ii such as glucose and xylose, and trisaccharides such as sucrose and maltose. The present invention was achieved by developing the following.

[Means to solve the problem]

That is, the present invention has vinyl alcohol units and crosslinkable monomer units, the amount of hydroxyl groups derived from the vinyl alcohol units per polymer weight is 0.5 to 4,0 meq/g, and the amount of anion exchange groups is 0. .02 to 5.0 meq/g, the amount of water that can be retained is 0.5 to 4.0 g/g, and the proportion (X) of crosslinkable monomer units is 0.45. ≦X
The present invention provides an anion exchanger characterized in that the ion exchanger is in the range of ≦0.8, and a liquid chromatography column for saccharide analysis characterized in that it is packed with the anion exchanger.

a+nb Here, a is the mole fraction of the monomer units excluding the crosslinkable monomer units in the crosslinked copolymer, b is the mole fraction of the crosslinkable monomer units, and n is the crosslinkable monomer 1 It is the number of ethylenic double bonds that a molecule has. ) The anion exchanger of the present invention contains 0.5 to 4.0 meq hydroxyl groups derived from vinyl alcohol units per polymer weight.
/ Included in the range of H. By containing hydroxyl groups within this range, the anion exchanger has appropriate hydrophilicity. When separating ta using the ion exchanger, a mixed solvent of a water-soluble organic solvent and water is used as an eluent. Although the polarity of the eluent changes when the mixing ratio of organic solvent and water changes, the anion exchanger of the present invention has appropriate hydrophilicity,
Practically speaking, the amount of hydroxyl groups is preferably 0.5 to 3.0, which is preferable because swelling and shrinkage are small even when the polarity of the solvent changes. 0meq
/g range.

The amount of hydroxyl groups can be determined by reacting the hydroxyl groups with acetic anhydride and measuring the amount of acetic anhydride consumed or the change in weight of the crosslinked anion exchanger. The amount of hydroxyl groups when 1 g of the dry crosslinked anion exchanger reacts with 1 smol of acetic anhydride is 1 meq/g.

If the anion exchanger has primary or secondary amino groups, the negative or secondary amino groups will also react with acetic anhydride, so subtract the amount of ion exchange groups determined separately from the amount of reacted acetic anhydride. By doing this, the amount of hydroxyl groups can be determined.

When the anion exchanger has a tertiary amino group, the tertiary amino group does not react with acetic anhydride, so it can be determined directly from the amount of acetic anhydride consumed.

The anion exchange group in the anion exchanger is 0.02 to 5.0
Exists within the range of Due to the presence of anion exchange groups in this range, the anion exchanger has the ability to separate Group Ii and has sufficient strength as a packing material for liquid chromatography. The anion exchange group is practically 0.1
~1.5 seq/g.

The amount of anion exchange groups can be determined by the usual method for measuring the exchange capacity of ion exchange resins (Reference: "Ion Exchange - A Guide to Theory and Applications - R,W, Grimshaw and C,E,
Harland, translated by Kuroda et al., p. 78, Maruzen).

Examples of the ion exchange group of the anion exchanger of the present invention include a -class amino group, a secondary amino group, and a tertiary amino group. One type of these ion exchange groups may be present alone in the anion exchanger, or two or more types may be present.

The anion exchanger of the present invention has a crosslinked structure.

Examples of the crosslinking unit in the present invention include a crosslinking unit derived from a crosslinking agent having two or more ethylenic double bonds and/or acetylenic triple bonds. A crosslinking unit derived from a crosslinking agent having two or more ethylenic double bonds and/or acetylenic triple bonds is
Examples of the crosslinking agent exhibiting a structure formed by polymerization or copolymerization include polymethacrylates such as mono- and polyethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, and polyunsaturated compounds such as divinyl adipate and divinyl phthalate. Examples include alcohol esters, polyunsaturated ethers such as divinyl ether, and crosslinking agents having a triazine ring such as triallyl isocyanurate, and preferred examples include triallyl isocyanurate and triallyl cyanurate.

The proportion (X) of crosslinkable monomer units in all monomer units forming the anion exchanger according to the present invention is 0.45≦X≦0.
80) is preferable.

