JPH11292903A - Surface modification of filler of polymer gel - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a highly selective filler for mold polymerization of a saccharide. SOLUTION: This filler of polymer gel for chromatography is obtained by (1) 2-stage swelling polymerization of a monomer (e.g. methacrylic acid or 4-vinylpyridine), in the presence of a crosslinking agent (e.g. ethylene dimethacrylate or methylene bisarcylamide) and solvent (e.g. toluene), (2) adsorption of a saccharide (e.g. galactose, glucose or mannose) on the polymer obtained by the above step (1), and (3) heat treatment of the saccharide-adsorbed polymer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing a polymer gel and a packing material for chromatography comprising the polymer gel.

[0002]

2. Description of the Related Art Active research is being conducted on high molecular weight hosts that specifically recognize saccharides, such as lectins, which are proteins. As one of the preparation methods, a method using a molecular imprint method has been proposed. The molecular imprinting method is receiving attention as a method for preparing a polymer having a specific molecular recognition ability. As a general preparation method, a so-called bulk polymerization method is generally used in which a monomer serving as a host and a print molecule serving as a guest are put into a test tube and polymerized. Specifically, first, a print molecule and a monomer are added. At this time, a non-covalent interaction is formed between the print molecule and the monomer. Next, add a crosslinking agent,
Initiate polymerization. After completion of the polymerization, the polymer is washed to remove the print molecules. In the obtained polymer, sites storing the size of the print molecule and the position of the functional group are prepared. FIG. 1 schematically shows such a molecular imprinting method.
This bulk polymerization method has the advantage that weak interaction such as hydrogen bond formed between the host monomer and the print molecule is not hindered because water does not exist in the system. However, this polymerization has a limitation that only an oil-soluble print molecule can be generally applied, and this is particularly problematic when preparing a synthetic polymer that specifically recognizes a water-soluble substance as a substitute for a biopolymer. . For example, a problem of the molecular imprinting method using a sugar as a template is that the template must be dissolved in a polar solvent such as water, and the interaction between the host monomer and the template is disadvantageous. . In recent years, a molecular imprint method in an aqueous medium using a monosaccharide as a template has been reported (Hosoya et al., Proceedings of the 45th Society of Polymer Science, 45
(1996) 1764 and K. Tsukagoshi et al., Che
m. Lett., 1994, 681). In addition, it has been reported that by heating the mold to 120 ° C. in a state where the mold is present in the host, the discrimination ability for the mold is increased (B. Sellergen).
et al., J. Chromatogr., 635 (1993) 31). However, the sugar used as a template has not yet developed sufficient discriminating ability. Furthermore, the polymer mass obtained by the bulk polymerization method can be used as a filler for HPLC (high-performance liquid chromatography) by pulverization, but operations such as classification are complicated, and the particle size is large. There was a disadvantage that the performance of the obtained column was not high due to the non-uniformity.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to overcome the problems of the conventional preparation method (bulk polymerization method) and to provide a polymer gel which specifically recognizes sugar.

[0004]

Means for Solving the Problems As a result of intensive studies, the present inventors conducted polymerization using methacrylic acid and a crosslinking agent,
Furthermore, it has been found that when heat treatment is performed in the presence of a sugar, which is a template molecule, selective discrimination with respect to saccharides is further enhanced. Furthermore, it has been found that the selective discrimination extends not only to the template but also to saccharides having a structure similar to the template. The present invention has been completed based on these findings.

That is, the present invention relates to a method for producing a polymer gel, wherein a polymer obtained by polymerization using a monomer, a crosslinking agent and a solvent as raw materials has a uniform particle size. In addition, the present invention relates to a method for producing a polymer gel, which comprises subjecting a polymer obtained by polymerization using a monomer, a cross-linking agent, and a solvent to a raw material, to adsorb sugar, and performing a heat treatment. Furthermore, the present invention relates to a packing material for chromatography comprising a polymer gel obtained by any one of the production methods described above. Furthermore, the present invention relates to a method for modifying the surface of a polymer gel filler, wherein the polymer obtained by polymerization using a monomer, a crosslinking agent and a solvent as raw materials has a uniform particle size. In addition, the present invention relates to a method for modifying the surface of a polymer gel filler, which comprises subjecting a polymer obtained by polymerization using a monomer, a cross-linking agent, and a solvent to a raw material and adsorbing a sugar to perform a heat treatment.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The sugar (print molecule) used in the present invention includes monosaccharides such as galactose (Gal), glucose (Glc), and mannose (Man). Examples of the monomer (host monomer) used in the present invention include methacrylic acid (MA) and vinylpyridine (VP). Further, as a crosslinking agent used in the present invention, ethylene dimethacrylate (ED
MA) or methylenebisacrylamide. Further, the solvent used in the present invention includes toluene.