Here, a is the mole fraction of monomer units excluding crosslinkable monomer units in the crosslinked copolymer, b is the mole fraction of crosslinkable monomer units, and n is one molecule of crosslinkable monomer. is the number of ethylenic double bonds that has. ) By having the crosslinkable monomer unit within this range, it is possible to obtain an anion exchanger that satisfies both the separation ability of Group Ii and the strength as a filler.

In order to balance the separation ability and strength of the packing material obtained from the anion exchanger, it is necessary to maintain the water retention amount within an appropriate range. Conventionally, fillers obtained from cross-linked agarose and cross-linked dextran, which have hydroxyl groups and anion exchange groups, have high water content and low mechanical strength, as described above.
This tendency was particularly strong for fillers with large pore sizes. The filler obtained from the anion exchanger of the present invention has a water content in the range of 0.5 to 4.0 g/g, preferably 0.5 to 3.0 g/g, regardless of the ear size, and is suitable for high performance liquid chromatography. It has sufficient strength to be used as a filler for industrial applications. The water retention capacity of an anion exchanger is expressed as the amount per anion exchange weight.The copolymer that has been sufficiently swollen in water is placed in a centrifuge tube equipped with a filter, and the water adhering to the surface of the copolymer is centrifuged. After that, the copolymer is dried, and the weight can be determined from the change in weight before and after drying.

The shape of the anion exchanger of the present invention is not particularly limited, and can take any shape such as granules, membranes, threads, and lumps depending on the method of use. When used as a packing material for liquid chromatography, it is preferably granular or spherical. In that case, the particle size is not particularly limited, but the weight average particle size is usually 1 to 1.
It is in the range of 500pm. When used as a packing material for high performance liquid chromatography, it is preferably in the range of 1 to 20 J111, more practically 3 to 15 #lI.

When separating and analyzing sugars using the anion exchanger of the present invention, the anion exchanger described above is used, for example, by filling a column made of stainless steel.

The column diameter can be selected depending on the purpose, but for analysis purposes, the inner diameter is 3ffI11-10mm and the length is 5cm.
A range of m to 100 CI is preferred.

A method for separating and analyzing sugars using the anion exchanger of the present invention is to use a separation column prepared as described above and use an organic solvent such as water or acetonitrile, or a mixture thereof as an eluent. It can be done by In that case, for example, by changing the eluent composition, column temperature, column size, etc., I! It is possible to adjust the separation status of the types.

Next, an example of a method for producing the anion exchanger of the present invention will be shown.

The anion exchanger of the present invention can be produced by, for example, saponifying or transesterifying a part of the ester group of a copolymer consisting of a carboxylic acid vinyl ester and a crosslinkable monomer, and then combining a part of the hydroxyl group with a halogenocarboxylic acid halide. It can be obtained by reacting dichlorohydrin, epichlorohydrin, etc., and then reacting ammonia or various amines.

Here, the carboxylic acid vinyl ester refers to a compound having one or more polymerizable carboxylic acid vinyl ester groups, and is selected from vinyl acetate, vinyl propionate, vinyl butyrate, vinyl valerate, and vinyl pivalate. They can be used alone or in combination of two or more. Among them, it is particularly preferable to use vinyl acetate and vinyl propionate from the viewpoint of ease of polymerization, transesterification, saponification, and availability.

Examples of the crosslinkable monomer include crosslinkable monomers having a triazine ring, and among them, triallyl isocyanurate is preferred as a crosslinking agent.

Polymerization to obtain a copolymer consisting of a carboxylic acid vinyl ester and a crosslinkable monomer having a triazine ring can be carried out by a conventional polymerization method such as suspension polymerization, bulk polymerization, or emulsion polymerization. Suspension polymerization is preferred when obtaining a packing material for liquid chromatography.

Note that the anion exchanger of the present invention can be obtained by copolymerizing a monomer other than a carboxylic acid vinyl ester or a crosslinkable monomer having a triazine ring to the extent that it hardly affects the physical properties of the copolymer. There is no problem with that.

In addition, when copolymerizing a carboxylic acid vinyl ester and a crosslinkable monomer having a triazine ring, a copolymer obtained by adding one or more organic solvents that dissolve the monomer to the monomer. The coalescence forms a permanent bore and controls the weight, pore size, or pore size distribution of the bore. Specific examples of organic solvents that dissolve monomers include aromatic hydrocarbons such as toluene and xylene; aliphatic hydrocarbons such as heptane, octane, cyclohexane, and decalin;
Aliphatic esters such as n-butyl acetate, 1so-butyl acetate, n-hexyl acetate, dioctyl adipate, aromatic esters such as dimethyl phthalate, dioctyl phthalate, methyl benzoate, butanol, heptatool, octatool Examples include alcohols such as In the case of suspension polymerization, it is preferable to use a solvent that is soluble in water. The amount of these organic solvents used is not particularly limited, but for example, 20 to 300 parts by weight per 100 parts by weight of the monomer.
Used in parts by weight. In particular, when producing a filler for high performance liquid chromatography that requires mechanical strength, the amount of organic solvent is preferably in the range of 30 to 100 parts by weight.

Linear polymers or rubbers that are soluble in the monomer mixture can be added to the monomer mixture to control the pore size or pore size distribution of the anion exchanger or to increase the flexibility of the anion exchanger. Examples of linear polymers and rubbers that can be dissolved in the monomer mixture include polyvinyl acetate, polystyrene, chloroprene rubber, butadiene rubber, etc., and these can be used in an amount of 20 parts by weight per 100 parts by weight of the monomer. parts by weight or less, preferably 10 parts by weight or less.

The type and amount of the initiator used during the polymerization can be arbitrarily selected according to the polymerization method. In normal suspension polymerization and bulk polymerization, a common radical polymerization initiator, such as 2.21-
Abbisisobutyronitrile, 2,2'-azobis-(
Azo-based initiators such as 2,4-dimethylvaleronitrile) and peroxide-based initiators such as benzoyl peroxide and lauroyl peroxide can be used.

Transesterification or saponification of the copolymer obtained by polymerization is carried out using an acid or alkali in water, alcohol, or a mixture thereof as a solvent. The saponification rate can be controlled by selecting appropriate conditions by changing the reaction solvent, reaction temperature, or reaction time.

Examples of methods for introducing anion exchange groups into the gel obtained by saponification and/or transesterification include the following methods.

That is, the hydroxyl groups of the gel obtained by saponification and/or transesterification are reacted with, for example, halogenocarboxylic acid halide, epichlorohydrin, butanediol divinyl ether, etc., and then ammonia, a primary amine such as ethylamine, or a secondary amine such as diethylamine are reacted with the hydroxyl groups of the gel obtained by saponification and/or transesterification. By reacting, it is possible to obtain a gel having primary amino groups, secondary amino groups, and tertiary amino groups as ion exchange groups, respectively.

〔Example〕

The present invention will be described in more detail below with reference to Examples, but it goes without saying that the technical scope of the present invention is not limited to these Examples.

Vinyl acetate 100g, triallyl isocyanurate 14
5g (X=0.6), 170g of n-butyl acetate and 2
．． A homogeneous mixed solution consisting of 7 g of 2-azobisisobutyronitrile and 1,400 g of water in which a small amount of polyvinyl alcohol and sodium phosphate were dissolved were placed in a 51 three-neck flask equipped with a reflux condenser, a nitrogen inlet tube, and a stirrer. and stirred thoroughly. Then, while stirring under a nitrogen stream, the temperature was increased to 60°C.
Polymerization was carried out for 16 hours to obtain a granular polymer. The polymer was filtered, washed, extracted with acetone, and then dried. Then, the polymer was mixed with water 32 in which 60 g of caustic soda was dissolved.
The polymer was placed in a 5P three-bottle flask equipped with a reflux condenser, a nitrogen inlet tube, and a stirrer, and stirred at 15°C under a nitrogen stream for 20 hours to perform a saponification reaction, followed by filtration, water washing, and further Dry. The density of hydroxyl groups in the polymer obtained by saponification was 1.7 s+eq/g.

Then, the polymer was added with 3.5 L of toluene, 230 g of 1-bromobutanoyl chloride, and 200 g of pyridine,
The polymer was placed in a 10 f three-bottle flask equipped with a reflux condenser, a nitrogen inlet tube, and a stirrer, and stirred at 15° C. under a nitrogen stream for 20 hours to introduce 1-bromobutanoyl groups into the polymer.