In the present invention, the polymerization conditions in the polymerization using a monomer, a crosslinking agent and a solvent are as follows. That is, the temperature is from room temperature to about 50 ° C., and the polymerization time is from 4 hours to 8 hours. In addition, photopolymerization can also be used as a polymerization method. After completion of the polymerization, the solvent is removed to obtain a desired polymer gel. Furthermore, after the polymerization is completed, the sugar is adsorbed on the polymer obtained by removing the solvent,
Heat treatment is performed. In this case, the heating temperature is generally 100 ° C.
The temperature is preferably in the range of -300 ° C, particularly preferably around 250 ° C.

[0008] The polymer gel of the present invention produced in this manner can be used for various purposes such as high performance liquid chromatography (HPLC) and capillary electrochromatography (CEC) for the purpose of carbohydrate analysis and separation of glycoconjugates in general. It can be used as a filler for chromatographies. When the polymer gel is used as a filler for chromatography, the particle size of the polymer particles of the polymer gel can be appropriately set according to the type of chromatography to be used, the type of substance to be separated, and the like. Desirable particle diameter is usually 2 μm to 300 μm, preferably 2 μm to 20 μm, but is not limited to this range.

[0009]

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

[0010]

Example 1 Ethylene dimethacrylate 2 as a crosslinking agent
The polymerization reaction was carried out at 50 ° C. by a two-stage swelling polymerization method using 4 ml of toluene as a raw material and 1 ml (12 mmol) of methacrylic acid as a host monomer, and methylene bis as a crosslinking agent on the surface of the obtained polymer gel. 0.5 g (3 mmol) of acrylamide was introduced by polymerization. After the polymerization reaction, the polymer gel is washed,
Glucose was adsorbed on the surface of the polymer gel and subjected to a heat treatment at 250 ° C. for 30 minutes. D-xylose (Xyl), L-arabinose (Ara), D-mannose (Man), N-acetyl D-glucosamine under the following conditions using high performance liquid chromatography using the obtained polymer particles as a filler. (GlcNAc), N-acetyl-
The saccharide identification was examined using a saccharide mixture of D-galactosamine (GalNAc), D-glucose (Glc) and D-galactose (Gal).

Chromatography conditions : Mobile phase: 95% acetonitrile aqueous solution and 90% acetonitrile aqueous solution Flow rate: 1 ml / min Column size: 4.6 mm (id) × 150 mm Column temperature: 30 ° C. Detection: RI Results: FIG. Shown in The following can be seen from FIG. (1) When heat treatment is performed without adding a template, the selectivity to saccharides does not change much from before heat treatment. (2) Comparing the effects of adding the template, it was shown that the heat treatment significantly changed the selectivity for saccharides having an amide group. (3) The selectivity does not identify only the template,
In particular, acetylhexosamine and aldopentose / aldohexose are identified as two groups. (4) By performing the heat treatment under the condition where the mold exists, some change occurs in the state of the carboxyl group on the filler surface,
It is presumed that the interaction with the amide group changed.

In FIG. 2, the symbols on the vertical and horizontal axes represent the following meanings.

[Table 1]

[0013]

Example 2 Using D-galactose (Gal) and L-galactose under the following conditions using vinylpyridine as the host monomer and high performance liquid chromatography using the polymer particles described in Example 1 as a filler under the following conditions: The saccharide identification was examined using a sugar mixture of arabinose (Ara), D-glucose (Glc), D-xylose (Xyl), D-mannose (Man) and D-lyxose (Lyx). Chromatography conditions : Mobile phase: 85% acetonitrile aqueous solution Flow rate: 1 ml / min Column size: 4.6 mm (id) × 150 mm Column temperature: 50 ° C. Detection: RI Injection amount: 140 nmol The results are shown in Table 2. As shown in Table 2 below, in the case where vinylpyridine was used as the host monomer, there was a tendency for the saccharides to be identified by the group as in the case where methacrylic acid was used as the host monomer.

[Table 2]

[0014]

Example 3 When the change in ion exchange capacity of a polymer using methacrylic acid as a host monomer was measured with the heating time, as shown in Table 3 below, a decrease in the ion exchange capacity was observed as the heating time was increased. Was done. The conditions for measuring the ion exchange capacity are as follows. The polymer was packed in a 4.6 mm inner diameter, 150 mm long column. Next, a 0.1N aqueous potassium chloride solution was passed through the column, and the eluted solution was titrated with a 0.1N aqueous potassium hydroxide solution to determine the ion exchange capacity.

[Table 3]

With respect to the polymer using methacrylic acid as the host monomer, it was confirmed by the FT-IR spectrum that no change in the composition of the polymer due to the heat treatment was observed.

[0016]

Example 4 An aqueous solution of acetic acid having a concentration of 1.7 μmol / L,
Using an aqueous solution containing 1.7 μmol / L of acetic acid and 0.17 μmol / L of D-glucose,
The V spectrum was measured. Further, a polymethacrylic acid aqueous solution having a concentration of 47 mg / L, and a polymethacrylic acid 47
Using an aqueous solution containing mg / L and D-glucose 58 μmol / L, the UV spectra of both were measured in the same manner. The results are shown in FIGS. The following can be seen from both figures. (1) In the absorption band due to the n → π ° transition of polymethacrylic acid, the presence of glucose in water shows a light color shift and a dark color effect, and such a remarkable difference is not seen in monomeric acetic acid. Was. (2) Polymethacrylic acid and glucose may interact with each other by hydrogen bonding even in water.

The effect of the heating time on the saccharides and functional groups on the discrimination ability was examined. As shown in Table 4, the heat treatment using a saccharide as a template was relatively advantageous with respect to the retention of monosaccharides and amines. Showed a tendency to become.
It is presumed that although the ion exchange group disappeared by the heat treatment, the decrease was suppressed by the presence of the template, or the ion exchange group remained concentrated at a certain site.

[Table 4] The measurement conditions of each substance in Table 4 are as follows. (1) In the case of D-xylose, D-glucose, N-acetyl-D-glucosamine Mobile phase: 98% acetonitrile aqueous solution Flow rate: 1 mL / min Column size: 4.6 mm (id) × 150 mm Column temperature: 30 C. Detection: RI Injection amount: 140 nmol (2) In the case of triethylamine and diethylenetriamine Mobile phase: pH 4.8 citrate buffer Flow rate: 1 mL / min Column size: 4.6 mm (id) × 150 mm Column temperature: 30 ° C. Detection: RI Injection amount: 140 nmol

[0018]

The packing material for chromatography comprising the polymer gel of the present invention prepared using sugar as a template exhibits greater selectivity for sugars. Moreover, the selectivity is not only for identifying the template, but for the aldpentose /
Aldohexose and N-acetylhexosamine were each identified as two groups. Therefore, it is considered that the present invention makes it possible to provide a filler having high selectivity in template polymerization of sugar, and greatly contributes to not only analysis of saccharide but also separation of complex saccharides in general.

[0019]

[Brief description of the drawings]

FIG. 1 is a diagram illustrating steps of a molecular imprint method.

FIG. 2 is a graph showing the ability of a polymer gel filler prepared by heat treatment using methacrylic acid as a host monomer to recognize sugar.

FIG. 3 is a UV spectrum showing recognition of a sugar by acetic acid.

FIG. 4 is a UV spectrum showing recognition of sugar by polymethacrylic acid.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C08F 226/06 C08F 226/06 C08J 3/12 C08J 3/12 Z

Claims (17)

[Claims] 1. A method for producing a polymer gel, wherein a polymer obtained by polymerization using a monomer, a crosslinking agent and a solvent as raw materials has a uniform particle size. 2. A method for producing a polymer gel, comprising subjecting a polymer obtained by polymerization using a monomer, a cross-linking agent and a solvent to a raw material, by adsorbing saccharide and subjecting the polymer to heat treatment. 3. The method according to claim 1, wherein the sugar is selected from the group consisting of galactose, glucose and mannose.
The production method described in 1. 4. The method according to claim 1, wherein the monomer is methacrylic acid or 4-vinylpyridine. 5. The method according to claim 1, wherein the crosslinking agent is ethylene dimethacrylate or methylene bisacrylamide. 6. The method according to claim 1, wherein the solvent is toluene. 7. A packing material for chromatography comprising a polymer gel obtained by the production method according to claim 1 or 2. 8. The packing material for chromatography according to claim 7, wherein the sugar is selected from the group consisting of galactose, glucose and mannose. 9. The packing material for chromatography according to claim 7, wherein the monomer is methacrylic acid or 4-vinylpyridine. 10. The packing material for chromatography according to claim 7, wherein the crosslinking agent is ethylene dimethacrylate or methylene bisacrylamide. 11. The packing material for chromatography according to claim 7, wherein the solvent is toluene. 12. A method for modifying the surface of a polymer gel filler, wherein the polymer obtained by polymerization using a monomer, a crosslinking agent and a solvent as raw materials has a uniform particle size. 13. A method for modifying the surface of a polymer gel filler, which comprises subjecting a polymer obtained by polymerization using a monomer, a crosslinking agent, and a solvent to a raw material, to adsorb sugar and subjecting the polymer to heat treatment. 14. The method according to claim 12, wherein the sugar is selected from the group consisting of galactose, glucose and mannose. 15. The surface modification method according to claim 12, wherein the monomer is methacrylic acid or 4-vinylpyridine. 16. The surface modification method according to claim 12, wherein the crosslinking agent is ethylene dimethacrylate or methylene bisacrylamide. 17. The surface modification method according to claim 12, wherein the solvent is toluene.