The polymer was then washed with ethanol and acetone and dried. Thereafter, the polymer was treated with dimethyl sulfoxide 3.
72.Put it together with 900 d of 28% ammonia water in a 102-meter three-necked flask equipped with a reflux condenser, nitrogen inlet tube, and stirrer, and stir at 20°C under a nitrogen stream for 20 hours to introduce primary amino groups into the polymer. did. After washing and drying the polymer, it was classified to obtain an anion exchanger having a particle size of 5 μm. The amount of amino groups in the anion exchanger is 0.4 meq/
g anion exchanger, the density of hydroxyl groups is 1.1 meq/
g anion exchanger, and the water retention amount was 1.3 g/g anion exchanger.

The anion exchanger of ions was packed into a stainless steel column with an inner diameter of 6 mm and a length of 150 mm, and the packed column was used to extract rhamnose, xylose, fructose, glucose,
A mixture of sucrose and lactose was analyzed and the chromatogram shown in Figure 1 was obtained. As shown in FIG. 1, six types of sugars could be separated well and efficiently using the anion exchanger of the present invention.

Eluent flow rate column manufactured by Kunigojo Eluent flow rate column Constant temperature bath Temperature flow rate ± Human HPLC pump Detector 80% acetonitrile 1,0 d/a + 1n 40°C 880PIJ (manufactured by JASCO Corporation) Refractometer St! -51 (manufactured by Showa Denko ■) SIC-70008 (manufactured by System Instruments) Data processing device m The saponified polymer of Example 1 was mixed with dimethyl sulfoxide 2
．． 54! , 1200 g of epichlorohydrin and 130 d of 10N caustic soda into a 51 three-neck flask equipped with a reflux condenser, a nitrogen inlet tube and a stirrer,
The reaction was carried out at 30° C. for 20 hours under a nitrogen stream. The polymer was then filtered and washed. The filtered and washed polymer was placed in a 51 three-bottle flask equipped with a reflux condenser, a nitrogen inlet tube, and a stirrer together with 2.5 g of 28% ammonia water and stirred at 60°C under a nitrogen stream for 20 hours to remove amino groups. was introduced into the polymer.

After washing and drying the polymer, it was classified to obtain an anion exchanger having a particle size of 5-. The amount of amino groups in the anion exchanger is 0.5 l1eq/g anion exchanger, the density of hydroxyl groups is 1.5 meq/g anion exchanger, and the water retention amount is 1.4g/g anion exchanger. there were.

The anion exchanger containing ion 6 was packed in a stainless steel column in the same manner as in Example 1, and a mixture of fructose, glucose, sucrose and lactose was analyzed using the packed column under the same conditions as in Example 1. Figure 2 B madogram was obtained. As shown in FIG. 2, four types of sugars were successfully separated using the anion exchanger of the present invention.

〔Effect of the invention〕

As explained above, the anion exchanger of the present invention is hard, and when packed in a column and used as a separation packing material for Class II, the eluent can be passed at a high flow rate and rapid analysis is possible. be. Furthermore, the anion exchanger of the present invention is chemically stable and can provide results with good reproducibility over a long period of time. In addition, since the anion exchanger of the present invention is highly crosslinked and has appropriate hydrophilicity, it is less likely to swell or shrink due to changes in the polarity of the eluent, and I! Suitable for separating species.

[Brief explanation of the drawing]

FIG. 1 is a chromatogram obtained in Example 1, in which peak A is rhamnose, peak B is xylose, peak C is fructose, peak D is glucose, peak E is sucrose, and peak F is lactose. FIG. 2 is a chromatogram obtained in Example 2, where peak A is fructose, peak B is glucose,
Peak C is sucrose and peak D is lactose.

Claims (1)

[Claims] 1. Having a vinyl alcohol unit and a crosslinkable monomer unit,
The amount of hydroxyl groups derived from vinyl alcohol units per polymer weight is 0.5 to 4.0 meq/g, the amount of anion exchange groups is 0.02 to 5.0 meq/g, and the amount of water that can be retained is 0.
．． 5 to 4.0 g/g of a crosslinked copolymer, characterized in that the proportion (X) of crosslinkable monomer units is in the range of 0.45≦X≦0.8, anion exchange body. (where, (n indicates the number of ethylenic double bonds that one molecule of the crosslinkable monomer has.) 2. For saccharide separation, characterized in that it is filled with the anion exchanger described in claim 1. Liquid chromatography column.